Staining In Microscopy - Stains And Techniques From Wikipedia 52403

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=494-38-2 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=62344 [3] http:/ / www. chemspider. com/ 56136 [4] http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=200-614-0 [5] http:/ / www. kegg. jp/ entry/ C19315 [6] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Acridine+ orange [7] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=234241 [8] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL81880 [9] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=n1c3c%28cc2c1cc%28N%28C%29C%29cc2%29ccc%28c3%29N%28C%29C [10] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=477241097& page2=%3AAcridine+ orange

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Azurophilic granule

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Azurophilic granule Azurophilic granule Latin granulum azurophilum Code TH H2.00.04.1.02011 [1] [1] TH H2.00.04.1.02014

An azurophil is an object readily stained with an azure stain. in white blood cells and hyperchromatin, imparting a burgundy or merlot coloration. Neutrophils in particular are known for containing azurophils loaded with a wide variety of anti-microbial defensins that fuse with phagocytic vacuoles. Azurophils may contain myeloperoxidase, phospholipase A2, Acid Hydrolases, Elastase, defensins, neutral serine proteases, [2] bactericidal/permeability-increasing protein, lysozyme, cathepsin G, proteinase 3, and proteoglycans. Azurophil granules are also known as "primary granules".[3]

References [1] http:/ / www. unifr. ch/ ifaa/ Public/ EntryPage/ ViewTH/ THh200. html [2] "Phagocytes-Neutrophils" (http:/ / www. dent. ucla. edu/ pic/ / neutrophils/ neutrophils2. html). . [3] John P. Greer; Maxwell Myer Wintrobe (1 December 2008). Wintrobe's clinical hematology (http:/ / books. google. com/ books?id=68enzUD7BVgC& pg=PA173). Lippincott Williams & Wilkins. pp. 173–. ISBN 978-0-7817-6507-7. . Retrieved 10 November 2010.

External links • Histology at ucsf.edu (http://missinglink.ucsf.edu/lm/IDS_101_histo_resource/images/neutrophil100x_copy. jpg-labelled.jpg)

Basophilic

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Basophilic Basophilic is a technical term used by histologists. It describes the microscopic appearance of cells and tissues, as seen down the microscope, after a histological section has been stained with a basic dye. The most common such dye is haematoxylin. Basophilic describes the appearance of structures seen in histological sections which take up basic dyes. The structures usually stained are those that contain nucleic acid such as the cell nucleus and ribosomes. Basophils are cells that "love" base, and which usually show up deep blue under standard staining techniques (H&E). Specifically, this term refers to: • basophil granulocytes • anterior pituitary basophils

Bismarck brown Bismarck brown may refer to: • Bismarck brown R, basic brown 4 • Bismarck brown Y, basic brown 1

A Basophil granulocyte stains dark purple upon H&E staining.

Bismarck brown Y

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Bismarck brown Y Bismarck brown Y

Identifiers [1]

CAS number

8005-77-4

PubChem

13981

ChemSpider

13374

Jmol-3D images

Image 1 [5] Image 2

[2] [3]

[4]

Properties Molecular formula

C21H24N8

Molar mass

388.47 g mol

−1

()

[6]

(what is: / ?) Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)

Infobox references

Bismarck brown Y is a diazo dye. It is used in histology for staining tissues. It stains acid mucins to yellow color. It can be used with live cells. It is also used to stain cartilage in bone specimens, as one of Kasten's Schiff-type reagents in the periodic acid-Schiff stain to stain cellulose, and in Feulgen stain to stain DNA. It was more common in the past; today it is partially replaced by other stains. Bismarck brown Y is a constituent of Papanicolaou stains. It can also be used as a counterstain for Victoria blue R for staining of acid-fast microorganisms.

References [1] [2] [3] [4]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=8005-77-4 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=13981 http:/ / www. chemspider. com/ 13374 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CC1%3DC%28C%3DC%28C%3DC1%29N%3DNC2%3DC%28C%3DC%28C%28%3DC2%29C%29N%29N%29N%3DNC3%3DC%28C%3DC% [5] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=N%28%3DN%2Fc1ccc%28cc1N%29N%29%5Cc3cccc%28%2FN%3DN%2Fc2ccc%28N%29cc2N%29c3 [6] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=459979781& page2=%3ABismarck+ brown+ Y

Carmine

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Carmine Carmine ( /ˈkɑrmɪn/ or /ˈkɑrmaɪn/), also called Crimson Lake, Cochineal, Natural Red 4[1], C.I. 75470[1], or E120, is a pigment of a bright-red color obtained from the aluminum salt of carminic acid, which is produced by some scale insects, such as the cochineal scale and the Polish cochineal, and is used as a general term for a particularly deep-red color of the same name. Carmine is used in the manufacture of artificial flowers, paints, Carminic acid crimson ink, rouge, and other cosmetics, and is routinely added to food products such as yogurt and certain brands of juice, the most notable ones being those of the ruby-red variety. To prepare carmine, the powdered scale insect bodies are boiled in ammonia or a sodium carbonate solution, the insoluble matter is removed by filtering, and alum is added to the clear salt solution of carminic acid to precipitate the red aluminium salt, called "carmine lake" or "crimson lake." Purity of color is ensured by the absence of iron. Stannous chloride, citric acid, borax, or gelatin may be added to regulate the formation of the precipitate. For shades of purple, lime is added to the alum; thus, the traditional crimson color is guaranteed not only by carminic acid but also by choice of its chelating metal salt ion.[2]

Carmine

Etymology The English word "carmine" is derived from the French word carmin (12 c.), from Medieval Latin carminium, from Arabic qirmiz "crimson," from Sanskrit krimiga "insect-produced", from krmi "worm, insect". Influenced in Latin by minium "red lead, cinnabar", said to be of Iberian origin.[3]

Production Carmine may be prepared from cochineal,[4] by boiling dried insects in water to extract the carminic acid and then treating the clear solution with alum. Other substances such as cream of tartar, stannous chloride, or potassium hydrogen oxalate can also be used to effect the precipitation, but aluminum is needed for the color. Use of these chemicals causes the coloring and animal matters present in the liquid to be precipitated to give a lake pigment. Aluminum from the alum gives the traditional crimson color to carminic acid precipitates, which are called carmine lakes or crimson lakes. This color is degraded by the presence of iron salts. Addition of lime (calcium) can give carminic acid lakes a purple cast.[2]

A cluster of Dactylopius coccus females growing in Barlovento, La Palma, Canary Islands.

Carmine

Other methods for the production of carmine dye are in use, in which egg white, fish glue, or gelatine is sometimes added before the precipitation. The carminic acid used to produce the pigment can also be extracted from various microbes engineered for the purpose. Microbes are dissolved in a containment structure separate from their cultivation vats, and then allowed to settle out. The liquid and suspended carminic acid is then siphoned off, and metal salts are then added to give a lake pigment in a procedure that is mostly identical to the procedure for acid extracted from insects. Zapotec nests on Opuntia ficus-indica host cacti. The quality of carmine is affected by the temperature and the degree of illumination during its preparation, sunlight being requisite for the production of a brilliant hue. It also differs according to the amount of alumina present in it. It is sometimes adulterated with cinnabar, starch and other materials; from these, the carmine can be separated by dissolving it in ammonia. Good carmine should crumble readily between the fingers when dry.

Carmine can be used as a staining agent in microbiology, as a Best's carmine to stain glycogen, mucicarmine to stain acidic mucopolysaccharides, and carmalum to stain cell nuclei. In these applications, it is applied together with a mordant, usually an Al(III) salt.

Allergy Carmine is used as a food dye in many different products such as juices, ice cream, yogurt, and candy, and as a dye in cosmetic products such as eyeshadow and lipstick. Although principally a red dye, it is found in many foods that are shades of red, pink, and purple. As a food dye it has been known to cause severe allergic reactions and anaphylactic shock in some people.[5][6]

Regulations for use in foods United States In January 2006, the United States Food and Drug istration evaluated a proposal that would require food products containing carmine to list it by name on the ingredient label.[7] It was also announced that the FDA will separately review the ingredient labels of prescription drugs that contain colorings derived from carmine. A request from the Center for Science in the Public Interest (article titled: "FDA Urged to Improve Labeling of or Ban Carmine Food Coloring"[8][9]) to require ingredient labels to explicitly state that carmine may cause severe allergic reactions and anaphylactic shock and that it is derived from insects was declined by the FDA. Food industries were aggressively opposed to the idea of writing "insect-based" on the label, and they finally agreed to putting simply "carmine." Carmine is approved as dye for foodstuffs. In January 2009, FDA ed a new regulation[10] requiring carmine and cochineal to be listed by name on the label. Although concerns over hazards from allergic reactions have been asserted,[11] the FDA has not banned the use of carmine and states it found no evidence of a "significant hazard" to the general population.[12] As with many chemical compounds, the dye may still pose an allergen hazard to a subset of the population.

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Carmine

European Union In the European Union, the use of carmine in foods is regulated under the European Commission's directives governing food additives in general[13][14] and food dyes in particular[15] and listed under the names Cochineal, Carminic acid, Carmines and Natural Red 4 as additive E 120 in the list of EU-approved food additives.[16] The directive governing food dyes approves the use of carmine for certain groups of foods only[17] and specifies a maximum amount which is permitted or restricts it to the quantum satis. The EU-Directive 2000/13/EC[18] on food labeling mandates that carmines (like all food additives) must be included in the list of ingredients of a food product with its additive category and listed name or additive number, that is either as Food colour carmines or as Food colour E 120 in the local language(s) of the market(s) the product is sold in. Although concerns of hazards from allergic reactions were raised, the use of carmine in foods is not banned in the EU. However, the use of carmine in foods has been discouraged by European Food Safety Authorities, and, although it is used predominantly as coloring in alcoholic beverages, it can still be found in foods such as supermarket Indian curries. A re-evaluation process of the approval status of several food colors (including carmine) was started by the on food additives, flavourings, processing aids and materials in with food of the EFSA in early 2006 and is scheduled to be completed by 2008.[19][20] As of January, 2012, the European Food Safety Authority (EFSA) has changed the way they manufacture Carmine E120 for pharmaceutical products. The EFSA had raised concerns, over the increasing number of allergic reactions to Carmine derived from insects (E120.360), when used within the British pharmacopeia. Pharmaceutical products which had previously contained insect-derived carmine, have been replaced with a synthesised version of the food colorant. Internal studies have shown that the new formulations, of popular anti-nausea and weight-gain liquid medication, had a significantly lower risk in of allergic reactions. The new formulation is known to be of plant origin, using calcium oxide in order to gauge colour depth.

References [1] Dapson, R.; Frank, M.; Penney, D.; Kiernan, J. (2007). "Revised procedures for the certification of carmine (C.I. 75470, Natural red 4) as a biological stain". Biotechnic & Histochemistry 82: 13. doi:10.1080/10520290701207364. [2] Threads In Tyme, LTD. "Time line of fabrics" (http:/ / web. archive. org/ web/ 20051028155009/ http:/ / threadsintyme. tripod. com/ id63. htm). Archived from the original (http:/ / threadsintyme. tripod. com/ id63. htm) on 2005-10-28. . Retrieved July 14, 2005. [3] http:/ / www. etymonline. com/ index. php?term=carmine [4] http:/ / www. food-info. net/ uk/ e/ e120. htm [5] Dr J. B. Greig. "WHO FOOD ADDITIVES SERIES 46:COCHINEAL EXTRACT, CARMINE, AND CARMINIC ACID" (http:/ / www. inchem. org/ documents/ jecfa/ jecmono/ v46je03. htm). Food Standards Agency. . Retrieved 2010-09-02. "The nature of the adverse reactions, e.g. urticaria, rhinitis, diarrhoea, and anaphylaxis, provides clear evidence that systemic reactions can follow exposure of a sensitized individual to cochineal colours." [6] A.I. Tabar et al.. "Asthma and allergy due to carmine dye; PMID 13679965; An Sist Sanit Navar. 2003;26 Suppl 2:65–73." (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 13679965). U.S. National Library of Medicine. . Retrieved 2010-09-02. [7] Dye may cause some consumers to bug out (http:/ / media. www. thecowl. com/ media/ storage/ paper493/ news/ 2006/ 02/ 02/ World/ Dye-May. Cause. Some. Consumers. To. Bug. Out-1597165. shtml). [8] http:/ / www. cspinet. org/ new/ 200605011. html [9] http:/ / www. cspinet. org/ new/ carmine_8_24_98. htm [10] http:/ / www. foodnavigator-usa. com/ Product-Categories/ Flavors-and-colors/ New-labeling-rules-for-cochineal-bug-coloring New labeling rules for cochineal bug coloring. [11] A.I. Tabar et al., Asthma and allergy due to carmine dye (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 13679965); PMID 13679965; An Sist Sanit Navar. 2003;26 Suppl 2:65–73. [12] . http:/ / web. archive. org/ web/ 20060210231346/ http:/ / www. cnn. com/ 2006/ US/ 01/ 27/ insect. dye. ap/ index. html. [13] http:/ / www. foodlaw. rdg. ac. uk/ additive. htm [14] http:/ / www. fsai. ie/ legislation/ food/ eu_docs/ Food_additives/ General_provisions/ Dir%20292. 97%20EC. pdf [15] http:/ / www. fsai. ie/ legislation/ food/ eu_docs/ Food_additives/ Dir94. 36. pdf [16] http:/ / www. food. gov. uk/ safereating/ chemsafe/ additivesbranch/ enumberlist [17] a list of approved uses is included in Annexes I and III of EU-Directive 94/36 (http:/ / www. fsai. ie/ legislation/ food/ eu_docs/ Food_additives/ Dir94. 36. pdf)

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Carmine [18] http:/ / www. fsai. ie/ legislation/ food/ eu_docs/ Labelling/ General%20Labelling%20Provisions%20for%20Foodstuffs/ Dir%202000. 13%20EC. pdf [19] (http:/ / www. efsa. europa. eu/ en/ science/ afc. html) Accessed on 2 January 2007 [20] http:/ / www. efsa. europa. eu/ etc/ medialib/ efsa/ science/ afc. Par. 0001. File. dat/ afc_food_colours_%20re-evaluation_%20call%20for%20data. pdf

This article incorporates text from a publication now in the public domain: Chisholm, Hugh, ed. (1911). Encyclopædia Britannica (11th ed.). Cambridge University Press.

Further reading • Greenfield, Amy Butler (2005). A perfect red: Empire, espionage, and the quest for the color of desire. New York: HarperCollins. ISBN 0-06-052275-5.

External links • Center for Science in the Public Interest press release Bug-Based Food Dye Should Be ... Exterminated, Says CSPI (http://www.cspinet.org/new/200605011.html) • LaVerne M. Dutton. "Cochineal: A Bright Red Animal Dye" (http://www.cochineal.info/). Master's Thesis for Baylor University. Retrieved November 13, 2010.

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Chromophobe cell

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Chromophobe cell Chromophobe cell Code TH H3.08.02.2.00018 [1]

The term chromophobe refers to histological structures which do not stain readily, and thus appear more relatively pale under the microscope—hence their "fear" ("phobia") of "color" ("chrome").

Cancer "Chromophobe" also refers to a type of renal cell carcinoma (distinct from "clear cell").[2]

References [1] http:/ / www. unifr. ch/ ifaa/ Public/ EntryPage/ ViewTH/ THh308. html [2] ACS :: What Is Kidney Cancer (Renal Cell Carcinoma)? (http:/ / www. cancer. org/ docroot/ cri/ content/ cri_2_4_1x_what_is_kidney_cancer_22. asp)

External links • Chromophobe (http://www.emedicinehealth.com/script/main/srchcont_dict.asp?src=Chromophobe) at eMedicine Dictionary

Coomassie Brilliant Blue Coomassie Brilliant Blue R-250

Identifiers CAS number

6104-59-2

PubChem

61365

Jmol-3D images

Image 1

[1]

[2] [3]


C H N NaO S (Sodium salt)

Molar mass

825.97 g/mol

Solubility in water

Insoluble in cold, slightly soluble in hot (bright red blue)

Solubility in ethanol

Slightly soluble

45 44 3

7 2

Coomassie Brilliant Blue

35

()

[4]


Infobox references

Coomassie Brilliant Blue G-250


6104-58-1

PubChem

6324599

Jmol-3D images

Image 1

[5]

[6]

[7]


C47H50N3NaO7S2 (Sodium salt)

Molar mass

856.03 g/mol

Solubility in water

Slightly soluble in cold, soluble in hot (bright blue)

Solubility in ethanol

Soluble

Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa) Infobox references

Coomassie Brilliant Blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups. The name "Coomassie" is a ed trademark of Imperial Chemical Industries.

Name and discovery The name Coomassie was adopted at the end of the 19th century as a trade name by the Blackley based dye manufacturer Levinstein Ltd, in marketing a range of acid wool dyes.[8] In 1896 during the Fourth Anglo-Ashanti War, British forces had occupied the town of Coomassie (modern-day Kumasi in Ghana). In 1918 Levinstein Ltd became part of British Dyestuffs which in 1926 became part of Imperial Chemical Industries.[9] Although ICI still owns the Coomassie trademark, the company no longer manufactures the dyes. The blue disulfonated triphenylmethane dyes were first produced in 1913 by Max Weiler who was based in Elberfeld, .[10] Various patents were subsequently taken out on the organic synthesis.[11][12][13] Papers published in biochemistry journals frequently refer to these dyes simply as "Coomassie" without specifying which dye was actually used. In fact the Colour Index lists over 40 dyes with "Coomassie" in their name. There are also other Coomassie "blue" dyes. For example, the Merck Index (10th edition) lists Coomassie Blue RL (Acid Blue 92, C.I. 13390) which has a completely different structure.


Dye colour The suffix "R" in the name of Coomassie Brilliant Blue R-250 is an abbreviation for Red as the blue colour of the dye has a slight reddish tint. For the "G" variant the blue colour has a more greenish tint. The "250" originally denoted the purity of the dye. The colour of the two dyes depends on the acidity of the solution. The "G" form of the dye has been studied in detail.[14] At a pH of less than 0 the dye has a red colour with an absorption maximum at a wavelength of 470 nm. At a pH of around 1 the dye is green with an absorption maximum at 620 nm while above pH 2 the dye is bright blue with a maximum at 595 nm. At pH 7 the dye has an extinction coefficient of 43,000 M−1cm−1.[14] The different colours are a result of the different charged states of the dye molecule. In the red form, all three nitrogen atoms carry a positive charge. The two sulfonic acid groups have extremely low pKa's and will normally be negatively charged, thus at a pH of around zero the dye will be a cation with an overall charge of +1. The green colour corresponds to a form of the dye with no net overall charge. At neutral pH (pH 7), only the nitrogen atom of the diphenylamine moiety carries a positive charge and the blue dye molecule is an anion with an overall charge of -1. The pKa's for the loss of the two protons are 1.15 and 1.82. The final proton is lost under alkaline conditions and the dye becomes pink in colour (pKa 12.4).[14] The dye molecules bind to proteins including wool (keratin) to form a protein-dye complex. The formation of the complex stabilises the negatively charged anionic form of the dye producing the blue colour, even under acid conditions when most of the molecules in solution are in the cationic form.[14] This is the basis of the Bradford assay which is used to quantify the concentration of protein in a solution. The dye also forms a complex with the anionic detergent sodium dodecylsulfate.[15] The formation of this complex stabilises the neutral green form of the dye. This effect can interfere with the estimation of protein concentration using the Bradford assay. It is also likely that the anionic detergent competes with the dye for binding to the protein.

Applications in biochemistry Coomassie Brilliant Blue R-250 was first used to visualise proteins in 1963 by Fazekas de St. Groth and colleagues.[16] Protein samples were separated electrophoretically on a cellulose acetate sheet. The sheet was then soaked in sulfosalicylic acid to fix the protein bands and then transferred to a solution of the dye. Two years later in 1965 Meyer and Lambert used Coomassie Brilliant Blue R-250 to stain protein samples after electrophoretic separation in a polyacrylamide gel.[17] They soaked the gel in a dye solution containing methanol, acetic acid and water. As the dye stained the polyacrylamide gel as well as the protein, to visualise the protein bands they needed to destain the gel which they did electrophoretically. Subsequent publications reported that polyacrylamide gels could be successfully destained using an acetic acid solution. The first report of the use of the "G" form of the dye to visualise protein bands in polyacrylamide gels came in 1967, where the dye was dissolved in an acetic acid solution containing methanol.[18] It was subsequently discovered that the protein bands could be stained without staining the polyacrylamide by using a colloid of the "G" form of the dye in a trichloroacetic acid solution containing no methanol. Using this procedure it was no longer necessary to destain the gel.[19] Modern formulations typically use a colloid of the "G" form of dye in a solution containing phosphoric acid, ethanol (or methanol) and ammonium sulfate (or aluminium sulfate).[20][21][22][23] The Bradford assay uses the spectral properties of Coomassie Brilliant Blue G-250 to estimate the amount of protein in a solution.[24] A protein sample is added to a solution of the dye in phosphoric acid and ethanol. Under the acid conditions the dye is normally a brownish colour but on binding to the protein the blue form of the dye is produced. The optical absorbance of the solution is measured at a wavelength of 595 nm. On binding to a protein the negatively charged Coomassie Brilliant Blue G-250 dye molecule will give an overall negative charge to the protein. This property can be used to separate proteins or protein complexes using polyacrylamide gel electrophoresis under non-denaturing conditions in a technique called Blue Native PAGE.[25][26]

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The mobility of the complex in the polyacrylamide gel will depend on both the size of the protein complex (i.e. the molecular weight) and on the amount of dye bound to the protein.

Medical uses Brilliant Blue G has recently been used in scientific experiments to treat spinal injuries in laboratory rats.[27] It acts by reducing the body's natural swelling response, which can cause neurons in the area to die of metabolic stress. Testing is still in progress to determine if this treatment can be used effectively in humans. The recent tests have istered the dye within 15 minutes of injury, but to be effective in a real-life setting, where it may take time for a patient to reach the emergency room, the treatment should be effective even when istered up to two hours after injury. The only reported side effect was that the rats temporarily turned blue.[28][29][27] Under the trade name Brilliant Peel, Brilliant Blue G is used as a stain to assist surgeons in retinal surgery.[30]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=6104-59-2 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=61365

[3] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CCN%28CC1%3DCC%28%3DCC%3DC1%29S%28%3DO%29%28%3DO%29%5BO-%5D%29C2%3DCC%3DC%28C%3DC2%29C%28%3DC3 %5BNa%2B%5D [4] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=455133631& page2=%3ACoomassie+ Brilliant+ Blue [5] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=6104-58-1 [6] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=6324599 [7] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CCN%28CC1%3DCC%28%3DCC%3DC1%29S%28%3DO%29%28%3DO%29%5BO-%5D%29C2%3DCC%28%3DC%28C%3DC2%29C%28%3 %5BNa%2B%5D [8] Fox, M. R. (1987). Dye-makers of Great Britain 1856-1976 : A History of Chemists, Companies, Products and Changes. Manchester: Imperial Chemical Industries. p. 38. [9] Fox, M. R. (1987). Dye-makers of Great Britain 1856-1976 : A History of Chemists, Companies, Products and Changes. Manchester: Imperial Chemical Industries. p. 259. [10] (pdf) Colour Index (http:/ / www. colour-index. org/ help/ 3121_Triarylmethane. pdf). 4 (3rd ed.). Bradford: Society of Dyers and Colourists. 1971. pp. 4397–4398. . [11] FR patent 474260 (http:/ / worldwide. espacenet. com/ textdoc?DB=EPODOC& IDX=FR474260), "Procédé de production de colorants de la série du triarylméthane", issued 1915-02-16, assigned to Bayer [12] US patent 1218232 (http:/ / worldwide. espacenet. com/ textdoc?DB=EPODOC& IDX=US1218232), Weiler, Max, "Blue Triphenylmethane Dye", issued 1917-03-06 [13] GB patent 275609 (http:/ / worldwide. espacenet. com/ textdoc?DB=EPODOC& IDX=GB275609), "Manufacture of Triarylmethane-dyestuffs", issued 1927-11-03, assigned to IG Farbenindustrie [14] Chial, H. J.; Thompson, H. B.; Splittgerber, A. G. (1993). "A spectral study of the charge forms of Coomassie Blue G". Analytical Biochemistry 209 (2): 258–266. doi:10.1006/abio.1993.1117. PMID 7682385. [15] Compton, S. J.; Jones, C. G. (1985). "Mechanism of dye response and interference in the Bradford protein assay". Analytical Biochemistry 151 (2): 369–374. doi:10.1016/0003-2697(85)90190-3. PMID 4096375. [16] Fazekas de St. Groth, S.; Webster, R. G.; Datyner, A. (1963). "Two new staining procedures for quantitative estimation of proteins on electrophoretic strips". Biochimica et Biophysica Acta 71: 377–391. doi:10.1016/0006-3002(63)91092-8. PMID 18421828. [17] Meyer, T. S.; Lambert, B. L. (1965). "Use of Coomassie brilliant blue R250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide-gel strips". Biochimica et Biophysica Acta 107 (1): 144–145. doi:10.1016/0304-4165(65)90403-4. PMID 4159310. [18] Altschul, A. M.; Evans, W. J. (1967). "Zone electrophoresis with polyacrylamide gel". Methods in Enzymology 11: 179–186. doi:10.1016/S0076-6879(67)11019-7.. Page 184 personal communication from W. J. Saphonov. [19] Diezel, W.; Kopperschläger, G.; Hofmann, E. (1972). "An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue". Analytical Biochemistry 48 (2): 617–620. doi:10.1016/0003-2697(72)90117-0. PMID 4115985. [20] Neuhoff, V.; Stamm, R.; Eibl, H. (1985). "Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis". Electrophoresis 6 (9): 427–448. doi:10.1002/elps.1150060905. [21] Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L. et al. (2004). "Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis". Electrophoresis 25 (9): 1327–1333. doi:10.1002/elps.200305844. PMID 15174055.

Coomassie Brilliant Blue [22] Steinberg, T. H. (2009). "Protein gel staining methods: an introduction and overview". Methods in Enzymology 463: 541–563. doi:10.1016/S0076-6879(09)63031-7. PMID 19892191. [23] Pink, M.; Verma, N.; Rettenmeier, A. W.; Schmitz-Spanke, S. (2010). "CBB staining protocol with higher sensitivity and mass spectrometric compatibility". Electrophoresis 31 (4): 593–598. doi:10.1002/elps.200900481. PMID 20162584. [24] Bradford, M. M. (1976). "Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding". Analytical Biochemistry 72: 248–254. doi:10.1016/0003-2697(76)90527-3. PMID 942051. [25] Schägger, H.; Jagow, G. (1991). "Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form". Analytical Biochemistry 199 (2): 223–231. doi:10.1016/0003-2697(91)90094-A. PMID 1812789. [26] Wittig, I.; Braun, H. P.; Schägger, H. (2006). "Blue native PAGE". Nature Protocols 1 (1): 418–428. doi:10.1038/nprot.2006.62. PMID 17406264. [27] Peng, W.; Cotrina, M. L.; Han, X.; Yu, H.; Bekar, L.; Blum, L.; Takano, T.; Tian, G. F. et al. (2009). "Systemic istration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury". Proceedings of the National Academy of Science of the United States of America 106 (30): 12489–12493. doi:10.1073/pnas.0902531106. PMC 2718350. PMID 19666625. [28] "Blue M&Ms 'mend spinal injuries'" (http:/ / www. telegraph. co. uk/ science/ science-news/ 5921266/ Blue-MandMs-mend-spinal-injuries. html). Telegraph. 2009-07-28. . Retrieved 2010-01-19. [29] "Blue Food Dye Treats Spine Injury in Rats" (http:/ / www. wired. com/ wiredscience/ 2009/ 07/ bluerats/ ). Wired.com. 2009-07-27. . Retrieved 2010-01-19. [30] Mennel, S.; Meyer, C. H.; Schmidt, J. C.; Kaempf, S.; Thumann, G. (2008). "Trityl dyes patent blue V and brilliant blue G - clinical relevance and in vitro analysis of the function of the outer blood-retinal barrier". Developments in Ophthalmology 42: 101–114. doi:10.1159/000138988. PMID 18535384.

Further reading • Gessner, T.; Mayer, U. (2002), "Triarylmethane and Diarylmethane Dyes", Ullmann's Encyclopedia of Industrial Chemistry 6th Edition, Weinheim: Wiley-VCH, doi:10.1002/14356007.a27_179

External links • BBC News - Food dye 'may ease spinal injury' (http://news.bbc.co.uk/1/hi/health/8170033.stm)

38

Counterstain

39

Counterstain A counterstain is a stain with color contrasting to the principal stain, making the stained structure more easily visible. An example is the malachite green counterstain to the fuchsine stain in the Gimenez staining technique. Another example is eosin counterstain to haematoxylin in the H&E stain. Also in Gram staining, crystal violet stains only Gram-positive bacteria, and safranin counterstain is applied which stains all cells, even allowing the identification of Gram-negative bacteria as well. An alternative method uses dilute carbofluozide.

Gram-positive anthrax bacteria with counterstained white blood cells

Cryostat A cryostat (from cryo meaning cold and stat meaning stable) is a device used to maintain cold cryogenic temperatures of samples or devices mounted within the cryostat. Low temperatures may be maintained within a cryostat by using various refrigeration methods, most commonly using cryogenic fluid bath such as liquid helium. Hence it is usually assembled into a vessel, similar in construction to a vacuum flask or Dewar. Cryostats have numerous applications within science, engineering, and medicine.

Types

NASA's WISE infrared instrument is kept cold by a cryostat. The cryostat can be seen at the top of the spacecraft.

Closed-cycle cryostats Closed-cycle cryostats consist of a chamber through which cold helium vapour is pumped. An external mechanical refrigerator extracts the warmer helium exhaust vapour, which is cooled and recycled. Closed-cycle cryostats consume a relatively large amount of electrical power, but need not be refilled with helium and can run continuously for an indefinite period. Objects may be cooled by attaching them to a metallic coldplate inside a vacuum chamber which is in thermal with the helium vapour chamber.

Continuous-flow cryostats Continuous-flow cryostats are cooled by liquid cryogens (typically liquid helium or nitrogen) from a storage dewar. As the cryogen boils within the cryostat, it is continuously replenished by a steady flow from the storage dewar. Temperature control of the sample within the cryostat is typically performed by controlling the flow rate of cryogen into the cryostat together with a heating wire attached to a PID temperature control loop. The length of time over which cooling may be maintained is dictated by the volume of cryogens available. Owing to the scarcity of liquid helium, some laboratories have facilities to capture and recover helium as it escapes from the cryostat, although these facilities are also costly to operate.

Cryostat

Bath cryostats Bath cryostats are similar in construction to vacuum flasks filled with liquid helium. A coldplate is placed in thermal with the liquid helium bath. The liquid helium may be replenished as it boils away, at intervals between a few hours and several months, depending on the volume and construction of the cryostat. The boil-off rate is minimised by shielding the bath with either cold helium vapour, or vacuum shield with walls constructed from so-called super insulator material. The helium vapour which boils away from the bath very effectively cools thermal shields around the outside of the bath. In the older designs there may be additional liquid nitrogen bath, or several concentric layers of shielding, with gradually increasing temperatures. However, the invention of super insulator materials has obsoleted this technology.

Multistage cryostats In order to achieve temperature lower than liquid helium additional cooler stages may be added to the cryostat. Temperatures down to 1K can be reached by attaching the coldplate to 1-K pot, which is a container of He-4 isotope which is connected to vacuum pump. Temperatures down to 1mK can be reached by employing dilution refrigerator or dry dilution refrigerator typically in addition to the main stage and 1K pot. Temperatures below that can be reached using magnetic refrigeration.

Applications Magnetic Resonance Imaging and Research magnet types Cryostats used in MRI machines are designed to hold a cryogen, typically helium, in a liquid state with minimal evaporation (boil-off). The liquid helium bath is designed to keep the superconducting magnet's bobbin of superconductive wire in its superconductive state. In this state the wire has no electrical resistance and very large currents are maintained with a low power input. To maintain superconductivity, the bobbin must be kept below its transition temperature by being immersed in the liquid helium. If, for any reason, the wire becomes resistive, i.e. loses superconductivity, a condition known as a "quench", the liquid helium evaporates, instantly raising pressure within the vessel. A burst disk, usually made of carbon, is placed within the chimney or vent pipe so that during a pressure excursion, the gaseous helium can be safely vented out of the MRI suite. Modern MRI cryostats use a mechanical refrigerator (cryocooler) to re-condense the helium gas and return it to the bath, to maintain cryogenic conditions and to conserve helium. Typically cryostats are manufactured with two vessels, one inside the other. The outer vessel is evacuated with the vacuum acting as a thermal insulator. The inner vessel contains the cryogen and is ed within the outer vessel by structures made from low-conductivity materials. An intermediate shield between the outer and inner vessels intercepts the heat radiated from the outer vessel. This heat is removed by a cryocooler. Older helium cryostats used a liquid nitrogen vessel as this radiation shield and had the liquid helium in an inner, third, vessel. Nowadays few units using multiple cryogens are made with the trend being towards 'cryogen-free' cryostats in which all heat loads are removed by cryocoolers.

40

Cryostat

Biological microtome type Cryostat are used in medicine to cut histological slides. They are usually used in a process called frozen section histology (see Frozen section procedure). The cryostat is essentially an ultrafine "deli-slicer", called a microtome, placed in a freezer. The cryostat is usually a stationary upright freezer, with an external wheel for rotating the microtome. The temperature can be varied, depending on the tissue being cut - usually from minus 20 to minus 30 degree Celsius. The freezer is either powered by electricity, or by a refrigerant like liquid nitrogen. Small portable cryostats are available and can run off Cryostat-microtome generators or vehicle inverters. To minimize unnecessary warming all necessary mechanical movements of the microtome can be achieved by hand via a wheel mounted outside the chamber. Newer microtomes have electric push button advancement of the tissue. The precision of the cutting is in micrometres. Tissue are sectioned as thin as 1 micrometre. Usual histology slides are mounted with a thickness of about 7 micrometres. Specimens that are soft at room temperature are mounted on a cutting medium (often made of egg white) on a metal "chuck", and frozen to cutting temperature (for example at -20 degrees C). Once frozen, the specimen on the chuck is mounted on the microtome. The crank is rotated and the specimen advances toward the cutting blade. Once the specimen is cut to a satisfactory quality, it is mounted on a warm (room temperature) clear glass slide, where it will instantaneously melt and adhere. The glass slide and specimen is dried with a dryer or air dried, and stained. The entire process from mounting to reading the slide takes from 10 to 20 minutes, allowing rapid diagnosis in the operating room, for the surgical excision of cancer. The cryostat can be used to cut histology and tissue slide outside of medicine, but the quality of the section is poor compared to standard fixed section wax mounted histology.

41

DAPI

42

DAPI DAPI

Identifiers [1]

CAS number

28718-90-3

PubChem

2954

ChemSpider

2848

ChEBI

CHEBI:51231

ChEMBL

CHEMBL48217

Jmol-3D images

Image 1 [7] Image 2

[2] [3]

[4]

[5]

[6]


C16H15N5

Molar mass

277.324 ()

[8]


Infobox references

DAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in fluorescence microscopy. DAPI can through an intact cell membrane therefore it can be used to stain both live and fixed cells, though it es through the membrane less efficiently in live cells and therefore the effectiveness of the stain is lower.

DAPI

43

History DAPI was first synthesised in 1971 in the laboratory of Otto Dann as part of a search for drugs to treat trypanosomiasis although it was unsuccessful as a drug. Further investigation indicated it bound strongly to DNA and became more fluorescent when it did so. This led to its use in identifying mitochondrial DNA in ultracentrifugation in 1975, the first recorded use of DAPI as a fluorescent DNA stain.[9] Strong fluorescence when bound to DNA led to the rapid adoption of DAPI for fluorescent staining of DNA for fluorescence microscopy. Its use for detecting DNA in plant, metazoa and bacteria cells and virus particles was demonstrated in the late 1970s, and quantitative staining of DNA inside cells was demonstrated in 1977. Use of DAPI as a DNA stain for flow cytometry was also demonstrated around this time.[9]

Fluorescence properties When bound to double-stranded DNA DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore for fluorescence microscopy DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad[10] DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.[11] DAPI's blue emission is convenient for microscopists who wish to use multiple fluorescent stains in a single sample. There is some fluorescence overlap between DAPI and green-fluorescent molecules like fluorescein and green fluorescent protein (GFP) but the effect of this is small. Use of spectral unmixing can for this effect if extremely precise image analysis is required.

DAPI (magenta) bound to the minor groove of DNA (green and [12] blue). From PDB 1D30 .

Outside of analytical fluorescence light microscopy DAPI is also popular for labeling of cell cultures to detect the DNA of contaminating mycoplasma or virus. The labelled mycoplasma or virus particles in the growth medium fluoresce once stained by DAPI making them easy to detect.

Live cells and toxicity DAPI can be used for both fixed and live cell staining, though the concentration of DAPI needed for live cell staining is generally much higher than for fixed cells.[13] It is labeled non-toxic in its MSDS[14] and though it was not shown to have mutagenicity to E. coli,[15] it is labelled as a known mutagen in manufacturer information.[10] As it is a DNA binding compound it is likely to have some low level mutagenic properties and care should be taken in its handling and disposal.

DAPI

44

Alternatives The Hoechst stains are similar to DAPI in that they are also blue-fluorescent DNA stains which are compatible with both live- and fixed-cell applications.

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=28718-90-3 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=2954 [3] http:/ / www. chemspider. com/ 2848 [4] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=51231 [5] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL48217 [6] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BN%40H%5D%3DC%28N%29c3ccc%28c2cc1ccc%28cc1n2%29C%28%3D%5BN%40H%5D%29N%29cc3 [7] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BH%5D%2FN%3DC%28%2Fc1ccc%28cc1%29c2cc3ccc%28cc3%5BnH%5D2%29%2FC%28%3DN%2F%5BH%5D%29%2FN%29%5CN Epithelial cells stained with DAPI (blue) and two antibodies (green [8] http:/ / en. wikipedia. org/ wiki/ and red) via immunofluorescence Special%3Acomparepages?rev1=443556310& page2=%3ADAPI [9] Kapuscinski J (September 1995). "DAPI: a DNA-specific fluorescent probe". Biotech Histochem 70 (5): 220–33. PMID 8580206. [10] Invitrogen, DAPI Nucleic Acid Stain (http:/ / probes. invitrogen. com/ media/ pis/ mp01306. pdf). accessed 2009-12-08. [11] Scott Prahl, DAPI (http:/ / omlc. ogi. edu/ spectra/ PhotochemCAD/ html/ dapi(H2O). html). accessed 2009-12-08. [12] http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1D30 [13] Zink D, Sadoni N, Stelzer E. (2003). "Visualizing Chromatin and Chromosomes in Living Cells.". Methods 29 (1): 42–50. doi:10.1016/S1046-2023(02)00289-X. PMID 12543070. [14] (http:/ / www. kpl. com/ docs/ msds/ 710301. pdf) [15] Ohta T, Tokishita S, Yamagata H. (2001). "Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli.". Mutat Res. 492 (1-2): 91–7. doi:10.1016/S1383-5718(01)00155-3. PMID 11377248.

Eosin

45

Eosin Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic.

Etymology The name Eosin comes from Eos, the Ancient Greek word for 'dawn' and the name of the Ancient Greek goddess of the dawn.

Variants

Eosin Y

There are actually two very closely related compounds commonly referred to as eosin. Most often used is Eosin Y (also known as eosin Y ws, eosin yellowish, Acid Red 87, C.I. 45380, bromoeosine, bromofluoresceic acid, D&C Red No. 22); it has a very slightly yellowish cast. The other eosin compound is eosin B (eosin bluish, Acid Red 91, C.I. 45400, Saffrosine, Eosin Scarlet, or imperial red); it has a very faint bluish cast. The two dyes are interchangeable, and the use of one or the other is a matter of preference and tradition. Eosin Y is a tetrabromo derivative of fluorescein. dibromo dinitro derivative of fluorescein. [2]

[1]

Eosin B is a Eosin B

Use in histology Eosin is most often used as a counterstain to haematoxylin in H&E (haematoxylin and eosin) staining. H&E staining is one of the most commonly used techniques in histology. Tissue stained with haematoxylin and eosin shows cytoplasm stained pink-orange and nuclei stained darkly, either blue or purple. Eosin also stains red blood cells intensely red. Eosin is an acidic dye and shows up in the basic parts of the cell, i.e. the cytoplasm. Haematoxylin however is a basic dye and shows up in the acidic part of the cell like the nucleus, where nucleic acids (DNA and RNA) are concentrated. For staining, eosin Y is typically used in concentrations of 1 to 5 percent weight by volume, dissolved in water or ethanol. For prevention of mold growth in aqueous solutions, thymol is sometimes added (The science of laboratory diagnosis By John Crocker, David Burnett (relevant citation for thymol inhibits growth of fungi). A small concentration (0.5 percent) of acetic acid usually gives a deeper red stain to the tissue. It is listed as an IARC class 3 carcinogen.

Eosin

References • Jocelyn H. Bruce-Gregorios, M.D.: Histopathologic Techniques, JMC Press Inc., Quezon City, Philippines, 1974. [1] Its CAS number is [ 17372-87-1 (http:/ / www. emolecules. com/ cgi-bin/ search?t=ss& q=17372-87-1& c=1& v=)] and its SMILES structure is O=C5C(Br)=C2O C1=C(Br)C([O-]) =C(Br)C=C1C(C4=C (C([O-])=O)C=C C=C4)=C2C=C3Br. [2] Its CAS number is [ 548-28-3 (http:/ / www. emolecules. com/ cgi-bin/ search?t=ss& q=548-28-3& c=1& v=)] and its SMILES structure is O=C5C(Br)=C2O C1=C(Br)C([O-]) =C([N+]([O-])=O) C=C1C(C4=C(C([O-]) =O)C=CC=C4)=C2 C=C3[N+]([O-])=O.

External links • Eosin Y - Applications information (http://www.abbeycolor.com/eosin-y.php) http://en.wikipedia.org/wiki/Eos

46

Eosin Y

47

Eosin Y Eosin Y

Identifiers [1]

CAS number

17372-87-1

PubChem

27020

ChemSpider

10580

UNII

TDQ283MPCW

MeSH

Eosine+Yellowish-(YS)

ChEBI

CHEBI:52053

ChEMBL

CHEMBL411675

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6] [7]

[8]


C20H6Br4Na2O5

Molar mass

691.85 g mol

−1

()

[9]


Infobox references

Eosin Y is a form of eosin.

References [1] [2] [3] [4] [5] [6] [7] [8]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=17372-87-1 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=27020 http:/ / www. chemspider. com/ 10580 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=TDQ283MPCW http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Eosine+ Yellowish-(YS) https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=52053 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL411675 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=c1ccc%28c%28c1%29c2c3cc%28c%28c%28c3oc-4c%28c%28%3DO%29c%28cc24%29Br%29Br%29Br%29%5BO-%5D%29Br%29C%28%3DO% %5BNa%2B%5D. %5BNa%2B%5D [9] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=435776474& page2=%3AEosin+ Y

Ethanol

48

Ethanol Ethanol

Identifiers [2]

CAS number

64-17-5

PubChem

702

ChemSpider

682

UNII

3K9958V90M

EC number

200-578-6

UN number

1170

DrugBank

DB00898

KEGG

D00068

MeSH

Ethanol

ChEBI

CHEBI:16236

ChEMBL

CHEMBL545

RTECS number

KQ6300000

ATC code

D01 AE06

Beilstein Reference

1718733

Gmelin Reference

787

3DMet

B01253

Jmol-3D images

Image 1

[3] [4]

[5]

[6]

[7]

[8]

[9] [10]

[11]

[12]

,D08 AX08

[13]

, V03 AB16

[14]

, V03 AZ01

[15]

[16] [17]


CHO

Molar mass

46.07 g mol−1

Appearance

Colorless liquid

Density

0.789 g/cm3

Melting point

−114 °C, unknown operator: u'\u2212' K, unknown operator: u'\u2212' °F

Boiling point

78.37 °C, 352 K, 173 °F

2 6

Ethanol

49 log P

-0.18

Vapor pressure

5.95 kPa (at 20 °C)

Acidity (pKa)

15.9

Basicity (pKb)

-1.9

Refractive index (nD)

1.36

Viscosity

0.0012 Pa s (at 20 °C)

Dipole moment

1.69 D

[18]

[19]

Pharmacology Routes of istration

Intramuscular Intravenous Oral Topical

Metabolism

Hepatic Hazards

EU Index

603-002-00-5

EU classification F R-phrases

R11

S-phrases

(S2), S7, S16

NFPA 704 Flash point

13–14 °C

Autoignition temperature

362 °C

LD50

5628 mg kg

−1

(oral, rat)

()

[20]


Infobox references

Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a solvent, and as a fuel. In common usage, it is often referred to simply as alcohol or spirits.

Chemical formula Ethanol is a 2-carbon alcohol with the molecular formula CH3CH2OH. Its empirical formula is C2H6O. An alternative notation is CH3–CH2–OH, which indicates that the carbon of a methyl group (CH3–) is attached to the carbon of a methylene group (–CH2–), which is attached to the oxygen of a hydroxyl group (–OH). It is a constitutional isomer of dimethyl ether. Ethanol is often abbreviated as EtOH, using the common organic chemistry notation of representing the ethyl group (C2H5) with Et.

Ethanol

Name Ethanol is the systematic name defined by the IUPAC nomenclature of organic chemistry for a molecule with two carbon atoms (prefix "eth-"), having a single bond between them (suffix "-ane"), and an attached -OH group (suffix "-ol").[21]

History The fermentation of sugar into ethanol is one of the earliest biotechnologies employed by humanity. The intoxicating effects of ethanol consumption have been known since ancient times. Ethanol has been used by humans since prehistory as the intoxicating ingredient of alcoholic beverages. Dried residue on 9,000-year-old pottery found in China imply that Neolithic people consumed alcoholic beverages.[22] Although distillation was well known by the early Greeks and Arabs, the first recorded production of alcohol from distilled wine was by the School of Salerno alchemists in the 12th century.[23] The first to mention absolute alcohol, in contrast with alcohol-water mixtures, was Raymond Lull.[23] In 1796, Johann Tobias Lowitz obtained pure ethanol by mixing partially purified ethanol (the alcohol-water azeotrope) with an excess of anhydrous alkali and then distilling the mixture over low heat.[24] Antoine Lavoisier described ethanol as a compound of carbon, hydrogen, and oxygen, and in 1807 Nicolas-Théodore de Saussure determined ethanol's chemical formula.[25][26] Fifty years later, Archibald Scott Couper published the structural formula of ethanol. It is one of the first structural formulas determined.[27] Ethanol was first prepared synthetically in 1825 by Michael Faraday. He found that sulfuric acid could absorb large volumes of coal gas.[28] He gave the resulting solution to Henry Hennell, a British chemist, who found in 1826 that it contained "sulphovinic acid" (ethyl hydrogen sulfate).[29] In 1828, Hennell and the French chemist Georges-Simon Sérullas independently discovered that sulphovinic acid could be decomposed into ethanol.[30][31] Thus, in 1825 Faraday had unwittingly discovered that ethanol could be produced from ethylene (a component of coal gas) by acid-catalyzed hydration, a process similar to current industrial ethanol synthesis.[32] Ethanol was used as lamp fuel in the United States as early as 1840, but a tax levied on industrial alcohol during the Civil War made this use uneconomical. The tax was repealed in 1906.[33] Original Ford Model T automobiles ran on ethanol until 1908.[34] With the advent of Prohibition in 1920, ethanol fuel sellers were accused of being allied with moonshiners,[33] and ethanol fuel fell into disuse until late in the 20th century. In modern times, ethanol intended for industrial use is also produced from ethylene.[35] Ethanol has widespread use as a solvent of substances intended for human or consumption, including scents, flavorings, colorings, and medicines. In chemistry, it is both an essential solvent and a feedstock for the synthesis of other products. It has a long history as a fuel for heat and light, and more recently as a fuel for internal combustion engines.

50

Ethanol

51

Physical properties Ethanol is a volatile, colorless liquid that has a slight odor.[36] It burns with a smokeless blue flame that is not always visible in normal light. The physical properties of ethanol stem primarily from the presence of its hydroxyl group and the shortness of its carbon chain. Ethanol's hydroxyl group is able to participate in hydrogen bonding, rendering it more viscous and less volatile than less polar organic compounds of similar molecular weight, such as propane. Ethanol is slightly more refractive than water, having a refractive index of 1.36242 (at λ=589.3 nm and 18.35 °C).[37] The triple point for ethanol is 150 K at a pressure of 4.3 * 10-4 Pa.[38]

Solvent properties Ethanol burning with its spectrum depicted

Ethanol is a versatile solvent, miscible with water and with many organic solvents, including acetic acid, acetone, benzene, carbon tetrachloride, chloroform, diethyl ether, ethylene glycol, glycerol, nitromethane, pyridine, and toluene.[37][39] It is also miscible with light aliphatic hydrocarbons, such as pentane and hexane, and with aliphatic chlorides such as trichloroethane and tetrachloroethylene.[39] Ethanol's miscibility with water contrasts with the immiscibility of longer-chain alcohols (five or more carbon atoms), whose water miscibility decreases sharply as the number of carbons increases.[40] The miscibility of ethanol with alkanes is limited to alkanes up to undecane, mixtures with dodecane and higher alkanes show a miscibility gap below a certain temperature (about 13 °C for dodecane[41]). The miscibility gap tends to get wider with higher alkanes and the temperature for complete miscibility increases. Ethanol-water mixtures have less volume than the sum of their individual components at the given fractions. Mixing equal volumes of ethanol and water results in only 1.92 volumes of mixture.[37][42] Mixing ethanol and water is exothermic. At 298 K, up to 777 J/mol[43] are set free. Mixtures of ethanol and water form an azeotrope at about 89 mole-% ethanol and 11 mole-% water[44] or a mixture of about 96 volume percent ethanol and 4% water at normal pressure and T = 351 K. This azeotropic composition is strongly temperature- and pressure-dependent and vanishes at temperatures below 303 K.[45]

Ethanol

52

Hydrogen bonding causes pure ethanol to be hygroscopic to the extent that it readily absorbs water from the air. The polar nature of the hydroxyl group causes ethanol to dissolve many ionic compounds, notably sodium and potassium hydroxides, magnesium chloride, calcium chloride, ammonium chloride, ammonium bromide, and sodium bromide.[39] Sodium and potassium chlorides are slightly soluble in ethanol.[39] Because the ethanol molecule also has a nonpolar end, it will also dissolve nonpolar substances, including most essential oils[46] and numerous flavoring, coloring, and medicinal agents.

Hydrogen bonding in solid ethanol at −186 °C

The addition of even a few percent of ethanol to water sharply reduces the surface tension of water. This property partially explains the "tears of wine" phenomenon. When wine is swirled in a glass, ethanol evaporates quickly from the thin film of wine on the wall of the glass. As the wine's ethanol content decreases, its surface tension increases and the thin film "beads up" and runs down the glass in channels rather than as a smooth sheet.

Flammability An ethanol-water solution that contains 40% ABV will catch fire if heated to about 79 °F (unknown operator: u'strong' °C) and if an ignition source is applied to it. This is called its flash point.[47] The flash point of pure ethanol is 61.88 °F (unknown operator: u'strong' °C), less than average room temperature.[48] The flash points of ethanol concentrations from 10% ABV to 96% ABV are shown below:[49] • • • • • • • • • • •

10% — 120 °F (unknown operator: u'strong' °C) 12.5% — about 125 °F (unknown operator: u'strong' °C) 20% — 97 °F (unknown operator: u'strong' °C) 30% — 84 °F (unknown operator: u'strong' °C) 40% — 79 °F (unknown operator: u'strong' °C) 50% — 75 °F (unknown operator: u'strong' °C) 60% — 72 °F (unknown operator: u'strong' °C) 70% — 70 °F (unknown operator: u'strong' °C) 80% — 68 °F (unknown operator: u'strong' °C) 90% — 63 °F (unknown operator: u'strong' °C) 96% — 63 °F (unknown operator: u'strong' °C)

Alcoholic beverages that have a low concentration of ethanol will burn if sufficiently heated and an ignition source (such as an electric spark or a match) is applied to them. For example, the flash point of ordinary wine containing 12.5% ethanol is about 125 °F (unknown operator: u'strong' °C).[50]

Ethanol

53

Production Ethanol is produced both as a petrochemical, through the hydration of ethylene and, via biological processes, by fermenting sugars with yeast.[51] Which process is more economical depends on prevailing prices of petroleum and grain feed stocks.

Ethylene hydration Ethanol for use as an industrial feedstock or solvent (sometimes referred to as synthetic ethanol) is made from petrochemical feed stocks, primarily by the acid-catalyzed hydration of ethylene, represented by the chemical equation C2H4 + H2O → CH3CH2OH The catalyst is most commonly phosphoric acid,[52] adsorbed onto a porous such as silica gel or diatomaceous earth. This catalyst was first used for large-scale ethanol production by the Shell Oil Company in 1947.[53] The reaction is carried out with an excess of high pressure steam at 300 °C. In the U.S., this process was used on an industrial scale by Union Carbide Corporation and others; but now only LyondellBasell uses it commercially.

94% denatured ethanol sold in a bottle for household use

In an older process, first practiced on the industrial scale in 1930 by Union Carbide,[54] but now almost entirely obsolete, ethylene was hydrated indirectly by reacting it with concentrated sulfuric acid to produce ethyl sulfate, which was hydrolysed to yield ethanol and regenerate the sulfuric acid:[55] C2H4 + H2SO4 → CH3CH2SO4H CH3CH2SO4H + H2O → CH3CH2OH + H2SO4

Fermentation Ethanol for use in alcoholic beverages, and the vast majority of ethanol for use as fuel, is produced by fermentation. When certain species of yeast (e.g., Saccharomyces cerevisiae) metabolize sugar they produce ethanol and carbon dioxide. The chemical equations below summarize the conversion: C6H12O6 → 2 CH3CH2OH + 2 CO2 C12H22O11 + H2O → 4 CH3CH2OH + 4 CO2 Fermentation is the process of culturing yeast under favorable thermal conditions to produce alcohol. This process is carried out at around 35–40 °C. Toxicity of ethanol to yeast limits the ethanol concentration obtainable by brewing; higher concentrations, therefore, are usually obtained by fortification or distillation. The most ethanol-tolerant strains of yeast can survive up to approximately 15% ethanol by volume.[56] To produce ethanol from starchy materials such as cereal grains, the starch must first be converted into sugars. In brewing beer, this has traditionally been accomplished by allowing the grain to germinate, or malt, which produces the enzyme amylase. When the malted grain is mashed, the amylase converts the remaining starches into sugars. For fuel ethanol, the hydrolysis of starch into glucose can be accomplished more rapidly by treatment with dilute sulfuric acid, fungally produced amylase, or some combination of the two.[57]

Ethanol

54

Cellulosic ethanol Sugars for ethanol fermentation can be obtained from cellulose.[58][59] Until recently, however, the cost of the cellulase enzymes capable of hydrolyzing cellulose has been prohibitive. The Canadian firm Iogen brought the first cellulose-based ethanol plant on-stream in 2004.[60] Its primary consumer so far has been the Canadian government, which, along with the United States Department of Energy, has invested heavily in the commercialization of cellulosic ethanol. Deployment of this technology could turn a number of cellulose-containing agricultural by-products, such as corncobs, straw, and sawdust, into renewable energy resources. Other enzyme companies are developing genetically engineered fungi that produce large volumes of cellulase, xylanase, and hemicellulase enzymes. These would convert agricultural residues such as corn stover, wheat straw, and sugar cane bagasse and energy crops such as switchgrass into fermentable sugars.[61] Cellulose-bearing materials typically also contain other polysaccharides, including hemicellulose. When undergoing hydrolysis, hemicellulose decomposes into mostly five-carbon sugars such as xylose. S. cerevisiae, the yeast most commonly used for ethanol production, cannot metabolize xylose. Other yeasts and bacteria are under investigation to ferment xylose and other pentoses into ethanol.[62] On January 14, 2008, General Motors announced a partnership with Coskata, Inc. The goal was to produce cellulosic ethanol cheaply, with an eventual goal of US$1 per US gallon ($0.30/L) for the fuel. The partnership planned to begin producing the fuel in large quantity by the end of 2008, and by 2011 to have a full-scale plant on line, capable of producing 50 million US gallons (unknown operator: u'strong' m3) to 100 million US gallons (unknown operator: u'strong' m3) of ethanol a year (200–400 ML/a).[63] In October 2011, an article on the Coskata website stated that a "semi-commercial" pilot plant in Madison, Pennsylvania, had been running successfully for 2 years and that a full scale facility was planned for Alabama.[64] Hydrocarbon-based ethanol production A process developed and marketed by Celanese Corporation under the name TCX Technology uses hydrocarbons such as natural gas or coal for ethanol production rather than using fermented crops such as corn or sugarcane.[65]

Prospective technologies The anaerobic bacterium Clostridium ljungdahlii, discovered in commercial chicken wastes, can produce ethanol from single-carbon sources including synthesis gas, a mixture of carbon monoxide and hydrogen that can be generated from the partial combustion of either fossil fuels or biomass. Use of these bacteria to produce ethanol from synthesis gas has progressed to the pilot plant stage at the BRI Energy facility in Fayetteville, Arkansas.[66] The BRI technology has been purchased by INEOS. The bacterium E.coli when genetically engineered with cow rumen genes and enzymes can produce ethanol from corn stover.[67]

Ethanol plant in Turner County, South Dakota

Another prospective technology is the closed-loop ethanol plant.[68] Ethanol produced from corn has a number of critics who suggest that it is primarily just recycled fossil fuels because of the energy required to grow the grain and convert it into ethanol. There is also the issue of competition with use of corn for food production. However, the closed-loop ethanol plant attempts to address this criticism. In a closed-loop plant, renewable energy for distillation comes from fermented manure, produced from cattle that have been fed the DDSG by-products from grain ethanol production. The concentrated compost nutrients from manure are then used to fertilize the soil and grow the next crop of grain to start the cycle again. Such a process is expected to lower the fossil fuel consumption used during conversion to ethanol by 75%.[69]

Ethanol

55

An alternative technology allows for the production of biodiesel from distillers grain as an additional value product.[70] Though in an early stage of research, there is some development of alternative production methods that use feed stocks such as municipal waste or recycled products, rice hulls, sugarcane bagasse, small diameter trees, wood chips, and switchgrass.[71]

Testing Breweries and biofuel plants employ two methods for measuring ethanol concentration. Infrared ethanol sensors measure the vibrational frequency of dissolved ethanol using the CH band at 2900 cm−1. This method uses a relatively inexpensive solid state sensor that compares the CH band with a reference band to calculate the ethanol content. The calculation makes use of the Beer-Lambert law. Alternatively, by measuring the density of the starting material and the density of the product, using a hydrometer, the change in specific gravity during fermentation indicates the alcohol content. This inexpensive and indirect method has a long history in the beer brewing industry.

Infrared reflection spectra of liquid ethanol, showing the -OH band centered at ~3300 cm−1 and C-H bands at ~2950 cm−1.

Purification Ethylene hydration or brewing produces an ethanol–water mixture. For most industrial and fuel uses, the ethanol must be purified. Fractional distillation can concentrate ethanol to 95.6% by volume (89.5 mole%). This mixture is an azeotrope with a boiling point of 78.1 °C, and cannot be further purified by distillation. Near infrared spectrum of liquid ethanol.

Common methods for obtaining absolute ethanol include desiccation using adsorbents such as starch, corn grits, or zeolites, which adsorb water preferentially, as well as azeotropic distillation and extractive distillation. Most ethanol fuel refineries use an adsorbent or zeolite to desiccate the ethanol stream. In another method to obtain absolute alcohol, a small quantity of benzene is added to rectified spirit and the mixture is then distilled. Absolute alcohol is obtained in the third fraction, which distills over at 78.3 °C (351.4 K).[40] Because a small amount of the benzene used remains in the solution, absolute alcohol produced by this method is not suitable for consumption, as benzene is carcinogenic.[72] There is also an absolute alcohol production process by desiccation using glycerol. Alcohol produced by this method is known as spectroscopic alcohol—so called because the absence of benzene makes it suitable as a solvent in spectroscopy.

Ethanol

56

Grades of ethanol Denatured alcohol Pure ethanol and alcoholic beverages are heavily taxed as a psychoactive drug, but ethanol has many uses that do not involve consumption by humans. To relieve the tax burden on these uses, most jurisdictions waive the tax when an agent has been added to the ethanol to render it unfit to drink. These include bittering agents such as denatonium benzoate and toxins such as methanol, naphtha, and pyridine. Products of this kind are called denatured alcohol.[73][74] Absolute alcohol Absolute or anhydrous alcohol refers to ethanol with a low water content. There are various grades with maximum water contents ranging from 1% to ppm levels. Absolute alcohol is not intended for human consumption. If azeotropic distillation is used to remove water, it will contain trace amounts of the material separation agent (e.g. benzene).[75] Absolute ethanol is used as a solvent for laboratory and industrial applications, where water will react with other chemicals, and as fuel alcohol. Spectroscopic ethanol is an absolute ethanol with a low absorbance in ultraviolet and visible light, fit for use as a solvent in ultraviolet-visible spectroscopy.[76] Pure ethanol is classed as 200 proof in the USA, equivalent to 175 degrees proof in the UK system.[77] Rectified spirits Rectified spirit, an azeotropic composition containing 4% water, is used instead of anhydrous ethanol for various purposes. Wine spirits are about 188 proof. The impurities are different from those in 190 proof laboratory ethanol.[78]

Reactions Ethanol is classified as a primary alcohol, meaning that the carbon its hydroxyl group attaches to has at least two hydrogen atoms attached to it as well. Many ethanol reactions occur at its hydroxyl group.

Ester formation In the presence of acid catalysts, ethanol reacts with carboxylic acids to produce ethyl esters and water: RCOOH + HOCH2CH3 → RCOOCH2CH3 + H2O This reaction, which is conducted on large scale industrially, requires the removal of the water from the reaction mixture as it is formed. Esters react in the presence of an acid or base to give back the alcohol and a salt. This reaction is known as saponification because it is used in the preparation of soap. Ethanol can also form esters with inorganic acids. Diethyl sulfate and triethyl phosphate are prepared by treating ethanol with sulfur trioxide and phosphorus pentoxide respectively. Diethyl sulfate is a useful ethylating agent in organic synthesis. Ethyl nitrite, prepared from the reaction of ethanol with sodium nitrite and sulfuric acid, was formerly a widely used diuretic.

Ethanol

57

Dehydration Strong acid desiccants cause the dehydration of ethanol to form diethyl ether and other byproducts. If the dehydration temperature exceeds around 160 °C, ethylene will be the main product. Millions of kilograms of diethyl ether are produced annually using sulfuric acid catalyst: 2 CH3CH2OH → CH3CH2OCH2CH3 + H2O (on 120 °C)

Combustion Complete combustion of ethanol forms carbon dioxide and water vapor: C2H5OH (l) + 3 O2 (g) → 2 CO2 (g) + 3 H2O (g); (ΔHc = −1371 kJ/mol[79]) specific heat = 2.44 kJ/(kg·K)

Acid-base chemistry Ethanol is a neutral molecule and the pH of a solution of ethanol in water is nearly 7.00. Ethanol can be quantitatively converted to its conjugate base, the ethoxide ion (CH3CH2O−), by reaction with an alkali metal such as sodium:[40] 2 CH3CH2OH + 2 Na → 2 CH3CH2ONa + H2 or a very strong base such as sodium hydride: CH3CH2OH + NaH → CH3CH2ONa + H2 The acidity of water and ethanol are nearly the same, as indicated by their pKa of 15.7 and 16 respectively. Thus, sodium ethoxide and sodium hydroxide exist in an equilbrium that is closely balanced: CH3CH2OH + NaOH

CH3CH2ONa + H2O

Halogenation Ethanol is not used industrially as a precursor to ethyl halides, but the reactions are illustrative. Ethanol reacts with hydrogen halides to produce ethyl halides such as ethyl chloride and ethyl bromide via an SN2 reaction: CH3CH2OH + HCl → CH3CH2Cl + H2O

These reactions require a catalyst such as zinc chloride.[55] HBr requires refluxing with a sulfuric acid catalyst.[55] Ethyl halides can, in principle, also be produced by treating ethanol with more specialized halogenating agents, such as thionyl chloride or phosphorus tribromide.[40][55] CH3CH2OH + SOCl2 → CH3CH2Cl + SO2 + HCl Upon treatment with halogens in the presence of base, ethanol gives the corresponding haloform (CHX3, where X = Cl, Br, I). This conversion is called the haloform reaction.[80] " An intermediate in the reaction with chlorine is the aldehyde called chloral: 4 Cl2 + CH3CH2OH → CCl3CHO + 5 HCl

Ethanol

58

Oxidation Ethanol can be oxidized to acetaldehyde and further oxidized to acetic acid, depending on the reagents and conditions.[55] This oxidation is of no importance industrially, but in the human body, these oxidation reactions are catalyzed by the enzyme liver alcohol dehydrogenase. The oxidation product of ethanol, acetic acid, is a nutrient for humans, being a precursor to acetyl CoA, where the acetyl group can be spent as energy or used for biosynthesis.

Uses As a fuel [81]

Energy content of some fuels compared with ethanol: Fuel type

MJ/L

Dry wood (20% moisture)

MJ/kg

Research octane number

~19.5 [82]

Methanol

17.9

19.9

Ethanol

21.2

26.8

108.6

E85 (85% ethanol, 15% gasoline)

25.2

33.2

105

Liquefied natural gas

25.3

~55

Autogas (LPG) (60% propane + 40% butane)

26.8

50.

[83]

108.7 [83]

[82]

Aviation gasoline 33.5 (high-octane gasoline, not jet fuel)

46.8

100/130 (lean/rich)

Gasohol (90% gasoline + 10% ethanol)

33.7

47.1

93/94

Regular gasoline

34.8

44.4

[84] min. 91

gasoline

max. 104

Diesel

38.6

45.4

Charcoal, extruded

50

23

25

The largest single use of ethanol is as a motor fuel and fuel additive. More than any other major country, Brazil relies on ethanol as a motor fuel. Gasoline sold in Brazil contains at least 25% anhydrous ethanol. Hydrous ethanol (about 95% ethanol and 5% water) can be used as fuel in more than 90% of new cars sold in the country. Brazilian ethanol is produced from sugar cane and noted for high carbon sequestration.[85] The US uses Gasohol (max 10% ethanol) and E85 (85% ethanol) ethanol/gasoline mixtures.

Ethanol

Ethanol may also be utilized as a rocket fuel, and is currently in lightweight rocket-powered racing aircraft.[86] Australian law limits of the use of pure ethanol sourced from sugarcane waste to up to 10% in automobiles. It has been recommended that older cars (and vintage cars designed to use a slower burning fuel) have their valves upgraded or replaced.[87] According to an industry advocacy group for promoting ethanol called the American Coalition for Ethanol, ethanol as a fuel reduces harmful tailpipe emissions of carbon monoxide, particulate matter, oxides of nitrogen, and other ozone-forming pollutants.[88] Argonne National Laboratory analyzed the greenhouse gas emissions of many different engine and fuel combinations. Comparing ethanol blends with gasoline alone, they showed reductions of 8% with the biodiesel/petrodiesel blend known as B20, 17% with the conventional E85 ethanol blend, and that using cellulosic ethanol lowers emissions 64%.[89] Ethanol combustion in an internal combustion engine yields many of the USP grade ethanol for laboratory products of incomplete combustion produced by gasoline and significantly larger use. amounts of formaldehyde and related species such as acetaldehyde.[90] This leads to a significantly larger photochemical reactivity that generates much more ground level ozone.[91] These data have been assembled into The Clean Fuels Report comparison of fuel emissions[92] and show that ethanol exhaust generates 2.14 times as much ozone as does gasoline exhaust. When this is added into the custom Localised Pollution Index (LPI) of The Clean Fuels Report the local pollution (pollution that contributes to smog) is 1.7 on a scale where gasoline is 1.0 and higher numbers signify greater pollution. The California Air Resources Board formalized this issue in 2008 by recognizing control standards for formaldehydes as an emissions control group, much like the conventional NOx and Reactive Organic Gases (ROGs).[93] World production of ethanol in 2006 was 51 gigalitres (expected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected operatorexpected 10 operatorexpected operator×10 US gal), with 69% of the world supply coming from Brazil and the United States.[94] More than 20% of Brazilian cars are able to use 100% ethanol as fuel, which includes Ethanol pump station in São Paulo, Brazil where ethanol-only engines and flex-fuel engines.[95] Flex-fuel engines in the fuel is available commercially. Brazil are able to work with all ethanol, all gasoline or any mixture of both. In the US flex-fuel vehicles can run on 0% to 85% ethanol (15% gasoline) since higher ethanol blends are not yet allowed or efficient. Brazil s this population of ethanol-burning automobiles with large national infrastructure that produces ethanol from domestically grown sugar cane. Sugar cane not only has a greater concentration of sucrose than corn (by about 30%), but is also much easier to extract. The bagasse generated by the process is not wasted, but is used in power plants to produce electricity.

59

Ethanol

A Ford Taurus "fueled by clean burning ethanol" owned by New York City.

60 The United States fuel ethanol industry is based largely on corn. According to the Renewable Fuels Association, as of October 30, 2007, 131 grain ethanol bio-refineries in the United States have the capacity to produce 7.0 billion US gallons (unknown operator: u'strong' m3) of ethanol per year. An additional 72 construction projects underway (in the U.S.) can add 6.4 billion US gallons (unknown operator: u'strong' m3) of new capacity in the next 18 months. Over time, it is believed that a material portion of the ≈150-billion-US-gallon (unknown operator: u'strong' m3) per year market for gasoline will begin to be replaced with fuel ethanol.[96]

One problem with ethanol is its high miscibility with water, which means that it cannot be efficiently shipped through modern pipelines, like liquid hydrocarbons, over long distances.[97] Mechanics also have seen increased cases of damage to small engines, in particular, the carburetor, attributable to the increased water retention by ethanol in fuel.[98] In 2011, the Open Fuel Standard Coalition introduced a bill into Congress that would mandate most cars sold in the United States to be warranted to run on ethanol, as well as methanol and gasoline. The bill aims to provide enough financial incentive to find better ways to make ethanol fuel so it could compete economically against gasoline.

United States Postal Service vehicle running on E85, a "flex-fuel" blend in Saint Paul, Minnesota.

Alcoholic beverages Ethanol is the principal psychoactive constituent in alcoholic beverages, with depressant effects on the central nervous system. It has a complex mode of action and affects multiple systems in the brain, the most notable one being its agonistic action on the GABA receptors.[99] Similar psychoactives include those that also interact with GABA receptors, such as benzodiazepines, barbiturates, gamma-hydroxybutyric acid (GHB).[100] Ethanol is metabolized by the body as an energy-providing nutrient, as it metabolizes into acetyl CoA, an intermediate common with glucose and fatty acid metabolism that can be used for energy in the citric acid cycle or for biosynthesis. Alcoholic beverages vary considerably in ethanol content and in foodstuffs they are produced from. Most alcoholic beverages can be broadly classified as fermented beverages, beverages made by the action of yeast on sugary foodstuffs, or distilled beverages, beverages whose preparation involves concentrating the ethanol in fermented beverages by distillation. The ethanol content of a beverage is usually measured in of the volume fraction of ethanol in the beverage, expressed either as a percentage or in alcoholic proof units. Fermented beverages can be broadly classified by the foodstuff they are fermented from. Beers are made from cereal grains or other starchy materials, wines and ciders from fruit juices, and meads from honey. Cultures around the world have made fermented beverages from numerous other foodstuffs, and local and national names for various fermented beverages abound. Distilled beverages are made by distilling fermented beverages. Broad categories of distilled beverages include whiskeys, distilled from fermented cereal grains; brandies, distilled from fermented fruit juices; and rum, distilled from fermented molasses or sugarcane juice. Vodka and similar neutral grain spirits can be distilled from any fermented material (grain and potatoes are most common); these spirits are so thoroughly distilled that no tastes from the particular starting material remain. Numerous other spirits and liqueurs are prepared by infusing flavors from

Ethanol fruits, herbs, and spices into distilled spirits. A traditional example is gin, which is created by infusing juniper berries into a neutral grain alcohol. The ethanol content in alcoholic beverages can be increased by means other than distillation. Applejack is traditionally made by freeze distillation, by which water is frozen out of fermented apple cider, leaving a more ethanol-rich liquid behind. Ice beer (also known by the German term Eisbier or Eisbock) is also freeze-distilled, with beer as the base beverage. Fortified wines are prepared by adding brandy or some other distilled spirit to partially fermented wine. This kills the yeast and conserves some of the sugar in grape juice; such beverages not only are more ethanol-rich but are often sweeter than other wines. Alcoholic beverages are used in cooking for their flavors and because alcohol dissolves hydrophobic flavor compounds. Just as industrial ethanol is used as feedstock for the production of industrial acetic acid, alcoholic beverages are made into vinegar. Wine and cider vinegar are both named for their respective source alcohols, whereas malt vinegar is derived from beer.

Feedstock Ethanol is an important industrial ingredient and has widespread use as a base chemical for other organic compounds. These include ethyl halides, ethyl esters, diethyl ether, acetic acid, ethyl amines, and to a lesser extent butadiene.

Antiseptic Ethanol is used in medical wipes and in most common antibacterial hand sanitizer gels at a concentration of about 62% v/v as an antiseptic. Ethanol kills organisms by denaturing their proteins and dissolving their lipids and is effective against most bacteria and fungi, and many viruses, but is ineffective against bacterial spores.[101]

Treatment for poisoning by other alcohols Ethanol is sometimes used to treat poisoning by other, more toxic alcohols, in particular methanol[102] and ethylene glycol. Ethanol competes with other alcohols for the alcohol dehydrogenase enzyme, lessening metabolism into toxic aldehyde and carboxylic acid derivatives,[103] and reducing one of the more serious toxic effect of the glycols to crystallize in the kidneys.

Solvent Ethanol is miscible with water and is a good general purpose solvent. It is found in paints, tinctures, markers, and personal care products such as perfumes and deodorants. It may also be used as a solvent or solute in cooking, such as in vodka sauce.

Historical uses Before the development of modern medicines, ethanol was used for a variety of medical purposes. It has been known to be used as a truth drug (as hinted at by the maxim "in vino veritas"), as medicine for depression and as an anesthetic. Ethanol was commonly used as fuel in early bipropellant rocket (liquid propelled) vehicles, in conjunction with an oxidizer such as liquid oxygen. The German V-2 rocket of World War II, credited with beginning the space age, used ethanol, mixed with 25% of water to reduce the combustion chamber temperature.[104][105] The V-2's design team helped develop U.S. rockets following World War II, including the ethanol-fueled Redstone rocket, which launched the first U.S. satellite.[106] Alcohols fell into general disuse as more efficient rocket fuels were developed.[105]

61

Ethanol

62

Pharmacology Ethanol binds to acetylcholine, GABA, serotonin, and NMDA receptors.[107] The removal of ethanol through oxidation by alcohol dehydrogenase in the liver from the human body is limited. Hence, the removal of a large concentration of alcohol from blood may follow zero-order kinetics. This means that alcohol leaves the body at a constant rate, rather than having an elimination half-life. Also, the rate-limiting steps for one substance may be in common with other substances. For instance, the blood alcohol concentration can be used to modify the biochemistry of methanol and ethylene glycol. Methanol itself is not highly toxic, but its metabolites formaldehyde and formic acid are; therefore, to reduce the concentration of these harmful metabolites, ethanol can be ingested to reduce the rate of methanol metabolism due to shared rate-limiting steps. Ethylene glycol poisoning can be treated in the same way.

Drug effects Pure ethanol will irritate the skin and eyes.[108] Nausea, vomiting and intoxication are symptoms of ingestion. Long-term use by ingestion can result in serious liver damage.[109] Atmospheric concentrations above one in a thousand are above the European Union Occupational exposure limits.[109]

Short-term [110]

BAC (g/L)

BAC (% v/v)

0.5

0.05%

Euphoria, talkativeness, relaxation

1

0.1 %

Central nervous system depression, nausea, possible vomiting, impaired motor and sensory function, impaired cognition

>1.4

>0.14% Decreased blood flow to brain

3

0.3%

Stupefaction, possible unconsciousness

4

0.4%

Possible death

>5.5

>0.55% Death

Symptoms

Effects on the central nervous system Ethanol is a central nervous system depressant and has significant psychoactive effects in sublethal doses; for specifics, see "Effects of alcohol on the body by dose". Based on its abilities to change the human consciousness, ethanol is considered a psychoactive drug.[111] Death from ethyl alcohol consumption is possible when blood alcohol level reaches 0.4%. A blood level of 0.5% or more is commonly fatal. Levels of even less than 0.1% can cause intoxication, with unconsciousness often occurring at 0.3–0.4%.[112] The amount of ethanol in the body is typically quantified by blood alcohol content (BAC), which is here taken as weight of ethanol per unit volume of blood. The table at right summarizes the symptoms of ethanol consumption. Small doses of ethanol, in general, produce euphoria and relaxation; people experiencing these symptoms tend to become talkative and less inhibited, and may exhibit poor judgment. At higher dosages (BAC > 1 g/L), ethanol acts as a central nervous system depressant, producing at progressively higher dosages, impaired sensory and motor function, slowed cognition, stupefaction, unconsciousness, and possible death. Ethanol acts in the central nervous system by binding to the GABA-A receptor, increasing the effects of the inhibitory neurotransmitter GABA (i.e., it is a positive allosteric modulator).[113] Prolonged heavy consumption of alcohol can cause significant permanent damage to the brain and other organs. See Alcohol consumption and health.

Ethanol According to the US National Highway Traffic Safety istration, in 2002 about "41% of people fatally injured in traffic crashes were in alcohol related crashes".[114] The risk of a fatal car accident increases exponentially with the level of alcohol in the driver's blood.[115] Most drunk driving laws governing the acceptable levels in the blood while driving or operating heavy machinery set typical upper limits of blood alcohol content (BAC) between 0.05% and 0.08%. Discontinuing consumption of alcohol after several years of heavy drinking can also be fatal. Alcohol withdrawal can cause anxiety, autonomic dysfunction, seizures, and hallucinations. Delirium tremens is a condition that requires people with a long history of heavy drinking to undertake an alcohol detoxification regimen. Effects on metabolism Ethanol within the human body is converted into acetaldehyde by alcohol dehydrogenase and then into the acetyl in acetyl CoA by acetaldehyde dehydrogenase. Acetyl CoA is the final product of both carbohydrate and fat metabolism, where the acetyl can be further used to produce energy or for biosynthesis. As such, ethanol is a nutrient. However, the product of the first step of this breakdown, acetaldehyde,[116] is more toxic than ethanol. Acetaldehyde is linked to most of the clinical effects of alcohol. It has been shown to increase the risk of developing cirrhosis of the liver[100] and multiple forms of cancer. Drug interactions Ethanol can intensify the sedation caused by other central nervous system depressant drugs such as barbiturates, benzodiazepines, opioids, phenothiazines, and anti-depressants.[112] Magnitude of effects Some individuals have less effective forms of one or both of the metabolizing enzymes, and can experience more severe symptoms from ethanol consumption than others. However, those having acquired alcohol tolerance have a greater quantity of these enzymes, and metabolize ethanol more rapidly.[117]

Long-term Birth defects Ethanol is classified as a teratogen. See fetal alcohol syndrome. Other effects Frequent drinking of alcoholic beverages has been shown to be a major contributing factor in cases of elevated blood levels of triglycerides.[118] Ethanol is not a carcinogen.[119][120] However, the first metabolic product of ethanol, acetaldehyde, is toxic, mutagenic, and carcinogenic. Ethanol is also widely used, clinically and over the counter, as an antitussive agent.[121]

Natural occurrence Ethanol is a byproduct of the metabolic process of yeast. As such, ethanol will be present in any yeast habitat. Ethanol can commonly be found in overripe fruit.[122] Ethanol produced by symbiotic yeast can be found in Bertam Palm blossoms. Although some species such as the Pentailed Treeshrew exhibit ethanol seeking behaviors, most show no interest or avoidance of food sources containing ethanol.[123] Ethanol is also produced during the germination of many plants as a result of natural anerobiosis.[124] Ethanol has been detected in outer space, forming an icy coating around dust grains in interstellar clouds.[125]

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Ethanol

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A. (1960). Journal of the American Chemical Society 82 (4): 795. doi:10.1021/ja01489a008. [19] Lide, David R., ed. (2008). CRC Handbook of Chemistry and Physics (89 ed.). Boca Raton: CRC Press. p. 9-55. [20] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=477167117& page2=%3AEthanol [21] USA (2012-03-06). "Ethanol - PubChem" (http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=702). Pubchem.ncbi.nlm.nih.gov. . Retrieved 2012-04-23. [22] Roach, J. (July 18, 2005). "9,000-Year-Old Beer Re-Created From Chinese Recipe" (http:/ / news. nationalgeographic. com/ news/ 2005/ 07/ 0718_050718_ancientbeer. html). National Geographic News. . Retrieved 2007-09-03. [23] Forbes, Robert James(1948) A short history of the art of distillation, p.89 [24] T. 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64

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[107] "The Brain from Top to Bottom – Alcohol (Intermediate, Molecular)" (http:/ / thebrain. mcgill. ca/ flash/ i/ i_03/ i_03_m/ i_03_m_par/ i_03_m_par_alcool. html). Canadian Institute of Neurosciences, Mental Health and Addiction. McGill University. . Retrieved 2010-02-13. [108] http:/ / www. nfpa. org/ Assets/ files/ AboutTheCodes/ 704/ CLA-AAA_ROPminutes_01-10. pdf [109] "Safety data for ethyl alcohol" (http:/ / msds. chem. ox. ac. uk/ ET/ ethyl_alcohol. html). Msds.chem.ox.ac.uk. 2008-05-09. . Retrieved 2011-01-03. [110] Pohorecky LA, Brick J (1988). "Pharmacology of ethanol". Pharmacol. Ther. 36 (2–3): 335–427. doi:10.1016/0163-7258(88)90109-X. PMID 3279433. [111] MedlinePlus Encyclopedia Alcohol Use (http:/ / www. nlm. nih. gov/ medlineplus/ ency/ article/ 001944. htm) [112] David A. Yost, MD (2002). Acute care for alcohol intoxication (http:/ / my. lecom. edu/ library/ internetresources/ journal articles/ Acute Care for Alcohol Intoxication. pdf). 112. Postgraduate Medicine Online. . Retrieved 2007-09-29. [113] Santhakumar V, Wallner M, Otis TS (2007). "Ethanol acts directly on extrasynaptic subtypes of GABAA receptors to increase tonic inhibition". Alcohol 41 (3): 211–21. doi:10.1016/j.alcohol.2007.04.011. PMC 2040048. PMID 17591544. [114] Hingson R, Winter M (2003). "Epidemiology and consequences of drinking and driving". Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism 27 (1): 63–78. PMID 15301401. [115] Naranjo CA, Bremner KE (1993). "Behavioural correlates of alcohol intoxication". Addiction 88 (1): 25–35. doi:10.1111/j.1360-0443.1993.tb02761.x. PMID 8448514. [116] Boggan, Bill. "Metabolism of Ethyl Alcohol in the Body" (http:/ / chemcases. com/ alcohol/ alc-06. htm). Chemases.com. . Retrieved 2007-09-29. [117] Agarwal DP, Goedde HW (1992). "Pharmacogenetics of alcohol metabolism and alcoholism". Pharmacogenetics 2 (2): 48–62. doi:10.1097/00008571-199204000-00002. PMID 1302043. [118] "Triglycerides" (http:/ / web. archive. org/ web/ 20070827102812/ http:/ / www. americanheart. org/ presenter. jhtml?identifier=4778). American Heart Association. Archived from the original (http:/ / americanheart. org/ presenter. jhtml?identifier=4778) on 2007-08-27. . Retrieved 2007-09-04. [119] "Fisher Scientific anydrous ethanol MSDS" (http:/ / fscimage. fishersci. com/ msds/ 89308. htm). Fscimage.fishersci.com. . Retrieved 2011-05-31. [120] Chavez, Pollyanna R.; Wang, Xiang-Dong; Meyer, Jean. "Animal Models for Carcinogenesis and Chemoprevention, abstract #C42: Effects of Chronic Ethanol Intake on Cyclin D1 Levels and Altered Foci in Diethylnitrosamine-initiated Rats" (http:/ / www. aacr. org/ PDF_files/ 2004Prevention/ Program/ 2004_Prevention_Abstracts. pdf) (PDF). USDA. . Retrieved 2007-10-24. [121] Calesnick, B.; H. Vernick (1971). "Antitussive activity of ethanol" (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 4932255). Q J Stud Alcohol 32 (2): 434-441. . [122] Robert Dudley (2004). "Ethanol, Fruit Ripening, and the Historical Origins of Human Alcoholism in Primate Frugivory" (http:/ / icb. oxfordjournals. org/ cgi/ reprint/ 44/ 4/ 315. pdf). Integrative Comparative Biology 44 (4): 315–323. doi:10.1093/icb/44.4.315. . Retrieved 2010-07-23. [123] Cynthia Graber (2008). "Fact or Fiction?: Animals Like to Get Drunk" (http:/ / www. scientificamerican. com/ article. cfm?id=animals-like-to-get-drunk). Scientific American. . Retrieved 2010-07-23. [124] Sylva Leblová, Eva Sinecká and Věra Vaníčková (1974). "Pyruvate metabolism in germinating seeds during natural anaerobiosis" (http:/ / www. springerlink. com/ content/ 9m775m527k2t1810/ ). Biologia Plantarum 16 (6): 406–411. doi:10.1007/BF02922229. . Retrieved 2010-07-23. [125] A. Schriver, L. Schriver-Mazzuoli, P. Ehrenfreund and L. d’Hendecourt (2007). "One possible origin of ethanol in interstellar medium: Photochemistry of mixed CO2–C2H6 films at 11 K. A FTIR study" (http:/ / www. sciencedirect. com/ science?_ob=ArticleURL&

67

Ethanol

68 _udi=B6TFM-4N68NMT-2& _=10& _coverDate=04/ 20/ 2007& _rdoc=1& _fmt=high& _orig=search& _sort=d& _docanchor=& view=c& _searchStrId=1410911507& _rerunOrigin=google& _acct=C000050221& _version=1& _urlVersion=0& _id=10& md5=5a8312d85b559afe8d56454167e8886a). Chemical Physics 334 (1–3): 128–137. doi:10.1016/j.chemphys.2007.02.018. . Retrieved 2010-07-23.

