Microbial Growth 6x1f6b

Prof. D.A. Chouhan resource Microbiology B.Sc I /Paper II/ Unit II

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B.Sc I Microbiology PAPER II: MICROBIAL PHYSIOLOGY AND MICROBIAL TECHNIQUES Unit II: Microbial Growth A. MICROBIAL GROWTH INTRODUCTION  Definition: In microbiology, microbial growth is defined as a process of increase in the

number of cells, cell mass and cell activity.  Growth is an essential component of the circle of life.  Any given cell has a finite life span, and the species is maintained only as a result of continued growth of the population.  The indicators of microbial growth were  Increase in both population size and population mass:  Microbial populations tend to increase in number and in cell mass simultaneously.  Increase in cell number and increase in cell population mass both usually occur in a measurably coordinated fashion.  The cell population and population mass typically increase with time (with growth).  Increase in cell number  An increase in cell number is an immediate consequence of cell division.  Because most bacteria grow by binary fission, doubling in cell number usually occurs at the same rate that individual cells grow and divide.  Increase in cell mass  Doubling in size: Individual cells of many species double in size between divisions.  Cell mass thus increases at the same rate as cell number.  Increase in Metabolic activity  Anabolic process: The increase in mass is a consequence of anabolism.  For anabolism to occur a cell must be situated in an environment that supplies all necessary nutrients and which physically falls into a range in which growth can occur.

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The bacterial cell is a living machine capable of duplicating itself. The process of bacterial cell growth involves as many as 2000 chemical reactions of a wide variety of types. Some of these reactions involve energy transformations. Other reactions involve the biosynthesis of small molecules-the building blocks of macromolecules. Still others provide the various cofactors and coenzymes needed for enzymatic reactions. However, the main reactions of cell synthesis are polymerization reactions, the processes by which macromolecules are made from monomers. As macromolecules accumulate in the cytoplasm of a cell, they are assembled into new structures, such as the cell wall, cytoplasmic membrane, flagella, ribosome’s, inclusion bodies, enzyme complexes, and so on, eventually leading to cell division.

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Department of Microbiology, D.B. Science College, Gondia.


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BACTERIAL REPRODUCTION  Definition: The process of regeneration of same type of progeny by parent cell is called

as reproductions.  In microorganisms reproduction takes place by sexual , asexual as well by vegetative

propagation.  In case of bacteria, reproduction is defined as regeneration of new cells from a parental

cell by cell division.  The bacterial cell reproduce or regenerate by four major processes  Binary fission: The normal reproductive method of bacteria is binary fission, in which a single cell divides into two identical cells.  Budding: Some bacteria reproduce by budding; they form a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates  Fragmentation: Some filamentous bacteria (certain actinomycetes) and fungi reproduce by simply fragment, and the fragments initiate the growth of new cell  Reproduction through specialized structure like conidia, cysts and Spores: Some filamentous bacteria (certain actinomycetes) reproduce by producing chains of conidiospores carried extremely at the tips of filaments.


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1. BINARY FISSION  The normal reproductive method of bacteria is binary fission, in which a single cell divides into two identical cells.  Binary fission is the process by which most procaryotes replicate.  Binary fission generally involves the separation of a single cell into two more or less identical daughter cells, each containing, among other things, at least one copy of the parental DNA.  Most bacteria reproduce by a relatively simple asexual process called binary fission: each cell increases in size and divides into two cells.  Stepwise process:  The first steps of binary fission include cell elongation and DNA replication.  The cell envelope then pinches inward, eventually meeting.  A cross wall is formed and ultimately two distinct cells are present, each essentially identical to the original parent cell.

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Fig: Schematic drawing of modes of cell division in various bacteria.(A) Transverse binary fission occurs in Bacillus subtilis (B) Streptococcus faecalis (C) Budding occurs in Rhodopseudomonas acidophila (D) Fragmentation occurs in filamentous cell of a Nocardia species. (E) Formation of conidiospores by Streptomyces species.


