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Gram Staining : Principle, Procedure, Interpretation and Animation Posted on January 19, 2016 by Dhurba Giri in Bacteriology, Microbiology // 0 Comments
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The Gram staining technique is the most important
and
microbiological technique.
It
widely
differential was
developed
used staining by
Dr.
Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). In addition
this
stain
also
allows
determination of cell morphology, size, and arrangement. It is typically the first differential brought
test into
run the
on
a
specimen
laboratory
for
identification. In some cases, a rapid, presumptive
identification
of
the
organism or elimination of a particular organism is possible.
PRINCIPLE OF GRAM STAINING
The structure of the organism’s cell wall determines whether the organism is gram psitive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while
others
get
decolorized
by
a
decolorizer. Those bacteria which retain the primary stain are called Gram positive and those bacteria which gets decolorized and then get counterstained are called Gram negative.
Crystal violet (CV) dissociates into CV+ and Cl– ions in aqueous solutions. 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), used as mordant interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. Since Gram negative
organism
peptidoglycan
layer(1-2
have layers)
thin and
have additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram-positive cell also due to the thick and multilayered (40 layers) nature of its peptidoglycan.
After decolorization, the Gram-positive cell
remains
negative
cell
purple loses
and its
the
Gram-
purple
color.
Counterstain, which is usually positivelycharged safranin or basic fuchsin, is applied last to give decolorized Gramnegative bacteria a pink or red color.
REQUIREMENTS AND PREPARATION OF REAGENTS 1. Primary Stain : Crystal violet Solution A : Crystal violet = 2 gm Ethyl alcohol= 20 ml Solution B : Ammonium
oxalate
=
0.8 gm Distilled water = 80 ml Mix solution A and B. Keep for 24 hours and filter. Store in an amber colored bottle.
2. Mordant : Gram’s Iodine Iodine = 1 gm Potassium iodide = 2 gm Distilled water = to 100 ml Mix and Store in an amber colored bottle. 3. Decolorizer : 95% Ethanol or 1:1 acetone with ethanol Acetone = 50 ml Ethanol (95%) = 50ml
4. Counterstain: safranin Safranin O = 0.34 gm Absolute alcohol = 10ml Distilled water = 90ml Mix, filter and store in ambered colored bottle.
PROCEDURE STAINING
OF
GRAM
Smear preparation : 1. Take a grease free dry slide. 2. Sterilize the inoculating loop on a flame of a Bunsen burner. 3. Transfer a loopful of culture (or the specimen) by sterile loop and make a smear at the center. Smear should not be very thin or very thick. 4. Allow the smeat to dry in the air. 5. Fix the dry smear by ing the slide 3-4 times through the flame quickly with the smear side facing up.
Gram Staining :
1. Place the slides on the staining rods.
2. Cover the smear with crystal violet stain and leave for 1 minute. 3. Wash carefully under running tap water. 4. Flood the smear with Gram’s iodine solution and leave for 1 minute. 5. Drain off the iodine Wash the slide for the again in a gentle stream of tap water. 6. Flood the slide with the decolorizing agent then wait for 20-30 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear. 7. Gently wash the slide under running tap water and drain completely. 8. Counterstain with safranin for and and wait for about 30 seconds to 1 minute. 9. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. 10. Observe under microscope.
INTERPRETATION STAINING
OF
GRAM
The staining results of gram stain are as follows : Gram Positive : Dark purple Gram Negative : Pale to dark red Yeasts : Dark purple Epithelial cells : Pale red
Examples of Gram Positive Organisms Bacillus,
Nocardia,
Propionibacterium,
Clostridium, Actinomyces,
Enterococcus,
Cornyebacterium,
Lactobacillus,
Gardnerella,
Listria,
Mycoplasma,
Staphylococcus, Streptomyces, Streptococcus etc
Examples of Gram Positive Organisms Escherichia,
Helicobcater,
Neisseria,
Klebsiella,
Enterobacter,
Vibrio,
Pseudomonas,
Chlamydia,
Hemophilus,
Salmonella, Shigella
ANIMATION STAINING
OF
GRAM
Se produjo un error. Intenta ver este video en www.youtube.com o habilita JavaScript en caso de que no lo tengas habilitado tu navegador.
Animation of Gram Staining (Visited 76 times, 75 visits today) Featured Gram Stain Gram Stain Protocol
Gram Staining
Gram Staining Principle Gram Staining Procedure Gram Staining Video
About Dhurba Giri Dhurba Giri is the author at LaboratoryInfo.com, a scientific blog dedicated for Medical Laboratory Professionals. He's currently studying Bachelor in Medical Laboratory Technology final year at Pokhara University, Nepal. Connect with him on Facebook !
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Gram Staining : Principle, Procedure, Interpretation and Animation Posted on January 19, 2016 by Dhurba Giri in Bacteriology, Microbiology // 0 Comments
Like
1
153 0
0 0
1
The Gram staining technique is the most important
and
microbiological technique.
It
widely
differential was
developed
used staining by
Dr.
Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). In addition
this
stain
also
allows
determination of cell morphology, size, and arrangement. It is typically the first differential brought
test into
run the
on
a
specimen
laboratory
for
identification. In some cases, a rapid, presumptive
identification
of
the
organism or elimination of a particular organism is possible.
PRINCIPLE OF GRAM STAINING
The structure of the organism’s cell wall determines whether the organism is gram psitive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while
others
get
decolorized
by
a
decolorizer. Those bacteria which retain the primary stain are called Gram positive and those bacteria which gets decolorized and then get counterstained are called Gram negative.
Crystal violet (CV) dissociates into CV+ and Cl– ions in aqueous solutions. 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), used as mordant interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. Since Gram negative
organism
peptidoglycan
layer(1-2
have layers)
thin and
have additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram-positive cell also due to the thick and multilayered (40 layers) nature of its peptidoglycan.