Further reading • The National Institute on Alcohol Abuse and Alcoholism maintains a database of alcohol-related health effects. ETOH Archival Database (1972–2003) (http://etoh.niaaa.nih.gov/Archive.htm) Alcohol and Alcohol Problems Science Database. • Boyce, John M., and Pittet Didier. (2003). "Hand Hygiene in Healthcare Settings." (http://cdc.gov/ handhygiene/) Centers for Disease Control, Atlanta, Georgia, United States. • Sci-toys website explanation of US denatured alcohol designations (http://sci-toys.com/ingredients/alcohol. html) • Smith, M.G., and M. Snyder. (2005). "Ethanol-induced virulence of Acinetobacter baumannii". American Society for Microbiology meeting. June 5 – June 9. Atlanta.

Appendix Thermophysical properties of mixtures of ethanol with water and dodecane

Excess volume of the mixture of ethanol and water (volume contraction)

Heat of mixing of the mixture of ethanol and water

Solid-liquid equilibrium of the mixture of ethanol and water (including eutecticum)

Miscibility gap in the mixture of dodecane and ethanol

Vapor-liquid equilibrium of the mixture of ethanol and water (including azeotrope)

Ethanol

External links • International Labour Organization (http://www.inchem.org/documents/icsc/icsc/eics0044.htm) ethanol safety information • National Pollutant Inventory – Ethanol Fact Sheet (http://www.npi.gov.au/substances/ethanol/index.html) • National Institute of Standards and Technology (http://webbook.nist.gov/cgi/cbook.cgi?Name=ethanol& Units=SI) chemical data on ethanol • ChEBI – biology related (http://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:16236) • Chicago Board of Trade (http://cbot.com/) news and market data on ethanol futures • Calculation of vapor pressure (http://ddbonline.ddbst.de/AntoineCalculation/AntoineCalculationCGI. exe?component=Ethanol), liquid density (http://ddbonline.ddbst.de/DIPPR105DensityCalculation/ DIPPR105CalculationCGI.exe?component=Ethanol), dynamic liquid viscosity (http://ddbonline.ddbst.de/ VogelCalculation/VogelCalculationCGI.exe?component=Ethanol), surface tension (http://ddbonline.ddbst.de/ DIPPR106SFTCalculation/DIPPR106SFTCalculationCGI.exe?component=Ethanol) of ethanol • U.S. National Library of Medicine: Drug Information Portal – Ethanol (http://druginfo.nlm.nih.gov/ drugportal/dpdirect.jsp?name=Ethanol) • Ethanol History (http://www.ethanolhistory.com/) A look into the history of ethanol • ChemSub Online: Ethyl alcohol (http://chemsub.online.fr/name/ethyl_alcohol.html)

69

Ethidium bromide

70

Ethidium bromide Ethidium bromide

Identifiers [1]

CAS number

1239-45-8

PubChem

14710

ChemSpider

14034

UNII

059NUO2Z1L

EC number

214-984-6

KEGG

C11161

ChEBI

CHEBI:4883

ChEMBL

CHEMBL284328

RTECS number

SF7950000

ATCvet code

QP51 AX06

Jmol-3D images

Image 1

[2] [3]

[6]

[10]

C H BrN 21 20

[4]

[5]


3

[7]

[9]

[8]

Ethidium bromide

71 Molar mass

394.294 g/mol

Appearance

Purple-red solid

Melting point

260 - 262 °C

Solubility in water

~ 40 g/l Hazards

R-phrases

R25 R36/37/38 R46

S-phrases

S22 S24/25 S26 S36/37/39 S45 S53


> 100 °C ()

[11]


Infobox references

Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomosis in cattle, a disease caused by trypanosomes.[12] The high incidence of Absorption spectrum of ethidium bromide antibiotic resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Ethidium bromide may be a mutagen, carcinogen or teratogen although this depends on the organism and the conditions.

Structure, chemistry, fluorescence As with most fluorescent compounds, ethidium bromide is aromatic. Its core heterocyclic moiety is generically known as a phenanthridine, an isomer of which is the fluorescent dye acridine. The reason for ethidium bromide's intense fluorescence after binding with DNA is probably not due to rigid stabilization of the phenyl moiety, because the phenyl ring has been shown to project outside the intercalated bases. In fact, the phenyl group is found to be almost perpendicular to the plane of the ring system, as it rotates about its single bond to find a position where it will impinge upon the ring system minimally. Instead, the hydrophobic environment found between the base pairs is believed to be responsible. By moving into this hydrophobic environment and away from the solvent, the ethidium cation is forced to shed any water molecules that were associated with it. As water is a highly efficient fluorescent quencher, the removal of these water molecules allows the ethidium to fluoresce.

Ethidium bromide

Applications Ethidium bromide is commonly used to detect nucleic acids in molecular biology laboratories. In the case of DNA this is usually double-stranded DNA from PCRs, restriction digests, etc. Single-stranded RNA can also be detected, since it usually folds back onto itself and thus provides local base pairing for the dye to intercalate. Detection typically involves a gel containing nucleic acids placed on or under a UV lamp. Since ultraviolet light is harmful to eyes and skin, gels stained with ethidium bromide are usually viewed indirectly using an enclosed camera, with the fluorescent images recorded as photographs. Where direct viewing is needed, the viewer's eyes and exposed skin should be protected. In the laboratory the intercalating properties have long been utilized to minimize chromosomal condensation when a culture is exposed to mitotic arresting agents during harvest. The resulting slide preparations permit a higher degree of resolution, and thus more confidence in determining structural integrity of chromosomes upon microscopic analysis. Ethidium bromide has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells.[13]

Alternatives There are alternatives to ethidium bromide which are d as being less dangerous and having better performance.[14][15] For example, several SYBR-based dyes are used by some researchers and there are other emerging stains such as Novel Juice. SYBR dyes are less mutagenic than EtBr by the Ames test with liver extract.[16] However, SYBR Green I was actually found to be more mutagenic than EthBr to the bacterial cells exposed to UV (which is used to visualize either dye).[17] This may be the case for other "safer" dyes, but whilst mutagenic and toxicity details are available [18] these have not been published in peer reviewed journals. The above article does find that DAPI is a completely nonmutagenic stain. MSDS for SYBR Safe reports a LD50 for rats of >5 g/kg, which is higher than that of EtBr (1.5g/kg), but both are many orders of magnitude higher than the concentrations used in molecular biology. Also, many alternative dyes are suspended in DMSO, which has health implications of its own including increased skin absorption of organic compounds.[16] Despite the performance advantage of using SYBR dyes instead of EtBr for staining purposes, many researchers still prefer EtBr since it is considerably less expensive.

Health risks Ethidium bromide is thought to act as a mutagen because it intercalates double stranded DNA (i.e., inserts itself between the strands), deforming the DNA.[19] This could affect DNA biological processes, like DNA replication and transcription. Ethidium bromide has been shown to be mutagenic to bacteria via the Ames test, but only after treatment with liver homogenate, which simulates the metabolic breakdown of the molecule being tested.[20] The lack of detected mutagenicity without liver homogenate indicates that ethidium bromide is not directly mutagenic, but that its metabolites are. The identity of these mutagenic metabolites are unknown. The National Toxicology Program states it is nonmutagenic in rats and mice.[21] These results are ed by a subchronic carcinogenicity study in mice conducted at the university of Düsseldorf where also no mutagenic effects could be detected.[22] Ethidium bromide (Homidium brand) use in animals to treat trypanosome infection suggests that toxicity and mutagenicity are not high. Studies have been conducted in animals to evaluate EtBr as a potential antitumorigenic chemotherapeutic agent.[23] Its chemotherapeutic use is due to its toxicity to mitochondria.[24] A more recent study shows that EtBr acts as a topoisomerase I poison, just like several anticancer drugs used in humans.[25] The above studies do not the commonly held idea that ethidium bromide is a potent mutagen in humans, but they do indicate that it can be toxic at high concentrations. Most use of ethidium bromide in the laboratory (0.25–1 microgram/ml) is below the level required for toxicity. The level is high enough that exposure may interfere with replication of mitochondrial DNA in some human cell lines, although the implications of that are not clear. Testing in humans and longer studies in any mammalian system would be required to fully understand the potential risk ethidium bromide poses to lab workers.[26]

72

Ethidium bromide Ethidium bromide can be added to YPD media and used as an inhibitor for cell growth.[27]

Handling and disposal Ethidium bromide is not regulated as hazardous waste at low concentrations,[28] but is treated as hazardous waste by many organizations. Material should be handled according to the material safety data sheet (MSDS). Wastes should always be treated in accordance with federal, state and local guidelines. The disposal of laboratory ethidium bromide remains a controversial subject.[29] Ethidium bromide can be degraded chemically, or collected and incinerated. It is common for ethidium bromide waste below a mandated concentration to be disposed of normally (e.g., pouring it down a drain). A common practice is to treat ethidium bromide with sodium hypochlorite (bleach) before disposal.[30] According to Lunn and Sansone, Chemical degradation using bleach yields compounds which are mutagenic by the Ames test. Data are lacking on the mutagenic effects of degradation products. Lunn and Sansone describe more effective methods for degradation.[31] EtBr can be removed from solutions with activated charcoal or amberlite ion exchange resin. Various commercial products are available for this use.[32]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=1239-45-8 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=14710 [3] http:/ / www. chemspider. com/ 14034 [4] http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=059NUO2Z1L [5] http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=214-984-6 [6] http:/ / www. kegg. jp/ entry/ C11161 [7] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=4883 [8] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL284328 [9] http:/ / www. whocc. no/ atcvet/ atcvet_index/ ?code=QP51AX06 [10] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CC%5Bn%2B%5D1c2cc%28ccc2c3ccc%28cc3c1c4ccccc4%29N%29N. %5BBr-%5D [11] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=443740742& page2=%3AEthidium+ bromide [12] Stevenson P, Sones KR, Gicheru MM, Mwangi EK. (1995). "Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya.". Acta Trop. 59 (2): 257–258. doi:10.1016/0001-706X(94)00080-K. PMID 7676909. [13] Diaz F, Bayona-Bafaluy MP, Rana M, Mora M, Hao H, Moraes CT. (November 2002). "Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control.". Nucleic Acids Res. 30 (21): 4626–33. doi:10.1093/nar/gkf602. PMC 135822. PMID 12409452. [14] Huang Q, Fu WL (2005). "Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis". Clin. Chem. Lab. Med. 43 (8): 841–2. doi:10.1515/CCLM.2005.141. PMID 16201894. [15] Dean Madden, Safer stains for DNA (http:/ / www. bioscience-explained. org/ ENvol1_2/ index. html#schollar_test). accessed 2009-12-08. [16] Singer VL, Lawlor TE, Yue S. (1999). "Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test).". Mutat Res. 439 (1): 37–47. PMID 10029672. [17] Ohta T, Tokishita S, Yamagata H. (2001). "Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli.". Mutat Res. 492 (1-2): 91–7. doi:10.1016/S1383-5718(01)00155-3. PMID 11377248. [18] Novel Juice testing report (http:/ / www. newmarketscientific. com/ datasheets/ Novel_Juice_Testing_Report_012011. pdf) [19] M.J. Waring (1965). "Complex formation between ethidium bromide and nucleic acids.". Journal of Molecular Biology 13 (1): 269–282. doi:10.1016/S0022-2836(65)80096-1. PMID 5859041. [20] J McCann and B N Ames (1975). "Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals". PNAS 72 (12): 5135–5139. doi:10.1073/pnas.72.12.5135. PMC 388891. PMID 1061098. [21] National Toxicology Program (August 15, 2005). "Ethidium Bromide: Genetic Toxicity." (http:/ / ntp. niehs. nih. gov/ index. cfm?objectid=BDAF3AE4-123F-7908-7BE09D1BEA25B435). . Retrieved September 30, 2009 [22] Marossek V (December 18, 2001). "Identifizierung und Charakterisierung molekularbiologischer Veränderungen am Beispiel des Tumorsuppressors p53 in der Tamoxifen- bzw. Bromdeoxyuridin-induzierten Karzinogenese im Labornager." (http:/ / docserv. uni-duesseldorf. de/ servlets/ DocumentServlet?id=2136). . Retrieved September 8, 2011 [23] M.J. Kramer, E. Grunberg. (1973). "Effect of Ethidium Bromide against Transplantable Tumors in Mice and Rats.". Experimental Chemotherapy 19 (4): 254–258. doi:10.1159/000221462.

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Ethidium bromide [24] von Wurmb-Schwark N, Cavelier L, Cortoi GA. (2006). "A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line.". Mutat Res. 596 (1-2): 57–63. doi:10.1016/j.mrfmmm.2005.12.003. PMID 16488450. [25] Gentry AC, Juul S, Veigaard C, Knudsen BR, Osheroff N. (2011). "The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I.". NUCLEIC ACIDS RESEARCH 39 (3): 1014–1022. doi:10.1093/nar/gkq822. [26] National Toxicology Program (August 15, 2005). "Executive Summary Ethidium Bromide: Evidence for Possible Carcinogenic Activity" (http:/ / ntp. niehs. nih. gov/ ?objectid=6F5F63F6-F1F6-975E-79965F7EE68AE7C0). . Retrieved September 30, 2009 [27] Caesar, Robert, Jonas Warringer, and Anders Blomberg. "Physiological Importance and Identification of Novel Targets for the N-Terminal Acetyltransferase NatB -- Caesar et al. 5 (2): 368 --." Eukaryotic Cell. 16 Dec. 2005. Web. 31 Jan. 2010. . [28] National Toxicology Program (August 15, 2005). "Executive Summary Ethidium Bromide: Table of Contents." (http:/ / ntp. niehs. nih. gov/ ?objectid=6F5EA06A-F1F6-975E-73079A5FE34F7E88). . Retrieved September 30, 2009 [29] HENGEN P. N. (1994). "Methods and Reagents: Disposal of Ethidium Bromide". Trends in Biochemical Sciences 19 (6): 257–258. doi:10.1016/0968-0004(94)90152-X. PMID 8073504. [30] Margaret-Ann Armour (2003). Hazardous laboratory chemicals disposal guide. CRC; 3 edition (February 27, 2003). pp. 222–223. ISBN 1-56670-567-3. [31] Lunn G, Sansone EB (May 1987). "Ethidium bromide: destruction and decontamination of solutions". Anal. Biochem. 162 (2): 453–8. doi:10.1016/0003-2697(87)90419-2. PMID 3605608. [32] http:/ / web. princeton. edu/ sites/ ehs/ chemwaste/ etbr. html

Further reading • Borst P (November 2005). "Ethidium DNA agarose gel electrophoresis: how it started". IUBMB Life 57 (11): 745–7. doi:10.1080/15216540500380855. PMID 16511967.

Fixation (histology) In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues.

Purposes of fixation Fixation of tissue is done for several reasons. One reason is to kill the tissue so that postmortem decay (autolysis and putrefaction) is prevented.[1] Fixation preserves a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination. To achieve this, several conditions usually must be met. First, a fixative usually acts to disable intrinsic biomolecules—particularly proteolytic enzymes—which otherwise digests or damages the sample. Second, a fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms. Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis. Even the most careful fixation does alter the sample and introduce artifacts that can interfere with interpretation of cellular ultrastructure. A prominent example is the bacterial mesosome, which was thought to be an organelle in gram-positive bacteria in the 1970s, but was later shown by new techniques developed for electron microscopy to be simply an artifact of chemical fixation.[2][3] Standardization of fixation and other tissue processing procedures takes

74

Fixation (histology) this introduction of artifacts into , by establishing what procedures introduce which kinds of artifacts. Researchers who know what types of artifacts to expect with each tissue type and processing technique can accurately interpret sections with artifacts, or choose techniques that minimize artifacts in areas of interest.

Fixation process Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry uses antibodies that bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used.

Types of fixation There are generally three types of fixation process: Heat fixation: After a smear has dried at room temperature, the slide is gripped by tongs or a clothespin and ed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the slide. Routinely used with bacteria and archaea. Heat fixation generally preserves overall morphology but not internal structures. Heat denatures the proteolytic enzyme and prevent autolysis. Heat fixation cannot be used in the capsular stain method as heat fixation will shrink or destroy the capsule (glycocalyx) and cannot be seen in stains. Perfusion: Fixation via blood flow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms) Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 20 times greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue to fix, so tissue size and density, as well as type of fixative must be considered. Using a larger sample means it takes longer for the fixative to reach the deeper tissue. Best in a slight vacuum.

Chemical Fixation In this process, structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative that can stabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble.

Types of Chemical Fixatives Crosslinking fixatives - Aldehydes Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. By far the most commonly used fixative in histology is formaldehyde. It is usually used as a 10% Neutral Buffered Formalin (NBF), that is approx. 3.7%-4.0% formaldehyde in phosphate buffered saline. Because formaldehyde is a gas at room temperature, formalin-formaldehyde gas dissolved in water (~37% w/v)-is used when making the former fixative. Paraformaldehyde is a polymerised form of formaldehyde, usually obtained as a fine white powder, which depolymerises back to formalin when heated. Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. Its effects are reversible by excess water and it avoids formalin pigmentation. Other benefits include: Long term storage and good tissue penetration. It is particularly good for

75

Fixation (histology) immunohistochemistry techniques. Also the formaldehyde vapour can be used as a fixatives for cell smears. Another popular aldehyde for fixation is glutaraldehyde. It operates in a similar way to formaldehyde by causing deformation of the alpha-helix structures in proteins. However glutaraldehyde is a larger molecule, and so its rate of diffusion across membranes is slower than formaldehyde. Consequently glutaraldehyde fixation on thicker tissue samples may be hampered, but this problem can be overcome by reducing the size of the tissue sample. One of the advantages of glutaraldehyde fixation is that it may offer a more rigid or tightly linked fixed product—its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules. It causes rapid and irreversible changes, fixes quickly, is well suited for electron microscopy, fixes well at 4oC, and gives best overall cytoplasmic and nuclear detail. However it is not ideal for immunohistochemistry staining. Some fixation protocols call for a combination of formaldehyde and glutaraldehyde so that their respective strengths complement one another. These crosslinking fixatives–especially formaldehyde–tend to preserve the secondary structure of proteins and may protect significant amounts of tertiary structure as well.

Precipitating fixatives - Alcohols Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with the aldehyde fixatives. The most common precipitating fixatives are ethanol and methanol. They are commonly used to fix frozen sections and smears. Acetone is also used and has been shown to produce better histological preservation than frozen sections when employed in the Acetone Methylbenzoate Xylene (AMEX) technique. The protein denaturants - methanol, ethanol and acetone - are rarely used alone for fixing blocks unless studying nucleic acids. Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives. The alcohols, by themselves, are known to cause considerable shrinkage and hardening of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology.

Oxidising agents The oxidising fixatives can react with various side chains of proteins and other biomolecules, allowing formation of crosslinks that stabilize tissue structure. However they cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary fixatives. Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. (It is not used for light microscopy as it penetrates thick sections of tissue very poorly.) Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.

76

Fixation (histology)

Mercurials Mercurials such as B-5 and Zenker's fixative have an unknown mechanism that increases staining brightness and give excellent nuclear detail. Despite being fast, mercurials penetrate poorly and produce tissue shrinkage. Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Also note that since they contain mercury care must be taken with disposal.

Picrates Picrates penetrate tissue well to react with histones and basic proteins to form crystalline picrates with amino acids and precipitate all proteins. It is a good fixative for connective tissue, preserves glycogen well, and extracts lipids to give superior results to formaldehyde in immunostaining of biogenic and polypeptide hormones However, it causes a loss of basophilia unless the specimen is thoroughly washed following fixation.

HOPE Fixative Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) gives formalin-like morphology, excellent preservation of protein antigens for immunohistochemistry and enzyme histochemistry, good RNA and DNA yields and absence of crosslinking proteins.

Frozen Sections Small pieces of tissue (5×5×3mm) are placed in a cryoprotective embedding medium—OCT, TBS, or Cryogel—then snap frozen in isopentane cooled by liquid nitrogen. Tissue is then sectioned in a freezing microtome or cryostat. Sections are then fixed in one of the following fixatives: Absolute acetone for 10–15 minutes, 95% ethanol for 10–15 minutes or Absolute acetone 10 minutes followed by 95% ethanol 10 minutes

Advantages • • • •

Give better preservation of antigenicity Minimal exposure to fixative Not exposed to the organic solvents Much faster than other forms of fixations.

Disadvantages • Lack morphological detail • Present a potential biohazard

Target and Chemical Fixative Do's and Don'ts

77


78

Target

Fixative of Choice

Fixative to Avoid

Proteins

Neutral Buffered Formalin, Paraformaldehyde

Osmium Tetroxide

Enzymes

Frozen Sections

Chemical Fixatives

Lipids

Frozen Sections*, Glutaraldehyde/Osmium Tetroxide Alcoholic fixatives, Neutral Buffered Formalin

Nucleic Acids

Alcoholic fixatives, HOPE

Mucopolysaccharides Frozen Sections Biogenic Amines

Bouin Solution~, Neutral Buffered Formalin

Glycogen

Alcoholic based fixatives

Aldehyde fixatives Chemical fixatives

Osmium Tetroxide

• Frozen Sections preserve RNA and Lipids despite poor morphology. Compare to Paraffin sections, synonymous to Chemical Fixatives in the table, which destroy RNA and affect some antigens BUT give good morphology. ~ A picrate

Factors Affecting Fixation pH Should be kept in the physiological range, between pH 4-9. The pH for the ultrastructure preservation should be buffered between 7.2 to 7.4

Osmolarity Hypertonic solutions give rise to cell shrinkage. Hypotonic solutions result in cell swelling and poor fixation. 10% neutral buffer formalin is 4% formaldehyde (1.33 osmolar) in PBS buffer (0.3 osmolar) sums to 1.63 osmolar. This is a very hypertonic solution yet it has worked well as a general tissue fixation condition for many years in pathology labs.

Size of the Specimen 1-4mm Thickness

Volume of the Fixative At least 15-20 times greater than tissue volume

Temperature Increasing the temperature increases speed of fixation. However, care is required to avoid cooking the specimen. Fixation is routinely carried out at room temperature.

Duration As a general rule 1hr per 1mm


Time from Removal to Fixation Fixation is a chemical process, and time must be allowed for the process to complete. Although "over fixation" can be detrimental, under-fixation has recently been appreciated as a significant problem and may be responsible for inappropriate results for some assays.

External links • Fixing specimens for making permanent slides [4] • Fixation strategies and formulations for immunohistochemical staining [5]

References [1] Carson, Freida L; Christa Hladik (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. p. 2. ISBN 978-0-89189-581-7. [2] Ryter A (1988). "Contribution of new cryomethods to a better knowledge of bacterial anatomy". Ann. Inst. Pasteur Microbiol. 139 (1): 33–44. doi:10.1016/0769-2609(88)90095-6. PMID 3289587. [3] Friedrich, CL; D Moyles, TJ Beveridge, REW Hancock (2000). "Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria". Antiomicrobial Agents and Chemotherapy 44 (8): 2086–2092. doi:10.1128/AAC.44.8.2086-2092.2000. PMC 90018. PMID 10898680. [4] http:/ / www. microbehunter. com/ 2010/ 08/ 05/ fixing-specimens-for-making-permanent-slides/ [5] http:/ / www. piercenet. com/ browse. cfm?fldID=B7273393-CD71-46C0-A8A7-E702C294529E

79

Formaldehyde

80

Formaldehyde Formaldehyde

Identifiers [1]

CAS number

50-00-0

PubChem

712

ChemSpider

692

UNII

1HG84L3525

EC number

200-001-8

UN number

2209

DrugBank

DB03843

KEGG

D00017

MeSH

Formaldehyde

ChEBI

CHEBI:16842

ChEMBL

CHEMBL1255

RTECS number

LP8925000

ATCvet code

QP53 AX19

Beilstein Reference

1209228

Gmelin Reference

445

3DMet

B00018

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]

[7]

[8] [9]

[10]

[11]

[12] [13]

[14]


CH O

Molar mass

30.03 g mol−1

2

Formaldehyde

81 Appearance

Colorless gas

Density

0.8153 g/cm³ (−20 °C)

Melting point

-92 °C, 181 K, -134 °F

Boiling point

-19 °C, 254 K, -2 °F (

Solubility in water

400 g dm−3

log P

0.350

Acidity (pKa)

13.3

Basicity (pKb)

0.7

Dipole moment

2.33 D

[15]

[15]

)

Structure Molecular shape

Trigonal planar Hazards [16]

MSDS

MSDS

EU Index

605-001-00-5

EU classification T R-phrases

R23/24/25 R34 R40 R43

S-phrases

(S1/2) S26 S36/37/39 S45 S51


64 °C (unknown operator: u'strong' °F)


430 °C (unknown operator: u'strong' °F)

Explosive limits

7–73%

LD50

100 mg/kg (oral, rat) Related compounds

Related aldehydes

Acetaldehyde Butyraldehyde Decanal Heptanal Hexanal Nonanal Octadecanal Octanal Pentanal Propionaldehyde

Related compounds

methanol formic acid ()

[17]


Infobox references

Formaldehyde is an organic compound with the formula CH2O. It is the simplest form of aldehyde, hence its systematic name methanal.

Formaldehyde A gas at room temperature, formaldehyde is colorless and has a characteristic pungent, irritating odor. It is an important precursor to many other chemical compounds, especially for polymers. In 2005, annual world production of formaldehyde was estimated to be 23 million tonnes (50 billion pounds).[18] Commercial solutions of formaldehyde in water, commonly called formalin, were formerly used as disinfectants and for preservation of biological specimens. In view of its widespread use, toxicity and volatility, exposure to formaldehyde is a significant consideration for human health.[19] On 10 June 2011, the US National Toxicology Program described formaldehyde as "known to be a human carcinogen".[20][21][22]

Forms Formaldehyde is more complicated than many simple carbon compounds because it adopts different forms. One important derivative is the cyclic trimer metaformaldehyde or trioxane (CH2O)3. There is also an infinite polymer called paraformaldehyde. When dissolved in water, formaldehyde combines with water to form methanediol or methylene glycol H2C(OH)2. The diol also exists in equilibrium with a series of oligomers (short polymers), depending on the concentration and temperature. A saturated water solution, that contains about 40% formaldehyde by volume or 37% by mass, is called "100% formalin". A small amount of stabilizer, such as methanol, is usually added to limit oxidation and polymerization. A typical commercial grade formalin may contain 10–12% methanol in addition to various metallic impurities.

Occurrence Formaldehyde is a naturally occurring substance in the environment made of carbon, hydrogen and oxygen. Natural processes in the upper atmosphere may contribute up to 90 percent of the total formaldehyde in the environment. Formaldehyde is an intermediate in the oxidation (or combustion) of methane as well as other carbon compounds, e.g. forest fires, in automobile exhaust, and in tobacco smoke. When produced in the atmosphere by the action of sunlight and oxygen on atmospheric methane and other hydrocarbons, it becomes part of smog. Formaldehyde has also been detected in outer space (see below). Formaldehyde, as well as its oligomers and hydrates, are rarely encountered in living organisms. Methanogenesis proceeds via the equivalent of formaldehyde, but this one-carbon species is masked as a methylene group in methanopterin. Formaldehyde is the primary cause of methanol's toxicity, since methanol is metabolised into toxic formaldehyde by alcohol dehydrogenase. Formaldehyde does not accumulate in the environment, because it is broken down within a few hours by sunlight or by bacteria present in soil or water. Humans metabolize formaldehyde quickly, so it does not accumulate, and is converted to formic acid in the body. Small amounts of formaldehyde are produced in case of incomplete combustion of methane gas.

Interstellar formaldehyde Formaldehyde was the first polyatomic organic molecule detected in the interstellar medium[23] and since its initial detection has been observed in many regions of the galaxy. Because of the widespread interest in interstellar formaldehyde it has recently been extensively studied, yielding new extragalactic sources.[24] A proposed mechanism for the formation is the hydrogenation of CO ice, shown below.[25] H + CO → HCO HCO + H → H2CO (rate constant=9.2×10−3 s−1) Formaldehyde appears to be a useful probe for astrochemists due to its low reactivity in the gas phase and to the fact that the 110←111 and 211←212 K-doublet transitions are rather clear.

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Formaldehyde

Synthesis and industrial production Formaldehyde was first reported in 1859 by the Russian chemist Aleksandr Butlerov (1828–86)[26] and was conclusively identified in 1869 by August Wilhelm von Hofmann.[27][28] Formaldehyde is produced industrially by the catalytic oxidation of methanol. The most common catalysts are silver metal or a mixture of an iron and molybdenum or vanadium oxides. In the commonly used formox process, methanol and oxygen react at ca. 250–400 °C in presence of iron oxide in combination with molybdenum and/or vanadium to produce formaldehyde according to the chemical equation:[18] 2 CH3OH + O2 → 2 CH2O + 2 H2O The silver-based catalyst usually operates at a higher temperature, about 650 °C. Two chemical reactions on it simultaneously produce formaldehyde: that shown above and the dehydrogenation reaction: CH3OH → H2CO + H2 In principle formaldehyde could be generated by oxidation of methane, but this route is not industrially viable because the formaldehyde is more easily oxidized than methane.[18]

Organic chemistry Formaldehyde is a building block in the synthesis of many other compounds of specialised and industrial significance. It exhibits most of the chemical properties of other aldehydes but is more reactive. For example it is more readily oxidized by atmospheric oxygen to formic acid (formic acid is found in ppm levels in commercial formaldehyde). Formaldehyde is a good electrophile, participating in electrophilic aromatic substitution reactions with aromatic compounds, and can undergo electrophilic addition reactions with alkenes and aromatics. Formaldehyde undergoes a Cannizzaro reaction in the presence of basic catalysts to produce formic acid and methanol.

Examples of organic synthetic applications Condensation with acetaldehyde affords pentaerythritol, a chemical necessary in synthesizing PETN, a high explosive.[29] Condensation with phenols gives phenol-formaldehyde resins. With 4-substituted phenols one obtains calixarenes.[30] When combined with hydrogen sulfide it forms trithiane.[31] 3CH2O + 3H2S → (CH2S)3 + 3H2O

Uses In photography, formaldehyde is used in low concentrations for process C-41 (color negative film) stabilizer in the final wash step,[32] as well as in the process E-6 pre-bleach step, to obviate the need for it in the final wash. Formaldehyde is used extensively in the woodworking and cabinet-making industries. Urea-formaldehyde is used in the glues that bond particle board together.[33] The particle board is used underneath wood veneer and plastic laminate. Cabinets, bank counters, and veneered and laminated woodwork all use particle board containing urea-formaldehyde under the plastic laminate and wood veneer.

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Formaldehyde

Industrial applications Formaldehyde is a common building block for the synthesis of more complex compounds and materials. In approximate order of decreasing consumption, products generated from formaldehyde include urea formaldehyde resin, melamine resin, phenol formaldehyde resin, polyoxymethylene plastics, 1,4-butanediol, and methylene diphenyl diisocyanate.[18] The textile industry uses formaldehyde-based resins as finishers to make fabrics crease-resistant.[34] Formaldehyde-based materials are key to the manufacture of automobiles, and used to make components for the transmission, electrical system, engine block, door s, axles and brake shoes. The value of sales of formaldehyde and derivative products was over $145 billion in 2003, about 1.2% of the Gross Domestic Product (GDP) of the United States and Canada. Including indirect employment, over 4 million work in the formaldehyde industry across approximately 11,900 plants in the U.S. and Canada.[35] When reacted with phenol, urea, or melamine, formaldehyde produces, respectively, hard thermoset phenol formaldehyde resin, urea formaldehyde resin, and melamine resin, which are commonly used in permanent adhesives such as those used in plywood or carpeting. It is used as the wet-strength resin added to sanitary paper products such as (listed in increasing concentrations injected into the paper machine headstock chest) facial tissue, table napkins, and roll towels. They are also foamed to make insulation, or cast into moulded products. Production of formaldehyde resins s for more than half of formaldehyde consumption. Formaldehyde is also a precursor to polyfunctional alcohols such as pentaerythritol, which is used to make paints and explosives. Other formaldehyde derivatives include methylene diphenyl diisocyanate, an important component in polyurethane paints and foams, and hexamine, which is used in phenol-formaldehyde resins as well as the explosive RDX. Formaldehyde has been found as a contaminant in several bath products, at levels from 54–610 ppm: it is thought to arise from the breakdown of preservatives in the products,[36] most frequently diazolidinyl urea.

Disinfectant and biocide An aqueous solution of formaldehyde can be useful as a disinfectant as it kills most bacteria and fungi (including their spores). Formaldehyde solutions are applied topically in medicine to dry the skin, such as in the treatment of warts. Many aquarists use formaldehyde as a treatment for the parasites Ichthyophthirius multifiliis and Cryptocaryon irritans.[37] Formaldehyde is used to inactivate bacterial products for toxoid vaccines (vaccines that use an inactive bacterial toxin to produce immunity). It is also used to kill unwanted viruses and bacteria that might contaminate the vaccine during production.[38] Urinary tract infections are also often treated using a derivative of formaldehyde (methenamine), a method often chosen because it prevents overuse of antibiotics and the resultant development of bacterial resistance to them. In an acid environment methenamine is converted in the kidneys to formaldehyde, which then has an antibacterial effect in the urinary tract. This is not safe for long term use due to the carcinogenic effect of formaldehyde. Some topical creams, cosmetics and personal hygiene products also contain derivatives of formaldehyde as the active ingredients that prevent the growth of potentially harmful bacteria.

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85

Tissue fixative and embalming agent Formaldehyde preserves or fixes tissue or cells by a mixture of reversible (short exposure time and low temperatures) and irreversible (long exposure time and higher temperatures) cross-linking of primary amino groups in proteins with other nearby nitrogen atoms in protein or DNA through a -CH2- linkage. This is exploited in ChIP-on-chip transcriptomics experiments, where nucleotide-binding proteins are cross-linked to their cognate binding sites on the chromosome and analyzed to determine what genes are regulated by the proteins. Formaldehyde is also used as a denaturing agent in RNA gel electrophoresis, preventing RNA from forming secondary structures. A solution of 4% formaldehyde fixes pathology tissue specimens at about one mm per hour at room temperature.

Octopodes preserved in formaldehyde solution

Formaldehyde solutions are used as a fixative for microscopy and histology, although the percentage formaldehyde used may vary based on the method of analysis. Additionally, the methanol used to stabilize formaldehyde may interfere with the ability to properly fix tissue or cells, and therefore commercial formaldehyde preparations are available that are packaged in glass ampules under an inert gas to prevent the use of contaminating methanol for stabilization. Formaldehyde-based solutions are also used in embalming to disinfect and temporarily preserve human and animal remains. It is the ability of formaldehyde to fix the tissue that produces the tell-tale firmness of flesh in an embalmed body. In post mortem examinations a procedure known as the "sink test" involves placing the lungs of an animal in an aqueous solution of formaldehyde; if the lungs float it suggests the animal was probably breathing or able to breathe at the time of death. Although formaldehyde solutions are commonly used as a biological preserving medium, usually for smaller specimens, it usually just prolongs the decaying process. Several European countries restrict the use of formaldehyde, including the import of formaldehyde-treated products and embalming. Starting September 2007, the European Union banned the use of formaldehyde due to its carcinogenic properties as a biocide (including embalming) under the Biocidal Products Directive (98/8/EC).[39][40] Countries with a strong tradition of embalming corpses, such as Ireland and other colder-weather countries, have raised concerns. Despite reports to the contrary,[41] no decision on the inclusion of formaldehyde on Annex I of the Biocidal Products Directive for product-type 22 (embalming and taxidermist fluids) had been made as of September 2009.[42]

Drug testing Formaldehyde, along with 18 M (concentrated) sulfuric acid makes Marquis reagent which can be used to identify alkaloids and other compounds.

Safety Formaldehyde is highly toxic to all animals, regardless of method of intake. Ingestion of as little as 30 mL (1 oz.) of a solution containing 37% formaldehyde has been reported to cause death in an adult human.[43] Water solution of formaldehyde is very corrosive and its ingestion can cause severe injury to the upper gastrointestinal tract. Occupational exposure to formaldehyde by inhalation is mainly from three types of sources: thermal or chemical decomposition of formaldehyde-based resins, formaldehyde emission from aqueous solutions (for example, embalming fluids), and the production of formaldehyde resulting from the combustion of a variety of organic compounds (for example, exhaust gases). Formaldehyde can be toxic, allergenic, and carcinogenic.[19] Because formaldehyde resins are used in many construction materials it is one of the more common indoor air pollutants.[44]

Formaldehyde At concentrations above 0.1 ppm in air formaldehyde can irritate the eyes and mucous membranes, resulting in watery eyes.[45] Formaldehyde inhaled at this concentration may cause headaches, a burning sensation in the throat, and difficulty breathing, and can trigger or aggravate asthma symptoms.[46][47] A 1988 Canadian study of houses with urea-formaldehyde foam insulation found that formaldehyde levels as low as 0.046 ppm were positively correlated with eye and nasal irritation.[48] Although many studies have failed to show a relationship between formaldehyde and asthma, a recent review of studies has shown a strong association between exposure to formaldehyde and the development of childhood asthma.[49] Chronic exposure at higher levels, starting at around 1.9 ppm, has been shown to result in significant damage to pulmonary function, resulting in reduced maximum mid-expiratory flow and forced vital capacity.[50] There is also research that s the theory that formaldehyde exposure contributes to reproductive problems in women. A study on Finnish women working in laboratories at least 3 days a week found a significant correlation between spontaneous abortion and formaldehyde exposure, and a study of Chinese women found abnormal menstrual cycles in 70% of the women occupationally exposed to formaldehyde compared to only 17% in the control group.[50] There have been no studies done on the effect of formaldehyde exposure on reproduction in men. The primary exposure concern is for the workers in the industries producing or using formaldehyde. As far back as 1987, the U.S. EPA classified it as a probable human carcinogen and after more studies the WHO International Agency for Research on Cancer (IARC), in 1995, also classified it as a probable human carcinogen. Further information and evaluation of all known data led the IARC to reclassify formaldehyde as a known human carcinogen [51] associated with nasal sinus cancer and nasopharyngeal cancer.[52] Recent studies have also shown a positive correlation between exposure to formaldehyde and the development of leukemia, particularly myeloid leukemia.[53][54] The formaldehyde theory of carcinogenesis was proposed in 1978. In the residential environment, formaldehyde exposure comes from a number of different routes; formaldehyde can off-gas from wood products, such as plywood or particle board, but it is produced by paints, varnishes, floor finishes, and cigarette smoking as well.[55] The United States Environmental Protection Agency (EPA) allows no more than 16 ppb formaldehyde in the air in new buildings constructed for that agency.[56] A U.S. Environmental Protection Agency study found a new home measured 0.076 ppm when brand new and 0.045 ppm after 30 days.[57] The Federal Emergency Management Agency (FEMA) has also announced limits on the formaldehyde levels in trailers purchased by that agency.[58] The EPA recommends the use of “exterior-grade” pressed-wood products with phenol instead of urea resin to limit formaldehyde exposure, since pressed-wood products containing formaldehyde resins are often a significant source of formaldehyde in homes.[52] For most people, irritation from formaldehyde is temporary and reversible, though formaldehyde can cause allergies and is part of the standard patch test series. People with formaldehyde allergy are advised to avoid formaldehyde releasers as well (e.g., Quaternium-15, imidazolidinyl urea, and diazolidinyl urea).[59] People who suffer allergic reactions to formaldehyde tend to display lesions on the skin in the areas that have had direct with the substance, such as the neck or thighs (often due to formaldehyde released from permanent press finished clothing) or dermatitis on the face (typically from cosmetics).[60] Formaldehyde has been banned in cosmetics in both Sweden and Japan. The eyes are most sensitive to formaldehyde exposure: The lowest level at which many people can begin to smell formaldehyde is about 0.05 ppm and the highest level is 1 ppm. The maximum concentration value at the workplace is 0.3 ppm.[61] In controlled chamber studies, individuals begin to sense eye irritation at about 0.5 ppm; 5 to 20 percent report eye irritation at 0.5 to 1 ppm; and greater certainty for sensory irritation occurred at 1 ppm and above. While some agencies have used a level as low as 0.1 ppm as a threshold for irritation, the expert found that a level of 0.3 ppm would protect against nearly all irritation. In fact, the expert found that a level of 1.0 ppm would avoid eye irritation—the most sensitive endpoint—in 75–95% of all people exposed.[62] Formaldehyde levels in building environments are affected by a number of factors. These include the potency of formaldehyde-emitting products present, the ratio of the surface area of emitting materials to volume of space,

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Formaldehyde environmental factors, product age, interactions with other materials, and ventilation condition. Formaldehyde emits from a variety of construction materials, furnishings, and consumer products. The three products that emit the highest concentrations are medium density fiberboard, hardwood plywood, and particle board. Environmental factors such as temperature and relative humidity can elevate levels because formaldehyde has a high vapor pressure. Formaldehyde levels from building materials are the highest when a building first opens because materials would have less time to off-gas. Formaldehyde levels decrease over time as the sources suppress. Formaldehyde levels in air can be sampled and tested in several ways, including impinger, treated sorbent, and ive monitors.[63] The National Institute for Occupational Safety and Health (NIOSH) has measurement methods numbered 2016, 2541, 3500, and 3800.[64] Studies on the interactions between formaldehyde and proteins at the molecular level have been reported on the effects of the body’s carrier protein, serum albumin. The binding of formaldehyde loosens the skeletal structure of albumin and exposure of aromatic ring amino acids in the internal hydrophobic region. Symptoms may affect personal awareness, making one feel tired or fatigue. Formaldehyde inhalation has also shown to cause oxidative stress and inflammation in animals. Mice studied over an exposure to a high dose of formaldehyde (3ppm), showed increased levels of NO levels in plasma. This result suggests that FA inhalation either decreased NO production or increased NO scavenging, which may be an anti-stress mechanism in the body. Formaldehyde inhalation changes the sensitivity of immune system, which influences oxidative stress. In June 2011, the twelfth edition of the National Toxicology Program (NTP) Report on Carcinogens (RoC) changed the listing status of formaldehyde from “reasonably anticipated to be a human carcinogen” to “known to be a human carcinogen”.[20][21][22] Concurrently, a National Academy of Sciences (NAS) committee was convened and issued an independent review of the draft United States Environmental Protection Agency IRIS assessment of formaldehyde, providing a comprehensive health effects assessment and quantitative estimates of human risks of adverse effects.[65]

International bans There are several web articles claiming that formaldehyde has been banned from manufacture or import into the European Union (EU) under REACH (Registration, Evaluation, Authorization, and restriction of Chemical substances) legislation. This appears to be misinformation, as official EU chemical databases contradict these claims as of February 19, 2010. This misconception has gained some ground. Formaldehyde is not listed in the Annex I of Regulation (EC) No 689/2008 (export and import of dangerous chemicals regulation), nor on a priority list for risk assessment. However, formaldehyde is banned from use in certain applications (preservatives for liquid-cooling and processing systems, slimicides, metalworking-fluid preservatives, and antifouling products) under the Biocidal Products Directive.[66][67] In the EU, the maximum allowed concentration of formaldehyde in finished products is 0.2%, and any product that exceeds 0.05% has to include a warning that the product contains formaldehyde.[60] In the United States, a bill was ed in congress on July 7, 2010 regarding the use of formaldehyde in hardwood plywood, particle board, and medium density fiberboard. The bill limited the allowable amount of formaldehyde emissions from these wood products to .09 ppm, a standard which companies will have to meet by January 2013.[68] Formaldehyde was declared a toxic substance by the 1999 Canadian Environmental Protection Act.[69]

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FEMA trailer incidents Hurricanes Katrina and Rita In the U.S. the Federal Emergency Management Agency (FEMA) provided travel trailers, recreational park trailers and manufactured homes starting in 2006 for habitation by residents of the U.S. gulf coast displaced by Hurricane Katrina and Hurricane Rita. Some of the people who moved into the trailers complained of breathing difficulties, nosebleeds, and persistent headaches. Formaldehyde-catalyzed resins were used in the production of these homes. The United States Centers For Disease Control and Prevention (CDC) performed indoor air quality testing for formaldehyde[70] in some of the units. On February 14, 2008 the CDC announced that potentially hazardous levels of formaldehyde were found in many of the travel trailers and manufactured homes provided by the agency.[71][72] The CDC's preliminary evaluation of a scientifically established random sample of 519 travel trailers and manufactured homes tested between December 21, 2007 and January 23, 2008 (2+ years after manufacture) showed average levels of formaldehyde in all units of about 77 parts per billion (ppb). Long-term exposure to levels in this range can be linked to an increased risk of cancer and, at levels above this range, there can also be a risk of respiratory illness. These levels are higher than expected in indoor air, where levels are commonly in the range of 10–20 ppb, and are higher than the Agency for Toxic Substance Disease Registry (ATSDR, division of the CDC) Minimal Risk Level (MRL) of 8 ppb.[73] Levels measured ranged from 3 ppb to 590 ppb.[74] FEMA, which requested the testing by the CDC, said it would work aggressively to relocate all residents of the temporary housing as soon as possible. Lawsuits are being filed against FEMA as a result of the exposures.[75] Iowa floods of 2008 Also in the U.S., problems arose in trailers again provided by FEMA to residents displaced by the Iowa floods of 2008. Several months after moving to the trailers, occupants reported violent coughing, headaches, as well as asthma, bronchitis, and other problems. Tests showed that in some trailers, levels of formaldehyde exceeded the limits recommended by the U.S. Environmental Protection Agency and American Lung Association.[76][77] The associated publicity has resulted in additional testing to begin in November.[78] 2008 Sichuan earthquake After an earthquake hit Sichuan, China, a large number of survivors were housed in trailers made with medium-density fiberboard that emitted up to 5 times China's maximum allowable formaldehyde levels. In April 2009, 100 miscarriages were recorded in this community, which may have been linked to exposure to high levels of formaldehyde found in the trailers used for housing after the disaster.[50]

Contaminant in food Scandals have broken in both the 2005 Indonesia food scare and 2007 Vietnam food scare regarding the addition of formaldehyde to foods to extend shelf life. After a four-year absence, in 2011 Indonesian authorities have again found some foods with formaldehyde being sold in markets in a number of regions across the country. Besides using formaldehyde, they also use borax, but not combined together.[79] In August 2011, at least at 2 Carrefour supermarkets, the Central Jakarta Livestock and Fishery Sub-Department found a sweet glutinous rice drink (cendol) contained 10 parts per million of formaldehyde.[80] Foods known to be contaminated include noodles, salted fish, tofu, and rumors of chicken and beer. In some places, such as China, formaldehyde is still used illegally as a preservative in foods, which exposes people to formaldehyde ingestion.[81] In humans, the ingestion of formaldehyde has been shown to cause vomiting, abdominal pain, dizziness, and in extreme cases can cause death; in addition, there is limited evidence of a carcinogenic effect.[19][50] Testing for formaldehyde is by blood and/or urine by gas chromatography-mass spectrometry. Other methods include infrared detection, gas detector tubes, etc., of which HPLC is the most sensitive [82] In the early 1900s, it was frequently added by US milk plants to milk bottles as a

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Formaldehyde method of pasteurization due to the lack of knowledge regarding formaldehyde's toxicity. In 2011 in Nakhon Ratchasima, Thailand, truckloads of rotten chicken were exposed to formaldehyde in which "a large network" including 11 slaughterhouses run by a criminal gang were implicated.[83] In 2012, 1 billion Rupiah (almost 1 million USD) of fish were imported from Pakistan to Batam, Indonesia were found laced with formaldehyde.[84]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=50-00-0 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=712 [3] http:/ / www. chemspider. com/ 692 [4] http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=1HG84L3525 [5] http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=200-001-8 [6] http:/ / www. drugbank. ca/ drugs/ DB03843 [7] http:/ / www. kegg. jp/ entry/ D00017 [8] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Formaldehyde [9] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=16842 [10] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL1255 [11] http:/ / www. whocc. no/ atcvet/ atcvet_index/ ?code=QP53AX19 [12] http:/ / www. 3dmet. dna. affrc. go. jp/ html/ B00018. html [13] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=C%3DO [14] Weast, Robert C., ed. (1981). CRC Handbook of Chemistry and Physics (62nd ed.). Boca Raton, FL: CRC Press. pp. C–301, E–61. ISBN 0-8493-0462-8. [15] Formaldehyde (PDF) (http:/ / www. inchem. org/ documents/ sids/ sids/ FORMALDEHYDE. pdf), SIDS Initial Assessment Report, International Programme on Chemical Safety [16] http:/ / www. einstein. yu. edu/ ehs/ PDF%20Files/ Formaldehyde. PDF [17] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=464368486& page2=%3AFormaldehyde [18] Günther Reuss, Walter Disteldorf, Armin Otto Gamer, Albrecht Hilt “Formaldehyde” in Ullmann's Encyclopedia of Industrial Chemistry, 2002, Wiley-VCH, Weinheim. doi:10.1002/14356007.a11_619 [19] "Formaldehyde" (http:/ / monographs. iarc. fr/ ENG/ Monographs/ vol88/ mono88-6. pdf) (PDF). Formaldehyde, 2-Butoxyethanol and 1-tert-Butoxypropan-2-ol. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 88. Lyon, : International Agency for Research on Cancer. 2006. pp. 39–325. ISBN 92-832-1288-6. . "Formaldehyde (gas)" (http:/ / ntp. niehs. nih. gov/ ntp/ roc/ eleventh/ profiles/ s089form. pdf), Report on Carcinogens, Eleventh Edition, U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program, 2005, [20] Harris, Gardiner (10 June 2011). "Government Says 2 Common Materials Pose Risk of Cancer" (http:/ / www. nytimes. com/ 2011/ 06/ 11/ health/ 11cancer. html). New York Times. . Retrieved 2011-06-11. [21] National Toxicology Program (10 June 2011). "12th Report on Carcinogens" (http:/ / ntp. niehs. nih. gov/ index. cfm?objectid=72016262-BDB7-CEBA-FA60E922B18C2540). National Toxicology Program. . Retrieved 2011-06-11. [22] National Toxicology Program (10 June 2011). "Report On Carcinogens – Twelfth Edition – 2011" (http:/ / ntp. niehs. nih. gov/ ntp/ roc/ twelfth/ roc12. pdf) (PDF). National Toxicology Program. . Retrieved 2011-06-11. [23] Zuckerman, B.; Buhl, D.; Palmer, P.; Snyder, L. E. (1970). "Observation of interstellar formaldehyde". Astrophys. J. 160: 485–506. Bibcode 1970ApJ...160..485Z. doi:10.1086/150449. [24] Mangum, Jeffrey G.; Darling, Jeremy; Menten, Karl M.; Henkel, Christian (2008). "Formaldehyde Densitometry of Starburst Galaxies". Astrophys. J. 673 (2): 832–46. Bibcode 2008ApJ...673..832M. doi:10.1086/524354. [25] Woon, David E. (2002). "Modeling Gas-Grain Chemistry with Quantum Chemical Cluster Calculations. I. Heterogeneous Hydrogenation of CO and H2CO on Icy Grain Mantles". Astrophys. J. 569: 541–48. Bibcode 2002ApJ...569..541W. doi:10.1086/339279. [26] A. Butlerow (1859) "Ueber einige Derivate des Jodmethylens" (http:/ / books. google. com/ books?id=NYs8AAAAIAAJ& pg=PA242#v=onepage& q& f=false) (On some derivatives of methylene iodide), Annalen der Chemie und Pharmacie, vol . 111, pages 242–252. In this paper, Butlerov discovered formaldehyde, which he called "Dioxymethylen" (methylene dioxide) [page 247] because his empirical formula for it was incorrect (C4H4O4). [27] In 1867, A. W. Hofmann first announced to the Royal Prussian Academy of Sciences the production of formaldehyde by ing methanol vapor in air over hot platinum wire. See: A. W. Hofmann (14 October 1867) "Zur Kenntnis des Methylaldehyds" (http:/ / books. google. com/ books?id=Vh4XAAAAYAAJ& pg=PA665#v=onepage& q& f=false) ([Contributions] to our knowledge of methylaldehyde), Monatsbericht der Königlich Preussischen Akademie der Wissenschaften zu Berlin (Monthly Report of the Royal Prussian Academy of Sciences in Berlin), vol. 8, pages 665–669. Reprinted in: •

A.W. Hofmann, (1868) "Zur Kenntnis des Methylaldehyds" (http:/ / books. google. com/ books?id=RgEuAAAAIAAJ& pg=PA357#v=onepage& q& f=false), Annalen der Chemie und Pharmacie (Annals of Chemistry and Pharmacy), vol. 145, no. 3, pages

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Formaldehyde 357–361. A.W. Hofmann (1868) "Zur Kenntnis des Methylaldehyds" (http:/ / books. google. com/ books?id=4hNLAAAAYAAJ& pg=PA246#v=onepage& q& f=false), Journal für praktische Chemie (Journal for Practical Chemistry), vol. 103, no. 1, pages 246–250. However, it was not until 1869 that Hofmann determined the correct empirical formula of formaldehyde. See: A.W. Hofmann (5 April 1869) "Beiträge zur Kenntnis des Methylaldehyds" (http:/ / books. google. com/ books?id=58YAAAAAYAAJ& pg=PA362#v=onepage& q& f=false), Monatsbericht der Königlich Preussischen Akademie der Wissenschaften zu Berlin, vol. ?, pages 362–372. Reprinted in: •

•

A.W. Hofmann (1869) "Beiträge zur Kenntnis des Methylaldehyds" (http:/ / books. google. ch/ books?id=2AIwAAAAIAAJ& pg=PA414#v=onepage& q& f=false), Journal für praktische Chemie, vol. 107, no. 1, pages 414–424. • A.W. Hofmann (1869) "Beiträge zur Kenntnis des Methylaldehyds," Berichte der Deutschen Chemischen Gesellschaft (Reports of the German Chemical Society), vol. 2, pages 152–159. [28] Read, J. (1935). Text-Book of Organic Chemistry. London: G Bell & Sons. [29] Schurink, H. B. J. (1925), "Pentaerythritol" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=cv1p0425), Org. Synth. 4: 53, ; Coll. Vol. 1: 425 [30] Gutsche, C. D.; Iqbal, M. (1993), "p-tert-Butylcalix[4]arene" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=cv8p0075), Org. Synth., ; Coll. Vol. 8: 75 [31] Bost, R. W.; Constable, E. W. (1936), "sym-Trithiane" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=cv2p0610), Org. Synth. 16: 81, ; Coll. Vol. 2: 610 [32] "Process C-41 Using Kodak Flexicolor Chemicals – Publication Z-131" (http:/ / www. kodak. com/ global/ en/ service/ Zmanuals/ z131. shtml). Kodak. . Retrieved 2009-09-01. [33] Antonio Pizzi, K. L. Mittal. Handbook of adhesive technology. [34] "Formaldehyde in Clothing and Other Textiles" (http:/ / www. nicnas. gov. au/ Publications/ Information_Sheets/ Existing_Chemical_Information_Sheets/ EC_IS_Formaldehyde_102007_PDF. pdf) (PDF). Existing Chemicals Information Sheet. Australian National Industrial Chemicals Notification and Assessment Scheme. October 2007. . Retrieved 2009-09-01. [35] Economic Importance (http:/ / www. formaldehyde. org/ economy/ economic_importance/ ), Formaldehyde Council. 2009. Accessed on April 14, 2010. [36] "No More Toxic Tub: Getting Contaminants Out Of Children’s Bath & Personal Care Products" (http:/ / safecosmetics. org/ s/ NoMoreToxicTub_Mar09Report. pdf) (PDF). Campaign for Safe Cosmetics. March 2009. . Retrieved 19 May 2012. [37] Francis-Floyd, Ruth (April 1996). "Use of Formalin to Control Fish Parasites" (http:/ / edis. ifas. ufl. edu/ VM061). Institute of Food and Agricultural Sciences, University of Florida. . [38] Center for Disease Control: Vaccines [39] Directive 98/8/EC of the European Parliament and of the Council of 16 February 1998 concerning the placing of biocidal products on the market (http:/ / eur-lex. europa. eu/ LexUriServ/ LexUriServ. do?uri=CELEX:31998L0008:EN:HTML). OJEU L123, 24.04.1998, pp. 1–63. ( consolidated version to 2008-09-26 (PDF) (http:/ / eur-lex. europa. eu/ LexUriServ/ LexUriServ. do?uri=CONSLEG:1998L0008:20080926:EN:PDF)) [40] Commission Regulation (EC) No 2032/2003 of 4 November 2003 on the second phase of the 10-year work programme referred to in Article 16(2) of Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market, and amending Regulation (EC) No 1896/2000 (http:/ / eur-lex. europa. eu/ LexUriServ/ LexUriServ. do?uri=CELEX:32003R2032:EN:HTML). OJEU L307, 24.11.2003, p. 1–96. ( consolidated version to 2007-01-04 (PDF) (http:/ / eur-lex. europa. eu/ LexUriServ/ LexUriServ. do?uri=CONSLEG:2003R2032:20070104:EN:PDF)) [41] Patel, Alkesh (2007-07-04). "Formaldehyde Ban set for 22 September 2007" (http:/ / www. webwire. com/ ViewPressRel. asp?aId=41468). WebWire. . Retrieved 19 May 2012. [42] "European chemical Substances Information System (ESIS) entry for formaldehyde" (http:/ / esis. jrc. ec. europa. eu/ ). . Retrieved 2009-09-01. [43] "Medical Management Guidelines for Formaldehyde" (http:/ / www. atsdr. cdc. gov/ mmg/ mmg. asp?id=216& tid=39). . [44] "Indoor Air Pollution in California" (http:/ / www. arb. ca. gov/ research/ indoor/ ab1173/ rpt0705. pdf) (PDF). Air Resources Board, California Environmental Protection Agency. July 2005. pp. 65–70. . Retrieved 19 May 2012. [45] "Formaldehyde" (http:/ / www. osha. gov/ SLTC/ formaldehyde/ index. html). Occupational Safety and Health istration. August 2008. . Retrieved 2009-09-01. [46] "Formaldehyde Reference Exposure Levels" (http:/ / www. oehha. ca. gov/ air/ hot_spots/ 2008/ AppendixD1_final. pdf#page=128) (PDF). California Office Of Health Hazard Assessment. December 2008. . Retrieved 19 May 2012. [47] Formaldehyde and Indoor Air (http:/ / www. hc-sc. gc. ca/ iyh-vsv/ environ/ formaldehyde_e. html). Health Canada. August 2005. ISBN 0-8155-1129-9. . Retrieved 2009-09-01. [48] Broder, I; Corey, P; Brasher, P; Lipa, M; Cole, P (1991). "Formaldehyde exposure and health status in households". Environmental health perspectives 95: 101–4. PMC 1568408. PMID 1821362. [49] McGwin, G; Lienert, J; Kennedy, JI (November 2009). "Formaldehyde Exposure and Asthma in Children: A Systematic Review" (http:/ / ehp03. niehs. nih. gov/ article/ info:doi/ 10. 1289/ ehp. 0901143). Environmental health perspectives (Environmental Health Perspectives) 118 (3) (3): 313–7. doi:10.1289/ehp.0901143. PMC 2854756. PMID 20064771. . [50] Formaldehyde in China: Production, Consumption, Exposure Levels, and Health Effects (http:/ / www. sciencedirect. com. silk. library. umass. edu:2048/ science?_ob=ArticleURL& _udi=B6V7X-4WR0CXG-1& _=1516330& _coverDate=11/ 30/ 2009& _rdoc=1&

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Formaldehyde _fmt=high& _orig=search& _origin=search& _sort=d& _docanchor=& view=c& _acct=C000053443& _version=1& _urlVersion=0& _id=1516330& md5=afb7ac57f3db99442955436d4e998eb7& searchtype=a#secx15). 35 (8). Environmental International. November 2009. pp. 1210–1224. . [51] (pdf, html) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Volume 88 (2006) Formaldehyde, 2-Butoxyethanol and 1-tert-Butoxypropan-2-ol (http:/ / monographs. iarc. fr/ ENG/ Monographs/ vol88/ index. php), WHO Press, 2006( English ), [52] "Formaldehyde and Cancer Risk" (http:/ / www. cancer. gov/ cancertopics/ factsheet/ Risk/ formaldehyde). . [53] Formaldehyde exposure and Leukemia: A New Meta-Analysis and Potential Mechanisms (http:/ / www. sciencedirect. com. silk. library. umass. edu:2048/ science?_ob=ArticleURL& _udi=B6T2G-4T0FFC4-1& _=1516330& _coverDate=06/ 30/ 2009& _rdoc=1& _fmt=high& _orig=search& _origin=search& _sort=d& _docanchor=& view=c& _acct=C000053443& _version=1& _urlVersion=0& _id=1516330& md5=5f211232d0a85078ff8db52d7f55a8c5& searchtype=a). 681 (2–3). Mutation Research/Reviews in Mutation Research. March–June 2009. pp. 150–168. . [54] Formaldehyde and Leukemia: Epidemiology, Potential Mechanisms, and Implications for Risk Assessment (http:/ / onlinelibrary. wiley. com. silk. library. umass. edu:2048/ doi/ 10. 1002/ em. 20534/ pdf). 51. Environmental and Molecular Mutagenesis. 2010. pp. 181–191. doi:10.1002/em.20534/pdf. . [55] Dales, R; Liu, L; Wheeler, AJ; Gilbert, NL (July 2008). "Quality of indoor residential air and health" (http:/ / www. cmaj. ca/ cgi/ content/ full/ 179/ 2/ 147). Canadian Medical Association Journal (Canadian Medical Association Journal) 179 (2) (2): 147–52. doi:10.1503/cmaj.070359. PMC 2443227. PMID 18625986. . [56] "Testing for Indoor Air Quality, Baseline IAQ, and Materials" (http:/ / www. epa. gov/ rtp/ new-bldg/ environmental/ s_01445. htm). Environmental Protection Agency. . [57] Residential Indoor Air Formaldehyde Testing Program: A Pilot Study," M. Koontz, et al, prepared for U.S. EPA, 1996 [58] Evans, Ben (2008-04-11). "FEMA limits formaldehyde in trailers" (http:/ / www. boston. com/ news/ nation/ washington/ articles/ 2008/ 04/ 11/ fema_limits_formaldehyde_in_trailers/ ). Boston.com. . Retrieved 2008-09-04. [59] "Formaldehyde allergy" (http:/ / dermnetnz. org/ dermatitis/ formaldehyde-allergy. html). DermNet NZ. New Zealand Dermatological Society. June 2009. . Retrieved 2009-09-01. [60] De Groot, Anton C; Flyvholm, Mari-Ann; Lensen, Gerda; Menné, Torkil; Coenraads, Pieter-Jan (2009). "Formaldehyde-releasers: relationship to formaldehyde allergy. allergy to formaldehyde and inventory of formaldehyde-releasers" (http:/ / onlinelibrary. wiley. com. silk. library. umass. edu:2048/ doi/ 10. 1111/ j. 1600-0536. 2009. 01582. x/ full). Dermatitis 61 (2): 63–85. doi:10.1111/j.1600-0536.2009.01582.x. PMID 19706047. . [61] Formaldehyde CAS 50-00-0 (PDF) (http:/ / www. inchem. org/ documents/ sids/ sids/ FORMALDEHYDE. pdf) [62] Formaldehyde and Facts About Health Effects (http:/ / www2. dupont. com/ Plastics/ en_US/ assets/ s/ processing/ FETEG_Facts. pdf) (PDF). Formaldehyde Epidemiology, Toxicology and Environmental Group. August 2002. Accessed on April 25, 2010. [63] When Sampling Formaldehyde, The Medium Matters (http:/ / galsonlabs. com/ services/ referenceinfo/ technical_bulletins. php?tb_id=18) [64] NIOSH Pocket Gide to Chemical Hazards: Formaldehyde (http:/ / www. cdc. gov/ niosh/ npg/ npgd0293. html) [65] Addendum to the 12th Report on Carcinogens (PDF) (http:/ / ntp. niehs. nih. gov/ ntp/ roc/ twelfth/ Addendum. pdf) National Toxicology Program, U.S. Department of Health and Human Services, retrieved 06-13-2011 [66] "European Union Bans formaldehyde/formalin within Europe" (http:/ / ec. europa. eu/ environment/ biocides/ pdf/ 070622_withdrawal_notice. pdf) (PDF). European Commission's Environment Directorate-General. September 2007. pp. 1–3. . Retrieved 19 May 2012. [67] "ESIS (European Chemical Substances Information System)" (http:/ / esis. jrc. ec. europa. eu/ ). European Commission t Research Centre Institute for Health and Consumer Protection. February 2009. . Retrieved 19 May 2012. [68] "S.1660 – Formaldehyde Standards for Composite Wood Products Act" (http:/ / www. opencongress. org/ bill/ 111-s1660/ text). OpenCongress. July 2010. . Retrieved 19 May 2012. [69] "Health Canada – Proposed residential indoor air quality guidelines for formaldehyde" (http:/ / www. hc-sc. gc. ca/ ewh-semt/ pubs/ air/ formaldehyde/ preamble-eng. php). Health Canada. April 2007. . [70] CFC.gov (PDF) (http:/ / www. cdc. gov/ niosh/ nmam/ pdfs/ 2016. pdf) [71] Formaldehyde Levels in FEMA-Supplied Trailers (PDF) (http:/ / www. cdc. gov/ nceh/ ehhe/ trailerstudy/ pdfs/ SummaryofStudyFindings. pdf) [72] Mike Brunker (2006-07-25). "Are FEMA trailers 'toxic tin cans'?" (http:/ / www. msnbc. msn. com/ id/ 14011193/ from/ ET/ #storyContinued). MSNBC. . Retrieved 19 May 2012. [73] ATSDR – Minimal Risk Levels for Hazardous Substances (MRLs) (http:/ / www. atsdr. cdc. gov/ mrls/ index. html) [74] FEMA: CDC Releases Results Of Formaldehyde Level Tests (http:/ / www. fema. gov/ news/ newsrelease. fema?id=42606) [75] Kunzelman, Michael (2007-08-08). "Suit Filed Over FEMA Trailer Toxins" (http:/ / www. washingtonpost. com/ wp-dyn/ content/ article/ 2007/ 08/ 08/ AR2007080801758. html). The Washington Post. . Retrieved 2010-05-02. [76] Megan Terlecky (2008-10-24). "How We Tested for Formaldehyde" (http:/ / www. kgan. com/ newsroom/ top_stories/ videos/ kgan_vid_1515. shtml). KGAN-TV. . [77] Nigel duara (2008-10-21). "FEMA disputes formaldehyde study of Iowa trailers" (http:/ / ap. google. com/ article/ ALeqM5jQTjw89qkEGgyRpUyURQLnyrPsMwD93V991G0). Associated Press. . [78] Cindy Hadish (2008-10-24). "FEMA meets with mobile home residents over health concerns" (http:/ / www. gazetteonline. com/ apps/ pbcs. dll/ article?AID=/ 20081025/ NEWS/ 710259952/ 1006). Cedar Rapids Gazette. .

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Formaldehyde [79] "Formaldehyde-laced foods reemerge in Indonesian markets" (http:/ / www. antaranews. com/ en/ news/ 74626/ formaldehyde-laced-foods-reemerge-in-indonesian-markets). August 10, 2011. . [80] "Formaldehyde-Tainted Rice Drinks Found at Carrefour Markets" (http:/ / www. thejakartaglobe. com/ health/ formaldehyde-tainted-rice-drinks-found-at-carrefour-markets/ 460829). August 22, 2011. . [81] Xiaojiang Tang et al., " Formaldehyde in China: Production, consumption, exposure levels, and health effects (PDF) (http:/ / superfund. berkeley. edu/ pdf/ 117. pdf)", Environment International 35 (2009): 1215–16, and other references cited on p. 1216; see also " Municipality sees red over bad blood processing (http:/ / www. chinadaily. com. cn/ china/ 2011-03/ 18/ content_12189671. htm)" (2011-03-18, China Daily, online English edition; retrieved on May 17, 2011). [82] Moise Ngwa (2010-10-25). "formaldehyde testing" (http:/ / monographs. iarc. fr/ ENG/ Monographs/ vol88/ mono88-6A. pdf) (PDF). Cedar Rapids Gazette. . Retrieved 19 May 2012. [83] Illegal business 'being run by a gang' - The Nation (http:/ / www. nationmultimedia. com/ 2011/ 06/ 16/ national/ Illegal-business-being-run-by-a-gang-30157928. html) [84] Import of formaldehyde fish from Pakistan foiled in Batam|The Jakarta Post (http:/ / www. thejakartapost. com/ news/ 2012/ 02/ 23/ import-formaldehyde-fish-pakistan-foiled-batam. html)

External links • International Chemical Safety Card 0275 (http://www.inchem.org/documents/icsc/icsc/eics0275.htm) (gas) • International Chemical Safety Card 0695 (http://www.inchem.org/documents/icsc/icsc/eics0695.htm) (solution) • NIOSH Pocket Guide to Chemical Hazards 0293 (http://www.cdc.gov/niosh/npg/npgd0293.html) • Entry for "Formaldehyde" (http://www.npi.gov.au/database/substance-info/profiles/45.html) on the Australian National Pollutant Inventory • Formaldehyde Council (http://www.formaldehyde.org/) (grouping of producers and industrial s in North America) • Formaldehyde (http://chemsub.online.fr/name/formaldehyde.html) from ChemSub Online • Prevention guide—Formaldehyde in the Workplace (PDF) (http://www.irsst.qc.ca/files/documents/ PubIRSST/RG-473.pdf) from the IRSST • Formaldehyde (http://www.cdc.gov/niosh/topics/formaldehyde/) from the National Institute for Occupational Safety and Health • IPCS Health and Safety Guide 57: Formaldehyde (http://www.inchem.org/documents/hsg/hsg/hsg057.htm) • IPCS Environmental Health Criteria 89: Formaldehyde (http://www.inchem.org/documents/ehc/ehc/ehc89. htm) • SIDS Initial Assessment Report for Formaldehyde (http://www.inchem.org/documents/sids/sids/ FORMALDEHYDE.pdf) from the Organisation for Economic Co-operation and Development (OECD) • Formaldehyde Added to "Known Carcinogens" List Despite Lobbying by Chemical Industry (http://www. democracynow.org/2011/6/14/formaldehyde_added_to_known_carcinogens_list) — video report by Democracy Now!

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Franz Nissl Franz Nissl (9 September 1860, Frankenthal – 11 August 1919, Munich) was a German medical researcher. He was a noted neuropathologist.

Early life Nissl was born in Frankenthal to Theodor Nissl and Maria Haas. Theodor taught Latin in a Catholic school and desired that Franz become a priest. However Franz entered the Ludwig Maximilian University of Munich to study medicine. One of Nissl's university professors was Bernhard von Gudden. His assistant, Sigbert Josef Maria Ganser suggested that Nissl write an essay on the pathology of the cells of the cortex of the brain. When the medical faculty offered a competition for a prize in neurology in 1884, Nissl did undertake the brain-cortex study. He used alcohol as a fixative and developed a staining technique that allowed demonstrating several new nerve-cell constituents. Nissl won the prize, and wrote his doctoral dissertation on the same topic in 1885.[1]

Career in medical research and education Portrait of Franz Nissl.

Professor von Gudden was the judge in Nissl's college-essay competition, and he was so impressed with the study that he offered Nissl an assistantship at the Oberbayerische Kreis-Irrenanstalt Haar in Munich which von Gudden headed. Nissl accepted, and remained in that post from 1885 until 1888. In 1888 Nissl moved to the Institution Blankenheim. In 1889 he went to Frankfurt as second in position under Emil Sioli (1852–1922) at the Städtische Irrenanstalt. There he met neurologist Ludwig Edinger and neuropathologist Karl Weigert, who was developing a neuroglial stain. This work motivated Nissl to study mental and nervous diseases by relating them to observable changes in glial cells, blood elements, blood vessels and brain tissue in general. In Frankfurt Nissl became acquainted with Alois Alzheimer, and they collaborated over seven years. They became close friends,[2] tly editing the Histologische und histopathologische Arbeiten über die Grosshirnrinde (1904–1921). In 1895 Emil Kraepelin invited Nissl to become assistant physician at the University of Heidelberg. By 1904 he was a full professor at that institution, and became director of the Department of Psychiatry when Kraepelin moved to Munich.

Franz Nissl

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Later life and death The burden of teaching and istration, combined with poor research facilities, forced Nissl to leave many scientific projects unfinished. He also suffered from a kidney disease. During World War I he was charged with istering a large military hospital. In 1918 Kraepelin again invited Nissl to accept a research position at the Deutsche Forschungsanstalt für Psychiatrie in Munich. After one year at that position, where he performed research alongside Korbinian Brodmann and Walther Spielmeyer, he died in 1919 of kidney disease.

Personal Nissl was of small stature, with poor posture. He had a birthmark on his left face. He never married, and his life revolved entirely around his work.[3] One day, for a practical joke, Nissl (who was an active campaigner against human consumption of alcohol) placed a row of empty beer bottles outside his laboratory and made sure that Kraepelin heard that he could be found lying under his desk, dead drunk. Nissl was a competent pianist. Hugo Spatz (1888–1969) told of his first meeting, when Spatz applied for a position in Nissl's laboratory. Nissl was busy that morning and asked the student to come to his home at twelve. When Spatz came to the house at noon, Nissl was not there, and the housekeeper finally opined that the Professor must have meant twelve midnight, so Spatz returned that night. Nissl was at home then, but Spatz had to wait in the anteroom for half an hour until Nissl had finished the piano sonata that he was playing. The conversation lasted until daybreak.

Legacy Nissl was possibly the greatest neuropathologist of his day and also a fine clinician who popularised the use of spinal puncture,[4] which had been introduced by Heinrich Quincke. Nissl also examined the neural connections between the human cortex and thalamic nuclei; he was in the midst of this study at the time of his death. An example of his research philosophy is taken from his 1896 writings: As soon as we agree to see in all mental derangements the clinical expression of definite disease processes in the cortex, we remove the obstacles that make impossible agreement among alienists.

Named histology concepts

Image of a Nissl-stained histological section through the rodent hippocampus showing various classes of neurons.

The Nissl method refers to staining of the cell body, and in particular endoplasmic reticulum. This is done by using various basic dyes (e.g. aniline, thionine, or cresyl violet) to stain the negatively charged RNA blue, and is used to highlight important structural features of neurons. The Nissl substance (rough endoplasmic reticulum) appears dark blue due to the staining of ribosomal RNA, giving the cytoplasm a mottled appearance. Individual granules of extranuclear RNA are named Nissl granules (ribosomes). DNA present in the nucleus stains a similar color.

External links • Nissl staining method and protocol link [5]

Franz Nissl

References [1] [2] [3] [4] [5]

http:/ / www. whonamedit. com/ doctor. cfm/ 2465. html Biography of Franz Nissl, accessed 01 November 2009 Nissl was Alzheimer's best man at the latter's wedding in April 1894 Biography Nissl's nickname among medical students of the day was "punctator maximus" http:/ / openwetware. org/ wiki/ Nissl_staining

• More information from the University of Illinois at Chicago (http://www.uic.edu/depts/mcne/founders/ page0067.html)

Frozen section procedure The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection. The quality of the slides produced by frozen section is of lower quality than formalin fixed, wax embedded tissue processing. While diagnosis can be rendered in many cases, fixed tissue processing is preferred in many conditions for more accurate diagnosis. The intraoperative consultation is the name given to the whole intervention by the pathologist, which includes not only frozen section but also gross evaluation of the specimen, examination of cytology preparations taken on the specimen (e.g. touch imprints), and aliquoting of the specimen for special studies (e.g. molecular pathology techniques, flow cytometry). The report given by the pathologist is usually limited to a "benign" or "malignant" diagnosis, and communicated to the surgeon operating via intercom. When operating on a previously confirmed malignancy, the main purpose of the pathologist is to inform the surgeon if the surgical margin is clear of residual cancer, or if residual cancer is present at the surgical margin. The method of processing is usually done with the bread loafing technique. But margin controlled surgery (CDMA) can be performed using a variety of tissue cutting and mounting methods, including mohs surgery.

Procedure The key instrument for cryosection is the cryostat, which is essentially a microtome inside a freezer. The microtome can be compared to a very accurate "deli" slicer, capable of slicing sections as thin as 1 micrometre. The usual histology slice is cut at 5 to 10 micrometres. The surgical specimen is placed on a metal tissue disc which is then secured in a chuck and frozen rapidly to about −20 to -30 °C. The specimen is embedded in a gel like medium consisting of poly ethylene glycol and polyvinyl alcohol; this compound is known by many names and when frozen has the same density as frozen tissue. At this Tissue embedded within OCT, mounted on a temperature, most tissues become rock-hard. Usually a lower chuck in a cryostat and ready for section temperature is required for fat or lipid rich tissue. Each tissue has a production preferred temperature for processing. Subsequently it is cut frozen with the microtome portion of the cryostat, the section is picked up on a glass slide and stained (usually with hematoxylin and eosin, the H&E stain). The preparation of the sample is much more rapid than with traditional histology technique (around 10 minutes vs 16 hours). However, the technical quality of the sections is much lower. The entire laboratory can occupy a space less than 9-square-foot (unknown operator: u'strong' m2), and minimal ventilation is required compared to a standard wax embedded specimen laboratory.

95

Frozen section procedure

Uses The principal use of the frozen section procedure is the examination of tissue while surgery is taking place. This may be for various reasons: • In the performance of Mohs surgery - a simple method for 100% margin control of a surgical specimen. • If a tumor appears to have metastasized, a sample of the suspected metastasis is sent for cryosection to confirm its identity. This will help the surgeon decide whether there is any point in continuing the operation. Usually, aggressive surgery is performed only if there is a chance to cure the patient. If the tumor has metastasized, surgery is usually not curative, and the surgeon will choose a more conservative surgery, or no resection at all. • If a tumor has been resected but it is unclear whether the surgical margin is free of tumor, an intraoperative consultation is requested to assess the need to make a further resection for clear margins. • In a sentinel node procedure, a sentinel node containing tumor tissue prompts a further lymph node dissection, while a benign node will avoid such a procedure. • If surgery is explorative, rapid examination of a lesion might help identify the possible cause of a patient's symptoms. It is important to note, however, that the pathologist is very limited by the poor technical quality of the frozen sections. A final diagnosis is rarely offered intraoperatively. • Rarely, cryosections are used to detect the presence of substances lost in the traditional histology technique, for example lipids. They can also be used to detect some antigens masked by formalin. • The cryostat is available in a small portable device weighing less than 80 lb (unknown operator: u'strong' kg), to a large stationary device 500 lb (unknown operator: u'strong' kg) or more. The entire histologic laboratory can be carried in one portable box, making frozen section histology a possible tool in primitive medicine.

History The frozen section procedure as practiced today in medical laboratories is based on the description by Dr Louis B. Wilson in 1905. Wilson developed the technique from earlier reports at the request of Dr William Mayo, surgeon and one of the founders of the Mayo Clinic. Earlier reports by Dr Thomas S. Cullen at Johns Hopkins Hospital in Baltimore also involved frozen section, but only after formalin fixation, and pathologist Dr William Welch, also at Hopkins, experimented with Cullen's procedure but without clinical consequences. Hence, Wilson is generally credited with truly pioneering the procedure (Gal & Cagle, 2005).

References • Gal AA, Cagle PT. The 100-year anniversary of the description of the frozen section procedure. [1] JAMA 2005;294:3135-7. • Wilson LB. A method for the rapid preparation of fresh tissues for the microscope. J Am Med Assoc 1905;45:1737.

External links • JAMA patient page [2] on frozen section procedure • Description of the frozen section procedure [3]

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Frozen section procedure

97

References [1] http:/ / jama. ama-assn. org/ cgi/ content/ full/ 294/ 24/ 3135 [2] http:/ / jama. ama-assn. org/ cgi/ content/ full/ 294/ 24/ 3200 [3] http:/ / www. medparse. com/ axsop/ axsop025. htm

Fuchsine Fuchsine

Identifiers [1]

CAS number

632-99-5

ChemSpider

21106314

UNII

8UUC89LHB2

RTECS number

8053-09-6

Jmol-3D images

Image 1

[2]

[3]

[4]


C20H20N3·HCl

Molar mass

337.86 g/mol (hydrochloride)

Melting point

200°C

Solubility in water

2650 mg/L (25°C)

log P

2.920

Vapor pressure

7.49E-10 mm Hg (25°C)

k

2.28E-15 atm-m3/mole (25°C)

Atmospheric OH rate constant

4.75E-10 cm3/molecule-sec (25°C)

H

Hazards Main hazards

Ingestion, inhalation, skin and eye , combustible at high temperature, slightly explosive around open flames and sparks.

NFPA 704 ()

[5]


Infobox references

Fuchsine

98

Fuchsine or rosaniline hydrochloride is a magenta dye with chemical formula C20H19N3·HCl.[6][7] There are other similar chemical formulations of products sold as fuchsine, and several dozen other synonyms of this molecule.[6] It becomes magenta when dissolved in water; as a solid, it forms dark green crystals. As well as dying textiles, fuchsine is used to stain bacteria and sometimes as a disinfectant. In the literature of biological stains the name of this dye is frequently misspelled, with omission of the terminal e, which indicates an amine[8] American and English dictionaries (Webster's, Oxford, Chambers etc.) give the correct spelling, which is also used in the literature of industrial dyeing.[9]

Picture of Basic Fuchsine Pieces, C.I. 42500.