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During this process there is an orderly increase in cellular structures and components, replication and segregation of the bacterial DNA, and formation of a septum or cross wall which divides the cell into two progeny cells The process is coordinated by the bacterial membrane perhaps by means of mesosomes. The DNA molecule is believed to be attached to a point on the membrane where it is replicated. The two DNA molecules remain attached at points side-by-side on the membrane while new membrane material is synthesized between the two points. This draws the DNA molecules in opposite directions while new cell wall and membrane are laid down as a septum between the two chromosomal compartments. When septum formation is complete the cell splits into two progeny cells. The time interval required for a bacterial cell to divide or for a population of bacterial cells to double is called the generation time. Generation times for bacterial species growing in nature may be as short as 15 minutes or as long as several days.

2. BUDDING:


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Budding is an another mode by which most of bacteria reproduce in which a parental cell forms an tuber like outgrowth called as bud, which after detachment from a parental cell gives rise to new cell with similar phenotypic and genotypic characters  The bacteria reproduce by budding is Rhodopseudomonas acidophila  The yeast reproduce by budding is S. cereviseae  They form a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates 3. FRAGMENTATION  Fragmentation is a mode of asexual reproduction common in filamentous microorganisms like fungi and filamentous bacteria.  During reproduction by fragmentation a small fragment is detached or dissociated from parental filament by mechanical damage.  This detached fragment then initiate process of reproduction and giving rise to same kind of filamentous structure similar to parental filament.  Example: Reproduction by Fragmentation occurs in filamentous cell of a Nocardia species Reproduction by Fragmentation occurs in filamentous fungi like Aspergillus spp.

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Cellular events in the cells of certain prokaryotes may change and lead to the formation of new cell types. This type of activity is called differentiation. In bacteria dormant or resting structures of four kinds can be produced 1. Heat resistant endospores: Endospores are formed asexually without union of nuclear material from two different types of cells. Usually one endospore is produced per cell. Several species of bacteria capable to produced heat resistant endospores. Example: Bacillus spp., Clostridium spp.,Desulfatomaculum, spororosarcina and Thermoactinomyces 2. Exospores: Kind of heat resistant dormant structures produced by bacteria and Cyanobacteria which were liberated by mother cell before cell lysis and which giving new vegetative structure after germination is called exospores. In certain bacterial species up to four exospores produced by single cell. Bacteria producing exospores include Methylosinus spp. and Rhodomicrobium vannielii. 3. Cysts: Cysts are formed asexually without union of nuclear material from two different types of cells. Usually one cyst is produced per cell. Several species of bacteria capable to produced heat cyst. Some of them are Azotobacter spp. , Myxococcus spp. and Sporocytophaga spp. 4. Conidia : The dormant structure of the fungus like Actinomycetes is an heat labile asexual spore that is formed at the end of special surface ( aerial) cells by aprocess of fragmentation. This dormant structure is called conidia. Microorganisms producing conidia includes Actinomyces spp., Micromonospora spp., Nocardia spp., Streptomyces spp., Streptosporangium spp. ===============================================================================

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STRUCTURE OF ENDOSPORE:



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========================================================================================= 4. SPORULATION: Sporulation is a process of formation of endospore inside the vegetative cell in nutrient deficient or adverse conditions. Sporulation is a multi step process takes place in multiple phases. Different stages in sporulation process are 1. Stage 0: 2. Stage I: Axial filament formation 3. Stage II : Spore septum formation: 4. Stage III: Engulfment of forespores: 5. Stage IV: Cortex synthesis: 6. Stage V: Early coat synthesis: 7. Stage VI : Maturation: 8. Stage VII: Lysis and spore liberation:

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Fig. 1. The morphological stages of sporulation.



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1. Stage 0:    

Represents vegetative cell. In this phase vegetative cell; showing duplication of genetic material. So vegetative cell shows presence of two copes of nuclear material. Cell sensitizing limitation of nutrients and unfavorable conditions.

2. Stage I: Axial filament formation:  This is an event which is observed in initial 0-1.5 hrs when cell phasing nutrient deficiency.  In this phase two nuclear bodies were fused and redistribution of nuclear material from end to end is seen.  This leading to formation of axial filament.  In this phase cell goes on elongation.  Some vegetative cells form granules of poly –B hydroxybutyric acid (PHB).  Such microorganism used this food reserve during sporulation. Such sporulation in which reserved food material is used during sporulation, is called endogenous sporulation.  Example: Bacillus spp.  In some bacteria no internal food reserve, which gain nutrients from exogenous sources for sporulation, such sporulation is called exogenous sporulation. Example: Clostridium ssp.  Protein synthesis takes place.  Synthesis of enzyme- amylase, protease and ribosidase.  Antibiotic synthesis takes place in this phase.