After decolorization, the Gram-positive cell
remains
negative
cell
purple loses
and its
the
Gram-
purple
color.
Counterstain, which is usually positivelycharged safranin or basic fuchsin, is applied last to give decolorized Gramnegative bacteria a pink or red color.
REQUIREMENTS AND PREPARATION OF REAGENTS 1. Primary Stain : Crystal violet Solution A : Crystal violet = 2 gm Ethyl alcohol= 20 ml Solution B : Ammonium
oxalate
=
0.8 gm Distilled water = 80 ml Mix solution A and B. Keep for 24 hours and filter. Store in an amber colored bottle.
2. Mordant : Gram’s Iodine Iodine = 1 gm Potassium iodide = 2 gm Distilled water = to 100 ml Mix and Store in an amber colored bottle. 3. Decolorizer : 95% Ethanol or 1:1 acetone with ethanol Acetone = 50 ml Ethanol (95%) = 50ml
4. Counterstain: safranin Safranin O = 0.34 gm Absolute alcohol = 10ml Distilled water = 90ml Mix, filter and store in ambered colored bottle.
PROCEDURE STAINING
OF
GRAM
Smear preparation : 1. Take a grease free dry slide. 2. Sterilize the inoculating loop on a flame of a Bunsen burner. 3. Transfer a loopful of culture (or the specimen) by sterile loop and make a smear at the center. Smear should not be very thin or very thick. 4. Allow the smeat to dry in the air. 5. Fix the dry smear by ing the slide 3-4 times through the flame quickly with the smear side facing up.
Gram Staining :
1. Place the slides on the staining rods.
2. Cover the smear with crystal violet stain and leave for 1 minute. 3. Wash carefully under running tap water. 4. Flood the smear with Gram’s iodine solution and leave for 1 minute. 5. Drain off the iodine Wash the slide for the again in a gentle stream of tap water. 6. Flood the slide with the decolorizing agent then wait for 20-30 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear. 7. Gently wash the slide under running tap water and drain completely. 8. Counterstain with safranin for and and wait for about 30 seconds to 1 minute. 9. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. 10. Observe under microscope.
INTERPRETATION STAINING
OF
GRAM
The staining results of gram stain are as follows : Gram Positive : Dark purple Gram Negative : Pale to dark red Yeasts : Dark purple Epithelial cells : Pale red
Examples of Gram Positive Organisms Bacillus,
Nocardia,
Propionibacterium,
Clostridium, Actinomyces,
Enterococcus,
Cornyebacterium,
Lactobacillus,
Gardnerella,
Listria,
Mycoplasma,
Staphylococcus, Streptomyces, Streptococcus etc
Examples of Gram Positive Organisms Escherichia,
Helicobcater,
Neisseria,
Klebsiella,
Enterobacter,
Vibrio,
Pseudomonas,
Chlamydia,
Hemophilus,
Salmonella, Shigella
ANIMATION STAINING
OF
GRAM
Se produjo un error. Intenta ver este video en www.youtube.com o habilita JavaScript en caso de que no lo tengas habilitado tu navegador.
Animation of Gram Staining (Visited 76 times, 75 visits today) Featured Gram Stain Gram Stain Protocol
Gram Staining
Gram Staining Principle Gram Staining Procedure Gram Staining Video
About Dhurba Giri Dhurba Giri is the author at LaboratoryInfo.com, a scientific blog dedicated for Medical Laboratory Professionals. He's currently studying Bachelor in Medical Laboratory Technology final year at Pokhara University, Nepal. Connect with him on Facebook !
← Previous article
Leave a comment Your email address will not be published. Comment
Name *
Email *
Website
Post Comment
To search, type and hit enter
Recent Posts Gram Staining : Principle, Procedure, Interpretation and Animation Fecal Occult Blood Test : Methods, Principle, Procedure, Clinical Significance and Interpretation Auramine-Rhodamine Staining for AFB : Principle, Procedure, Reporting and Limitations Ziehl-Neelsen Stain (ZN-Stain) : Principle, Procedure, Reporting and Modifications Erythrocyte Sedimentation Rate (ESR) : Principle, Methods of Determination and Clinical Significance Anti-Streptolysin O (ASO) Test : Principle, Procedure, Interpretation and Clinical Significance ELISA : Principle, Procedure, Types, Applications and Animation
C-Reactive Protein (CRP) Test : Introduction, Principle, Procedure and Interpretation CLED (Cystine Lactose Electrolyte Deficient) Agar : Composition, Preparation, Method of Use and Characteristics of Colonies High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications
Popular Posts Differences between Hepatitis A, B, C, D and E Types of Crystals Found In Human Urine and Their Clinical Significance Variations in Red Blood Cell Morphology : Size, Shape, Color and Inclusion Bodies High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications
Types Of Casts Found In Urine And Their Clinical Significance Coombs Test : Types, Principle, Procedure and Interpretation Cross-Matching : Types, Purpose, Principle, Procedure and Interpretation Common blood collection tubes, their additives and laboratory uses Indole Test : principle, procedure and interpretation List of Laboratory Safety Symbols and Their Meanings
Like Us on Facebook Medical Laboratory … 19,030 likes
Like Page
Share
Be the first of your friends to like this
Categories Bacteriology Biochemistry General Informations Hematology Histopathology Immunology Microbiology Mycology Parasitology Virology
Menu
Us Disclaimer Policy
Meta Entries RSS Comments RSS WordPress.org
Connect With Us RSS Twitter Facebook Youtube
Post Archives Post Archives Select Month
Copyright © 2016 | Designed by Dhurba Giri
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