History Fuchsine was first prepared by August Wilhelm von Hofmann from aniline and carbon tetrachloride in 1858.[10][11] François-Emmanuel Verguin discovered the substance independent of Hofmann the same year and patented it.[12] Fuchsine was named by its original manufacturer Renard frères et Franc,[13] is usually cited with one of two etymologies: from the color of the flowers of the plant genus Fuchsia,[14] named in honor of botanist Leonhart Fuchs, or as the German translation Fuchs of the French name Renard, which means fox.[15] An 1861 article in Répertoire de Pharmacie said that the name was chosen for both reasons.[16][17]

Acid fuchsine Acid fuchsine is a mixture of homologues of basic fuchsin, modified by addition of sulfonic groups. While this yields twelve possible isomers, all of them are satisfactory despite slight differences in their properties.

Basic fuchsine Basic fuchsine is a mixture of rosaniline, pararosaniline, new fuchsine and Magenta II.[18] Formulations usable for making of Schiff reagent must have high content of pararosanilin. The actual composition of basic fuchsine tends to somewhat vary by vendor and batch, making the batches differently suitable for different purposes. In solution with phenol (also called carbolic acid) as an accentuator[19] it is called carbol fuchsin and is used for the Ziehl-Neelsen and other similar acid fast staining of the mycobacteria which cause tuberculosis, leprosy etc.[20] Basic fuchsine is widely used in biology to stain the nucleus.

Properties The 'crystals', pictured at the right are of basic fuchsine, also known as basic violet 14, basic red 9, pararosanaline or CI 42500. Their structure differs from the structure shown above by the absence of the methyl group on the upper ring, otherwise they are quite similar. They are soft, with a hardness of less than 1, about the same as or less than talc. They possess a strong metallic lustre and a green yellow colour. They leave dark greenish streaks on paper and when these are moistened with a solvent, the strong magenta colour appears. The two magenta stains on the paper were made by placing one drop of ethanol-water azeotope, centre, and water, right, on the streaks remaining on the paper after the 'crystals' were removed. The 'crystals' were then replaced and the photograph taken.

Fuchsine

99

Notes on Chemical Structure This compound is an amine salt, and the structure depicted above may be a little unclear as to the relationship between the organic part and the hydrogen chloride, in green. Just as ammonia can bind a hydrogen ion to its lone pair to form the ammonium ion, any amine can basically do the same as long as no electron-withdrawing groups are positioned near the amine group to retract the lone pair. As an amine is simply ammonia in which one or more of the hydrogen atoms have been replaced by 'R' groups, usually organic groups containing carbon where a carbon atom binds to the amine nitrogen instead of hydrogen as in ammonia. This compound possesses three amine groups, blue, two primary amines and a secondary amine. If one of these is protonated to form ABCNH+, the positive charge is delocalized across the whole symmetrical molecule due to pi cloud electron movement. The positive charge can be thought of as residing on the central carbon atom and all three 'wings' becoming identical aromatic rings terminated by a primary amine group. Other resonance structures can be thought of, where the positive charge 'moves' from one amine group to the next, or one third of the positive charge resides on each amine group. It is this ability to be protonated by a stronger acid which gives this compound its basic property. The positive charge is neutralized by the negative charge on the chloride ion. The positive 'basic fuchsinium ions' and negative chloride ions stack to form the salt 'crystals' depicted above. Many other amine compounds, such as many alkaloids, drugs and pesticides form salts in the same manner with strong acids like hydrochloric acid, just as this compound does.

References [1] [2] [3] [4]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=632-99-5 http:/ / www. chemspider. com/ 21106314 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=8UUC89LHB2 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=Cl. %5BNH2%2B%5D%3DC%5C1%2FC%3DC%5CC%28C%3DC%2F1%29%3DC%28%5Cc2ccc%28N%29c%28C%29c2%29c3ccc%28N%29cc3 [5] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=425367120& page2=%3AFuchsine [6] "Basic chemical data" (http:/ / dtp. nci. nih. gov/ dtpstandard/ servlet/ ChemData?queryHOLD=& searchtype=namestarts& chemnameboolean=and& outputformat=html& searchlist=Rosaniline& Submit=Submit). Discovery Series online database, Developmental Therapeutics Program, U.S. National Institutes of Health. Retrieved on 2007-10-08. [7] Goyal, S.K. "Use of rosaniline hydrochloride dye for atmospheric SO2 determination and method sensitivity analysis" (http:/ / www. rsc. org/ ej/ EM/ 2001/ b106209n. pdf). Journal of Environmental Monitoring, 3, 666-670, doi:10.1039/b106209n. Retrieved on 2007-10-08. [8] Baker JR (1958) Principles of Biological Microtechnique. London: Methuen. [9] Hunger K (2003) Industrial Dyes. Chemistry, Properties, Applications. Weinheim: wiley-VHC. [10] von Hofmann, August Wilhelm (1859). "Einwirkung des Chlorkohlenstoffs auf Anilin. Cyantriphenyldiamin". Journal für Praktische Chemie 77: 190. doi:10.1002/prac.18590770130. [11] von Hofmann, August Wilhelm (1858). "Action of Bichloride of Carbon on Aniline" (http:/ / zs. thulb. uni-jena. de/ receive/ jportal_jpvolume_00057523?XSL. view. objectmetadata=false& jumpback=true& maximized=true& page=PMS_1859_Bd17_ 0089. tif). Philosophical Magazine: 131–142. . [12] . pp. 42–46. http:/ / books. google. de/ books?id=z-MEAAAAQAAJ& pg=PA42. [13] Béchamp, M. A. (January–June 1860.) "Comptes rendus hebdomadaires des séances de l'Académie des sciences. 1860. (T. 50)." (http:/ / gallica. bnf. fr/ ark:/ 12148/ bpt6k3007r) French Academy of Sciences, Mallet-Bachelier: Paris, tome 50, page 861. Retrieved on 2007-09-25. [14] (2004.) "Fuchsin" (http:/ / dictionary. reference. com/ browse/ fuchsin) The American Heritage Dictionary of the English Language, Fourth Edition, Houghton Mifflin Company, via dictionary.com. Retrieved on 2007-09-20 [15] "Fuchsine." (http:/ / humanities. uchicago. edu/ orgs/ ARTFL/ forms_unrest/ webster. form. html) (Website.) ARTFL Project: 1913 Webster's Revised Unabridged Dictionary. Retrieved on 2007-09-25 [16] Chevreul, M. E. (July 1860). "Note sur les étoffes de soie teintes avec la fuchsine, et réflexions sur le commerce des étoffes de couleur." (http:/ / books. google. com/ books?id=lfIKAAAAYAAJ& pg=PA62& dq=fuchsine+ renard+ fuchsia& as_brr=3#PPA5,M1) Répertoire de Pharmacie, tome XVII, p. 62. Retrieved on 2007-09-25. [17] Belt, H. V. D.; Hornix, Willem J.; Bud, Robert; Van Den Belt, Henk (1992). "Why Monopoly Failed: The Rise and Fall of Société La Fuchsine". The British Journal for the History of Science 25 (1): 45–63. doi:10.1017/S0007087400045325. JSTOR 4027004. [18] Horobin RW & Kiernan JA 20002. Conn's Biological Stains, 10th ed. Oxford: BIOS, p.184-191

Fuchsine

100

[19] http:/ / stainsfile. info/ StainsFile/ theory/ accent. htm [20] Clark G 1973 Staining Procedures Used by the Biological Stain Commission, 3rd ed. Baltimore: Williams & Wilkins, pp. 252-254

Giemsa stain Giemsa stain, named after Gustav Giemsa, an early German microbiologist, is used in cytogenetics and for the histopathological diagnosis of malaria and other parasites.[1]

Uses It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. Giemsa stain is used in Giemsa banding, commonly called G-banding, to stain chromosomes and often used to create an idiogram. It can identify chromosomal aberrations such as translocations and rearrangements.

Giemsa stained Trypanosoma parasites (Chagas disease pathogen)

Giemsa stain is also a differential stain. It can be used to study the adherence of pathogenic bacteria to human cells. It differentially stains human and bacterial cells purple and pink respectively. It can be used for histopathological diagnosis of malaria[2] and some other spirochete and protozoan blood parasites. It is also used in Wolbach's tissue stain. Giemsa stain is a classic blood film stain for peripheral blood smears and bone marrow specimens. Whirling disease section stained with Giemsa Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta. Giemsa stain is also used to visualize chromosomes. Giemsa stain also stains the fungus histoplasma and chlamydia. It can also be used to identify Mast cells.[3]

Generation Giemsa's solution is a mixture of methylene blue, eosin, and azure B. The stain is usually prepared from commercially available Giemsa powder. A thin film of the specimen on a microscope slide is fixed in pure methanol for 30 seconds, by immersing it or by putting a few drops of methanol on the slide. The slide is immersed in a freshly prepared 5% Giemsa stain solution for 20–30 minutes (in emergencies 5–10 minutes in 10% solution can be used), then flushed with tap water and left to dry.[4]

Giemsa stain

References [1] Giemsa G (1904 Eine Vereinfachung und Vervollkommnung meiner Methylenblau-Eosin-Färbemethode zur Erzielung der Romanowsky-Nocht’schen Chromatinfärbung. Centralblatt für Bakteriologie I Abteilung 32, 307–313. [2] Shapiro HM, Mandy F (September 2007). "Cytometry in malaria: moving beyond Giemsa". Cytometry. Part A : the journal of the International Society for Analytical Cytology 71 (9): 643–5. doi:10.1002/cyto.a.20453. PMID 17712779. [3] Damsgaard TE, Olesen AB, Sørensen FB, Thestrup-Pedersen K, Schiøtz PO (April 1997). "Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin" (http:/ / link. springer. de/ link/ service/ journals/ 00403/ bibs/ 7289005/ 72890256. htm). Arch. Dermatol. Res. 289 (5): 256–60. doi:10.1007/s004030050189. PMID 9164634. . [4] "4.2.2.2" (http:/ / www. impact-malaria. com/ FR/ EPS/ Formations_et_cours_internationaux/ Formation_de_la_Liverpool_School_LSTMH/ cours_liverpool/ Unit_4/ 4_2_2_2. html). .

Gimenez stain The Gimenez staining technique uses biological stains to detect and identify bacterial infections in tissue samples. Although largely superseded by techniques like Giemsa staining, the Gimenez technique may be valuable for detecting certain slow-growing or fastidious bacteria. Basic fuchsin stain in aqueous solution with phenol and ethanol colours many bacteria (both gram positive and Gram negative) red, magenta, or pink. A malachite green counterstain gives a blue-green background cast to the surrounding tissue.

References • P. Bruneval et al.. "Detection of fastidious bacteria in cardiac valves in cases of blood culture negative endocarditis. [1]" Journal of Clinical Pathology. 54:238-240 (2001). • D.F. Gimenez. "Staining Rickettsiae in yolksack cultures". Stain Technol 39:135–40 (1964).

References [1] http:/ / j. bmjjournals. com/ cgi/ content/ full/ 54/ 3/ 238

101

Glutaraldehyde

102

Glutaraldehyde Glutaraldehyde

Identifiers [1]

CAS number

111-30-8

PubChem

3485

ChemSpider

3365

UNII

T3C89M417N

DrugBank

DB03266

KEGG

D01120

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]

[7]


C5H8O2

Molar mass

100.12 g mol

Appearance

Clear liquid

Density

1.06 g/mL

Melting point

-14 °C, 259 K, 7 °F

Boiling point

187 °C, 460 K, 369 °F

Solubility in water

Miscible

−1

()

[8]


Infobox references

Glutaraldehyde is an organic compound with the formula CH2(CH2CHO)2. A pungent colorless oily liquid, glutaraldehyde is used to disinfect medical and dental equipment (though it is no longer in use by tattooing and piercing artists due to being either ineffective, have not been used properly, or have been found to be inappropriate for use in the industry). It is also used for industrial water treatment and as a chemical preservative.[9]

Glutaraldehyde

Production and structure Glutaraldehyde is produced industrially by the oxidation of cyclopentene and by the Diels-Alder reaction of acrolein and methyl vinyl ether followed by hydrolysis. Like other dialdehydes (e.g., glyoxal), it does not exist typically as the dialdehyde but as the hydrate.[9] Monomeric glutaraldehyde can polymerize by aldol condensation reaction yielding alpha, beta-unsaturated poly-glutaraldehyde. This reaction usually occurs at alkaline pH values.

Uses Fixative A glutaraldehyde solution of 0.1 % to 1.0 % concentration may be used for system disinfection and as a preservative for long term storage. Glutaraldehyde is used in biological electron microscopy as a fixative. It kills cells quickly by crosslinking their proteins and is usually employed alone or mixed with formaldehyde[10] as the first of two fixative processes to stabilize specimens such as bacteria, plant material, and human cells. A second fixative procedure uses osmium tetroxide to crosslink and stabilize cell and organelle membrane lipids. Fixation is usually followed by dehydration of the tissue in ethanol or acetone, followed by embedding in an epoxy resin or acrylic resin. Another example of an application for treatment of proteins with glutaraldehyde is the inactivation of bacterial toxins to create toxoid vaccines, e.g., the pertussis (whooping cough) toxoid component in the Boostrix Tdap vaccine produced by GlaxoSmithKline. [11] In a related application, glutaraldehyde is sometimes employed in the tanning of leather.

Biochemical reagent Glutaraldehyde is frequently used in biochemistry applications as an amine-reactive homobifunctional crosslinker. The oligomeric state of proteins can be examined through this application. Glutaraldehyde is also used in SDS-PAGE to fix proteins and peptides prior to staining. Typically, a gel is treated with a 5 % solution for approximately one half hour, after which it must be thoroughly washed to remove the yellow stain brought about by reacting with free tris.

Wart treatment A solution of 10 % w/w glutaraldehyde is sold under the name "Diswart Solution" to remove common and plantar warts. Product claims include: "Inactivates viruses and bacteria. Dries the wart surface. Stains the area treated brown, but will not harm the surrounding skin."

Algaecidal activity A polymerized isomer of glutaraldehyde known as polycycloglutaracetal is a fertilizer for aquatic plants . It is claimed that it provides a bioavailable source of carbon for higher plants that is not available to algae. Though not marketed as such due to federal regulations, the biocidal effect of glutaraldehyde kills most algae at concentrations of 0.5–5.0 ppm. These levels are not harmful to most aquatic fauna and flora. Adverse reactions have been observed by some aquarists at these concentrations in some aquatic mosses, liverworts, and vascular plants.

103

Glutaraldehyde

Safety As a strong disinfectant, glutaraldehyde is toxic and can cause severe eye, nose, throat, and lung irritation, along with headaches, drowsiness and dizziness. It is a main source of occupational asthma among health care providers.[12]

References [1] [2] [3] [4] [5] [6] [7] [8] [9]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=111-30-8 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=3485 http:/ / www. chemspider. com/ 3365 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=T3C89M417N http:/ / www. drugbank. ca/ drugs/ DB03266 http:/ / www. kegg. jp/ entry/ D01120 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=O%3DCCCCC%3DO http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=450655655& page2=%3AGlutaraldehyde Christian Kohlpaintner, Markus Schulte, Jürgen Falbe, Peter Lappe, Jürgen Weber (2005), "Aldehydes, Aliphatic", Ullmann's Encyclopedia of Industrial Chemistry, Weinheim: Wiley-VCH, doi:10.1002/14356007.a01_321.pub2 [10] Karnovsky, M.J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell Biology 27: 137A–138A [11] Boostrix prescribing information (http:/ / www. gsksource. com/ gskprm/ en/ US/ adirect/ gskprm?cmd=ProductDetailPage& product_id=1244167203071& featureKey=600532), ©2009, GlaxoSmithKline [12] Canadian Centre for Occupational Health and Safety (CCOHS) (a federal government site) > OSH Answers > Diseases, Disorders & Injuries > Asthma (http:/ / www. ccohs. ca/ oshanswers/ diseases/ asthma. html) Document last updated on February 8, 2005

External links • National Pollutant Inventory - Glutaraldehyde Fact Sheet (http://www.npi.gov.au/substances/glutaraldehyde/ index.html) • National Institute for Occupational Safety and Health - Glutaraldehyde (http://www.cdc.gov/niosh/topics/ glutaraldehyde/) • NIST WebBook (http://webbook.nist.gov/cgi/cbook.cgi?Name=glutaraldehyde&Units=SI) • Hazardous Chemical Information (http://www.tedpella.com/msds_html/18411msd.htm) • Glutaraldehyde at OpenWetWare (the life science wiki) (http://openwetware.org/wiki/ Glutaraldehyde_(Pentanedial))

104

Golgi's method

105

Golgi's method Golgi's method is a nervous tissue staining technique discovered by Italian physician and scientist Camillo Golgi (1843–1926) in 1873. It was initially named the black reaction (la reazione nera) by Golgi, but it became better known as the Golgi stain or later, Golgi method. Golgi' staining was famously used by Spanish neuroanatomist Santiago Ramón y Cajal (1852–1934) to discover a number of novel facts about the organization of the nervous system, inspiring the birth of the neuron doctrine. Ultimately, Ramon y Cajal improved the technique by using a method he termed "double impregnation." Ramon y Cajal's staining technique, still in use, is called Cajal's Stain.

Drawing by Camillo Golgi of a hippocampus stained with the silver nitrate method

Mechanism The cells in nervous tissue are densely packed and little information on their structures and interconnections can be obtained if all the cells are stained. Furthermore, the thin filamentary extensions of neural cells, including the axon and the dendrites of neurons, are too slender and transparent to be seen with normal staining techniques. Golgi's method stains a limited number of cells at random in their entirety. The mechanism by which this happens is still largely unknown.[1] Dendrites, as well as the cell soma, are clearly stained in brown and black and can be followed in their entire length, which allowed neuroanatomists to track connections between neurons and to make visible the complex networking structure of many parts of the brain and spinal cord. Golgi's staining is achieved by impregnating fixed nervous tissue with potassium dichromate and silver nitrate. Cells thus stained are filled by microcrystallization of silver chromate.

Technique

Drawing of a Purkinje cell in the cerebellum cortex done by Santiago Ramón y Cajal, clearly demonstrating the power of Golgi's staining method to reveal fine detail

According to SynapseWeb,[2] this is the recipe for Golgi's staining technique: 1. Immerse a block (approx. 10x5 mm) of formol-fixed (or paraformaldehyde- glutaraldehyde-perfused) brain tissue into a 2% aqueous solution of potassium dichromate for 2 days 2. Dry the block shortly with filter paper. 3. Immerse the block into a 2% aqueous solution of silver nitrate for another 2 days. 4. Cut sections approx. 20-100 µm thick. 5. Dehydrate quickly in ethanol, clear and mount (e.g., into Depex or Enthalan).

Golgi's method

106

This technique has since been refined to substitute the silver precipitate with gold by immersing the sample in gold chloride then oxalic acid, followed by removal of the silver by sodium thiosulphate. This preserves a greater degree of fine structure with the ultrastructural details marked by small particles of gold. [3]

Quote Ramón y Cajal said of the Golgi method: I expressed the surprise which I experienced upon seeing with my own eyes the wonderful revelatory powers of the chrome-silver reaction and the absence of any excitement in the scientific world aroused by its discovery.

A human neocortical pyramidal neuron stained via Golgi technique. Notice the apical dendrite extending vertically above the soma and the numerous basal dendrites radiating laterallypene from the base of the cell body.

Recuerdos de mi vida, Vol. 2, Historia de mi labor científica. Madrid: Moya, 1917, p. 76.

Notes [1] Nicholls, J. G. (2001). From neuron to brain. Sinauer Associates. pp. 5. ISBN 0878934391/ISSN. [2] Spacek, J., Fiala, J. (2002-06-28). "Visualization of Dendritic Spines" (http:/ / synapses. clm. utexas. edu/ learn/ visualize/ visualize. stm). SynapseWeb. . Retrieved 2010-06-17. [3] http:/ / www. springerlink. com/ (f0cb2ybwsglgtnuip5efub55)/ app/ home/ contribution. asp?referrer=parent& backto=issue,5,11;journal,215,243;linkingpublicationresults,1:100182,1

External links • Photomicrograph of a cortex cell stained with Golgi's (http://www.ihcworld.com/imagegallery/images/ special-stain/Golgi-g.jpg). IHC Image Gallery. • Golgi impregnations (http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/golgi/). Images of the brain of flies. • Visualization of dendritic spines using Golgi Method (http://synapses.clm.utexas.edu/learn/visualize/golgi2. stm#GolgiTime). SynapseWeb. Includes a time-lapse study of Golgi impregnation. • Berrebi, Albert: Cell Biology of Neurons: Structure and Methods of Study (http://www.ihcworld.com/ imagegallery/displayimage.php?album=4&pos=13). (in PDF) • BrainMaps at UCDavis Golgi-stained neurons (http://brainmaps.org/index.php?q=Golgi-stained)

Gram staining

107

Gram staining Gram staining (or Gram's method) is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). It is based on the chemical and physical properties of their cell walls. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram positive bacteria.[1] A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color. The Gram stain is almost always the first step in the identification of a bacterial organism, and is the default stain performed by laboratories over a sample when no specific culture is referred. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram-variable and Gram-indeterminate groups as well.

Gram-positive anthrax bacteria (purple rods) and white blood cells (round) in cerebrospinal fluid. Gram-negative bacteria would have appeared pink.

The word Gram is always spelled with a capital, referring to Hans Christian Gram, the inventor of Gram staining.

History The method is named after its inventor, the Danish scientist Hans Christian Gram (1850–1938), who developed the technique while A Gram stain of mixed Staphylococcus aureus working with Carl Friedländer in the morgue of the city hospital in (Gram positive cocci) and Escherichia coli (Gram Berlin. Gram devised his technique not for the purpose of negative bacilli), the most common Gram stain reference bacteria distinguishing one type of bacterium from another but to enable [2] bacteria to be seen more readily in stained sections of lung tissue. He published his method in 1884, and included in his short report the observation that the Typhus bacillus did not retain the stain.[3]

Uses Gram staining is a bacteriological laboratory technique[4] used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell walls.[5] Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.[6] The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of extremely limited use in environmental microbiology. It has been largely superseded by molecular techniques even in the medical microbiology lab. Some organisms are Gram-variable (that means, they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and information-rich than differential staining. Gram-staining has proven to be as effective a diagnostic tool as PCR. Particularly with regards to gonorrhoea diagnosis in Kuwait.The similarity of the results of both Gram stain and PCR for diagnosis of gonorrhea was 99.4% in Kuwait.[7]

Gram staining

Medical Gram stains are performed on body fluid or biopsy when infection is suspected. Gram stains yield results much more quickly than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.[4][8] .

Staining mechanism Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell envelope), which are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell envelope), which are stained pink by the counter-stain. There are four basic steps of the Gram stain: • Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixing kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure. • The addition of a mordant, which binds to crystal violet and traps it in the cell (Gram's iodine) • Rapid decolorization with alcohol or acetone, and • Counterstaining with safranin.[9] Carbol fuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.[10] Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl−) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I− or I) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, color the cell.[11] When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell will lose its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.[12][13] Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative.[14] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

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Examples Gram-positive bacteria Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is followed by two phyla — Firmicutes (except for the classes Mollicutes and Negativicutes) and the Actinobacteria.[5][15] In contrast, of the Chloroflexi (green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative, positive or indeterminate.[5][15] of the Deinococcus-Thermus group, stain positive but are diderms with a thick peptidoglycan.[5][15] Historically, the Gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

Gram-negative bacteria Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phyla are Gram-negative, including the cyanobacteria, spirochaetes and green sulfur bacteria, and most Proteobacteria (exceptions being some of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).[5][15]

Gram-indeterminate bacteria Gram-indeterminate bacteria do not respond to Gram staining and, therefore, cannot be determined as either Gram-positive or Gram-negative. Examples of them, but not limited to, are Gram-variable and acid fast bacteria.

References [1] Bergey, David H.; John G. Holt; Noel R. Krieg; Peter H.A. Sneath (1994). Bergey's Manual of Determinative Bacteriology (9th ed.). Lippincott Williams & Wilkins. ISBN 0-683-00603-7. [2] Austrian, R. (1960). "The Gram stain and the etiology of lobar pneumonia, an historical note". Bacteriol. Rev. 24 (3): 261–265. PMC 441053. PMID 13685217. [3] Gram, HC (1884). "Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten" (in German). Fortschritte der Medizin 2: 185–189. English translation in: Brock, T.D. (1999). Milestones in Microbiology 1546-1940 (http:/ / books. google. com/ ?id=q5JHcs8w21gC& lpg=PP1& dq=Milestones in Microbiology& pg=PA215#v=onepage& q) (2 ed.). ASM Press. pp. 215–218. ISBN 1-55581-142-6. . Translation is also at: Brock, T.D.. Pioneers in Medical Laboratory Science: Christian Gram 1884 (http:/ / www. hoslink. com/ history2. htm#gram). Hoslink. . Retrieved 2010-07-27 [4] Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 232–3. ISBN 0-8385-8529-9. [5] Madigan, MT; Martinko J, Parker J (2004). Brock Biology of Microorganisms (10th ed.). Lippincott Williams & Wilkins. ISBN 0-13-066271-2. [6] Beveridge TJ (2001). "Use of the Gram stain in microbiology". Biotech Histochem 76 (3): 111–8. doi:10.1080/714028139. PMID 11475313. [7] El-Garnal, A.H., Al-Otaibi, S.R., Alshamali, A., Abdulrazzaq, A., Najem, N., & Fouzan, A.A. Polymerase chain reaction is no better than Gram stain for diagnosis of gonococcal urethritis. Indian Journal of Dermatology, Venereology, and Leprology,(2009); 75, 101. [8] Søgaard M, Nørgaard M, Schønheyder H (2007). "First notification of positive blood cultures: high accuracy of the Gram stain report (Epub ahead of publication)". J Clin Microbiol 45 (4): 1113. doi:10.1128/JCM.02523-06. PMC 1865800. PMID 17301283. [9] Microbiology; J.G. Black Prentice Hall, 1993 [10] http:/ / www. med-chem. com/ procedures/ GRAMSTAIN. pdf [11] Llewellyn, Brian D. (May, 2005). http:/ / stainsfile. info/ StainsFile/ theory/ notmr ayikho yonke l;ento. Retrieved 2009-09-10. [12] Beveridge TJ, Davies JA (November 1983). "Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain" (http:/ / jb. asm. org/ cgi/ pmidlookup?view=long& pmid=6195148). Journal of bacteriology 156 (2): 846–58. PMC 217903. PMID 6195148. . [13] Davies JA, Anderson GK, Beveridge TJ, Clark HC (November 1983). "Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy, which replaces the iodine mordant of the stain" (http:/ / jb. asm. org/ cgi/ pmidlookup?view=long& pmid=6195147). Journal of bacteriology 156 (2): 837–45. PMC 217902. PMID 6195147. .

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[14] Beveridge TJ (March 1990). "Mechanism of Gram variability in select bacteria" (http:/ / jb. asm. org/ cgi/ pmidlookup?view=long& pmid=1689718). Journal of bacteriology 172 (3): 1609–20. PMC 208639. PMID 1689718. . [15] George M. Garrity, ed. (July 26, 2005) [1984(Williams & Wilkins)]. Introductory Essays (http:/ / www. springer. com/ life+ sciences/ book/ 978-0-387-24143-2). Bergey's Manual of Systematic Bacteriology. 2A (2nd ed.). New York: Springer. pp. 304. ISBN 978-0-387-24143-2. British Library no. GBA561951. .

External links • Gram staining technique video (http://www.tgw1916.net/movies.html) • Gram staining procedure (http://www.microbeid.com/Methods/gramstain.html)(dead link)

Gram-negative bacteria Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol.[1] In a Gram stain test, a counterstain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their bacterial cell walls. Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution. The pathogenic capability of Gram-negative bacteria is often Microscopic image of Gram-negative associated with certain components of Gram-negative cell envelope, in Pseudomonas aeruginosa bacteria (pink-red rods). particular, the lipopolysaccharide layer (also known as LPS or [1] endotoxin layer). In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation. Inflammation is a common result of cytokine (from the Greek cyto, cell and kinesis, movement) production, which can also produce host toxicity.

Characteristics The following characteristics are displayed for Gram-negative bacteria: 1. Cytoplasmic membrane 2. Thin peptidoglycan layer (which is much thinner than in Gram-positive bacteria) 3. Outer membrane containing lipopolysaccharide (LPS, which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet

Structure of gram-negative cell wall

4. Porins exist in the outer membrane, which act like pores for particular molecules

Gram-negative bacteria

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5. There is a space between the layers of peptidoglycan and the secondary cell membrane called the periplasmic space 6. The S-layer is directly attached to the outer membrane, rather than the peptidoglycan 7. If present, flagella have four ing rings instead of two 8. No teichoic acids or lipoteichoic acids are present 9. Lipoproteins are attached to the polysaccharide backbone. 10. Most of them contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond 11. Most do not sporulate (Coxiella burnetii, which produces spore-like structures, is a notable exception)

Gram-positive- and negative bacteria are chiefly differentiated by their cell wall structure.

Classification Along with cell shape, Gram staining is a rapid diagnostic tool and used to be used to group species at the subdivision of Bacteria. In fact, historically the kingdom Monera was divided into four divisions based on Gram staining: Firmacutes (+), Gracillicutes (-), Mollicutes (0) and Mendocutes (var.).[2] Since 1987, the monophyly of the Gram-negative bacteria has been disproven with molecular studies.[3] However some authors, such as Cavalier-Smith still treat them as a monophyletic clade and refer to the group as subkingdom "Negibacteria".[4]

Species identification hierarchy in clinical settings.

It is worth stressing here that the description of bacteria as Gram-positive or Gram-negative is ambiguous as it can refer to three distinct aspects (staining result, cell-envelope organization, taxonomic group), which do not necessarily coalesce for some bacterial species.[5] When referring to the type of bacterial cell envelope, the of monoderm and diderm bacteria are more appropriate.[5] The diderm bacteria can be further differentiated between diderm–LPS and diderm–mycolate, at least.[6]


Example species The proteobacteria are a major group of Gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella and numerous others. Other notable groups of Gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria. Medically relevant Gram-negative cocci include three organisms, which cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis). Medically relevant Gram-negative bacilli include a multitude of species. Some of them primarily cause respiratory problems (Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella typhi). Gram-negative bacteria associated with nosocomial infections include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in intensive-care units of hospital establishments.

Medical treatment One of the several unique characteristics of Gram-negative bacteria is the structure of the outer membrane. The outer leaflet of the membrane comprises a complex lipopolysaccharide whose lipid portion acts as an endotoxin. If endotoxin enters the circulatory system, it causes a toxic reaction, with the sufferer developing a high temperature, high respiration rate, and low blood pressure. This may lead to endotoxic shock, which may be fatal. This outer membrane protects the bacteria from several antibiotics, dyes, and detergents that would normally damage the inner membrane or cell wall (peptidoglycan). The outer membrane provides these bacteria with resistance to lysozyme and penicillin. However, alternative medicinal treatments such as lysozyme with EDTA and the antibiotic ampicillin have been developed to combat the protective outer membrane of some pathogenic Gram-negative organisms. Other drugs can be used, significant ones being chloramphenicol, streptomycin, and nalidixic acid.

References •

This article incorporates public domain material from the NCBI document "Science Primer" [7].

Notes [1] Salton MRJ , Kim KS (1996). Structure. in: Baron's Medical Microbiology (Baron S et al., eds.) (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=mmed. section. 289) (4th ed.). Univ of Texas Medical Branch. ISBN 0-9631172-1-1. . [2] Gibbons, N. E.; Murray, R. G. E. (1978). "Proposals Concerning the Higher Taxa of Bacteria". International Journal of Systematic Bacteriology 28 (1): 1–6. doi:10.1099/00207713-28-1-1. [3] Woese, C. R. (1987). "Bacterial evolution". Microbiological reviews 51 (2): 221–271. PMC 373105. PMID 2439888. [4] Cavalier-Smith T (2006). "Rooting the tree of life by transition analyses" (http:/ / www. biology-direct. com/ content/ 1/ / 19). Biol. Direct 1: 19. doi:10.1186/1745-6150-1-19. PMC 1586193. PMID 16834776. . [5] , Desvaux M, Hébraud M, Talon R, Henderson IR. 2009. Secretion and subcellular localizations of bacterial proteins: a semantic awareness issue. Trends Microbiol. 17:139-145. doi:10.1016/j.tim.2009.01.004 [6] , Sutcliffe IC. 2010. A phylum level perspective on bacterial cell envelope architecture. Trends Microbiol. 18:464-470. doi:10.1016/j.tim.2010.06.005 [7] http:/ / www. ncbi. nlm. nih. gov/ About/ primer/ index. html

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External links • 3D structures of proteins from inner membranes of Gram-negative bacteria (http://opm.phar.umich.edu/ localization.php?localization=Bacterial gram-negative inner membrane) • Gram Staining Procedure and Images (http://microbeid.com/Methods/gramstain.html)

Gram-positive bacteria Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria.

Gram-positive bacteria, stained purple, of both the bacillus (“rod-shaped”) and coccus (spherical) forms. A few Gram-negative bacteria are also present, stained pink. Numbered ticks are eleven (11) microns apart.

Gram-positive bacteria

114

Characteristics The following characteristics are generally present in a Gram-positive bacterium:[1] 1. cytoplasmic lipid membrane 2. thick peptidoglycan layer • teichoic acids and lipoids are present, forming lipoteichoic acids, which serve to act as chelating agents, and also for certain types of adherence. 3. capsule polysaccharides (only in some species) 4. flagellum (only in some species) • if present, it contains two rings for as opposed to four in Gram-negative bacteria because Gram-positive bacteria have only one membrane layer. 5. The individual peptidoglycan molecules are cross-linked by pentaglycine chains by a DD-transpeptidase enzyme. In gram-negative bacteria, the transpeptidase creates a covalent bond directly between peptidoglycan molecules, with no intervening bridge. Both Gram-positive and Gram-negative bacteria may have a membrane called an S-layer. In Gram-negative bacteria, the S-layer is attached directly to the outer membrane. In Gram-positive bacteria, the S-layer is attached to the peptidoglycan layer. Unique to Gram-positive bacteria is the presence of teichoic acids in the cell wall. Some particular teichoic acids, lipoteichoic acids, have a lipid component and can assist in anchoring peptidoglycan, as the lipid component is embedded in the membrane.

Gram-positive and -negative cell wall structure

Structure of Gram-positive cell wall

Classification Along with cell shape, Gram staining is a rapid diagnostic tool of use to group species of Bacteria. In traditional and even some areas of contemporary microbiological practice, such staining, alongside growth requirement and antibiotic susceptibility testing, and other macroscopic and physiologic tests, forms the full basis for classification and subdivision of the Bacteria (e.g., see Figure, and pre-1990 versions of Bergey's Manual).

Gram-positive bacteria

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Species identification hierarchy in clinical settings.

As such, historically, the kingdom Monera was divided into four divisions based primarily on Gram staining: Firmicutes (positive in staining), Gracillicutes (negative in staining), Mollicutes (neutral in staining) and Mendocutes (variable in staining).[2] Since 1987 and the seminal 16S ribosomal RNA phylogenetic studies of Carl Woese (Department of Microbiology, University of Illinois) and collaborators and colleagues, the monophyly of the Gram-positive bacteria has been challenged,[3] with striking productive implications for the therapeutic and general study of these organisms. Based on molecular studies of 16S sequences, Woese recognised 12 bacterial phyla, two being Gram-positive: high-GC Gram-positives and low-GC Gram-positives (where G and C refer to the guanine and cytosine content in their genomes),[3] which are now referred to by these names, or as Actinobacteria and Firmicutes. The former, the Actinobacteria, are the high GC content Gram-positive bacteria and contains genera such as Corynebacterium, Mycobacterium, Nocardia and Streptomyces. The latter, the Firmicutes are the "low-GC" Gram-positive bacteria, which actually have 45%–60% GC content but lower than that of the Actinobacteria,.[1] The Firmicutes contain the well-known genera that are majority of Gram-positives of medical interest: Staphylococcus, Streptococcus, Enterococcus (cocci), Bacillus, Clostridium and Listeria (bacilli/rods). This group also been expanded to include the Mycoplasma, or Mollicutes, bacteria-like organisms that lack cell walls and cannot be Gram-stained, but appear to have derived evolutionarily from such forms. Despite the wide acceptance and practical record of utility of the new molecular phylogeny, a small group, including Cavalier-Smith, still treat the Monera as a monophyletic clade and refer to the group as division "Posibacteria".[4] It is worth stressing here that the description of bacteria as Gram-positive or Gram-negative is ambiguous as it can refers to three distinct aspects (staining result, cell-envelope organization, taxonomic group), which do not necessarily coalesce for some bacterial species.[5] When referring to the type of bacterial cell envelope, the of monoderm and diderm bacteria are much more appropriate,[5] where the diderm bacteria can even be further

Gram-positive bacteria differentiated between diderm–LPS and diderm–mycolate, at least.[6]

Exceptions In general, Gram-positive bacteria have a single lipid bilayer (monoderms), whereas Gram-negative have two (diderms). Some taxa lack peptidoglycan (such as the domain Archaea, the class Mollicutes, some of the Rhickettsiales, and the insect-endosymbionts of the Enterobacteriales) and are Gram-variable. This, however, does not always hold true. The Deinococcus-Thermus bacteria have Gram-positive stains, although they are structurally similar to Gram-negative bacteria with two layers (diderms). The Chloroflexi have a single layer, yet (with some exceptions[7]) stain negative.[8] Two related phyla to the Chloroflexi, the TM7 clade and the Ktedonobacteria, are also monoderms.[9][10] Some Firmicute species are not Gram-positive; these belong to the class Mollicutes (alternatively considered a class of the phylum Tenericutes), which lack peptidoglycan (Gram-indeterminate), and the class Negativicutes, which includes Selenomonas and which stain Gram-negative.[11]

Pathogenesis Most pathogens in humans are Gram-positive organisms. In the classical sense, six Gram-positive genera are typically pathogenic in humans. Two of these, Streptococcus and Staphylococcus, are cocci (sphere-shaped bacteria). The remaining organisms are bacilli (rod-shaped bacteria) and can be subdivided based on their ability to form spores. The non-spore formers are Corynebacterium and Listeria (a coccobacillus), whereas Bacillus and Clostridium produce spores.[12] The spore-forming bacteria can again be divided based on their respiration: Bacillus is a facultative anaerobe, while Clostridium is an obligate anaerobe.

References [1] Madigan M; Martinko J (editors). (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1. [2] Gibbons, N. E.; Murray, R. G. E. (1978). "Proposals Concerning the Higher Taxa of Bacteria". IJSEM 28 (1): 1–6. doi:10.1099/00207713-28-1-1. [3] Woese, C. R. (1987). "Bacterial evolution". Microbiological reviews 51 (2): 221–271. PMC 373105. PMID 2439888. [4] Cavalier-Smith T (2006). "Rooting the tree of life by transition analyses" (http:/ / www. biology-direct. com/ content/ 1/ / 19). Biol. Direct 1: 19. doi:10.1186/1745-6150-1-19. PMC 1586193. PMID 16834776. . [5] , Desvaux M, Hébraud M, Talon R, Henderson IR. 2009. Secretion and subcellular localizations of bacterial proteins: a semantic awareness issue. Trends Microbiol. 17:139-145. doi:10.1016/j.tim.2009.01.004 [6] , Sutcliffe IC. 2010. A phylum level perspective on bacterial cell envelope architecture. Trends Microbiol. 18:464-470. doi:10.1016/j.tim.2010.06.005 [7] Yabe, S.; Aiba, Y.; Sakai, Y.; Hazaka, M.; Yokota, A. (2010). "Thermogemmatispora onikobensis gen. nov., sp. nov. And Thermogemmatispora foliorum sp. nov., isolated from fallen leaves on geothermal soils, and description of Thermogemmatisporaceae fam. Nov. And Thermogemmatisporales ord. Nov. Within the class Ktedonobacteria". International Journal of Systematic and Evolutionary Microbiology 61 (4): 903–910. doi:10.1099/ijs.0.024877-0. PMID 20495028. [8] Sutcliffe, I. C. (2011). "Cell envelope architecture in the Chloroflexi: A shifting frontline in a phylogenetic turf war". Environmental Microbiology 13 (2): 279–282. doi:10.1111/j.1462-2920.2010.02339.x. PMID 20860732. [9] Hugenholtz, P.; Tyson, G. W.; Webb, R. I.; Wagner, A. M.; Blackall, L. L. (2001). "Investigation of Candidate Division TM7, a Recently Recognized Major Lineage of the Domain Bacteria with No Known Pure-Culture Representatives". Applied and Environmental Microbiology 67 (1): 411. doi:10.1128/AEM.67.1.411-419.2001. PMC 92593. PMID 11133473. [10] Cavaletti, L.; Monciardini, P.; Bamonte, R.; Schumann, P.; Rohde, M.; Sosio, M.; Donadio, S. (2006). "New Lineage of Filamentous, Spore-Forming, Gram-Positive Bacteria from Soil". Applied and Environmental Microbiology 72 (6): 4360–4369. doi:10.1128/AEM.00132-06. PMC 1489649. PMID 16751552. [11] Marchandin, H.; Teyssier, C.; Campos, J.; Jean-Pierre, H.; Roger, F.; Gay, B.; Carlier, J. -P.; Jumas-Bilak, E. (2009). "Negativicoccus succinicivorans gen. Nov., sp. Nov., isolated from human clinical samples, emended description of the family Veillonellaceae and description of Negativicutes classis nov., Selenomonadales ord. Nov. And Acidaminococcaceae fam. Nov. In the bacterial phylum Firmicutes". International Journal of Systematic and Evolutionary Microbiology 60 (6): 1271–1279. doi:10.1099/ijs.0.013102-0. PMID 19667386.

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Gram-positive bacteria [12] Gladwin, Mark; Bill Trattler (2007). Clinical Microbiology made ridiculously simple. Miami, FL: MedMaster, Inc. pp. 4–5. ISBN 978-0-940780-81-1.

External links •

This article incorporates public domain material from the NCBI document "Science Primer" (http://www. ncbi.nlm.nih.gov/About/primer/index.html).

• 3D structures of proteins associated with plasma membrane of Gram-positive bacteria (http://opm.phar.umich. edu/localization.php?localization=Bacterial gram-positive plasma membrane) • 3D structures of proteins associated with cell wall of Gram-positive bacteria (http://opm.phar.umich.edu/ localization.php?localization=Bacterial gram-positive acid-fast cell wall) • Gram Staining Procedure and Images (http://microbeid.com/Methods/gramstain.html)

H&E stain H&E stain, HE stain or hematoxylin and eosin stain is a popular staining method in histology. It is the most widely used stain in medical diagnosis; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed H&E section, H+E section, or HE section. The staining method involves application of hemalum, which is a complex formed from aluminum ions and oxidized haematoxylin. This colors nuclei of cells (and a few other objects, such as keratohyalin granules) blue. The nuclear staining is followed by Histologic specimen of human lung tissue stained with hematoxylin and eosin. counterstaining with an aqueous or alcoholic solution of eosin Y, which colors other, eosinophilic structures in various shades of red, pink and orange. The staining of nuclei by hemalum does not require the presence of DNA and is probably due to binding of the dye-metal complex to arginine-rich basic nucleoproteins such as histones. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue. Staining by basic dyes is prevented by chemical or enzymatic extraction of nucleic acids. Such extractions do not prevent staining of nuclei by hemalum. The eosinophilic structures are generally composed of intracellular or extracellular protein. The Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm is eosinophilic. Red blood cells are stained intensely red. The structures do not have to be acidic or basic to be called basophilic and eosinophilic. The terminology is based on the affinity to the dyes. Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments, e.g. melanin. Some structures do not stain well. Basal laminae need to be stained by PAS stain or some silver stains, if they have to be well visible. Reticular fibers also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, e.g. adipocytes, myelin around neuron axons, and Golgi apparatus membranes.

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H&E stain

References • Godwin Avwioro (2011). Histochemical Uses Of Haematoxylin - A Review. JPCS 1:24-34. PDF [1] • Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion. • Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear stains with soluble metachrome mordant lake dyes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23-35. • Llewellyn BD (2009) Nuclear staining with alum-hematoxylin. Biotech. Histochem. 84: 159-177. • Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353-364.

External links • SIGMA-ALDRICH H&E Informational Primer [2]

Protocol • Routine Mayer's Hematoxylin and Eosin Stain (H&E) [3] • Hematoxylin & Eosin (H&E) Staining Protocol [4] • Rosen Lab, Department of Molecular and Cellular Biology, Baylor College of Medicine) Step by step protocol [5]

References [1] [2] [3] [4] [5]

http:/ / www. arpapress. com/ Volumes/ JPCS/ Vol1/ JPCS_1_05. pdf http:/ / www. sigmaaldrich. com/ img/ assets/ 7361/ Primer-H& Emay04. pdf http:/ / ccm. ucdavis. edu/ bcancercd/ 52/ prcl_HandE. html http:/ / www. ihcworld. com/ _protocols/ special_stains/ h& e_ellis. htm http:/ / www. bcm. edu/ mcb/ rosenlab/ index. cfm?pmid=12983

118

Haematoxylin

119

Haematoxylin Haematoxylin

Identifiers [1]

CAS number

517-28-2

PubChem

10603

ChemSpider

21106443

UNII

YKM8PY2Z55

MeSH

Hematoxylin

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]


C16H14O6

Molar mass

302.28 g mol

−1

()

[7]


Infobox references

Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the heartwood of the logwood tree.[8] When oxidized it forms haematein, a compound that forms strongly coloured complexes with certain metal ions, the most notable ones being Fe(III) and Al(III) salts. Metal-haematein complexes are used to stain cell nuclei prior to examination under a microscope. Structures that stain with iron- or aluminium-haematein are often called basophilic, even though the mechanism of the staining is different from that of staining with basic dyes.

Haematein

Haematoxylin and eosin stain is one of the most commonly used stains in histology. It is a permanent stain as opposed to temporary stains (e.g. iodine solution in KI). Another common stain is phosphotungstic acid haematoxylin, a mix of haematoxylin with phosphotungstic acid. In 1970s, due to clear felling of forests in Brazil and Central America, there was a shortage of logwood and therefore of haematoxylin. Its price went to record heights, which affected the cost of diagnostic histopathology, and prompted a search for alternative nuclear stains. Before the use of any alternatives became firmly established, haematoxylin returned to the market, though at a higher price, and resumed its place in histopathology. There were several dyes recommended as replacements: Celestine blue B (CI 51050), Gallocyanin (CI 51030), Gallein (CI 45445) and Solochrome cyanin (CI 43820). All four used Fe(III) as the mordant. Another alternative is the red dye brazilin,

Haematoxylin which differs from haematoxylin by only one hydroxyl group.

Haematoxylin staining solutions These stains are commonly employed for histological studies. The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or coloured complexes (dye-mordant-tissue complexes), the colour of which will depend on the salt used. Aluminium salt lakes are usually coloured blue-white, whereas ferric salt lakes are coloured blue-black.

Aluminium haematoxylin solutions The three main alum haematoxylin solutions employed are Ehrlich's haematoxylin, Harris's haematoxylin, and Mayer's haematoxylin. The name haemalum is preferable to "haematoxylin" for these solutions because haematein, a product of oxidation of haematoxylin, is the compound that combines with aluminium ions to form the active dye-metal complex. Alum haematoxylin solutions impart to the nuclei of cells a light transparent red stain that rapidly turns blue on exposure to any neutral or alkaline liquid. Alum or potassium aluminium sulfate used as the mordant usually dissociates in an alkaline solution, combining with OH− of water to form insoluble aluminium hydroxide. In the presence of excess acid, aluminium hydroxide cannot be formed, thus causing failure of aluminium haematoxylin dye-lake to form, due to lack of OH− ions. Hence, acid solutions of alum haematoxylin become red. During staining, alum haematoxylin-stained sections are usually ed on to a neutral or alkaline solution (e.g., hard tap water or 1% ammonium hydroxide) in order to neutralize the acid and form an insoluble blue aluminium haematin complex. This procedure is known as blueing. When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing haematoxylin, a tap water substitute consisting of 3.5 g NaHCO3 and 20 g MgSO4.7H2O in one litre of water with thymol (to inhibit formation of moulds), is used to accelerate blueing of thin paraffin sections. Addition of a trace of any alkali to tap or distilled water also provides an effective blueing solution; a few drops of strong ammonium hydroxide or of saturated aqueous lithium carbonate, added immediately before use, are sufficient for a 400 ml staining dish full of water. Use of very cold water slows down the blueing process, whereas warming accelerates it. In fact, the use of water below 10 °C for blueing sections may even produce pink artifact discolourations in the tissue.

External links • Stainsfile [9] • Mayer's Hematoxylin Preparation [10]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=517-28-2 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=10603 [3] http:/ / www. chemspider. com/ 21106443 [4] http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=YKM8PY2Z55 [5] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Hematoxylin [6] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=Oc2cc3C%5BC%40%5D4%28O%29COc1c%28O%29c%28O%29ccc1C4c3cc2O [7] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=446217709& page2=%3AHaematoxylin [8] Cooksey 2010 [9] http:/ / stainsfile. info/ StainsFile/ dyes/ 75290. htm [10] http:/ / www. histochem. net/ buffers. htm

• Godwin Avwioro (2011). Histochemical Uses Of Haematoxylin - A Review. JPCS 1:24-34. PDF (http://www. arpapress.com/Volumes/JPCS/Vol1/JPCS_1_05.pdf) • Cooksey C (2010) Hematoxylin and related compounds - an annotated bibliography concerning their origin, properties, chemistry and certain applications. Biotechnic & Histochemistry 85(1): 65-82. PMID 19568968

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Haematoxylin • Brown, G. G. (1978). An Introduction to Histotechnology. Appleton-Century-Crofts, New York. • Jocelyn H. Bruce-Gregorios, M.D.: Histopathologic Techniques, JMC Press Inc., Quezon City, Philippines, 1974. • Meloan, S. M. & Puchtler, H. 1987. "Harris hematoxylin," what Harris really wrote and the mechanism of hemalum stains. Journal of Histotechnology 10: 257-261. • Puchtler, H., Meloan, S.N. & Waldrop, F.S. 1986. Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353-364.

Heidenhain's AZAN trichrome stain Trichrome stains are staining methods in which three anionic dyes are used, in conjunction with either phosphomolybdic acid (PMA), phosphotungstic acid (PTA), or a mixture of these heteropolyacids. Probably the first trichrome method was that of Frank B Mallory, an American pathologist, first published in 1900[1]. Unfortunately, none of Mallory's publications (they go from 1891[2] to 1938[3]) provide any explanation of the rationales of either his trichrome or his phosphotungstic acid-haematoxylin (PTAH) method. Nobody knows why Mallory introduced heteropolyacids into microtechnique. Mallory's trichrome method, using acid fuchsine followed by a solution containing PTA, orange G and aniline blue, provides dark red nuclei, orange erythrocytes, and blue collagen fibres, cartilage matrix and mucus.[4]. In 1915, M. Heidenhain introduced azocarmine G in place of the acid fuchsine of Mallory's method. Heidenhain also introduced visually controlled destaining to provide for different colours in cell nuclei (dark red), collagen (blue) and a variety of colours in cytoplasm.

References [1] [2] [3] [4]

Mallory FB (1900) A contribution to staining methods. Journal of Experimental Medicine 5: 15-20. Mallory FB (1891) Phospho-molybdic acid-haematoxylin. Anatomischer Anzeiger 6: 375-376. Mallory FB (1938) Pathological Technique. New York: Hafner. Kiernan JA (2008) Histological and Histochemical Methods. Theory and Practice. 4th ed. Bloxham, UK: Scion. ISBN 978-1-904842-42-2

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Histological section

Histological section Histological section refers to thin slices of tissue applied to a microscopic slide, usually around 5 to 10 micrometres thick, which are viewed under a microscope. For further discussion of histological section and staining methods, one should review histology article.

Method for making histological sections Gross cutting The specimen is cut into the correct size and configuration prior to fixation and microtome cutting. The specimen is stained and positioned for proper orientation. With Mohs surgery or the CDMA method of cutting, the specimen is cut in a manner to allow mounting all of the surgical margins on one plane. With standard bread loafing, the specimen is usually cut into multiple sections with the surgical margin stained. Some technologist will stain the edge to be oriented toward the microtome. The cut specimen is then transferred directly to frozen medium for frozen section processing, or placed in small cassettes for dehydration and paraffin embedding.

Fixation Fixation is done either by the fixed tissue method with paraffin, or by frozen section. With fixed tissue method, the tissue specimen is preserve in either formaldehyde or an acidic solution until it is processed. The tissue is then removed from the preservative, dehydrated with multiple solvent baths, and fixed in hot liquid parafin. The hardened parafin block with the fixed tissue is then cut with the microtome. With frozen tissue sectioning, the tissue is immediately frozen prior to processing (frozen section).

Microtome Cutting The frozen tissue block embedded in a frozen cutting medium, or the paraffin fixed tissue is cut using a very fine knife called a microtome. A cryostat is a micotome mounted inside a freezer for processing frozen tissue.

Mounting The frozen thin slices of tissue are mounted on a warm glass slide at room temperature, or the paraffin embedded slides are mounted on a heated glass. This allow them to be stained and ready for staining. The tissue mounted slides are then dry in open air or in a drying oven.

Staining and coverslipping Multiple stain baths are used to make the tissue more visible to the naked eye. Please see histology for discussion of the stains used. Sections usually have a very thin piece of glass applied over the surface called a cover slip. The glass cover slip is glued onto the slide with a special optical grade transparent glue.

Common laboratory stains

122

Histological section

Stain

123

Common use

Nucleus

Cytoplasm

Red blood cell (RBC)

Collagen fibers

Specifically stains

Haematoxylin

General staining when paired with eosin (i.e. H&E)

Blue

N/A

N/A

N/A

Nucleic acids—blue ER (endoplasmic reticulum)—blue

Eosin

General staining when paired with haematoxylin (i.e. H&E)

N/A

Pink

Orange/red

Pink

Elastic fibers—pink Collagen fibers—pink Reticular fibers—pink

Toluidine blue

General staining

Blue

Blue

Blue

Blue

Mast cells granules—purple

Masson's trichrome stain

Connective tissue

Black

Red/pink

Red

Blue/green

Cartilage—blue/green Muscle fibers—red

Mallory's trichrome stain

Connective tissue

Red

Pale red

Orange

Deep blue

Keratin—orange Cartilage—blue Bone matrix—deep blue Muscle fibers—red

Weigert's elastic Elastic fibers stain

Blue/black

N/A

N/A

N/A

Elastic fibers—blue/black

Heidenhain's AZAN trichrome stain

Distinguishing cells from extracellular components

Red/purple

Pink

Red

Blue

Muscle fibers—red Cartilage—blue Bone matrix—blue

Silver stain

Reticular fibers, nerve fibers, fungi

N/A

N/A

N/A

Reticular fibers—brown/black Nerve fibers—brown/black

Wright's stain

Blood cells

Bluish/purple

Bluish/gray Red/pink

N/A

Neutrophil granules—purple/pink Eosinophil granules—bright red/orange Basophil granules—deep purple/violet Platelet granules—red/purple

Orcein stain

Elastic fibres

Deep blue [or crazy red]

N/A

Bright red

Pink

Elastic fibres—dark brown Mast cells granules—purple Smooth muscle—light blue

Blue

N/A

N/A

Pink

Glycogen and other carbohydrates—magenta

Periodic Basement acid-Schiff stain membrane, (PAS) localizing carbohydrates

Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3. The Nissl method and Golgi's method are useful in identifying neurons.

Histology

124

Histology Histology (compound of the Greek words: ἱστός "tissue", and -λογία -logia) is the study of the microscopic anatomy of cells and tissues of plants and animals. It is commonly performed by examining cells and tissues by sectioning and staining, followed by examination under a light microscope or electron microscope. Histological studies may be conducted via tissue culture, where live cells can be isolated and maintained in a proper environment outside the body for various research projects. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of histological stains. Histology is an essential tool of biology and medicine.

A stained histologic specimen, sandwiched between a glass microscope slide and coverslip, mounted on the stage of a light microscope.

Histopathology, the microscopic study of diseased tissue, is an important tool in anatomical pathology, since accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples. Trained medical doctors, frequently board-certified as pathologists, are the personnel who perform histopathological examination and provide diagnostic information based on their observations. The trained scientists who perform the preparation of histological sections are histotechnicians, histology technicians Microscopic view of a histologic specimen of human lung tissue stained with hematoxylin and eosin. (HT), histology technologists (HTL), medical scientists, medical laboratory technicians, or biomedical scientists. Their field of study is called histotechnology.

Histology Fixing Chemical fixation with formaldehyde or other chemicals Chemical fixatives are used to preserve tissue from degradation, and to maintain the structure of the cell and of sub-cellular components such as cell organelles (e.g., nucleus, endoplasmic reticulum, mitochondria). The most common fixative for light microscopy is 10% neutral buffered formalin (4% formaldehyde in phosphate buffered

Histology saline). For electron microscopy, the most commonly used fixative is glutaraldehyde, usually as a 2.5% solution in phosphate buffered saline. These fixatives preserve tissues or cells mainly by irreversibly cross-linking proteins. The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of CH2 (methylene) linkage, in the case of formaldehyde, or by a C5H10 cross-links in the case of glutaraldehyde. This process, while preserving the structural integrity of the cells and tissue can damage the biological functionality of proteins, particularly enzymes, and can also denature them to a certain extent. This can be detrimental to certain histological techniques. Further fixatives are often used for electron microscopy such as osmium tetroxide or uranyl acetate Formalin fixation leads to degradation of mRNA, miRNA and DNA in tissues. However, extraction, amplification and analysis of these nucleic acids from formalin-fixed, paraffin-embedded tissues is possible using appropriate protocols.[1] Frozen section fixation Frozen section is a rapid way to fix and mount histology sections. It is used in surgical removal of tumors, and allow rapid determination of margin (that the tumor has been completely removed). It is done using a refrigeration device called a cryostat. The frozen tissue is sliced using a microtome, and the frozen slices are mounted on a glass slide and stained the same way as other methods. It is a necessary way to fix tissue for certain stain such as antibody linked immunofluorescence staining. It can also be used to determine if a tumour is malignant when it is found incidentally during surgery on a patient.

Processing - dehydration, clearing, and infiltration The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Biological tissue must be ed in a hard matrix to allow sufficiently thin sections to be cut, typically 5 μm (micrometres; 1000 micrometres = 1 mm) thick for light microscopy and 80-100 nm (nanometre; 1,000,000 nanometres = 1 mm) thick for electron microscopy. For light microscopy, paraffin wax is most frequently used. Since it is immiscible with water, the main constituent of biological tissue, water must first be removed in the process of dehydration. Samples are transferred through baths of progressively more concentrated ethanol to remove the water. This is followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax, the infiltration agent, which replaces the xylene. Paraffin wax does not provide a sufficiently hard matrix for cutting very thin sections for electron microscopy. Instead, resins are used. Epoxy resins are the most commonly employed embedding media, but acrylic resins are also used, particularly where immunohistochemistry is required. Thicker sections (0.35μm to 5μm) of resin-embedded tissue can also be cut for light microscopy. Again, the immiscibility of most epoxy and acrylic resins with water necessitates the use of dehydration, usually with ethanol.

Embedding After the tissues have been dehydrated, cleared, and infiltrated with the embedding material, they are ready for external embedding. During this process the tissue samples are placed into molds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned. Because Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, FFPE tissues are an important resource for historical studies in medicine. Embedding can also be accomplished using frozen, non-fixed tissue in a water-based medium. Pre-frozen tissues are placed into molds with the liquid embedding material, usually a water-based glycol, OCT, TBS, Cryogel, or resin, which is then frozen to form hardened blocks.

125

Histology

Sectioning Sectioning can be done in limited ways. Vertical sectioning perpendicular to the surface of the tissue is the usual method. Horizontal sectioning is often done in the evaluation of the hair follicles and pilosebaceous units. Tangential to horizontal sectioning is done in Mohs surgery and in methods of CDMA. For light microscopy, a steel knife mounted in a microtome is used to cut 10-micrometer-thick tissue sections which are mounted on a glass microscope slide. For transmission electron microscopy, a diamond knife mounted in an ultramicrotome is used to cut 50-nanometer-thick tissue sections which are mounted on a 3-millimeter-diameter copper grid. Then the mounted sections are treated with the appropriate stain. Frozen tissue embedded in a freezing medium is cut on a microtome in a cooled machine called a cryostat.

Staining Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. Where the underlying mechanistic chemistry of staining is understood, the term histochemistry is used. Hematoxylin and eosin (H&E stain) is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope. Special staining: There are hundreds of various other techniques that have been used to selectively stain cells and cellular components. Other compounds used to color tissue sections include safranin, oil red o, Congo red, fast green FCF, silver salts, and numerous natural and artificial dyes that were usually originated from the development dyes for the textile industry. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in diseases like hemochromatosis. Histology samples have often been examined by radioactive techniques. In historadiography, a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is used to visualize the locations to which a radioactive substance has been transported within the body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with dark field microscopy. Recently, antibodies have been used to specifically visualize proteins, carbohydrates, and lipids. This process is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially alkaline phosphatase and tyramide signal amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail. Digital cameras are increasingly used to capture histological and histopathological image

126

Histology

127

Common laboratory stains Stain

Common use

Nucleus

Cytoplasm Red blood cell (RBC)

Collagen fibers

Specifically stains

Haematoxylin

General staining when paired with eosin (i.e. H&E)

Blue

N/A

N/A

N/A

Nucleic acids—blue ER (endoplasmic reticulum)—blue

Eosin

General staining when paired N/A with haematoxylin (i.e. H&E)

Pink

Orange/red

Pink

Elastic fibers—pink Collagen fibers—pink Reticular fibers—pink

Toluidine blue

General staining

Blue

Blue

Blue

Blue

Mast cells granules—purple


Connective tissue

Black

Red/pink

Red

Blue/green

Cartilage—blue/green Muscle fibers—red

Mallory's trichrome stain

Connective tissue

Red

Pale red

Orange

Deep blue

Keratin—orange Cartilage—blue Bone matrix—deep blue Muscle fibers—red

Weigert's elastic stain

Elastic fibers

Blue/black

N/A

N/A

N/A

Elastic fibers—blue/black

Heidenhain's AZAN trichrome stain

Distinguishing cells from extracellular components

Red/purple

Pink

Red

Blue

Muscle fibers—red Cartilage—blue Bone matrix—blue

Silver stain

Reticular fibers, nerve fibers, fungi

N/A

N/A

N/A

N/A

Reticular fibers—brown/black Nerve fibers—brown/black Fungi—black

Wright's stain

Blood cells

Bluish/purple Bluish/gray Red/pink

N/A

Neutrophil granules—purple/pink Eosinophil granules—bright red/orange Basophil granules—deep purple/violet Platelet granules—red/purple

Orcein stain

Elastic fibres

Deep blue

N/A

Bright red

Pink

Elastic fibres—dark brown Mast cells granules—purple Smooth muscle—light blue

Periodic acid-Schiff stain (PAS)

Basement membrane, localizing carbohydrates

Blue

N/A

N/A

Pink

Glycogen and other carbohydrates—magenta

Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3. The Nissl method and Golgi's method are useful in identifying neurons.

Alternative techniques Alternative techniques include cryosection. The tissue is frozen using a cryostat, and cut. Tissue staining methods are similar to those of wax sections. Plastic embedding is commonly used in the preparation of material for electron microscopy. Tissues are embedded in epoxy resin. Very thin sections (less than 0.1 micrometer) are cut using diamond or glass knives. The sections are stained with electron dense stains (uranium and lead) so that they can possibly be seen with the electron microscope.

Histology

128

History In the 19th century, histology was an academic discipline in its own right. The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had dueling interpretations of the neural structure of the brain based in differing interpretations of the same images. Cajal won the prize for his correct theory and Golgi for the staining technique he invented to make it possible.

Histological classification of animal tissues There are four basic types of tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissue. All tissue types are subtypes of these four basic tissue types (for example, blood cells are classified as connective tissue, since they generally originate inside bone marrow). • • • • • • • •

•

Santiago Ramón y Cajal in his laboratory

Epithelium: the lining of glands, bowel, skin, and some organs like the liver, lung, and kidney Endothelium: the lining of blood and lymphatic vessels Mesothelium: the lining of pleural and pericardial spaces Mesenchyme: the cells filling the spaces between the organs, including fat, muscle, bone, cartilage, and tendon cells Blood cells: the red and white blood cells, including those found in lymph nodes and spleen Neurons: any of the conducting cells of the nervous system Germ cells: reproductive cells (spermatozoa in men, oocytes in women) Placenta: an organ characteristic of true mammals during pregnancy, ing mother and offspring, providing endocrine secretion and selective exchange of soluble, but not particulate, blood-borne substances through an apposition of uterine and trophoblastic vascularised parts Stem cells: cells with the ability to develop into different cell types

Note that tissues from plants, fungi, and microorganisms can also be examined histologically. Their structure is very different from animal tissues.

Related sciences • Cell biology is the study of living cells, their DNA and RNA and the proteins they express. • Anatomy is the study of organs visible by the naked eye. • Morphology studies entire organisms.

Artifacts Artifacts are structures or features in tissue that interfere with normal histological examination. These are not always present in normal tissue and can come from outside sources. Artifacts interfere with histology by changing the tissues appearance and hiding structures. These can be divided into two categories:

Histology

Pre-histology These are features and structures that have been introduced prior to the collection of the tissues. A common example of these include: ink from tattoos and freckles (melanin) in skin samples.

Post-histology Artifacts can result from tissue processing. Processing commonly leads to changes like shrinkage, washing out of particular cellular components, color changes in different tissues types and alterations of the structures in the tissue. Because these are caused in a laboratory the majority of post histology artifacts can be avoided or removed after being discovered. A common example is mercury pigment left behind after using Zenker's fixative to fix a section.

Notes [1] Weiss AT, Delcour NM, Meyer A, Klopfleisch R. (2010). "Efficient and Cost-Effective Extraction of Genomic DNA From Formalin-Fixed and Paraffin-Embedded Tissues". Veterinary Pathology 227 (4): 834–8. doi:10.1177/0300985810380399. PMID 20817894.

References 1. Merck Source (2002). Dorland's Medical Dictionary. Retrieved 2005-01-26. 2. Stedman's Medical Dictionaries (2005). Stedman's Online Medical Dictionary (http://stedmans.com/). Retrieved 2005-01-26. 3. 4,000‫ﻱ‬online histology images (2007). (http://histology-online.com)

External links • • • • • • • • • • • • • •

Meyer's Histology - a complete online histology course (http://meyershistology.moodle.com.au/) Histology-online (http://histology-online.com/) Histology Protocols (http://www.ihcworld.com/protocol_database.htm) Histology atlas and more (http://sites.google.com/site/estudehistologia/) Histoweb (http://www.kumc.edu/instruction/medicine/anatomy/histoweb) SIU SOM Histology (http://www.siumed.edu/~dking2/index.htm) Visual Histology Atlas (http://www.visualhistology.com/Visual_Histology_Atlas/) Histology Glossary (http://www.histology-world.com/glossary/glossary1.htm) Histology Group of Victoria Incorporated (http://www.hgv.org.au) Histology Photomicrographs (http://www.histology-world.com/photoalbum/) Virtual Slidebox (http://www.path.uiowa.edu/virtualslidebox) Blue Histology (http://www.lab.anhb.uwa.edu.au/mb140/) BiMed - 20.000 histology images of fundamental tissues (http://www.informed.unal.edu.co/) The Histology Image Dataset (histologyDS) (http://www.informed.unal.edu.co/histologyDS/)

129

Histopathology

130

Histopathology Histopathology (compound of three Greek words: ἱστός histos "tissue", πάθος pathos "disease-suffering", and -λογία -logia) refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue fragments.

Micrograph showing contraction band necrosis, a histopathologic finding of myocardial infarction (heart attack).

Collection of tissues Histopathological examination of tissues starts with surgery, biopsy, or autopsy. The tissue is removed from the body or plant, and then placed in a fixative which stabilizes the tissues to prevent decay. The most common fixative is formalin (10% formaldehyde in water).

Preparation for histology The tissue is then prepared for viewing under a microscope using either chemical fixation or frozen section.

Chemical fixation In chemical fixation, the samples are transferred to a cassette, a container designed to allow reagents to freely act on the tissue inside. This cassette is immersed in multiple baths of progressively more concentrated ethanol, to dehydrate the tissue, followed by toluene or xylene, and finally extremely hot liquid (usually paraffin). During this 12 to 16 hour process, paraffin will replace the water in the tissue, turning soft, moist tissues into a sample miscible with paraffin, a type of wax. This process is known as tissue processing. The processed tissue is then taken out of the cassette and set in a mold. Through this process of embedding, additional paraffin is added to create a paraffin block which is attached to the outside of the cassette. The process of embedding then allows the sectioning of tissues into very thin (2 - 7 micrometer) sections using a microtome. The microtome slices the tissue ready for microscopic examination. The slices are thinner than the average cell, and are layered on a glass slide for staining.

Frozen section processing The second method of histology processing is called frozen section processing. In this method, the tissue is frozen and sliced thinly using a microtome mounted in a below-freezing refrigeration device called the cryostat. The thin frozen sections are mounted on a glass slide, fixed immediately & briefly in liquid fixative, and stained using the similar staining techniques as traditional wax embedded sections. The advantages of this method is rapid processing time, less equipment requirement, and less need for ventilation in the laboratory. The disadvantage is the poor

Histopathology quality of the final slide. It is used in intra-operative pathology for determinations that might help in choosing the next step in surgery during that surgical session (for example, to preliminarily determine clearness of the resection margin of a tumor during surgery).

Staining of the Processed Histology Slides This can be done to slides processed by the chemical fixation or frozen section slides. To see the tissue under a microscope, the sections are stained with one or more pigments. The aim of staining is to reveal cellular components; counterstains are used to provide contrast. The most commonly used stain in histopathology is a combination of hematoxylin and eosin (often abbreviated H&E). Hematoxylin is used to stain nuclei blue, while eosin stains cytoplasm and the extracellular connective tissue matrix pink. There are hundreds of various other techniques which have been used to selectively stain cells. Other compounds used to color tissue sections include safranin, Oil Red O, congo red, silver salts and artificial dyes. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls' Prussian blue reaction, used to demonstrate iron deposits in diseases like Hemochromatosis. [1] Recently, antibodies have been used to stain particular proteins, lipids and carbohydrates. Called immunohistochemistry, this technique has greatly increased the ability to specifically identify categories of cells under a microscope. Other advanced techniques include in situ hybridization to identify specific DNA or RNA molecules. These antibody staining methods often require the use of frozen section histology. Digital cameras are increasingly used to capture histopathological images.

Interpretation The histological slides are examined under a microscope by a pathologist, a medically qualified specialist who has completeted a recognised training programme (5 - 5.5 years in the United Kingdom). This medical diagnosis is formulated as a pathology report describing the histological findings and the opinion of the pathologist. In the case of cancer, this represents the tissue diagnosis required for most treatment protocols. In the removal of cancer, the pathologist will indicate whether the surgical margin is cleared, or is involved (residual cancer is left behind). This is done using either the bread loafing or CDMA method of processing.

In myocardial infarction Further information: Timeline of myocardial infarction pathology After a myocardial infarction, no histopathology is seen the first ~30 minutes. The only possible sign the first 4 hours is waviness of fibres at border. Later, however, a coagulation necrosis is initiated, with edema and hemorrhage. After 12 hours, there can be seen karyopyknosis and hypereosinophilia of myocytes with contraction band necrosis in margins, as well as beginning of neutrophil infiltration. At 1 – 3 days there is continued coagulation necrosis with loss of nuclei and striations and an increased infiltration of neutrophils to interstitium. Until the end of the first week after infarction there is beginning of disintegration of dead muscle fibres, necrosis of neutrophils and beginning of macrophage removal of dead cells at border, which increases the succeeding days. After a week there is also beginning of granulation tissue formation at margins, which matures during the following month, and gets increased collagen deposition and decreased cellularity until the myocardial scarring is fully mature at approximately 2 months after infarction.[2]

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Histopathology

References [1] http:/ / www. idoub. com/ doc/ 4448747/ Perl Perls' Prussian blue original formula and uses. Accessed April 2, 2009. [2] Chapter 11 in: Mitchell, Richard Sheppard; Kumar, Vinay; Abbas, Abul K.; Fausto, Nelson. Robbins Basic Pathology. Philadelphia: Saunders. ISBN 1-4160-2973-7. 8th edition.

External links • Virtual Histology Course - University of Zurich (http://www.pathol.uzh.ch/histologiekurs/) (German, English version in preparation) • Histopathology of the uterine cervix - digital atlas (IARC Screening Group) (http://screening.iarc.fr/atlashisto. php) • PathologyPics.com (http://www.PathologyPics.com): An interactive histology database for the Practicing Anatomic Pathologist as well as Pathology Trainees. • Researcher's Portfolio (http://www.jshalliop.co.uk/) Janina Sokolowska-Halliop: Histology, Pathomorfology, Pathologic Anatomy. • Histopathology Virtual Slidebox (http://www.path.uiowa.edu/virtualslidebox/iowa_histopathology/ content_index_db.html) - University of Iowa

Hoechst stain Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA.[1][2] These Bis-benzimides were originally developed by the Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Chemical structure of Hoechst dyes. Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra.

132

Hoechst stain

133

Molecular characteristics Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescent light around an emission maximum at 461 nm. Unbound dye has its maximum fluorescence emission in the 510-540 nm range. Hoechst stains can be excited with a xenon- or mercury-arc lamp or with an ultraviolet laser. There is a considerable Stokes shift between the excitation and emission spectra that makes Hoechst dyes useful in experiments in which multiple fluorophores are used. The fluorescence intensity of Hoechst dyes also increases with the pH of the solvent.[3]

Excitation/Emission spectra of Hoechst dyes.

Hoechst dyes are soluble in water and in organic solvents such as dimethyl formamide or dimethyl sulfoxide. Concentrations can be achieved of up to 10 mg/mL. Aqueous solutions are stable at 2-6 °C for at least six months when protected from light. For long-term storage the solutions are instead frozen at ≤-20 °C.[3] The dyes bind to the minor groove of double-stranded DNA with a preference for sequences rich in adenine and thymine. Although the dyes can bind to all nucleic acids, AT-rich double-stranded DNA strands enhance fluorescence [5] considerably. Hoechst dyes are cell-permeable and can bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. Cells that express specific ATP-binding cassette transporter proteins can also actively transport these stains out of their cytoplasm.

Hoechst (magenta) bound to the minor groove of DNA (green and blue). From [4] PDB 264D .

Hoechst stain

134

Applications A concentration of 0.1-12 µg/ml is commonly used to stain DNA in bacteria or eukaryotic cells. Cells are stained for 1-30 min at room temperature or 37 °C and then washed to remove unbound dye. A green fluorescence of unbound Hoechst dye may be observed on samples which were stained with too much dye or which were not washed thoroughly.[3] Hoechst dyes are often used as substitutes for another nucleic acid stain called DAPI. Key differences between Hoechst dyes and DAPI are: • Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells[6]

Transmission image of HeLa cells, with overlay of Hoechst 33258 staining (blue). The leftmost cell is in the prometaphase stage of mitosis; its chromosomes fluoresce brightly because they contain highly compacted DNA.

• The additional ethyl group of the Hoechst dyes renders them more cell-permeable. Hoechst 33342 and 33258 are quenched by Bromodeoxyuridine (BrdU), which is commonly used to detect dividing cells. Hoechst 33342 exhibits a 10 fold greater cell-permeability than H 33258. Cells can integrate BrdU in newly synthesized DNA as a substitute for thymidine. When BrdU is integrated into DNA, it is supposed that the bromine deforms the minor groove so that Hoechst dyes cannot reach their optimal binding site. Binding of Hoechst dyes is even stronger to BrdU-substituted DNA; however, no fluorescence ensues. Hoechst dyes can be used together with BrdU to monitor cell cycle progression.[7][8] Hoechst dyes are commonly used to stain genomic DNA in the following applications: • Fluorescence microscopy and immunohistochemistry, often together with other fluorophores[9] • Flow cytometry to count or sort out cells. An example is the use of Hoechst dyes to analyse how many cells of a population are in which phase of the cell cycle[10] • Detection of DNA in the presence of RNA in agarose gels[11] • Automated DNA determination[12] • Chromosome sorting.[11]

Toxicity and safety Because Hoechst stains bind to DNA, they interfere with DNA replication during cell division. Consequently, they are potentially mutagenic and carcinogenic, so care should be taken in their handling and disposal. Hoechst stain is used to sort sperm in livestock and humans. Its safety has been debated.[13][14]

External links • Spectral traces for fluorescent dyes [15] • Manual for Hoechst stains [16] • An online guide to fluorescent probes and commercial labeling technologies [17]

Hoechst stain

References [1] Latt, SA; Stetten, G, Juergens, LA, Willard, HF, Scher, CD (1975 Jul). "Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence.". The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 23 (7): 493–505. doi:10.1177/23.7.1095650. PMID 1095650. [2] Latt, SA; Stetten, G (1976 Jan). "Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis". The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 24 (1): 24–33. doi:10.1177/24.1.943439. PMID 943439. [3] "Hoechst Stains" (http:/ / probes. invitrogen. com/ media/ pis/ mp21486. pdf). Invitrogren (Molecular Probes). . [4] http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=264D [5] Portugal, J; Waring, MJ (1988 Feb 28). "Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study". Biochimica et Biophysica Acta 949 (2): 158–68. doi:10.1016/0167-4781(88)90079-6. PMID 2449244. [6] BD Bioscience (2009). Techniques for Immune Function Analysis (http:/ / www. bdbiosciences. com/ eu/ documents/ Immune-Function-Application-Handbook-2ndEd. pdf) (2 ed.). Becton, Dickinson and Company. . [7] Kubbies, M; Rabinovitch, PS (1983 Jan). "Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes". Cytometry 3 (4): 276–81. doi:10.1002/cyto.990030408. PMID 6185287. [8] Breusegem, SY; Clegg, RM, Loontiens, FG (2002 Feb 1). "Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex". Journal of Molecular Biology 315 (5): 1049–61. doi:10.1006/jmbi.2001.5301. PMID 11827475. [9] Iain Johnson, Michelle T.Z. Spence, ed. (2011). Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies (11 ed.). Invitrogen. ISBN 0-9829279-1-6. [10] Kubbies, M (1990). "Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding". Cytometry 11 (3): 386–94. doi:10.1002/cyto.990110309. PMID 1692786. [11] Mocharla, R; Mocharla, H, Hodes, ME (1987 Dec 23). "A novel, sensitive fluorometric staining technique for the detection of DNA in RNA preparations". Nucleic Acids Research 15 (24): 10589. doi:10.1093/nar/15.24.10589. PMC 339970. PMID 2447564. [12] Sterzel, W; Bedford, P, Eisenbrand, G (1985 Jun). "Automated determination of DNA using the fluorochrome Hoechst 33258". Analytical Biochemistry 147 (2): 462–7. doi:10.1016/0003-2697(85)90299-4. PMID 2409841. [13] Ashwood-Smith, M.J. (1994). "Safety of human sperm selection by flow cytometry" (http:/ / humrep. oxfordjournals. org/ cgi/ pdf_extract/ 9/ 5/ 757). Human Reproduction (Oxford University Press) 9 (5): 757–759. PMID 7929716. . [14] Parrilla, I; Vázquez, J M; Cuello, C; Gil, MA; Roca, J; Di Berardino, D; Martínez, EA (2004). "Hoechst 33342 stain and u.v. laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa" (http:/ / www. reproduction-online. org/ cgi/ content/ abstract/ 128/ 5/ 615). Reproduction 128 (5): 615–621. doi:10.1530/rep.1.00288. PMID 15509707. . [15] http:/ / www. mcb. arizona. edu/ IPC/ spectra_page. htm [16] http:/ / probes. invitrogen. com/ media/ pis/ mp21486. pdf [17] http:/ / www. invitrogen. com/ site/ us/ en/ home/ References/ Molecular-Probes-The-Handbook. html

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Hydroquinone

136

Hydroquinone Hydroquinone

Identifiers [1]

CAS number

123-31-9

ChemSpider

764

UNII

XV74C1N1AE

KEGG

D00073

ChEBI

CHEBI:17594

ChEMBL

CHEMBL537

RTECS number

MX3500000

ATC code

D11 AX11

Jmol-3D images

Image 1

[2]

[3]

[4]

[5]

[6]

[7]

[8]


CHO

Molar mass

110.11 g mol−1

Appearance

white solid

Density

1.3 g cm−3, solid

Melting point

172 °C, 445 K, 342 °F

Boiling point

287 °C, 560 K, 549 °F

6 6 2

Hydroquinone

137 Solubility in water

5.9 g/100 mL (15 °C) Structure

Dipole moment

0D Hazards

EU classification

Harmful (Xn) Carc. Cat. 3 Muta. Cat. 3 Dangerous for the environment (N)

R-phrases

R22 R40 R41 R43 R50 R68

S-phrases

(S2) S26 S36/37/39 S61


165 °C Related compounds

Related benzenediols

Pyrocatechol Resorcinol

Related compounds

1,4-benzoquinone ()

[9]


Infobox references

Hydroquinone, also benzene-1,4-diol or quinol, is an aromatic organic compound that is a type of phenol, having the chemical formula C6H4(OH)2. Its chemical structure, shown in the table at right, features two hydroxyl groups bonded to a benzene ring in a para position. It is a white granular solid. Substituted derivatives of this parent compound are also referred to as hydroquinones.

Production Hydroquinone is produced industrially by three routes, two of which are dominant.[10] Similar to the cumene process in reaction mechanism, the most widely used route involves the dialkylation of benzene with propene to give 1,4-diisopropylbenzene. This compound reacts with air to afford the bis(hydroperoxide), which is structurally similar to cumene hydroperoxide and rearranges to give acetone and hydroquinone in acid. A second route involves hydroxylation of phenol. The conversion uses hydrogen peroxide and affords a mixture of hydroquinone and catechol: C6H5OH + H2O2 → C6H4(OH)2 + H2O The third method, practiced only in China, is the oxidation of aniline by manganese dioxide followed by reduction of the resulting 1,4-benzoquinone. The process is conducted batchwise and generates a substantial waste stream.

Reactions In term of the reactivity of its O-H groups, hydroquinone resembles other phenols, being weakly acidic. The resulting conjugate base undergoes easy O-alkylation to give mono- and diethers. Similarly, hydroquinone is highly susceptible to ring substitution by Friedel-Crafts reactions such as alkylation. This reaction is exploited en route to popular antioxidants such as 2-tert-butyl-4-methoxyphenol ("BHA"). The useful dye quinizarin is produced by diacylation of hydroquinone with phthalic anhydride[10]

Hydroquinone

Redox Hydroquinone undergoes oxidation under mild conditions to give benzoquinone. This process can be reversed. Some naturally occurring hydroquinone derivatives exhibit this sort of reactivity, one example being coenzyme Q. Industrially this reaction is exploited both with hydroquinone itself but more often with its derivatives where one OH has been replaced by an amine.