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3. Stage II : Spore septum formation:  Inward growth of a double layer of the cell membrane takes place from the mesosome , takes place near one end of the cell.  Cell is asymmetrically partitioned by doubled membrane wall called as spore septum.  Nuclear material is symmetrically divided, so each compartment bear single copy of genetic material.  Asymmetric cytoplasmic division generating two compartment.  Largest one is called as mother cell while smaller one is called forespore.  Septum does not have peptidoglycan.  Alanine dehydrogenase enzyme synthesized in this phase. 4. Stage III: Engulfment of forespores:  The membrane of larger cell invaginates towards the pole of the cell and engulf the forespore  Forspore thus engulfed by two concentric layers i.e inner forespore membrane and outer forespore membrane.  Protein synthesis is continued in this phase.  Enzymes involved in TCA and glyoxylate cycle synthesized in this phase.  Catalase, alkaline phoaphatase and sulpholactic acid also synthesized in this phase.



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5. Stage IV: Cortex synthesis:  In this phase cortex is develop in between outer and inner forespore membrane.  This occurs in two sub stages, deposition of primordial cell wall on inner forespore membrane and deposition of specific thick layer of peptidoglycan outside the primordial cell wall.  Forespore stars to accumulates calcium and dipicolinic acid and become grey in color.  Dipicolinic acid to Calcium ration in most of spores is 1:1.  Total DPA only accumulated in forespore.  Enzymes like adenine deaminase and ribosidase synthesized in this phase. 6. Stage V: Early coat synthesis:  In this phase multilayered proteinaceous coat is synthesized outside of outer forespore membrane.  This spore coat is synthesized of about 80% keratin like proteins.  It is electron dense and rich in Cysteine and hydrophobic amino acids.  Responsible for the resistance of spore to chemicals.  Accumulation of DPA and calcium continues.  So forespore becomes phase white. In B. cereus a thin exosporium is formed around the coat5 in this phase. 7. Stage VI : Maturation:  In this stage spore become refractile.  Water is withdrawn during this phase and spore become dehydrated.  Dehydration takes place due to contractile nature of cortex. 8. Stage VII: Lysis and spore liberation:  In this phase spore is liberated by autolysis of the mother cell.  No metabolic activity reported within spore.

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a. GROWTH PHASES: Whenever microbial cell subjected to growth in batch cultivation mode, with respect to time conc. Of growth limiting nutrients were decreasing, so growth of microorganism also follows a perticular patter with respect to time. The plot in between Log cell number and time giving a curve called as growth curve. If during batch cultivation single growth limiting nutrient is present in medium, growth curve generated in such cultivation is called monophasic growth curve and growth is called monophasic growth. While if during batch cultivation two growth limiting nutrient is present in medium, growth curve generated in such cultivation is called biphasic growth curve and growth is called diauxic growth.


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Growth Phases of a Simple Batch Culture are 1. Lag phase 2. Accelerating growth phase 3. Logarithmic or exponential growth phase 4. Declining growth phase 5. Stationary phase 6. Increasing death phase 7. Logarithmic death phase 8. Survival phase

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1. THE BACTERIAL GROWTH CURVE INTRODUCTION  During favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations). This is called exponential growth.  In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature.  When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve.  When bacteria are grown in a closed system (also called a batch culture), like a test tube, the population of cells almost always exhibits these growth dynamics: cells initially adjust to the new medium (lag phase) until they can start dividing regularly by the process of binary fission (exponential phase). When their growth becomes limited, the cells stop dividing (stationary phase), until eventually they show loss of viability (death phase). Note the parameters of the x and y axes. Growth is expressed as change in the number viable cells vs time. Generation times are calculated during the exponential phase of growth. Time measurements are in hours for bacteria with short generation times. Figure : The typical bacterial growth curve.

1. Lag Phase.:  Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged.  Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.  The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.  Transfers of bacteria from one medium to another, where there exist chemical differences between the two media, typically results in a lag in cell division.  This lag in division is associated with a physiological adaptation to the new environment, by the cells, prior to their resumption of division.  Cells may increase in size during this time, but simply do not undergo binary fission.  In this phase cell goes on adaptation to new environment. So this phase also called Adaptation or acclimation period:  The lag phase is the initial phase which represents the period (time) required for bacteria to adapt to their new environment.  Constant number of cells: During this phase, the individual bacterial cells increase in size, but the number of cells remains unchanged.  Cells are physiologically active: Cells are very active physiologically and are synthesizing new enzymes and activating factors.  Increase in mass: there is no change in number, but an increase in mass.  The length of the lag phase is determined in part by characteristics of the bacterial species and in part by conditions in the media 2. Accelerating Growth Phase:  Transition period from the lag phase to the log phase.  Cell is beginning to grow (increase in numbers) noticeably as enzyme systems are gearing up. 3. Exponential or logarithmic growth Phase:  The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression.  The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation.  The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations).  Exponential growth is a physiological state marked by back-to-back division cycles such that the population doubles in number every generation time.  During exponential growth there is no change in average cell mass. Because individuals cells are constantly changing in mass as they increase in mass, then divide thus rapidly decreasing in mass (while increasing in number).  During exponential growth the number of cells present at any given time is a multiplicative function of the number of cells present at a previous time.


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4. Declining growth phase:  Transition period from the log phase to the stationary phase.  Decreasing growth rate  Exhaustion of essential nutrients  Accumulation of toxic metabolic products  The growth rate can be limited either by the exhaustion of essential nutrients or by the accumulation of toxic metabolic products.  Food becomes limiting factor and therefore growth rate and mass of bacteria are dependent on the amount of food present. 5. Stationary Phase:  Stationary phase is classically defined as a physiological point where the rate of cell division equals the rate of cell death, hence viable cell number remains constant.  Growth of new cells is balanced by the death of old cells.  No increase in cell mass  Population is "stable" because net growth rate = 0  A way to distinguish these possibilities is to compare viable count with total count.  If both total counts and viable counts don't change then you know that there is both no cell division and no cell death.  If total count increases while viable counts remain constant, then you know that you are observing a true balance between ongoing cell division and cell death  Population growth is limited by one of three factors: 1. Exhaustion of available nutrients; 2. Accumulation of inhibitory metabolites or end products; 3. Exhaustion of space, in this case called a lack of "biological space".  During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing.  The stationary phase, like the lag phase, is not necessarily a period of quiescence.


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Under constant conditions the multiplicative increase in cell number consequently is constant for any given interval of the same duration. Exponential growth: during this phase, the bacterial cells divide regularly at a constant rate. Growth rate is constant and maximum. Straight line on semilog scale: The logarithms of the number of cells plotted against time results in a straight line. Maximum Rate of Substrate utilization: A maximum growth rate occurs under optimal conditions, and substrate is removed from the medium at the maximum rate. The growth rate is limited only by the bacteria's ability to process the substrate. Food is in excess (not limiting) so that the rate of growth is only limited by the ability to process the food Sometimes called "0-order growth" Examples: If a log phase culture goes from 2 cells to 4 cells during a 20 minute interval, then the culture will go from 4 cells to 8 cells during the next 20 minutes.

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Prof. D.A. Chouhan resource Microbiology B.Sc I /Paper II/ Unit II     

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Bacteria produce secondary metabolites, such as antibiotics during the stationary phase of the growth cycle. It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process. Growth of new cells is balanced by the death of old cells. No increase in cell mass Population is "stable" because net growth rate = 0

6. Increasing death phase:  The number of viable cells decreases slowly while the total mass may remain constant due to the fact that the death rate exceeds the production rate of new cells.  Depletion of essential nutrients  Accumulation of inhibitory products  Death occurs primarily as a result of depletion of essential nutrients and/or the accumulation of inhibitory products. 7. Logarithmic Death Phase:.  If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines.  During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.  Death phase is a physiological point at which cell deaths exceed cell births.  More specifically, viable count declines.  During the decline phase, many cells undergo involution---that is, they assume a variety of unusual shapes, which makes them difficult to identify."


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REMARKS:  Growth curve is for a batch system (fed once) and pure culture.  Real biological treatment systems are typically continuously fed (and wasted).  Growth of bacteria is dependent upon many other factors .  Activated sludge and other BWT system contain many types of organisms.  - i.e., biological treatment systems are ecosystems and as such the growth characteristics of one organism may be affected by other organisms.  In general, cells settle much better when they are in endogenous phases.