Amination An important reaction is the conversion of hydroquinone to the mono- and diamino derivatives. Methylaminophenol, used in photography, is produced in this way:[10] C6H4(OH)2 + CH3NH2 → C6H4(OH)(N(H)CH3) + H2O Similarly diamines, useful in the rubber industry as antiozone agents, are produced similarly from aniline: C6H4(OH)2 + 2 C6H5NH2 → C6H4(N(H)CH6H5)2 + 2 H2O

Uses Hydroquinone has a variety of uses principally associated with its action as a reducing agent that is soluble in water. It is a major component in most photographic developers for film and paper where, with the compound Metol, it reduces silver halides to elemental silver. There are a variety of other uses associated with its reducing power. As a polymerization inhibitor, hydroquinone prevents polymerization of acrylic acid, methyl methacrylate, cyanoacrylate, and other monomers that are susceptible to radical-initiated polymerization. This application exploits the antioxidant properties of hydroquinone. Hydroquinone can undergo mild oxidation to convert to the compound parabenzoquinone, C6H4O2, often called p-quinone or simply quinone. Reduction of quinone reverses this reaction back to hydroquinone. Some biochemical compounds in nature have this sort of hydroquinone or quinone section in their structures, such as Coenzyme Q, and can undergo similar redox interconversions. Hydroquinone can lose an H+ from both to form a diphenolate ion. The disodium diphenolate salt of hydroquinone is used as an alternating comonomer unit in the production of the polymer PEEK.

Skin depigmentation In human medicine, hydroquinone is used as a topical application in skin whitening to reduce the color of skin. It does not have the same predisposition to cause dermatitis as metol does. This use is banned in some countries, including the member states of the European Union under Directive 76/768/EEC:1976.[11][12] In 2006, the United States Food and Drug istration revoked its previous approval of hydroquinone and proposed a ban on all over-the-counter preparations.[13] The FDA stated that hydroquinone cannot be ruled out as a potential carcinogen. This conclusion was reached based on the extent of absorption in humans and the incidence of neoplasms in rats in several studies where adult rats were found to have increased rates of tumours, including thyroid follicular cell hyperplasias, anisokaryosis, mononuclear cell leukemia, hepatocellular adenomas and renal tubule cell adenomas. The Campaign for Safe Cosmetics has also highlighted concerns.[14] Numerous studies have revealed that hydroquinone can cause exogenous ochronosis, a disfiguring disease in which blue-black pigments are deposited onto the skin, if taken orally; however, skin preparations containing the ingredient are istered topically. The FDA has classified hydroquinone currently as a safe product, as currently used.[13][15] While using hydroquinone as lightening agent can be effective with proper use, it can also cause skin sensitivity. Using a daily sunscreen with a high PPD (persistent pigment darkening) rating reduces the risk of further damage. Hydroquinone is sometimes combined with alpha hydroxy acids that exfoliate the skin to quicken the lightening

138

Hydroquinone process. In the United States, topical treatments usually contain up to 2% in hydroquinone. Otherwise, higher concentrations (up to 4%) should be prescribed and used with caution.

Natural occurrences Hydroquinones are one of the two primary reagents in the defensive glands of bombardier beetles, along with hydrogen peroxide (and perhaps other compounds, depending on the species), which collect in a reservoir. The reservoir opens through a muscle-controlled valve onto a thick-walled reaction chamber. This chamber is lined with cells that secrete catalases and peroxidases. When the contents of the reservoir are forced into the reaction chamber, the catalases and peroxidases rapidly break down the hydrogen peroxide and catalyze the oxidation of the hydroquinones into p-quinones. These reactions release free oxygen and generate enough heat to bring the mixture to the boiling point and vaporize about a fifth of it, producing a hot spray from the beetle's abdomen.[16] Farnesyl hydroquinone derivatives are the principal irritants exuded by the poodle-dog bush, which can cause severe dermatitis in humans. Hydroquinone is thought to be the active toxin in Agaricus hondensis mushrooms.[17]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=123-31-9 [2] http:/ / www. chemspider. com/ 764 [3] http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=XV74C1N1AE [4] http:/ / www. kegg. jp/ entry/ D00073 [5] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=17594 [6] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL537 [7] http:/ / www. whocc. no/ atc_ddd_index/ ?code=D11AX11 [8] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=c1cc%28ccc1O%29O [9] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=443862076& page2=%3AHydroquinone [10] Phillip M. Hudnall "Hydroquinone" in Ullmann's Encyclopedia of Industrial Chemistry 2002, Wiley-VCH, Weinheim. 2005 Wiley-VCH, Weinheim. doi:10.1002/14356007.a13_499. [11] 76/768/EEC:1976 Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products : http:/ / eur-lex. europa. eu/ LexUriServ/ LexUriServ. do?uri=CELEX:31976L0768:EN:HTML [12] Example of a product recall in Ireland (http:/ / www. consumerconnect. ie/ eng/ News_+ _Research/ Product_Recalls/ Clear_N_Smooth_cream_withdrawn. html) [13] United States Food and Drug istration (2006). Skin Bleaching Drug Products for Over-the-Counter Product Use; Proposed Rule (http:/ / www. fda. gov/ OHRMS/ DOCKETS/ 98fr/ 78n-0065-npr0003. pdf) (Report). 1978N-0065. . [14] Campaign For Safe Cosmetics - Hydroquinone (http:/ / www. safecosmetics. org/ article. php?id=289) [15] Olumide, YM; Akinkugbe, AO; Altraide, D; Mohammed, T; Ahamefule, N; Ayanlowo, S; Onyekonwu, C; Essen, N (April 2008). "Complications of chronic use of skin lightening cosmetics". International Journal of Dermatology 47 (4): 344–53. doi:10.1111/j.1365-4632.2008.02719.x. PMID 18377596. [16] Organic Chemistry, Solomon and Fryhle, 10th edition, Wiley Publishing, 2010. [17] Joval, E; Kroeger, P; N (April 1996). "Hydroquinone: the toxic compound of Agaricus hondensis". Planta Medica 62 (2): 185. doi:10.1055/s-2006-957852. PMID 17252436.

139

Hydroquinone

External links • International Chemical Safety Card 0166 (http://www.inchem.org/documents/icsc/icsc/eics0166.htm) • NIOSH Pocket Guide to Chemical Hazards (http://www.cdc.gov/niosh/npg/npgd0338.html) • IARC Monograph: "Hydroquinone" (http://www-cie.iarc.fr/htdocs/monographs/vol71/024-hydroquinone. html) • IUPAC Nomenclature of Organic Chemistry (http://www.acdlabs.com/iupac/nomenclature/) (online version of the "Blue Book")

Immunofluorescence Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualise the location of the antibodies.[1] Immunofluorescence can be used on tissue sections, cultured cell Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an lines, or individual cells, and may be used to analyse the anti-IgA antibody. The skin is from a patient with distribution of proteins, glycans, and small biological and Henoch-Schonlein purpura: IgA deposits are found in non-biological molecules. Immunofluoresence can be used in the walls of small superficial capillaries (yellow combination with other, non-antibody methods of fluorescent arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis. staining, for example, use of DAPI to label DNA. Several microscope designs can be used for analysis of immunofluorescence samples; the simplest is the epifluorescence microscope, and the confocal microscope is also widely used. Various super-resolution microscope designs that are capable of much higher resolution can also be used.[2]

140

Immunofluorescence

141

Types of immunofluorescence There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).

Primary (direct) Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy. This technique has several advantages over the secondary (or indirect) protocol below because of the direct conjugation of the antibody to the fluorophore. This reduces the number of steps in the staining procedure making the process faster and can reduce background signal by avoiding some issues with antibody cross-reactivity or non-specificity. However, since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence.[3]

Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places: The first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive). The second is within the nuclei of the epidermal cells (anti-nuclear antibodies).

Secondary (indirect) Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognises the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen.[3] This protocol is more complex and time consuming than the primary (or direct) protocol above, but it allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.[3] This protocol is possible because an antibody consists of two parts, a variable region (which recognizes the antigen) and constant region (which makes up the structure of the antibody molecule). It is important to realize that this division is artificial and in reality the antibody molecule is four polypeptide chains: two heavy chains and two light chains. A researcher can generate several primary antibodies that recognize various antigens (have different variable regions), but all share the same constant region. All these antibodies may therefore be recognized by a single secondary antibody. This saves the cost of modifying the primary antibodies to directly carry a fluorophore. Different primary antibodies with different constant regions are typically generated by raising the antibody in different species. For example, a researcher might create primary antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit secondary antibodies that recognize the goat antibody constant region ("rabbit anti-goat" antibodies). The researcher may then create a second set of primary antibodies in a mouse that could be recognized by a separate "donkey anti-mouse" secondary antibody. This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.

Immunofluorescence

Limitations As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g., Alexa Fluors, Seta Fluors, or DyLight Fluors). Immunofluorescence is only limited to fixed (i.e., dead) cells when structures within the cell are to be visualized because antibodies cannot cross the cell membrane. Proteins in the supernatant or on the outside of the cell membrane can be bound by the antibodies; this allows for living cells to be stained. Depending on the fixative that is being used, proteins of interest might become cross-linked and this could result in either false positive or false negative signals due to unspecific binding. An alternative approach is using recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). Use of such "tagged" proteins allows determination of their localization in live cells. Even though this seems to be an elegant alternative to immunofluorescence, the cells have to be transfected or transduced with the GFP-tag, and as a consequence they become at least S1 or above organisms that require stricter security standards in a laboratory.

References [1] Immunology/Immunofluorescence Protocol at Protocal-online.org (http:/ / www. protocol-online. org/ prot/ Immunology/ Immunofluorescence/ ) [2] Immunofluorescence Method (http:/ / www. bio. davidson. edu/ Courses/ genomics/ method/ IMF. html) [3] Fritschy, J.-M. and Härtig, W. 2001. Immunofluorescence. eLS.

External links • Images associated with autoimmune diseases (http://www.ii.bham.ac.uk/clinicalimmunology/ CISimagelibrary/) at University of Birmingham • Immunofluorescence protocol and confocal microscopy resources (http://www.confocal-microscopy.org/ Protocols - Immunofluorescence.htm) at confocal-microscopy.org • Immunofluorescence Staining Protocol (http://www.prosci-inc.com/Immunofluorescence-Protocol.html) • Overview (http://www.bio.davidson.edu/COURSES/genomics/method/IMF.html) at Davidson College • Immunofluorescence (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=& term=Immunofluorescence) at the US National Library of Medicine Medical Subject Headings (MeSH) • SynD - Automatic synapse and neurite detection in immuno-fluorescence images (http://www.johanneshjorth. se/SynD/)

142

Immunohistochemistry

143

Immunohistochemistry Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.[1] IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

Immunohistochemistry labels individual proteins, such as TH (green) in the axons of sympathetic autonomic neurons.

Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see immunoperoxidase staining). Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine (see immunofluorescence).

Sample preparation While using the right antibodies to target the correct antigens and amplify the signal is important for visualization, complete preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. Paraformaldehyde is usually used with fixation. Depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 μm) sections are sliced from the tissue of interest, or if the tissue is not very thick and is penetrable it is used whole. The slicing is usually accomplished through the use of a microtome, and slices are mounted on slides. "Free-floating IHC" uses slices that are not mounted; these slices are normally produced using a vibrating microtome. Because of the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval (microwave method, enzyme method, hot incubation method); these steps often make the difference between staining and no staining. Additionally, depending on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining. Unlike immunocytochemistry, the tissue does not need to be permeabilized because this has already been accomplished by the microtome blade during sample preparation. Detergents like Triton X-100 are generally used in immunohistochemistry to reduce surface tension, allowing less reagent to be used to achieve better and more even coverage of the sample. Although antibodies show preferential avidity for specific epitopes, they may partially or weakly bind to sites on nonspecific proteins (also called reactive sites) that are similar to the cognate binding sites on the target antigen. In the context of antibody-mediated antigen detection, nonspecific binding causes high background staining that can mask the detection of the target antigen. To reduce background staining in IHC, ICC and any other immunostaining application, the samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. Commercial blocking buffers with proprietary formulations are available for greater efficiency.


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Sample Labeling Antibody types The antibodies used for specific detection can be polyclonal or monoclonal. Polyclonal antibodies are made by injecting animals with peptide Ag and, after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes. Monoclonal antibodies show specificity for a single epitope and are therefore considered more specific to the target antigen than polyclonal antibodies. For IHC detection strategies, antibodies are classified as primary or secondary reagents. Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against immunoglobulins of the primary antibody species. The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then recruits reporter molecules, or the secondary antibody is directly bound to the reporter molecule itself.

IHC reporters Reporter molecules vary based on the nature of the detection method, and the most popular methods of detection are with enzyme- and fluorophore-mediated chromogenic and fluorescence detection, respectively. With chromogenic reporters, an enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel, producing a deep purple/black staining. Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semiand fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.

Target antigen detection methods The direct method is a one-step staining method and involves a labeled antibody (e.g. FITC-conjugated antiserum) reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, such as with indirect methods, and is less commonly used than indirect methods.

The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.

The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled


secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.

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The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.

Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin or NeutrAvidin proteinbound-enzyme. The difference between these three biotin-binding proteins is their individual binding affinity to endogenous tissue targets leading to nonspecific binding and high background; the ranking of these proteins based on their nonspecific binding affinities, from highest to lowest, is: 1) avidin, 2) streptavidin and 3) Neutravidin protein. The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be generated. For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit. With the direct method, it would be necessary to label each primary antibody for every antigen of interest.

Counterstains After immunohistochemical staining of the target antigen, a second stain is often applied to provide contrast that helps the primary stain stand out. Many of these stains show specificity for discrete cellular compartments or antigens, while others will stain the whole cell. Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design, and include: hematoxylin, Hoechst stain and DAPI are commonly used.

IHC Troubleshooting In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, and many potential problems affect the outcome of the procedure. The major problem areas in IHC staining include strong background staining, weak target antigen staining and autofluorescence. Endogenous biotin or reporter enzymes or primary/secondary antibody cross-reactivity are common causes of strong background staining, while weak staining may be caused by poor enzyme activity or primary antibody potency. Furthermore, autofluorescence may be due to the nature of the tissue or the fixation method. These aspects of IHC tissue prep and antibody staining must be systematically addressed to identify and overcome staining issues.


Diagnostic IHC markers IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. It is also an effective way to examine the tissues .This has made it a widely used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures. Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. For this reason, primary antibodies must be well-validated in a Western Blot or similar Immunohistochemical staining of normal kidney procedure. The technique is even more widely used in diagnostic with CD10. surgical pathology for typing tumors (e.g. immunostaining for e-cadherin to differentiate between DCIS (ductal carcinoma in situ: stains positive) and LCIS (lobular carcinoma in situ: does not stain positive)[2]). • • • • • • • • • •

Carcinoembryonic antigen (CEA): used for identification of adenocarcinomas. Not specific for site. Cytokeratins: used for identification of carcinomas but may also be expressed in some sarcomas.[3] CD15 and CD30 : used for Hodgkin's disease Alpha fetoprotein: for yolk sac tumors and hepatocellular carcinoma CD117 (KIT): for gastrointestinal stromal tumors (GIST) CD10 (CALLA): for renal cell carcinoma and acute lymphoblastic leukemia Prostate specific antigen (PSA): for prostate cancer estrogens and progesterone staining for tumour identification Identification of B-cell lymphomas using CD20 Identification of T-cell lymphomas using CD3

Directing therapy A variety of molecular pathways are altered in cancer and some of the alterations can be targeted in cancer therapy. Immunohistochemistry can be used to assess which tumors are likely to respond to therapy, by detecting the presence or elevated levels of the molecular target.

Chemical inhibitors Tumor biology allows for a number of potential intracellular targets. Many tumors are hormone dependent. The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antihormonal therapy. One of the first therapies was the antiestrogen, tamoxifen, used to treat breast cancer. Such hormone receptors can be detected by immunohistochemistry.[4] Imatinib, an intracellualar tyrosine kinase inhibitor, was developed to treat chronic myelogenous leukemia, a disease characterized by the formation of a specific abnormal tyrosine kinase. Imitanib has proven effective in tumors, that express other tyrosine kinases, most notably KIT. Most gastrointestinal stromal tumors express KIT, which can be detected by immunohistochemistry.[5]

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Monoclonal antibodies Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are potential targets for therapies utilising monoclonal antibodies. Monoclonal antibodies, due to their size, are utilized against cell surface targets. Among the overexpressed targets, the of the epidermal growth factor receptor (EGFR) family, transmembrane proteins with an extracellular receptor domain regulating an intracellular tyrosine kinase,[6] Of these, HER2/neu (also known as Erb-B2) was the first to be developed. The molecule is highly expressed in a variety of cancer cell types, most notably breast cancer. As such, antibodies against HER2/neu have been FDA approved for clinical treatment of cancer under the drug name Herceptin. There are commercially available immunohistochemical tests, Dako HercepTest [7] and Ventana Pathway.[8] Similarly, EGFR (HER-1) is overexpressed in a variety of cancers including head and neck and colon. Immunohistochemistry is used to determine patients who may benefit from therapeutic antibodies such as Erbitux (cetuximab).[9] Commercial systems to detect EGFR by immunohistochemistry include the Dako pharmDx [10].

References [1] Ramos-Vara, JA (2005). "Technical Aspects of Immunohistochemistry" (http:/ / www. vetpathology. org/ cgi/ content/ short/ 42/ 4/ 405). Vet Pathol 42 (4): 405–426. doi:10.1354/vp.42-4-405. PMID 16006601. . [2] O'Malley F and Pinder S, Breast Pathology, 1st. Ed. Elsevier 2006. ISBN 978-0-443-06680-1 [3] Leader M, Patel J, Makin C, Henry K (December 1986). "An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial tumours. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas". Histopathology 10 (12): 1315–24. doi:10.1111/j.1365-2559.1986.tb02574.x. PMID 2434403. [4] Jørgensen, Jan Trøst; Kirsten Vang Nielsen, Bent Ejlertsen (April 2007). "Pharmacodiagnostics and targeted therapies - a rational approach for individualizing medical anticancer therapy in breast cancer" (http:/ / theoncologist. alphamedpress. org/ cgi/ content/ full/ 12/ 4/ 397). The Oncologist (United States: AlphaMed Press) 12 (4): 397–405. doi:10.1634/theoncologist.12-4-397. ISSN 1083-7159. PMID 17470682. . Retrieved 2008-03-14. [5] Gold JS, Dematteo RP (August 2006). "Combined Surgical and Molecular Therapy: The Gastrointestinal Stromal Tumor Model". Ann. Surg. 244 (2): 176–84. doi:10.1097/01.sla.0000218080.94145.cf. PMC 1602162. PMID 16858179. [6] Harari, P M (December 2004). "Epidermal growth factor receptor inhibition strategies in oncology" (http:/ / erc. endocrinology-journals. org/ cgi/ content/ full/ 11/ 4/ 689?ijkey=9caa7985e4396550fdc851b303ea7958513e070e). Endocrine-Related Cancer (England: Society for Endocrinology) 11 (4): 689–708. doi:10.1677/erc.1.00600. ISSN 1351-0088. PMID 15613446. . Retrieved 2008-03-14. [7] http:/ / www. dakousa. com/ index/ prod_search/ prod_baseproducts. htm?productareaid=1& productgroupid=3& productsubgroupid=1003000 [8] Press, Michael F.; Guido Sauter, Leslie Bernstein, Ivonne E.Villalobos, MartinaMirlacher, Jian-Yuan Zhou, RoobaWardeh, Yong-Tian Li, Roberta Guzman, Yanling Ma, Jane Sullivan-Halley, Angela Santiago, Jinha M. Park, Alessandro Riva, Dennis J.Slamon (September 15, 2005). "Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials" (http:/ / clincancerres. aacrjournals. org/ cgi/ content/ full/ 11/ 18/ 6598). Clinical Cancer Research (United States: American Association for Cancer Research.) 2005 15;11(18): (18): 6598–6607. doi:10.1158/1078-0432.CCR-05-0636. ISSN 1078-0432. PMID 16166438. . Retrieved 2008-03-14. [9] Bibeau F, Boissière-Michot F, Sabourin JC, et al. (September 2006). "Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray". Virchows Arch. 449 (3): 281–7. doi:10.1007/s00428-006-0247-9. PMC 1888717. PMID 16865406. [10] http:/ / www. dakousa. com/ index/ prod_search/ prod_groups. htm?productareaid=1

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External links • Overview of Immunohistochemistry--describes all aspects of IHC including sample prep, staining and troubleshooting (http://www.piercenet.com/browse. cfm?fldID=F95B91A9-3DC1-4B56-8E8D-59CA044A8BA7) • Yale Core Tissue Microarray Facility (http://tissuearray.org/yale/) • Histochemical Staining Methods (http://www.urmc.rochester.edu/path/zqu/Histostain/index.html) University of Rochester Department of Pathology • Immunohistochemistry Staining Protocol (http://www.prosci-inc.com/Immunohistochemistry-Protocol.html) • HistoWiki entry for Immunohistochemistry (http://www.ihcworld.com/histowiki/doku. php?id=immunohistochemistry#immunohistochemistry) • Burnett R, Guichard Y, Barale E (1997). "Immunohistochemistry for light microscopy in safety evaluation of therapeutic agents: an overview". Toxicology 119 (1): 83–93. doi:10.1016/S0300-483X(96)03600-1. PMID 9129199. • Immunohistochemistry (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=& term=Immunohistochemistry) at the US National Library of Medicine Medical Subject Headings (MeSH) • Joyner, A.; Wall, N. (2008). "Immunohistochemistry of Whole-Mount Mouse Embryos". Cold Spring Harbor Protocols 2008 (2): pdb.prot4820. doi:10.1101/pdb.prot4820.

India ink India ink (or Indian ink in British English) is a simple black ink once widely used for writing and printing and now more commonly used for drawing, especially when inking comic books and comic strips.

Composition Basic India ink is composed of a variety of fine soot known as lampblack, combined with water to form a liquid. A binding agent such as gelatin or, more commonly, shellac may also be added, to make the ink more durable once dried. India ink is occasionally sold not as a liquid, but in solid form (most commonly, a stick), which must be moistened before use.

History The process of making India ink was known in China as far back as the middle of the 3rd millennium BC, during Neolithic China.[1] India ink was first invented in China,[2][3] although the source of materials to make the carbon pigment in India ink was later often traded from India, thus the term India ink was coined.[2][3] The traditional Chinese method of making the ink was to grind a mixture of hide glue, carbon black, lampblack, and bone black pigment with a pestle and mortar before pouring it into a ceramic dish where it could dry.[2] In order to use the dry mixture, a wet brush would be applied until it reliquified.[2] The manufacture of India ink was well-established by the Cao Wei Dynasty (220–265 AD).[4] Historically the ink used in China were in the form of ink sticks made of lampblack and animal glue. The Chinese had used India ink derived from pine soot prior to the 11th century AD, when the polymath official Shen Kuo (1031–1095) of the mid Song Dynasty became troubled by deforestation (due to the demands of charcoal for the iron industry) and desired making ink from a source other than pine soot. He believed that petroleum (which the Chinese called 'rock oil') was produced inexhaustibly within the earth and so decided to make an ink from the soot of burning petroleum, which the later pharmacologist Li Shizhen (1518–1593) wrote was as lustrous as lacquer and was superior to pine soot ink.[5][6][7][8]

148

India ink India ink has been in use in India since at least the 4th century BC, where it was called masi, an ixture of several substances.[9] Indian documents written in Kharosthi with this ink have been unearthed in as far as Xinjiang, China.[10] The practice of writing with ink and a sharp-pointed needle was common practice since antiquity in South India.[11] Several ancient Buddhist and Jain scripts in India were also compiled in ink.[12] In India, the carbon black from which India ink is formulated was obtained indigenously by burning bones, tar, pitch and other substances.[13]

Uses other than writing • Hanetsuki (羽根突き, 羽子突き) is a Japanese traditional game, similar to bton, played by girls at the New Year with a rectangular wooden paddle called a hagoita and a brightly-colored shuttlecock. The shuttlecock must be kept in the air as long as possible. Girls who fail to hit the shuttlecock get marked on the face with India ink.[14] • Amateur tattoo artists will sometimes use India ink for tattooing the skin. Non-medical grade India ink should not be used for homemade tattoos because it contains chemicals which could cause poisoning. • In pathology laboratories, India ink is applied to surgically removed tissue specimens to maintain orientation and indicate tumor resection margins. The painted tissue is sprayed with acetic acid, which acts as a mordant, "fixing" the ink so it doesn't track. This ink is used because it survives tissue processing, during which tissue samples are bathed in alcohol and xylene and then embedded in paraffin wax. When viewed under the microscope, the ink at the tissue edge informs the pathologist of the surgical resection margin or other point of interest. • Microbiologists use India ink to stain a slide containing micro-organisms. The background is stained while the organisms remain clear. This type of staining is called a negative stain. India ink, along with other stains, can be used to determine if a cell has a gelatinous capsule.[15] A common application of this procedure in the clinical microbiology laboratory is to confirm the morphology of the encapsulated yeast Cryptococcus spp. • Model Railroaders use a mixture of India ink and Isopropyl Alcohol as a wood stain, graying wood to appear aged, and to bring out detail. • India ink is used diluted as an ultra fine polishing medium for making precise optical surfaces on metals.[16][17] • In ophthalmology, it was and still is used to some extent in corneal tattooing. • Once dry, its conductive properties make it useful for electrical connections to difficult substrates, such as glass. Although relatively low in conductivity, surfaces can be made suitable for electroplating, low frequency shielding, or for creating large conductive geometries for high voltage apparatuses. A piece of paper impregnated with India ink serves as a grid leak resistor in some tube radio circuits. • Zoological museum specimens were often tagged in India ink, either directly or on a piece of tracing paper stored along the specimen, because of its durability even when submerged in preservative fluids.

Notes [1] Woods & Woods, 51–52. [2] Gottsegen, page 30. [3] Smith, page 23. [4] Sung, Sun & Sun, page 286-288. [5] Sivin, III, page 24. [6] Menzies, page 24. [7] Needham, Volume 5, Part 7, pages 75–76. [8] Deng, page 36. [9] Banerji, page 673 [10] Sircar, page 206 [11] Sircar, page 62 [12] Sircar, page 67 [13] "India ink." in Encyclopædia Britannica. 2008 Encyclopædia Britannica Inc. [14] http:/ / www. englishjapaneseonlinedictionary. com/ Japanese%20textbook/ pages/ Japanese_textbook_76. htm [15] Woeste and Demchick, Volume 57, Part 6, pages 1858-1859

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India ink [16] http:/ / www. techbriefs. com/ component/ content/ article/ 1831 [17] NASA Technical Brief

References • Banerji, Sures Chandra (1989). A Companion to Sanskrit Literature. Motilal Banarsidass. ISBN 81-208-0063-X. • Deng, Yinke (2005). Ancient Chinese Inventions. Translated by Wang Pingxing. Beijing: China Intercontinental Press. ISBN 7-5085-0837-8. • Gottsegen, Mark D. (2006). The Painter's Handbook: A Complete Reference. New York: Watson-Guptill Publications. ISBN 0-8230-3496-8. • Menzies, Nicholas K. (1994). Forest and Land Management in Imperial China. New York: St. Martin's Press, Inc. ISBN 0-312-10254-2. • Needham, Joseph (1986). Science and Civilization in China: Volume 5, Chemistry and Chemical Technology, Part 7, Military Technology; the Gunpowder Epic. Taipei: Caves Books, Ltd. • Sircar, D.C. (1996). Indian epigraphy. Motilal Banarsidass. ISBN 81-208-1166-6. • Sivin, Nathan (1995). Science in Ancient China: Researches and Reflections. Brookfield, Vermont: Variorum, Ashgate Publishing. • Smith, Joseph A. (1992). The Pen and Ink Book: Materials and Techniques for Today's Artist. New York: Watson-Guptill Publications. ISBN 0-8230-3986-2. • Sung, Ying-hsing; Sun, E-tu Zen; Sun, Shiou-chuan (1997). Chinese Technology in the Seventeenth Century: T'ien-kung K'ai-wu. Mineola: Dover Publications. ISBN 0-486-29593-1. • Woods, Michael; Woods, Mary (2000). Ancient Communication: Form Grunts to Graffiti. Minneapolis: Runestone Press; an imprint of Lerner Publishing Group..... • Woeste S.; Demchick, P. (1991). Appl Environ Microbiol. 57(6): 1858-1859 ASM.org (http://aem.asm.org/cgi/ content/abstract/57/6/1858)

150

Intercalation (chemistry)

151

Intercalation (chemistry) In chemistry, intercalation is the reversible inclusion of a molecule (or group) between two other molecules (or groups). Examples include DNA intercalation and graphite intercalation compounds.

Intercalation induces structural distortions. Left: unchanged DNA strand. Right: DNA strand intercalated at three locations (red areas).

DNA intercalation There are several ways molecules (in this case, also known as ligands) can interact with DNA. Ligands may interact with DNA by covalently binding, electrostatically binding, or intercalating.[1] Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA. These ligands are mostly polycyclic, aromatic, and planar, and therefore often make good nucleic acid stains. Intensively studied DNA intercalators include berberine, ethidium bromide, Ethidium intercalated between two adenine-thymine base pairs. proflavine, daunomycin, doxorubicin, and thalidomide. DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells. Examples include doxorubicin (adriamycin) and daunorubicin (both of which are used in treatment of Hodgkin's lymphoma), and dactinomycin (used in Wilm's tumour, Ewing's Sarcoma, rhabdomyosarcoma). In order for an intercalator to fit between base pairs, the DNA must dynamically open a space between its base pairs by unwinding. The degree of unwinding varies depending on the intercalator; for example, ethidium cation (the ionic form of ethidium bromide found in aqueous solution) unwinds DNA by about 26°, whereas proflavine unwinds it by about 17°. This unwinding causes the base pairs to separate, or "rise", creating an opening of about 0.34 nm (3.4 Å). This unwinding induces local structural changes to the DNA strand, such as lengthening of the DNA strand or twisting of the base pairs. These structural modifications can lead to functional changes, often to the inhibition of transcription and replication and DNA repair processes, which makes intercalators potent mutagens. For this reason, DNA intercalators are often carcinogenic, such as the exo (but not the endo) 8,9 epoxide of aflatoxin B1, acridines such as proflavine or quinacrine, or ethidium bromide. Intercalation as a mechanism of interaction between cationic, planar, polycyclic aromatic systems of the correct size (on the order of a base pair) was first proposed by Leonard Lerman in 1961.[2][3][4] One proposed mechanism of

Intercalation (chemistry)

152

intercalation is as follows: In aqueous isotonic solution, the cationic intercalator is attracted electrostatically to the surface of the polyanionic DNA. The ligand displaces a sodium and/or magnesium cation present in the "condensation cloud" of such cations that surrounds DNA (to partially balance the sum of the negative charges carried by each phosphate oxygen), thus forming a weak electrostatic association with the outer surface of DNA. From this position, the ligand diffuses along the surface of the DNA and may slide into the hydrophobic environment found between two base pairs that may transiently "open" to form an intercalation site, allowing the ethidium to move away from the hydrophilic (aqueous) environment surrounding the DNA and into the intercalation site. The base pairs transiently form such openings due to energy absorbed during collisions with solvent molecules.

Materials science Many layered solids intercalate guest molecules. A famous example is the intercalation of potassium into graphite.[5] Intercalation expands the "van der Waals gap" between sheets, which requires energy. Usually this energy is supplied by charge transfer between the guest and the host solid, i.e., redox. Aside from graphite, well-known intercalation hosts are the layered dichalcogenides such as tantalum disulfide and iron oxychloride.[6] In characteristic manner, intercalation is analyzed by X-ray diffraction, since the spacing between sheets increases, and by electrical conductivity, since charge transfer alters the number of charge carriers.

Quantum-Mechanical Modelling of Intercalated Fluorescent Probes Model of intercalation of potassium into graphite

Recently the light-switch properties of the DNA fluorescent probe, Thiazole Orange (TO) [7] have been effectively modelled, using the Density Functional Theory (DFT) with the M06-2X functional [8] coupled with the Polarisable Continuum model (PCM) [9].

Notes [1] Richards, A.D. & Rodgers, A. (2007). "Synthetic metallomolecules as agents for the control of DNA structure". Chem. Soc. Rev. 36: 471–483. doi:10.1039/b609495c. PMID 17325786. [2] Lerman L. S. “Structural considerations in the interactions of deoxyribonucleic acid and acridines” Journal of Molecular Biolology 1961, 3, 18-30. [3] Luzzati V., Masson F., Lerman L. S. “Interaction of DNA and proflavine: a small-angle x-ray scattering study” J. Mol. Biol. 1961, 3, 634-639. [4] Lerman L. S. “The structure of the DNA-acridine complex” Proc. Natl. Acad. Sci. USA, 1963, 49, 94-102. [5] Holleman, A. F.; Wiberg, E. "Inorganic Chemistry" (http:/ / books. google. com/ books?id=vEwj1WZKThEC& pg=PA794) Academic Press: San Diego, 2001. p. 794 ISBN 0-12-352651-5. [6] S. Kikkawa, F. Kanamaru, M. Koizumi "Layered Intercalation Compounds" Inorganic Syntheses, 1983, Volume 22, 86. doi:10.1002/9780470132531.ch17 [7] A. Biancardi , T. Biver , A. Marini , B. Mennucci F. Secco (2011). "Thiazole orange (TO) as a light-switch probe: a combined quantum-mechanical and spectroscopic study". Phys. Chem. Chem. Phys. 13: 12595. doi:10.1039/C120812H. [8] Y. Zhao, D. G. Truhlar (2008). "The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals". Theor. Chem. Acc. 102: 215. [9] J. Tomasi, B. Mennucci, R> Cammi (2005). "Quantum Mechanical Continuum Solvation Models". Chem. Rev. 105: 2999.

Iodine

153

Iodine Iodine Appearance lustrous metallic gray, violet as a gas

General properties Name, symbol, number iodine, I, 53 Pronunciation

/ˈaɪ.ɵdaɪn/ EYE-o-dyne, /ˈaɪ.ɵdɪn/ EYE-o-dən, or /ˈaɪ.ɵdiːn/ EYE-o-deen

Element category

halogen

Group, period, block

17, 5, p

Standard atomic weight 126.90447 g·mol−1 Electron configuration

[Kr] 4d 5s 5p

Electrons per shell

2, 8, 18, 18, 7 (Image)

10

2

5

Physical properties Phase

solid

Density (near r.t.)

4.933 g·cm

Melting point

386.85 K,113.7 °C,236.66 °F

Boiling point

457.4 K,184.3 °C,363.7 °F

Triple point

386.65 K (113°C), 12.1 kPa

Critical point

819 K, 11.7 MPa

Heat of fusion

(I ) 15.52 kJ·mol−1

−3

2

Heat of vaporization

(I ) 41.57 kJ·mol−1 2

Specific heat capacity

(25 °C) (I ) 54.44 J·mol−1·K−1 2

Vapor pressure (rhombic) P/Pa

1

10 100 1 k 10 k 100 k

at T/K 260 282 309 342 381

457

Atomic properties Oxidation states

7, 5, 3, 1, -1 (strongly acidic oxide)

Electronegativity

2.66 (Pauling scale)

Iodine

154 Ionization energies

1st: 1008.4 kJ·mol−1 2nd: 1845.9 kJ·mol−1 3rd: 3180 kJ·mol−1

Atomic radius

140 pm

Covalent radius

139±3 pm

Van der Waals radius

198 pm

Miscellanea Crystal structure

orthorhombic

Magnetic ordering

diamagnetic

Electrical resistivity

(0 °C) 1.3×107Ω·m

Thermal conductivity

(300 K) 0.449 W·m ·K

Bulk modulus

7.7 GPa

CAS registry number

7553-56-2

[1]

−1

−1

Most stable isotopes iso

NA

half-life

123

syn

13 h

I

I 100%

127

127

131

I

syn

ε, γ

0.16

DP 123

Te

I is stable with 74 neutron

6 I trace 15.7×10 y

129

DM DE (MeV)

−

β

8.02070 d β−, γ

0.194

129

0.971

131

Xe Xe

Iodine ( /ˈaɪ.ɵdaɪn/ EYE-o-dyne, /ˈaɪ.ɵdɪn/ EYE-o-dən, or /ˈaɪ.ɵdiːn/ EYE-o-deen in both American[2] and British[3] English[4]) is a chemical element with symbol I and atomic number 53. The name is from Greek ἰοειδής ioeidēs, meaning violet or purple, due to the color of elemental iodine vapor.[5] Iodine and its compounds are primarily used in nutrition, and industrially in the production of acetic acid and certain polymers. Iodine's relatively high atomic number, low toxicity, and ease of attachment to organic compounds have made it a part of many X-ray contrast materials in modern medicine. Iodine has only one stable isotope. A number of iodine radioisotopes are also used in medical applications. Iodine is found on Earth mainly as the highly water-soluble iodide I-, which concentrates it in oceans and brine pools. Like the other halogens, free iodine occurs mainly as a diatomic molecule I2, and then only momentarily after being oxidized from iodide by an oxidant like free oxygen. In the universe and on Earth, iodine's high atomic number makes it a relatively rare element. However, its presence in ocean water has given it a role in biology. It is the heaviest essential element utilized widely by life in biological functions (only tungsten, employed in enzymes by a few species of bacteria, is heavier). Iodine's rarity in many soils, due to initial low abundance as a crust-element, and also leaching of soluble iodide by rainwater, has led to many deficiency problems in land animals and inland human populations. Iodine deficiency affects about two billion people and is the leading preventable cause of intellectual disabilities.[6] Iodine is required by higher animals, which use it to synthesize thyroid hormones, which contain the element. Because of this function, radioisotopes of iodine are concentrated in the thyroid gland along with nonradioactive iodine. The radioisotope iodine-131, which has a high fission product yield, concentrates in the thyroid, and is one of

Iodine

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the most carcinogenic of nuclear fission products.

Characteristics Iodine under standard conditions is a bluish-black solid. It can be seen apparently sublimating at standard temperatures into a violet-pink gas that has an irritating odor. This halogen forms compounds with many elements, but is less reactive than the other of its Group VII (halogens) and has some metallic light reflectance. Elemental iodine dissolves easily in most organic solvents such as hexane or chloroform owing to its lack of polarity, but is only slightly soluble in water. However, the solubility of elemental iodine in water can be increased by the addition of potassium iodide. The molecular iodine reacts reversibly with the negative ion, generating the triiodide anion I3− in equilibrium, which is soluble in water. This is also the formulation of some types of medicinal (antiseptic) iodine, although tincture of iodine classically dissolves the element in aqueous ethanol.

In the gas phase, iodine shows its violet color.

The colour of solutions of elemental iodine changes depending on the polarity of the solvent. In non-polar solvents like hexane, solutions are violet; in moderately polar dichloromethane, the solution is dark crimson, and, in strongly polar solvents such as acetone or ethanol, it appears orange or brown. This effect is due to the formation of adducts.

Iodine melts at the relatively low temperature of 113.7 °C, although the liquid is often obscured by a dense violet vapor of gaseous iodine.

Occurrence Iodine is rare in the solar system and Earth's crust (47–60th in abundance); however, iodide salts are often very soluble in water. Iodine occurs in slightly greater concentrations in seawater than in rocks, 0.05 vs. 0.04 ppm. Minerals containing iodine include caliche, found in Chile. The brown algae Laminaria and Fucus found in temperate zones of the Northern Hemisphere contain 0.028–0.454 dry weight percent of iodine. Aside from tungsten, iodine is the heaviest element to be essential in living organisms. About 19,000 tonnes are produced annually from natural sources.[7] Organoiodine compounds are produced by marine life forms, the most notable being iodomethane (commonly called methyl iodide). About 214 kilotonnes/year of iodomethane is produced by the marine environment, by microbial activity in rice paddies and by the burning of biological material.[8] The volatile iodomethane is broken up in the atmosphere as part of a global iodine cycle.[8][9] Iodomethane

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Structure and bonding Iodine normally exists as a diatomic molecule with an I-I bond length of 270 pm,[10] one of the longest single bonds known. The I2 molecules tend to interact via the weak van der Waals force called the London Forces, and this interaction is responsible for the higher melting point compared to more compact halogens, which are also diatomic. Since the atomic size of Iodine is larger, its melting point is higher. The solid crystallizes as orthorhombic crystals. The crystal motif in the Hermann–Mauguin notation is Cmca (No 64), Pearson symbol oS8. The I-I bond is relatively weak, with a bond dissociation energy of 36 kcal/mol, and most bonds to iodine are weaker than for the lighter halides. One consequence of this weak bonding is the relatively high tendency of I2 molecules to dissociate into atomic iodine.

Structure of solid iodine

Production Of the several places in which iodine occurs in nature, only two sources are useful commercially: the caliche, found in Chile, and the iodine-containing brines of gas and oil fields, especially in Japan and the United States. The caliche contains sodium nitrate, which is the main product of the mining activities, and small amounts of sodium Crystalline iodine iodate and sodium iodide. In the extraction of sodium nitrate, the [11] sodium iodate and sodium iodide are extracted. The high concentration of iodine in the caliche and the extensive mining made Chile the largest producer of iodine in 2007. Most other producers use natural occurring brine for the production of iodine. The Japanese Minami Kanto gas field east of Tokyo and the American Anadarko Basin gas field in northwest Oklahoma are the two largest sources for iodine from brine. The brine has a temperature of over 60°C owing to the depth of the source. The brine is first purified and acidified using sulfuric acid, then the iodide present is oxidized to iodine with chlorine. An iodine solution is produced, but is dilute and must be concentrated. Air is blown into the solution, causing the iodine to evaporate, then it is ed into an absorbing tower containing acid where sulfur dioxide is added to reduce the iodine. The hydrogen iodide (HI) is reacted with chlorine to precipitate the iodine. After filtering and purification the iodine is packed.[11][12] 2 HI + Cl2 → I2↑ + 2 HCl I2 + 2 H2O + SO2 → 2 HI + H2SO4 2 HI + Cl2 → I2↓ + 2 HCl The production of iodine from seawater via electrolysis is not used owing to the sufficient abundance of iodine-rich brine. Another source of iodine is kelp, used in the 18th and 19th centuries, but it is no longer economically viable.[13]

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Commercial samples often contain high concentrations of impurities, which can be removed by sublimation. The element may also be prepared in an ultra-pure form through the reaction of potassium iodide with copper(II) sulfate, which gives copper(II) iodide initially. That decomposes spontaneously to copper(I) iodide and iodine: Cu2+ + 2 I– → CuI2

Iodine output in 2005

2 CuI2 → 2 CuI + I2 There are also other methods of isolating this element in the laboratory, for example, the method used to isolate other halogens: oxidation of the iodide in hydrogen iodide (often made in situ with an iodide and sulfuric acid) by manganese dioxide (see below in Descriptive chemistry).

Isotopes and their applications Of the 37 known (characterized) isotopes of iodine, only one, 127I, is stable. The longest-lived radioisotope, 129I, has a half-life of 15.7 million years. This is long enough to make it a permanent fixture of the environment on human time scales, but far too short for it to exist as a primordial isotope today. Instead, iodine-129 is an extinct radionuclide, and its presence in the early solar system is inferred from the observation of an excess of its daughter xenon-129. This nuclide is also newly-made by cosmic rays and as a byproduct of human nuclear fission, which it is used to monitor as a very long-lived environmental contaminant. The next-longest-lived radioisotope, iodine-125, has a half-life of 59 days. It is used as a convenient gamma-emitting tag for proteins in biological assays, and a few nuclear medicine imaging tests where a longer half-life is required. It is also commonly used in brachytherapy implanted capsules, which kill tumors by local short-range gamma radiation (but where the isotope is never released into the body). Iodine-123 (half-life 13 hours) is the isotope of choice for nuclear medicine imaging of the thyroid gland, which naturally accumulates all iodine isotopes. Iodine-131 (half-life 8 days) is a beta-emitting isotope, which is a common nuclear fission product. It is preferably istered to humans only in very high doses which destroy all tissues that accumulate it (usually the thyroid), which in turn prevents these tissues from developing cancer from a lower dose (paradoxically, a high dose of this isotope appears safer for the thyroid than a low dose). Like other radioiodines, I-131 accumulates in the thyroid gland, but unlike the others, in small amounts it is highly carcinogenic there, it seems, owing to the high local cell mutation due to damage from beta decay. Because of this tendency of 131I to cause high damage to cells that accumulate it and other cells near them (0.6 to 2 mm away, the range of the beta rays), it is the only iodine radioisotope used as direct therapy, to kill tissues such as cancers that take up artificially iodinated molecules (example, the compound iobenguane, also known as MIBG). For the same reason, only the iodine isotope I-131 is used to treat Grave's disease and those types of thyroid cancers (sometimes in metastatic form) where the tissue that requires destruction, still functions to naturally accumulate iodide. Nonradioactive ordinary potassium iodide (iodine-127), in a number of convenient forms (tablets or solution) may be used to saturate the thyroid gland's ability to take up further iodine, and thus protect against accidental contamination from iodine-131 generated by nuclear fission accidents, such as the Chernobyl disaster and more recently the Fukushima I nuclear accidents, as well as from contamination from this isotope in nuclear fallout from nuclear weapons.

Iodine

History Iodine was discovered by Bernard Courtois in 1811.[14][15] He was born to a manufacturer of saltpeter (a vital part of gunpowder). At the time of the Napoleonic Wars, was at war and saltpeter was in great demand. Saltpeter produced from French niter beds required sodium carbonate, which could be isolated from seaweed collected on the coasts of Normandy and Brittany. To isolate the sodium carbonate, seaweed was burned and the ash washed with water. The remaining waste was destroyed by adding sulfuric acid. Courtois once added excessive sulfuric acid and a cloud of purple vapor rose. He noted that the vapor crystallized on cold surfaces, making dark crystals. Courtois suspected that this was a new element but lacked funding to pursue it further. Courtois gave samples to his friends, Charles Bernard Desormes (1777–1862) and Nicolas Clément (1779–1841), to continue research. He also gave some of the substance to chemist Joseph Louis Gay-Lussac (1778–1850), and to physicist André-Marie Ampère (1775–1836). On 29 November 1813, Dersormes and Clément made public Courtois's discovery. They described the substance to a meeting of the Imperial Institute of . On December 6, Gay-Lussac announced that the new substance was either an element or a compound of oxygen.[16][17][18] It was Gay-Lussac who suggested the name "iode", from the Greek word ιώδες (iodes) for violet (because of the color of iodine vapor).[14][16] Ampère had given some of his sample to Humphry Davy (1778–1829). Davy did some experiments on the substance and noted its similarity to chlorine.[19] Davy sent a letter dated December 10 to the Royal Society of London stating that he had identified a new element.[20] Arguments erupted between Davy and Gay-Lussac over who identified iodine first, but both scientists acknowledged Courtois as the first to isolate the element.

Applications Catalysis The major application of iodine is as a co-catalyst for the production of acetic acid by the Monsanto and Cativa processes. In these technologies, which the world's demand for acetic acid, hydroiodic acid converts the methanol feedstock into methyl iodide, which undergoes carbonylation. Hydrolysis of the resulting acetyl iodide regenerates hydroiodic acid and gives acetic acid.[7]

Animal feed The production of ethylenediammonium diiodide (EDDI) consumes a large fraction of available iodine. EDDI is provided to livestock as a nutritional supplement.[7]

Disinfectant and water treatment Elemental iodine is used as a disinfectant in various forms. The iodine exists as the element, or as the water-soluble triiodide anion I3- generated in situ by adding iodide to poorly water-soluble elemental iodine (the reverse chemical reaction makes some free elemental iodine available for antisepsis). In alternative fashion, iodine may come from iodophors, which contain iodine complexed with a solubilizing agent (iodide ion may be thought of loosely as the iodophor in triiodide water solutions). Examples of such preparations include:[21] • Tincture of iodine: iodine in ethanol, or iodine and sodium iodide in a mixture of ethanol and water. • Lugol's iodine: iodine and iodide in water alone, forming mostly triiodide. Unlike tincture of iodine, Lugol's has a minimized amount of the free iodine (I2) component. • Povidone iodine (an iodophor)

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Health, medical, and radiological use In most countries, table salt is iodized. Iodine is required for the essential thyroxin hormones produced by and concentrated in the thyroid gland. Potassium iodide has been used as an expectorant, although this use is increasingly uncommon. In medicine, potassium iodide is used to treat acute thyrotoxicosis, usually as a saturated solution of potassium iodide (SSKI). It is also used to block uptake of iodine-131 in the thyroid gland (see isotopes section above), when this isotope is used as part of radiopharmaceuticals (such as iobenguane) that are not targeted to the thyroid or thyroid type tissues. Iodine-131 (in the chemical form of iodide) is a component of nuclear fallout and a particularly dangerous one owing to the thyroid gland's propensity to concentrate ingested iodine, where it is kept for periods longer than this isotope's radiological half-life of eight days. For this reason, if people are expected to be exposed to a significant amount of environmental radioactive iodine (iodine-131 in fallout), they may be instructed to take non-radioactive potassium iodide tablets. The typical adult dose is one 130 mg tablet per 24 hours, supplying 100 mg (100,000 micrograms) iodine, as iodide ion. (Note: typical daily dose of iodine to maintain normal health is of order 100 micrograms; see "Dietary Intake" below.) By ingesting this large amount of non-radioactive iodine, radioactive iodine uptake by the thyroid gland is minimized. See the main article above for more on this topic.[22]

Radiocontrast agent Iodine, as a physically dense element with high electron density and high atomic number, is quite radio-opaque (i.e., it absorbs X-rays well). This property can be fully exploited by filtering imaging X-rays so that they are more energetic than iodine's "K-edge" at 33.3 keV, or the energy where the iodine begins to absorb X-rays strongly due to the photoelectric effect from electrons in its K shell.[23] Organic compounds of a certain type (typically iodine-substituted benzene derivatives) are thus used in medicine as X-ray radiocontrast agents for intravenous injection. This is often in conjunction with advanced X-ray techniques such as angiography and CT scanning. At present, all water-soluble radiocontrast agents rely on iodine.

Diatrizoic acid, a radiocontrast agent

Other uses Inorganic iodides find specialized uses. Hafnium, zirconium, titanium are purified by the van Arkel Process, which involves the reversible formation of the tetraiodides of these elements. Silver iodide is a major ingredient to traditional photographic film. Thousands of kilograms of silver iodide are consumed annually for cloud seeding.[7] The organoiodine compound erythrosine is an important food coloring agent. Perfluoroalkyl iodides are precursors to important surfactants, such as perfluorooctanesulfonic acid.[7]

Iodine chemistry Iodine adopts a variety of oxidation states, commonly ranging from (formally) I7+ to I-, and including the intermediate states of I5+, I3+ and I+. Practically, only the 1- oxidation state is of significance, being the form found in iodide salts and organoiodine compounds. Iodine is a Lewis acid. With electron donors such as triphenylphosphine and pyridine it forms a charge-transfer complex. With the iodide anion it forms the triiodide ion.[24] Iodine and the iodide ion form a redox couple. I2 is easily reduced and I- is easily oxidized.

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Solubility Being a nonpolar molecule, iodine is highly soluble in nonpolar organic solvents, including ethanol (20.5 g/100 ml at 15 °C, 21.43 g/100 ml at 25 °C), diethyl ether (20.6 g/100 ml at 17 °C, 25.20 g/100 ml at 25 °C), chloroform, acetic acid, glycerol, benzene (14.09 g/100 ml at 25 °C), carbon tetrachloride (2.603 g/100 ml at 35 °C), and carbon disulfide (16.47 g/100 ml at 25 °C).[25] Elemental iodine is poorly soluble in water, with one gram dissolving in 3450 ml at 20 °C and 1280 ml at 50 °C. Aqueous and ethanol solutions are brown reflecting the role of these solvents as Lewis bases. Solutions in chloroform, carbon tetrachloride, and carbon disulfide are violet, the color of iodine vapor.

Redox reactions In everyday life, iodides are slowly oxidized by atmospheric oxygen in the atmosphere to give free iodine. Evidence for this conversion is the yellow tint of certain aged samples of iodide salts and some organoiodine compounds.[7] The oxidation of iodide to iodine in air is also responsible for the slow loss of iodide content in iodized salt if exposed to air.[26] Some salts use iodate to prevent the loss of iodine. Iodine is easily reduced. Most common is the interconversion of I- and I2. Molecular iodine can be prepared by oxidizing iodides with chlorine: 2 I− + Cl2 → I2 + 2 Cl− or with manganese dioxide in acid solution:[27] 2 I− + 4 H+ + MnO2 → I2 + 2 H2O + Mn2+ Iodine is reduced to hydroiodic acid by hydrogen sulfide and hydrazine:[28] 8 I2 + 8 H2S → 16 HI + S8 2 I2 + N2H4 → 4 HI + N2 When dissolved in fuming sulfuric acid (65% oleum), iodine forms an intense blue solution. The blue color is due to I cation, the result of iodine being oxidized by SO3:[29] 2 I2 + 2 SO3 + H2SO4 → 2 I The I

+ SO2 + 2 HSO

cation is also formed in the oxidation of iodine by SbF5 or TaF5. The resulting I

Sb2F or I

Ta2F can be

isolated as deep blue crystals. The solutions of these salts turn red when cooled below −60°C, owing to the formation of the I cation:[29] 2I

I

Under slightly more alkaline conditions, I can then react with I

to form I

disproportionates into I

(green) and I

and an iodine(III) compound. Excess iodine

[29]

(black).

Oxides of iodine The best-known oxides are the anions, IO3– and IO4–, but several other oxides are known, such as the strong oxidant iodine pentoxide. By contrast with chlorine, the formation of the hypohalite ion (IO–) in neutral aqueous solutions of iodine is negligible. I2 + H2O H+ + I− + HIO (K = 2.0×10−13)[27] In basic solutions (such as aqueous sodium hydroxide), iodine converts in a two stage reaction to iodide and iodate:[27] I2 + 2 OH− → I− + IO− + H2O (K = 30) 3 IO− → 2 I− + IO3−

(K = 1020)

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Organic derivatives of hypoiodate (2-Iodoxybenzoic acid, and Dess-Martin periodinane) are used in organic chemistry. Iodic acid (HIO3), periodic acid (HIO4) and their salts are strong oxidizers and are of some use in organic synthesis. Iodine is oxidized to iodate by nitric acid as well as by chlorates:[30] I2 + 10 HNO3 → 2 HIO3 + 10 NO2 + 4 H2O I2 + 2 ClO3− → 2 IO3− + Cl2

Inorganic iodine compounds Iodine forms compounds with all the elements except for the noble gases. From the perspective of commercial applications, an important compound is hydroiodic acid, used as a co-catalyst in the Cativa process for the production of acetic acid. Titanium and aluminium iodides are used in the production of butadiene, a precursor to rubber tires.[7] Alkali metal salts are common colourless solids that are highly soluble in water. Potassium iodide is a convenient source of the iodide anion; it is easier to handle than sodium iodide because it is not hygroscopic. Both salts are mainly used in the production of iodized salt. Sodium iodide is especially useful in the Finkelstein reaction, because it is soluble in acetone, whereas potassium iodide is less so. In this reaction, an alkyl chloride is converted to an alkyl iodide. This relies on the insolubility of sodium chloride in acetone to drive the reaction: R-Cl (acetone) + NaI (acetone) → R-I (acetone) + NaCl (s) Despite having the lowest electronegativity of the common halogens, iodine reacts violently with some metals, such as aluminium: 3 I2 + 2 Al → 2 AlI3 This reaction produces 314 kJ per mole of aluminium, comparable to thermite's 425 kJ. Yet the reaction initiates spontaneously, and if unconfined, causes a cloud of gaseous iodine due to the high temperature. Interhalogen compounds Interhalogen compounds are well known; examples include iodine monochloride and trichloride; iodine pentafluoride and heptafluoride.

Organic compounds Many organoiodine compounds exist; the simplest is iodomethane, approved as a soil fumigant. Iodinated organic compounds are used as synthetic reagents. Organic synthesis Organoiodine compounds can be made in many ways. For example, methyl iodide can be prepared from methanol, red phosphorus, and iodine.[31] The iodinating reagent is phosphorus triiodide that is formed in situ: 3 CH3OH + PI3 → 3 CH3I + H3PO3 The iodoform test uses an alkaline solution of iodine to react with methyl ketones to give the labile triiodomethide leaving group, forming iodoform, which precipitates. Aryl and alkyl iodides both form Grignard reagents. Iodine is sometimes used to activate magnesium when preparing Grignard reagents. Alkyl iodides such as iodomethane are good alkylating agents. Some drawbacks to use of organoiodine compounds in chemical synthesis are: • iodine compounds are more expensive than the corresponding bromides and chlorides, in that order • iodides are much stronger alkylating agents, and so are more toxic (e.g., methyl iodide is very toxic (T+).[32]

Iodine • low-molecular-weight iodides tend to have a much higher equivalent weight, compared to other alkylating agents (e.g., methyl iodide versus dimethyl carbonate), owing to the atomic mass of iodine.

Analytical chemistry and bioanalysis Iodine is useful in analytical chemistry because of its reactions with alkenes, starch and oxidizing and reducing agents. The highly colored species involved in these reactions make it easy to detect the endpoints in many analytical determinations. Iodine is a common general stain used in thin-layer chromatography. Iodine forms an intense blue complex with the glucose polymers starch and glycogen. Several analytical methods rely on this property: • Iodometry. The concentration of an oxidant can be determined by adding it to an excess of iodide, to destroy elemental Testing a seed for starch with a solution of iodine iodine/triiodide as a result of oxidation by the oxidant. A starch indicator is then used as the indicator close to the end-point, in order to increase the visual contrast (dark blue becomes colorless, instead of the yellow of dilute triiodide becoming colorless). • An Iodine test may be used to test a sample substance for the presence of starch. The Iodine clock reaction is an extension of the techniques in iodometry. • Iodine solutions are used in counterfeit banknote detection pens; the premise being that counterfeit banknotes made using commercially available paper contain starch. • Starch-iodide paper are used to test for the presence of oxidants such as peroxides. The oxidants convert iodide to iodine, which shows up as blue. A solution of starch and iodide can perform the same function.[33] • During colposcopy, Lugol's iodine is applied to the vagina and cervix. Normal vaginal tissue stains brown owing to its high glycogen content (a color-reaction similar to that with starch), while abnormal tissue suspicious for cancer does not stain, and thus appears pale compared to the surrounding tissue. Biopsy of suspicious tissue can then be performed. This is called a Schiller's Test. Iodine value or iodine number is used to indicate the number of carbon-carbon double bonds in vegetable oils and fatty acids.

Clandestine synthetic chemical use In the United States, the Drug Enforcement istration (DEA) regards iodine and compounds containing iodine (ionic iodides, iodoform, ethyl iodide, and so on) as reagents useful for the clandestine manufacture of methamphetamine.[34][35]

Biological role Iodine is an essential trace element for life, the heaviest element commonly needed by living organisms. Only tungsten, a component of a few bacterial enzymes, has a higher atomic number and atomic weight.

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Iodine's main role in animal biology is as a constituent of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). These are made from addition condensation products of the amino acid tyrosine, and are stored prior to release in an iodine-containing protein called thyroglobulin. T4 and T3 contain four and three atoms of iodine per molecule, respectively. The thyroid gland actively absorbs iodide from the blood to make and release these hormones into the blood, actions that are regulated by a second hormone TSH from the pituitary. Thyroid hormones are phylogenetically very old molecules that are synthesized by most multicellular organisms, and that even have some effect on unicellular organisms.

163

Thyroxines are iodine-containing hormones that justify the widespread use of iodised salt.

Thyroid hormones play a basic role in biology, acting on gene transcription to regulate the basal metabolic rate. The total deficiency of thyroid hormones can reduce basal metabolic rate up to 50%, while in excessive production of thyroid hormones the basal metabolic rate can be increased by 100%. T4 acts largely as a precursor to T3, which is (with minor exceptions) the biologically active hormone. Iodine has a nutritional relationship with selenium. A family of selenium-dependent enzymes called deiodinases converts T4 to T3 (the active hormone) by removing an iodine atom from the outer tyrosine ring. These enzymes also convert T4 to reverse T3 (rT3) by removing an inner ring iodine atom, and convert T3 to 3,3'-diiodothyronine (T2) also by removing an inner ring atom. Both of the latter are inactivated hormones that are ready for disposal and have, in essence, no biological effects. A family of non-selenium-dependent enzymes then further deiodinates the products of these reactions. Iodine s for 65% of the molecular weight of T4 and 59% of the T3. Fifteen to 20 mg of iodine is concentrated in thyroid tissue and hormones, but 70% of the body's iodine is distributed in other tissues, including mammary glands, eyes, gastric mucosa, arterial walls, the cervix, and salivary glands. In the cells of these tissues, iodide enters directly by sodium-iodide symporter (NIS). Its role in mammary tissue is related to fetal and neonatal development, but its role in the other tissues is unknown.[36]

Dietary intake The daily Dietary Reference Intake recommended by the United States Institute of Medicine is between 110 and 130 µg for infants up to 12 months, 90 µg for children up to eight years, 130 µg for children up to 13 years, 150 µg for adults, 220 µg for pregnant women and 290 µg for lactating mothers.[37] The Tolerable Upper Intake Level (UL) for adults is 1,100 μg/day (1.1 mg/day).[38] The tolerable upper limit was assessed by analyzing the effect of supplementation on thyroid-stimulating hormone.[36] The thyroid gland needs no more than 70 micrograms/day to synthesize the requisite daily amounts of T4 and T3. The higher recommended daily allowance levels of iodine seem necessary for optimal function of a number of body systems, including lactating breast, gastric mucosa, salivary glands, oral mucosa, arterial walls[39][40][41], thymus, epidermis, choroid plexus, etc.[42][43][44] The high iodide-concentration of thymus tissue in particular suggests an anatomical rationale for this role of iodine in the immune system.[45] The trophic, antioxidant and apoptosis-inductor actions and the presumed anti-tumour activity of iodides has been suggested to also be important for prevention of oral and salivary glands diseases.[46] Natural sources of iodine include sea life, such as kelp and certain seafood, as well as plants grown on iodine-rich soil.[47][48] Iodized salt is fortified with iodine.[48] As of 2000, the median intake of iodine from food in the United States was 240 to 300 μg/day for men and 190 to 210 μg/day for women.[38] In Japan, consumption is much higher, owing to the frequent consumption of seaweed or kombu kelp.[36]

Iodine After iodine fortification programs (e.g., iodized salt) have been implemented, some cases of iodine-induced hyperthyroidism have been observed (so called Jod-Basedow phenomenon). The condition seems to occur mainly in people over forty, and the risk appears higher when iodine deficiency is severe and the initial rise in iodine intake is high.[49] It should also be noted that information processing, fine motor skills, and visual problem solving are improved by iodine repletion in moderately iodine-deficient children.[50]

Deficiency In areas where there is little iodine in the diet,[9] typically remote inland areas and semi-arid equatorial climates where no marine foods are eaten, iodine deficiency gives rise to hypothyroidism, symptoms of which are extreme fatigue, goitre, mental slowing, depression, weight gain, and low basal body temperatures.[51] Iodine deficiency is the leading cause of preventable mental retardation, a result that occurs primarily when babies or small children are rendered hypothyroidic by a lack of the element. The addition of iodine to table salt has largely eliminated this problem in the wealthier nations, but, as of March 2006, iodine deficiency remained a serious public health problem in the developing world.[52] Iodine deficiency is also a problem in certain areas of Europe. Other possible health effects being investigated as being related to deficiency include: • Breast cancer. The breast strongly and actively concentrates iodine into breast-milk for the benefit of the developing infant, and may develop a goiter-like hyperplasia, sometimes manifesting as fibrocystic breast disease, when iodine level are low. • Stomach cancer. Some researchers have found an epidemiologic correlation between iodine deficiency, iodine-deficient goitre and gastric cancer.[53][54][55] A decrease of the incidence of death rate from stomach cancer after implementation of the effective iodine-prophylaxis has been reported also.[56]

Precautions and toxicity of elemental iodine Elemental iodine (I2) is mildly toxic if taken orally. The lethal dose for an adult human is 30 mg/kg, which is about 2,1-2,4 grams (even if experiments on rats demostrated that this animals could survive after eating a 14000 mg/kg dose). Excess iodine can be more cytotoxic in the presence of selenium deficiency.[57] Iodine supplementation in selenium-deficient populations is, in theory, problematic, partly for this reason.[36] Its toxicity derives from its oxidizing properties, which make it able to denaturate proteins (and so also enzymes). Elemental iodine is an oxidizing irritant and direct with skin can cause lesions, so iodine crystals should be handled with care. Solutions with high elemental iodine concentration such as tincture of iodine and Lugol's solution are capable of causing tissue damage if their use for cleaning and antiseptics is prolonged.

Iodine sensitivity Some people develop a sensitivity to iodine. Application of tincture of iodine can cause a rash. Some cases of reaction to Povidone-iodine (Betadine) resulted in chemical burns.[58] Eating iodine-containing foods can cause hives . Medical use of iodine (i.e. as a contrast agent, see above) can cause anaphylactic shock in highly iodine-sensitive patients. Some cases of sensitivity to iodine can be formally classified as iodine allergies. Iodine sensitivity is rare but has a considerable effect given the extremely widespread use of iodine-based contrast media.[59]

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Iodine

References [1] Magnetic susceptibility of the elements and inorganic compounds (http:/ / www-d0. fnal. gov/ hardware/ cal/ lvps_info/ engineering/ elementmagn. pdf), in Handbook of Chemistry and Physics 81st edition, CRC press. [2] Iodine (http:/ / www. merriam-webster. com/ dictionary/ iodine). Merriam-Webster Dictionary. Retrieved on 2011-12-23. [3] Iodine (http:/ / oxforddictionaries. com/ definition/ iodine?view=uk) – Oxford Dictionaries Online (World English)]. Retrieved on 2011-12-23. [4] All three pronunciations are used in both British and American English, but /ˈaɪ.ɵdiːn/ EYE-o-deen is the most common British one and /ˈaɪ.ɵdaɪn/ EYE-o-dyne is the most common American one. [5] Online Etymology Dictionary, s.v. iodine (http:/ / www. etymonline. com/ index. php?term=iodine). Retrieved 2012-02-07. [6] McNeil, Donald G. Jr (2006-12-16). "In Raising the World’s I.Q., the Secret’s in the Salt" (http:/ / www. nytimes. com/ 2006/ 12/ 16/ health/ 16iodine. html?fta=y). New York Times. . Retrieved 2008-12-04. [7] Lyday, Phyllis A. "Iodine and Iodine Compounds" in Ullmann's Encyclopedia of Industrial Chemistry, 2005, Wiley-VCH, Weinheim, ISBN 978-3-527-30673-2 doi:10.1002/14356007.a14_381 Vol. A14 pp. 382–390. [8] Bell, N. et al. (2002). "Methyl iodide: Atmospheric budget and use as a tracer of marine convection in global models". Journal of GeophysicalResearch 107: 4340. Bibcode 2002JGRD..107.4340B. doi:10.1029/2001JD001151. [9] Dissanayake, C. B.; Chandrajith, Rohana; Tobschall, H. J. (1999). "The iodine cycle in the tropical environment — implications on iodine deficiency disorders". International Journal of Environmental Studies 56 (3): 357. doi:10.1080/00207239908711210. [10] Wells, A.F. (1984) Structural Inorganic Chemistry, Oxford: Clarendon Press. ISBN 0-19-855370-6. [11] Kogel, Jessica Elzea et al. (2006). Industrial Minerals & Rocks: Commodities, Markets, and Uses (http:/ / www. google. com/ books?id=zNicdkuulE4C). SME. pp. 541–552. ISBN 978-0-87335-233-8. . [12] Maekawa, Tatsuo; Igari, Shun-Ichiro and Kaneko, Nobuyuki (2006). "Chemical and isotopic compositions of brines from dissolved-in-water type natural gas fields in Chiba, Japan". Geochemical Journal 40 (5): 475. doi:10.2343/geochemj.40.475. [13] Stanford, Edward C. C. (1862). "On the Economic Applications of Seaweed" (http:/ / books. google. com/ ?id=wW8KAAAAIAAJ& pg=PA185). Journal of the Society of Arts: 185–189. . [14] Courtois, Bernard (1813). "Découverte d'une substance nouvelle dans le Vareck" (http:/ / books. google. com/ books?id=YGwri-w7sMAC& pg=RA2-PA304). Annales de chimie 88: 304. . In French, seaweed that had been washed onto the shore was called "varec", "varech", or "vareck", whence the English word "wrack". Later, "varec" also referred to the ashes of such seaweed: The ashes were used as a source of iodine and salts of sodium and potassium. [15] Swain, Patricia A. (2005). "Bernard Courtois (1777–1838) famed for discovering iodine (1811), and his life in Paris from 1798" (http:/ / www. scs. uiuc. edu/ ~mainzv/ HIST/ awards/ OPA Papers/ 2007-Swain. pdf). Bulletin for the History of Chemistry 30 (2): 103. . [16] Gay-Lussac, J. (1813). "Sur un nouvel acide formé avec la substance décourverte par M. Courtois" (http:/ / books. google. com/ books?id=YGwri-w7sMAC& pg=RA2-PA511). Annales de chimie 88: 311. . [17] Gay-Lussac, J. (1813). "Sur la combination de l'iode avec d'oxigène" (http:/ / books. google. com/ books?id=YGwri-w7sMAC& pg=RA2-PA519). Annales de chimie 88: 319. . [18] Gay-Lussac, J. (1814). "Mémoire sur l'iode" (http:/ / books. google. com/ books?id=Efms0Fri1CQC& pg=PA5). Annales de chimie 91: 5. . [19] Davy, H. (1813). "Sur la nouvelle substance découverte par M. Courtois, dans le sel de Vareck" (http:/ / books. google. com/ books?id=YGwri-w7sMAC& pg=RA2-PA522& lpg=RA2-PA522). Annales de chemie 88: 322. . [20] Davy, Humphry (January 1, 1814). "Some Experiments and Observations on a New Substance Which Becomes a Violet Coloured Gas by Heat". Phil. Trans. R. Soc. Lond. 104: 74. doi:10.1098/rstl.1814.0007. [21] Block, Seymour Stanton (2001). Disinfection, sterilization, and preservation. Hagerstwon, MD: Lippincott Williams & Wilkins. p. 159. ISBN 0-683-30740-1. [22] U.S. Centers for Disease Control "CDC Radiation Emergencies" (http:/ / www. bt. cdc. gov/ radiation/ ki. asp), U.S. Centers for Disease Control, October 11, 2006, accessed November 14, 2010. [23] (http:/ / ric. uthscsa. edu/ personalpages/ lancaster/ DI-II_Chapters/ DI_chap4. pdf) Determinants of X-ray opacity in elements and and principles of use of radiocontrast agents in medicine. [24] Küpper F. C., Feiters M. C., Olofsson B., Kaiho T., Yanagida S., Zimmermann M. B., Carpenter L. J., Luther G. W., Lu Z. et al. (2011). "Commemorating Two Centuries of Iodine Research: An Interdisciplinary Overview of Current Research". Angewandte Chemie International Edition 50: 11598–11620. doi:10.1002/anie.201100028. [25] Windholz, Martha; Budavari, Susan; Stroumtsos, Lorraine Y. and Fertig, Margaret Noether, ed. (1976). Merck Index of Chemicals and Drugs, 9th ed. J A Majors Company. ISBN 0-911910-26-3. [26] Waszkowiak, Katarzyna; Szymandera-Buszka, Krystyna (2008). "Effect of storage conditions on potassium iodide stability in iodised table salt and collagen preparations". International Journal of Food Science & Technology 43 (5): 895–899. doi:10.1111/j.1365-2621.2007.01538.x. [27] Cotton, F. A. snd Wilkinson, G. (1988). Advanced Inorganic Chemistry, 5th ed.. John Wiley & Sons. ISBN 0-471-84997-9. [28] Glinka, N.L. (1981). General Chemistry (volume 2). Mir Publishing. [29] Wiberg, Egon; Wiberg, Nils and Holleman, Arnold Frederick (2001). Inorganic chemistry. Academic Press. pp. 419–420. ISBN 0-12-352651-5. [30] Linus Pauling (1988). General Chemistry. Dover Publications. ISBN 0-486-65622-5.

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Iodine [31] King, C. S.; Hartman, W. W. (1943), "Methyl Iodide" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=CV2P0399), Org. Synth., ; Coll. Vol. 2: 399 [32] "Safety data for iodomethane" (http:/ / msds. chem. ox. ac. uk/ IO/ iodomethane. html). Oxford University. . [33] Toreki, R.. "Peroxide" (http:/ / www. ilpi. com/ msdS/ ref/ peroxide. html). The MSDS HyperGlossary. . [34] 21 "USC Sec. 872 2007-01-03" (http:/ / www. deadiversion. usdoj. gov/ 21cfr/ 21usc/ 872. htm). 21. [35] Federal agents say 88-year-old Saratoga man's invention is being used by meth labs (http:/ / www. mercurynews. com/ saratoga/ ci_19385037). Mercurynews.com. Retrieved on 2011-12-23. [36] Patrick L (2008). "Iodine: deficiency and therapeutic considerations" (http:/ / www. thorne. com/ altmedrev/ . fulltext/ 13/ 2/ 116. pdf). Altern Med Rev 13 (2): 116. PMID 18590348. . [37] "Dietary Reference Intakes (DRIs): Recommended Intakes for Individuals, Vitamins" (http:/ / iom. edu/ en/ Global/ News Announcements/ ~/ media/ Files/ Activity Files/ Nutrition/ DRIs/ DRISummaryListing2. ashx). Institute of Medicine. 2004. . Retrieved 2010-06-09. [38] United States National Research Council (2000). Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (http:/ / books. nap. edu/ openbook. php?record_id=10026& page=258). National Academies Press. pp. 258–259. . [39] Pellerin P (1961). "La tecnique d’autoradiographie anatomique a la temperature de l’azote liquide.". Path Biol 232 (9): 233–252. [40] Ullberg S, Ewaldsson B (1964). "Distribution of radio-iodine studied by whole-body autoradiography.". Acta Radiologica Therapy Physics Biology 41: 24–32. [41] Venturi, Sebastiano (2011). "Evolutionary Significance of Iodine". Current Chemical Biology- 5 (3): 155–162. doi:10.2174/187231311796765012. ISSN 1872-3136. [42] Brown-Grant, K. (1961). "Extrathyroidal iodide concentrating mechanisms" (http:/ / physrev. physiology. org/ cgi/ reprint/ 41/ 1/ 189. pdf). Physiol Rev. 41 (1): 189. . [43] Spitzweg, C., Joba, W., Eisenmenger, W. and Heufelder, A.E. (1998). "Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acid from salivary gland, mammary gland, and gastric mucosa". J Clin Endocrinol Metab. 83 (5): 1746. doi:10.1210/jc.83.5.1746. PMID 9589686. [44] Banerjee, R.K., Bose, A.K., Chakraborty, T.K., de, S.K. and Datta, A.G. (1985). "Peroxidase catalysed iodotyrosine formation in dispersed cells of mouse extrathyroidal tissues". J Endocrinol. 2 (2): 159. PMID 2991413. [45] Venturi S, Venturi M (September 2009). "Iodine, thymus, and immunity". Nutrition 25 (9): 977–9. doi:10.1016/j.nut.2009.06.002. PMID 19647627. [46] Venturi S.; Venturi M. (2009). "Iodine in evolution of salivary glands and in oral health". Nutrition and Health 20 (2): 119–134. doi:10.1177/026010600902000204. PMID 19835108. [47] "Sources of iodine" (http:/ / www. iccidd. org/ pages/ iodine-deficiency/ sources-of-iodine. php). International Council for the Control of Iodine Deficiency Disorders. . [48] "MedlinePlus Medical Encyclopedia: Iodine in diet" (http:/ / www. nlm. nih. gov/ medlineplus/ ency/ article/ 002421. htm). . [49] Wu T, Liu GJ, Li P, Clar C (2002). Wu, Taixiang. ed. "Iodised salt for preventing iodine deficiency disorders". Cochrane Database Syst Rev (3): CD003204. doi:10.1002/14651858.CD003204. PMID 12137681. [50] Michael B Zimmermann, Kevin Connolly, Maksim Bozo, John Bridson, Fabian Rohner, Lindita Grimci (2006). "Iodine supplementation improves cognition in iodine-deficient schoolchildren in Albania: a randomized, controlled, double-blind study" (http:/ / www. ajcn. org/ content/ 83/ 1/ 108. long). American Journal of Clinical Nutrition 83 (1): 108–114. . [51] Felig, Philip; Frohman, Lawrence A. (2001). "Endemic Goiter" (http:/ / books. google. com/ ?id=AZUUGrp6yUgC& pg=RA1-PA351). Endocrinology & metabolism. McGraw-Hill Professional. ISBN 978-0-07-022001-0. . [52] "Micronutrients – Iodine, Iron and Vitamin A" (http:/ / www. unicef. org/ nutrition/ index_iodine. html). UNICEF. . [53] Josefssson, M.; Ekblad, E. (2009). "Sodium Iodide Symporter (NIS) in Gastric Mucosa: Gastric Iodide Secretion". In Preedy, Victor R.; Burrow, Gerard N.; Watson, Ronald. Comprehensive Handbook of Iodine: Nutritional, Biochemical, Pathological and Therapeutic Aspects. [54] Abnet CC, Fan JH, Kamangar F, Sun XD, Taylor PR, Ren JS, Mark SD, Zhao P, Fraumeni JF Jr, Qiao YL, Dawsey SM (2006). "Self-reported goiter is associated with a significantly increased risk of gastric noncardia adenocarcinoma in a large population-based Chinese cohort". International Journal of Cancer 119 (6): 1508–1510. doi:10.1002/ijc.21993. PMID 16642482. [55] Behrouzian, R.; Aghdami, N. (2004). "Urinary iodine/creatinine ratio in patients with stomach cancer in Urmia, Islamic Republic of Iran". East Mediterr Health J. 10 (6): 921–924. PMID 16335780. [56] Golkowski F, Szybinski Z, Rachtan J, Sokolowski A, Buziak-Bereza M, Trofimiuk M, Hubalewska-Dydejczyk A, Przybylik-Mazurek E, Huszno B. (2007). "Iodine prophylaxis—the protective factor against stomach cancer in iodine deficient areas". Eur J Nutr. 46 (5): 251. doi:10.1007/s00394-007-0657-8. PMID 17497074. [57] Smyth, PP (2003). "Role of iodine in antioxidant defence in thyroid and breast disease". BioFactors (Oxford, England) 19 (3-4): 121–30. PMID 14757962. [58] Lowe, D. O. et al. (2006). "Povidone-iodine-induced burn: case report and review of the literature". Pharmacotherapy 26 (11): 1641–5. doi:10.1592/phco.26.11.1641. PMID 17064209. [59] Katelaris, Constance (2009). "'Iodine Allergy' label is misleading" (http:/ / www. australianprescriber. com/ magazine/ 32/ 5/ 125/ 8/ ). Australian Prescriber 32 (5): 125–128. .

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External links • "Micronutrient Research for Optimum Health", Linus Pauling Institute, OSU Oregon State University (http://lpi. oregonstate.edu/infocenter/minerals/iodine/) • ATSDR – CSEM: Radiation Exposure from Iodine 131 (http://www.atsdr.cdc.gov/csem/iodine/) U.S. Department of Health and Human Services (public domain) • ChemicalElements.com – Iodine (http://chemicalelements.com/elements/i.html) • who.int, WHO Global Database on Iodine Deficiency (http://whqlibdoc.who.int/publications/2004/ 9241592001.pdf) • Oxidizing Agents > Iodine (http://www.organic-chemistry.org/chemicals/oxidations/iodine.shtm)

Jenner's stain Jenner's Stain (methylene blue eosinate) is used in microscopy for staining blood smears.

Leishman stain Leishman's stain (CAS:12627-53-1, EC: 235-732-1, MFCD:00131498[1]), also Leishman stain, is used in microscopy for staining blood smears. It provides excellent stain quality. It is generally used to differentiate and identify leucocytes, malaria parasites, and trypanosomas. It is based on a methanolic mixture of "polychromed" methylene blue (i.e. demethylated into various azures) and eosin. The methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step. If a working solution is made by dilution with an aqueous buffer the resulting mixture is very unstable and cannot be used for long. Leishman stain is named after its inventor, the Scottish pathologist William Boog Leishman. It is similar to and partially replaceable with Giemsa stain, Jenner's stain, and Wright's stain (See more details in Advantages disadvantages and comparisons section below). Like them, it is a version of Romanowsky stain.

Preparation[2] from Commercial source Many companies are certified sell good quality commercial Leishman Stain in the form of a dry powder. The following cautions should be taken while preparing. 1. All glassware (the measuring cylinder and amber storage bottle) should be dried completely free of water before use. 2. Both the Methanol bottle and the newly prepared Stain solution should be in containers with appropriate labels and precautions as Flammable and Toxic chemicals (Methanol if ingested can cause blindness and also is dangerous if directly s the eye, thus idally Eyeshields, Gloves, type N95 (US) or type P1 (EN143) respirator filter, may be recommended when a lot of methanol is being used, as during making reagents in bulk. U*nder the above precautions the powder is to be mixed well into good quality anhydrous methanol in a proportion of 0.6g powder into 400 ml methanol. Using glass beads or a magnetic stirrer or carefully warming to 37 degree C may help in dissolving. An aliquot of the stain (e.g. usually 50-100 ml) should be filtered into a dispensing unit for daily use, and the following storage conditions should be followed.

167

Leishman stain

Storage[3] Bright light and heat oxidize the stain, especially when in aqueous solution and will cause precipitation of insoluble precipitates e.g. of methylene violet (Bernthsen). Evaporation of methanol, absorption of moisture and precipitation of Azure-Eosinate salts are also additional problems during storage that require filtering the stock while aliquoting for daily use. For daily use, store the stain in an airtight (prevent moisture entering the stain) amber (semi-opaque) container. Closable dropper bottle e.g. TK dropper bottle can be used that should be kept tightened when not in use. Keep in a cool place (not refrigerated) and never in direct sunlight. The stock stain should be kept in a tightly stoppered light opaque (e.g. amber) container in a cool dark place. Renew every 3 months or earlier if indicated. To obtain optimum colour reaction, some suggest that 3–5 days should be allowed before using freshly made stain.

Staining Methods There are several ways of doing this stain. A common method[4] is: 1 Take an air dried blood smear on and glass slide and cover the smear with the undiluted stain. Take care not to overflow with excess stain. Preferably add just the enough number of drops to cover the smear. To standardize, count the number of drops (usually 7-10) required to cover the film (so that double the number of water can be added) and adjust the incubation time according to the result (usually 1–2 minutes, 3 minutes per WHO). The undiluted stain both acts as a fixative and also partially stains the smear. Still, since the moisture content can vary it is better to fix the slide in methanol before staining. 2 Add twice the volume of pH 6.8 buffered water (i.e. if e.g. 7 drops of stain was used, then use 14 drops water) to dilute the stain, taking caution that the stain should not overflow (which will make the dilution inaccurate). However, the standard operating procedure published by WHO suggests adding equal (instead of twice) volume of water.[5] Mix the water with the stain underneath by gently blowing with a straw or using a plastic bulb pipette. Allow to stain for 10–12 minutes (time may require adjusting). In this method, better ionization during the dilution by the aqueous buffer in this step is necessary to complete the staining. The WHO protocol also clarifies that during this incubation "the appearance of a polychromatic 'scum' on the surface of the slide is merely a result of oxidation of the dye components and can be ignored." 3. Wash off the stain with clean (or filtered) tap water . If the stain is tiped off instead of washing, this will leave a fine deposit covering the film. Wipe the back of the slide clean and stand it in a draining rack to dry. The stained smear should grossly appear neither too pink nor too blue ( final results microscopically). If the tap water is highly acidic, resulting in smear turning grossly pink too fast or highly alkaline, resulting in the smear remaining too blue, try using boiled cooled water or filtered rain water or pH 6.8 buffered water which can be used as an additional flooding step after washing in running water. 4. The slide should be air dried and can be viewed under microscope either directly or optionally (per WHO) a nonaqueous mounting medium such as DPX (Dibutyl phthalate in Xylene) may be used.