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8. Survival phase or Endogenous Phase:  Near-starvation condition: That portion of the bacterial growth curve encoming parts of the stationary and declining phases in which microorganisms are in near-starvation is more frequently called the endogenous phase.  Most activated sludge treatment systems are operated in this phase since the microorganisms flocculate rapidly and settle out of solution by gravity.  Food is scarce and the bacteria are forced to metabolize storage products and dead, lysed cells  Growth does not cease, but death exceeds it, net growth rate is negative.


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DIAUXIC GROWTH  Diauxie is a Greek word coined by Jacques Monod to mean two growth phases. The word is used in English in cell biology to describe the growth phases of a microorganism in batch culture as it metabolizes a mixture of two sugars. Rather than metabolizing the two available sugars simultaneously, microbial cells commonly consume them in a sequential pattern, resulting in two separate growth phases.  A diauxic growth curve refers to the growth curve generated by an organism which has two growth peaks. Jacques Monod discovered diauxic growth in 1941 during his experiments with Escherichia coli and Bacillus subtilis.  The bacterium is grown on a growth media containing two types of sugars, one of which is easier to metabolize than the other (for example glucose and lactose).  When a bacterial population subjected to batch cultivation in a medium containing two different sugars as an growth limiting nutrients while other nutrients were present in higher concentrations, then growth pattern of microorganism in such environment is called as diauxic growth and plot in between log of cell number and time is called as diauxic growth curve. Diauxic growth curve shows following phases 1. Initial lag phase 2. 1st Exponential phase 3. 2nd lag phase 4. 2nd exponential phase 5. Stationary phase 6. Death phase

Figure: The Diauxic Growth Curve of E. coli grown in limiting concentrations of a mixture of glucose and lactose

1. Initial lag phase: In this phase cell goes on adaptation to new environment. So this phase

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2. 1st Exponential phase: A plot of the bacterial growth rate resulted in a diauxic growth curve which showed two distinct phases of active growth. During the first phase of exponential growth, the bacteria utilize glucose as a source of energy until all the glucose is exhausted. During the period of glucose utilization, lactose is not utilized because the cells are unable to transport and cleave the disaccharide lactose. Glucose is always metabolized first in preference to other sugars. Only after glucose is completely utilized is lactose degraded. Glucose is a better source of energy than lactose since its utilization requires two less enzymes.

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also called Adaptation or acclimation period: The lag phase is the initial phase which represents the period (time) required for bacteria to adapt to their new environment.



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3. 2nd lag phase: Only after glucose is exhausted are the enzymes for lactose utilization synthesized. The secondary lag during diauxic growth represents the time required for the complete induction of the lac operon and synthesis of the enzymes necessary for lactose utilization (lactose permease and beta-galactosidase). Only then does bacterial growth occur at the expense of lactose. 4. 2nd exponential phase: Then, after a secondary lag phase, the lactose is utilized during a second stage of exponential growth. Eventually, when all the glucose has been consumed, the bacterium will begin the process of expressing the genes to metabolize the lactose. This will only occur when all glucose in the media has been consumed. For these reasons, diauxic growth occurs in multiple phases. In this phase bacteria using lactose sugar and showing exponential phase. 5. Stationary Phase:Lactose is also exhausted and due to nutrient deficiency some bacterial

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cells goes on death. Stationary phase is classically defined as a physiological point where the rate of cell division equals the rate of cell death, hence viable cell number remains constant. Growth of new cells is balanced by the death of old cells. No increase in cell mass Population is "stable" because net growth rate = 0 6. Death Phase: If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. During the death phase, the number of viable cells decreases. Death phase is a physiological point at which cell deaths exceed cell births.