168

Leishman stain

Advantages, disadvantages and comparisons Advantages Ease of technique According to Sir John Vivian Dacie's famous text book of Practical Haematology: "Amongst the Romanowsky stains now in use, Jenner's is the simplest and Giemsa's the most complex. Leishman's stain, which occupies an intermediate position, is still widely used in the routine staining." The WHO protocol [6] mentions: "There are a number of different combinations of these dyes which vary in their staining characteristics. May-Grunwald-Giemsa is a good method for routine work. Wright's stain is a simpler method, whilst Leishman's is also a simple method which is especially suitable when a stained blood film is required urgently or the routine stain is not available (e.g. at night). Field's stain is a rapid stain used primarily on thin films for malarial parasites. Whichever method is used, it is important to select dyes that are not contaminated with other dyes or metallic salts." Sir William Boog Leishman of London and Karl Reuter of independently discovered in 1901 what is considered "Perhaps the most practical modifi cations of Malachowski’s stain"[7] They adopted the best aspects of the stains developed by Malachowski and Jenner, i.e., they used both polychromed methylene blue (accidentally done by Romanowsky who got the name, systematically done by Ernst Malachowski even before Romanowsky, and rediscovered by Bernhard Nocht, but unknown to Jenner, May, Grunwald and many others who used simple methylene blue) and filtering the Azure Eosinate precipipate from the aqueous mixture and redissolving in an alcoholic solvent (Jenner's wisdom). Differences between Leishman's and Reuter's methods were: Leishman used methanol (like Jenner) and substituted eosin B for eosin Y, whereas Reuter used ethyl alcohol and rightly stressed the importance of using an absolutely pure solvent. Both methods produced a stable stain and the desired purple color.

Fairly good contrast Leishman stain generally shows the brilliant violet color of the nucleus and the neutrophil granules for which differential count becomes convenient and makes the quality of staining better than the stains that are simple methylene blue and eosin based which does not produce enough contrast between the cytoplasma dn the nucleus. Since Leishman like other Romanowsky stains, stain cytoplasmic details and granules better hematologists generally prefer them (However see below about cytologists). Compared to the costly and toxic pure synthetic AZure B and Eosin Y based reagents used by the ICSH reference methods which are also not free from the disadvantage of getting oxidized and eventually giving greyish instead of the optimal blue colors for cytoplasm, Leishman is a cheap easily available and easy to do technique which gives a fairly acceptable contrast.

Good sensitivity for Malaria parasite It has been documented that Leishman staining is more sensitive than Field's stain and as good as fluorescent stains for detection of malaria parasite.[8]

Difficulty controling molar ratio Composition of polychromed methylene blue mixed with Eosin is never as good as the directly weighed and mixed proportions in Giemsa type stains. Albert Plehn in 1890[9] had figured out that the molar ratio of basic to acidic dyes had to be increased from 2:1 to 3:1, however from Jenners time (1899) the use of the Azure Eosinate crystals brought the ratio back to the unsatisfactory 2:1 and the depth of color became less. It was only the work by Gustav Giemsa and the likes who again manually controlled the proportion of these two components that brought the depth of

169

Leishman stain staining back. However Giemsa and others why artificially controlled the proportion sometimes went to the other extreme (a large molar excess of Azure up to a ratio of 16.1) which was probably unnecessary. per ICSH the optimal ratio is 6.5 to 7.3.[10]

Instability The Leishman stain if reconstituted with buffer becomes very unstable (in contrast with Giemsa which is relatively more stable due to Glycerol, or the ICSH reference stains which use methanol+DMSO in 6:4 v/v ratio) and starts precipitating and needs repeated filtering. If not carefully supervised, water absorption, methanol evaporation from an opened container and the repeated filtering changes the composition and required frequent changing and makes this method wasteful. If not filtered, the precipitate deposits on the smear may be confused with platelets.

Cytoplasmic contrast good but nuclear contrast not as good as H&E Like all other Malachowski-Romanowsky-Giemsa methods, it fades with time and cannot be stably archived for long. Also like its above counterparts it stains the nuclei dark purple and the nuclear feature details are not as clear as Hematoxylene and Eosin, which are thus preferred by some cytopathologists. But that way it is a complementary method (since it stains cytoplasmic details granules etc. better than H&E stain)

References [1] http:/ / www. sigmaaldrich. com/ catalog/ product/ sigma/ l6254?lang=en& region=IN [2] District Laboratory Practice in Tropical Countries, Part 2 By Monica Cheesbrough, Appendix I, Preparation of Reagents and Culture Media, p396 [3] District Laboratory Practice in Tropical Countries, Part 2 By Monica Cheesbrough, Section 8 Hematological Tests, subsection 8.7 Blood Films, p322 [4] District Laboratory Practice in Tropical Countries, Part 2 By Monica Cheesbrough, Section 8 Hematological Tests, subsection 8.7 Blood Films [5] Blood Safety and Clinical Technology Guidelines on Standard Operating Procedures for HAEMATOLOGY Chapter 11 - Preparation and Staining of Blood Films, (Last update: 27 April 2006) (http:/ / www. searo. who. int/ en/ Section10/ Section17/ Section53/ Section480_1732. htm) [6] Blood Safety and Clinical Technology Guidelines on Standard Operating Procedures for HAEMATOLOGY Chapter 11 - Preparation and Staining of Blood Films, (Last update: 27 April 2006) (http:/ / www. searo. who. int/ en/ Section10/ Section17/ Section53/ Section480_1732. htm) [7] Biotech Histochem. 2011 Feb;86(1):7-35. The color purple: from royalty to laboratory, with apologies to Malachowski. Krafts KP, Hempelmann E, Oleksyn BJ. [8] Indian Journal of Medical Microbiology, (2006) 24 (1):49-51 (http:/ / www. bioline. org. br/ pdf?mb06009) [9] Plehn F (1890b) Zur Aetiologie der Malaria. Berl. Klin. Wochenschr. 27: 292–294. [10] ICSH reference method for staining of blood and bone marrow films by azure B and eosin Y (Romanowsky stain) INTERNATIONAL COMMITTEE FOR STANDARDIZATION IN HAEMATOLOGY Article first published online: 12 MAR 2008 DOI: 10.1111/j.1365-2141.1984.tb02949.x

170

Light Green SF yellowish

171

Light Green SF yellowish Light Green SF yellowish


5141-20-8

PubChem

21223

ChemSpider

19952

KEGG

C19439

Jmol-3D images

Image 1

[1]

[2] [3]

[4]

[5]


C37H37N2O9S3+

Molar mass

749.893 g/mol ()

[6]


Infobox references

Light Green SF yellowish, or Light Green, Acid Green, Lissamine green SF, Acid Green 5, Food Green 2, FD&C Green no. 2, Green No. 205, Acid Brilliant Green 5, Pencil Green SF, or C.I. 42095, is a green triarylmethane dye. It is used in histology for staining collagen; for that purpose it is a standard dye in North America. In Masson's trichrome it is used as a counterstain to acid fuchsin. It is a critical component of Papanicolaou stains together with eosin Y and bismarck brown Y. It usually comes as a disodium salt. Its maximum absorption is at 630 (422) nm. The dye is not very durable — it has a tendency to fade. When fading is to be avoided, it is replaced with Fast Green FCF, which also has more brilliant color. Fast Green FCF can also substitute Light Green SF yellowish in many other procedures. Lissamine green dye can be used to check the health of the anterior surfaces of the eye. It is available on a steret. The steret is wet with saline and then the dye is dropped into the lower fornix. The dye shows up conjunctival staining similar to rose bengal dye but it does not sting like rose bengal does.

Light Green SF yellowish

172

References [1] [2] [3] [4] [5]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=5141-20-8 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=21223 http:/ / www. chemspider. com/ 19952 http:/ / www. kegg. jp/ entry/ C19439 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BNa%2B%5D. %5BNa%2B%5D. %5BO-%5DS%28%3DO%29%28%3DO%29c1cccc%28c1%29CN%28c2ccc%28cc2%29%5CC%28%3DC%5C4%2FC%3DC%5CC%28%3D%5BN%2B%5D% [6] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=400142566& page2=%3ALight+ Green+ SF+ yellowish

Lipophilicity Lipophilicity, (Gr. fat-liking), refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene.[1] These non-polar solvents are themselves lipophilic — the axiom that like dissolves like generally holds true. Thus lipophilic substances tend to dissolve in other lipophilic substances, while hydrophilic (water-loving) substances tend to dissolve in water and other hydrophilic substances. Lipophilicity, hydrophobicity, and non-polarity can describe the same tendency towards participation in the London dispersion force as the are often used interchangeably. However, the "lipophilic" and "hydrophobic" are not synonymous, as can be seen with silicones and fluorocarbons, which are hydrophobic but not lipophilic.

Chemical bonding Lipophilic substances interact within themselves and with other substances through the London dispersion force. They have little to no capacity to form hydrogen bonds. When a molecule of a lipophilic substance is enveloped by water, surrounding water molecules enter into an 'ice-like' structure over the greater part of its molecular surface, the thermodynamically unfavourable event that drives oily substances out of water. Being 'driven out of water' is the quality of a substance referred to as hydrophobic (water-avoiding or water-fearing). Thus lipophilic substances tend to be water insoluble. They invariably have large o/w (oil/water) partition coefficients.

Surfactants Hydrocarbon-based surfactants are compounds that are amphiphilic (or amphipathic), having a hydrophilic, water interactive 'end', referred to as their 'head group', and a lipophilic 'end', usually a long chain hydrocarbon fragment, referred to as their 'tail'. They congregate at low energy surfaces, including the air-water interface (lowering surface tension) and the surfaces of the water-immiscible droplets found in o/w emulsions (lowering interfacial tension). At these surfaces they naturally orient themselves with their head groups in water and their tails either sticking up and largely out of water (as at the air-water interface) or dissolved in the water-immiscible phase that the water is in with (e.g. as the emulsified oil droplet). In both these configurations the head groups strongly interact with water while the tails avoid all with water. Surfactant molecules also aggregate in water as micelles with their head groups sticking out and their tails bunched together. Micelles draw oily substances into their hydrophobic cores, explaining the basic action of soaps and detergents used for personal cleanliness and for laundering clothes. Micelles are also biologically important for the transport of fatty substances in the small intestine surface in the first step that leads to the absorption of the components of fats (largely fatty acids and 2-monoglycerides). Cell membranes are bilayer structures principally formed from phospholipids, molecules which have a highly water interactive, ionic phosphate head groups attached to two long alkyl tails. By contrast, fluorosurfactants are not amphiphilic or detergents because fluorocarbons are not lipophilic.

Lipophilicity

References [1] Compendium of Chemical Terminology, lipophilic (http:/ / goldbook. iupac. org/ L03572. html), accessed 15 Jan 2007.

Lugol's iodine Lugol's iodine, also known as Lugol's solution, first made in 1829, is a solution of elemental iodine and potassium iodide in water, named after the French physician J.G.A. Lugol. Lugol's iodine solution is often used as an antiseptic and disinfectant, for emergency disinfection of drinking water, and as a reagent for starch detection in routine laboratory and medical tests. These uses are possible since the solution is a source of effectively free elemental iodine, which is readily generated from the equilibration between elemental iodine molecules and triiodide ion in the solution. It has been used more rarely to replenish iodine deficiency.[1] However, pure potassium iodide, containing the relatively benign iodide ion without the more toxic elemental iodine, is strongly preferred for this purpose. Likewise, in the Chernobyl disaster some Lugol's solution was used as an emergency source of iodide to block radioactive iodine uptake, simply because it was widely available as a drinking water decontaminant, and pure potassium iodide without iodine (the preferred agent) was not available.

Formula and manufacture Lugol's solution is available in different potencies of 1%, 2%, or 5% Iodine. The 5% solution consists of 5% (wt/v) iodine (I2) and 10% (wt/v) potassium iodide (KI) mixed in distilled water and has a total iodine content of 130 mg/mL.[2] Potassium iodide renders the elementary iodine soluble in water through the formation of the triiodide (I) ion. It is not to be confused with tincture of iodine solutions, which consist of elemental iodine, and iodide salts dissolved in water and alcohol. Lugol's solution contains no alcohol. Other names for Lugol's solution are I2KI (iodine-potassium iodide); Markodine, Strong solution (Systemic); and Aqueous Iodine Solution B.[3] Lugol's is obtained over the counter from drug stores or health food stores. This indicator, also called a stain, is used in many different fields.

Applications • As a mordant when performing a Gram Stain. It is applied for 1 minute after staining with crystal violet, but before ethanol to ensure that gram positive organisms' peptidoglycan remains stained, easily identifying it as a gram positive in microscopy. • This solution is used as an indicator test for the presence of starches in organic compounds, with which it reacts by turning a dark-blue/black. Elemental iodine solutions like Lugol's will stain starches due to iodine's interaction with the coil structure of the polysaccharide. Starches include the plant starches amylose and amylopectin and glycogen in animal cells. Lugol's solution will not detect simple sugars such as glucose or fructose. In the pathologic condition amyloidosis, amyloid deposits (i.e., deposits that stain like starch, but are not) can be so abundant that affected organs will also stain grossly positive for the Lugol reaction for starch. • It can be used as a cell stain, making the cell nuclei more visible and for preserving phytoplankton samples. • During colposcopy, Lugol's iodine is applied to the vagina and cervix. Normal vaginal tissue stains brown due to its high glycogen content, while tissue suspicious for cancer does not stain, and thus appears pale compared to the surrounding tissue. Biopsy of suspicious tissue can then be performed. This is called a Schiller's Test. • Lugol's iodine may also be used to better visualize the mucogingival junction in the mouth. Similar to the method of staining mentioned above regarding a colposcopy, alveolar mucosa has a high glycogen content that gives a

173

Lugol's iodine positive iodine reaction vs. the keratinized gingiva.[4] • Lugol's solution can also be used in various experiments to observe how a cell membrane uses osmosis and diffusion. • Lugol's iodine may also be used as an oxidizing germicide, however it is somewhat undesirable in that it may lead to scarring and discolors the skin temporarily. One way to avoid this problem is by using a solution of 70% ethanol to wash off the iodine later. • Lugol's solution is also used in the marine aquarium industry. Lugol's solution provides a strong source of free iodine and iodide to reef inhabitants and macroalgae. Although the solution is thought to be effective when used with stony corals, systems containing xenia and soft corals are assumed to be particularly benefited by the use of Lugol's solution. Used as a dip for stony and soft or leather corals, Lugol's may help rid the animals of unwanted parasites and harmful bacteria. The solution is thought to foster improved coloration and possibly prevent bleaching of corals due to changes in light intensity, and to enhance coral polyp expansion. The blue colors of Acropora spp. are thought to be intensified by the use of potassium iodide. Specially packaged supplements of the product intended for aquarium use can be purchased at specialty stores and online. • Preoperative istration of Lugol's solution decreases intraoperative blood loss during thyroidectomy in patients with Grave's disease.[5] However, it appears ineffective in patients who are already euthyroid on anti-thyroid drugs and levothyroxine.[6]

Historical applications Lugol's was often used in the treatment of gout. It was also used at one time as a first line treatment for hyperthyroidism, as the istration of pharmacologic amounts of iodine leads to temporary inhibition of iodine organification in the thyroid gland, a phenomenon called the Wolff-Chaikoff effect. However it is not used to treat certain autoimmune causes of thyroid disease as iodine-induced blockade of iodine organification may result in hypothyroidism. They are not considered as a first line therapy because of possible induction of resistant hyperthyroidism but may be considered as an adjuvant therapy when used together with other hyperthyrodism medications. Because of its wide availability as a drinking-water decontaminant, and high content of potassium iodide, emergency use of it was at first recommended to the Polish government in 1986, after the Chernobyl disaster to replace and block any intake of radioactive 131I, even though it was known to be a non-optimal agent, due to its somewhat toxic free-iodine content.[7] Other sources state that pure potassium iodide solution in water (SSKI) was eventually used for most of the thyroid protection after this accident.[8] There is "strong scientific evidence" for potassium iodide thyroid protection to help prevent thyroid cancer. Potassium iodide does not provide immediate protection but can be a component of a general strategy in a radiation emergency.[9] Historically, Lugol's iodine solution has been widely available and used for a number of health problems with some precautions.[10] Lugol's is sometimes prescribed in a variety of alternative medical treatments.[11][12] Until 2007, in the United States of America, Lugol's solution was unregulated and available over the counter as a general reagent, an antiseptic, a preservative,[13] or as a medicament for human or veterinary application. However, effective August 1, 2007, the DEA now regulates Lugol's solution (and, in fact, all iodine solutions containing greater than 2.2% iodine) as a List I precursor because it may potentially be used in the illicit production of methamphetamine.[14] However, transactions of up to one fluid ounce (30 ml) of Lugol's solution are exempt from this regulation. By contrast, Lugol's iodine solution is available over the counter in Canada and Mexico.

174

Lugol's iodine

Toxicity Because it contains free iodine, Lugol's solution at 2% or 5% concentration without dilution is irritating and destructive to mucosa, such as the lining of the esophagus and stomach. Doses of 10 mL of 5% solution have been reported to cause gastric lesions when used in endoscopy. [15] The lethal dose of free iodine for an adult human of 2 to 3 grams (2000-3000 mg) free iodine represents 40 to 60 mL (less than 2 fluid ounces) of 5% Lugol's solution.

References [1] http:/ / lpi. oregonstate. edu/ infocenter/ minerals/ iodine/ Higdon, J., "Micronutrient Information Center: Iodine," Linus Pauling Institute/Oregon State University; April, 2003 (revised by Drake, V.J., July, 2007). [2] http:/ / www. quailwoodherbal. com/ Lugol. html [3] http:/ / www. quailwoodherbal. com/ Lugol. html [4] Han, J. Changes in Gingival Dimensions Following Connective Tissue Grafts for Root Coverage: Comparison of Two Procedures. J Perio 2008;79:1346-1354. [5] Erbil Y, Ozluk Y, Giriş M, et al. (June 2007). "Effect of lugol solution on thyroid gland blood flow and microvessel density in the patients with Graves' disease". J. Clin. Endocrinol. Metab. 92 (6): 2182–9. doi:10.1210/jc.2007-0229. PMID 17389702. [6] Kaur S, Parr JH, Ramsay ID, Hennebry TM, Jarvis KJ, Lester E (May 1988). "Effect of preoperative iodine in patients with Graves' disease controlled with antithyroid drugs and thyroxine". Ann R Coll Surg Engl 70 (3): 123–7. PMC 2498739. PMID 2457351. [7] Rotkiewicz, Marcin; Henryk Suchar and Ryszard Kamiñski (14 January 2001). "Chernobyl: the Biggest BLUFF of the 20th Century" (http:/ / www. wonuc. org/ xfiles/ chern_02. html). Polish weekly Wprost. pp. no 2. . Retrieved 2008-06-18. [8] (http:/ / www. birdflumanual. com/ resources/ Self_Defense/ files/ Guidance for use of KI for nuclear emergency USG. pdf) US FDA, "Potassium Iodide as a Thyroid Blocking Agent in Radiation Emergencies," U.S. Department of Health and Human Services Food and Drug istration Center for Drug Evaluation and Research (CDER); December, 2001. [9] "Iodine." (http:/ / www. nlm. nih. gov/ medlineplus/ druginfo/ natural/ patient-iodine. html) MedlinePlus. [10] (http:/ / www. drugs. com/ cons/ Lugol_s_solution. html) Drugs.com, "Lugol's Solution." [11] (http:/ / www. optimox. com/ pics/ Iodine/ IOD-05/ IOD_05. html) Optimox.com, "Iodine." [12] (http:/ / www. jcrows. com/ iodine. html) Jcrows.com, "Iodine." [13] (http:/ / dx. doi. org/ 10. 1016/ j. hal. 2005. 03. 001) Hawkins et al., "Change in cyanobacterial biovolume due to preservation by Lugol's Iodine," Harmful Algae, Volume 4, Issue 6, pp. 1033-1043; November, 2005. [14] (http:/ / www. deadiversion. usdoj. gov/ fed_regs/ rules/ 2007/ fr0702. htm) US DEA, "Final Rule: Changes in the Regulation of Iodine Crystals and Chemical Mixtures Containing Over 2.2 Percent Iodine," Federal , Volume 72, Number 126; July 2, 2007 (FR Doc E7-12736). [15] (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC1774547/ ) Direct toxicity of Lugol's solution.

175

Malachite green

176

Malachite green Malachite green

Identifiers [1]

CAS number

569-64-2

PubChem

11294

ChemSpider

10820

UNII

12058M7ORO

ChEMBL

CHEMBL186357

ATCvet code

QP53 AX16

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]

[7]


C H ClN (chloride)

Molar mass

364.911 g/mol (chloride)

23 25

()

2

[8]


Infobox references

Malachite green is an organic compound that is used as a dyestuff and has emerged as a controversial agent in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper. Although called malachite green, the compound is not related to the mineral malachite — the name just comes from the similarity of color.

Malachite green

177

Structures and properties Malachite green is classified in the dyestuff industry as a triarylmethane dye. Formally, Malachite green refers to the chloride salt [C6H5C(C6H4N(CH3)2)2]Cl, although the term Malachite green is used loosely and often just refers to the colored cation. The oxalate salt is also marketed. The chloride and oxalate anions have no effect on the color. The intense green color of the cation results from a strong absorption band at 621 nm (extinction coefficient of 105 M−1cm−1). Malachite green (first transition) (pH indicator) below pH 0.2 0.2

above pH 1.8 ↔

1.8

Malachite green (second transition) (pH indicator) below pH 11.5 11.5

above pH 13.2 ↔

13.2

Malachite green is prepared by the condensation of benzaldehyde and dimethylaniline to give leuco malachite green (LMG): C6H5CHO + 2 C6H5N(CH3)2 → C6H5CH(C6H4N(CH3)2)2 + H2O Second, this colorless leuco compound, a relative of triphenylmethane, is oxidized to the cation that is MG: C6H5CH(C6H4N(CH3)2)2 + HCl + 1/2 O2 → [C6H5C(C6H4N(CH3)2)2]Cl + H2O A typical oxidizing agent is manganese dioxide. Hydrolysis of MG gives the carbinol form:[9]

On the left is leuco-Malachite Green (LMG) and on the right are the two equivalent resonance structures of the MG cation. The carbinol derivative of MG is derived from LMG by replacement of the unique C-H by C-OH.

[C6H5C(C6H4N(CH3)2)2]Cl + H2O → C6H5C(OH)(C6H4N(CH3)2)2 + HCl

This alcohol is important because it, not MG, traverses cell membranes. Once inside the cell, it is metabolized into LMG. Only the cation MG is deeply colored, whereas the LMG and carbinol derivatives are not. This difference arises because only the cationic form has extended pi-delocalization, which allows the molecule to absorb visible light.

Malachite green

178

Preparation Malachite green was first prepared by Fischer in 1877 by condensing benzaldehyde and dimethylaniline in the molecular ratio 1:2 and in the presence of a dehydrating agent[10]

Uses Malachite green is traditionally used as a dye. Millions of kilograms of MG and related triarylmethane dyes are produced annually for this purpose.[11] MG is active against the oomycete Saprolegnia, which infects fish eggs in commercial aquaculture, and other fungi. Furthermore, MG is also used as [12] a parasiticide and antibacterial. It is a very popular treatment against ichthyophthirius in freshwater aquaria. The principal metabolite, LMG, is found in fish treated with malachite green, and this finding is the basis of controversy and government regulation.

Niche uses Numerous niche applications exploit the intense color of MG. It is used as a biological stain for microscopic analysis of cell biology and tissue samples. In the Gimenez staining method, basic fuchsin stains bacteria red or magenta, and malachite green is used as a blue-green counterstain. Malachite green can also directly stain endospores within cells; here a safranin counterstain is often used. Malachite green can also be used as a saturable absorber in dye lasers, or as a pH indicator between pH 0.2–1.8. However this use is relatively rare. Leuco-malachite green (LMG) is used as a detection method for latent blood in forensic science. Hemoglobin catalyzes the A preparation of Bacillus subtilis showing endospores stained with reaction between LMG and hydrogen peroxide, malachite green (vegetative cells are stained red) converting the colorless LMG into malachite green. Therefore, the appearance of a green color indicates the presence of blood.[13]

Regulation In 1992 Canadian authorities determined that eating fish contaminated with malachite green posed a significant health risk.[14] Malachite green was classified a Class II Health Hazard. Due to its low manufacturing cost, malachite green is still used in certain countries with less restrictive laws for non-aquaculture purposes. In 2005, analysts in Hong Kong found traces of malachite green in eels and fish imported from China and Taiwan. In 2006 the United States Food and Drug istration (FDA) detected malachite green in seafood imported from China, among others, where the substance is also banned for use in aquaculture. In June 2007, the FDA blocked the importation of several varieties of seafood due to continued malachite green contamination.[15] The substance has been banned in the United States since 1983 in food-related applications. It is banned in the UK also.[16]

Malachite green Aquatic animals metabolize malachite green to its leuco form. Being non-polar, LMG is retained in catfish muscle longer (t1/2 = 10 days) than is MG (t1/2 = 2.8 days).

Toxicity The LD50 (oral, mouse) is 80 mg/kg. Rats fed malachite green experience “a dose-related increase in liver DNA adducts” along with lung adenomas. Leuco-malachite green causes an “increase in the number and severity of changes”. As leuco-malachite green is the primary metabolite of malachite green and is retained in fish muscle much longer, most intake of malachite green would be in the leuco form. During the experiment, rats were fed up to 543 ppm of leuco-malachite green, an extreme amount compared to the average 5 ppb discovered in fish. After a period of two years, an increase in lung adenomas in male rats was discovered but no incidences of liver tumors. Therefore it could be concluded that malachite green caused carcinogenic symptoms, but a direct link between malachite green and liver tumor was not established.[17]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=569-64-2 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=11294 [3] http:/ / www. chemspider. com/ 10820 [4] [5] [6] [7]

http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=12058M7ORO https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL186357 http:/ / www. whocc. no/ atcvet/ atcvet_index/ ?code=QP53AX16 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CN%28C%29c1ccc%28cc1%29C%28%3DC2C%3DCC%28%3D%5BN%2B%5D%28C%29C%29C%3DC2%29c3ccccc3. %5BCl-%5D [8] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=438218601& page2=%3AMalachite+ green [9] Adina Raducan, Alexandra Olteanu, Mihaela Puiu, Dumitru Oancea "Influence of surfactants on the fading of malachite green" Central European Journal of Chemistry, 2008, Volume 6, pp 1895-1066 (Print) 1644-3624 (Online). doi:10.2478/s11532-007-0066-0 [10] Dr. M Vishwanathan. Principles of organic chemistry. Jai Sai Publications. pp. 2/37. [11] Thomas Gessner and Udo Mayer "Triarylmethane and Diarylmethane Dyes" in Ullmann's Encyclopedia of Industrial Chemistry 2002, Wiley-VCH, Weinheim.doi:10.1002/14356007.a27_179 [12] Srivastava S, Sinha R, Roy D (2004). "Toxicological effects of malachite green". Aquatic Toxicology 66 (3): 319–29. PMID 15129773. [13] Protocol 2.18 Leucomalachite Green Presumptive Test for Blood (http:/ / static. dna. gov/ lab-manual/ Linked Documents/ Protocols/ pdi_lab_pro_2. 18. pdf), National Forensic Science Technology Center, July 8, 2010, Retrieved on July 8, 2010. [14] Wendy C. Andersen, Sherri B. Turnipseed, and José E. Roybal "Quantitative and Confirmatory Analyses of Malachite Green and Leucomalachite Green Residues in Fish and Shrimp" J. Agric. Food Chem. 2006, volume 54, pp 4517–4523.doi:10.1021/jf0532258 and references therein [15] Chinese fish crisis shows seafood safety challenges (http:/ / www. usatoday. com/ money/ industries/ food/ 2007-06-28-fish-cover-usat_N. htm), USA Today, 7/1/2007 [16] Veterinary Residues Committee. Annual Report on Surveillance for Veterinary Residues in Food in the UK for 2001, 2002, and 2003. http:/ / www. vmd. defra. gov. uk/ vrc/ Reports/ annual. htm. [17] S.J. Culp et al. (2002). "Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green". Mutation Research 506-507: 55–63. doi:10.1016/S0027-5107(02)00152-5. PMID 12351145.

Further reading • Bongsup P. Cho et al. (2003). "Synthesis and Characterization of N-Demethylated Metabolites of Malachite Green and Leucomalachite Green". Chem. Res. Toxicol. 16 (3): 285–294. doi:10.1021/tx0256679. PMID 12641428. • S. M. Plakas, K. R. El Said, G. R. Stehly, W. H. Gingerich and J. H. Allen (1996). "Uptake, tissue distribution, and metabolism of malachite green in the channel catfish (Ictalurus punctatus)". Can. J. Fish. Aquat. Sci. 53 (6): 1427–1433. doi:10.1139/cjfas-53-6-1427. • Schoettger, 1970; Smith and Heath, 1979; Gluth and Hanke, 1983. Bills et al. (1977)

179

Malachite green

180

• Steven C. DeFina, Thorsten Dieckmann (2002). "Synthesis of selectively 15N- or 13C-labelled malachite green". Journal of Labelled Compounds and Radiopharmaceuticals 45 (3): 241. doi:10.1002/jlcr.554.

External links • U.S. National Institutes of Health (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve& db=PubMed&list_uids=15213768&dopt=Abstract) • University of Guelph (http://www.chembio.uoguelph.ca/preuss/Historical UK perspective on MG.pdf) • U.S. Food and Drug istration (http://www.cfsan.fda.gov/~frf/lib4363.html) • U.K. Department of Health (http://www.advisorybodies.doh.gov.uk/com/malachit.htm) • Malachite green - endospore staining technique (video) (http://www.tgw1916.net/video_pages/Spore.html)

Masson's trichrome stain Masson's trichrome is a three-colour staining protocol used in histology. The recipes evolved from Claude L. Pierre Masson's original formulation to different specific applications, but all are suited for distinguishing cells from surrounding connective tissue. Most recipes produce red keratin and muscle fibers, blue or green collagen and bone, light red or pink cytoplasm, and dark brown to black cell nuclei. The trichrome is applied by immersion of the fixated sample into Weigert's iron hematoxylin, and then three different solutions, labeled A, B, and C:

Masson's trichrome stain of rat airway. Connective tissue is stained blue, nuclei are stained dark red/purple, and cytoplasm is stained red/pink.

• Weigert's hematoxylin is a sequence of three solutions: ferric chloride in diluted hydrochloric acid, hematoxylin in 95% ethanol, and potassium ferricyanide solution alkalized by sodium borate. It is used to stain the nuclei. • Solution A, also called plasma stain, contains acid fuchsin, Xylidine Ponceau, glacial acetic acid, and distilled water. Other red acid dyes can be used, e.g. the Biebrich scarlet in Lillie's trichrome. • Solution B contains phosphomolybdic acid in distilled water. • Solution C, also called fibre stain, contains Light Green SF yellowish, or alternatively Fast Green FCF. It is used to stain collagen. If blue is preferred to green, methyl blue, water blue or aniline blue can be substituted.


Variants A common variant is Lillie's trichrome. It is often erroneously called Masson's trichrome. It differs in the dyes used, their concentrations, and the immersion times. Another common variant is the Masson trichrome & Verhoeff stain, which combines the Masson trichrome stain and Verhoeff stain.[1] It is sometimes just referred to as a Masson trichrome . A reference is needed for the previous statement, since the Masson's trichrome method on its own does not stain elastin fiber black. This combination is useful for the examination of blood vessels; the Verhoeff stain highlights elastin (black) and allows one to easily differentiate small arteries (which typically have two elastic laminae) and veins (which have one elastic lamina).

References [1] Masson Trichrome & Verhoeff Stain. vetmed.vt.edu. URL: (http:/ / education. vetmed. vt. edu/ Curriculum/ VM8054/ Labs/ Lab2/ Examples/ exvrmass. htm). Accessed on: August 20, 2009.

External links • Stainsfile: Masson's Trichrome (http://stainsfile.info/StainsFile/stain/conektv/masson.htm) • Masson's Trichrome Protocol (http://www.idoub.com/doc/49699318/Masson-Protocol)

181

Methanol

182

Methanol Methanol

Identifiers [1]

CAS number

67-56-1

PubChem

887

ChemSpider

864

UNII

Y4S76JWI15

EC number

200-659-6

UN number

1230

KEGG

D02309

MeSH

Methanol

ChEBI

CHEBI:17790

ChEMBL

CHEMBL14688

RTECS number

PC1400000

Beilstein Reference

1098229

Gmelin Reference

449

3DMet

B01170

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]

[7] [8]

[9]

[10] [11]


CH O

Molar mass

32.04 g mol−1

Appearance

Colorless liquid

Density

0.7918 g cm−3

Melting point

−98--97 °C, unknown operator: u'\u2212'-176 K, unknown operator: u'\u2212'--143 °F

Boiling point

65 °C, 338 K, 149 °F

log P

-0.69

Vapor pressure

13.02 kPa (at 20 °C)

4

Methanol

183 [12]

Acidity (pKa)

15.5

Viscosity

5.9×10−4 Pa s (at 20 °C)

Dipole moment

1.69 D [13]

Hazards EU Index

603-001-00-X

EU classification F

T

R-phrases

R11, R23/24/25, R39/23/24/25

S-phrases

(S1/2), S7, S16, S36/37, S45


11–12 °C


385 °C

Explosive limits

36% Related compounds

Related compounds

Methanethiol Silanol ()

[14]


Infobox references

Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH (often abbreviated MeOH). It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol (drinking alcohol).[15] At room temperature, it is a polar liquid, and is used as an antifreeze, solvent, fuel, and as a denaturant for ethanol. It is also used for producing biodiesel via transesterification reaction. Methanol is produced naturally in the anaerobic metabolism of many varieties of bacteria, and is ubiquitous in the environment. As a result, there is a small fraction of methanol vapor in the atmosphere. Over the course of several days, atmospheric methanol is oxidized with the help of sunlight to carbon dioxide and water. Methanol burns in air, forming carbon dioxide and water: 2 CH3OH + 3 O2 → 2 CO2 + 4 H2O Because of its toxic properties, methanol is frequently used as a denaturant additive for ethanol manufactured for industrial uses — this addition of methanol exempts industrial ethanol from liquor excise taxation. Methanol is often called wood alcohol because it was once produced chiefly as a byproduct of the destructive distillation of wood.

Methanol

History In their embalming process, the ancient Egyptians used a mixture of substances, including methanol, which they obtained from the pyrolysis of wood. Pure methanol, however, was first isolated in 1661 by Robert Boyle, when he produced it via the distillation of buxus (boxwood). It later became known as "pyroxylic spirit". In 1834, the French chemists Jean-Baptiste Dumas and Eugene Peligot determined its elemental composition. They also introduced the word "methylene" to organic chemistry, forming it from Greek methy = "wine" + hȳlē = wood (patch of trees), with Greek language errors: "wood (substance)" (Greek xylon) was intended, and the components in the wrong order for Greek. The term "methyl" was derived in about 1840 by back-formation from "methylene", and was then applied to describe "methyl alcohol". This was shortened to "methanol" in 1892 by the International Conference on Chemical Nomenclature. The suffix -yl used in organic chemistry to form names of carbon groups, was extracted from the word "methyl". In 1923, the German chemists Alwin Mittasch and Mathias Pier, working for BASF, developed a means to convert synthesis gas (a mixture of carbon monoxide, carbon dioxide, and hydrogen) into methanol. A patent was filed Jan 12 1926 (reference no. 1,569,775). This process used a chromium and manganese oxide catalyst, and required extremely vigorous conditions—pressures ranging from 50 to 220 atm, and temperatures up to 450 °C. Modern methanol production has been made more efficient through use of catalysts (commonly copper) capable of operating at lower pressures, the modern low pressure methanol (LPM) was developed by ICI in the late 1960s with the technology now owned by Johnson Matthey, which is a leading licensor of methanol technology. Methanol is one of the most heavily traded chemical commodities in the world, with an estimated global demand of around 27 to 29 million metric tons. In recent years, production capacity has expanded considerably, with new plants coming on-stream in South America, China and the Middle East, the latter based on access to abundant supplies of methane gas. Even though nameplate production capacity (coal-based) in China has grown significantly, operating rates are estimated to be as low as 50 to 60%. No new production capacity is scheduled to come on-stream until 2015. The main applications for methanol are the production of formaldehyde (used in construction and wooden boarding), acetic acid (basis for a.o. PET-bottles), MTBE (fuel component and replacement for the very volatile diethyl ether) and more recently for the formation of methyl esters in the production of bio-diesel. In China, demand is expected to grow exponentially, not only caused by a growing internal market of the traditional applications, but accelerated by new applications, such as direct blending (with gasoline), Methanol-To-Olefins (e.g. propylene) and DME. Methanol can also be used to produce gasoline. The use of methanol as a motor fuel received attention during the oil crises of the 1970s due to its availability, low cost, and environmental benefits. By the mid-1990s, over 20,000 methanol "flexible fuel vehicles" capable of operating on methanol or gasoline were introduced in the U.S. In addition, low levels of methanol were blended in gasoline fuels sold in Europe during much of the 1980s and early-1990s. Automakers stopped building methanol FFVs by the late-1990s, switching their attention to ethanol-fueled vehicles. While the methanol FFV program was a technical success, rising methanol pricing in the mid- to late-1990s during a period of slumping gasoline pump prices diminished the interest in methanol fuels. [16] In 2006, astronomers using the MERLIN array of radio telescopes at Jodrell Bank Observatory discovered a large cloud of methanol in space, 288 billion miles across.[17][18]

184

Methanol

185

Production Production of synthesis gas Today, synthesis gas is most commonly produced from the methane component in natural gas rather than from coal, because it contains hydrogen. Three processes are commercially practiced. At moderate pressures of 4 MPa (40 atm) and high temperatures (around 850 °C), methane reacts with steam on a nickel catalyst to produce syngas according to the chemical equation: CH4 + H2O → CO + 3 H2 This reaction, commonly called steam-methane reforming or SMR, is endothermic, and the heat transfer limitations place limits on the size of and pressure in the catalytic reactors used. Methane can also undergo partial oxidation with molecular oxygen (at atmospheric pressure) to produce syngas, as the following equation shows: 2 CH4 + O2 → 2 CO + 4 H2 This reaction is exothermic, and the heat given off can be used in-situ to drive the steam-methane reforming reaction. When the two processes are combined, it is referred to as autothermal reforming. The high pressures and high temperatures needed for steam-reforming require a greater capital investment in equipment than is needed for a simple partial-oxidation process; however, the energy-efficiency of steam-reforming is higher than for partial-oxidation, unless the waste-heat from partial-oxidation is used. Stoichiometry Stoichiometry for Methanol production requires the ratio of H2 / CO to equal 2. The partial oxidation process yields a ratio of 2, and the steam reforming process yields a ratio of 3. The H2 / CO ratio can be adjusted to some extent by the water-gas shift reaction, CO + H2O → CO2 + H2, to provide the appropriate stoichiometry for methanol synthesis.

Production of methanol from synthesis gas Carbon monoxide and hydrogen react on a second catalyst to produce methanol. Today, the most widely used catalyst is a mixture of copper, zinc oxide, and alumina first used by ICI in 1966. At 5–10 MPa (50–100 atm) and 250 °C, it can catalyze the production of methanol from carbon monoxide and hydrogen with high selectivity (>99.8%): CO + 2 H2 → CH3OH It is worth noting that the production of synthesis gas from methane produces three moles of hydrogen gas for every mole of carbon monoxide, while the methanol synthesis consumes only two moles of hydrogen gas per mole of carbon monoxide. One way of dealing with the excess hydrogen is to inject carbon dioxide into the methanol synthesis reactor, where it, too, reacts to form methanol according to the equation: CO2 + 3 H2 → CH3OH + H2O Some chemists believe that the certain catalysts synthesize methanol using CO2 as an intermediary, and consuming CO only indirectly. CO2 + 3 H2 → CH3OH + H2O where the H2O byproduct is recycled via the water-gas shift reaction CO + H2O → CO2 + H2, This gives an overall reaction, which is the same as listed above. CO + 2 H2 → CH3OH

Methanol

186

Feedstock materials Although natural gas is the most economical and widely used feedstock for methanol production, many other feedstocks can be used to produce syngas via steam reforming.[19] Steam-reformed coal is sometimes used as a feedstock for methanol production, particularly in China. In addition, mature technologies available for biomass gasification are being used for methanol production. For instance, woody biomass can be gasified to water gas (a hydrogen-rich syngas), by introducing a blast of steam in a blast furnace. The water-gas / syngas can then be synthesized to methanol using standard methods. The net process is carbon neutral, since the CO2 byproduct is required to produce biomass via photosynthesis. 2 C16H23O11 + 19 H2O + O2 → 42 H2 + 21 CO + 11 CO2 → 21 CH3OH + 11 CO2

Applications Methanol, a common laboratory solvent, is especially useful for HPLC, UV/VIS spectroscopy, and LCMS due to its low UV cutoff.

Feedstock The largest use of methanol by far is in making other chemicals. About 40% of methanol is converted to formaldehyde, and from there into products as diverse as plastics, plywood, paints, explosives, and permanent press textiles. Also in the early 1970s, a methanol to gasoline process was developed by Mobil for producing gasoline ready for use in vehicles. One such industrial facility was built at Motunui in New Zealand in the 1980s. In the 1990s, large amounts of methanol were used in the United States to produce the gasoline additive methyl tert-butyl ether (MTBE). While MTBE is no longer marketed in the U.S., it is still widely used in other parts of the world. In addition to direct use as a fuel, methanol (or less commonly, ethanol) is used as a component in the transesterification of triglycerides to yield a form of biodiesel. Other chemical derivatives of methanol include dimethyl ether, which has replaced chlorofluorocarbons as an aerosol spray propellant, and acetic acid. Dimethyl ether (DME) also can be blended with liquified petroleum gas (LPG) for home heating and cooking, and can be used as a diesel replacement for transportation fuel.

Fuel for vehicles Methanol is used on a limited basis to fuel internal combustion engines. Pure methanol is required by rule to be used in Champcars, Monster Trucks, USAC sprint cars (as well as midgets, modifieds, etc.), and other dirt track series, such as World of Outlaws, and Motorcycle Speedway. Methanol is also used, as the primary fuel ingredient since the late 1940s, in the powerplants for radio control, control line and free flight airplanes (as methanol is required in the engines that primarily power them), cars and trucks, from such an engine's use of a platinum filament glow plug being able to ignite the methanol vapor through a catalytic reaction. Drag racers and mud racers, as well as heavily modified tractor pullers, also use methanol as their primary fuel source. Methanol is required with a supercharged engine in a Top Alcohol dragster and, until the end of the 2006 season, all vehicles in the Indianapolis 500 had to run methanol. Mud racers have mixed methanol with gasoline and nitrous oxide to produce more power than gasoline and nitrous oxide alone. One of the potential drawbacks of using high concentrations of methanol (and other alcohols, such as ethanol) in fuel is the corrosivity to some metals of methanol, particularly to aluminium. Methanol, although a weak acid, attacks the oxide coating that normally protects the aluminum from corrosion: 6 CH3OH + Al2O3 → 2 Al(OCH3)3 + 3 H2O The resulting methoxide salts are soluble in methanol, resulting in a clean aluminium surface, which is readily oxidized by dissolved oxygen. Also, the methanol can act as an oxidizer:

Methanol

187 6 CH3OH + 2 Al → 2 Al(OCH3)3 + 3 H2

This reciprocal process effectively fuels corrosion until either the metal is eaten away or the concentration of CH3OH is negligible. Concerns with methanol's corrosivity have been addressed by using methanol-compatible materials, and fuel additives that serve as corrosion inhibitors. When produced from wood or other organic materials, the resulting organic methanol (bioalcohol) has been suggested as renewable alternative to petroleum-based hydrocarbons. Low levels of methanol can be used in existing vehicles, with the use of proper cosolvents and corrosion inhibitors. The European Fuel Quality Directive allows up to 3% methanol with an equal amount of cosolvent to be blending in gasoline sold in Europe. Today, China uses more than one billion gallons of methanol per year as a transportation fuel in both low level blends used in existing vehicles, and as high level blends in vehicles designed to accommodate the use of methanol fuels. Because of climate change, alternatives to fossil fuels have been sought to run ground vehicles. Various alternatives have been proposed. Biofuels are carbon-neutral, but they require a great deal of fresh water to produce and are not practical in most climates. If a source of renewable or sustainable energy becomes widely available (such as wind, solar or nuclear power), various chemical alternatives have been proposed to power ground vehicles in places of batteries. An example is a hydrogen economy. However, various alcohol-based economies, including a methanol based economy has been proposed in which artificially produced methanol stores all power which cannot be directly used from sustainable sources, and also is used for ground transportation. The chief advantage of a methanol economy is that it could be adapted to present internal combustion engines with a minimum of modification in both engines and infrastructure to store and deliver liquid fuel. In 2011, the Open Fuel Standard Act of 2011 was introduced into Congress to encourage car manufacturers to warrant their cars to burn methanol as a fuel in addition to gasoline and ethanol. The bill is being championed by the Open Fuel Standard Coalition.

Other applications Methanol is a traditional denaturant for ethanol, the product being known as "methylated spirit".[20] Methanol is also used as a solvent, and as an antifreeze in pipelines and windshield washer fluid. In some wastewater treatment plants, a small amount of methanol is added to wastewater to provide a carbon food source for the denitrifying bacteria, which convert nitrates to nitrogen to reduce the nitrification of sensitive aquifers. During World War II, methanol was used as a fuel in several German military rocket designs, under the name M-Stoff, and in a mixture known as C-Stoff. Methanol was used as an automobile coolant antifreeze in the early 1900s.[21] Methanol is used as a denaturing agent in polyacrylamide gel electrophoresis. Direct-methanol fuel cells are unique in their low temperature, atmospheric pressure operation, allowing them to be miniaturized to an unprecedented degree. This, combined with the relatively easy and safe storage and handling of methanol, may open the possibility of fuel cell-powered consumer electronics, such as for laptop computers and mobile phones.[22] Methanol is also a widely used fuel in camping and boating stoves. Methanol burns well in an unpressurized burner, so alcohol stoves are often very simple, sometimes little more than a cup to hold fuel. This lack of complexity makes them a favorite of hikers who spend extended time in the wilderness. Similarly, the alcohol can also be gelled to reduce risk of leaking or spilling, as with the brand "Sterno". Methanol is mixed with water and injected into high performance diesel engines for an increase of power and a decrease in exhaust gas temperature in a process known as water methanol injection.

Methanol

Health and safety Toxicity Methanol has a high toxicity in humans. If ingested, for example, as little as 10 mL of pure methanol can cause permanent blindness by destruction of the optic nerve, and 30 mL is potentially fatal,[23] although the median lethal dose is typically 100 mL (4 fl oz) (i.e. 1–2 mL/kg of pure methanol[24]). Toxic effects take hours to start, and effective antidotes can often prevent permanent damage.[23] Because of its similarities to ethanol (the alcohol in beverages), it is difficult to differentiate between the two (such is the case with denatured alcohol). Methanol is toxic by two mechanisms. First, methanol (whether it enters the body by ingestion, inhalation, or absorption through the skin) can be fatal due to its CNS depressant properties in the same manner as ethanol poisoning. Second, in a process of toxication, it is metabolized to formic acid (which is present as the formate ion) via formaldehyde in a process initiated by the enzyme alcohol dehydrogenase in the liver.[25] Methanol is converted to formaldehyde via alcohol dehydrogenase (ADH) and formaldehyde is converted to formic acid (formate) via aldehyde dehydrogenase (ALDH). The conversion to formate via ALDH proceeds completely, with no detectable formaldehyde remaining.[26] Formate is toxic because it inhibits mitochondrial cytochrome c oxidase, causing the symptoms of hypoxia at the cellular level, and also causing metabolic acidosis, among a variety of other metabolic disturbances.[27] Fetal tissue will not tolerate methanol. Methanol poisoning can be treated with the antidotes ethanol or fomepizole.[25][28][29] Both drugs act to reduce the action of alcohol dehydrogenase on methanol by means of competitive inhibition, so it is excreted by the kidneys rather than being transformed into toxic metabolites.[25] Further treatment may include giving sodium bicarbonate for metabolic acidosis, and hemodialysis or hemodiafiltration can be used to remove methanol and formate from the blood.[25] Folinic acid or folic acid is also istered to enhance the metabolism of formate.[25] The initial symptoms of methanol intoxication include central nervous system depression, headache, dizziness, nausea, lack of coordination, and confusion. Sufficiently large doses can cause unconsciousness and death. The initial symptoms of methanol exposure are usually less severe than the symptoms resulting from the ingestion of a similar quantity of ethanol.[15] Once the initial symptoms have ed, a second set of symptoms arises, 10 to as many as 30 hours after the initial exposure to methanol, including blurring or complete loss of vision and acidosis.[25] These symptoms result from the accumulation of toxic levels of formate in the blood, and may progress to death by respiratory failure. Physical examination may show tachypnea, and opthalmologic examination may show dilated pupils with hyperemia of the optic disc and retinal edema. Small amounts of methanol are produced by the metabolism of food and are generally harmless, being metabolized quickly and completely. Ethanol is sometimes denatured (adulterated), and thus made undrinkable, by the addition of methanol. The result is known as methylated spirit, "meths" (U.K. use) or "metho" (Australian slang). These are not to be confused with "meth", a common U.S. abbreviation for methamphetamine, and U.K. abbreviation for methadone.

Safety in automotive fuels Pure methanol has been used in open wheel auto racing since the mid-1960s. Unlike petroleum fires, methanol fires can be extinguished with plain water. A methanol-based fire burns invisibly, unlike gasoline, which burns with a visible flame. If a fire occurs on the track, there is no flame or smoke to obstruct the view of fast approaching drivers, but this can also delay visual detection of the fire and the initiation of fire suppression. The decision to permanently switch to methanol in American IndyCar racing was a result of the devastating crash and explosion at the 1964 Indianapolis 500, which killed drivers Eddie Sachs and Dave MacDonald.[30] In 2007 IndyCars switched to ethanol.[31] Methanol is readily biodegradable in both aerobic (oxygen present) and anaerobic (oxygen absent) environments. Methanol will not persist in the environment. The half-life for methanol in groundwater is just one to seven days, while many common gasoline components have half-lives in the hundreds of days (such as benzene at 10–730 days).

188

Methanol Since methanol is miscible with water and biodegradable, it is unlikely to accumulate in groundwater, surface water, air or soil.[32]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=67-56-1 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=887 [3] http:/ / www. chemspider. com/ 864 [4] http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=Y4S76JWI15 [5] http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=200-659-6 [6] http:/ / www. kegg. jp/ entry/ D02309 [7] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Methanol [8] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=17790 [9] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL14688 [10] http:/ / www. 3dmet. dna. affrc. go. jp/ html/ B01170. html [11] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CO [12] Ballinger, P.; Long, F.A. (1960). "Acid Ionization Constants of Alcohols. II. Acidities of Some Substituted Methanols and Related Compounds". J. Am. Chem. Soc. 82 (4): 795–798. doi:10.1021/ja01489a008. [13] "The Emergency Response Safety and Health Database: Systematic Agent: METHANOL" (http:/ / www. cdc. gov/ niosh/ ershdb/ EmergencyResponseCard_29750029. html#er). Centers for Disease Control and Prevention. . Retrieved 26 August 2009. [14] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=409752739& page2=%3AMethanol [15] National Institute for Occupational Safety and Health (August 22, 2008). "The Emergency Response Safety and Health Database: Methanol" (http:/ / www. cdc. gov/ niosh/ ershdb/ EmergencyResponseCard_29750029. html). . Retrieved March 17, 2009. [16] James D. Halderman; Tony Martin (2009). Hybrid and alternative fuel vehicles (http:/ / books. google. com/ books?id=LqgeAQAAIAAJ). Pearson/Prentice Hall. ISBN 978-0-13-504414-8. . Retrieved 21 February 2011. [17] "Upgraded MERLIN spies cloud of alcohol spanning 288 billion miles" (http:/ / www. jodrellbank. manchester. ac. uk/ news/ 2006/ cloud/ ) (Press release). Jodrell Bank Centre for Astrophysics. 2006-04-19. . [18] Jonathan Amos (2006-04-05). "Merlin sees vast alcohol stream" (http:/ / news. bbc. co. uk/ 2/ hi/ science/ nature/ 4878048. stm). BBC News. . [19] http:/ / www. afdc. energy. gov/ afdc/ fuels/ methanol_basics. html [20] Blum, Deborah (2010-02-19). "The little-told story of how the U.S. government poisoned alcohol during Prohibition. – By Deborah Blum – Slate Magazine" (http:/ / www. slate. com/ id/ 2245188/ ). Slate.com. . Retrieved 2010-06-10. [21] "Methanol Antifreeze and Methanol Poisoning – Industrial & Engineering Chemistry (ACS Publications)" (http:/ / pubs. acs. org/ doi/ abs/ 10. 1021/ ie50257a020). Pubs.acs.org. 2002-05-01. . Retrieved 2010-06-10. [22] Sandy Berger (September 30, 2006). "Methanol Laptop Fuel" (http:/ / www. compukiss. com/ populartopics/ tech_gadgetshtm/ article887. htm). Compu·Kiss. . Retrieved 2007-05-22. [23] Vale A (2007). "Methanol". Medicine 35 (12): 633–4. doi:10.1016/j.mpmed.2007.09.014. [24] "Methanol Poisoning Overview" (http:/ / www. antizol. com/ mpoisono. htm). Antizol. . Retrieved 4/10/11. [25] Schep LJ, Slaughter RJ, Vale JA, Beasley DM (Sep 30 2009). "A seaman with blindness and confusion" (http:/ / www. bmj. com/ cgi/ content/ full/ 339/ sep30_1/ b3929). BMJ 339: b3929. doi:10.1136/bmj.b3929. PMID 19793790. . [26] McMartin KE, Martin-Amat G, Noker PE, Tephly TR (March 1979). "Lack of a role for formaldehyde in methanol poisoning in the monkey" (http:/ / linkinghub. elsevier. com/ retrieve/ pii/ 0006-2952(79)90149-7). Biochem. Pharmacol. 28 (5): 645–9. doi:10.1016/0006-2952(79)90149-7. PMID 109089. . [27] Liesivuori J, Savolainen H (September 1991). "Methanol and formic acid toxicity: biochemical mechanisms". Pharmacol. Toxicol. 69 (3): 157–63. doi:10.1111/j.1600-0773.1991.tb01290.x. PMID 1665561. [28] Casavant MJ (Jan 2001). "Fomepizole in the treatment of poisoning" (http:/ / pediatrics. aappublications. org/ cgi/ content/ full/ 107/ 1/ 170). Pediatrics 107 (1): 170. doi:10.1542/peds.107.1.170. PMID 11134450. . [29] Brent J (May 2009). "Fomepizole for ethylene glycol and methanol poisoning". N Engl J Med 360 (21): 2216–23. doi:10.1056/NEJMct0806112. PMID 19458366. [30] McDonald, Norris (2007-04-21). "Green no longer bad luck at Indy" (http:/ / www. thestar. com/ comment/ columnists/ article/ 205088). Toronto Star. . Retrieved 2010-05-12. [31] "IndyCar Series Teams Begin Use Of Ethanol-Blended Fuel" (http:/ / www. indycar. com/ news/ archive/ show/ 55-izod-indycar-series/ 28531-indycar-series-teams-begin-use-of-ethanol-blended-fuel/ ). Indycar.com. 2005-12-01. . Retrieved 2010-11-07. [32] Reference: Evaluation of the Fate and Transport of Methanol in the Environment, Malcolm Pirnie, January 1999.

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Methanol

Further reading • Robert Boyle, The Sceptical Chymist (1661) – contains of distillation of wood alcohol.

External links • International Chemical Safety Card 0057 (http://www.inchem.org/documents/icsc/icsc/eics0057.htm) • The Methanol Institute (http://www.methanol.org) Industry trade group, lots of information on methanol's use in fuel cells and as an alternative fuel. • China Takes Gold in Methanol Fuel (http://www.ensec.org/index.php?option=com_content&view=article& id=148:chinatakesgoldinmethanolfuel&catid=82:asia&Itemid=324) • The methanol story: a sustainable fuel for the future (http://www.setamericafree.org/Rnichols.pdf) article by Ford Motor's Roberta Nichols, the mother of the flexible fuel vehicle, discussing Gasoline-Ethanol-Methanol flexibility in the Journal of Scientific & Industrial Research • National Pollutant Inventory – Methanol Fact Sheet (http://www.npi.gov.au/database/substance-info/ profiles/54.html) • Methanol Discovered in Space (http://www.labnews.co.uk/laboratory_article.php/1017/2/ giant-cloud-of-space-alcohol-found) • Calculation of vapor pressure (http://ddbonline.ddbst.de/AntoineCalculation/AntoineCalculationCGI. exe?component=Methanol), liquid density (http://ddbonline.ddbst.de/DIPPR105DensityCalculation/ DIPPR105CalculationCGI.exe?component=Methanol), dynamic liquid viscosity (http://ddbonline.ddbst.de/ VogelCalculation/VogelCalculationCGI.exe?component=Methanol), surface tension (http://ddbonline.ddbst. de/DIPPR106SFTCalculation/DIPPR106SFTCalculationCGI.exe?component=Methanol) of methanol

190

Methyl violet

191

Methyl violet Methyl violet 2B

Identifiers [1]

CAS number

8004-87-3

ChemSpider

170606

ChEMBL

CHEMBL64894

[2]

[3]


C24H28N3Cl

Appearance

Green to dark-green powder

Melting point

137 °C (279 °F) – decomposes

Solubility in water

Soluble in water, ethanol, insoluble in xylene

[4] [4] [4]

()

[5]


Infobox references

Methyl violet is a family of organic compounds that are mainly used as dyes. Depending on the amount of attached methyl groups, the color of the dye can be altered. Its main use is as a purple dye for textiles and to give deep violet colors in paint and ink. Methyl violet 10B is also known as crystal violet (and many other names) and has medical uses.[6]

Structure The term methyl violet encomes three compounds that differ in the number of methyl groups attached to the amine functional group. They are all soluble in water, ethanol, diethylene glycol and dipropylene glycol.

Methyl violet

192

Name

Methyl violet 2B

Methyl violet 10B (Crystal violet)

C24H28N3Cl

C25H30N3Cl

8004-87-3

548-62-9

Structure

Formula

C23H26N3Cl

CAS no ChemSpider ID

21164086

[7]

PubChem ID Formula

[8]

170606

[10]

196986 C23H26N3

2006225

PubChem ID

2724053

[12]

[16]

[11]

11057

C24H28N3

ChemSpider ID

[9]

10588

C25H30N3 3349

[13]

, 9080056

[14]

3468

, 10354393

[15]

[17]

Methyl violet 2B Methyl violet 2B (IUPAC name:N-(4-(bis(4-(dimethylamino)phenyl)methylene)cyclohexa-2,5-dien-1-ylidene)methanaminium chloride) is a green powder which is soluble in water in ethanol and water, but not in xylene. It appears yellow in solution of low pH (~0.15) and changes to violet with pH increasing toward 3.2.[4]

Methyl violet 10B Methyl violet 10B has six methyl groups. It is known in medicine as Gentian violet (or crystal violet or pyoctanin(e)[6]) and is the active ingredient in a Gram stain, used to classify bacteria. It is used as a pH indicator, with a range between 0 and 1.6. The protonated form (found in acidic conditions) is yellow, turning blue-violet above pH levels of 1.6.[18] Gentian violet destroys cells and can be used as a disinfectant.[19] Compounds related to methyl violet are potential carcinogens. Methyl violet 10B also inhibits the growth of many Gram positive bacteria, except streptococci. When used in conjunction with nalidixic acid (which destroys gram-negative bacteria), it can be used to isolate the streptococci bacteria for the diagnosis of an infection. Methyl violet 10B also binds to DNA. This means it can be used in cell viability assays in biochemistry. However, this binding to DNA will cause replication errors in living tissue, possibly leading to mutations and cancer.

Methyl violet

Degradation Methyl violet is a mutagen and mitotic poison, therefore concerns exist regarding the ecological impact of the release of methyl violet into the environment. Methyl violet has been used in vast quantities for textile and paper dyeing, and 15% of such dyes produced worldwide are released to environment in wastewater. Numerous methods have been developed to treat methyl violet pollution. The three most prominent are chemical bleaching, biodegradation, and photodegradation.

Chemical bleaching Chemical bleaching is achieved by oxidation or reduction. Oxidation can destroy the dye completely, e.g. through the use of sodium hypochlorite (NaClO, common bleach) or hydrogen peroxide.[20][21] Reduction of methyl violet occurs in microorganisms but can be attained chemically using sodium dithionite.

Biodegradation Biodegradation has been well investigated because of its relevance to sewage plants with specialized microorganisms. Two microorganisms that have been studied in depth are the White Rot Fungus and the bacterium Nocardia Corallina.[22][23]

Photodegradation Light alone does not rapidly degrade methyl violet,[24] but the process is accelerated upon the addition of large band-gap semiconductors, TiO2 or ZnO.[25][26]

Other methods Many others methods have been developed to treat the contamination of dyes in a solution, including electrochemical degradation,[27] ion exchange,[28] laser degradation, and absorption onto various solids such as activated charcoal.

References [1] [2] [3] [4]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=8004-87-3 http:/ / www. chemspider. com/ 170606 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL64894 R. W. Sabnis (29 March 2010). Handbook of Biological Dyes and Stains: Synthesis and Industrial Applications (http:/ / books. google. com/ books?id=M59Kw54ehwAC& pg=PA309). John Wiley and Sons. pp. 309–. ISBN 978-0-470-40753-0. . Retrieved 27 June 2011. [5] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=462241694& page2=%3AMethyl+ violet [6] Gorgas, Ferdinand J. S. (1901). Pyoctanin – Methyl-Violet – Pyoctanine (http:/ / chestofbooks. com/ health/ materia-medica-drugs/ Manual-Of-Dental-Materia-Medica-And-Therapeutics/ Pyoctanin-Methyl-Violet-Pyoctanine. html). chestofbooks.com. Archived (http:/ / www. webcitation. org/ 5xD3LZSj4) from the original on 2011-03-15. . Retrieved 2011-03-15. [7] http:/ / www. chemspider. com/ Chemical-Structure. 21164086. html [8] http:/ / www. chemspider. com/ Chemical-Structure. 170606. html [9] http:/ / www. chemspider. com/ Chemical-Structure. 10588. html [10] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=196986 [11] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=11057 [12] http:/ / www. chemspider. com/ Chemical-Structure. 2006225. html [13] http:/ / www. chemspider. com/ Chemical-Structure. 3349. html [14] http:/ / www. chemspider. com/ Chemical-Structure. 9080056. html [15] http:/ / www. chemspider. com/ Chemical-Structure. 10354393. html [16] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=2724053 [17] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=3468 [18] Kristallviolett – ein pH-Indikator (http:/ / www. uni-regensburg. de/ Fakultaeten/ nat_Fak_IV/ Organische_Chemie/ Didaktik/ Keusch/ D-KV-e. htm)

193

Methyl violet [19] WHO Model Lists of Essential Medicines, March 2007 (http:/ / www. who. int/ entity/ medicines/ publications/ 08_ENGLISH_indexFINAL_EML15. pdf) [20] Pizzolato, T (2002). "Colour removal with NaClO of dye wastewater from an agate-processing plant in Rio Grande do Sul, Brazil". International Journal of Mineral Processing 65 (3–4): 203. doi:10.1016/S0301-7516(01)00082-5. [21] XP-Chloro Degradation Malachite green U.S. Patent 2755202 (http:/ / www. google. com/ patents?vid=2755202) [22] Bumpus, JA; Brock, BJ (1988). "Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium". Applied and environmental microbiology 54 (5): 1143–50. PMC 202618. PMID 3389809. [23] Yatome, Chizuko; Yamada, Shigeyuki; Ogawa, Toshihiko; Matsui, Masaki (1993). "Degradation of Crystal violet by Nocardia corallina". Applied Microbiology and Biotechnology 38 (4). doi:10.1007/BF00242956. [24] Bhasikuttan, A; Sapre, A.V.; Shastri, L.V. (1995). "Oxidation of crystal violet and malachite green in aqueous solutions — a kinetic spectrophotometric study". Journal of Photochemistry and Photobiology A: Chemistry 90 (2–3): 177. doi:10.1016/1010-6030(95)04094-V. [25] Senthilkumaar, S; Porkodi, K (2005). "Heterogeneous photocatalytic decomposition of Crystal Violet in UV-illuminated sol-gel derived nanocrystalline TiO2 suspensions". Journal of colloid and interface science 288 (1): 184–9. doi:10.1016/j.jcis.2005.02.066. PMID 15927578. [26] Sahoo, C; Gupta, A; Pal, A (2005). "Photocatalytic degradation of Crystal Violet (C.I. Basic Violet 3) on silver ion doped TiO". Dyes and Pigments 66 (3): 189. doi:10.1016/j.dyepig.2004.09.003. [27] Sanroman, M; Pazos, M; Ricart, M; Cameselle, C (2004). "Electrochemical decolourisation of structurally different dyes". Chemosphere 57 (3): 233–9. doi:10.1016/j.chemosphere.2004.06.019. PMID 15312740. [28] Wu, J; Liu, C; Chu, K; Suen, S (2008). "Removal of cationic dye methyl violet 2B from water by cation exchange membranes". Journal of Membrane Science 309: 239. doi:10.1016/j.memsci.2007.10.035.

194

Methylene blue

195

Methylene blue Methylene blue


C16H18N3SCl

Molar mass

319.85 g/mol

Melting point

100-110 °C (with decomposition)

Boiling point

Decomposes ()

[1]


Infobox references

Methylene blue (CI 52015) is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl. It has many uses in a range of different fields, such as biology and chemistry. At room temperature it appears as a solid, odorless, dark green powder, that yields a blue solution when dissolved in water. The hydrated form has 3 molecules of water per molecule of methylene blue.[2] Methylene blue should not be confused with methyl blue, another histology stain, new methylene blue, nor with the methyl violets often used as pH indicators. The International Nonproprietary Name (INN) of methylene blue is methylthioninium chloride.[3][4]

Light absorption properties Methylene blue is a potent cationic dye with maximum absorption of light around 670 nm. The specifics of absorption depend on a number of factors, including protonation, adsorption to other materials, and metachromasy - the formation of dimers and higher-order aggregates depending on concentration and other interactions:[5]

Absorption spectrum of methylene blue, in of the molar extinction coefficient (base 10 logarithm). In this dataset a peak absorbance of 1.7 (i.e. 98% of transmitted light absorbed) was observed with 665 nm light ing through 1 cm of 10 micromolar methylene blue solution.

Methylene blue

196

Species

Absorption peak Extinction coefficient (dm3/mole·cm)

MB+ (solution)

664

95000

MBH2+ (solution)

741

76000

(MB+)2 (solution)

605

132000

(MB+)3 (solution)

580

110000

MB+ (adsorbed on clay)

673

116000

MBH2+ (adsorbed on clay) 763

86000

(MB+)2 (adsorbed on clay) 596

80000

(MB+)3 (adsorbed on clay) 570

114000

Uses Redox indicator Methylene blue is widely used as a redox indicator in analytical chemistry. Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent. The redox properties can be seen in a classical demonstration of chemical kinetics in general chemistry, the "blue bottle" experiment. Typically, a solution is made of glucose (dextrose), methylene blue, and sodium hydroxide. Upon shaking the bottle, oxygen oxidizes methylene blue, and the solution turns blue. The dextrose will gradually reduce the methylene blue to its colorless, reduced form. Hence, when the dissolved dextrose is entirely consumed, the solution will turn blue again.

Peroxide generator A volumetric flask of a methylene blue solution

Methylene blue is also a photosensitizer used to create singlet oxygen when exposed to both oxygen and light. It is used in this regard to make organic peroxides by a Diels-Alder reaction which is spin forbidden with normal atmospheric triplet oxygen.

Sulfide analysis The formation of methylene blue after the reaction of hydrogen sulfide with dimethyl-p-phenylenediamine and iron(III) at pH 0.4 – 0.7 is used to determine by photometric measurements sulfide concentration in the range 0.020 to 1.50 mg/L (20 ppb to 1.5 ppm). The test is very sensitive and the blue coloration developing upon of the reagents with dissolved H2S is stable for 60 min. Ready-to-use kits such as the Spectroquant sulfide test[6] facilitate routine analyses. The methylene blue sulfide test is a convenient method often used in soil microbiology to quickly detect in water the metabolic activity of sulfate reducing bacteria (SRB). It should be observed that in this test, methylene blue is a product of reaction and not a reagent. The addition of a strong reducing agent, such as ascorbic acid, to a sulfide-containing solution is sometimes used to prevent sulfide oxidation from atmospheric oxygen. Although it is certainly a sound precaution for the determination

Methylene blue of sulfide with an ion selective electrode, it might however hamper the development of the blue color if the freshly formed methylene blue is also reduced, as described here above in the paragraph on redox indicator.

Water testing A color reaction in an acidified, aqueous methylene blue solution containing chloroform can detect anionic surfactants in a water sample. Such a test is known as an MBAS assay (methylene blue active substances assay). The MBAS assay cannot distinguish between specific surfactants, however. Some examples of anionic surfactants are carboxylates, phosphates, sulfates, and sulfonates.

Biology In biology methylene blue is used as a dye for a number of different staining procedures, such as Wright's stain and Jenner's stain. Since it is a temporary staining technique, methylene blue can also be used to examine RNA or DNA under the microscope or in a gel: as an example, a solution of methylene blue can be used to stain RNA on hybridization membranes in northern blotting to the amount of nucleic acid present. While methylene blue is not as sensitive as ethidium bromide, it is less toxic and it does not intercalate in nucleic acid chains, thus avoiding interference with nucleic acid retention on hybridization membranes or with the hybridization process itself. It can also be used as an indicator to determine if a cell such as yeast is alive or not. The blue indicator turns colorless indicating living cells. However, if it stays blue it doesn't mean that the cell is dead or there are no cells. Methylene blue can inhibit the respiration of the yeast as it picks up hydrogen ions made during the process and the yeast cell cannot then use those ions to release energy. In neuroscience, methylene blue can also serve as a non-selective inhibitor of NO synthase.

Medical uses Methylene blue is a monoamine oxidase inhibitor (MAOI),[7] and if infused intravenously at doses exceeding 5 mg/kg, may precipitate serious serotonin toxicity, serotonin syndrome, if combined with any selective serotonin reuptake inhibitors (SSRIs) or other serotonin reuptake inhibitor (e.g., duloxetine, sibutramine, venlafaxine, clomipramine, imipramine).[8] Methylene blue is also structurally similar to the chlorpromazine and the typical antipsychotics. It is the basic compound from which chlorpromazine and many other antipsychotics are made.[9] Methylene blue is a component of a frequently prescribed urinary analgesic/anti-infective/anti-spasmodic known as "Prosed", a combination of drugs which also contains phenyl salicylate, benzoic acid, hyoscyamine sulfate, and methenamine (aka hexamethylenetetramine and not to be confused with 'methanamine').[10]

Malaria Methylene blue was identified by Paul Ehrlich about 1891 as a successful treatment for malaria. It disappeared as an anti-malarial during the Pacific War in the tropics, since American and Allied soldiers disliked its two prominent, but reversible side effects: turning the urine green, and the sclera (the whites of the eyes) blue. Interest in its use as an anti-malarial has recently been revived,[11] especially due to its low price. Several clinical trials are in progress, trying to find a suitable drug combination. Initial attempts to combine methylene blue with chloroquine were disappointing;[12] however, more recent attempts have appeared more promising.

197

Methylene blue

Cancer Recent research suggests that methylene blue, toluidine blue, and other 3,7-diaminophenothiazinium-based redox cyclers induce selective cancer cell apoptosis by NAD(P)H:quinone oxidoreductase (NQO1)-dependent bioreductive generation of cellular oxidative stress.[13] Combined with plant auxin (indole-3-acetic acid), methylene blue is being investigated for the photodynamic treatment of cancer.[14]

Combined with light Methylene blue combined with light has been used to treat resistant plaque psoriasis,[15] AIDS-related Kaposi's sarcoma,[16] West Nile virus,[17] and to inactivate staphylococcus aureus,[18] HIV-1,[19] Duck hepatitis B,[20] adenovirus vectors,[21] and hepatitis C.[22] Phenothiazine dyes and light have been known to have virucidal properties for over 80 years.[23] In some circumstances, the combination can cause DNA damage that may lead to cancer.[24][25]

Methemoglobinemia While many texts indicate that methylene blue has oxidizing agent properties, its effects as an oxidizing agent occurs only at very high doses. At pharmacologic doses it has reducing agent properties. It is owing to this reason that methylene blue is employed as a medication for the treatment of methemoglobinemia. This can arise from ingestion of certain pharmaceuticals, toxins, or broad beans.. Normally, through the NADH or NADPH dependent methemoglobin reductase enzymes, methemoglobin is reduced back to hemoglobin. When large amounts of methemoglobin occur secondary to toxins, methemoglbin reductases are overwhelmed. Methylene blue, when injected intravenously as an antidote, is itself first reduced to leucomethylene blue, which then reduces the heme group from methemoglobin to hemoglobin. Methylene blue can reduce the half life of methemoglobin from hours to minutes. [26] At high doses, however, methylene blue actually induces methemoglobinemia, reversing this pathway.[26] Methylene blue also blocks accumulation of cyclic guanosine monophosphate (cGMP) by inhibiting the enzyme guanylate cyclase: this action results in reduced responsiveness of vessels to cGMP-dependent vasodilators like nitric oxide and carbon monoxide. Cardiac surgical teams have found this very useful in the treatment of extremely low blood pressure (hypotension)which may occur during heart surgery requiring cardiac by.[27] Similar use is noted in the treatment of hypotension associated with overwhelming infections (sepsis).[28]

Cyanide poisoning Since its reduction potential is similar to that of oxygen and can be reduced by components of the electron transport chain, large doses of methylene blue are sometimes used as an antidote to potassium cyanide poisoning, a method first successfully tested in 1933 by Dr. Matilda Moldenhauer Brooks in San Francisco.[29]

Carbon monoxide poisoning Methylene blue was also used in the mid-twentieth century in the treatment of carbon monoxide poisoning.[29]

Dye/Stain Methylene blue is used in endoscopic polypectomy as an adjunct to saline or epinephrine, and is used for injection into the submucosa around the polyp to be removed. This allows the submucosal tissue plane to be identified after the polyp is removed, which is useful in determining if more tissue needs to be removed, or if there has been a high risk for perforation. Methylene blue is also used as a dye in chromoendoscopy, and is sprayed onto the mucosa of the gastrointestinal tract in order to identify dysplasia, or pre-cancerous lesions. Intravenously injected methylene blue is readily released into the urine and thus can be used to test the urinary tract for leaks or fistulas.

198

Methylene blue In surgeries such as sentinel lymph node dissections, methylene blue can be used to visually trace the lymphatic drainage of pertinent tissues. Similarly, methylene blue is added to bone cement in orthopedic operations to provide easy discrimination between native bone and cement. Additionally, methylene blue accelerates the hardening of bone cement, increasing the speed at which bone cement can be effectively applied. It can also be used to stain lymph nodes. When methylene blue is "polychromed" (Oxidized in solution or "ripened" by fungal metabolism [30], as originally noted in the thesis of Dr D L Romanowsky in 1890's), it gets serially demethylated and forms all the tri, di, mono and non methyl intermediates - which are Azure B, Azure A, Azure C and thionine respectively.[31]. This is the basis of the basophilic part of the spectrum of Romanowski-Giemsa effect. If only synthetic Azure B and Eosin Y is used, it may serve as a standardized Giemsa stain; but, without Methylene Blue, the normal neutrophilic granules tend to overstain and look like toxic granules. On the other hand, if methylene blue is used it might help to give the normal look of neutrophil granules and may additionally also enhances the staining of nucleoli and polychromatophilic RBCs (reticulocytes)[32].

Placebo Methylene blue has been used as a placebo; physicians would tell their patients to expect their urine to change color and view this as a sign that their condition had improved.[33] This same side effect makes methylene blue difficult to test in traditional placebo-controlled clinical studies.[34]

Ifosfamide neurotoxicity Another, less well-known use of methylene blue is its utility for treating ifosfamide neurotoxicity. Methylene blue was first reported for treatment and prophylaxis of ifosfamide neuropsychiatric toxicity in 1994. A toxic metabolite of ifosfamide, chloroacetaldehyde (CAA), disrupts the mitochondrial respiratory chain, leading to an accumulation of nicotinamide adenine dinucleotide hydrogen (NADH). Methylene blue acts as an alternative electron acceptor, and reverses the NADH inhibition of hepatic gluconeogenesis while also inhibiting the transformation of chloroethylamine into chloroacetaldehyde, and inhibits multiple amine oxidase activities, preventing the formation of CAA.[35] The dosing of methylene blue for treatment of ifosfamide neurotoxicity varies, depending upon its use simultaneously as an adjuvant in ifosfamide infusion, versus its use to reverse psychiatric symptoms that manifest after completion of an ifosfamide infusion. Reports suggest that methylene blue at 50–60 mg up to six doses a day have resulted in improvement of symptoms within 10 minutes to several days.[36] Alternatively, it has been suggested that intravenous methylene blue 50 mg every six hours for prophylaxis during ifosfamide treatment in patients with history of ifosfamide neuropsychiatric toxicity.[37] Prophylactic istration of 50 mg of methylene blue the day before initiation of ifosfamide, and 50 mg three times daily during ifosfamide chemotherapy has been recommended to lower the occurrence of ifosfamide neurotoxicity.[38]

Clinical trials TauRx Therapeutics has reported that methylene blue (methylthioninium chloride), under the tradename Rember, may provide a way of halting or slowing the progression of Alzheimer's dementia.[39] However, the formulation used was different from that commonly available as a medicine and caution has been expressed e of methylene blue as a treatment for Alzheimer's.[40] TauRx Therapeutics has suggested that the mechanism by which methylene blue might delay or reverse neurodegeneration in Alzheimer's disease is as an inhibitor of Tau protein aggregation. While methylene blue arguably has an effect on Tau aggregation, it has been shown to have a great effect in dissociation of amyloids[41] and also has an effect on mitochondrial function which are both likely targets that result in its therapeutic effect. In vitro studies suggest that methylene blue might be an effective remedy for both Alzheimer's and Parkinson's disease by enhancing key mitochondrial biochemical pathways. It can disinhibit and increase complex IV, whose inhibition correlates with Alzheimer's disease.

199

Methylene blue Methylene blue might also delay senescence as one study has shown that it extended the lifespan of IMR90 fibroblasts by more than 20 population doublings.[42] These findings are highly controversial, and a clear dosage response curve has not been found.

Aquaculture Methylene blue is used in aquaculture and by tropical fish hobbyists as a treatment for fungal infections. It can also be effective in treating fish infected with ich, the parasitic protozoa Ichthyophthirius multifiliis. It is usually used to protect newly laid fish eggs from being infected by fungus or bacteria. This is useful when the hobbyist wants to artificially hatch the fish eggs. Methylene Blue is also very effective when used as part of a "medicated fish bath" for treatment of ammonia, nitrite, and cyanide poisoning as well as for topical and internal treatment of injured or sick fish as a "first response".[43]

History Methylene blue has been described as "the first fully synthetic drug used in medicine." Its use in the treatment of malaria was pioneered by Paul Guttman and Paul Ehrlich in 1891. During this period before the first World War, researchers like Ehrlich believed that drugs and dyes worked in the same way, by preferentially staining pathogens and possibly harming them. Methylene blue continued to be used in the second World War, where it was not well liked by soldiers, who observed, "Even at the loo, we see, we pee, navy blue." Antimalarial use of the drug has recently been revived.[44] The blue urine was used to monitor psychiatric patients' compliance with medication regimes. This led to interest - from the 1890s to the present day - in the drug's antidepressant and other psychotropic effects. It became the lead compound in research leading to the discovery of chlorpromazine.[11]

Popular culture Prank It is, or was at one time, a common prank among college students in biomedical fields to spike someone's drink with methylene blue, thus creating amusement at the victim's expense when he reacts with alarm to his urine turning blue. With concern over date rape drugs, spiking someone's drink is considered far more serious than it used to be, and the prank has somewhat gone out of fashion. In literature An episode of M*A*S*H, "Sons and Bowlers", showed Major Winchester using a dose of methylene blue to take down a rival camp's bowling champion—who had been a high-ranked professional bowler in civilian life—during a contest. The champ panics when his urine turns blue, and listens to Winchester's advice to refrain from all exercise – including bowling, which allows the 4077th to win. In the 1946 film noir, Decoy, the chemical is portrayed in an entirely different manner - as having resuscitative qualities – in that it is used successfully to bring a criminal back to life after execution by hydrocyanic gas.[45][46] The tactic of using methylene blue to monitor medication compliance has been noted by authors such as Kurt Vonnegut in "Welcome to the Monkey House".

200

Methylene blue

201

Adverse reactions Cardiovascular Central Nervous System • Hypertension • Dizziness • Precordial pain • Mental confusion • Headache • Fever

Dermatologic

Gastrointestinal

• Staining of skin • Fecal • Injection site necrosis (SC) discoloration • Nausea • Vomiting • Abdominal pain

Genito-urinary • Discoloration of urine • Bladder irritation

Hematologic • Anemia

[47][48]

Causes hemolytic anemia in carriers of the G6PD (favism) enzymatic deficiency.

References [1] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=476992187& page2=%3AMethylene+ blue [2] http:/ / www. methylene-blue. com/ substance. php [3] Adams V., Marley J., McCarroll C. (November 2007). "Prilocaine induced methaemoglobinaemia in a medically compromised patient. Was this an inevitable consequence of the dose istered?". Br. Dent. J. 203 (10): 585–7. doi:10.1038/bdj.2007.1045. PMID 18037845. [4] Linz A.J., Greenham R.K., Fallon L.F. (May 2006). "Methemoglobinemia: an industrial outbreak among rubber molding workers" (http:/ / meta. wkhealth. com/ pt/ pt-core/ template-journal/ lwwgateway/ media/ landingpage. htm?an=00043764-200605000-00010). J. Occup. Environ. Med. 48 (5): 523–8. doi:10.1097/01.jom.0000201815.32098.99. PMID 16688009. . [5] J. Cenens and R. A. Schoonheydt (1988). "VISIBLE SPECTROSCOPY OF METHYLENE BLUE ON HECTORITE, LAPONITE B, AND BARASYM IN AQUEOUS SUSPENSION" (http:/ / www. clays. org/ journal/ archive/ volume 36/ 36-3-214. pdf). Clay and Clay Minerals 36 (3): 214-224. . [6] Spectroquant 114779 Sulfide Test. Method: photometric 0.020 - 1.50 mg/l S2- (http:/ / photometry. merck. de/ ) [7] Gillman P.K.; Ng, Bradley K.W.; Cameron, Andrew J.D.; Liang, Rhea W.Y. (May 2008). "Methylene blue is a potent monoamine oxidase inhibitor". Can. J. Anaesth. 55 (5): 311–312. doi:10.1007/BF03017212. PMID 18451123. [8] Gillman P.K. (October 2006). "Methylene blue implicated in potentially fatal serotonin toxicity". Anaesthesia 61 (10): 1013–4. doi:10.1111/j.1365-2044.2006.04808.x. PMID 16978328. [9] Healy, David (2002). The creation of psychopharmacology. Harvard University Press. ISBN 0-674-00619-4. [10] http:/ / www. rxlist. com/ prosed-ds-drug. htm [11] Schirmer H., Coulibaly B., Stich A. et al. (2003). "Methylene blue as an antimalarial agent—past and future". Redox Rep 8 (5): 272–276. doi:10.1179/135100003225002899. PMID 14962363. [12] Meissner P.E., Mandi G., Coulibaly B. et al. (2006). "Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine". Malaria Journal 5: 84. doi:10.1186/1475-2875-5-84. PMC 1617109. PMID 17026773. [13] NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis. Wondrak GT. Free Radic Biol Med. 2007 Jul 15;43(2):178-90 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 17603928?itool=EntrezSystem2. PEntrez. Pubmed. Pubmed_Results. Pubmed_RVDocSum& ordinalpos=1) [14] http:/ / cancerres. aacrjournals. org/ cgi/ content/ full/ 63/ 4/ 776 [15] Salah M., Samy N., Fadel M. (January 2009). "Methylene blue mediated photodynamic therapy for resistant plaque psoriasis". J. Drugs Dermatol. 8 (1): 42–9. PMID 19180895. [16] Tardivo J.P., Del Giglio A., Paschoal L.H., Baptista M.S. (August 2006). "New photodynamic therapy protocol to treat AIDS-related Kaposi's sarcoma". Photomed Laser Surg 24 (4): 528–31. doi:10.1089/pho.2006.24.528. PMID 16942436. [17] Papin J.F., Floyd R.A., Dittmer D.P. (November 2005). "Methylene blue photoinactivation abolishes West Nile virus infectivity in vivo". Antiviral Res. 68 (2): 84–7. doi:10.1016/j.antiviral.2005.07.001. PMID 16118025. [18] Zolfaghari P.S., Packer S., Singer M., Nair S.P., Bennett J., Street C., Wilson M. (2009). "In vivo killing of Staphylococcus aureus using a light-activated antimicrobial agent". BMC Microbiol. 9: 27. doi:10.1186/1471-2180-9-27. PMC 2642833. PMID 19193212. [19] Floyd R.A., Schneider J.E., Dittmer D.P. (March 2004). "Methylene blue photoinactivation of RNA viruses". Antiviral Res 61 (3): 141–51. doi:10.1016/j.antiviral.2003.11.004. PMID 15168794. [20] Wagner S.J., Skripchenko A., Pugh J.C., Suchmann D.B., Ijaz M.K. (September 2001). "Duck hepatitis B photoinactivation bydimethylmethylene blue in RBC suspensions". Transfusion 41 (9): 1154–8. doi:10.1046/j.1537-2995.2001.41091154.x. PMID 11552074. [21] Schagen F.H., Moor A.C., Cheong S.C., Cramer S.J., van Ormondt H., van der Eb A.J., Dubbelman T.M., Hoeben R.C. (May 1999). "Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation". Gene Ther. 6 (5): 873–81. doi:10.1038/sj.gt.3300897. PMID 10505113. [22] Müller-Breitkreutz K., Mohr H. (November 1998). "Hepatitis C and human immunodeficiency virus RNA degradation by methylene blue/light treatment of human plasma". J. Med. Virol. 56 (3): 239–45. doi:10.1002/(SICI)1096-9071(199811)56:3<239::AID-JMV11>3.0.CO;2-9. PMID 9783692.

Methylene blue [23] Wagner S.J., Skripchenko A., Robinette D., Mallory D.A., Hirayama J., Cincotta L., Foley J. (2000). "The use of dimethylmethylene blue for virus photoinactivation of red cell suspensions". Dev. Biol. (Basel) 102: 125–9. PMID 10794099. [24] Sturmey R.G., Wild C.P., Hardie L.J. (May 2009). "Removal of red light minimizes methylene blue-stimulated DNA damage in oesophageal cells: implications for chromoendoscopy". Mutagenesis 24 (3): 253–8. doi:10.1093/mutage/gep004. PMID 19218330. [25] Olliver J.R., Wild C.P., Sahay P., Dexter S., Hardie L.J. (August 2003). "Chromoendoscopy with methylene blue and associated DNA damage in Barrett's oesophagus". Lancet 362 (9381): 373–4. doi:10.1016/S0140-6736(03)14026-3. PMID 12907012. [26] Brent J. (2005). Critical care toxicology: diagnosis and management of the critically poisoned patient.. Elsevier Health Sciences. [27] http:/ / jtcs. ctsnetjournals. org/ cgi/ content/ abstract/ 125/ 6/ 1426 [28] http:/ / www. medscape. com/ viewarticle/ 409626 [29] Matilda Moldenhauer Brooks (1936). "Methylene blue as an antidote for cyanide and carbon monoxide poisoning". The Scientific Monthly 43 (6): 585–586. JSTOR 16280. [30] Dako Education Guide - Special Stains and H & E ” second edition Chapter 19: On Chemical Reactions and Staining Mechanisms by John A. Kiernan, Subsection What is Giemsa’s stain and how does it color blood cells, bacteria and chromosomes? p172 (http:/ / www. dako. com/ 08066_12may10_webchapter19. pdf) [31] J Exp Med. 1907 Nov 1;9(6):645-70. ON THE CHEMISTRY AND STAINING PROPERTIES OF CERTAIN DERIVATIVES OF THE METHYLENE BLUE GROUP WHEN COMBINED WITH EOSIN. Wilson TM. (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC2124692/ pdf/ 645. pdf) [32] Dacie and Lewis Practical Haematology 10th ed, p61 [33] Novella Steve. "The ethics of deception in medicine" (http:/ / www. sciencebasedmedicine. org/ ?p=29). Science Based Medicine. . Retrieved 2008-01-24. [34] "Methylene blue for cognitive dysfunction in bipolar disorder" (http:/ / clinicaltrials. gov/ ct2/ show/ NCT00214877). United States National Library of Medicine. September 20, 2005. . Retrieved 2009-02-15. [35] Alici-Evcimen Y., Breitbart W.S. (October 2007). "Ifosfamide neuropsychiatric toxicity in patients with cancer". Psychooncology 16 (10): 956–960. doi:10.1002/pon.1161. PMID 17278152. [36] Patel P.N. (2006). "Methylene blue for management of ifosfamide induced encephalopathy". Ann. Pharmacother. 40 (2): 266–303. doi:10.1345/aph.1G114. PMID 16391008. [37] Dufour C., Grill J., Sabouraud P. et al. (February 2006). "Ifosfamide induced encephalopathy: 15 observations" (in French). Arch. Pediatr. 13 (2): 140–145. doi:10.1016/j.ared.2005.10.021. PMID 16364615. [38] Aeschlimann T.; Cerny, T; Küpfer, A (1996). "Inhibition of (mono)amine oxidase activity and prevention of ifosfamide encephalopathy by methylene blue". Drug. Metab. Dispos. 24 (12): 1336–1339. PMID 8971139. [39] "Alzheimer's drug 'halts' decline" (http:/ / news. bbc. co. uk/ 1/ hi/ health/ 7525115. stm). BBC News. 2008-07-30. . Retrieved 2008-07-30. [40] "Slowing disease's mental ravages" (http:/ / web. archive. org/ web/ 20090130013724/ http:/ / archives. chicagotribune. com/ 2008/ jul/ 30/ health/ chi-alzheimers-wedjul30). Chicago Tribune. 2008-07-30. . [41] Medina DX, C. A., Oddo S (2011). "Methylene blue reduces aβ levels and rescues early cognitive deficit by increasing proteasome activity." (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC2992595/ ) Brain Pathology 21(2): 140-149. [42] Atamna H., Nguyen A., Schultz C., Boyle K., Newberry J., Kato H., Ames B.N. (March 2008). "Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways". FASEB J. 22 (3): 703–712. doi:10.1096/fj.07-9610com. PMID 17928358. [43] (http:/ / www. americanaquariumproducts. com/ AquariumMedication3. html) Aquarium Chemical Treatments [44] http:/ / www. plosone. org/ article/ info:doi/ 10. 1371/ journal. pone. 0005318 [45] Internet Movie Database entry for Decoy (http:/ / www. imdb. com/ title/ tt0038462/ ) [46] Film Noir of the Week entry for Decoy (http:/ / www. noiroftheweek. com/ 2007/ 08/ decoy-1946. html) [47] Mokhlesi B., Leikin J.B., Murray P., Corbridge T.C. (March 2003). "Adult toxicology in critical care: Part II: specific poisonings". Chest 123 (3): 897–922. doi:10.1378/chest.123.3.897. PMID 12628894. [48] Harvey J.W., Keitt A.S. (May 1983). "Studies of the efficacy and potential hazards of methylene blue therapy in aniline-induced methaemoglobinaemia". Br. J. Haematol. 54 (1): 29–41. doi:10.1111/j.1365-2141.1983.tb02064.x. PMID 6849836.

External links • NIH - Methylene blue test (http://www.nlm.nih.gov/medlineplus/ency/article/003412.htm) • Methylene blue (http://stainsfile.info/StainsFile/dyes/52015.htm) at stainsfile

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Microtome A microtome (from the Greek mikros, meaning "small", and temnein, meaning "to cut") is a tool used to cut extremely thin slices of material, known as sections. Important in science, microtomes are used in microscopy, allowing for the preparation of samples for observation under transmitted light or electron radiation. Microtomes use steel, glass, or diamond blades depending upon the specimen being sliced and the desired thickness of the sections being cut. Steel blades are used to prepare sections of animal or plant tissues for light microscopy histology. Glass knives are used to slice sections for light microscopy and to slice very thin sections for electron microscopy. Industrial grade An ultramicrotome used in microscopy. diamond knives are used to slice hard materials such as bone, teeth and plant matter for both light microscopy and for electron microscopy. Gem quality diamond knives are used for slicing thin sections for electron microscopy. Microtomy is a method for the preparation of thin sections for materials such as bones, minerals and teeth, and an alternative to electropolishing and ion milling. Microtome sections can be made thin enough to section a human hair across its breadth, with section thickness between 50 nm and 100 µm.

History In the beginnings of light microscope development, sections from plants and animals were manually prepared using razor blades. It was found that to observe the structure of the specimen under observation it was important to make clean reproducible cuts on the order of 100 µm, through which light can be transmitted. This allowed for the observation of samples using light microscopes in a transmission mode. One of the first devices for the preparation of such cuts was invented in 1770 by George Adams, Jr. (1750–1795) and further developed by Alexander Cummings.[2] The device was hand operated, and the sample held in a cylinder and sections created from the top of the sample using a hand crank.[1][3] In 1835, Andrew Prichard developed a table based model which allowed for the vibration to be isolated by affixing the device to the table, separating the operator from the knife.[4] Occasionally, attribution for the invention of the microtome is given to the anatomist Wilhelm His, Sr. (1865),[5][6] In his Beschreibung eines Mikrotoms (German for Description of a Microtome), Wilhelm wrote: The apparatus has enabled a precision in work by which I can achieve sections that by hand I cannot possibly create. Namely it has enabled the possibility of achieving unbroken sections of objects in the course of research.

A diagram of a microtome drawn by [1] Cummings in 1770.

Other sources further attribute the development to a Czech physiologist Jan Evangelista Purkyně. [7] Several sources describe the Purkyne model as the first in practical use.[8][9]

Microtome The obscurities in the origins of the microtome are due to the fact that the first microtomes were simply cutting apparatuses, and the developmental phase of early devices is widely undocumented. At the end of the 1800s, the development of very thin and consistently thin samples by microtomy, together with the selective staining of important cell components or molecules allowed for the visualisation of microscope details.[10][11] Today, the majority of microtomes are a knife-block design with a changeable knife, a specimen holder and an advancement mechanism. In most devices the cutting of the sample begins by moving the sample over the knife, where the advancement mechanism automatically moves forward such that the next cut for a chosen thickness can be made. The section thickness is controlled by an adjustment mechanism, allowing for precise control.

Applications The most common applications of microtomes are: • Traditional Histology Technique: tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 50 µm. From there the tissue can be mounted on a microscope slide, stained with appropriate aqueous dye(s) after prior removal of the paraffin, and examined using a light microscope. Microtome (C. Reichert, Vienna, 1905–1915). • Cryosectioning Technique: water-rich tissues are hardened by freezing and cut in the frozen state with a freezing microtome or microtome-cryostat; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs 16 hours) and is used in conjunction with medical procedures to achieve a quick diagnosis. Cryosections can also be used in immunohistochemistry as freezing tissue stops degradation of tissue faster than using a fixative and does not alter or mask its chemical composition as much.

• Electron Microscopy Technique: after embedding tissues in epoxy resin, a microtome equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometer). Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission electron microscope. This instrument is often called an ultramicrotome. The ultramicrotome is also used with its glass knife or an industrial grade diamond knife to cut survey sections prior to thin sectioning. These survey sections are generally 0.5 to 1 µm thick and are mounted on a glass slide and stained to locate areas of interest under a light microscope prior to thin sectioning for the TEM. Thin sectioning for the TEM is often done with a gem quality diamond knife. Complementing traditional TEM techniques ultramicrotomes are increasingly found mounted inside an SEM chamber so the surface of the block face can be imaged and then removed with the microtome to uncover the next surface for imaging. This technique is called Serial Block-Face Scanning Electron Microscopy (SBFSEM). • Botanical Microtomy Technique: hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier blades and cannot cut as thin as a regular microtome. • Spectroscopy (especially FTIR or Infrared spectroscopy) Technique: thin polymer sections are needed in order that the infra-red beam will penetrate the sample under examination. It is normal to cut samples to between 20 and 100 µm in thickness. For more detailed analysis of much smaller areas in a thin section, FTIR microscopy can be used for sample inspection. A recent development is the laser microtome, which cuts the target specimen with a femtosecond laser instead of a mechanical knife. This method is -free and does not require sample preparation techniques. The laser microtome has the ability to slice almost every tissue in its native state. Depending on the material being processed, slice thicknesses of 10 to 100 µm are feasible.

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Microtome types Sledge microtome

A sled microtome.

A sledge microtome is a device where the sample is placed into a fixed holder (shuttle), which then moves backwards and forwards across a knife. Modern sled microtomes have the sled placed upon a linear bearing, a design that allows for the microtome to readily cut many coarse sections.[12] By adjusting the angles between the sample and the microtome knife, the pressure applied to the sample during the cut can be reduced.[12] Typical applications for this design of microtome are of the preparation of large samples, such as those embedded in paraffin for biological preparations. Typical cut thickness achievable on a sledge microtome is between 1 and 60 µm.

Rotary microtome This instrument is a common microtome design. This device operates with a staged rotary action such that the actual cutting is part of the rotary motion. In a rotary microtome, the knife is typically fixed in a horizontal position.[13] In the figure to the left, the principle of the cut is explained. Through the motion of the sample holder, the sample is cut by the knife position 1 to position 2), at which point the fresh section remains on the knife. At the highest point of the rotary motion, the sample holder is advanced by the same thickness as the section that is to be made, allowing for the next section to be made. The flywheel in many microtomes can be operated by hand. This has the advantage that a clean cut can be made, as the relatively large mass of the flywheel prevents the sample from being stopped during the A rotary microtome of older construction. sample cut. The flywheel in newer models is often integrated inside the microtome casing. The typical cut thickness for a rotary microtome is between 1 and 60 µm. For hard materials, such as a sample embedded in a synthetic resin, this design of microtome can allow for good "Semi-thin" sections with a thickness of as low as 0.5 µm.

Microtome

206

Cryomicrotome For the cutting of frozen samples, many rotary microtomes can be adapted to cut in a liquid nitrogen chamber, in a so-called cryomicrotome setup. The reduced temperature allows for the hardness of the sample to be increased, such as by undergoing a glass transition, which allows for the preparation of semi-thin samples.[12] However the sample temperature and the knife temperature must be controlled in order to optimise the resultant sample thickness

A cryomicrotome.

Ultramicrotome An ultramicrotome is a main tool of ultramicrotomy. It can allow for the preparation of extremely thin sections, with the device functioning in the same manner as a rotational microtome, but with very tight tolerances on the mechanical construction. As a result of the careful mechanical construction, the linear thermal expansion of the mounting is used to provide very fine control of the thickness.[12] These extremely thin cuts are important for use with transmission electron microscope (TEM) and Serial Block-Face Scanning Electron Microscopy (SBFSEM), and are sometimes also A ribbon of ultrathin sections prepared by room temperature ultramicrotomy, floating on water in the important for light-optical microscopy.[13] The typical thickness of boat of a diamond knife used to cut the sections. The these cuts is between 40 and 100 nm for transmission electron knife blade is the edge at the upper end of the trough of microscopy and often between 30 and 50 nm for SBFSEM. water. Thicker sections up to 500 nm thick are also taken for specialized TEM applications or for light microscopy survey sections to select an area for the final thin sections. Diamond knives (preferably) and glass knives are used with ultramicrotomes. To collect the sections they are floated on top of a liquid as they are cut and are carefully picked up onto grids suitable for TEM specimen viewing. The thickness of the section can be estimated by the thin-film interference colors of reflected light that are seen as a result of the extremely low sample thickness.[14]

Vibrating microtome The vibrating microtome operates by cutting using a vibrating blade, allowing the resultant cut to be made with less pressure than would be required for a stationary blade. The vibrating microtome is usually used for difficult biological samples.[12] The cut thickness is usually around 30-500 µm for live tissue and 10-500 µm for fixed tissue.

Saw microtome The saw microtome is especially for hard materials such as teeth or bones. The microtome of this type has a recessed rotating saw, which slices through the sample. The minimal cut thickness is approximately 30 µm, and can be made for comparatively large samples.[12]

Microtome

Laser microtome The laser microtome is an instrument for free slicing.[15] Prior preparation of the sample through embedding, freezing or chemical fixation is not required, thereby minimizing the artifacts from preparation methods. Alternately this design of microtome can also be used for very hard materials, such as bones or teeth as well as some ceramics. Dependant upon the properties of the sample material, the thickness achievable is between 10 and 100 µm. The device operates using a cutting action of an infra-red laser. As the A conceptual diagram of laser microtome operation. laser emits a radiation in the near infra-red, in this wavelength regime the laser can interact with biological materials. Through sharp focusing of the probe within the sample, a focal point of very high intensity, up to TW/cm2, can be achieved. Through the non-linear interaction of the optical penetration in the focal region a material separation in a process known as photo-disruption is introduced. By limiting the laser pulse durations to the femtoseconds range, the energy expended at the target region is precisely controlled, thereby limiting the interaction zone of the cut to under a micrometre. External to this zone the ultra-short beam application time introduces minimal to no thermal damage to the remainder of the sample. The laser radiation is directed onto a fast scanning mirror based optical system which allows for three dimensional positioning of the beam crossover, whilst allowing for beam traversal to the desired region of interest. The combination of high power with a high raster rate allows the scanner to cut large areas of sample in a short time. In the laser microtome the laser-microdissection of internal areas in tissues, cellular structures, and other types of small features is also possible.

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Microtome knives The design of a microtome knife is dependant upon the material and preparation of the samples, as well as the final sample requirements (e.g. cut thickness and quality).

Knife design and cut types Generally, knives are characterized by the profile of the knife blade, which falls under the categories of planar concave, wedge shaped or chisel shaped designs. Planar concave microtome knives are extremely sharp, but are also very delicate and are therefore only used with very soft samples.[13] The wedge profile knives are somewhat more stable and find use in moderately hard materials, such as in epoxy or cryogenic sample cutting. Finally, the chisel profile with its blunt edge, raises the stability of the knife, whilst requiring significantly more force to achieve the cut. For ultramicrotomes, glass and diamond knives are required, the cut breadth of the blade is therefore on the order of a few millimetres and is therefore significantly smaller than for classical microtome knives. Glass knives are usually manufactured by the fracture of glass bars using special "knife-maker" fracturing devices. Glass knives may be used for initial sample preparations even where diamond knives may be used for final sectioning. Glass knives usually have small troughs, made with plastic tape, which are filled with water to allow the sample to float for later collection.[12] Diamond blades may be built into such an existing trough, allowing for the same collection method.

Profiles of microtome knives.

Sectioning Prior to cutting by microtomy, biological materials are usually placed in a more rigid fixative, in a process known as embedding. This is achieved by the inflow of a liquid substance around the sample, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a "block" which is readily cut. The declination is the angle of between the sample vertical and knife blade. If the knife blade is at right angles (declination=90) the cut is made directly using a pressure based mode, and the forces are therefore proportionally larger. If however the knife is tilted, the relative motion of the knife is increasingly parallel to sample motion, allowing for a slicing action. This behaviour is very important for large or hard samples The inclination of the knife is the angle between the knife face and the sample. For an optimal result, this angle must be chosen appropriately. The optimal angle depends upon the knife geometry, the cut speed and many other parameters. If the angle is adjusted to zero, the knife cut can often become erratic, and a new location of the knife must be used to smooth this out. If the angle is too large, the sample can crumple and the knife can induce periodic thickness variations in the cut. By further increasing the angle such that it is too large one can damage the knife blade itself.

Microtome

References [1] Hill, John (1770). The Construction of Timber, from its early growth; Explained by Microscope, and proven from Experiments, in a great Variety of Kinds. (http:/ / www. archive. org/ details/ constructiontim00hillgoog). London. pp. 5–11, Plate I. . [2] Quekett, John (1848). A Practical Treatise on the use of the Microscope (http:/ / www. archive. org/ details/ practicaltreatis00quekuoft). London: Hippolyte Bailliere. pp. 306, Chapter XII (Microtomes and Microtome Knives). . [3] Anonymous (1910). "An eighteenth century Microtome". Journal of the Royal Microscopical Society (Oxford, England: The Royal Microscopical Society): 779–782. [4] Gilbert Morgan Smith: The Development of Botanical Microtechnique. In: Transactions of the American Microscopical Society 34, Nr. 2. 1915, S. 71–129, ( PDF-Version of the article) (http:/ / scientificobjects. mpiwg-berlin. mpg. de/ scientificobjects/ dms/ ResearchNetworkDocuments/ basicdocuments/ V1_Smith--technique1915/ V1_Smith, technique1915. pdf) [5] "Wilhelm His" (http:/ / www. britannica. com/ EBchecked/ topic/ 266898/ Wilhelm-His). Encyclopædia Britannica Online. Encyclopædia Britannica. . Retrieved 24. März 2009. [6] Loukas, Marios; Pamela Clarke, R. Shane Tubbs, Theodoros Kapos and Margit Trotz (2008). "The His family and their contributions to cardiology" (http:/ / linkinghub. elsevier. com/ retrieve/ pii/ S0167527307003907). International Journal of Cardiology (Ireland: Elsevier) 123 (2): 75–78. doi:10.1016/j.ijcard.2006.12.070. ISSN 0167-5273. PMID 17433467. . Retrieved 24 März 2009. [7] "Histology" (http:/ / encarta. msn. com/ encyclopedia_761573144/ histology. html). msn Encarta. . Retrieved 18 March 2009. [8] Detlev Ganten: Handbuch der molekularen Medizin (Handbook of molecular medicine), Springer, ISBN 3-540-64552-7, ( Google-Books (http:/ / books. google. de/ books?id=DjwuzN4XvLMC& printsec=frontcover#PPA548,M1)) [9] Werner Gerabek, Bernhard D. Haage, Gundolf Keil, Wolfgang Wegner (2005): Enzyklopädie Medizingeschichte (Encyclopaedia of medical history), Walter de Gruyter, ISBN 3-11-015714-4, ( Google-Books (http:/ / books. google. de/ books?id=LLoOUP-y54YC& printsec=frontcover#PPA1203,M1)) [10] Ernst Mayr (2002). [ Google-Books (http:/ / books. google. de/ books?id=Y_HvUDa4OqwC& pg=PA533) Die Entwicklung der biologischen Gedankenwelt. (The evolution of the biological thought )]. Springer. ISBN 3-540-43213-2. . [11] Werner Linß, Werner Linb, Jochen Fanghänel: Histologie: Zytologie, allgemeine Histologie, mikroskopische Anatomie. (Histology: Cytology, general Histology, microscopial anatomy) Walter de Gruyter, 1998, ISBN 3-11-014032-2 ( Google-Books (http:/ / books. google. de/ books?id=S1HRxeGOQfMC& pg=PA1& dq=Purkinjes+ mikrotom& as_brr=3)) [12] Gudrun Lang (2006). Histotechnik. Praxislehrbuch für die Biomedizinische Analytik. (Histology : practical textbook for analytical biomedicine). Springer, Wien/New York. ISBN 3-211-33141-7. [13] Klaus Henkel: Das Schneiden mit dem Mikrotom (http:/ / www. mikroskopie-muenchen. de/ cut-mikrotom. html). Mikrobiologische Vereinigung München e. V., 2006, accessed 15 February 2009 [14] Peachey Lee D. (1958). "Thin Sections: A study of section thickness and physical distortion produced during microtomy" (http:/ / jcb. rupress. org/ cgi/ reprint/ 4/ 3/ 233. pdf). J. Biophysic. & Biochem. Cytol. 4 (3): 233–242. doi:10.1083/jcb.4.3.233. . [15] Holger Lubatschowski 2007: Laser Microtomy, WILEY-VCH Verlag GmbH, Biophotonics, S. 49-51, ( PDF (http:/ / www. photonicnet. de/ Aktuelles/ partner/ 2007/ 06/ laser_microtomy_optik-photonik_juni_2007. pdf)).

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Mordant A mordant is a substance used to set dyes on fabrics or tissue sections by forming a coordination complex with the dye which then attaches to the fabric or tissue.[1] It may be used for dyeing fabrics, or for intensifying stains in cell or tissue preparations. The term mordant comes from the Latin word, "mordere", to bite. In the past, it was thought that a mordant helped the dye bite onto the fiber so that it would hold fast during washing. A mordant is often a polyvalent metal ion.[2] The resulting coordination complex of dye and ion is colloidal and can be either acidic or alkaline.

Common dye mordants

A French Indienne, a printed or painted textile in the manner of Indian productions, which used mordants to fix the dyes.

Mordants include tannic acid, alum, urine, chrome alum, sodium chloride, and certain salts of aluminium, chromium, copper, iron, iodine, potassium, sodium, and tin. Iodine is often referred to as a mordant in Gram stains but is in fact a trapping agent.[3]

Dyeing methods The three methods used for mordanting are: • Pre-mordanting (onchrome): The substrate is treated with the mordant and then dyed. • Meta-mordanting (metachrome): The mordant is added in the dye bath itself. • Post-mordanting (afterchrome): The dyed material is treated with a mordant. The type of mordant used changes the shade obtained after dyeing and also affects the fastness property of the dye. The application of mordant, either pre-, meta- or post-mordant methods, is influenced by:

Dye rot from iron mordant

• The action of the mordant on the substrate: if the mordant and dye methods are harsh (e.g. an acidic mordant with an acidic dye), pre- or post- mordanting limits the potential for damage to the substrate. • The stability of the mordant and/or dye lake: the formation of a stable dye lake means that the mordant can be added in the dye without risk of losing the dye properties (meta-mordanting). Dye results can also rely on the mordant chosen as the introduction of the mordant into the dye will have a marked effect on the final color. Each dye can have different reactions to each mordant. For example, cochineal scarlet, or Dutch scarlet as it came to be known, used cochineal along with a tin mordant to create a brilliant orange-hued red.[4] Residual iron mordant can damage or fade fabric, producing "dye rot".[5]

Mordant

The dye lake The dye lake is an insoluble molecule formed when the complex of dye and mordant are combined, which then attaches to the substrate. Mordants increase the fastness of the dye since the larger molecule is now bonded to the fibre.[2] The term "lake" is derived from the term lac, the secretions of the Indian wood insect Laccifer lacca (formerly known as the Coccus lacca). This is the same insect from which shellac is obtained.[6] The type of mordant used can change the colour of both the dye-plus-mordant solution and influence the shade of the final product.

Wool Unlike cotton, wool is highly receptive toward mordants. Due to its amphoteric nature wool can absorb acids and bases with equal efficiency. When wool is treated with a metallic salt it hydrolyses the salt into an acidic and basic component. The basic component is absorbed at –COOH group and the acidic component is removed during washing. Wool also has a tendency to absorb fine precipitates from solutions; these cling to the surface of fibres and dye particles attached to these contaminants result in poor rubbing fastness.

Silk Like wool, silk is also amphoteric and can absorb both acids as well as bases. However, wool has thio groups (-SH) from the cystine amino acid, which act as reducing agent and can reduce hexavalent chromium of potassium dichromate to trivalent form. The trivalent chromium forms the complex with the fibre and dye. Therefore potassium dichromate cannot be used as mordant effectively.

Animal and plant Tissues In Histology, mordants are indispensable in adhering dyes to tissues for microscopic examination. Methods for mordant application depend on the desired stain and tissues under study; pre-, meta- and post-mordanting techniques are used as required. The most commonly used stain used in diagnostic histology of animal tissues is Harris' haematoxylin as part of a haematoxylin and eosin (H&E) stain.

References [1] Nic, M.; Jirat, J.; Kosata, B., eds. (2006–). "mordant" (http:/ / goldbook. iupac. org/ M04029. html). IUPAC Compendium of Chemical Terminology (Online ed.). doi:10.1351/goldbook.{{{file}}}. ISBN 0-9678550-9-8. . [2] Llewellyn, Bryan D. (May, 2005). "Stain Theory – How mordants work" (http:/ / web. archive. org/ web/ 20070814015208/ stainsfile. info/ StainsFile/ theory/ mordant. htm). . [3] Llewellyn, Bryan D. (May, 2005). "Stain Theory – Trapping agents" (http:/ / web. archive. org/ web/ 20080620165727/ stainsfile. info/ StainsFile/ theory/ trapping. htm). . [4] Phipps, Elena (2010). Cochineal Red The Art History of a Color, p34. Yale University Press, New Haven and London. ISBN 978-0-300-15513-6. [5] (http:/ / www. quilthistory. com/ cleaning. htm) [6] Llewellyn, Bryan D. (May, 2005). "Stain Theory – Lac" (http:/ / web. archive. org/ web/ 20070813220846/ stainsfile. info/ StainsFile/ dyes/ 75450. htm). .

211

Negative stain

Negative stain Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. For bright field microscopy, negative staining is typically performed using a black ink fluid such as nigrosin. The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark surrounding background. An alternative method has been developed using an ordinary waterproof marking pen to deliver the negative stain.[1] In the case of transmission electron microscopy, opaqueness to electrons is related to the atomic number, i.e., the number of protons. Some suitable negative stains include ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osmium ferricyanide[2] and auroglucothionate. These have been chosen because they scatter electrons well and also adsorb to biological matter well. The structures which can be negatively stained are much smaller than those studied with the light microscope. Here, the method is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power. Some stains, such as osmium tetroxide and Electron micrograph of (organic) plant tissue osmium ferricyanide, are very chemically active. As strong oxidants, without (top) and with OsO4 staining they cross-links lipids mainly by reacting with unsaturated carbon-carbon bonds, and thereby both fix biological membranes in place in tissue samples and simultaneously stain them.[3][4] The choice of negative stain in electron microscopy can be very important. A study of plant viruses from negatively-stained leaf dips of a diseased plant showed only spherical viruses with one stain and only rod-shaped viruses with another. The verified conclusion was that this plant suffered from a mixed infection by two separate viruses. Negative staining at both light microscope and electron microscope level should never be performed with infectious organisms unless stringent safety precautions are followed. Negative staining is a very mild preparation method and does not reduce the possibility of operator infection.

212

Negative stain

References [1] S. Woeste and P. Demchick (1991). Appl Environ Microbiol. 57(6): 1858-1859 http:/ / aem. asm. org/ cgi/ content/ abstract/ 57/ 6/ 1858 [2] D. Chadwick (2002). Role of the sarcoplasmic reticulum in smooth muscle. John Wiley and Sons. pp. 259–264. ISBN 0-470-84479-5. [3] Bozzola, John J.; Russell, Lonnie D. (1999). "Specimen Preparation for Transmission Electron Microscopy" (http:/ / books. google. de/ books?id=RqSMzR-IXk0C& pg=PA21). Electron microscopy : principles and techniques for biologists. Sudbury, Mass.: Jones and Bartlett. pp. 21–31. ISBN 978-0-7637-0192-5. . [4] M. A. Hayat (2000). Principles and techniques of electron microscopy: biological applications (http:/ / books. google. co. jp/ books?id=nfsVMH8it1kC& hl=en). Cambridge University Press. pp. 45–61. ISBN 0-521-63287-0. .

External links • "Negative staining for dummies" (http://www.snaggledworks.com/em_for_dummies/negative_stain.html). Retrieved 2009-06-06. • "Negative staining" (http://web.uct.ac.za/depts/mmi/stannard/negstain.html). Retrieved 2009-06-06.

213

Neutral red

214

Neutral red Neutral red


553-24-2

ChemSpider

10634

Jmol-3D images

Image 1

[2]

[1]

[3]


C15H17ClN4

Molar mass

288.78 g/mol

Melting point

290°C

Boiling point

°C ()

[4]


Infobox references

Neutral red (pH indicator) below pH 6.8 6.8

above pH 8.0 ↔

8.0

Neutral Red (or toluylene red, Basic Red 5, or C.I. 50040) is a eurhodin dye used for staining in histology. It stains lysosomes red.[5] It is used as a general stain in histology, as a counterstain in combination with other dyes, and for many staining methods. Together with Janus Green B, it is used to stain embryonal tissues and supravital staining of blood. Can be used for staining Golgi apparatus in cells and Nissl granules in neurons. Neutral red can be used as a vital stain, to stain living cells. It is used to stain cell cultures for plate titration of viruses. Neutral Red is added to some growth media for bacteria and cell cultures. It usually comes as a chloride salt. Neutral Red acts as a pH indicator, changing from red to yellow between the pH 6.8-8.0.

Neutral red

References [1] [2] [3] [4] [5]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=553-24-2 http:/ / www. chemspider. com/ 10634 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=Cl. n1c3c%28nc2c1cc%28c%28c2%29N%29C%29cc%28N%28C%29C%29cc3 http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=448562119& page2=%3ANeutral+ red Winckler, J. Vital staining of lysosomes and other cell organelles of the rat with neutral Red. Prog. Histochem. Cytochem. 6, 1–89 (1974).

215

Nile blue

216

Nile blue Nile blue

Identifiers [1]

CAS number

3625-57-8

PubChem

422690

ChemSpider

374117

ChEMBL

CHEMBL1185333

Jmol-3D images

Image 1

[2] [3]

[4]

, CHEMBL407862

[5]

[6]


C20H20ClN3O

Molar mass

353.845 g/mol ()

[7]


Infobox references

Nile blue (or Nile blue A) is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei. It may also be used in conjunction with fluorescence microscopy to stain for the presence of polyhydroxybutyrate granules in prokaryotic or eukaryotic cells. Boiling a solution of Nile blue with sulfuric acid produces Nile red (Nile blue oxazone).

Nile blue

217

Chemical and physical properties Nile blue is a fluorescent dye. The fluorescence shows especially in nonpolar solvents with a high quantum yield.[8]

Nile blue hydrochloride in water. Concentrations, left to right: 1000 ppm, 100 ppm, 10 ppm, 1 ppm, 100 ppb.

Nile blue in water. Left to right: pH 0, pH 4, pH 7, pH 10, pH 14.

Nile blue in water (lower phase) and ethyl acetate (upper phase) in daylight. Left to right: pH 0, pH 4, pH 7, pH 10, pH 14

Nile blue in water (lower phase) and ethyl acetate (upper phase) in UV light (366 nm). Left to right: pH 0, pH 4, pH 7, pH 10, pH 14

The absorption and emission maxima of Nile blue are strongly dependent on pH and the solvents used:[8]

Nile blue

218

Nile blue (free base) in daylight (top row) and UV light (366 nm, bottom row) in different solvents. Left to right: 1. methanol, 2. ethanol, 3. methyl-tert-butylether, 4. cyclohexane, 5. n-hexane, 6. acetone, 7. tetrahydrofuran, 8. ethyl acetate, 9. dimethyl formamide, 10. acetonitrile, 11. toluene, 12. chloroform

Solvent

Absorption λ max (nm) Emission λ max (nm)

Toluene

493

574

Acetone

499

596

Dimethylformamide

504

598

Chloroform

624

647

1-Butanol

627

664

2-propanol

627

665

Ethanol

628

667

Methanol

626

668

Water

635

674

1.0 N hydrochloric acid (pH = 1.0)

457

556

0.1 N sodium hydroxide solution (pH = 11.0) 522

668

Ammonia water (pH = 13.0)

668

524

The duration of Nile blue fluorescence in ethanol was measured as 1.42 ns. This is shorter than the corresponding value of Nile red with 3.65 ns. The fluorescence duration is independent on dilution in the range 10−3–10−8 mol/L.[8]

Nile blue staining Nile blue is used for histological staining of biological preparations. It highlights the distinction between neutral lipids (triglycerides, cholesteryl esters, steroids) which are stained pink and acids (fatty acids, chromolipids, phospholipids) which are stained blue.[9] The Nile blue staining, according to Kleeberg, uses the following chemicals: • Nile Blue A • 1% acetic acid • Glycerol or glycerol gelatin

Nile blue

Workflow The sample or frozen sections is/are fixated in formaldehyde, then immersed for 20 minutes in the Nile blue solution and rinsed with water. For better differentiation, it is dipped in 1% acetic acid for 10–20 minutes until the colors are pure. This might take only 1–2 minutes. Then the sample is thoroughly rinsed in water (for one to two hours). Afterwards, the stained specimen is taken on a microscope slide and excess water is removed. The sample can be embedded in glycerol or glycerol gelatin.

Results Unsaturated glycerides are pink, nuclei and elastins(?) dark, fatty acids and various fatty substances and fat mixtures are purple blue.[10] Example: Detection of poly-β-hydroxybutyrate granules (PHB) The PHB granules in the cells of Pseudomonas solanacearum can be visualized by Nile blue A staining. The PHB granules in the stained smears are observed with an epifluorescence microscope under oil immersion, at a 1000 times magnification; under 450 nm excitation wavelength they show a strong orange fluorescence.[11]

Nile blue in DNA Electrophoresis Nile blue is also apparently used in a variety of commercial DNA staining formulations used for DNA electrophoresis.[12] As it does not require UV trans-illumination in order to be visualised in an agarose gel as with ethidium bromide, it can be used to observe DNA as it is separated and also as a dye to aid in gel-extraction of DNA fragments without incurring damage by UV-irradiation.

Nile blue in oncology Derivatives of Nile blue are potential photosensitizers in photodynamic therapy of malignant tumors. These dyes aggregate in the tumor cells, especially in the lipid membranes, and/or are sequestered and concentrated in subcellular organelles.[13] With the Nile blue derivative N-Ethyl-Nile Blue (EtNBA), normal and premalignant tissues in animal experiments can be distinguished by fluorescence spectroscopy in fluorescence imaging. EtNBA shows no phototoxic effects.[14]

References [1] [2] [3] [4] [5] [6]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=3625-57-8 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=422690 http:/ / www. chemspider. com/ 374117 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL1185333 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL407862 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=N%3D1c3c%28OC%3D2C%3D1%5CC%3DC%2FC%28N%28CC%29CC%29C%3D2%29cc%28c4c3cccc4%29N [7] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=476996698& page2=%3ANile+ blue [8] Jose, Jiney; Burgess, Kevin (2006). "Benzophenoxazine-based fluorescent dyes for labeling biomolecules" (http:/ / www. chem. tamu. edu/ cfi/ Publications/ 221. pdf). Tetrahedron 62 (48): 11021. doi:10.1016/j.tet.2006.08.056. . [9] Roche Lexikon, accessed 25 June 2007 (http:/ / gripsdb. dimdi. de/ rochelexikon/ ro25000/ r27033. html). [10] Benno Romeis, Mikroskopische Technik, 15. Aufl., R. Oldenbourg Verlag, München, 1948 [11] 97/647/EG: Entscheidung der EU-Kommission vom 9. September 1997 über ein vorläufiges Versuchsprogramm für Diagnose, Nachweis und Identifizierung von Pseudomonas solanacearum (Smith) Smith in Kartoffeln, accessed 27 June 2007 (http:/ / eur-lex. europa. eu/ LexUriServ/ LexUriServ. do?uri=CELEX:31997D0647:DE:NOT). [12] PDF DNA staining protocol for schools, University of Reading (http:/ / www. ncbe. reading. ac. uk/ ncbe/ PROTOCOLS/ DNA/ PDF/ DNA14. pdf) [13] Lin, CW; Shulok, JR; Kirley, SD; Cincotta, L; Foley, JW (1991). "Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells". Cancer Research 51 (10): 2710–9. PMID 2021950.

219

Nile blue

220

[14] Van Staveren, HJ; Speelman, OC; Witjes, MJ; Cincotta, L; Star, WM (2001). "Fluorescence imaging and spectroscopy of ethyl nile blue a in animal models of (pre)malignancies". Photochemistry and photobiology 73 (1): 32–8. doi:10.1562/0031-8655(2001)073<0032:FIASOE>2.0.CO;2. PMID 11202363.

Nile red Nile red

Identifiers [1]

CAS number

7385-67-3

PubChem

65182

ChemSpider

58681

ChEBI

CHEBI:52169

ChEMBL

CHEMBL144472

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]


C H NO

Molar mass

318.369 g/mol

20 18 2 2

()

[7]


Infobox references

Nile red

Nile red under visible and ultraviolet (366 nm) light in different solvents From left to right: 1. water, 2. methanol, 3. ethanol, 4. acetonitrile, 5. dimethylformamide, 6. acetone, 7. ethyl acetate, 8. dichloromethane, 9. n-hexane, 10. methyl-tert-butylether, 11. cyclohexane, 12. toluene.

221 Nile red (also known as Nile blue oxazone) is a lipophilic stain. It is produced by boiling a solution of Nile blue with sulfuric acid.[8] As can be seen from the structural formulae, this process replaces an amino group with a carbonyl group. Nile red stains intracellular lipid droplets red. In most polar solvents Nile Red will not fluoresce, however when in a lipid-rich environment can be intensely fluorescent, with varying colours from deep red to strong yellow-gold emission. Whilst it generally excites at 485 nm, and emits at 525 nm (552/636 nm in methanol), the fluorescence of the dye is heavily dependent on the solvent used, and in some cases does not fluoresce at all.[9] Since the reaction to generate Nile red does not usually completely exhaust the supply of Nile blue, additional separation steps are required if pure Nile red is needed.

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=7385-67-3 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=65182 [3] http:/ / www. chemspider. com/ 58681 [4] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=52169 [5] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL144472 [6] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=CCN%28CC%29c1ccc2c%28c1%29oc-3cc%28%3DO%29c4ccccc4c3n2 [7] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=476992673& page2=%3ANile+ red [8] SD Fowler and P Greenspan (1985). "Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O". Journal of Histochemistry and Cytochemistry 33 (8): 833–836. [9] P Greenspan, E. P. Mayer and S. D. Fowler (1985). "Nile Red, A Selective Fluorescent Stain for Intracellular Lipid Droplets". Journal of Cell Biology 100 (1): 965–973.

Oil Red O

222

Oil Red O Oil Red O


1320-06-5

PubChem

5841742

ChemSpider

14217961

MeSH

oil+red+O

Jmol-3D images

Image 1

[1]

[2] [3]

[4]

[5]


C26H24N4O

Molar mass

408.49496 ()

[6]


Infobox references

Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N4O) is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance of a red powder with maximum absorption at 518 (359)nm.

Uses Oil Red O is one of the dyes used for Sudan staining. Similar dyes include Sudan III, Sudan IV, and Sudan Black B. The staining has to be performed on fresh samples, as alcohol fixation removes the lipids. Oil Red O largely replaced Sudan III and Sudan IV, as it provides much deeper red color and the stains are therefore much easier to see. In pyrotechnics, Oil Red O is used in some compositions of red colored smokes. It is also used to dye some plastics, e.g. polystyrene resins.

Oil Red O

Forensic When staining, Oil Red O can make fat more visible in various cuts in pathology.[7] It is also used in a technique (the method is called as the dye: Oil Red O), discovered in 2004 by Alexandre Beaudoin, for staining latent fingerprints.[8] This technique allows the development of latent fingerprints on porous exhibits (such as paper, cardboard, etc.) that are dry or wet. It mainly targets fat deposits on the surface of porous exhibits.[9] It is a non-destructive technique (which does not destroy the exhibit and doesn’t prevent the use of other techniques). It is a safe alternative to the Physical Developer method,[10] and is also used in sequence with other methods of fingerprints development.[11]

References [1] [2] [3] [4] [5]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=1320-06-5 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=5841742 http:/ / www. chemspider. com/ 14217961 http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=oil+ red+ O http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=Cc4cc%28%2FN%3DN%2Fc1cc%28C%29c%28cc1C%29%2FN%3DN%2Fc2c3ccccc3ccc2O%29c%28C%29cc4

[6] [7] [8] [9]

http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=401252700& page2=%3AOil+ Red+ O "Forensic Pathology" (http:/ / library. med. utah. edu/ WebPath/ FORHTML/ FOR002. html). . Triplett M, Fingerprint Dictionary, Two Rings Publishing, Bellevue, Washington. Beaudoin, A. New technique for revealing latent fingerprints on wet, porous surfaces: Oil Red O. Journal of Forensic Identification, 2004, 54 (4), 413-421. [10] Rawji, A. ; Beaudoin, A. Oil Red O versus Physical Developer on wet papers: a comparitive study. Journal of Forensic Identification, 2006, 56 (1), 33-54. [11] Guigui, K.; Beaudoin, A. The use of Oil Red O in sequence with other methods of fingerprint development. Journal of Forensic Identification, 2007, 57 (4), 550-581.

External links • Stains File (http://stainsfile.info/StainsFile/dyes/26125.htm) entry

223

Orange G

224

Orange G Orange G

Identifiers [1]

CAS number

1936-15-8

PubChem

9566064

ChemSpider

10468647

KEGG

C19372

ChEMBL

CHEMBL410263

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

, CHEMBL1615565

[6]

[7]


C16H10N2Na2O7S2

Molar mass

452.38 g/mol Hazards

Main hazards

R36/37/38, S26, S36 ()

[8]


Infobox references

Orange G or orange gelb[9] is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.

Orange G

225

Staining The main use of Orange G is in the OG-6 Papanicolaou stain, to stain keratin, however it is also a major component of the Alexander test for pollen staining. It is often combined with other yellow dyes and used to stain erythrocytes in the trichrome methods.

Color marker Orange G can be used as a color marker to monitor the process of 1% agarose gel electrophoresis, running approximately at the size of a 50 Base pair (bp) DNA molecule, and polyacrylamide gel electrophoresis. Bromophenol blue and xylene cyanol can also be used for this purpose. (However, the apparent "size" of all these dyes varies according to the concentration of agarose in the gel and the buffer system used, so one should look up the appropriate reference before using the dyes to determine how far a gel has run.)

pH indicator Despite its two ionizable groups, it shows only two colors in aqueous solution, brilliant orange in neutral and acidic pH or red in pH greater than 9.

References [1] [2] [3] [4] [5] [6] [7]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=1936-15-8 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=9566064 http:/ / www. chemspider. com/ 10468647 http:/ / www. kegg. jp/ entry/ C19372 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL410263 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL1615565 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BNa%2B%5D. %5BNa%2B%5D. %5BO-%5DS%28%3DO%29%28%3DO%29c3cc2ccc%28O%29c%28%2FN%3DN%2Fc1ccccc1%29c2c%28c3%29S%28%5BO-%5D%29%28%3DO%29%3D [8] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=462265517& page2=%3AOrange+ G [9] Carson, Freida L; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. p. 362. ISBN 978-0-89189-581-7.

Orcein

226

Orcein Orcein, also archil, orchil, lacmus, Citrus Red 2, and C.I. Natural Red 28, are names for dyes extracted from several species of lichen, commonly known as "orchella weeds", found in various parts of the world. A major source is the archil lichen, Roccella tinctoria. Orcinol is extracted from such lichens. It is then converted to orcein by ammonia and air. In traditional dye-making methods, urine was used as the ammonia source. If the conversion is carried out in the presence of potassium carbonate, calcium hydroxide, and calcium sulfate (in the form of potash, lime, and gypsum in traditional dye-making methods), the result is litmus, a more complex molecule.[1] The manufacture was described by Cocq in 1812 [2] and in the UK in 1874.[3] Commercial archil is either a powder (called cudbear) or a paste. It is red in acidic pH and blue in alkaline pH. Orcein is approved as a food dye, with E number E121. Its CAS number is [1400-62-0 [4]]. Its chemical formula is C28H24N2O7. It forms dark brown crystals. The chemical components of orcein were elucidated only in the 1950s by Hans Musso.[5] The structures are shown below. A single alternative structure, possibly incorrect, is given by the National Library of Medicine [6] and Emolecules [4]. Orcein is a reddish-brown dye, orchil is a purple-blue dye. Orcein is also used as a stain in microscopy to visualize elastic fibers, Hepatitis B surface antigens, and copper-associated proteins. It is a mixture of phenoxazone derivates hydroxyorceins, aminoorceins, and aminoorceinimines.

Cudbear Cudbear is a dye extracted from orchil lichens that produces colours in the purple range. It can be used to dye wool and silk, without the use of mordant. Cudbear was developed by Dr Cuthbert Gordon of Scotland: production began in 1758, and it was patented in 1758, British patent 727 [7]. The lichen is first boiled in a solution of ammonium carbonate. The mixture is then cooled and ammonia is added and the mixture is kept damp for 3–4 weeks. Then the lichen is dried and ground to powder. The manufacture details were carefully protected, with a ten-feet high wall being built around the manufacturing facility, and staff consisting of Highlanders sworn to secrecy. The lichen consumption soon reached 250 tons per year and import from Norway and Sweden had to be arranged.[8] Cudbear was the first dye to be invented in modern times, and one of the few dyes to be credited to a named individual. Similar process was invented in . The lichen is extracted by urine or ammonia. Then the extract is acidified, the dissolved dye precipitates and is washed. Then it is dissolved in ammonia again, the solution is heated in air until it becomes purple, then it is precipitated with calcium chloride; the resulting insoluble purple solid is known as French purple, a fast lichen dye that did not fade in light like the other lichen dyes.

Gallery

α-amino orcein

α-hydroxy orcein

β-amino orcein

β-hydroxy orcein

Orcein

227

β-amino orceinimine

γ-amino orcein

γ-hydroxy orcein

γ-amino orceinimine

References [1] Beecken, H; E-M Gottschalk, U v Gizycki, et al. (2003). "Orcein and litmus". Biotechnic & Histochemistry 78 (6): 289–302. doi:10.1080/10520290410001671362. [2] Cocq M. (1812). Mémoire sur la fabrication et l'emploi de l'orseille. Annales de Chimie 81:258–278. Cited in: Chevreul ME. (1830). Leçons de chimie appliquée à la teinture (http:/ / www. archive. org/ details/ leonsdechimiea02chev). Paris: Pichon et Didier. p 114–116. [3] A Leeds Workman. 1874. Manufacture of Archil and Cudbear (http:/ / books. google. com/ books?id=NAYAAAAAMAAJ& pg=RA1-PA143#v=onepage& q& f=false). Chemical News 30(173):143. [4] http:/ / www. emolecules. com/ cgi-bin/ search?t=ss& q=1400-62-0& c=1& v= [5] Musso H. 1960. Orcein- und Lackmusfarbstoffe: Konstitutionsermittlung und Konstitutionsbeweis durch die Synthese. (Orcein and litmus pigments: constitutional elucidation and constitutional proof by synthesis.) Planta Medica 8:431–446. doi:10.1055/s-0028-1101580. [6] http:/ / chem. sis. nlm. nih. gov/ chemidplus/ jsp/ common/ ChemInfo. jsp?calledFrom=lite& type=formulas [7] http:/ / www. chriscooksey. demon. co. uk/ lichen/ bp727. html [8] http:/ / gdl. cdlr. strath. ac. uk/ stecit/ stecit05. htm

External links • Orchil, the poor person's purple (http://www.ravensgard.org/gerekr/Orchil.html)

Osmium tetroxide

228

Osmium tetroxide Osmium tetroxide

Identifiers [1]

CAS number

20816-12-0

PubChem

30318 , 56370778 [5] (monotemediate)

ChemSpider

28158

EC number

244-058-7

UN number

UN 2471

MeSH

Osmium+tetroxide

RTECS number

RN1140000

Jmol-3D images

Image 1

[2]

[6]

[3]

(monopotassiate) , 75811001

[4]

(monoquinuclidiniate) , 53113021

[7]

[8]

[9]


OsO

Molar mass

254.23 g/mol

Appearance

pale yellow solid

Odor

acrid, chlorine-like

Density

4.9 g/cm3

Melting point

40.25 °C

Boiling point

129.7 °C

Solubility in water

5.70 g/100 mL (10 °C) 6.23 g/100 mL (25 °C)

Solubility

375 g/100 mL (CCl ) 4 soluble in most organic solvents, ammonium hydroxide, phosphorus oxychoride

4

[10]

Structure Crystal structure

Monoclinic, mS20

Osmium tetroxide

Space group

229 [11]

C2/c; a = 0.4515 nm, b = 0.52046 nm, c = 0.80838 nm, α = 77.677°, β = 73.784°, γ = 64.294° Hazards [12]

MSDS

ICSC 0528

EU Index

076-001-00-5

EU classification

Very toxic (T+) Corrosive (C)

R-phrases

R26/27/28, R34

S-phrases

(S1/2), S7/9, S26, S45

NFPA 704 Related compounds Other cations

Ruthenium tetroxide

Related osmium oxides

Osmium(IV) oxide

()

[13]


Infobox references

Osmium tetroxide (also called osmium tetraoxide) is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite the rarity of osmium. It also has a number of interesting properties, one being that the solid is volatile.

Physical properties Osmium tetroxide exists as a pale yellow-brown crystalline solid (monoclinic crystal symmetry[11]) with a characteristic acrid chlorine-like odor.[14] The element name osmium is derived from osme, Greek for odor. OsO4 is volatile: it sublimes at room temperature. It is soluble in a wide range of organic solvents, and moderately soluble in water, with which it reacts reversibly to form osmic acid (see below).[15] Pure osmium tetroxide is probably [11] colourless[16] and it has been suggested that its yellow hue is due to Crystal structure of OsO4 osmium dioxide (OsO2) impurities.[17] The Osmium tetroxide molecule is tetrahedral and therefore non-polar. This nonpolarity helps OsO4 penetrate charged cell membranes. OsO4 is 518 times more soluble in CCl4 than in water.

Structure and electron configuration With a d0 configuration, Os(VIII) is expected to form tetrahedral complexes when bound to four ligands. Tetrahedral structures are seen for the electronically related oxides MnO4– and CrO42–. The osmium of OsO4 has a formal oxidation state of +8, the highest oxidation state known for a transition metal. The osmium atom has eight valence electrons. If one assumes that two electrons are donated by each of the four oxide ligands, the total electron count for the complex is 16, as also seen for the isoelectronic species permanganate and chromate.

Osmium tetroxide The high oxidation state of osmium in this compound can be rationalized by comparison of main-group and transition-metal chemistry. Just as the elements in groups 3 through 7 form compounds analogous to those formed by elements in groups 13 through 17 (e.g. TiCl4 and GeCl4, VF5 and AsF5, CrO42− and SeO42−, etc.), we might expect the elements in group 8 to form compounds analogous to those formed by the noble gases. This is the case, as demonstrated by the existence of compounds like OsO4 and XeO4.

Synthesis OsO4 is formed slowly when osmium powder reacts with O2 at ambient temperature. Reaction of bulk solid requires heating to 400 °C.[18] Os + 2 O2 → OsO4

Reactions Oxofluorides Osmium forms several oxofluorides, all of which are very sensitive to moisture. Purple cis-OsO2F4 forms at 77 K in an anhydrous HF solution:[19] OsO4 + 2 KrF2 → cis-OsO2F4 + 2 Kr + O2 OsO4 also reacts with F2 to form yellow OsO3F2:[20] 2 OsO4 + 2 F2 → 2 OsO3F2 + O2 OsO4 reacts with one equivalent of [Me4N]F at 298 K and 2 equivalents at 253 K:[18] OsO4 + [Me4N]F → [Me4N][OsO4F] OsO4 + 2 [Me4N]F → [Me4N]2[cis-OsO4F2]

Oxidation of alkenes OsO4 catalyzes the cis-dihydroxylation of alkenes by hydrogen peroxide or related sources of oxygen atoms in the presence of water. The reaction that is catalyzed is[21] R2C=CR2 + H2O2 → R2C(OH)-C(OH)R2.

In of mechanism, OsVIIIO4 adds to alkenes R2C=CR2 to afford cyclic "esters" R4C2O2OsVIO2, which undergo hydrolysis to give the vicinal diol and release a reduced osmium oxide (OsVI):

Lewis bases such as tertiary amines and pyridines increase the reaction rate. This "ligand-acceleration" arises via the formation of adduct OsO4L, which adds more rapidly to the alkene. If the amine is chiral, then the dihydroxylation can proceed with enantioselectivity (see Sharpless asymmetric dihydroxylation).[21] OsO4 is used in catalytic amounts due to its toxicity and high cost. The osmium catalyst is regenerated by oxidizing agents, such as H2O2, N-methylmorpholine N-oxide (NMO, see Upjohn dihydroxylation), and K3Fe(CN)6. These

230

Osmium tetroxide oxidizing reagents do not react with the alkenes on their own. Other sources of osmium tetroxide include potassium osmate(VI) dihydrate (K2OsO4·2H2O) and osmium(III) chloride hydrate (OsCl3·xH2O) which oxidise to osmium(VIII) in the presence of such oxidants.[22]

Miscellaneous reactions OsO4 does not react with most carbohydrates.[23] It dissolves in alkaline aqueous solution to give the osmate anion OsO2(OH)42−.[24] OsO4 is a Lewis acid, and when the Lewis bases are amines, the oxides can undergo substitution. Thus with NH3 one obtains the nitrido-oxide: OsO4 + NH3 + KOH → K[Os(N)O3] + 2 H2O The [Os(N)O3]- anion is isoelectronic and isostructural with OsO4. Using primary amine tert-BuNH2 one obtains the corresponding imido derivative: OsO4 + 4 Me3CNH2 → Os(NCMe3)4 + 4 H2O OsO4 is very soluble in tert-butanol and in solution is readily reduced by molecular hydrogen to osmium metal. The suspended osmium metal can be used to catalyze hydrogenation of a wide variety of organic chemicals containing double or triple bonds. OsO4 + 4 H2 (g) → Os (s) + 4 H2O OsO4 undergoes "reductive carbonylation" with carbon monoxide in methanol at 400 K and 200 bar of pressure to produce the triangular cluster Os3(CO)12: 3 OsO4 + 24 CO → Os3(CO)12 + 12 CO2[] In this reaction osmium changes oxidation state by eight units.

Uses Organic synthesis In organic synthesis OsO4 is widely used to oxidise alkenes to the vicinal diols, adding two hydroxyl groups at the same side (syn addition). See reaction and mechanism above. This reaction has been made both catalytic (Upjohn dihydroxylation) and asymmetric (Sharpless asymmetric dihydroxylation). Osmium tetroxide is also used in catalytic amount in the Sharpless oxyamination to give vicinal amino-alcohols. In combination with sodium periodate, OsO4 is used for the oxidative cleavage of alkenes (Lemieux-Johnson oxidation) when the periodate serves both to cleave the diol formed by dihydroxylation, and to reoxidize the OsO3 back to OsO4. The net transformation is identical to that produced by ozonolysis. Below an example from the total synthesis of Isosteviol.[25]

231

Osmium tetroxide

Biological staining OsO4 is a widely used staining agent used in transmission electron microscopy (TEM) to provide contrast to the image.[26] As a lipid stain, it is also useful in scanning electron microscopy (SEM) as an alternative to sputter coating. It embeds a heavy metal directly into cell membranes, creating a high secondary electron emission without the need for coating the membrane with a layer of metal, which can obscure details of the cell membrane. In the staining of the plasma membrane, osmium tetroxide binds phospholipid head regions, thus creating contrast with the neighbouring protoplasm (cytoplasm). Additionally, osmium tetroxide is also used for fixing biological samples in conjunction with HgCl2. Its rapid killing abilities are used to quickly kill specimen like protozoa. OsO4 stabilizes many proteins by transforming them into gels without destroying structural features. Tissue proteins that are stabilized by OsO4 are not coagulated by alcohols during dehydration.[23] Osmium tetroxide is also used as a stain for lipids in optical microscopy.[27] OsO4 also stains the human cornea (see safety considerations).

Polymer staining It is also used to stain copolymers preferentially, the best known example being block copolymers where one phase can be stained so as to show the microstructure of the material. For example, Electron micrograph of (organic) plant tissue without (top) and with OsO4 staining styrene-butadiene block copolymers have a central polybutadiene chain with polystyrene end caps. When treated with OsO4, the butadiene matrix reacts preferentially and so absorbs the oxide. The presence of a heavy metal is sufficient to block the electron beam, so the polystyrene domains are seen clearly in thin films in TEM.

Osmeth OsO4 can be recycled and stored in the form of osmeth, a golden crystalline solid. Osmeth is OsO4 complexed with hexamine and does not emit toxic fumes as opposed to pure OsO4. It can be dissolved in tetrahydrofuran (THF) and diluted in an aqueous buffer solution to make a dilute (0.25%) working solution of OsO4.[28]

Osmium ore refining OsO4 is an intermediate in osmium ore refining. Osmium residues are reacted with Na2O2 forming [OsO4(OH)2]2− anions, which, when reacted with chlorine (Cl2) gas and heated, form OsO4. The oxide is dissolved in alcoholic NaOH forming [OsO2(OH)4]2− anions, which, when reacted with NH4Cl, forms OsO2Cl2(NH4)4. This is ignited under hydrogen (H2) gas leaving behind pure osmium (Os).[15]

232

Osmium tetroxide

Buckminsterfullerene adduct OsO4 allowed for the confirmation of the soccer ball model of buckminsterfullerene, a 60 atom carbon allotrope. The adduct, formed from a derivative of OsO4, was C60(OsO4)(4-tert-butylpyridine)2. The adduct broke the fullerene's symmetry allowing for crystallization and confirmation of the structure of C60 by X-ray crystallography.[29]

Safety considerations OsO4 is highly poisonous, even at low exposure levels, and must be handled with appropriate precautions. In particular, inhalation at concentrations well below those at which a smell can be perceived can lead to pulmonary edema, and subsequent death. Noticeable symptoms can take hours to appear after exposure. OsO4 also stains the human cornea, which can lead to blindness if proper safety precautions are not observed. The permissible exposure limit for osmium tetroxide (8 hour Label with poison warning 3 [14] time-weighted average) is 2 µg/m . Osmium tetroxide can penetrate plastics and therefore is stored in glass in a cold place.[23] On April 6, 2004 British intelligence sources believed they had foiled a plot to detonate a bomb involving OsO4.[30] Experts interviewed by New Scientist affirmed osmium tetroxide's toxicity, though some highlighted the difficulties of using it in a weapon: osmium tetroxide is very expensive. The osmium tetroxide may be destroyed by the blast; what remaining toxic fumes may also be dispersed by the blast as well.[31]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=20816-12-0 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=30318 [3] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=56370778 [4] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=75811001 [5] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=53113021 [6] http:/ / www. chemspider. com/ 28158 [7] http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=244-058-7 [8] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Osmium+ tetroxide [9] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=O%3D%5BOs%5D%28%3DO%29%28%3DO%29%3DO [10] "Osmium tetroxide ICSC: 0528" (http:/ / www. inchem. org/ documents/ icsc/ icsc/ eics0528. htm). InChem. . [11] Krebs, B.; Hasse, K. D. (1976). "Refinements of the Crystal Structures of KTcO4, KReO4 and OsO4. The Bond Lengths in Tetrahedral Oxo-Anions and Oxides of d0 Transition Metals". Acta Crystallographica B 32 (5): 1334–1337. doi:10.1107/S056774087600530X. [12] http:/ / www. inchem. org/ documents/ icsc/ icsc/ eics0528. htm [13] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=451133444& page2=%3AOsmium+ tetroxide [14] "Osmium tetroxide (as Os)" (http:/ / www. cdc. gov/ niosh/ idlh/ 20816120. html). Documentation for Immediately Dangerous to Life or Health Concentrations (IDLHs). Centers for Disease Control. . [15] Thompson, M.. "Osmium tetroxide (OsO4)" (http:/ / www. chm. bris. ac. uk/ motm/ oso4/ oso4h. htm). Bristol University. . Retrieved 2012-04-07. [16] Butler, I. S.; Harrod, J. F. (1989). Inorganic Chemistry: Principles and Applications (http:/ / books. google. com/ books?id=Nd3vAAAAMAAJ). Benjamin / Cummings. p. 343. ISBN 978-0-8053-0247-9. . Retrieved 2012-04-07. [17] Cotton, F. A. (2007). Advanced Inorganic Chemistry (http:/ / books. google. com/ books?id=U3MWRONWAmMC) (6th ed.). New Delhi, India: J. Wiley. p. 1002. ISBN 978-81-265-1338-3. . [18] Housecroft, C. E.; Sharpe, A. G. (2004). Inorganic Chemistry (2nd ed.). Prentice Hall. pp. 671–673, 710. ISBN 978-0130399137.

233

Osmium tetroxide [19] Christe, K. O.; Dixon, D. A.; Mack, H. G.; Oberhammer, H.; Pagelot, A.; Sanders, J. C. P.; Schrobilgen, G. J. (1993). "Osmium tetrafluoride dioxide, cis-OsO2F4". Journal of the American Chemical Society 115 (24): 11279–11284. doi:10.1021/ja00077a029. [20] Cotton, S. A. (1997). Chemistry of Precious Metals. London: Chapman and Hall. ISBN 0-7514-0413-6. [21] Berrisford, D. J.; Bolm, C.; Sharpless, K. B. (1995). "Ligand-Accelerated Catalysis". Angewandte Chemie International Edition 34 (10): 1059–1070. doi:10.1002/anie.199510591. [22] Ogino, Y.; Chen, H.; Kwong, H.-L.; Sharpless, K. B. (1991). "On the timing of hydrolysis / reoxidation in the osmium-catalyzed asymmetric dihydroxylation of olefins using potassium ferricyanide as the reoxidant". Tetrahedron Letters 32 (32): 3965–3968. doi:10.1016/0040-4039(91)80601-2. [23] Hayat, M. A. (2000). Principles and Techniques of Electron Microscopy: Biological Applications (http:/ / books. google. com/ ?id=nfsVMH8it1kC). Cambridge University Press. pp. 45–61. ISBN 0-521-63287-0. . [24] Dulski, T. R. (1996). A Manual for the Chemical Analysis of Metals (http:/ / books. google. com/ books?id=ViOMjoLKB1gC& pg=PA130& lpg=PA130). ASTM International. p. 130. ISBN 0-8031-2066-4. . [25] Snider, B. B.; Kiselgof, J. Y.; Foxman, B. M. (1998). "Total Syntheses of (±)-Isosteviol and (±)-Beyer-15-ene-3β,19-diol by Manganese(III)-Based Oxidative Quadruple Free-Radical Cyclization". Journal of Organic Chemistry 63 (22): 7945–7952. doi:10.1021/jo981238x. [26] Bozzola, J. J.; Russell, L. D. (1999). "Specimen Preparation for Transmission Electron Microscopy" (http:/ / books. google. com/ ?id=RqSMzR-IXk0C& pg=PA21). Electron Microscopy : Principles and Techniques for Biologists. Sudbury, MA: Jones and Bartlett. pp. 21–31. ISBN 978-0-7637-0192-5. . [27] Di Scipio, F.; Raimondo, S.; Tos, P.; Geuna, S. (2008). "A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation". Microscopy Research and Technique 71 (7): 497–502. doi:10.1002/jemt.20577. PMID 18320578. [28] Kiernan, J. A.. "Re: "Disposal" of Osmium Tetroxide "Waste"" (http:/ / www. histosearch. com/ histonet/ Nov00A/ Re. quotDisposalquotofOsmi. html). Department of Anatomy & Cell Biology, The University of Western Ontario. . [29] Hawkins, J. M.; Meyer, A.; Lewis, T. A.; Loren, S.; Hollander, F. J. (1991). "Crystal Structure of Osmylated C60: Confirmation of the Soccer Ball Framework". Science 252 (5003): 312–313. doi:10.1126/science.252.5003.312. PMID 17769278. [30] "Chemical 'bomb plot' in UK foiled" (http:/ / news. bbc. co. uk/ 1/ hi/ uk/ 3603961. stm). BBC News. 2004-04-06. . [31] Bhattacharya, S. (2004-04-07). "Experts divided over poison bomb claim" (http:/ / technology. newscientist. com/ article/ dn4863-experts-divided-over-poison-bomb-claim. html). New Scientist. .

External links • • • • • •

International Chemical Safety Card 0528 (http://www.inchem.org/documents/icsc/icsc/eics0528.htm) NIOSH Pocket Guide to Chemical Hazards (http://www.cdc.gov/niosh/npg/npgd0473.html) BBC report on bomb plot (http://news.bbc.co.uk/1/hi/uk/3603961.stm) BBC What is Osmium tetroxide article (http://news.bbc.co.uk/1/hi/uk/3604857.stm) Osmium Tetroxide: Molecule of the Month (http://www.chm.bris.ac.uk/motm/oso4/oso4h.htm) Chemical Reactions (http://www.organic-chemistry.org/chemicals/oxidations/osmiumtetroxide.shtm)

234

Papanicolaou stain

235

Papanicolaou stain Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic staining cytological technique developed by George Papanikolaou, the father of cytopathology. Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells.

Papanicolaou stain showing a low-grade squamous intraepithelial lesion (LSIL). Pap test.

Pap staining is a very reliable technique. As such, it is used for cervical cancer screening in gynecology. The entire procedure is known as Pap smear.

The classic form of Pap stain involves five dyes in three solutions:[1] • A nuclear stain, haematoxylin, is used to stain cell nuclei. The unmordanted haematein may be responsible for the yellow color imparted to glycogen. • First OG-6 counterstain (-6 denotes the used concentration of phosphotungstic acid; other variants are OG-5 and OG-8). Orange G is used. It stains keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum. • Second EA (Eosin Azure) counterstain, comprising three dyes; the number denotes the proportion of the dyes, e.g. EA-36, EA-50, EA-65. • Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells. • Light Green SF yellowish stains the cytoplasm of other cells, including non-keratinized squamous cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, however it produces visually different results and is not considered satisfactory by some. • Bismarck brown Y stains nothing and in contemporary formulations it is often omitted. When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin patterns are well visible, the cells from borderline lesions are easier to interpret and the photomicrographs are better. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted. On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once. Pap stain is not fully standardized; it comes in several versions, subtly differing in the exact dyes used, their ratios, and timing of the process. The EA stain contains two mutually incompatible chemicals, Bismarck brown and phosphotungstic acid, which precipitate each other, impairing the useful life of the mixture and compromising the differential staining of eosin and light green. The descriptions of the compositions of the staining solutions vary by source and differ even in Papanicolaou's own publications. Mixtures of the same name from different vendors therefore can differ in composition, occasionally producing different or poor results.

Papanicolaou stain

236

Ultrafast Papanicolaou stain Ultrafast Papanicolaou stain is an alternative for the fine needle aspiration samples, developed to achieve comparable visual clarity in significantly shorter time. The process differs in rehydration of the air-dried smear with saline, use 4% formaldehyde in 65% ethanol fixative, and use of Richard-Allan Hematoxylin-2 and Cyto-Stain, resulting in a 90-second process yielding transparent polychromatic stains.[2]

References [1] Carson, Freida L; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. pp. 361-3363. ISBN 978-0-89189-581-7. [2] (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Retrieve& db=PubMed& list_uids=7531380& dopt=Abstract)

Paraffin In chemistry, paraffin is a term that can be used synonymously with "alkane", indicating hydrocarbons with the general formula CnH2n+2. Paraffin wax refers to a mixture of alkanes that falls within the 20 ≤ n ≤ 40 range; they are found in the solid state at room temperature and begin to enter the liquid phase past approximately 37 °C (unknown operator: [1] u'strong' °F). The simplest paraffin molecule is that of methane, CH4, a gas at room temperature. Heavier of the series, such as octane, C8H18, and mineral oil appear as liquids at room temperature. The solid forms of paraffin, called paraffin wax, are from the heaviest molecules from C20H42 to C40H82. Paraffin wax was identified by Carl Reichenbach in 1830.[2]

Paraffin wax

Paraffin, or paraffin hydrocarbon, is also the technical name for an alkane in general, but in most cases it refers specifically to a linear, or normal alkane — whereas branched, or isoalkanes are also called isoparaffins. It is distinct from the fuel known in the United Kingdom, Ireland and South Africa as paraffin oil or just paraffin, which is called kerosene in most of the U.S., Canada, Australia and New Zealand. The name is derived from Latin parum ("barely") + affinis, meaning "lacking affinity" or "lacking reactivity" indicating paraffin's unreactive nature [3])

Paraffin

Paraffin wax Paraffin wax (or simply "paraffin", but see alternative name for kerosene, above) is mostly found as a white, odorless, tasteless, waxy solid, with a typical melting point between about 46 and 68 °C (115 and 154 °F),[4] and having a density of around 0.9 g/cm3.[5] It is insoluble in water, but soluble in ether, benzene, and certain esters. Paraffin is unaffected by most common chemical reagents but burns readily.[6] Pure paraffin wax is an excellent electrical insulator, with an electrical resistivity of between 1013 and 1017 ohm metre.[7] This is better than nearly all other materials except some plastics (notably Teflon). It is an effective neutron and was used in James Chadwick's 1932 experiments to identify the neutron.[8][9] Paraffin wax is an excellent material to store heat, having a specific heat capacity of 2.14–2.9 J g−1 K−1 (joule per gram kelvin) and a heat of fusion of 200–220 J g−1.[10] This property is exploited in modified drywall for home building material: a certain type (with the right melting point) of wax is infused in the drywall during manufacture so that, when installed, it melts during the day, absorbing heat, and solidifies again at night, releasing the heat.[11] Paraffin wax phase change cooling coupled with retractable radiators was used to cool the electronics of the Lunar Rover.[12] Wax expands considerably when it melts and this allows its use in wax thermostatic elementthermostats for industrial, domestic and, particularly, automobile purposes.[13][14] In industrial applications, it is often useful to modify the crystal properties of the paraffin wax, typically by adding branching to the existing carbon backbone chain. The modification is usually done with additives, such as EVA copolymers, microcrystalline wax, or forms of polyethylene. The branched properties result in a modified paraffin with a higher viscosity, smaller crystalline structure, and modified functional properties. Pure paraffin wax is rarely used for carving original models for casting metal and other materials in the lost wax process, as it is relatively brittle at room temperature and presents the risks of chipping and breakage when worked. Soft and pliable waxes, like beeswax, may be preferred for such sculpture, but "investment casting waxes," often paraffin-based, are expressly formulated for the purpose.

Mineral oil Liquid paraffin, or mineral oil, is a mixture of heavier alkanes, and has a number of names, including nujol, adepsine oil, alboline, glymol, medicinal paraffin, or saxol. It has a density of around 0.8 g/cm3.[5] Medicinal liquid paraffin is used to aid bowel movement in persons suffering chronic constipation; it es through the gastrointestinal tract without itself being taken into the body, but it limits the amount of water removed from the stool. In the food industry, where it may be called "wax", it can be used as a lubricant in mechanical mixing, applied to baking tins to ensure that loaves are easily released when cooked and as a coating for fruit or other items requiring a "shiny" appearance for sale.[15] It is often used in infrared spectroscopy, as it has a relatively uncomplicated IR spectrum. When the sample to be tested is made into a mull (a very thick paste), liquid paraffin is added so it can be spread on the transparent (to infrared) mounting plates to be tested. Mineral oil has also seen widespread use in biotechnology for preventing the evaporation of small volumes of liquid during heating. Polymerase chain reaction samples may need to be overlaid with a layer of mineral oil to prevent evaporation[16] during the high heat (95 °C) required to denature DNA.

237

Paraffin

Uses Gaseous • Fuels

Liquids • • • • • • • • • •

Fuels Paints, Pigments, Dyes and Inks In leather industry for "pull up finish"[17] Medicine (Laxative) Biomedical science (evaporation control during PCR) Culinary Fire breathing and fire juggling Used in toiletries and cosmetics as a moisturiser or emollient. Used in some pesticides Cooling media for liquid submergence cooling of mainframe and home computers.

Paraffin wax • Candle-making • Coatings for waxed paper or cloth • Food-grade paraffin wax:

• • • • • • • • • • • • • •

•

• Shiny coating used in candy-making; although edible, it is nondigestible, ing right through the body without being broken down • Coating for many kinds of hard cheese, like Edam cheese • Sealant for jars, cans, and bottles • Chewing gum additive Investment casting Anti-caking agent, moisture repellent, and dustbinding coatings for fertilizers Agent for preparation of specimens for histology Bullet lubricant – with other ingredients, such as olive oil and beeswax Phlegmatizing agent, commonly used to stabilise/desensitize high explosives such as RDX Crayons Solid propellant for hybrid rocket motors[18] Component of surfwax, used for grip on surfboards in surfing Component of glide wax, used on skis and snowboards Friction-reducer, for use on handrails and cement ledges, commonly used in skateboarding Ink. Used as the basis for solid ink different color blocks of wax for thermal printers. The wax is melted and then sprayed on the paper producing images with a shiny surface Microwax[19]: food additive, a glazing agent with E number E905 Forensics aid: the nitrate test uses paraffin wax to detect nitrates and nitrites on the hand of a shooting suspect Antiozonant agents: blends of paraffin and micro waxes are used in rubber compounds to prevent cracking of the rubber; the ixture of wax migrates to the surface of the product and forms a protective layer. The layer can also act as a release agent, helping the product separate from its mould.[20] Mechanical thermostats and actuators, as an expansion medium for activating such devices[14]

• "Potting" guitar pickups, which reduces microphonic caused from the subtle movements of the pole pieces

238

Paraffin • • • • •

"Potting" of local oscillator coils to prevent microphonic frequency modulation in low end FM radios. Wax baths for beauty and therapy purposes Thickening agent in many Paintballs, as used by Crayola An effective, although comedogenic, moisturiser in toiletries and cosmetics such as Vaseline Prevents oxidation on the surface of polished steel and iron[21]

References Notes [1] Freund, Mihály; Mózes, Gyula; Jakab, E. (trans) (1982). Paraffin products: properties, technologies, applications. Amsterdam, Netherlands: Elsevier. p. 121. ISBN 0-444-99712-1. [2] Britannica (1911) [3] "Paraffin, n". Oxford English Dictionary. Oxford, England: Oxford University Press. March 2009. [4] Nasser, William E (1999). "Waxes, Natural and Synthetic". In McKetta, John J. Encyclopedia of Chemical Processing and Design. 67. New York: Marcel Dekker. p. 17. ISBN 0-8247-2618-9. This can vary widely, even outside the quoted range, according to such factors as oil content and crystalline structure. [5] Kaye, George William Clarkson; Laby,Thomas Howell. "Mechanical properties of materials" (http:/ / www. kayelaby. npl. co. uk/ general_physics/ 2_2/ 2_2_1. html). Kaye and Laby Tables of Physical and Chemical Constants. National Physical Laboratory. . Retrieved 2008-03-06. [6] Seager, Spencer L.; Slabaugh, Michael. "Alkane reactions". Chemistry for Today: General, Organic, and Biochemistry. Belmont, CA: Cengage. p. 364. ISBN 978-0-538-73332-8. [7] "Electrical insulating materials" (http:/ / www. kayelaby. npl. co. uk/ general_physics/ 2_6/ 2_6_3. html). Kaye and Laby Tables of Physical and Chemical Constants. National Physical Laboratory. 1995. . Retrieved 2007-04-23. [8] "Attenuation of fast neutrons: neutron moderation and diffusion" (http:/ / www. kayelaby. npl. co. uk/ atomic_and_nuclear_physics/ 4_7/ 4_7_3. html). Kaye and Laby Tables of Physical and Chemical Constants. National Physical Laboratory. . Retrieved 2007-04-23. [9] Rhodes, Richard (1986). The Making of the Atomic Bomb. New York: Simon and Schuster. pp. 163. ISBN 0-671-44133-7. [10] "Specific Heat Capacity" (http:/ / www. diracdelta. co. uk/ science/ source/ s/ p/ specific heat capacity/ source. html). Diracdelta.co.uk Science and Engineering Encyclopedia. Dirac Delta Consultants Ltd, Warwick, England. . Retrieved 2007-08-18. [11] "Micronal PCM SmartBoard" (http:/ / www. micronal. de/ portal/ basf/ ien/ dt. jsp?setCursor=1_290798). . [12] Dean, W. G.; Karu; Karu, Z. S. (February 1993). "Space Station thermal storage/refrigeration system research and development". Final Report Lockheed Missiles and Space Co. (NASA). Bibcode 1993lock.rept.....D. [13] Wax-pellet thermostat (http:/ / www. freepatentsonline. com/ 4948043. html) United States Patent 4948043 [14] Bodén, Roger. "Paraffin Microactuator" (http:/ / hermes. material. uu. se/ ~klas/ Paraffin_lab_eng. pdf). Materials Science Sensors and Actuators. University of Uppsala. . Retrieved 2007-04-23. [15] "Mineral Oil (Food Grade)" (http:/ / www. inchem. org/ documents/ jecfa/ jecmono/ v10je08. htm). WHO Food Additives Series 10. Food and Agriculture Organization of the United Nations; World Health Organization. 1976. . Retrieved 2007-08-21. [16] Hayat, M. A. (2005). Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 3: Molecular Genetics: Liver and Pancreatic Carcinomas. Amsterdam: Elsevier. p. 342. ISBN 0-12-088404-6. "Failure to use the required mineral oil overlay will result in the evaporation of samples..." [17] "Dyed saddle leather—German pull up finish" (http:/ / www. je-sedgwick. co. uk/ products/ saddle-leather/ dyed-saddle-leather-german-pull-up-finish/ ). Walsall, England: J & E Sedgwick. . Retrieved 14 April 2010. "applying specially formulated mineral oils to the open grain of the leather" [18] Staff (Fall 2004). "Rocket motor uses common household product for fuel" (http:/ / www. nasa. gov/ centers/ stennis/ pdf/ 69281main_fall. pdf). OASIS Ocean Air Space Industry Site (Stennis Space Center Pearlington, MS: NASA) 1 (3): 6. . Retrieved 28 November 2008. [19] http:/ / www. microcrystallinewax. net [20] Freund, etc., (1982:272) [21] Dick, William B. "Encyclopedia Of Practical Receipts And Processes" (http:/ / chestofbooks. com/ reference/ Encyclopedia-Of-Practical-Receipts-And-Processes/ Steel-Part-6. html). . Retrieved 2008-04-27.

239

Periodic acid-Schiff stain

240

Periodic acid-Schiff stain Periodic acid-Schiff (PAS) is a staining method used to detect polysaccharides such as glycogen, and neutral mucosubstances such as glycoproteins, glycolipids and neutral mucins in tissues. The reaction of periodic acid oxidizes the vicinal diols in these sugars, usually breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide units that are parts of the long polysaccharides, and creating a pair of aldehydes at the two free tips of each broken monosaccharide ring. The oxidation condition has to be sufficiently regulated so as to not oxidize the aldehydes further. These aldehydes then react with the Schiff reagent to give a purple-magenta color. A suitable basic stain is often used as a counterstain.

Periodic acid

Uses PAS staining is mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in e.g. connective tissues, mucus, the glycocalyx, and basal laminae. PAS staining can be used to assist in the diagnosis of several medical conditions: • Glycogen storage disease (versus other storage disorders); • Adenocarcinomas, which often secrete neutral mucins; • Paget disease of the breast[1]; • Alveolar soft part sarcoma [2];

Gastric signet ring cell carcinoma histopatholgy, PAS stain.

• staining macrophages in Whipple's disease[3]; • It can be used to diagnose α1-antitrypsin deficiency if periportal liver hepatocytes stain positive; • aggregates of PAS positive lymphocytes are present in epidermis in Mycosis fungoides and Sezary syndrome, called Pautrier microabscesses; • erythroleukemia, a leukemia of immature red blood cells. These cells stain a bright fuchsia.[4]; • Pulmonary alveolar proteinosis; • Fungal infection, the cell walls of fungi stain magenta. This only works on living fungi; in contrast, Grocott's methenamine silver stain(GMS) will stain both living and dead fungal organisms.