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GENERATION TIME  Definition: Generation time is defined as the time required for a population of microbial cells to double i.e to complete one generation. OR  The time required for a cell to divide (and its population to double) is called the generation time. It varies considerably among organisms and with environmental conditions such as temperature.  A bacterial generation time is also known as its doubling time.  Doubling time is the time it takes a bacterium to do one binary fission starting from having just divided.  Generation times vary with organism and environment and can range from 20 minutes for a fast growing bacterium under ideal conditions, to hours and days for less than ideal conditions or for slowly growing bacteria.  The bacterial growth rates during the phase of exponential growth, under standard nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium's generation time.  Generation times for bacteria vary from about 12 minutes to 24 hours or more.  The generation time for E. coli in the laboratory is 15-20 minutes, but in the intestinal tract, the coliform's generation time is estimated to be 12-24 hours.  For most known bacteria that can be cultured, generation times range from about 15 minutes to 1 hour.  Symbionts such as Rhizobium tend to have longer generation times.  Many lithotrophs, such as the nitrifying bacteria, also have long generation times.  Some bacteria that are pathogens, such as Mycobacterium tuberculosis and Treponema pallidum, have especially long generation times, and this is thought to be an advantage in their virulence.Generation times for a few bacteria are shown in Table . 



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MATHEMATICAL EXPRESSION OF GROWTH EXPONENTIAL GROWTH: CALCULATION OF GENERATION TIME  When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression.  The generation time is the time interval required for the cells (or population) to divide.  Thus, if we start with a single bacterium, the increase in population is geometric progression: 1 2 22 23 24……………..2n  Where n= the number of generations. Each succeeding generation, assuming no cell death, doubles the population. The total population N at the end of a given time period would be expressed  To calculate the generation time of individual microorganisms the following experimental data are required: 1. The number of organisms present at the beginning. 2. The number of organisms present at the end of a given time interval. 3. The time interval.  The relationship of cell numbers and generations can be expressed in a series of equations. Starting with a single cell, the total population N at the end of a given time period would be expressed as N = 1 x 2n………………………….(1) Where 2n is the bacterial population after n generations.  However, under practical conditions several thousands of bacteria are introduced into the medium at zero time and not one, so the formula now becomes. N = N0 x 2n…………………………(2) Where No = number of organisms at zero time. N = number of organisms after n generations. n = number of generations.  Solving equation (2) for n, we have log10N = log10N0 +nlog102 n = [log10N - log10N0] log102…………………..(3) If we now substitute the value of log102, which is 0.301, in the above equation, we can simplify the equation to n = [log10N - log10N0]  0.301 n = 3.3 [log10N - log10N0]………………… (4)

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Thus by equation (4) we can calculate the number of generations if we know the initial population No and the population N after time t. The generation time G is equal to t (the time which elapsed between No and N) divided by the number of generations n, The generation time G (the time required for the population to double) can be determined from the number of generations n that occur in particular time interval t. Using Eq. (4) for n, the generation time can be calculated by the following formula: G= t/n = t / [3.3(log10N - log10No)]



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Relationship of generation time and growth

. Example: What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth?

t_____ 3.3 log b/B G = 240 minutes 3.3 log 107/104 G = 240 minutes 3.3 x 3 G = 24 minutes


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GROWTH RATE CONSTANT The constant of proportionality is an index of the rate of growth and is called the exponential growth rate constant (K). It is defined as number of doublings in unit time, and is usually expressed as the number of doubling in an hour. It is calculated from the following equation: N = Population at time t. No = Population at time zero. Taking the logarithms log N = log No + Kt log 2, and Solving the equation for K K = (log N-log No) / t log2 The exponential growth rate constant is therefore reciprocal to generation time, i.e. G=1/K For example, generation time of E. coli is 20 minutes, i.e. t hour. 1/3=1/K K = 3 doublings per hour.

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G=


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CONTINUOUS GROWTH: CONTINUOUS CULTURE Contrary to the studies in batch culture where the exponential growth of bacterial population is restricted only for a few generations, it is often desirable to maintain prolonged exponential growth of bacterial population for genetical and biochemical studies, and in industrial processes. This condition is obtained by growing bacteria in a continuous culture, a culture in which nutrients are supplied and end products continuously removed. A continuous culture, therefore, is that in which the exponential growth phase of bacterial population can be maintained at a constant rate (steady state growth) for over a long period of time by continuously supplying fresh medium from a reservoir to, growth chamber and continuously removing excess volume of culture medium of growth chamber through a siphon overflow. By doing so the microbes never reach stationary phase because the end products do not accumulate to work as inhibitory to growth and nutrients are not completely expended. Continuous culture systems can be operated as chemostat or as turbidostat. Chemostat In a chemostat the flow rate is set at a particular value with the help of a flow rate regulator and the rate of growth of the culture adjusts to this flow rate. That is, the sterile medium is fed-into the vessel at the same rate as the media containing microorganisms is removed. In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium is allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are removed by the overflow. The rate of addition of the fresh medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient. Thus, the chemostat relieves the insufficiency of nutrients, the accumulation of toxic substances, and the accumulation of excess cells in the culture, which are the parameters that initiate the stationary phase of the growth cycle. The bacterial culture can be grown and maintained at relatively constant conditions, depending on the flow rate of the nutrients. Chemostat, a continuous culture system