Esophageal candidiasis, PAS stain.

Presence of glycogen can be confirmed on a section of tissue by using diastase to digest the glycogen from a section, then comparing a diastase digested PAS section with a normal PAS section. The diastase negative slide will show a magenta staining where glycogen is present within a section of tissue. The slide that has been treated with diastase will lack any positive PAS staining in those locations on the slide PAS staining is also used for staining cellulose. One example would be looking for implanted medical devices composed of nonoxidized cellulose.

Periodic acid-Schiff stain

241

If the PAS stain will be performed on tissue, the recommended fixative is 10% neutral-buffered formalin or Bouin solution. For blood smears, the recommended fixative is methanol. Glutaraldehyde is not recommended because free aldehyde groups may be available to react with the Schiff reagent, which may result in false positive staining.[5]

References [1] Thomas J. Lawton (27 April 2009). Breast (http:/ / books. google. com/ books?id=z17R70VGhnsC& pg=PA55). Cambridge University Press. pp. 55–. ISBN 978-0-521-88159-3. . Retrieved 16 November 2010. [2] ( Ladanyi et al 2002 (http:/ / ajp. amjpathol. org/ cgi/ content/ full/ 160/ 4/ 1215) [3] C. Ha (29 August 2005). Mayo Clinic Gastroenterology and Hepatology Board Review (http:/ / books. google. com/ books?id=nStxzRQlNaAC& pg=PA108). CRC Press. pp. 108–. ISBN 978-0-203-50274-7. . Retrieved 16 November 2010. [4] http:/ / www. answers. com/ topic/ leukemia-stains-1 [5] Carson, Freida L.; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. pp. 137–139. ISBN 978-0-89189-581-7.

External links • PAS Reaction (http://stainsfile.info/StainsFile/stain/schiff/reaction-pas.htm)

Phosphate buffered saline Phosphate buffered saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer's phosphate groups help to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

Applications PBS has many uses because it is isotonic and non-toxic to cells. These uses include substance dilution and cell container rinsing. PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. For these types of applications, Good's buffers are recommended.

Preparation There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium[1]. Generally, PBS contains the following constituents:

One of the common composition of PBS Salt (—)

Concentration Concentration (mmol/L)

(g/L)

NaCl

137

8.01

KCl

2.7

0.20

Na2HPO4 • 2 H2O

10

1.78

KH2PO4

2.0

0.27

pH

7.4

7.4

Phosphate buffered saline The simplest way to prepare a PBS solution is to use PBS buffer tablets. They are formulated to give a ready to use PBS solution upon dissolution in a specified quantity of distilled water. They are available in the standard volumes: 100, 200, 500 and 1000 mL [2]. If used in cell culturing, the solution can be dispensed into aliquots and sterilized by autoclaving (20 min, 121°C, liquid cycle). Sterilization may not be necessary depending on its use. PBS can be stored at room temperature, but may warrant refrigeration to prevent bacterial growth if solution is not sterile and is kept for long periods of time. However, concentrated stock solutions may precipitate when cooled and should be kept at room temperature until precipitate has completely dissolved before use.

References [1] Dulbecco, R. et al. (1954): Plaque formation and isolation of pure lines with poliomyelitis viruses. In: J. Exp. Med. vol. 99 (2), pp. 167-182. doi:10.1084/jem.99.2.167 PMID 13130792 [2] Medicago AB, (2010) Phosphate bufered saline specification sheet (http:/ / www. medicago. se/ sites/ default/ files/ pdf/ productsheets/ PBS_Buffer_v. _01. pdf)

242

Phosphotungstic acid

243

Phosphotungstic acid Phosphotungstic acid

Identifiers [1]

CAS number

1343-93-7 , 12501-23-4 (hydrate) Properties

Molecular formula

H3PW12O40

Molar mass

2880.2 g/mol (anhydrous)

Melting point

89 °C (hydrate) ()

[2]


Infobox references

Phosphotungstic acid (PTA), tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula H3PW12O40. It is normally present as a hydrate. EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly yellow-green crystals, with melting point 89 °C (24 H2O hydrate). It is odorless and soluble in water (200 g/100 ml). It is not especially toxic, but is a mild acidic irritant. The compound is known by a variety of different names and acronyms (see 'other names' section of infobox). In these names the "12" or "dodeca" reflects the fact that the anion contains 12 tungsten atoms. Some early workers who did not know the structure, such as Hsien Wu,[3] called it phospho-24-tungstic acid, formulating it as 3H2O.P2O5 24WO3.59H2O, (P2W24O80H6).29H2O, which correctly identifies the atomic ratios of P, W and O. This formula was still quoted in papers as late as 1970.[4] Phosphotungstic acid is used in histology as a component for staining of cell specimens, often together with haematoxylin as PTAH. It binds to fibrin, collagen, and fibres of connective tissues, and replaces the anions of dyes from these materials, selectively decoloring them. Phosphotungstic acid is electron dense, opaque for electrons. It is a common negative stain for viruses, nerves, polysaccharides, and other biological tissue materials for imaging by a transmission electron microscope.

Phosphotungstic acid

244

Structure Gouzerh[5] summarises the historical views on the structure of phosphotungstic acid leading up to Keggin's determination of the structure as: • H7[P(W2O7)6] proposed by Miolati and further developed by Rosenheim • H3[PO4W12O18(OH)36] (Pauling) The structure was determined by J.F Keggin first published in 1933[6] and then in 1934[7] and is generally known as the Keggin structure. The anion has full tetrahedral symmetry and comprises a cage of twelve tungsten atoms linked by oxygen atoms with the phosphorus atom at its centre. The picture on the right shows the octahedral coordination of oxygen atoms around the tungsten atoms, and that the surface of the anion has both bridging and terminal oxygen atoms. Further investigation showed that the compound was a hexahydrate not a pentahydrate as Keggin had proposed.[8]

Structure of the phosphotungstate anion

Preparation and chemical properties Phosphotungstic acid can be prepared by the reaction of sodium tungstate, Na2WO4.2H2O, with phosphoric acid, H3PO4, acidified with hydrochloric acid, HCl.[3] Phosphotungstic acid solutions decompose as the pH is increased. A step-wise decomposition has been determined and the approximate compositions at various pH values are as follows:[9] pH

principal components

1.0 [PW O ]3− 12 40 2.2 [PW O ]3−, [P W O ]6−, [PW O ]7− 12 40 2 21 71 11 39 3.5 [PW O ]3−, [P W O ]6−, [PW O ]7−, [P W O ]6−, [P W O ]10− 12 40 2 21 71 11 39 2 18 62 2 19 67 5.4 [P W O ]6−, [PW O ]7−, [P W O ]6− 2 21 71 11 39 2 18 62 7.3 [PW O ]9− 9 34 8.3 PO 3−, WO 2− 4 4

The species [PW11O39]7− is a lacunary, or defective Keggin ion. The [P2W18O62]6− has a Dawson structure. At pH less than 8, the presence of ethanol or acetone stabilises the anion, [PW12O40]3−, reducing decomposition.[9] Tungstophosphoric acid is thermally stable up to 400 °C, and is more stable than the analogous silicotungstic acid, H4SiW12O40.[10] Large quantities of polar molecules such as pyridine are absorbed into the bulk phase and not simply on the surface. Solid state NMR studies of ethanol absorbed in the bulk phase show that both protonated dimers, ((C2H5OH)2H+) and monomers, (C2H5OH2+) are present. Phosphotungstic acid is less sensitive to reduction than phosphomolybdic acid. Reduction with uric acid or iron(II) sulfate produces a brown coloured compound. the related silicotungstic acid when reduced forms a similar brown

Phosphotungstic acid compound where one of the four W3 units in the Keggin structure becomes a metal-metal bonded cluster of three edge shared W(IV) octahedra.[11] Phosphotungstic acid is the strongest of heteropolyacids. Its conjugate base is the PW12O403− anion.[12] Its acidity in acetic acid has been investigated and shows that the three protons dissociate independently rather than sequentially, and the acid sites are of the same strength.[13] One estimate of the acidity is that the solid has an acidity stronger than H0 =−13.16,[10] which would qualify the compound as a superacid. This acidic strength means that even at low pH the acid is fully dissociated.

Uses Catalyst In common with the other heteropolyacids phosphotungstic acid is a catalyst and its high acidity and thermal stability make it a catalyst of choice according to some researchers.[14] It is in solution as a homogeneous catalyst, and as a heterogeneous catalyst "ed" on a substrate e.g. alumina, silica. Some acid catalysed reactions include: • the homogeneous catalysis of the hydrolysis of propene to give 2-propanol • the homogeneous catalysis of the Prins reaction • the heterogeneous catalysis of the dehydration of 2-propanol to propene and methanol to hydrocarbons.

Dyeing and pigments Phosphotungstic acid has been used to precipitate different types of dyes as "lakes".[15] Examples are basic dyes and triphenylmethane dyes, e.g. pararosaniline derivatives.[16]

Histology Phosphotungstic acid is used in histology for staining specimens, as a component of phosphotungstic acid haematoxylin, PTAH, and “trichrome” reagents, and as a negative stain for imaging by a transmission electron microscope. Phosphotungstic acid haematoxylin (PTAH) Mallory described the reagent now generally known as PTAH in 1897.[17] PTAH stains tissues either reddish brown or blue depending on their type. This property of simultaneously staining two different colours is different from other haematoxylin reagents e.g. alum-haematoxylin. The role of phosphotungstic acid and the mechanism of staining is not fully understood. Interestingly the active component of haematoxylin is the oxidised form, haematin, although this rarely acknowledged in the literature which refer to haematoxylin staining. Phosphotungstic acid forms a lake with haematin.[18] The make –up of the reagent is uncertain, examination of a year old sample showed there to be three coloured components, blue, red and yellow.[19] These were not identified. Some investigations of “model” systems, reacting various compounds such as amino acids, purines, pyrimidines and amines with PTAH show that they give rise to different colours.[20] Trichrome reagents In these reagents two or three basic dyes are used with phosphotungstic acid, in either a one step or multi-stage procedure. These reagents colour different tissue types different colours. Again the mechanism of staining is not fully understood. Some explanations include the proposal that phosphotungstic acid acts as a mordant to bind the dye to the tissue[21] or that alternatively it binds to tissue blocking it to dye molecules.[22] Negative staining Adsorption onto tissue or the surface of viruses and its electron density are the bases of phosphotungstic acids action as a negative stain. This electron density arises from the presence of the 12 tungsten atoms which each have an atomic number of 74. The mechanism of the adsorption onto tissue has been proposed as being

245

Phosphotungstic acid electrostatic rather than involving hydrogen bonding, as adsorption is not affected by pH.[4]

Analysis The potassium salt is only slightly soluble, unlike most other phosphotungstate salts, and has been proposed as a method for the gravimetric analysis of potassium.[23]

Precipitation of proteins In a number of analytical procedures one of the roles of phosphotungstic acid is to precipitate out proteins. It has been termed a "universal" precipitant for polar proteins.[24] Further studies showed that no precipitation occurred with α-amino groups but did occur with guanidino, ε-amino and imidazole groups.[25]

Medicinal Very little work appears to have been carried out in this area. One example relates to liver necrosis in rats.[26]

Composite proton exchange membranes The heteropoly acids, including phosphotungstic acid, are being investigated as materials in composite proton exchange membranes, such as Nafion. The interest lies in the potential of these composite materials in the manufacture of fuel cells as they have improved operating characteristics.[27]

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=1343-93-7 [2] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=428739813& page2=%3APhosphotungstic+ acid [3] Contribution to the chemistry of phosphomolybdic acids, phosphotungstic acids and allied substances H Wu The Journal of Biological Chemistry 43, 1, (1920), 189 [4] On phosphotungstic staining, I G Quintarelli, R Zito, J.A Cifonelli The Journal of Histochemistry and Cytochemistry 19, 11, (1971, 641 [5] From Scheele and Berzelius to Müller: polyoxometalates (POMs) revisited and the "missing link" between the bottom up and top down approaches P. Gouzerh, M. Che; L’Actualité Chimique, 2006, 298, 9 [6] Structure of the Molecule of 12-Phosphotungstic Acid J. F. Keggin, Nature 1933, 131, 908. [7] The Structure and Formula of 12-Phosphotungstic Acid J.F. Keggin. Proc. Roy. Soc., A, 144, 851, 75-100 (1934) doi:10.1098/rspa.1934.0035 [8] Dodecatungstophosphoric acid hexahydrate, (H5O2+)3(PW12O403−). The true structure of Keggin's `pentahydrate' from single-crystal X-ray and neutron diffraction data Brown G.M., Noe-Spirlet M.-R., Busing W.R., Levy H.A., Acta Crystallogr., 1977, B33, 1038 doi:10.1107/S0567740877005330 [9] A study of the decomposition behaviour of 12-tungstophosphate heteropolyacid in solution Zhu Z., Tain R., Rhodes C. Canadian Journal of Chemistry, 81,10, 1, (2003), 1044-1050 [10] Oxide catalysts in solid state chemistry T Okuhara, M Misono Encyclopedia of Inorganic chemistry Editor R Bruce King (1994) John Wiley and Sons ISBN 0-471-93620-0 [11] Polyoxoanions M.T.Pope, Encyclopedia of Inorganic Chemistry Editor R Bruce King (1994) John Wiley and Sons ISBN 0-471-93620-0 [12] Acid Catalysis (http:/ / www. people. virginia. edu/ ~davis/ acid catalysis. htm), Davis Group, Department of Chemical Engineering, University of Virginia. Retrieved 2009-06-02. [13] Acidity measurements on a heteropolyacid hydrate in acetic acid solution : a case of three hydrons ionizing independently, rather than consecutively Farcasiu D. ; Jing Qi Li ; Journal of Catalysis 1995, 152, 1, 198-203 [14] Zirconia-ed 12-tungstophosphoric acid as a solid catalyst for the synthesis of linear alkyl benzenesBiju M. Devassy, F. Lefebvre and S.B. Halligudi, Journal of Catalysis 231,1,(2005),1-10 doi:10.1016/j.jcat.2004.09.024 [15] Non-staining pigments and their use US patent: 2999026 Issue date: Sep 1961, Inventor: Chester Davis [16] Pigments, Organic K Hunger, W Herbst Ullmans Encyclopedia of Industrial Chemistry [17] On certain improvements in histological technique: I. A differential stain for amoeligbæ coli. II. phosphotungstic-acid-hæmatoxylin stain for certain tissue elements. III. A method of fixation for neuroglia fibres. F. B. Mallory J. Exp. Med., 2, 5, (1897) 529-533 [18] Phosphotungstic acid- hematoxylin; spectrophotometry of the lake in solution and in stained tissue Terner JY, Gurland J, Gaer F. Stain Technol (1964),39, 141-53 [19] On the mechanism of Mallory's phosphotungstic acid - haematoxylin stain Puchtler H, Waldrop FS, Meloan S.N. J Microsc 1980, 119, 3, 383 [20] Phosphotungstic acid-hematoxylin. Reactivity in vitro J. Y. Terner Journal of Histochemistry and Cytochemistry, 1966, 4, 345

246

Phosphotungstic acid [21] Dyes and other colorants in microtechnique and biomedical research J A Kiernan Color. Technol. 122, 1–21 doi:10.1111/j.1478-4408.2006.00009.x [22] The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies M.M. Everett and W.A. Miller The Histochemical Journal, 6, 1, (1974), 25-34, doi:10.1007/BF01011535 [23] Gravimetric determination of potassium as phospho-12-tungstate W.K. Rieben, D.D. Van Slyke, Journal of Biological Chemistry, 156, (1944), 2, 765 [24] Phosphotungstate: a" universal" (nonspecific) precipitant for polar polymers in acid solution JE Scott - Journal of Histochemistry and Cytochemistry, 197119, 11, 689 [25] Precipitation of proteins: The separation of proteins with heteropolyacids M. Z. Sternberg, Biotechnology and Bioengineering 12, 1, (1970), 1 - 17 [26] Protective effects of tungstophosphoric acid and sodium tungstate on chemically induced liver necrosis in Wistar rats Snežana Uskoković-Marković1, Marina Milenković, Aleksandra Topić, Jelena Kotur-Stevuljević, Aleksandra Stefanović, Jelena Antić-Stanković, J Pharm Pharmaceut Sci 10 (3): 340-349, 2007 [27] Composite membranes for medium temperature PEM fuel cells G. Alberti and M. Casciola Annual Review of Materials Research 33, (2003), 129-154 doi:10.1146/annurev.matsci.33.022702.154702

247

Picric acid

248

Picric acid Picric acid

Identifiers [1]

CAS number

88-89-1

PubChem

6954

ChemSpider

6688

UNII

A49OS0F91S

DrugBank

DB03651

ChEBI

CHEBI:46149

ChEMBL

CHEMBL108541

RTECS number

TJ7875000

Jmol-3D images

Image 1

[2] [3]

[4]

[5] [6]

[7]

[8]


CHNO

Molar mass

229.10 g·mol−1

Appearance

Colorless to yellow solid

Density

1.763 g·cm−3, solid

Melting point

122.5 °C

Boiling point

> 300 °C (Explodes)

Solubility in water

12.7 g·L−1

Acidity (pK )

0.38

a

6 3 3 7

Hazards EU classification

Explosive (E), Toxic (T)

R-phrases

R1 R4 R11 R23 R24 R25

S-phrases

S28 S35 S37 S45

Picric acid

249 NFPA 704 Explosive data Explosive velocity

7,350 m·s−1 at ρ 1.70 ()

[9]


Infobox references

Picric acid is the chemical compound formally called 2,4,6-trinitrophenol (TNP). This yellow crystalline solid is one of the most acidic phenols. Like other highly nitrated compounds such as TNT, picric acid is an explosive. Its name comes from Greek πικρος (pik' ros), meaning "bitter", reflecting its bitter taste.

History Picric acid was probably first mentioned in the alchemical writings of Johann Rudolf Glauber in 1742. Initially, it was made by nitrating substances such as animal horn, silk, indigo, and natural resin, the synthesis from indigo first being performed by Peter Woulfe in 1779. Its synthesis from phenol, and the correct determination of its formula, were successfully accomplished in 1841. Not until 1830 did chemists think to use picric acid as an explosive. Before then, chemists assumed that only the salts of picric acid were explosive, not the acid itself. In 1873 Hermann Sprengel proved it could be detonated and most military powers used picric acid as their primary high explosive material. Picric acid is also used in the analytical chemistry of metals, ores, and minerals. Picric acid was the first high explosive nitrated organic compound widely considered suitable to withstand the shock of firing in conventional artillery. Nitroglycerine and guncotton were available earlier but shock sensitivity sometimes caused detonation in the artillery barrel at the time of firing. In 1885, based on research of Hermann Sprengel, French chemist Eugène Turpin patented the use of pressed and cast picric acid in blasting charges and artillery shells. In 1887 the French government adopted a mixture of picric acid and guncotton under the name melinite. In 1888, Britain started manufacturing a very similar mixture in Lydd, Kent, under the name lyddite. Japan followed with an "improved" formula known as shimose powder. In 1889, a similar material, a mixture of ammonium cresylate with trinitrocresol, or an ammonium salt of trinitrocresol, started to be manufactured under the name ecrasite in Austria-Hungary. By 1894 Russia was manufacturing artillery shells filled with picric acid. Ammonium picrate (known as Dunnite or explosive D) was used by the United States beginning in 1906. However, shells filled with picric acid become highly unstable if the compound reacts with metal shell or fuze casings to form metal picrates which are more sensitive than the parent phenol. The sensitivity of picric acid was demonstrated in the Halifax Explosion. Picric acid was used in the Battle of Omdurman,[10] Second Boer War,[11] the Russo-Japanese War,[12] and World War I.[13] began filling artillery shells with TNT in 1902. Toluene was less readily available than phenol, and TNT is less powerful than picric acid, but improved safety of munitions manufacturing and storage caused replacement of picric acid by TNT for most military purposes between the World Wars.[10]

Synthesis The aromatic ring of phenol is highly activated towards electrophilic substitution reactions, and attempted nitration of phenol, even with dilute nitric acid, results in the formation of high molecular weight tars. In order to minimize these side reactions, anhydrous phenol is sulfonated with fuming sulfuric acid, and the resulting p-phenolsulfonic acid is then nitrated with concentrated nitric acid. During this reaction, nitro groups are introduced, and the sulfonic acid group is displaced. The reaction is highly exothermic, and careful temperature control is required.

Picric acid

250

Uses By far, the largest use has been in munitions and explosives. It has found some use in organic chemistry for the preparation of crystalline salts of organic bases (picrates) for the purpose of identification and characterization. In metallurgy, a picric acid etch has been commonly used in optical metallography to reveal prior austenite grain boundaries in ferritic steels. The hazards associated with picric acid has meant it has largely been replaced with other chemical etchants. However, it is still used to etch magnesium alloys, such as AZ31. Bouin solution is a common picric acid-containing fixative solution used for histology specimens.[14] Workplace drug testing utilizes picric acid for the Jaffe Reaction to test for creatinine. It forms a colored complex that can be measured using spectroscopy.[15] Much less commonly, wet picric acid has been used as a skin dye or temporary branding agent. It reacts with proteins in the skin to give a dark brown color that may last as long as a month. In the early 20th century, picric acid was stocked in pharmacies as an antiseptic and as a treatment for burns, malaria, herpes, and smallpox. It was most notably used for the treatment of burns suffered by victims of the Hindenburg disaster in 1937. Picric acid emits a high-pitched whine during combustion in air and this has led to its widespread use in fireworks. Picric acid has been used for many years by fly tyers to dye mole skins and feathers dark olive green. Its popularity has been tempered by its toxic nature.

Safety Modern safety precautions recommend storing picric acid wet. Dry picric acid is relatively sensitive to shock and friction, so laboratories that use it store it in bottles under a layer of water, rendering it safe. Glass or plastic bottles are required, as picric acid can easily form metal picrate salts that are even more sensitive and hazardous than the acid itself. Industrially, picric acid is especially hazardous because it is volatile and slowly sublimes even at room temperature. Over time, the buildup of picrates on exposed metal surfaces can constitute a grave hazard.[16] Bomb disposal units are often called to dispose of picric acid if it has dried out.[17][18][19][20][21][22]

References [1] [2] [3] [4] [5] [6] [7] [8]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=88-89-1 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=6954 http:/ / www. chemspider. com/ 6688 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=A49OS0F91S http:/ / www. drugbank. ca/ drugs/ DB03651 https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=46149 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL108541 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=O%3D%5BN%2B%5D%28%5BO-%5D%29c1cc%28cc%28%5BN%2B%5D%28%5BO-%5D%29%3DO%29c1O%29%5BN%2B%5D%28%5BO-% [9] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=464206375& page2=%3APicric+ acid [10] Brown, G.I. (1998) The Big Bang: a History of Explosives Sutton Publishing ISBN 0-7509-1878-0 pp.151-163 [11] John Philip Wisser (1901). The second Boer War, 1899-1900 (http:/ / www. archive. org/ stream/ secondboerwar18900wissrich#page/ 243/ mode/ 1up). Hudson-Kimberly. p. 243. . Retrieved 2009-07-22. [12] Dunnite Smashes Strongest Armor (http:/ / query. nytimes. com/ mem/ archive-free/ pdf?res=9A0CEEDD1F30E233A2575BC1A96E9C946697D6CF), The New York Times, August 18, 1907 [13] Marc Ferro. The Great War. London and New York: Routeladge Classics, p. 98. [14] Carson, Freida L.; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. p. 19. ISBN 978-0-89189-581-7. [15] Creatinine Direct Procedure, on CimaScientific (http:/ / www. cimascientific. com/ 2600. htm) [16] JT Baker MSDS (http:/ / hazard. com/ msds/ mf/ baker/ baker/ files/ p4556. htm)

Picric acid [17] "Bomb squad called to Dublin lab" (http:/ / www. irishtimes. com/ newspaper/ breaking/ 2010/ 1001/ breaking49. html). irishtimes.com. Irish Times. 1 October 2010. . Retrieved 22 July 2011. [18] "Unstable chemicals made safe by army" (http:/ / www. rte. ie/ news/ 2010/ 1103/ teagasc. html). rte.ie. RTÉ New. 3 November 2010. . Retrieved 22 July 2011. [19] "Army bomb disposal team make Kerry scene safe" (http:/ / www. irishexaminer. com/ breakingnews/ ireland/ army-bomb-disposal-team-make-kerry-scene-safe-482831. html). irishexaminer.com. Irish Examiner. 22 November 2010. . Retrieved 22 July 2011. [20] "Dangerous chemicals made safe" (http:/ / www. irishtimes. com/ newspaper/ breaking/ 2010/ 1123/ breaking57. html). irishtimes.com. Irish Times. 23 November 2010. . Retrieved 22 July 2011. [21] "Unstable chemicals made safe" (http:/ / www. irishtimes. com/ newspaper/ breaking/ 2011/ 0531/ breaking38. html). irishtimes.com. Irish Times. 31 May 2011. . Retrieved 22 July 2011. [22] "Chemicals Blown Up At Transfer Station" (http:/ / www. kcra. com/ r/ 26394685/ detail. html). kcra.com. KCRA. 6 January 2011. . Retrieved 22 July 2011.

• Cooper, Paul W., Explosives Engineering, New York: Wiley-VCH, 1996. ISBN 0-471-18636-8 • Safety Information (http://ptcl.chem.ox.ac.uk/MSDS/PI/picric_acid.html)

251

Potassium dichromate

252

Potassium dichromate Potassium dichromate

Identifiers [1]

CAS number

7778-50-9

PubChem

24502

ChemSpider

22910

EC number

231-906-6

UN number

3288

ChEMBL

CHEMBL1374101

RTECS number

HX7680000

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]


K Cr O

Molar mass

294.185 g/mol

Appearance

red-orange crystalline solid

Odor

odorless

Density

2.676 g/cm3, solid

Melting point

398 °C

Boiling point

500 °C decomp.

Solubility in water

4.9 g/100 mL (0 °C) 102 g/100 mL (100 °C)

Solubility

insoluble in alcohol

2

Refractive index (n ) D

2 7

1.738 Structure

Crystal structure

Triclinic (α-form, <241.6 °C)


Coordination geometry

253 Tetrahedral (for Cr) Thermochemistry

Std enthalpy of formation ΔfHo298

-2033 kJ/mol

Standard molar entropy So298

291.2 J K−1 mol−1 Hazards [7]

MSDS

ICSC 1371

EU Index

024-002-00-6

EU classification

Oxidant (O) Carc. Cat. 2 Muta. Cat. 2 Repr. Cat. 2 Highly toxic (T+) Harmful (Xn) Corrosive (C) Dangerous for the environment (N)

R-phrases

R45, R46, R60, R61, R8, R21, R25, R26, R34, R42/43, R48/23, R50/53

S-phrases

S53, S45, S60, S61


non-flammable Related compounds

Other anions

Potassium chromate Potassium molybdate Potassium tungstate

Other cations

Ammonium dichromate Sodium dichromate

Related compounds

Potassium permanganate ()

[8]


Infobox references

Potassium dichromate, K2Cr2O7, is a common inorganic chemical reagent, most commonly used as an oxidising agent in various laboratory and industrial applications. As with all hexavalent chromium compounds, it is potentially harmful to health and must be handled and disposed of appropriately. It is a crystalline ionic solid with a very bright, red-orange color. It is also known as potassium bichromate; bichromate of potash; dipotassium dichromate; dichromic acid, dipotassium salt; chromic acid, dipotassium salt; and lopezite.[9]


Chemistry Production Potassium dichromate is usually prepared by the reaction of potassium chloride on sodium dichromate. Alternatively, it can be obtained from potassium chromate by roasting chrome ore with potassium hydroxide.

Reactions Potassium dichromate is an oxidant (oxidising agent). The reduction half-equation can be seen: Cr2O72−(aq) + 14H+ + 6e− → 2Cr3+(aq) + 7H2O (E = +1.33 V) In organic chemistry, potassium dichromate is a mild oxidizer compared with potassium permanganate. It is used to oxidize alcohols. It converts primary alcohols into aldehydes, or into carboxylic acids if heated under reflux. In contrast, with permanganate, carboxylic acids are the sole products. Secondary alcohols are converted into ketones — no further oxidation is possible. For example, menthone may be prepared by oxidation of menthol with acidified dichromate.[10] Tertiary alcohols are not oxidized by potassium dichromate. In an aqueous solution the color change exhibited can be used to test whether an aldehyde or ketone is present. When an aldehyde is present the chromium ions will be reduced from the +6 to the +3 oxidation state, changing color from orange to green. This is because the aldehyde can be further oxidized to the corresponding carboxylic acid. A ketone will show no such change because it cannot be oxidized further, and so the solution will remain orange.

Uses Cleaning Like other chromium(VI) compounds (chromium trioxide, sodium dichromate), potassium dichromate may be used to prepare "chromic acid", which can be used for cleaning glassware and etching materials.

Construction It is used as an ingredient in cement in which it retards the setting of the mixture and improves its density and texture. This usage commonly causes dermatitis in construction workers.[11]

Ethanol determination The concentration of ethanol in a sample can be determined by back titration with acidified potassium dichromate. Reacting the sample with an excess of potassium dichromate, all ethanol is oxidized to acetic acid: C2H5OH + [O] → CH3COOH The excess dichromate is determined by titration against sodium thiosulfate. Subtracting the amount of excess dichromate from the initial amount, gives the amount of ethanol present. Accuracy can be improved by calibrating the dichromate solution against a blank. One major application for this reaction is in old police breathalyzer tests. When alcohol vapor makes with the yellow dichromate-coated crystals, the color changes from yellow to green. The degree of the color change is directly related to the level of alcohol in the suspect's breath.

254


Leather It is used to tan leather which is used for footwear.[12]

Photography Potassium dichromate has important uses in photography and in photographic screen printing, where it is used as an oxidising agent together with a strong mineral acid. Gum bichromate printing was one of the very first stable photographic printing processes, dating back to about 1850. A solution of gum arabic and potassium dichromate, once applied to paper and dried, will harden when exposed to ultraviolet light. Chromium intensification or Photochromos uses potassium dichromate together with equal parts of concentrated hydrochloric acid diluted down to approximately 10% v/v to treat weak and thin negatives of black and white photograph roll. This solution reconverts the elemental silver particles in the film to silver chloride. After thorough washing and exposure to actinic light, the film can be redeveloped to its end-point yielding a stronger negative which is able to produce a more satisfactory print. A potassium dichromate solution in sulfuric acid can be used to produce a reversal negative (i.e., a positive transparency from a negative film). This is effected by developing a black and white film but allowing the development to proceed more or less to the end point. The development is then stopped by copious washing and the film then treated in the acid dichromate solution. This converts the silver metal to silver sulfate, a compound that is insensitive to light. After thorough washing and exposure to actinic light, the film is developed again allowing the previously unexposed silver halide to be reduced to silver metal. The results obtained can be unpredictable, but sometimes excellent results are obtained producing images that would otherwise be unobtainable. This process can be coupled with solarisation so that the end product resembles a negative and is suitable for printing in the normal way. CrVI compounds have the property of tanning animal proteins when exposed to strong light. This quality is used in photographic screen-printing. In screen-printing a fine screen of bolting silk or similar material is stretched taut onto a frame similar to the way canvas is prepared before painting. A colloid sensitized with a dichromate is applied evenly to the taut screen. Once the dichromate mixture is dry, a full-size photographic negative is attached securely onto the surface of the screen, and the whole assembly exposed to strong light - typically about half an hour in bright sunlight - hardening the exposed colloid. When the negative is removed, the unexposed mixture on the screen can be washed off with warm water, leaving the hardened mixture intact, acting as a precise mask of the desired pattern, which can then be printed with the usual screen-printing process.

Silver test When dissolved in an approximately 35% nitric acid solution it is called Schwerter's solution and is used to test for the presence of various metals, notably for determination of silver purity. Pure silver will turn the solution bright red, sterling silver will turn it dark red, low grade coin silver (0.800 fine) will turn brown (largely due to the presence of copper which turns the solution brown) and even green for 0.500 silver.

Sulfur dioxide test Potassium dichromate paper can be used to test for sulfur dioxide, as it turns distinctively from orange to green. This is typical of all redox reactions where hexavalent chromium is reduced to the less harmful trivalent chromium. Therefore, it is not a conclusive test for sulfur dioxide.

255


256

Wood treatment Potassium dichromate is used to stain certain types of wood by darkening the tannins in the wood. It produce deep, rich browns that cannot be achieved with modern color dyes. It is a particularly effective treatment on mahogany.[13]

Natural occurrence Potassium dichromate occurs naturally as the rare mineral lopezite. It has only been reported as vug fillings in the nitrate deposits of the Atacama desert of Chile and in the Bushveld igneous complex of South Africa.[14]

Safety

A ~10 mm crystal of potassium dichromate in the same form as the mineral lopezite

Potassium dichromate is one of the most common causes of chromium dermatitis;[15] chromium is highly likely to induce sensitization leading to dermatitis, especially of the hand and fore-arms, which is chronic and difficult to treat. Toxicological studies have further illustrated its highly toxic nature. With rabbits and rodents, concentrations as low as 14 mg/kg have shown a 50% fatality rate amongst test groups. [16] Aquatic organisms are especially vulnerable if exposed, and hence responsible disposal according local environmental regulations is advised. As with other CrVI compounds, potassium dichromate is carcinogenic and should be handled with gloves and appropriate health and safety protection. The compound is also corrosive and exposure may produce severe eye damage or blindness.[17] Human exposure further encomes impaired fertility, heritable genetic damage and harm to unborn children.

References [1] [2] [3] [4] [5]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=7778-50-9 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=24502 http:/ / www. chemspider. com/ 22910 http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=231-906-6 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL1374101

[6] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BK%2B%5D. %5BK%2B%5D. %5BO-%5D%5BCr%5D%28%3DO%29%28%3DO%29O%5BCr%5D%28%5BO-%5D%29%28%3DO%29%3DO [7] http:/ / www. inchem. org/ documents/ icsc/ icsc/ eics1371. htm [8] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=406377229& page2=%3APotassium+ dichromate [9] "POTASSIUM DICHROMATE LISTING" (http:/ / www. epa. gov/ osw/ hazard/ wastetypes/ wasteid/ inorchem/ docs/ pot-dich. pdf). US EPA. . [10] L. T. Sandborn, "l-Menthone" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=cv1p0340), Org. Synth., ; Coll. Vol. 1: 340 [11] Pekka Roto, Hannele Sainio, Timo Reunala, Pekka Laippala (January 1996). "Addition of ferrous sulfate to cement and risk of chromium dermatitis among construction workers" (http:/ / www. blackwell-synergy. com/ doi/ abs/ 10. 1111/ j. 1600-0536. 1996. tb02111. x). Dermatitis 34 (1): 43–50. doi:10.1111/j.1600-0536.1996.tb02111.x. PMID 8789225. [12] M. Saha, C. R. Srinivas, S. D. Shenoy, C. Balachandran (May 1993). "Footwear dermatitis" (http:/ / www. blackwell-synergy. com/ doi/ abs/ 10. 1111/ j. 1600-0536. 1993. tb03428. x). Dermatitis 28 (5): 260–264. doi:10.1111/j.1600-0536.1993.tb03428.x. PMID 8365123. [13] Jewitt, Jeff (1997). Hand-Applied Finishes. Newtown, CT USA: The Taunton Press, Inc. ISBN 1-56158-154-2. [14] http:/ / www. mindat. org/ min-2433. html Mindat [15] Farokh J. Master (2003). Diseases of Skin (http:/ / books. google. com/ ?id=OBAnewDc9CYC). New Delhi: B Jain Pub Pvt Ltd. p. 223. ISBN 81-7021-136-0. . [16] "Potassium dichromate MSDS" (http:/ / www. sigmaaldrich. com/ catalog/ ProductDetail. do?D7=0& N5=SEARCH_CONCAT_PNO|BRAND_KEY& N4=60190|FLUKA& N25=0& QS=ON& F=SPEC). Sigma-Aldrich. . Retrieved 2011-07-20. [17] "Potassium dichromate MSDS" (http:/ / hazard. com/ msds/ mf/ baker/ baker/ files/ p5719. htm). JT Baker. .


External links • International Chemical Safety Card 1371 (http://www.inchem.org/documents/icsc/icsc/eics1371.htm) • National Pollutant Inventory - Chromium VI and compounds fact sheet (http://www.npi.gov.au/database/ substance-info/profiles/25.html) • NIOSH Pocket Guide to Chemical Hazards (http://www.cdc.gov/niosh/npg/npgd0138.html) • IARC Monograph "Chromium and Chromium compounds" (http://www-cie.iarc.fr/htdocs/monographs/ vol49/chromium.html)

257

Prussian blue

258

Prussian blue Prussian blue

Identifiers [1]

CAS number

14038-43-8

PubChem

2724251

ChemSpider

20074656

UNII

TLE294X33A

EC number

237-875-5

ChEBI

CHEBI:30069

ChEMBL

CHEMBL1200325

RTECS number

V03AB31

Gmelin Reference

1093743

Jmol-3D images

Image 1

[2] [3]

[4]

[5] [6]

[7]

[8]


C18Fe7N18

Molar mass

859.23 g mol

Appearance

Blue opaque crystals

−1

Pharmacology Routes of istration

Oral Hazards

MSDS

MSDS prussian blue

[9]

Related compounds Other cations

Potassium ferrocyanide Sodium ferrocyanide ()

[10]


Infobox references

Prussian blue is a dark blue pigment with the idealized formula Fe7(CN)18. Another name for the color Prussian blue is Berlin blue or, in painting, Parisian blue. Turnbull's blue is the same substance but is made from different reagents. Prussian blue was one of the first synthetic pigments. It is employed as a very fine colloidal dispersion, as the compound itself is not soluble in water. It is famously complex,[11] owing to the presence of variable amounts of

Prussian blue

259

other ions and the sensitive dependence of its appearance on the size of the colloidal particles formed when it is made. The pigment is used in paints, and it is the traditional "blue" in blueprints. In medicine, Prussian blue is used as an antidote for certain kinds of heavy metal poisoning (caesium and thallium). Prussian blue lent its name to prussic acid, which was derived from it, and to ferrocyanide (originally meaning "blue compound of iron", from Latin ferrum and Greek κυανεος). As ferrocyanide is made of iron and CN ligands, reinterpreting the component "-cyanide" in the compound word produced the word "cyanide" for compounds containing the CN radical.

History Prussian blue [Fe4[Fe(CN)6]3] was probably synthesized for the first time by the paint maker Diesbach in Berlin around the year 1706.[12] Most historical sources do not mention a first name of Diesbach. Only Berger refers to him as Johann Jacob Diesbach.[13] It was named "Preußisch blau" and "Berlinisch Blau" in 1709 by its first trader.[14] The pigment replaced the expensive Lapis lazuli and was an important topic in the letters exchanged between Johann Leonhard Frisch[15] and the president of the Royal Academy of Sciences, Gottfried Wilhelm Leibniz, between 1708 and 1716.[14] It is first mentioned in a letter written by Frisch to Leibniz, from March 31, 1708. Not later than 1708, Frisch began to promote and sell the pigment across Europe. By August 1709, the pigment had been termed "Preussisch blau"; by November 1709, the German name "Berlinisch Blau" had been used for the first time by Frisch. Frisch himself is the author of the first known publication of Prussian blue in the paper Notitia Coerulei Berolinensis nuper inventi in 1710, as can be deduced from his letters. Diesbach had been working for Frisch since about 1701. In 1731, Georg Ernst Stahl published an of the first synthesis of Prussian blue.[16] The story involves not only Diesbach but also Johann Konrad Dippel. Diesbach was attempting to create a red lake pigment from cochineal but obtained the blue instead as a result of the contaminated potash he was using. He borrowed the potash from Dippel, who had used it to produce his "animal oil". No other known historical source mentions Dippel in this context. It is therefore difficult to judge the reliability of this story today. In 1724, the recipe was finally published by John Woodward.[17][18][19] To date, the "Entombment of Christ", dated 1709 by Pieter van der Werff (Picture Gallery, Sanssouci, Potsdam) is the oldest known painting where Prussian blue was used. Around 1710, painters at the Prussian court were already using the pigment. At around the same time, Prussian blue arrived in Paris, where Antoine Watteau and later his successors Nicolas Lancret and Jean-Baptiste Pater used it in their paintings.[20] This Prussian blue pigment is significant since it was the first stable and relatively lightfast blue pigment to be widely used following the loss of knowledge regarding the synthesis of Egyptian Blue. European painters had previously used a number of pigments such as indigo dye, smalt, and Tyrian purple, which tend to fade, and the extremely expensive ultramarine made from lapis lazuli. Japanese painters and woodblock print artists likewise did not have access to a long-lasting blue pigment until they began to import Prussian blue from Europe. "The Great Wave off Kanagawa" by Hokusai, a

In 1752 the French chemist Pierre J. Macquer made the important step famous artwork which makes extensive use of of showing the Prussian blue could be reduced to a salt of iron and a Prussian blue. new acid, which could be used to reconstitute the dye.[21] The new acid, hydrogen cyanide, first isolated from Prussian blue in pure form and characterized about 1783 by the Swedish chemist Carl Wilhelm Scheele, was eventually given the name Blausäure (literally "Blue acid") because of its derivation from Prussian blue, and in English became known popularly as Prussic acid. Prussian blue would also give the name to the ferrocyanide and cyanide family of compounds. Ferrocyanide (which is yellow) was coined as

Prussian blue Neo Latin for "iron-containing blue material", since it was first isolated from Prussian blue. Cyanide, a colorless anion that forms in the process of making Prussian Blue, was named, in turn, for hydrogen cyanide (also colorless), and ultimately from ferrocyanide. It is for this reason that cyanide, even though the name of a colorless radical, is a Latinized form of the Greek word for "dark blue."

Production Prussian blue is produced by oxidation of ferrous ferrocyanide salts. These white solids have the formula M2Fe[Fe(CN)6] where M+ = Na+ or K+. The iron in this material is all ferrous, hence the absence of deep color associated with the mixed valency. Oxidation of this white solid with hydrogen peroxide or sodium chlorate produces ferricyanide and affords Prussian Blue.[22] A "soluble" form of PB, K[FeIIIFeII(CN)6], which is really colloidal, can be made from potassium ferrocyanide and iron(III): K+ + Fe3+ + [FeII(CN)6]4- → KFeIII[FeII(CN)6] The similar reaction of potassium ferricyanide and iron(II) results in the same colloidal solution, because [FeIII(CN)6]3- is converted into ferrocyanide. "Insoluble" Prussian blue is produced if in the reactions above an excess of Fe3+ or Fe2+, respectively, is added. In the first case: 4Fe3+ + 3[FeII(CN)6]4- → FeIII[FeIIIFeII(CN)6]3 [23]

"Turnbull's blue" In former times, it was thought that addition of Fe(II) salts to a solution of ferricyanide affords a material different from Prussian blue. The product was traditionally named "Turnbull's Blue" (TB). It has been shown, however, by means of X-ray diffraction and electron diffraction methods, that the structures of PB and TB are identical.[24][25] The differences in the colors for TB and PB reflect subtle differences in the method of precipitation, which strongly affects particle size and impurity content.

Properties Ferricyanide ion, used to make 'Turnbull's blue'. Prussian blue is a microcrystalline blue powder. It is insoluble, but the crystallites tend to form a colloid. Such colloids can through fine filters.[11] Despite being one of the oldest known synthetic compounds, the composition of Prussian blue remained uncertain for many years. The precise identification of Prussian blue was complicated by three factors:

1. Prussian blue is extremely insoluble but also tends to form colloids; 2. Traditional syntheses tend to afford impure compositions; 3. Even pure Prussian blue is structurally complex, defying routine crystallographic analysis.

260

Prussian blue

Crystal structure The chemical formula of insoluble Prussian blue is Fe7(CN)18·xH2O, where x = 14–16. The structure was determined by using IR spectroscopy, Moessbauer spectroscopy, X-ray crystallography, and neutron crystallography. Since X-ray diffraction cannot distinguish carbon from nitrogen, the location of these lighter elements is deduced by spectroscopic means as well as by observing the distances from the iron atom centers. PB has a cubic lattice structure. Soluble PB crystals contain interstitial K+ ions; insoluble PB has interstitial water instead. In ideal insoluble PB crystals, the cubic framework is built from Fe(II)-C-N-Fe(III) sequences, with Fe(II)-carbon distances of 1.92 Å and Fe(III)-nitrogen distances of 2.03 Å. One-fourth of the sites of Fe(CN)6 subunits are vacant (empty), leaving three such groups. The empty nitrogen sites are filled with water molecules instead, which are coordinated to Fe(III). The Fe(II) centers, which are low spin, are surrounded by six carbon ligands in an octahedral configuration. The Fe(III) centers, which are high spin, are octahedrally surrounded on average by 4.5 nitrogen atoms and 1.5 oxygen atoms (the oxygen from the six coordinated water molecules). Additional eight (interstitial) water molecules are present in the unit cell, either as isolated molecules or hydrogen bonded to the coordinated water. The composition is notoriously variable due to the presence of lattice defects, allowing it to be hydrated to various degrees as water molecules are incorporated into the structure to occupy cation vacancies. The variability of Prussian blue's composition is attributable to its low solubility, which leads to its rapid precipitation without the time to achieve full equilibrium between solid and liquid.[26] [27]

Color Prussian blue is strongly colored and tends towards black and dark blue when mixed into oil paints. The exact hue depends on the method of preparation, which dictates the particle size. The intense blue color of Prussian blue is associated with the energy of the transfer of electrons from Fe(II) to Fe(III). Many such mixed-valence compounds absorb certain wavelengths of visible light resulting from intervalence charge transfer. In this case, orange-red light around 680 nanometers in wavelength is absorbed, and the reflected light appears blue as a result. Like most high chroma pigments, Prussian blue cannot be accurately displayed on a computer display. PB is electrochromic—changing from blue to colorless upon reduction. This change is caused by reduction of the Fe(III) to Fe(II) eliminating the intervalence charge transfer that causes Prussian blue's color.

Uses Pigment Because it is easily made, cheap, non-toxic, and intensely colored, Prussian blue has attracted many applications. The dominant uses are for pigments: approximately 12,000 tonnes of Prussian blue are produced annually for use in black and bluish inks. A variety of other pigments also contain the material.[22] Engineer's blue and the pigment formed on cyanotypes—giving them their common name blueprints. Certain crayons were once colored with Prussian blue (later relabeled Midnight Blue). It is also a popular pigment in paints. Similarly, Prussian blue is the basis for laundry bluing.

261

Prussian blue

262

Medicine Prussian blue's ability to incorporate monocations makes it useful as a sequestering agent for certain heavy metal poisons. Pharmaceutical-grade Prussian blue in particular is used for patients who have ingested thallium or radioactive caesium. According to the International Atomic Energy Agency, an adult male can eat at least 10 grams of Prussian blue per day without serious harm. The U.S. Food and Drug istration (FDA) has determined that the "500 mg Prussian blue capsules, when manufactured under the conditions of an approved New Drug Application (NDA), can be found safe and effective therapy" in certain poisoning cases.[28] Radiogardase (Prussian blue in soluble capsules [29]) is a commercial product for the removal of caesium-137 from the intestine and so indirectly from the bloodstream by intervening in the enterohepatic circulation of caesium-137,[30] reducing the internal residency time (and exposure) by about two-thirds.

Laboratory histopathology stain for iron Prussian blue is a common histopathology stain used by pathologists to detect the presence of iron in biopsy specimens, such as in bone marrow samples. The original stain formula, known historically (1867) as "Perls' Prussian blue" after its inventor, German pathologist Max Perls (1843–1881), used separate solutions of potassium ferrocyanide and acid to stain tissue (these are now used combined, just before staining). Iron deposits in tissue then form the purple Prussian blue dye in place, and are visualized as blue or purple deposits.[31] The formula is also known as Perls Prussian blue and (incorrectly) as Perl's Prussian blue.

Prussian blue stain

By machinists and toolmakers Prussian blue in oil paint is the traditional material used for spotting metal surfaces such as surface plates and bearings for hand scraping. A thin layer of non-drying paste is applied to a reference surface and transfers to the high spots of the workpiece. The toolmaker then scrapes, stones, or otherwise removes the marked high spots. Prussian blue is preferable because it will not abrade the extremely precise reference surfaces as many ground pigments may.

Analytical chemistry Prussian blue is formed in the Prussian blue assay for total phenols. Samples and phenolic standards are given acidic ferric chloride and ferricyanide which is reduced to ferrocyanide by the phenols. The ferric chloride and ferrocyanide react to form Prussian blue. Comparing the absorbance at 700 nm of the samples to the standards allows for the determination of total phenols.[32]

Prussian blue

Safety Despite the fact that it is prepared from cyanide salts, Prussian blue is nontoxic because the cyanide groups are tightly bound to Fe. Other polymeric cyanometalates are similarly stable with low toxicity.

References [1] [2] [3] [4] [5] [6] [7] [8]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=14038-43-8 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=2724251 http:/ / www. chemspider. com/ 20074656 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=TLE294X33A http:/ / esis. jrc. ec. europa. eu/ lib/ einecs_IS_reponse. php?genre=ECNO& entree=237-875-5 https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=30069 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL1200325 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BFe%2B3%5D. %5BFe%2B3%5D. %5BFe%2B3%5D. %5BFe%2B3%5D. N%23C%5BFe-4%5D%28C%23N%29%28C%23N%29%28C%23N%29%28C%23N%29C%23N. N%23C%5BFe-4%5D%28C%23N%29%28C%23N%29%28C%23N%29%28C%23N%29C%23N. N%23C%5BFe-4%5D%28C%23N%29%28C%23N%29%28C%23N%29%28C%23N%29C%23N [9] https:/ / fscimage. fishersci. com/ msds/ 62402. htm [10] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=464375726& page2=%3APrussian+ blue [11] Dunbar, K. R. and Heintz, R. A. (1997). "Chemistry of Transition Metal Cyanide Compounds: Modern Perspectives". Progress in Inorganic Chemistry 45: 283–391. [12] Jens Bartoll. "The early use of prussian blue in paintings" (http:/ / www. ndt. net/ article/ art2008/ papers/ 029bartoll. pdf) (PDF). 9th International Conference on NDT of Art, Jerusalem Israel, 25–30 May 2008. . Retrieved 2010-01-22. [13] J. E. Berger: Kerrn aller Fridrichs=Städtschen Begebenheiten Manuskript, Berlin, ca.1730 (Berlin, Staatsbibliothek zu Berlin – Preußischer Kulturbesitz, Handschriftenabteilung, Ms. Boruss. quart. 124) [14] J. L. Frisch: Briefwechsel mit Gottfried Wilhelm Leibniz L. H. Fischer (ed.), Berlin, Stankiewicz Buchdruck, 1896, reprint Hildesheim/New York: Georg Olms Verlag, 1976 [15] There is a Wikipedia article in German [16] G. E. Stahl: Experimenta, Observationes, Animadversiones CCC Numero, Chymicae et Physicae, (Berlin, 1731), pp. 281–283 (http:/ / books. google. com/ books?id=wro5AAAAcAAJ& pg=PA281). [17] Woodward, J. (1724–1725). "Praeparatio coerulei Prussiaci es Germanica missa ad Johannem Woodward.. [Preparation of Prussian blue sent from to John Woodward...]". Philosophical Transactions of the Royal Society of London 33 (381): 15–17. doi:10.1098/rstl.1724.0005. [18] Brown, John (1724–1725). "Observations and Experiments upon the Foregoing Preparation". Philosophical Transactions 33 (381): 17–24. Bibcode 1724RSPT...33...17B. doi:10.1098/rstl.1724.0006. JSTOR 103734.. The recipe was subsequently published in [Étienne-François] Geoffroy, "Observations sur la Preparation de Bleu de Prusse ou Bleu de Berlin," Mémoires de l'Académie royale des Sciences année 1725 (Paris, 1727), pp. 153–172. [19] Sarah Lowengard, The Creation of Color in Eighteenth-Century Europe (New York, New York: Columbia University Press, 2008), Chapter 23: Prussian Blue (http:/ / www. gutenberg-e. org/ lowengard/ C_Chap32. html). [20] J.Bartoll, B. Jackisch, M. Most, E. Wenders de Calisse, C. M. Vogtherr: Early Prussian Blue. Blue and green pigments in the paintings by Watteau, Lancret and Pater in the collection of Frederick II of Prussia In: TECHNE 25, 2007, pp. 39–46 [21] Pierre-Joseph Macquer (1752) "Éxamen chymique de bleu de Prusse," (http:/ / gallica. bnf. fr/ ark:/ 12148/ bpt6k35505/ f242) Mémoires de l'Académie royale des Sciences année 1752 . . . (Paris, 1756), pp. 60–77. This article was reviewed in "Sur le bleu de Prusse," (http:/ / gallica. bnf. fr/ ark:/ 12148/ bpt6k35505/ f86. tableDesMatieres) Histoire de l'Académie royale des Sciences... (1752), (Paris, 1756), pp. 79–85. [22] Völz, Hans G. et al. "Pigments, Inorganic" in Ullmann's Encyclopedia of Industrial Chemistry, 2006 Wiley-VCH, Weinheim. doi:10.1002/14356007.a20_243.pub2. [23] Egon Wiberg,Nils Wiberg,Arnold Frederick Holleman: Inorganic chemistry (http:/ / books. google. com/ books?id=vEwj1WZKThEC& pg=PA1444), p.1444. Academic Press, 2001; Google books [24] Ozeki, Toru.; Matsumoto, Koichi.; Hikime, Seiichiro. (1984). "Photoacoustic spectra of prussian blue and photochemical reaction of ferric ferricyanide". Analytical Chemistry 56 (14): 2819. doi:10.1021/ac00278a041. [25] Izatt, Reed M.; Watt, Gerald D.; Bartholomew, Calvin H.; Christensen, James J. (1970). "Calorimetric study of Prussian blue and Turnbull's blue formation". Inorganic Chemistry 9 (9): 2019. doi:10.1021/ic50091a012. [26] Herren, F.; Fischer, P.; Ludi, A.; Haelg, W. (1980). "Neutron diffraction study of Prussian Blue, Fe4[Fe(CN)6]3.xH2O. Location of water molecules and long-range magnetic order". Inorganic Chemistry 19 (4): 956. doi:10.1021/ic50206a032. [27] Lundgren, C. A.; Murray, Royce W. (1988). "Observations on the composition of Prussian blue films and their electrochemistry". Inorganic Chemistry 27 (5): 933. doi:10.1021/ic00278a036. [28] "Questions and Answers on Prussian Blue" (http:/ / www. fda. gov/ Drugs/ EmergencyPreparedness/ BioterrorismandDrugPreparedness/ ucm130337. htm). . Retrieved 2009-06-06.

263

Prussian blue [29] [30] [31] [32]

Radiogardase: Package insert with formula (http:/ / www. heyltex. com/ radiogardasePackageInsert. php) Heyltex Corporation – Toxicology (http:/ / www. heyltex. com/ toxicology. php) Formula for Perls' Prussian blue stain (http:/ / www. idoub. com/ doc/ 4448747/ Perl). Accessed April 2, 2009. Tannin Chemistry (http:/ / www. s. muohio. edu/ hagermae/ tannin. pdf)PDF (1.41 MB)Accessed December 19, 2009

External links • The FDA's page on prussian blue (http://www.fda.gov/Drugs/EmergencyPreparedness/ BioterrorismandDrugPreparedness/ucm130337.htm) • The CDC's page on prussian blue (http://www.bt.cdc.gov/radiation/prussianblue.asp) • National Pollutant Inventory – Cyanide compounds fact sheet (http://www.npi.gov.au/database/ substance-info/profiles/29.html) • Heyltex Corporation distributors of Radiogardase (Prussian blue insoluble capsules) (http://www.heyltex.com/ ) • Sarah Lowengard, "Prussian Blue" in The Creation of Color in Eighteenth Century Europe Columbia University Press, 2006 (http://www.gutenberg-e.org/lowengard/C_Chap32.html)

Reducing agent A reducing agent (also called a reductant or reducer) is the element or compound in a reduction-oxidation (redox) reaction that donates an electron to another species; however, since the reducer loses an electron we say it is "oxidized". This means that there must be an "oxidizer"; because if any chemical is an electron donor (reducer), another must be an electron recipient (oxidizer). Thus reducers are "oxidized" and oxidizers are "reduced". For example, consider the following reaction: 2 [Fe(CN)6]4− + Cl2 → 2 [Fe(CN)6]3− + 2 Cl− The reducing agent in this reaction is ferrocyanide ([Fe(CN)6]4-). It donates an electron, becoming oxidized to ferricyanide ([Fe(CN)6]3-). Simultaneously, the oxidizer chlorine is reduced to chloride. In organic chemistry, reduction more specifically refers to the addition of hydrogen to a molecule, though the aforementioned definition still applies. For example, benzene is reduced to cyclohexane in the presence of a platinum catalyst: C6H6 + 3 H2 → C6H12 In organic chemistry, good reducing agents are reagents that deliver H2.

Characteristics of reducing agents Strong reducing agents easily lose (or donate) electrons. An atom with a relatively large atomic radius tends to be a better reductant. In such species, the distance from the nucleus to the valence electrons is so long that these electrons are not strongly attracted. These elements tend to be strong reducing agents. Good reducing agents tend to consist of atoms with a low electronegativity, the ability of an atom or molecule to attract bonding electrons, and species with relatively small ionization energies serve as good reducing agents too. "The measure of a material to oxidize or lose electrons is known as its oxidation potential".[1] The table below shows a few reduction potentials that could easily be changed to oxidation potential by simply reversing the sign. Reducing agents can be ranked by increasing strength by ranking their oxidation potentials. The reducing agent is stronger when it has a more positive oxidation potential and weaker when it has a negative oxidation potential. The following table provides the reduction potentials of the indicated reducing agent at 25 °C.

264

Reducing agent

265

Oxidizing agent

Reducing agent Reduction potential (V)

Li+ + e− =

Li

−3.04

Na+ + e− =

Na

−2.71

Mg2+ + 2e− =

Mg

−2.38

Al3+ + 3e− =

Al

−1.66

2H2O(l) + 2e− =

H2(g) + 2OH −

−0.83

Cr3+ + 3e− =

Cr

−0.74

Fe2+ + 2e− =

Fe

−0.44

2H+ + e− =

H2

0.00

Sn4+ + 2e− =

Sn2+

+0.15

Cu2+ + e− =

Cu+

+0.16

Ag+ + e− =

Ag

+0.80

Br2 + 2e− =

2Br−

+1.07

Cl2 + 2e− =

2Cl−

+1.36

MnO4− + 8H+ + 5e− = Mn2+ + 4H2O

+1.49

To tell which is the strongest reducing agent, one can change the sign of its respective reduction potential to make it oxidation potential. The bigger the number, the stronger the reducing agent. For example, among Na, Cr, Cu and Cl−, Na is the strongest reducing agent and Cl− is the weakest one. Common reducing agents include metals potassium, calcium, barium, sodium and magnesium, and also compounds that contain the H− ion, those being NaH, LiH,[2] LiAlH4 and CaH2. Some elements and compounds can be both reducing or oxidizing agents. Hydrogen gas is a reducing agent when it reacts with non-metals and an oxidizing agent when it reacts with metals. 2 Li(s) + H2(g) → 2 LiH(s) Hydrogen acts as an oxidizing agent because it accepts an electron donation from lithium, which causes Li to be oxidized. Half reactions: 2 Li(s)0 → 2 Li(s)+ + 2 e−::::: H20(g) + 2 e− → 2 H−(g) H2(g) + F2(g) → 2 HF(g) Hydrogen acts as a reducing agent because it donates its electrons to fluorine, which allows fluorine to be reduced. Half reactions: H20(g) → 2 H+(g) + 2 e−::::: F20(g) + 2 e− → 2 F−(g)

Importance of reducing and oxidizing agents Reducing agents and oxidizing agents are the ones responsible for corrosion, which is the “degradation of metals as a result of electrochemical activity”.[1] Corrosion requires an anode and cathode to take place. The anode is an element that loses electrons (reducing agent), thus oxidation always occurs in the anode, and the cathode is an element that gains electrons (oxidizing agent), thus reduction always occurs in the cathode. Corrosion occurs whenever there’s a difference in oxidation potential. When this is present, the anode metal begins deteriorating, given there is an electrical connection and the presence of an electrolyte.

Reducing agent

266

Example of redox reaction The formation of iron(III) oxide; 4Fe + 3O2 → 2Fe23+O36In the above equation, the Iron (Fe) has an oxidation number of 0 before and 3+ after the reaction. For oxygen (O) the oxidation number began as 0 and decreased to 2−. These changes can be viewed as two "half-reactions" that occur concurrently: 1. Oxidation half reaction: Fe0 → Fe3+ + 3e− 2. Reduction half reaction: O2 + 4e− → 2 O2− Iron (Fe) has been oxidized because the oxidation number increased. Iron is the reducing agent because it gave electrons to the oxygen (O2). Oxygen (O2) has been reduced because the oxidation number has decreased and is the oxidizing agent because it took electrons from iron (Fe).

Common reducing agents • Lithium aluminium hydride (LiAlH4) • Nascent (atomic) hydrogen • Sodium amalgam • • • • • • • • • • • • •

Sodium borohydride (NaBH4) Compounds containing the Sn2+ ion, such as tin(II) chloride Sulfite compounds Hydrazine (Wolff-Kishner reduction) Zinc-mercury amalgam (Zn(Hg)) (Clemmensen reduction) Diisobutylaluminum hydride (DIBAH) Lindlar catalyst Oxalic acid (C2H2O4) Formic acid (HCOOH) Ascorbic acid (C6H8O6) Phosphites, hypophosphites, and phosphorous acid Dithiothreitol (DTT) – used in biochemistry labs to avoid S-S bonds Compounds containing the Fe2+ ion, such as iron(II) sulfate

Several common reducing agents and their products Agent

Product

Hydrogen

H+, H2O

NADH

NAD+

Metals

metal ions

Hydrocarbons CO2 carbon dioxide, H2O

Reducing agent

267

References [1] Electrode Reduction and Oxidation Potential (http:/ / www. siliconfareast. com/ ox_potential. htm) [2] Aufray M, Menuel S, Fort Y, Eschbach J, Rouxel D, Vincent B (2009). "New Synthesis of Nanosized Niobium Oxides and Lithium Niobate Particles and Their Characterization by XPS Analysis". Journal of Nanoscience and Nanotechnology 9 (8): 4780–4789. doi:10.1166/jnn.2009.1087.

Further reading • "Chemical Principles: The Quest for Insight", Third Edition. Peter Atkins and Loretta Jones p. F76

External links • Table summarizing strength of reducing agents (http://www.usm.maine.edu/~newton/Chy251_253/Lectures/ OxidtionReduction/OxiationReduction.html)

Rhodamine Rhodamine ( /ˈroʊdəmiːn/) is a family of related chemical compounds, fluorone dyes. Examples are Rhodamine 6G and Rhodamine B. They are used as a dye and as a dye laser gain medium.[1][2] They are often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with instruments called fluorometers. Rhodamine dyes are used extensively in biotechnology applications such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA.

Rhodamine core structure

Rhodamine dyes are generally toxic, and are soluble in water, methanol and ethanol.

Rhodamine 123 The laser dye rhodamine 123 is also used in biochemistry to inhibit mitochondrion function. Rhodamine 123 seems to bind to the mitochondrion membranes and inhibit transport processes, especially the electron transport chain, thus slowing down inner respiration. It is a substrate of P-glycoprotein (Pgp), which is usually overexpressed in Rhodamine in water. cancer cells. Recent reports indicate that rhodamine 123 may be also a substrate of multidrug resistance-associated protein (MRP), or more specifically, MRP1.

Other rhodamine derivatives There are many rhodamine derivatives used for imaging purposes, for example Carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and its isothiocyanate derivative (TRITC) and, sulforhodamine 101 (and its sulfonyl chloride form Texas Red) and Rhodamine Red. TRITC is the base rhodamine molecule functionalized with an isothiocyanate group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure. This derivative is reactive towards amine groups on proteins inside cells. A succinimidyl-ester functional group attached to the rhodamine core, creating NHS-rhodamine, forms another common amine-reactive derivative.

Rhodamine Other derivatives of rhodamine include newer fluorophores such as Alexa 546, Alexa 555, Alexa 633, DyLight 550 and DyLight 633, have been tailored for various chemical and biological applications where higher photostability, increased brightness, different spectral characteristics, or different attachment groups are needed.

References [1] F. P. Schäfer (Ed.), Dye Lasers, 3rd Ed. (Springer-Verlag, Berlin, 1990). [2] F. J. Duarte and L. W. Hillman (Eds.), Dye Laser Principles (Academic, New York, 1990).

External links • Absorption and Emission Spectra of Rhodamine B (http://omlc.ogi.edu/spectra/PhotochemCAD/html/ rhodamineB.html) • Absorption and Emission Spectra of Rhodamine 6G (http://omlc.ogi.edu/spectra/PhotochemCAD/html/ rhodamine6G.html) • Absorption and Emission Spectra of Rhodamine 123 (http://omlc.ogi.edu/spectra/PhotochemCAD/html/ rhodamine123.html) • Berlier et al. 2003 J. Histochem Cytochem refers to Alexa 633 as a rhodamine derivative.

Romanowsky stain Romanowsky staining is a prototypical staining technique that was the forerunner of several distinct but similar methods, including Giemsa, Jenner, Wright, Field, and Leishman stains, which are used to differentiate cells in pathologic specimens.

Microscopic examination of stained blood films Paul Ehrlich had used mixtures of acidic and basic dyes for this purpose in 1879: e.g. Fuchsine (acid dye) and methylene blue (basic dye).[2] In 1891 Ernst Malachowski [3][4][5] and Dmitry Leonidovich Romanowsky[6][7][8] independently developed techniques using a mixture of Eosin Y and modified Methylene blue that produced a surprising hue unattributable to either staining component: a beautiful, distinctive shade of purple.[9][10] Requirement for the occurrence of the Malachowski-Romanowsky-Giemsa effect are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used.[11] Because the aqueous dye solutions were unstable, methanol was introduced as a solvent, and William Ernst Malachowski Boog Leishman [12] y James Homer Wright[13] advocated use of methanol as a fixative prior to staining. Gustav Giemsa improved this technique by standardizing the dye solutions and adding glycerol to increase stability.[14]

268

Romanowsky stain

269

The demethylation of Methylene Blue in aqueous solution using heat and alkali produces a mixture of Azure A, Azure B, Methylene Violet and Methylene Blue. Eosin Y is then added to produce a "neutral" dye. The precipitate is then dissolved in a mixture of methanol and glycerol to form a stock solution; this is diluted with water or an aqueous buffer to form a 'working' solution that is used in the staining of pathology specimens. The 'working' solution is stable for 3 hours.[15] Immunochromatographic capture procedures (Rapid Diagnostic Tests) are nonmicroscopic diagnostic options for the laboratory that may not have appropriate microscopy expertise available.[16]

Giemsa-stained thin blood film showing Plasmodium [1] falciparum infections

References [1] Hempelmann E, Tesarowicz I, Oleksyn BJ (2008). "Malaria wciąż groźna". Wszechświat 109: 180–189. [2] Ehrlich P (1880). "Methodologische Beiträge zur Physiologie und Pathologie der verschiedenen Formen der Leukocyten" (http:/ / www. pei. de/ nn_157292/ SharedDocs/ s/ paul-ehrlich/ 1877-1885/ 1880-methodologisch-beitraege-physiogie-pathologie,templateId=raw,property=publicationFile. pdf/ 1880-methodologisch-beitraege-physiogie-pathologie. pdf). Z klin Med 1: 553–560. . [3] Malachowski E (1891). "Zur Morphologie des Plasmodium malariae". Centbl f klin Med 31: 601–603. [4] Krafts KP, Hempelmann E, Oleksyn BJ (2011). "The color purple: from royalty to laboratory, with apologies to Malachowski". Biotech Histochem 86 (1): 7–35. doi:10.3109/10520295.2010.515490. PMID 21235291. [5] Krafts K, Hempelmann E, Oleksyn BJ (2011). "In search of the malarial parasite : Biographical sketches of the blood stain contributors". Parasitol Research 109 (3): 521–529. doi:10.1007/s00436-011-2475-4. PMID 21660627. [6] Романовскiй Д.Л. (1890). "Къ вопросу о строенiи чужеядныхъ малярiи". Врачъ 52: 1171–1173. [7] Романовскiй Д.Л. Къ вопросу о паразитологiи и терапiи болотной лихорадки. Диссертацiя на степень доктора медицины. Спб. 1891 г., 118 с. [8] Romanowsky D (1891). "Zur Frage der Parasitologie und Therapie der Malaria". St Petersburg Med Wochenschr 16: 297–302, 307–315. [9] Horobin RW, Walter KJ (1987). "Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears" (https:/ / www. researchgate. net/ publication/ 20719192_Understanding_Romanowsky_staining. _I_The_Romanowsky-Giemsa_effect_in_blood_smears). Histochemistry 86 (3): 331–336. PMID 2437082. . [10] Woronzoff-Dashkoff KK. (2002). "The wright-giemsa stain. Secrets revealed" (https:/ / www. researchgate. net/ publication/ 11431311_The_wright-giemsa_stain. _Secrets_revealed). Clin Lab Med. 22 (1): 15–23. doi:10.1016/S0272-2712(03)00065-9. PMID 11933573. . [11] Wittekind D (1979). "On the nature of Romanowsky dyes and the Romanowsky-Giemsa effect" (https:/ / www. researchgate. net/ publication/ 23039385_On_the_nature_of_Romanowsky_dyes_and_the_Romanowsky-Giemsa_effect). Clin Lab Haematol 1 (4): 247–262. doi:10.1111/j.1365-2257.1979.tb01090.x. PMID 94558. . [12] Leishman W (1901). "Note on a Simple and Rapid Method of Producing Romanowsky Staining in Malarial and other Blood Films". Br Med J 2 (2125): 757–758. doi:10.1136/bmj.2.2125.757. PMC 2507168. PMID 20759810. [13] Wright JH (1902). "A Rapid Method for the Differential Staining of Blood Films and Malarial Parasites". J Med Res. 7 (1): 138–144. PMC 2105822. PMID 19971449. [14] Giemsa G (1904). "Eine Vereinfachung und Vervollkommnung meiner Methylenazur-Methylenblau-Eosin-Färbemethode zur Erzielung der Romanowsky-Nochtschen Chromatinfärbung". Centralbl f Bakt etc 37: 308–311. [15] Marshall PN, Bentley SA, Lewis SM. (1978). "Staining properties and stability of a standardised Romanowsky stain" (http:/ / j. bmjjournals. com/ content/ 31/ 3/ 280. abstract). J Clin Pathol 31 (3): 280–282. doi:10.1136/j.31.3.280. PMC 1145244. PMID 76638. . [16] Hempelmann E, Wilson RJM (1982). "Immunoprecipitation of malarial enzymes". Protozoology 29: 637.

Romanowsky stain

External links • Diagnosis and Treatment of Malaria (http://www.cdc.gov/malaria/diagnosis_treatment/diagnosis.htm) CDC website on malaria diagnosis and treatment • Field stain (http://www.btinternet.com/~ukneqas.parasitologyscheme/Blood_Scheme/Teaching_Information/ Stains_for_malaria_parasites/stains_for_malaria_parasites.html) Staining methods for the identification of malaria parasites • Laboratory Diagnosis of Malaria (http://www.rph.wa.gov.au//malaria.html) This Royal Perth Hospital (Australia) website provides an excellent overview of malaria diagnostics • UK malaria advisory, diagnostic and reference service for malaria (http://www.malaria-reference.co.uk) The Malaria Reference Laboratory of the Public Health Laboratory Service in the UK • Chenzinsky and Romanowsky (http://www.romanowsky.ru/English)(Russian)(English) On the 120th Anniversary of the Discovery of the Romanowsky Effect

270

Ruthenium tetroxide

271

Ruthenium tetroxide Ruthenium tetroxide


20427-56-9

PubChem

119079

[1]

[2]


RuO4

Molar mass

165.07 g/mol

Appearance

colorless liquid

Odor

pungent

Density

3.29 g/cm

Melting point

25.4 °C

Boiling point

40.0 °C

Solubility in water

2% w/v at 20°C

Solubility in other solvents

Soluble in Carbon tetrachloride Chloroform

3

Structure Molecular shape

tetrahedral

Dipole moment

zero Hazards

MSDS

external MSDS sheet

[3]

NFPA 704 Related compounds Related compounds

RuO 2 RuCl

3

[4]

() (what is: / ?) Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)

Infobox references

Ruthenium tetroxide Ruthenium tetroxide (RuO4) is a diamagnetic tetrahedral ruthenium compound. As expected for a charge-neutral symmetrical oxide, it is quite volatile. The analogous OsO4 is more widely used and better known. One of the few solvents in which it forms stable solutions is CCl4.

Preparation RuO4 is prepared by oxidation of ruthenium(III) chloride with NaIO4. 8 Ru3+(aq) + 5 IO4−(aq) + 12 H2O(l) → 8 RuO4(s) + 5 I−(aq) + 24 H+(aq) In typical reactions featuring RuO4 as the oxidant, many forms of ruthenium usefully serve as precursors to RuO4, such as oxide hydrates or hydrated chloride. Because RuO4 will readily decompose explosively at slightly elevated temperatures, most laboratories do not synthesize it directly, nor is it commercially available through major chemical vendors. Most laboratories instead use an anionic derivative from a salt of "TPAP" (tetrapropylammonium perruthenate), [N(C3H7)4]RuO4. TPAP is synthesized by oxidizing RuCl3 to RuO4− by NaBrO3 and countering it with the tetrapropylamine cation.

Properties and uses RuO4 oxidizes virtually any hydrocarbon. For example, it will oxidize adamantane to 1-adamantanol. It is used in organic synthesis to oxidize terminal alkynes to 1,2-diketones and primary alcohols to carboxylic acids. When used in this fashion, the ruthenium tetroxide is used in catalytic amounts and regenerated by the addition of sodium periodate to ruthenium(III) chloride and a solvent mixture of acetonitrile, water and carbon tetrachloride. Because it is such an aggressive oxidant, reaction conditions are mild, generally room temperature. Although a strong oxidant, RuO4 oxidations do not perturb stereocenters that are not oxidized. Illustrative is the oxidation of the following diol to a carboxylic acid:

Oxidation of epoxy alcohols also occurs without degradation of the epoxide ring:

Under milder condition, oxidative reaction yields aldehydes instead. RuO4 readily converts secondary alcohols into ketones. Although similar results can be achieved with other cheaper oxidants such as PCC- or DMSO-based oxidants, RuO4 is ideal when a very vigorous oxidant is needed but mild conditions must be maintained. RuO4 readily cleaves double bonds to yield carbonyl products, in a manner similar to ozonolysis. Osmium tetroxide, a more familiar oxidant that is structurally similar to RuO4, does not cleave double bonds, instead producing vicinal diol products. In of practical details, the substrate to be oxidized is typically dissolved in solvent such as CCl4, and acetonitrile is added as an aiding ligand to the catalytic cycle. Ether can then be added to precipitate and recover the ruthenium pre-catalyst.

272

Ruthenium tetroxide

Oxidative catalyst and mechanism Although used as a direct oxidant, due to the relatively high cost of RuO4 it is also used catalytically with an associated re-oxidant. For an oxidation of cyclic alcohols with RuO4 as a catalyst and bromate as a base, RuO4 is first activated by hydroxide: RuO4 + OH− → HRuO5− Then HRuO5− complexes with the cyclic alcohol and form a metal coordination complex (denoted C1 here): HRuO5− + (CH2CH2)nCHOH → C1 + OH− The Ru complex is then attracted by a bromate in which the oxidation of the coordinated alcohol take place: C1 + BrO3− → (CH2CH2)nC=O + HRuO5− In which HRuO5− is reformed as the catalyst, and the cyclic alcohol is oxidized into a cyclic ketone.

Staining Ruthenium tetroxide can be used as an even more aggressive form of staining for the study of polymers by Transmission Electron Microscopy than osmium tetroxide. It has the advantage of staining even polyethylene, the disadvantage is that it is not very selective in what it stains.

References [1] [2] [3] [4]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=20427-56-9 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=119079 http:/ / www. polysciences. com/ shop/ assets/ datasheets/ 320. pdf http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=428796357& page2=%3ARuthenium+ tetroxide

• Cotton, S.A. (1997). Chemistry of Precious Metals. London: Chapman and Hall. ISBN 978-0-7514-0413-5. • Martín, V. S.; Palazón, J. M.; Rodríguez, C. M.; Nevill, C. R. (2006). "Ruthenium(VIII) Oxide". Encyclopedia of Reagents for Organic Synthesis. doi:10.1002/047084289X.rr009.pub2. ISBN 0471936235. • Farmer, V.; Welton, T. (2002). "The oxidation of alcohols in substituted imidazolium ionic liquids using ruthenium catalysts". Green Chemistry 4 (2): 97. doi:10.1039/B109851A. • Singh, B.; Srivastava, S. (1991). "Kinetics and mechanism of ruthenium tetroxide catalysed oxidation of cyclic alcohols by bromate in a base". Transition Metal Chemistry 16 (4): 466. doi:10.1007/BF01129466. • Courtney, J.L.; Swansbor, K.F. (1972). "Ruthenium tetroxide oxidation". Reviews of Pure and Applied Chemistry 22: 47.

273

Safranin

274

Safranin Safranin


477-73-6

PubChem

2723800

ChemSpider

2005991

Jmol-3D images

Image 1

[1]

[2] [3]

[4]


C20H19N4 , Cl

Molar mass

350,84 g/mol

+

()

-

[5]


Infobox references

Safranin (also Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain. It can also be used for the detection of cartilage,[6] mucin and mast cell granules. Safranin typically has the chemical structure shown at right (sometimes described as dimethyl safranin). There is also trimethyl safranin, which has an added methyl group in the ortho- position of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin don't distinguish between the two. Commercial safranin preparations often contain a blend of both types. Safranin is also used as redox indicator in analytical chemistry.

Safranin

Safranines Safranines are the azonium compounds of symmetrical 2,8-dimethyl-3,7-diamino-phenazine. They are obtained by the t oxidation of one molecule of a para-diamine with two molecules of a primary amine; by the condensation of para-aminoazo compounds with primary amines, and by the action of para-nitrosodialkylanilines with secondary bases such as diphenylmetaphenylenediamine. They are crystalline solids showing a characteristic green metallic luster; they are readily soluble in water and dye blue or violet. They are strong bases and form stable monacid salts. Their alcoholic solution shows a yellow-red fluorescence. Phenosafranine is not very stable in the free state; its chloride forms green plates. It can be readily diazotized, and the diazonium salt when boiled with alcohol yields aposafranine or benzene induline, C18H12N3. F. Kehrmann showed that aposafranine could be diazotized in the presence of cold concentrated sulfuric acid, and the diazonium salt on boiling with alcohol yielded phenylphenazonium salts. Aposafranone, C18H12N2O, is formed by heating aposafranine with concentrated hydrochloric acid. These three compounds are perhaps to be represented as ortho- or as para-quinones. The "safranine" of commerce is a ortho-tolusafranine. The first aniline dye-stuff to be prepared on a manufacturing scale was mauveine, which was obtained by Sir William Henry Perkin by heating crude aniline with potassium bichromate and sulfuric acid. Mauveine was converted to parasafranine (1,8-dimethyl Safranine) by Perkin in 1878 by oxidative/reductive loss of the 7-N-para-tolyl group.[7]

References This article incorporates text from a publication now in the public domain: Chisholm, Hugh, ed. (1911). Encyclopædia Britannica (11th ed.). Cambridge University Press. [1] [2] [3] [4]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=477-73-6 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=2723800 http:/ / www. chemspider. com/ 2005991 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BCl-%5D. n1c4c%28%5Bn%2B%5D%28c2c1cc%28c%28N%29c2%29C%29c3ccccc3%29cc%28c%28c4%29C%29N [5] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=464386081& page2=%3ASafranin [6] Rosenberg L (1971). "Chemical Basis for the Histological Use of Safranin O in the Study of Articular Cartilage" (http:/ / www. ejbjs. org/ cgi/ content/ abstract/ 53/ 1/ 69) (abstract). J Bone t Surg Am. 53 (1): 69–82. PMID 4250366. . [7] W. H. Perkin F.R.S. (1879). "LXXIV.—On mauveine and allied colouring matters". J. Chem. Soc., Trans. 35: 717–732. doi:10.1039/CT8793500717.

275

Silver nitrate

276

Silver nitrate Silver nitrate

Identifiers [1]

CAS number

7761-88-8

PubChem

24470

ChemSpider

22878

UNII

95IT3W8JZE

ChEBI

CHEBI:32130

ChEMBL

CHEMBL177367

ATC code

D08 AL01

Jmol-3D images

Image 1

[2] [3]

[4]

[5]

[6]

[7]

[8]


AgNO

Molar mass

169.87 g mol−1

Appearance

white solid

Density

4.35 g/cm3

Melting point

212 °C, 485 K, 414 °F

Boiling point

444 °C, 717 K, 831 °F (decomp.)

Solubility in water

1.22 kg/L (0 °C) 2.16 kg/L (20 °C) 4.40 kg/L (60 °C) 7.33 kg/L (100 °C)

Solubility

soluble in ethanol and acetone

3

Hazards

Silver nitrate

277 EU classification C N R-phrases

R8, R34, R50/53

S-phrases

(S1/2), S26, S45, S60, S61

NFPA 704 ()

[9]


Infobox references

Silver nitrate is an inorganic compound with chemical formula AgNO3. This compound is a versatile precursor to many other silver compounds, such as those used in photography. It is far less sensitive to light than the halides. It was once called lunar caustic because silver was called luna by the ancient alchemists, who believed that silver was associated with the moon.[10] In solid silver nitrate, the silver ions are three-coordinated in a trigonal planar arrangement.[11]

Discovery Albertus Magnus, in the 13th century, documented the ability of nitric acid to separate gold and silver by dissolving the silver.[12] Magnus noted that the resulting solution of silver nitrate could blacken skin. Its common name at the time was nitric acid silver.

Synthesis Silver nitrate can be prepared by reacting silver, such as a silver bullion or silver foil, with nitric acid, resulting in silver nitrate, water, and oxides of nitrogen. 3 Ag + 4 HNO3 → 3 AgNO3 + 2 H2O + NO 3 Ag + 6 HNO3 → 3 AgNO3 + 3 H2O + 3 NO2 etc This is performed under a fume hood because of toxic nitrogen oxide(s) evolved during the reaction.[13]

Reactions A typical reaction with silver nitrate is to suspend a rod of copper in a solution of silver nitrate and leave it for a few hours. The silver nitrate reacts with copper to form hairlike crystals of silver metal and a blue solution of copper nitrate: 2 AgNO3 + Cu → Cu(NO3)2 + 2 Ag Silver nitrate(aq) also decomposes(g) when heated: 2 AgNO3 → 2 Ag + O2 + 2 NO2 Most metal nitrates thermally decompose to the respective oxides, but silver oxide decomposes at a lower temperature than silver nitrate, so the decomposition of silver nitrate yields elemental silver instead.

Silver nitrate

Uses Precursor to other silver compounds Silver nitrate is the least expensive salt of silver; it offers several other advantages as well. It is non-hygroscopic, in contrast to silver fluoroborate and silver perchlorate. It is relatively stable to light. Finally, it dissolves in numerous solvents, including water. The nitrate can be easily replaced by other ligands, rendering AgNO3 versatile. Treatment with solutions of halide ions gives a precipitate of AgX (X = Cl, Br, I). When making photographic film, silver nitrate is treated with halide salts of sodium or potassium to form insoluble silver halide in situ in photographic gelatin, which is then applied to strips of tri-acetate or polyester. Similarly, silver nitrate is used to prepare some silver-based explosives, such as the fulminate, azide, or acetylide, through a precipitation reaction. Treatment of silver nitrate with base gives dark grey silver oxide:[14] 2 AgNO3 + 2 NaOH → Ag2O + 2 NaNO3 + H2O

Halide abstraction The silver cation, Ag+, reacts quickly with halide sources to produce the insoluble silver halide, which is a cream precipitate if Br- is used, a white precipitate if Cl- is used and a yellow precipitate if I- is used. This reaction is commonly used in inorganic chemistry to abstract halides: Ag+ + X− (aq) → AgX where X− = Cl−, Br−, or I−. Other silver salts with non-coordinating anions, namely silver tetrafluoroborate and silver hexafluorophosphate are used for more demanding applications. Similarly, this reaction is used in analytical chemistry to confirm the presence of chloride, bromide, or iodide ions can be tested by adding silver nitrate solution. Samples are typically acidified with dilute nitric acid to remove interfering ions, e.g. carbonate ions and sulfide ions. This step avoids confusion of silver sulfide or silver carbonate precipitates with that of silver halides. The color of precipitate varies with the halide: white (silver chloride), pale yellow/cream (silver bromide), yellow (silver iodide). AgBr and especially AgI photo-decompose to the metal, as evidence by a grayish color on exposed samples.

Organic synthesis Silver nitrate is used in many ways in organic synthesis, e.g. for deprotection and oxidations. Ag+ binds alkenes reversibly, and silver nitrate has been used to separate mixtures of alkenes by selective absorption. The resulting adduct can be decomposed with ammonia to release the free alkene.[15]

Biology In histology, silver nitrate is used for silver staining, for demonstrating reticular fibers, proteins and nucleic acids. For this reason it is also used to demonstrate proteins in PAGE gels. It is also used as a stain in scanning electron microscopy..

278

Silver nitrate

Antimicrobial uses • Coating on catheters to prevent infection[16]

Medicine Silver salts have antiseptic properties. Until the development and widespread adoption of antibiotics, dilute solutions of AgNO3 used to be dropped into newborn babies' eyes at birth to prevent contraction of gonorrhea from the mother. Eye infections and blindness of newborns was reduced by this method; incorrect dosage, however, could cause blindness in extreme cases. This protection was first used by Credé in 1881.[17][18][19] Fused silver nitrate, shaped into sticks, was traditionally called "lunar caustic". It is used as a cauterizing agent, for example to remove granulation tissue around a stoma. General Sir James Abbott noted in his journals that in India in 1827 it was infused by a British surgeon into wounds in his arm resulting from the bite of a mad dog to cauterize the wounds and prevent the onset of rabies. [20] Dentists sometimes use silver nitrate infused swabs to heal oral ulcers. Silver nitrate is also used by some podiatrists to kill cells located in the nail bed. Silver nitrate is also used to cauterize superficial blood vessels in the nose to help prevent nose bleeds. The Canadian physician C. A. Douglas Ringrose researched the use of Micrograph showing a silver nitrate (brown) marked surgical margin. silver nitrate for sterilization procedures on women. A specialist in obstetrics and gynaecology, Ringrose believed that the corrosive properties of silver nitrate could be used to block and corrode the fallopian tubes, in a process that he called "office tubal sterilization".[21] The technique was ineffective; in fact at least two women underwent abortions. Ringrose was sued for malpractice, although these suits were unsuccessful.[22]

Disinfection Much research has been done in evaluating the ability of the silver ion at inactivating E. coli, a microorganism commonly used as an indicator for fecal contamination and as a surrogate for pathogens in drinking water treatment. Concentrations of silver nitrate evaluated in inactivation experiments range from 10–200 micrograms per liter as Ag+. The antimicrobial properties of silver was first observed thousands of years ago when silver containers were used to store water for preservation. Its disinfection ability has been scientifically studied for over a century. Silver's antimicrobial activity saw many applications prior to the discovery of pharmaceutical antibiotics, when it fell into near disuse. Its association with argyria made consumers wary and led them to turn away from it when given an alternative. Since that time, as antibiotic-resistant microorganisms have emerged, interest in using the silver ion for anti-microbial purposes has resumed.[23] Kinetics Before a disinfectant can be effectively used as a water disinfectant, its inactivation kinetics must be established. Kinetics generally depend on both the dosage of disinfectant and the time of application. It is important to understand the kinetics so that the minimum dosage of disinfectant can be applied for the minimum amount of time while still effectively inactivating any pathogens in the water. Because there are many microorganisms present in water, the inactivation kinetics of each one cannot be studied extensively. Therefore, indicator organisms generally more resistant to inactivation than others are used to estimate the kinetics of microorganisms as a whole. Escherichia

279

Silver nitrate coli, also referred to as E. coli, is a commonly used indicator organism. It is well documented that the silver ion is effective in the inactivation of E. coli.[24][25][26][27][28][29][30][31][32][33][34][35] However, there are many inconsistencies in the literature regarding the kinetics of the inactivation of E. coli by the silver ion. With inconsistent data, it is impossible to tell what the true inactivation kinetics are, and therefore impossible to implement any sort of large-scale water treatment. The inconsistencies may be due to several factors. First, the kinetics may depend on the source of the silver ion being used. In recent years, research has focused largely on electrolytically generated silver ions or colloidal silver. Most studies in which the inactivation kinetics of E. coli by silver nitrate were explored extensively date back several decades. Even within this smaller group of studies, vast inconsistencies exist, likely due to inaccurate analytical methods for measuring the concentration of silver in solution.[36] Monitoring the decay of the silver ion in solution is imperative as silver tends to both adsorb readily to organic matter in the water and to be light reactive.[37] Furthermore, silver tends to adsorb to glassware, which can lead not only to a decrease in the silver concentration within a given experiment but also to a release of the silver in subsequent experiments unless measures further than general glassware washing are taken for the removal of silver from the glassware surface.[34] Therefore studies must both minimize the external factors affecting the concentration and to measure the changes in concentration that take place throughout the experiment. Effects of various parameters Despite the inconsistencies in the literature regarding the kinetics of the inactivation of E. coli by silver nitrate, important information can still be taken from the work. A study by Wuhrmann and Zobrist investigated the effect of various parameters upon the kinetics. First, they studied the effect of several ions in the water, including calcium, phosphates and chloride, all of which were found to decrease the bactericidal effect of silver.[33] These effects are important to consider when deg an experiment. Because of the effect of phosphates, it is undesirable to use phosphate buffer to run experiments, as this creates a phosphate concentration much higher than that found in natural waters and will falsely slow the inactivation kinetics. Furthermore, it is important to avoid touching any glassware with bare hands, as chloride from sweat may contaminate the glassware, again slowing inactivation. Chambers, Proctor and Kabler established the importance of using an effective neutralizer solution made of a combination of sodium thioglycolate and sodium thiosulfate, rather than sodium thiosulfate alone, which though it is effective in neutralizing other disinfectants does not sufficiently stop the bactericidal action of silver nitrate.[34] Both tested the effect of pH on the kinetics, finding that a higher pH increased the bactericidal action.[34][37] Wuhrmann and Zobrist further established that at a higher temperature, inactivation occurs faster.[33] Kinetic models A further complication of the inactivation kinetics by silver is the question of which model to use. With most disinfectants, the inactivation is effectively modeled using a first-order Chick-Watson model, which states that a certain level of disinfection will occur at a certain CT Value (concentration * time).[38] According to this model, the same amount of inactivation should take place when a concentration of 0.2 mg/L is applied for 10 minutes as when 0.02 mg/L is applied for 100 minutes. Wuhrmann and Zobrist found rate kinetics that followed this model for all conditions, which agrees fairly well with a study by Chambers and Proctor, while another study by Renn and Chesney found curves that did not follow this law.[36] It is therefore unclear whether this law sufficiently models inactivation by the silver ion. Most recent papers regarding the disinfection of E. coli by silver nitrate have simply plotted the level of disinfection against time.[24][27][29][31][32] While this method of data analysis does not risk making false assumptions about first-order kinetics, it does nothing to for the applied concentration, which is essential to any kinetics. Therefore, different curves need to be generated for each concentration that might be applied. Furthermore, it does not for changes in concentration that might take place during the experiment, and which may vary based on many factors.

280

Silver nitrate

281

A third model which has been suggested for the inactivation kinetics by silver nitrate is that of Cs*T, or chemisorbed silver onto the cell body times time. This model suggests that the rate of inactivation depends not on the concentration in the water at a given time, but rather on the silver that has been chemisorbed by the bacteria. It is assumed, according to this model that C0 = C1 + C2 + C3, where C0 is the initial concentration, C1 is the silver still in solution, C2 is the silver lost to adsorption to glassware or other factors in the solution, and C3 is the silver chemisorbed to the bacteria. C0 is measured at the beginning of the experiment, C1 is measured throughout the experiment, and C2 is determine in a control experiment without bacteria. C3, or the Cs value, is then determined to be C0-C1-C2.[25][39] According to Hwang, et al., this model was successful in estimating inactivation of E. coli by silver nitrate.[25] Although it is possible that this model does not sufficiently for all of the possible fates of the initial silver nitrate added to the solution, it is certainly a compelling method of data analysis. Because it is a new model, it has not been extensively studied by various researchers.

Against warts Repeated daily application of silver nitrate can induce adequate destruction of cutaneous warts, but occasionally pigmented scars may develop. In a placebo-controlled study of 70 patients, silver nitrate given over nine days resulted in clearance of all warts in 43% and improvement in warts in 26% one month after treatment compared to 11% and 14%, respectively, in the placebo group.[40]

Safety As an oxidant, silver nitrate should be properly stored away from organic compounds. Despite being used in low concentrations to prevent gonorrhea and control nose bleeds, silver nitrate is toxic and corrosive.[41] Brief exposure to the chemical will not produce immediate or even any side effects other than the purple, brown or black skin stains; but with more exposure, side effects will become more noticeable, including burns. Long-term exposure may cause eye damage. Short can lead to deposition of black silver stains on the skin. Besides being very destructive of mucous membranes, it is a skin and eye irritant.

Skin of the hand stained by silver nitrate

Although silver nitrate is currently not regulated in water sources by the Environmental Protection Agency, when between 1-5 g of silver have accumulated in the body, a condition called argyria can develop. Argyria is a permanent cosmetic condition in which the skin and internal organs turn a blue-gray color. The United States Environmental Protection Agency had a maximum contaminant limit for silver in water until 1990, but upon determination that argyria did not impact the function of organs affected, removed the regulation.[37] Argyria is more often associated with the consumption of colloidal silver solutions than with silver nitrate, especially at the extremely low concentrations present for the disinfection of water. However, it is still important to consider before ingesting any sort of silver-ion solution.

Silver nitrate

References [1] http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=7761-88-8 [2] http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=24470 [3] http:/ / www. chemspider. com/ 22878 [4] http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=95IT3W8JZE [5] https:/ / www. ebi. ac. uk/ chebi/ searchId. do?chebiId=32130 [6] https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL177367 [7] http:/ / www. whocc. no/ atc_ddd_index/ ?code=D08AL01 [8] http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BN%2B%5D%28%3DO%29%28%5BO-%5D%29%5BO-%5D. %5BAg%2B%5D [9] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=464362299& page2=%3ASilver+ nitrate [10] "Definition of Lunar Caustic" (http:/ / dictionary. die. net/ lunar caustic). . [11] P. Meyer, A. Rimsky et R. Chevalier (1978). "Structure du nitrate d'argent à pression et température ordinaires. Example de cristal parfait". Acta Crystallographica Section B 34 (5): 1457–1462. doi:10.1107/S0567740878005907. [12] Szabadváry, Ferenc (1992). History of analytical chemistry (http:/ / books. google. com/ ?id=53APqy0KDaQC). Taylor & Francis. pp. 17. ISBN 2-88124-569-2. . [13] "Making silver nitrate (youtube)" (http:/ / www. youtube. com/ watch?v=d6hPgGV_qAg& feature=channel_page). . [14] Campaigne, E.; LeSuer, W. M. (1963), "3-Thiophenecarboxylic (Thenoic) Acid" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=cv4p0919), Org. Synth., ; Coll. Vol. 4: 919 (preparation of Ag2O, used in oxidation of an aldehyde) [15] Cope, A. C.; Bach, R. D. (1973), "trans-Cyclooctene" (http:/ / www. orgsyn. org/ orgsyn/ orgsyn/ prepContent. asp?prep=cv5p0315), Org. Synth., ; Coll. Vol. 5: 315 [16] Gupta, Amit; Silver, Simon (1998). "Silver As a Biocide: Will Resistance Become a Problem?". Nature Biotechnology 16 (10): 888. doi:10.1038/nbt1098-888. PMID 9788326. [17] Peter.H (2000). "Dr Carl Credé (1819-1892) and the prevention of ophthalmia neonatorum". Arch Dis Child Fetal Neonatal 83 (2): F158–F159. doi:10.1136/fn.83.2.F158. PMC 1721147. PMID 10952715. [18] Credé C. S. E. (1881). "Die Verhürtung der Augenentzündung der Neugeborenen". Archiv für Gynaekologie 17 (1): 50–53. doi:10.1007/BF01977793. [19] Bulletin of the WHO: Credé's method still valid? (http:/ / www. scielosp. org/ scielo. php?script=sci_arttext& pid=S0042-96862001000300017& lng=es& nrm=iso& tlng=en) [20] British Library, India Office Records, European Manuscripts, MSS EUR F171/33/3, page 109. [21] Ringrose CA. (1973). "Office tubal sterilization". Obstetrics and Gynecology 42 (1): 151–5. PMID 4720201. [22] Cryderman v. Ringrose (1978), 89 D.L.R. (3d) 32 (Alta S.C.) and Zimmer et al. v. Ringrose (1981) 4 W.W.R. 75 (Alta C.A.). [23] "A Brief History of the Health Uses of Silver" (http:/ / www. silver-colloids. com/ Pubs/ history-silver. html). Silver Colloids. . [24] Potapchenko, N. G., L. V. Grigor'eva, O. S. Savluk, and L. A. Kul'skii. "Dosage-Time Dependency of Effect of Silver in Water on Pathogenic Escherichia." Soviet Journal of Water Chemistry and Technology 10 (1988): 101-104. [25] Hwang, Myoung Goo; Katayama, Hiroyuki; Ohgaki, Shinichiro (2007). "Inactivation of Legionella pneumophila and Pseudomonas aeruginosa: Evaluation of the bactericidal ability of silver cations". Water Research 41 (18): 4097. doi:10.1016/j.watres.2007.05.052. PMID 17606286. [26] Jung, WK; Koo, HC; Kim, KW; Shin, S; Kim, SH; Park, YH (2008). "Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli.". Applied and environmental microbiology 74 (7): 2171–8. doi:10.1128/AEM.02001-07. PMC 2292600. PMID 18245232. [27] Kim, JY; Lee, C; Cho, M; Yoon, J (2008). "Enhanced inactivation of E. coli and MS-2 phage by silver ions combined with UV-A and visible light irradiation.". Water research 42 (1-2): 356–62. doi:10.1016/j.watres.2007.07.024. PMID 17692890. [28] Yamanaka, M.; Hara, K.; Kudo, J. (2005). "Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis". Applied and Environmental Microbiology 71 (11): 7589. doi:10.1128/AEM.71.11.7589-7593.2005. PMC 1287701. PMID 16269810. [29] Matsumura, Y; Yoshikata, K; Kunisaki, S; Tsuchido, T (2003). "Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate.". Applied and environmental microbiology 69 (7): 4278–81. doi:10.1128/AEM.69.7.4278-4281.2003. PMC 165194. PMID 12839814. [30] Khaydarov, R. A., R. R. Khaydarov, R. L. Olsen, and S. E. Rogers. "Water Disinfection using electrolytically generated silver, copper and gold ions." (http:/ / www. iwaponline. com/ jws/ 053/ jws0530567. htm) Journal of Water Supply: Research and Technology—AQUA 53, 8 (2004) 567-572. [31] Zhao, G; Stevens Jr, SE (1998). "Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion.". Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 11 (1): 27–32. PMID 9450315. [32] Pedahzur, R (1997). "Silver and hydrogen peroxide as potential drinking water disinfectants: their bactericidal effects and possible modes of action". Water Science and Technology 35 (11-12): 87. doi:10.1016/S0273-1223(97)00240-0. [33] Wuhrmann, Von K. and F. Zobrist. "Untersuchengen uber die bakterizide Wirkung von Silvber in Wasser." Schweizerische Zeitschrift fur Hydrologie 20 (1958) 218-255.

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[34] Chambers, Cecil W., Charles M. Proctor, and Paul W. Kabler. "Bactericidal Effect of Low Concentrations of Silver." Journal of the American Water Works Association 54 (1962) 208-216. [35] Pedahzur, R (1995). "The interaction of silver ions and hydrogen peroxide in the inactivation of E. coli: a preliminary evaluation of a new long acting residual drinking water disinfectant". Water Science and Technology 31 (5-6): 123. doi:10.1016/0273-1223(95)00252-I. [36] Woodward, Richard L. "Review of the Bactericidal Effectiveness of Silver." Journal of the American Water Works Association. 55.7 (1963) 881-886. [37] "Silver Compounds." Encyclopedia of Chemical Technology. Vol. 22. Fourth Ed. Excec. Ed. Jaqueline I. Kroschwitz. New York: John Wiley and Sons, 1997. [38] Masters, Gilbert M. and Wendell P. Ela. "Introduction to Environmental Engineering and Science." Fifth Edition. Upper Saddle River: Prentice Hall, 2006. [39] Hwang, MG; Katayama, H; Ohgaki, S (2006). "Accumulation of copper and silver onto cell body and its effect on the inactivation of Pseudomonas aeruginosa.". Water science and technology : a journal of the International Association on Water Pollution Research 54 (3): 29–34. PMID 17037129. [40] (http:/ / www. huidziekten. nl/ richtlijnen/ BADguidelineCutaneousWarts2001. pdf) Sterling, J. C.; Handfield-Jones, S.; Hudson, P. M.; British Association of Dermatologists (2001). "Guidelines for the management of cutaneous warts". British Journal of Dermatology 144 (1): 4–11. doi:10.1046/j.1365-2133.2001.04066.x. PMID 11167676. [41] "Safety data for silver nitrate (MSDS)" (http:/ / msds. chem. ox. ac. uk/ SI/ silver_nitrate. html). Oxford University Chemistry department. .

External links • International Chemical Safety Card 1116 (http://www.inchem.org/documents/icsc/icsc/eics1116.htm) • NIOSH Pocket Guide to Chemical Hazards (http://www.cdc.gov/niosh/npg/npgd0557.html)

Silver stain Silver staining is the use of silver to selectively alter the appearance of the target.

Use in medicine It is used to stain histologic sections. This kind of staining is important especially to show proteins (for example type III collagen) and DNA. It is used to show both substances inside and outside cells. Silver staining is also used in temperature gradient gel electrophoresis and in polyacrylamide gels.

Some cells are argentaffin. These reduce silver solution to metallic silver after formalin fixation. Other cells are argyrophilic. These reduce silver solution to metallic silver after being exposed to the stain that contains a reductant, for example hydroquinone or formalin. Silver nitrate forms insoluble silver phosphate with phosphate ions; this method is known as the Von Kossa Stain. When subjected to a reducing agent, usually hydroquinone, it forms black elementary silver. This is used for study of formation of calcium phosphate particles during bone growth.

A silver stain (GMS) demonstrating the fungus histoplasma (black round balls) in a liver biopsy.

A number of DNA samples from specimens of Littorina plena amplified using polymerase chain reaction with primers targeting a variable simple sequence repeat (SSR, a.k.a. microsatellite) locus. Samples have been run on a 5% polyacrylamide gel and visualized using silver staining.

Silver staining is used in light microscopy. The metallic silver particles are deposited on sensitised reticulin fibres and are then easily seen in the microscopic preparations. Silver stain aids in the perception of reticular fibers.[1]

Silver stain

284

Karyotype analysis Silver staining is also used in karyotype analysis. Silver nitrate stains the nucleolar organization region-associated protein. This yields a dark region where the silver is deposited, denoting the activity of rRNA genes within the NOR. Chromosomes 13, 14, 15, 21, and 22 have NORs. Furthermore, NORs increase the silver stain activity from about 50 times. This protein was discovered by Watson and Crick.

Organisms highlighted Pseudomonas,[2] Legionella, Leptospira, H. pylori, and fungi like Pneumocystis and Candida.

Methenamine silver stains There are several silver stains incorporating methenamine, including: • Grocott's methenamine silver stain, used widely as a screen for fungal organisms. • Jones' stain, a methenamine silver-Periodic acid-Schiff that stains for basement membrane, availing to view the "spiked" GBM associated with membranous glomerulonephritis.

Silver staining of SDS Polyacrylamide gels Camillo Golgi perfected the silver staining for the study of the nervous system. Golgi's method stains a limited number of cells at random in their entirety.[4] The exact chemical mechanism by which this happens is still largely unknown.[5] Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels.[6] The technique has been extended to the study of other biological macromolecules that have been separated in a variety of s.[7] Classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band, Silver staining increases the sensitivity typically 50 times. Many variables can influence the colour intensity and every protein has its own staining characteristics; clean glassware, pure reagents and water of highest purity are the key points to successful staining.[8] Red blood cell membrane proteins separated by [3] SDS-Page and silverstained

Use in art

Silver staining is also a technique in traditional stained glass to produce the yellow, brown or amber shading when painting on glass. It is a technique that is often used for realistic hair colors. It was discovered in the 14th Century but was not originally used very frequently.

Silver stain

References [1] Schwint OA, Labraga M, Cervino CO, Haffar M, Sequeiros PH, Marcos HJ (2004). "A modification of the staining technique of reticular fibres for image analysis of the cardiac collagen network" (http:/ / linkinghub. elsevier. com/ retrieve/ pii/ S1054880703001534). Cardiovasc. Pathol. 13 (4): 213–20. doi:10.1016/S1054-8807(03)00153-4. PMID 15210137. . [2] Barnini S, Dodi C, Campa M (2004). "Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa" (http:/ / www3. interscience. wiley. com/ resolve/ openurl?genre=article& sid=nlm:pubmed& issn=0266-8254& date=2004& volume=39& issue=3& spage=274). Lett. Appl. Microbiol. 39 (3): 274–7. doi:10.1111/j.1472-765X.2004.01576.x. PMID 15287874. . [3] Hempelmann E, Götze O (1984). "Characterization of membrane proteins by polychromatic silver staining". Hoppe Seyler's Z Physiol Chem 365: 241–242. [4] Grant G (Oct 2007). "How the 1906 Nobel Prize in Physiology or Medicine was shared between Golgi and Cajal". Brain Res Rev 55 (2): 490–498. doi:10.1016/j.brainresrev.2006.11.004. PMID 17306375. [5] Golgi C (1873). "Sulla struttura della sostanza grigia del cervello.". Gazzetta Medica Italiana (Lombardia) 33: 244–246. [6] Kerenyi L, Gallyas F (1973). "Über Probleme der quantitiven Auswertung der mit physikalischer Entwicklung versilberten Agarelektrophoretogramme". Clin. Chim. Acta 47 (3): 425–436. doi:10.1016/0009-8981(73)90276-3. PMID 4744834. [7] Switzer RC 3rd, Merril CR, Shifrin S (Sep 1979). "A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.". Anal Biochem. 98 (1): 231–237. doi:10.1016/0003-2697(79)90732-2. PMID 94518. [8] Hempelmann E, Schulze M, Götze O (1984). "Free SH-groups are important for the polychromatic staining of proteins with silver nitrat". Neuhof V (ed)Electrophoresis '84, Verlag Chemie Weinheim 1984: 328–330.

External links • MedEd at Loyola Histo/practical/stains/hp2-55.html (http://www.meddean.luc.edu/Lumen/MedEd/Histo/ practical/stains/hp2-55.html) • (http://www.biomalpar.org/updatedMethods_In_Malaria_Research_5thedition.pdf) Hempelmann E. SDS-Protein PAGE and Proteindetection by Silverstaining and Immunoblotting of Plasmodium falciparum proteins. in: Moll K, Ljungström J, Perlmann H, Scherf A, Wahlgren M (eds) Methods in Malaria Research, 5th edition, 2008, 263-266

285

Sudan Black B

286

Sudan Black B Sudan Black B

Identifiers [1]

CAS number

4197-25-5

PubChem

61336

ChemSpider

55272

MeSH

Sudan+Black+B

Jmol-3D images

Image 1

[2] [3]

[4]

[5]


C29H24N6

Molar mass

456.54 g/mol

Melting point

120–124 °C ()

[6]


Infobox references

Sudan Black B (C26H24N4O) is a nonfluorescent, relatively thermostable lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance of a dark brown to black powder with maximum absorption at 596-605 nm and melting point 120–124 °C. It stains blue-black. Sudan Black B is one of the dyes used for Sudan staining. Similar dyes include Oil Red O, Sudan III, and Sudan IV. Sudan Black B can be used to stain some other materials than the other Sudan dyes, as it is not so specific to lipids. A use of Sudan Black B is in fingerprint enhancement. It is useful for detecting fats that are contaminated with oil and grease. In differentiating haematological disorders Sudan black will stain myeloblasts but not lymphoblasts.

Production and composition Sudan Black is formed by coupling of diazotized 4-phenylazonaphthalenamine-1 with [7] 2,3-dihydro-2,2-dimethyl-1H-perimidine. Therefore the main product expected was 2,3-dihydro-2,2dimethyl-6-[(4-phenylazo-1-naphthalenyl)-azo]-1H-perimidine. However the dye resulting from the above reaction product actually contains many, up to 42 colored and colorless by products that can be fractionated. The two major products were blue in color confirmed by various chromato-graphic (TLC and column etc) separation and spectroscopic (IR, NMR, Mass) identification were named SSB-I & SSB-II (Rf values of 0.49 and 0.19 in thin Layer Chromatography)[8]. The above described product indeed turned out to be SSB-II which comprises up to 60% of the mixture, and the SSB-I was 2,3-dihydro-2,2dimethyl-4-[(4-phenylazo-1-naphthalenyl)-azo]-1H-perimidine.

Sudan Black B

287

Is there an "Acetylated" Sudan Black? Although there are abundant applications of a Sudan black derivative named acetylated sudan black, chemical analysis shows that thare is no addition acetylation but rather a loss of some of the components of the mixture.[9]

References [1] [2] [3] [4] [5] [6] [7] [8] [9]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=4197-25-5 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=61336 http:/ / www. chemspider. com/ 55272 http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Sudan+ Black+ B http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=N%28%3DN%2Fc5c1c%28cccc1%29c%28%2FN%3DN%2Fc4c2cccc3NC%28Nc%28c23%29cc4%29%28C%29C%29cc5%29%5Cc6ccccc6 http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=401068273& page2=%3ASudan+ Black+ B "Histochemistry. 1977 Dec 7;54(3):237-50. Sudan Black B: chemical structure and histochemistry of the blue main components. Pfüller U, Franz H, Preiss A" (http:/ / www. springerlink. com/ content/ v02ux861236872v3/ ). Springerlink.com. . Retrieved 2012-05-25. HISTOCHEMISTRY AND CELL BIOLOGY Volume 16, Number 1 (1968), 68-84, DOI: 10.1007/BF00306212 Thin layer chrornatography and histochemistry of Sudan Black B A. G. W. Lansink (http:/ / www. springerlink. com/ content/ mp14q1344066v104/ ) "Histochemistry. 1989;93(2):213-6. Evidence against the existence of acetylated Sudan Black B. Lauder RM, Beynon AD" (http:/ / www. springerlink. com/ content/ n0565147671271w6/ ). Springerlink.com. . Retrieved 2012-05-25.


Sudan III

288

Sudan III Sudan III

Identifiers [1]

CAS number

85-86-9

ChemSpider

16736189

UNII

ND733RX3JN

KEGG

C19527

Jmol-3D images

Image 1

[2]

[4]

[3]

[5]


C22H16N4O

Molar mass

352.39 g/mol

Melting point

199 °C ()

[6]


Infobox references

Sudan III is a lysochrome (fat-soluble dye) diazo dye used for staining of triglycerides in frozen sections, and some protein bound lipids and lipoproteins on paraffin sections. It has the appearance of reddish brown crystals and a maximum absorption at 507(304) nm. Sudan III is a dye used for Sudan staining. Similar dyes include Oil Red O, Sudan IV, and Sudan Black B. Sudan I, Sudan III, and Sudan IV have been classified as category 3 carcinogens by the International Agency for Research on Cancer.[7] Its risk and safety phrases are S22-S24/S25. Its other names are Sudan Red BK, Fat Ponceau G, Cerasin Red, C.I. 26100, Solvent Red 23, Sudan Red, Sudan Red III, Sudan V, Sudan Red B, Sudan G, Scarlet B, and Tony Red. In industry, it is used to color nonpolar substances like oils, fats, waxes, greases, various hydrocarbon products, and acrylic emulsions.

Sudan III

References [1] [2] [3] [4] [5] [6] [7]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=85-86-9 http:/ / www. chemspider. com/ 16736189 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=ND733RX3JN http:/ / www. kegg. jp/ entry/ C19527 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=Oc4ccc1ccccc1c4%2FN%3DN%2Fc3ccc%28%5CN%3DN%2Fc2ccccc2%29cc3 http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=401067109& page2=%3ASudan+ III Refat NA, Ibrahim ZS, Moustafa GG, Sakamoto KQ, Ishizuka M, Fujita S (2008). "The induction of cytochrome P450 1A1 by sudan dyes". J. Biochem. Mol. Toxicol. 22 (2): 77–84. doi:10.1002/jbt.20220. PMID 18418879.

• R. D. Lillie. Conn's Biological Stains. Williams & Wilkins, Baltimore, MD., U.S.A. • Aldrich chemical catalogue, 1992. Aldrich Chemical Company, Milwaukee, WI, USA. • Susan Budavari, Editor, (1996). The Merck Index, Ed. 12. Merck & Co., Inc., Whitehouse Station, NJ, USA • Edward Gurr, (1971). Synthetic dyes in biology, medicine and chemistry. Academic Press, London, England.


289

Sudan IV

290

Sudan IV Sudan IV

Identifiers [1]

CAS number

85-83-6

ChemSpider

11252033

KEGG

C19520

Jmol-3D images

Image 1

[2]

[3]

[4]


C24H20N4O ()

[5]


Infobox references

Sudan IV (C24H20N4O) is a lysochrome (fat-soluble dye) diazo dye used for the staining of lipids, triglycerides and lipoproteins on frozen paraffin sections. It has the appearance of reddish brown crystals with melting point 199 °C and maximum absorption at 520(357) nm. Sudan IV is one of the dyes used for Sudan staining. Similar dyes include Oil Red O, Sudan III, and Sudan Black B. Staining is an important biochemical technique, offering the ability to visually qualify the presence of the fatty compound of interest without isolating it. For staining purposes Sudan IV can be made up in propylene glycol[6]. Alternatively, authors have reported using the dye saturated in isopropyl alcohol, 95% ethanol, or 0.05% by weight in acetone:ethanol:water (50:35:15). The idea is to use a moderately apolar solvent to solubilize the dye allowing it to partition into the highly apolar fat without the solvent solubilizing the fat to be stained. Sudan I, Sudan III, and Sudan IV have been classified as category 3 carcinogens by the International Agency for Research on Cancer.[7] In its purified form it is called Biebrich scarlet R, which should not be confused with the water-soluble Biebrich scarlet. In industry, it is used to color nonpolar substances like oils, fats, waxes, greases, various hydrocarbon products, and acrylic emulsions. Sudan IV is also used in United Kingdom as a fuel dye to dye lower-taxed heating oil; because of that it is also known as Oil Tax Red. As a food dye, Sudan IV is considered an illegal dye, mainly because of its harmful effect over a long period of time, as it is a carcinogen. It was ruled unsafe in the 1995 food safety regulations report.

Sudan IV

References [1] [2] [3] [4]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=85-83-6 http:/ / www. chemspider. com/ 11252033 http:/ / www. kegg. jp/ entry/ C19520 http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=Cc4ccccc4%2FN%3DN%2Fc3ccc%28%2FN%3DN%2Fc1c2ccccc2ccc1O%29c%28C%29c3 [5] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=434006584& page2=%3ASudan+ IV [6] http:/ / www. ihcworld. com/ _protocols/ special_stains/ oil_red_o. htm [7] Refat NA, Ibrahim ZS, Moustafa GG, Sakamoto KQ, Ishizuka M, Fujita S (2008). "The induction of cytochrome P450 1A1 by sudan dyes". J. Biochem. Mol. Toxicol. 22 (2): 77–84. doi:10.1002/jbt.20220. PMID 18418879.


Sudan stain Sudan staining is the use of Sudan dyes to stain sudanophilic substances, usually lipids. Sudan lysochromes (Sudan II, Sudan III, Sudan IV, Oil Red O, and Sudan Black B) are used. Sudan dyes have high affinity to fats, therefore they are used to demonstrate triglycerides, lipids, and lipoproteins. Alcoholic solutions of Sudan dyes are usually used, however pyridine solutions can be used in some situations as well. Sudan stain test is often used to determine the level of fecal fat to diagnose steatorrhea. A small sample is dissolved in water or saline, glacial acetic acid is added to hydrolyze the insoluble salts of fatty acids, a few drops of alcoholic solution of Sudan III are added, the sample is spread on a microscopic slide, and heated twice to boil. Normally a stool sample should show only a few drops of red-orange stained fat under the microscope. The method is only semiquantitative, however due to its simplicity it is used for screening.

291

Surfactant

292

Surfactant Surfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants.

Etymology The term surfactant is a blend of surface active agents.[1] In Index Medicus and the United States National Library of Medicine, surfactant is reserved for the meaning pulmonary surfactant. For the more general meaning, surface active agent is the heading.

Micelle in aqueous solution

Definition Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups (their tails) and hydrophilic groups (their heads)[2]. Therefore, a surfactant molecule contains both a water insoluble (or oil soluble) component and a water soluble component. Surfactant molecules will diffuse in water and adsorb at interfaces between air and water or at the interface between oil and water, in the case where water is mixed with oil. The insoluble hydrophobic group may extend out of the bulk water phase, into the air or into the oil phase, while the water soluble head group remains in the water phase. This alignment of surfactant molecules at the surface modifies the surface properties of water at the water/air or water/oil interface.

Structure of surfactant phases in water

A micelle—the lipophilic tails of the surfactant molecules remain on the inside of the micelle due to unfavourable interactions. The polar "heads" of the micelle, due to favourable interactions with water, form a hydrophilic outer layer that in effect protects the hydrophobic core of the micelle. The compounds that make up a micelle are typically amphiphilic in nature, meaning that micelles are soluble not only in protic solvents such as water but also in aprotic solvents as a reverse micelle.

In the bulk aqueous phase, surfactants form aggregates, such as micelles, where the hydrophobic tails form the core of the aggregate and the hydrophilic heads are in with the surrounding liquid. Other types of aggregates such as spherical or cylindrical micelles or bilayers can be formed. The shape of the aggregates depends on the chemical structure of the surfactants, depending on the balance of the sizes of the hydrophobic tail and hydrophilic head. This is known as the HLB, Hydrophilic-lipophilic balance.

Adsorbed layers of surfactants at equilibrium

Surfactant Surfactants reduce the surface tension of water by adsorbing at the liquid-gas interface. The decrease of the surface tension depends on the number of adsorbed molecules per unit area, called the surface excess. The relation that links the surface tension and the surface excess is known as the Gibbs isotherm.

Dynamics of surfactants at interfaces The dynamics of adsorption of surfactants is of great importance for practical applications such as foaming, emulsifying or coating processes, where bubbles or drops are rapidly generated and need to be stabilized. The dynamics of adsorption depends on the diffusion coefficient of the surfactants. Indeed, as the interface is created, the adsorption is limited by the diffusion of the surfactants to the interface. In some cases, there exists a barrier of energy for the adsorption or the desorption of the surfactants, then the adsorption dynamics is known as ‘kinetically limited'. Such energy barrier can be due to steric or electrostatic repulsions. The surface rheology of surfactant layers, including the elasticity and viscosity of the surfactant layers plays a very important role in foam or emulsion stability.

Characterization of interfaces and surfactant layers Interfacial and surface tension can be characterized by classical methods such as the -pendant or spinning drop method Dynamic surface tensions, i.e. surface tension as a function of time, can be obtained by the Maximum Bubble Pressure apparatus The structure of surfactant layers can be studied by ellipsometry or X-Ray reflectivity. Surface rheology can be characterized by the oscillating drop method or shear surface rheometers such as double-cone, double-ring or magnetic rod shear surface rheometer.

Applications Surfactants play an important role as cleaning, wetting, dispersing, emulsifying, foaming and anti-foaming agents in many practical applications and products, including: • • • • • • • • • • •

Detergents Fabric softeners Emulsions Paints Adhesives Inks Anti-fogs Ski waxes, snowboard wax Deinking of recycled papers, in flotation, washing and enzymatic processes Laxatives Agrochemical formulations

• Herbicides (some) • Insecticides • Quantum dot coatings • Biocides (sanitizers) • Cosmetics: • Shampoos • Hair conditioners (after shampoo) • Toothpastes • Spermicides (nonoxynol-9)

293

Surfactant • • • • •

Firefighting Pipelines, liquid drag reducing agent Alkali Surfactant Polymers (used to mobilize oil in oil wells) Ferrofluids Leak Detectors

Detergents in biochemistry and biotechnology In solution, detergents help solubilize molecules by dissociating aggregates and unfolding proteins, including SDS, CTAB. Detergents are key reagents to extract protein by lysis of the cells and tissues: They disorganize the membrane's lipidic bilayer (SDS, Triton X-100, X-114, CHAPS, DOC, and NP-40), and solubilize proteins. Milder detergents such as (OctylThioGlucosides) are used to solubilize sensible proteins (enzymes, receptors). Non-solubilized material is harvested by centrifugation or other means. For electrophoresis, for example, proteins are classically treated with SDS to denature the native tertiary and quaternary structures, allowing the separation of proteins according to their molecular weight. Detergents have also been used to decellularise organs. This process maintains a matrix of proteins that preserves the structure of the organ and often the microvascular network. The process has been successfully used to prepare organs such as the liver and heart for transplant in rats.[3] Pulmonary surfactants are also naturally secreted by type II cells of the lung alveoli in mammals.

Classification of surfactants Surfactants can have a cationic, anionic or neutral head. Several types of hydrophobic tails exist. see classification of surfactants

Current market and forecast The annual global production of surfactants was 13 million metric tons in 2008, and the annual turnover reached US$24.33 billion in 2009, nearly 2% up from the previous year. The market is expected to experience quite healthy growth by 2.8% annually to 2012 and by 3.5 – 4% thereafter.[4][5] Specialists expect the global surfactant market to generate revenues of more than US$41 billion in 2018 – translating to an average annual growth of 4.5%[6]

Health and environmental controversy Some surfactants are known to be toxic to animals, ecosystems, and humans, and can increase the diffusion of other environmental contaminants.[7][8][9] Despite this, they are routinely deposited in numerous ways on land and into water systems, whether as part of an intended process or as industrial and household waste. Some surfactants have proposed or voluntary restrictions on their use. For example, PFOS is a persistent organic pollutant as judged by the Stockholm Convention. Additionally, PFOA has been subject to a voluntary agreement by the U.S. Environmental Protection Agency and eight chemical companies to reduce and eliminate emissions of the chemical and its precursors.[10] The two major surfactants used in the year 2000 were linear alkylbenzene sulfonates (LAS) and the alkyl phenol ethoxylates (APE). They break down in the aerobic conditions found in sewage treatment plants and in soil.[11] Ordinary dishwashing detergent, for example, will promote water penetration in soil, but the effect would last only a few days (many standard laundry detergent powders contain levels of chemicals such as alkali and chelating agents that can be damaging to plants and should not be applied to soils). Commercial soil wetting agents will continue to work for a considerable period, but they will eventually be degraded by soil micro-organisms. Some can, however, interfere with the life-cycles of some aquatic organisms, so care should be taken to prevent run-off of these products into streams, and excess product should not be washed down.

294

Surfactant Anionic surfactants can be found in soils as the result of sludge application, wastewater irrigation, and remediation processes. Relatively high concentrations of surfactants together with multimetals can represent an environmental risk. At low concentrations, surfactant application is unlikely to have a significant effect on trace metal mobility.[12][13]

Biosurfactants Biosurfactants are surface-active substances synthesised by living cells; they are generally non-toxic and biodegradable. Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, environmentally friendly nature, possibility of large-scale production, selectivity, performance under extreme conditions, and potential applications in environmental protection.[14][15] Biosurfactants enhance the emulsification of hydrocarbons, have the potential to solubilise hydrocarbon contaminants and increase their availability for microbial degradation. The use of chemicals for the treatment of a hydrocarbon polluted site may contaminate the environment with their by-products, whereas biological treatment may efficiently destroy pollutants, while being biodegradable themselves. Hence, biosurfactant-producing microorganisms may play an important role in the accelerated bioremediation of hydrocarbon-contaminated sites.[16][17][18] These compounds can also be used in enhanced oil recovery and may be considered for other potential applications in environmental protection.[18][19] Other applications include herbicides and pesticides formulations, detergents, healthcare and cosmetics, pulp and paper, coal, textiles, ceramic processing and food industries, uranium ore-processing, and mechanical dewatering of peat.[14][15][20] Several microorganisms are known to synthesise surface-active agents; most of them are bacteria and yeasts.[21][22] When grown on hydrocarbon substrate as the carbon source, these microorganisms synthesise a wide range of chemicals with surface activity, such as glycolipid, phospholipid, and others.[23][24] These chemicals are synthesised to emulsify the hydrocarbon substrate and facilitate its transport into the cells. In some bacterial species such as Pseudomonas aeruginosa, biosurfactants are also involved in a group motility behavior called swarming motility.

Biosurfactants and Deepwater Horizon The use of biosurfactants as a way to remove petroleum from contaminated sites has been questioned, and criticized as irresponsible and environmentally unsafe. Biosurfactants were not used by BP after the Deepwater Horizon offshore drilling rig went down on April 20, 2010, on the resulting Deepwater Horizon oil spill. However, unprecedented amounts of Corexit, a surfactant solution produced by Nalco Holding Company (whose active ingredient is Tween-80), were sprayed directly into the ocean at the leak and on the sea-water's surface, the theory being that the surfactants would isolate individual molecules of oil, making it easier for petroleum-consuming microbes to digest the oil. However, some scientists say that, rather than helping the situation, the surfactants have managed only to disperse and sink the oil below the surface and out of sight . Naturally occurring petroleum-consuming microbes have evolved on the bottom of the ocean, where they have adapted to live in areas where oil seeps naturally from the ocean floor.

295

Surfactant

References [1] Rosen MJ and Kunjappu JT (2012). Surfactants and Interfacial Phenomena (http:/ / books. google. com/ books?id=1rCdNIzB78AC& printsec=frontcover) (4th ed.). Hoboken, New Jersey: John Wiley & Sons. p. 1. ISBN 1-118-22902-9. . [2] "Bubbles, Bubbles, Everywhere, But Not a Drop to Drink" (http:/ / www. samuelfurse. com/ 2011/ 11/ bubbles-bubbles-everywhere-but-not-a-drop-to-drink/ ). The Lipid Chronicles. . Retrieved 08/01/2012. [3] Wein, Harrison (28 June 2010). "Progress Toward an Artificial Liver Transplant – NIH Research Matters" (http:/ / www. nih. gov/ researchmatters/ june2010/ 06282010liver. htm). National Institutes of Health (NIH). . [4] "Market Report: World Surfactant Market" (http:/ / www. acmite. com/ market-reports/ chemicals/ world-surfactant-market. html). Acmite Market Intelligence (http:/ / www. acmite. com). . [5] Reznik, Gabriel O.; Vishwanath, Prashanth; Pynn, Michelle A.; Sitnik, Joy M.; Todd, Jeffrey J.; Wu, Jun; Jiang, Yan; Keenan, Brendan G. et al. (2010). "Use of sustainable chemistry to produce an acyl amino acid surfactant". Applied Microbiology and Biotechnology 86 (5): 1387–97. doi:10.1007/s00253-009-2431-8. PMID 20094712. [6] Market Study on Surfactants by Ceresana Research (http:/ / www. ceresana. com/ en/ market-studies/ chemicals/ surfactants) [7] Metcalfe, Tracy L.; Dillon, Peter J.; Metcalfe, Chris D. (2008). "DETECTING THE TRANSPORT OF TOXIC PESTICIDES FROM GOLF COURSES INTO WATERSHEDS IN THE PRECAMBRIAN SHIELD REGION OF ONTARIO, CANADA". Environmental Toxicology and Chemistry 27 (4): 811–8. doi:10.1897/07-216.1. PMID 18333674. [8] Emmanuel, E; Hanna, K; Bazin, C; Keck, G; Clement, B; Perrodin, Y (2005). "Fate of glutaraldehyde in hospital wastewater and combined effects of glutaraldehyde and surfactants on aquatic organisms". Environment International 31 (3): 399–406. doi:10.1016/j.envint.2004.08.011. PMID 15734192. [9] Murphy, M; Alkhalidi, M; Crocker, J; Lee, S; Oregan, P; Acott, P (2005). "Two formulations of the industrial surfactant, Toximul, differentially reduce mouse weight gain and hepatic glycogen in vivo during early development: effects of exposure to Influenza B Virus". Chemosphere 59 (2): 235–46. doi:10.1016/j.chemosphere.2004.11.084. PMID 15722095. [10] USEPA: "2010/15 PFOA Stewardship Program" (http:/ / www. epa. gov/ opptintr/ pfoa/ pubs/ pfoastewardship. htm) Accessed October 26, 2008. [11] Scott, M; Jones, Malcolm N (2000). "The biodegradation of surfactants in the environment". Biochimica et Biophysica Acta (BBA) – Biomembranes 1508: 235–251. doi:10.1016/S0304-4157(00)00013-7. [12] Hernández-Soriano Mdel, C; Degryse, F; Smolders, E (2011). "Mechanisms of enhanced mobilisation of trace metals by anionic surfactants in soil". Environmental pollution (Barking, Essex : 1987) 159 (3): 809–16. doi:10.1016/j.envpol.2010.11.009. PMID 21163562. [13] Hernández-Soriano Mdel, C; Peña, A; Dolores Mingorance, M (2010). "Release of metals from metal-amended soil treated with a sulfosuccinamate surfactant: effects of surfactant concentration, soil/solution ratio, and pH". Journal of environmental quality 39 (4): 1298–305. doi:10.2134/jeq2009.0242. PMID 20830918. [14] Banat, I. M., Makkar, R. S., Cameotra, S. S. (2000). "Potential commercial applications of microbial surfactants". Appl. Microbiol. Biotechnol. 53 (5): 495–508. doi:10.1007/s002530051648. PMID 10855707. [15] Rahman, K. S. M., Thahira-Rahman, J., McClean, S., Marchant, R., Banat, I.M (2002). "Rhamnolipid biosurfactants production by strains of Pseudomonas aeruginosa using low cost raw materials". Biotechnol Prog. 18 (6): 1277–1281. doi:10.1021/bp020071x. PMID 12467462. [16] Rosenberg, E., Ron, E. Z (1999). "High and low molecular mass microbial surfactants". Appl. Microbiol. Biotechnol. 52 (2): 154–162. doi:10.1007/s002530051502. PMID 10499255. [17] Del ‘Arco, J. P., De Franca, F. P (2001). "Influence of oil contamination levels on hydrocarbon biodegradation in sandy sediments". Environ. Pollut. 110: 515–519. [18] Rahman, K. S. M., Banat, I.M., Thahira-Rahman, J., Thayumanavan, T., Lakshmanaperumalsamy, P (2002). "Bioremediation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant". Bioresource Technol. 81: 25–32. doi:10.1016/S0960-8524(01)00105-5. [19] Shulga, A., Karpenko, E., Vildanova-Martsishin, R., Turovsky, A., Soltys, M (1999). "Biosurfactant enhanced remediation of oil-contaminated environments". Adsorpt. Sci. Technol. 18: 171–176. [20] Ron, E. Z., Rosenberg, E (2001). "Natural roles of biosurfactants". Environ. Microbiol. 3 (4): 229–236. doi:10.1046/j.1462-2920.2001.00190.x. PMID 11359508. [21] Banat, I. M (1995). "Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: a review". Bioresource Technol. 51: 1–12. doi:10.1016/0960-8524(94)00101-6. [22] Kim, S.E., Lim, E. J., Lee, S.O., Lee , J. D., Lee, T.H (2000). "Purification and characterisation of biosurfactants from Nocardia sp. L-417". Biotechnol. Appl. Biochem. 31 (3): 249–253. doi:10.1042/BA19990111. [23] Muriel, J.M., Bruque, J.M., Olias, J.M., Sanchez, A. J (1996). "Production of biosurfactants by Cladosporium resinae". Biotechnol. Lett. 18 (3): 235–240. doi:10.1007/BF00142937. [24] Desai, J.D., Banat, I.M (1997). "Microbial production of surfactants and their commercial potential". Microbiol. Mol. Biol. Rev. 61 (1): 47–64. PMC 232600. PMID 9106364.

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External links • Surfactants explained for parents (http://www.curoservice.com/parents_visitors/surfactant/ surfactant_composition_action.asp)

Temperature gradient gel electrophoresis Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. TGGE and DGGE can be applied to nucleic acids such as DNA and RNA, and (less commonly) proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE was the original technique, and TGGE a refinement of it.

History DGGE was invented by Leonard Lerman, while he was a professor at SUNY Albany.[1][2][3] The same equipment can be used for analysis of protein, which was first done by Thomas E. Creighton of the MRC Laboratory of Molecular Biology, Cambridge, England.[4] Similar looking patterns are produced by proteins and nucleic acids, but the fundamental principles are quite different.

Negative image of an ethidium bromide-stained DGGE gel

TGGE was first described by Thatcher and Hodson [5] and by Roger Wartell of Georgia Tech. Extensive work was done by the group of Riesner in . Commercial equipment for DGGE is available from Bio-Rad, INGENY and CBS Scientific; a system for TGGE is available from Biometra.

Temperature gradient gel electrophoresis DNA has a negative charge and so will move to the positive electrode in an electric field. A gel is a molecular mesh, with holes roughly the same size as the diameter of the DNA string. When an electric field is applied, the DNA will begin to move through the gel, at a speed roughly proportional to the length of the DNA molecule — this is the basis for size dependent separation in standard electrophoresis. However, in TGGE, there is also a temperature gradient across the gel. At room temperature, the DNA will exist stably in a double-stranded form. As the temperature is increased, the strands begin to separate (melting), and the speed at which they move through the gel decreases drastically. Critically, the temperature at which melting occurs depends on the sequence (GC basepairs are more stable then AT due to stacking interactions, not, as commonly thought, due to the difference in hydrogen bonds (there are three hydrogen bonds between a cytosine and guanine base pair, but only two between adenine and thymine)), so TGGE provides a "sequence dependent, size independent method" for separating DNA molecules. TGGE not only separates molecules, but gives additional information about melting behavior and stability (Biometra, 2000).

Temperature gradient gel electrophoresis

Denaturing gradient gel electrophoresis Denaturing gradient gel electrophoresis (DGGE) works by applying a small sample of DNA (or RNA) to an electrophoresis gel that contains a denaturing agent. Researchers have found that certain denaturing gels are capable of inducing DNA to melt at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components, even those as small as 200-700 base pairs. What is unique about the DGGE technique is that as the DNA is subjected to increasingly extreme denaturing conditions, the melted strands fragment completely into single strands. The process of denaturation on a denaturing gel is very sharp: "Rather than partially melting in a continuous zipper-like manner, most fragments melt in a step-wise process. Discrete portions or domains of the fragment suddenly become single-stranded within a very narrow range of denaturing conditions" (Helms, 1990). This makes it possible to discern differences in DNA sequences or mutations of various genes: sequence differences in fragments of the same length often cause them to partially melt at different positions in the gradient and therefore "stop" at different positions in the gel. By comparing the melting behavior of the polymorphic DNA fragments side-by side on denaturing gradient gels, it is possible to detect fragments that have mutations in the first melting domain (Helms, 1990). Placing two samples side-by-side on the gel and allowing them to denature together, researchers can easily see even the smallest differences in two samples or fragments of DNA. There are a number of disadvantages to this technique: "Chemical gradients such as those used in DGGE are not as reproducible, are difficult to establish and often do not completely resolve heteroduplexes" (Westburg, 2001). These problems are addressed by TGGE, which uses a temperature, rather than chemical, gradient to denature the sample.

Method of TGGE To separate nucleic acids by TGGE, the following steps must be performed: • Preparing and pouring the Gels - Because a buffered system must be chosen, it is important that the system remain stable within the context of increasing temperature. Thus, urea is typically utilized for gel preparation; however, researchers need to be aware that the amount of urea used will have an impact on the overall temperature required to separate the DNA (Biometra, 2000). • Electrophoresis - The gel is loaded, the sample is placed on the gel according to the type of gel that is being run—i.e. parallel or perpendicular—the voltage is adjusted and the sample can be left to run (Biometra, 2000). Depending on which type of TGGE is to be run, either perpendicular or parallel, varying amounts of sample need to be prepared and loaded. A larger amount of one sample is used with perpendicular, while a smaller amount of many samples are used with parallel TGGE. • Staining – Once the gel has been run, the gel must be stained to visualize the results. While there are a number of stains that can be used for this purpose, silver staining has proven to be the most effective tool (Biometra, 2000). • Elution of DNA – the DNA can be eluted from the silver stain for further analysis through PCR amplification (Biometra, 2000).

Applications TGGE and DGGE are broadly useful in biomedical and ecological research; selected applications are described below.

Mutations in mtDNA According to a recent investigation by Wong, Liang, Kwon, Bai, Alper and Gropman, TGGE can be utilized to examine the mitochondrial DNA of an individual. According to these authors, TGGE was utilized to determine two novel mutations in the mitochondrial genome: "A 21-year-old woman who has been suspected of mitochondrial cytopathy, but negative for common mitochondrial DNA (mtDNA) point mutations and deletions, was screened for

298

Temperature gradient gel electrophoresis unknown mutations in the entire mitochondrial genome by temperature gradient gel electrophoresis" (Wong et al., 2002).

p53 mutation in pancreatic juices Lohr and coworkers (2001) report that in a comprehensive study of pancreatic juices of individuals without pancreatic carcinoma, p53 mutations could be found in the pancreatic juices of a small percentage of participants. Because mutations of p53 has been extensively found in pancreatic carcinomas, the researchers for this investigation were attempting to determine if the mutation itself can be linked to the development of pancreatic cancer. While Lohr was able to find p53 mutations via TGGE in a few subjects, none subsequently developed pancreatic carcinoma. Thus, the researchers conclude by noting that the p53 mutation may not be the sole indicator of pancreatic carcinoma oncogenesis.

Microbial ecology DGGE of small ribosomal subunit coding genes was first described by Gerard Muyzer,[6] while he was Post-doc at Leiden University, and has become a widely used technique in microbial ecology. PCR amplification of DNA extracted from mixed microbial communities with PCR primers specific for 16S rRNA gene fragments of Bacteria and Archaea, and 18S rRNA gene fragments of Eukaryotes results in mixtures of PCR products. Because these amplicons all have the same length, they cannot be separated from each other by agarose gel electrophoresis. However, sequence variations (i.e. differences in GC content and distribution) between different microbial rRNAs result in different denaturation properties of these DNA molecules. Hence, DGGE banding patterns can be used to visualize variations in microbial genetic diversity and provide a rough estimate of the richness and abundance of predominant microbial community . This method is often referred to as community fingerprinting. Recently, several studies have shown that DGGE of functional genes (e.g. genes involved in sulfur reduction, nitrogen fixation, and ammonium oxidation) can provide information about microbial function and phylogeny simultaneously. For instance, Tabatabaei et al. (2009) applied DGGE and managed to reveal the microbial pattern during the anaerobic fermentation of palm oil mill effluent (POME) for the first time.[7]

References [1] Cell. 1979 Jan;16(1):191-200. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Fischer SG, Lerman LS [2] Fischer S. G. and Lerman L. S. "Separation of random fragments of DNA according to properties of their sequences" Proc. Natl. Acad. Sci. USA, 1980, 77, 4420-4424. [3] Fischer S. G. and Lerman L. S. "DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory" Proc. Natl. Acad. Sci. USA, 1983, 80, 1579-1583. [4] J Mol Biol. 1994 Oct 7;242(5):670-82. Electrophoretic characterization of the denatured states of staphylococcal nuclease. Creighton TE, Shortle D. [5] Bioochem. J. (1981) 197, 105-109 [6] Muyzer G, de Waal EC, Uitterlinden AG. (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 59:695-700. (http:/ / www. pubmedcentral. nih. gov/ articlerender. fcgi?tool=pubmed& pubmedid=7683183) [7] PCR-Based DGGE and FISH Analysis of Methanogens in Anaerobic Closed Digester Tank Treating Palm Oil Mill Effluent. Meisam Tabatabaei, Mohd Rafein Zakaria, Raha Abdul Rahim, André-Denis G. Wright, Yoshihito Shirai, Norhani Abdullah, Kenji Sakai, Shinya Ikeno, Masatsugu Mori, Nakamura Kazunori, Alawi Sulaiman and Mohd Ali Hassan, 2009, Electronic Journal of Biotechnology, Vol.12 No.3, Issue of 15 July 2009, ISSN: 0717-3458

• Charles J. Sailey, M.D., M.S. Parts taken from a summary paper entitled "TGGE." 2003. The University of the Sciences in Philadelphia.

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Tolonium chloride

300

Tolonium chloride Tolonium chloride

Identifiers [1]

CAS number

92-31-9

PubChem

7084

ChemSpider

11239098

MeSH

Tolonium+chloride

Jmol-3D images

Image 1

[2] [3]

[4]

[5]


C15H16N3S

Molar mass

270.374 g/mol

+

()

[6]


Infobox references

Tolonium chloride (INN, also known as toluidine blue) is a blue cationic (basic) dye used in histology.

Test for lignin Toluidine blue solution is used in testing for lignin, a complex organic molecule that bonds to cellulose fibres and strengthens and hardens the cell walls in plants. A positive toluidine blue test causes the solution to turn from blue to blue-green. A similar test can be performed with phloroglucinol-HCl solution, which turns red.

Other histological uses Toluidine Blue is often used to identify mast cells, by virtue of the heparin in their cytoplasmic granules.[7] It is also used to stain proteoglycans and glycosaminoglycans in tissues such as cartilage. The strongly acidic macromolecular carbohydrates of mast cells and cartilage are coloured red by the blue dye, a phenomenon called metachromasia. Alkaline solutions of toluidine blue are commonly used for staining semi-thin (0.5 to 1 μm) sections of resin-embedded tissue. At high pH (about 10) the dye binds to nucleic acids and all proteins. Although everything in the tissue is stained, structural details are clearly visible because of the thinness of the sections. Semi-thin sections are used in conjunction with ultra-thin sections examined by electron microscopy.

Tolonium chloride

301

References [1] [2] [3] [4] [5]

http:/ / www. commonchemistry. org/ ChemicalDetail. aspx?ref=92-31-9 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=7084 http:/ / www. chemspider. com/ 11239098 http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2007/ MB_cgi?mode=& term=Tolonium+ chloride http:/ / chemapps. stolaf. edu/ jmol/ jmol. php?model=%5BCl-%5D. CN%28C%29c1ccc2nc3cc%28C%29c%28N%29cc3%5Bs%2B%5Dc2c1 [6] http:/ / en. wikipedia. org/ wiki/ Special%3Acomparepages?rev1=401636920& page2=%3ATolonium+ chloride [7] Carson, Freida L; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. p. 188. ISBN 978-0-89189-581-7.

Carson FL (1997) Histotechnology. A Self-Instructional Text. 2nd ed. American Society of Clinical Pathologists, Chicago. Green FJ (1990) The Sigma-Aldrich Handbook of Stains, Dyes and Indicators. Aldrich Chemical Company, Milwaukee, Wisconsin. Horobin RW, Kiernan JA, Eds (2002) Conn's Biological Stains. A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine. 10th ed. BIOS, Oxford. Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Scion, Bloxham, UK.

Weigert's elastic stain Weigert's elastic stain is a combination of stains used in histology which is useful in identifying elastic fibers. Often orcein or a combination of resorcinol and fuchsine are used for staining. For counterstaining cell nuclei nuclear fast red or hematoxylin is also used. After applying elastic fibers show up blue coloured while cell nuclei gets red or blue.

External links • http://stainsfile.info/StainsFile/stain/elastic/elasweig.htm

Blue coloured elastic fibers

Wright's stain

302

Wright's stain Wright's stain is a histologic stain that facilitates the differentiation of blood cell types. It is used primarily to stain peripheral blood smears and bone marrow aspirates which are examined under a light microscope. In cytogenetics it is used to stain chromosomes to facilitate diagnosis of syndromes and diseases. It is named for James Homer Wright, who devised the stain, a modification of the Romanowsky stain, in 1902. Because it distinguishes easily between blood cells, it became widely used for performing differential white blood cell counts, which are routinely ordered when infections are suspected. There are related stains known as the buffered Wright stain, the Wright-Giemsa stain, and the buffered Wright-Giemsa stain, and specific instructions depend on the solutions being used, which may include Eosin Y, Azure B, and Methylene Blue (some commercial preparations combine solutions to simplify staining). The May-Grünwald stain, which produces a more intense coloration, also takes a longer time to perform. White blood cells stained with Wright's stain:

lymphocyte

basophil

External links • Wright+stain [1] at eMedicine Dictionary

References [1] http:/ / www. emedicinehealth. com/ script/ main/ srchcont_dict. asp?src=Wright+ stain

eosinophil

neutrophil

ZiehlNeelsen stain

Ziehl–Neelsen stain The Ziehl–Neelsen stain, also known as the acid-fast stain, was first described by two German doctors: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898). It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group because it is responsible for tuberculosis (TB). Other important Mycobacterium species involved in human disease are Mycobacterium kansasii, Mycobacterium marinum, and of Mycobacterium tuberculosis visualization using the Mycobacterium avium complex. Acid fast organisms like the Ziehl–Neelsen stain. Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain.[1] It can also be used to stain a few other bacteria, such as Nocardia. The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol, and methylene blue. Acid-fast bacilli will be bright red after staining. A variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular hyphae of certain species of fungi in the genus Russula.[2][3] It is also useful in the identification of some protozoa, namely Cryptosporidium and Isospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in a ovum and parasite sputum sample (OnP) can be dissolved by the stain, and is often used in this clinical setting because signs and symptoms of paragonimiasis closely resemble those of TB.

Procedure 1. Drop suspension onto slide 2. Air dry slide 10 minutes at 60 °C, heat-fix slide 10 minutes at 90 °C 3. Flood slide with Carbol Fuchsin 4. Hold a flame beneath the slide until steam appears but do not allow it to boil 5. Allow hot slide to sit for 3 to 5 minutes, rinse with tap water 6. Flood slide with 30% hydrochloric acid in isopropyl alcohol 7. Allow to sit 1 minute, rinse with tap water 8. Flood slide with Methylene Blue 9. Allow to sit 1 minute, rinse with tap water 10. Blot dry 11. View under oil immersion lens Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB Culture should be performed along with an AFB stain; this has a much higher negative predictive value.

303

ZiehlNeelsen stain

Modifications • 5% sulfuric acid is used for destaining Mycobacterium leprae instead of the 20% used for Mycobacterium tuberculosis. • Kinyoun modification (or cold Ziehl–Neelsen technique) is also available. • A protocol in which a detergent is substituted for the highly toxic phenol in the fuchsin staining solution.[4]

References • "Microbiology with Diseases by Body System", Robert W. Bauman, 2009, Pearson Education, Inc. • Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient Car. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print.

Online protocol examples • Ziehl–Neelsen [5] protocol (PDF format).

References [1] Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient Care. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print. [2] Romagnesi, H. (1967). Les Russules d'Europe et d'Afrique du Nord. Bordas. ISBN 0-934454-87-6. [3] Largent, D; D Johnson, R Watling. (1977). How to identify fungi to genus III: microscopic features. Mad River Press. ISBN 0-916422-09-7. p 25. [4] Ellis, RC; LA Zabrowarny. (1993). "Safer staining method for acid fast bacilli" (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC501296/ pdf/ jclinpath00207-0071. pdf). Journal of Clinical Pathology 46: 559–560. doi:10.1136/j.46.6.559. PMC 501296. PMID 7687254. . [5] http:/ / www-medlib. med. utah. edu/ WebPath/ HISTHTML/ MANUALS/ AFB. PDF

304

Article Sources and Contributors

Article Sources and Contributors Staining Source: http://en.wikipedia.org/w/index.php?oldid=500118147 Contributors: 5 albert square, Aa77zz, Academic Challenger, Allmightyduck, Amp, Arcadian, Arony muhit, Beetstra, Benbest, Biolenz, Bubbletruble, CTZMSC3, Cacycle, Caesura, Christopherlin, Cleared as filed, Clicketyclack, Control.valve, Dbenbenn, Diderot, DoktorDec, Dr. Sunglasses, Dr.gurpreet.randhawa, Dysprosia, EKhan, Ediacara, Eleassar777, Enqueror, Epbr123, EscapingLife, Frosted14, GraemeL, Guanaco, Hu12, Itai, J. Spencer, JAKiernan, Jc37, Jcwf, Jeanloujustine, Jeffq, JigPu, Jimp, John of Reading, Kalaiarasy, Kalamkaar, Karol Langner, Kay Dekker, Ketiltrout, Kjkolb, Kntrabssi, Kzollman, MacGyverMagic, Mad Max, Mandarax, MarcoTolo, Mariana Splendor, Marshman, Martpol, MassimoScipio, Materialscientist, Matt.T, Mayfare, Mdphddr, Microbiologyprocedure.com, Mikael Häggström, Moormand, NawlinWiki, Nephron, Nymph, Optigan13, PaddyM, Pkatz, Plantsurfer, Poccil, Praveenrajkani, Prototypefahad, Pupunwiki, R. Baley, Rami radwan, Raven1977, Reedijk, Rholton, Robert M. Hunt, Rsabbatini, Ryan Postlethwaite, SCEhardt, Sarindam7, Shaddack, Shakiestone, Sinecostan, Small-town hero, Snek01, Someones life, TenOfAllTrades, TestPilot, Thumperward, Tide rolls, Timur lenk, Timurite, Tjmayerinsf, Tonyrex, V8rik, Vmenkov, WahreJakob, Who, Wimvandorst, Woohookitty, 161 anonymous edits Acetic acid Source: http://en.wikipedia.org/w/index.php?oldid=501047913 Contributors: *Kat*, 3Jane, 4giron, 66664yyiotjwoier, A - J - Hadley, A30N, A876, ABCD, Acetic Acid, Adashiel, AeonicOmega, Agne27, Akamad, Alansohn, Aleenf1, Alex LaPointe, Allens, Alpha1337Saint, Ambix, Andres, Anna Lincoln, Antonin, Antonio Lopez, Aoso0ck, Arcadian, Arina222, Arjayay, Arkwatem, ArnoldReinhold, Astronaut, Atlant, Aurumali, Aussie Alchemist, AxelBoldt, B.S. Lawrence, BAYAREEEUH, Badon, Banes, Barek, Barneyboo, Beetstra, Bender235, Bendono, Benjah-bmm27, Berserker79, Bfesser, Bhadani, Bjohns86, Bletch, Bo, Bobblewik, BoomerAB, Boomshadow, Bradeos Graphon, Brighterorange, Brossow, Bry9000, Bryan Derksen, Burntsauce, CMBJ, Cacycle, Camw, Can't sleep, clown will eat me, Canadian-Bacon, CanisRufus, Capricorn42, Captain Climber, spec-zero, of Ion Team, CarinaT, Carlosp123, Casforty, CatherineMunro, Cbacc, Ceyockey, Charlene.vickers, Ched Davis, Chem-awb, ChemNerd, Chempedia, ChickenPlow, Christian List, Christian75, Cimorcus, Cometstyles, CommonsDelinker, CoolKoon, Cormorant 80, Cwkmail, Cyc1009, D, Dachshund, Dan2097, Darth Panda, Darthgriz98, David spector, Deckiller, Decltype, Denelson83, Derktar, Dina, Dinosaur puppy, Dirac1933, Dmehring, Dmol, Doc glasgow, DoctorKnockersMD, Dorgan, DrKiernan, Dratman, Drbreznjev, Drilnoth, Drphilharmonic, Durin, ERcheck, ESkog, Ebe123, Edgar181, Edsanville, EdwinHJ, EgonWillighagen, El C, Eleland, 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Article Sources and Contributors Ethanol Source: http://en.wikipedia.org/w/index.php?oldid=500990233 Contributors: 00AgentBond93, 100DashSix, 16@r, 24.93.53.xxx, 28421u2232nfenfcenc, 84, A Softer Answer, A8UDI, ABF, AGeorgas, Aaaaabbb, Abcaemdkwm, Abe Shackleton, Abjs, AboutWeezer, Acegikmo1, AdamWeeden, AdjustShift, Adrian, Agateller, Agne27, Agyle, Aishwarykgupta, Aitias, Akin67, Aland38, Alansohn, Alchemyspirit34, Aleksa Lukic, AlexiusHoratius, Alfred, Algebraist, Alikebabay, Alison, Aloveless, Altermike, Ambix, Amckern, AmiDaniel, Amierzw2, Amplitude101, Anarchangel, Anders Torlind, Andrew Nutter, Andrew73, Andrewpmk, Anlace, AnonGuy, Anonymous Dissident, Antandrus, Antodav2007, Anyeverybody, Ap234, Aptak, Arcadian, Arthena, Ashlandchemist, Athaler, Athene cunicularia, AtomicZebra, AustinChamberlin, Avicennasis, Awesomebitch, AxelBoldt, AySz88, Azzie69, B, B9 hummingbird hovering, Babbage, BabuBhatt, Babybacca, Backin72, Bakabaka, Balabiot, Band123, Bansalr, Banus, Barberio, BarrelProof, BarretB, Basawala, Bathysiderodrome, Bcai388, Beano, Beetstra, Beland, Belovedfreak, Ben-Zin, BenFrantzDale, Benjah-bmm27, Benjamindrew9, BenpV2, Benplowman, Berthelsen, BesigedB, BesselDekker, Betacommand, Bettername, Bevo, Bfesser, Bfigura, Bhargavshivarthy, Billwhittaker, Birdman1, Bjohns86, Blanchardb, Bletch, Blood sliver, Bluerhinos, BoNoMoJo (old), Bob234, Bobblewik, Bobo192, Bogey97, Bonewah, Booyabazooka, BorgHunter, Bpburns88, Brian Crawford, Brianga, BridgeBurner, BritishWatcher, Brookie, Bryan Derksen, Buchanan-Hermit, Buckyboy314, Butros, C.Fred, C6541, CDN99, CHINTUROWAIL, CMBJ, CWenger, Cacycle, Calabe1992, Calibas, Can't sleep, clown will eat me, CanadianLinux, Canberra , CanisRufus, Canley, Canterbury Tail, Cantus, CapitalR, Capricorn42, CaptainVindaloo, Carey Evans, Carlosp123, Carlwfbird, Carnun, Casforty, CatherineMunro, Cbarreyre, Celarnor, Cenarium, Cengelbart, Centrx, Century0, Cfrjlr, ChadThomson, Chaos386, CharlotteWebb, Chasingsol, Chatzaras, Chem-awb, ChemNerd, Chicocvenancio, Chmyr, ChrisHodgesUK, Chrislk02, Christian75, Chuck Carroll, Chuunen Baka, Citicat, Cjflash, Cjthellama, Ckatz, Click23, Cloak&Dagger, Coasterlover1994, Coemgenus, Cointyro, ColesE85, Cometstyles, Comfychaos, ConcernedVancouverite, Cool Hand Luke, CosineKitty, Costyn, l Syx, Cquan, Ctbolt, Ctjf83, Cwkmail, Cybercobra, D.c.camero, DARTH SIDIOUS 2, DJ Clayworth, DMacks, DMahalko, DVD R W, Dan100, Daniel Case, Dar-Ape, Darth Panda, Darthveda, Das Nerd, David Justin, Davumaya, Dawn Bard, Dcooper, Dean Star Wars, Defeatedfear, Deglr6328, Deli nk, Deltabeignet, Demicx, Denisarona, Deor, DerHexer, Derek.cashman, DestinyKeyboardWarrior, Devon Fyson, Devourer09, Dextrose, Dgw, Digitalme, Dimadnk, Discospinster, Diwas, Djhyperman, Dlgiffen, Dmbrown00, Dnf27, DocWatson42, Doctor Biofuel, Dodgese, Dominus, Doorswaves, Dr bab, Dr. Christian, DrKees, Dragana666, Dragonflyspit, Draicone, Drakevz, Drcaldev, Drmies, Drmotley, Drphilharmonic, DrunkenSmurf, DuncanHill, Dwrmal, Dylan Lake, Dysprosia, E. 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Wang, Angesee, Animeronin, Arcadian, ArcadianOnUnsecuredLoc, Bender235, Bobo192, Bomac, Caltas, CanisRufus, Canthusus, Cburnett, CnorthMSU, Comcc, Conversion script, DaveR514, David Gerard, DavidArnold, Diberri, Dodger27, Draeco, Drphilharmonic, Dycedarg, ESkog, Eleassar, Eleassar777, Endemyon, Epastore, Eug, Falcon Kirtaran, Franklinjefferson, Freedomlinux, G3pro, Gigemag76, Gionnico, Gioto, Graevemoore, Graham87, Happysailor, Heritagefarm, Hibernian, Hodja Nasreddin, Huckfinne, II MusLiM HyBRiD II, InvictaHOG, Isirr, IvanLanin, J.delanoy, Jeff Dahl, JeremiahJohnson, Jonny-mt, Joseph Solis in Australia, Josh Grosse, Julianonions, KeyStroke, KingTT, Kmoksy, KnightRider, Kweetal, Kweetal nl, Llywelyn, Lopez50, Lstanley, LuftWaffle0, Macrakis, Magnus Manske, MarcoTolo, Maury Markowitz, Mav, Michael Hardy, Mr.Microbe, Mushin, N00bpwner333, Nagarajan paramasivam, Naprsnaprs, Neparis, Nikkimaria, Nunh-huh, Ompanju, Osm agha, Perkeleperkele, Phil Boswell, Pion, QuentinUK, R'n'B, Rdsmith4, Rich Farmbrough, Rjwilmsi, Rocketboy50, RodC, Sabedon, Sav vas, Scharks, Shaddack, Someone else, Sprightliful, Squidonius, Stemonitis, Stephen B Streater, Sugarskane, Swf5002, T.J.V., Things That Will Bite, Troytsm98, Twooars, Violethughes, Vssailu, Wasell, Wereon, Wickey-nl, Wikid77, Wikimachine, Ww, Yamamoto Ichiro, 178 anonymous edits Gram-positive bacteria Source: http://en.wikipedia.org/w/index.php?oldid=501007085 Contributors: Aitias, Alan Liefting, Alison.philp, AnnaJune, Arcadian, ArcadianOnUnsecuredLoc, Areyoucontagious, Arjun01, Aydin6, BD2412, Bender235, Blaxthos, Bob Blaylock, Bomac, Bryan Derksen, Busrider85, CWenger, Cathalgarvey, Cburnett, Conversion script, Daniil naumoff, David Gerard, Dawnseeker2000, Diberri, Don4of4, Drchazz, Dricherby, Drphilharmonic, Elassint, Eleassar, Eleassar777, Endemyon, Eog1916, Eric Kvaalen, FaerieInGrey, Figma, Flopster2, Funandtrvl, Gabbe, Gabefarkas, Gene Nygaard, Giacmele, Gigemag76, Glane23, Graevemoore, Grafen, Graham87, Hellbus, Hodja Nasreddin, Huckfinne, Intelati, Iridescence, Itai, IvanLanin, J. Spencer, J.delanoy, Jaganath, JeremiahJohnson, Josh Grosse, Julianonions, Karmosin, KnightRider, Kupirijo, Leszek Jańczuk, Liometopum, Lstanley, Mac, Macrakis, Magnus Manske, Manscher, MarcoTolo, Masparasol, Meduban, Michael Hardy, Mormegil, Mr. Toker, Mushin, Ompanju, Osm agha, Paul venter, Perkeleperkele, Phil Boswell, Pkj61, PleaseStand, Pol098, Rjwilmsi, Roadnottaken, Rror, Rswarbrick, Sabedon, Scharks, Sidhekin, Silivrenion, Someone else, Soobrickay, Squidonius, Sugarskane, TedE, Thingg, Tlroche, ToNToNi, Tremello, Twooars, Unused0026, Vgranucci, Violethughes, Westminsterboy, Wickey-nl, Xetheare, Yamamoto Ichiro, Zack2007, Zokie, 170 anonymous edits H&E stain Source: http://en.wikipedia.org/w/index.php?oldid=500443125 Contributors: A. Parrot, AJim, Arcadian, Chefyingi, Dirrival, Ediacara, Enzymes-GMU, Flyguy649, Graham87, Hoffmeier, JAKiernan, Kauczuk, Lb.at.wiki, Robert M. Hunt, Schmiteye, Shaddack, TenOfAllTrades, Timur lenk, VMS Mosaic, 18 anonymous edits Haematoxylin Source: http://en.wikipedia.org/w/index.php?oldid=492279885 Contributors: Arcadian, Beetstra, Chefyingi, Chem-awb, ChemNerd, Chris the speller, Daviesje, Drphilharmonic, Eleassar777, EncycloPetey, Ewen, Gene Nygaard, Hoffmeier, Ilikeeatingwaffles, InvictaHOG, JAKiernan, Jeff Dahl, Jfdwolff, Jfraser, Kereish, King of Hearts, Lamialestat, Markbri16, Mav, Max Schwarz, Merovingian, Mushin, Nono64, Nunh-huh, P-kun80, PhilUK, Physchim62, RJFJR, Rjwilmsi, Seglea, Shaddack, Sinatheory, VMS Mosaic, ‫ﺣﺴﻦ ﻋﻠﻲ ﺍﻟﺒﻂ‬, 22 anonymous edits Heidenhain's AZAN trichrome stain Source: http://en.wikipedia.org/w/index.php?oldid=382443970 Contributors: Bearcat, JAKiernan, Xezbeth, 1 anonymous edits Histological section Source: http://en.wikipedia.org/w/index.php?oldid=469195437 Contributors: Akerbeltz, Armando-Martin, Arthursilva, Bobblewik, Ceyockey, CheekyMonkey, Eisnel, Haruth, Icairns, InvictaHOG, Martpol, Northerncedar, Osquar F, Rustavo, SCEhardt, Tassedethe, WhatamIdoing, 6 anonymous edits Histology Source: http://en.wikipedia.org/w/index.php?oldid=493173577 Contributors: 13alexander, 2004-12-29T22:45Z, 217.99.96.xxx, APH, Aacruzr, Abulkasam, AdjustShift, Ahoerstemeier, Alansohn, Alessandro f2001, Alex.tan, Anclation, Andre Engels, Andres, Antandrus, Arcadian, Benjiboi, Beowulf.1000, Bio geekz, Bobblewik, CDN99, CTPhilosopher, Cellpath, Ceyockey, Chale14, Chmod007, Chris the speller, Chrisscrewball, Clicketyclack, Cncs wikipedia, Coffee, Conversion script, Correogsk, Ctalab, Cuahl, Cutler, DARTH SIDIOUS 2, DRosenbach, DavidMuskett, Davidiad, Delirium, Delldot, Demicx, Digital Tsukasa, DocWatson42, Dr. Kasem, Eleassar777, EmmanuelM, Emmanuelm, Emperorbma, Enchanter, Eran of Arcadia, Fenkys, Galaxiaad, Glenn, Glossologistsucks, Gogo Dodo, HaroldLHammond, Hgvinc, Hovea, Hu12, Hveziris, Iridescent, JAKiernan, JeremyA, Jfraser, Jhomtaz, Jll, John Vandenberg, Jonkerz, Juliancolton, Just James, Kaboytes, Kalaiarasy, KaurJmeb, Kjkolb, Klopfleisch, Kostisl, Kpjas, Kricxjo, LLDMart, Lexor, LuK3, MER-C, Magnus Manske, Mattopaedia, Mav, Metju, Meyer's Histology, Michi zh, Mickchy2k, Mike Dillon, Mike Rosoft, Mike Serfas, Mild Bill Hiccup, Mr.Bip, Mrskristyn, Mycroft7, NawlinWiki, Ncurrier, Nihola, Nk, Nlu, Northerncedar, Novangelis, Nqn, Nuujinn, Obafgkm, Origamiemensch, P. 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Hunt, Robert brand, Roberta F., Rodrigja, Rogerva, Rotational, Ryan, Ryomaandres, SCEhardt, ST47, ScAvenger, Schnurrbart, Selket, Shoeofdeath, Shportin1, Silvamad, Sitarius, Sjschen, SkyWalker, Snek01, Sp33dyphil, SpuriousQ, Staphylococcus314, SunCreator, Sunray, Tastyecats, Technidata, TenOfAllTrades, The Thing That Should Not Be, Thehumorous, Tiddly Tom, Tjcheckley, Tobby72, Toddst1, Treisijs, Trigger hippie77, Troberts 1234, Tryptofish, Uranium-junkie, Valodzka, Vinnie Della Speranza, WahreJakob, Wavelength, WikHead, Wimt, Wolfkeeper, Yerpo, Zfr, Zonethree, 257 anonymous edits Histopathology Source: http://en.wikipedia.org/w/index.php?oldid=497722732 Contributors: 13alexander, AndrewHowse, Avocado, Bk0, CDN99, Carissa921, Creidieki, Darklilac, Davidiad, DerHexer, Dkojic, El C, Eliz81, Emmanuelm, Graham87, Gtk, HaroldLHammond, J04n, Jack007, Jaeger5432, Jfdwolff, Johnnboy, Kembangraps, Lg1223, Lucaseric, Lysy, MER-C, MJM74, Macholl, Mikael Häggström, Ncurrier, Nephron, Nick Number, Northerncedar, NorwegianBlue, Orangeroof, Oreo Priest, Ozmaweezer, Pearle, Poptop43, RJFJR, Rodrigja, Rustavo, SCEhardt, Sbharris, Scrawlspacer, Tarale, TenOfAllTrades, TheTrojanHought, Роман Беккер, 66 anonymous edits

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edits Reducing agent Source: http://en.wikipedia.org/w/index.php?oldid=500386363 Contributors: Andres, Antifamilymang, Arcadian, BeefRendang, Bens, Br77rino, CarrotMan, Charles Matthews, Chemicalinterest, Cmdrjameson, Cyanos, DMacks, Dcirovic, Dwmyers, Frankenpuppy, H Padleckas, HexaChord, Hgrosser, IceCreamAntisocial, InverseHypercube, Isnow, Itub, Johndheathcote, Joseph Solis in Australia, Kaimiddleton, Kaverin, King Of The Juice, Kkmurray, LarryMorseDCOhio, Latobada, Lawrenceuniversity1, Leflyman, Materialscientist, Mihirgk, Morgana The Argent, Mr d logan, Northumbrian, OldakQuill, Pere prlpz, Rrburke, SavinSav, Sazzad Hossen, ScAvenger, Securiger, Smokefoot, Taoster, Tetracube, Torkel.bjornson, Tsemii, VikOlliver, Vrenator, Vsmith, Wikiredox, Xetxo, ~K, 93 anonymous edits Rhodamine Source: http://en.wikipedia.org/w/index.php?oldid=495207285 Contributors: Altenmann, AngelHerraez, Arjen Dijksman, Armando-Martin, Be4u92, Beetstra, Bluemoose, Brockert, Chem-awb, Closedmouth, 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Patriotic dissent, Pengo, Pgcote, Philip Trueman, Physchim62, PinchasC, Pitxulin1, Plantsurfer, Puzzlechef, Qxz, R. 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