Reservoir of Sterile Medium (Fresh) Flow Rate Regulator Air inlet Air Filter age for Inoculation and Air Outlet Siphon Overflow Growth Chamber Receptacle

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1. 2. 3. 4. 5. 6. 7. 8.



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Turbidostat In a turbidostat the system includes an optical sensing device (photoelectric device) which continuously monitors the culture density in the growth vessel and controls the dilution rate to maintain the culture density at a constant rate. If the culture density becomes too high the dilution rate is increased, and if it becomes too low the dilution rate is decreased. The turbidostat differs from the chemostat in many ways. The dilution rate in a turbidostat varies rather then remaining constant, and its culture medium lacks a limiting nutrient. The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at low dilution rates. Turbidostat

4. Photo cell

2. Valve Controlling Flow of Medium

5. Light Source

3. Outlet for Spent Medium

6. Turbidostat

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1. Reservoir of Sterile Medium



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CONCEPT OF SYNCHRONOUS CULTURE: DEFINITION AND USES Definition: Synchronous culture is population of bacterial cells which are physiologically identical, in the same stage of cell division cycle at a given time , has same age and size..  Synchronous growth of a bacterial population is that during which all bacterial cells of the population are physiologically identical divide at a same time.  Synchronous cultures of bacteria can be obtained by a number of techniques.  Two fundamental approaches were used  Synchronous populations can be sorted out according to age and size by physical separation method  Culture is induced by manipulating physical environment or chemical composition of the medium. A. Selection by size and age An excellent and most widely used method to obtain synchronous cultures is the Helmstetter-Cummings Technique in which an unsynchronized bacterial culture is filtered through cellulose nitrate membrane filter.

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Helmstetter-Cummings technique  Bacterial population from batch culture shows microbial populations with different age, size, physiological activities and different stage of growth.  Smallest cells were youngest while largest cells which just ready to divide.  Such mixed population when ed through membrane filters of pore size just enough to trap largest cells and filter small cells.  The filter is then inverted and fresh medium is allowed to flow through it.  The loosely bound bacterial cells are washed from the filter, leaving some cells tightly associated with the filter. The filter is now inverted and fresh medium is allowed to flow through it. New bacterial cells, which are produced by cell division and are not tightly associated with the filter, are washed into the effluent. Hence, all cells in the effluent are newly formed and are, therefore, at the same stage of growth and division cycle.  The effluent thus represents a synchronous culture



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B. Selection by induction technique:  A synchronous culture can be obtained by synchronization of bacterial population to a same stage of growth by shock treatment.  The shock treatments include temperature, starvation, drugs etc.  Example 1: In commonly used technique an exponentially growing culture at 370C is subjected to incubation at 200C. At lower temperature cells unable to divide so during incubation at 200C all cells mature to the point of fission (enlarged cell) at 30 minutes. When revert incubation temperature to 370C all cells divide at same time giving a synchronous culture.  Example 1: Auxotrophic microbial strain when subjected to growth in a minimal media which lacking growth factors. Auxotrophic strain goes on phase of starvation and not able to divide. But all cells were synchronized at a same stage of growth. When such culture reinoculated in a fresh compete media with growth factor, all cells divide at a same time generating synchronous culture.

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Uses of synchronous culture  Synchronous growth helps studying particular stages or the cell division cycle and their interrelations.  Studying the behavior of individual cells during growth of bacterial populations in batch or continuous cultures.  Information about the growth behavior of individual bacteria can, however, is obtained by the study of synchronous cultures.  These cultures are used in industry for production of primary and secondary microbial metabolites at industrial scale.  Synchronous cultures are used to check effectiveness of antibiotics.  Also used to assay antibiotic concentration.



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MEASUREMENT OF BACTERIAL GROWTH Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosomes, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission. For unicellular organisms such as the bacteria, growth can be measured in of two different parameters: changes in cell mass , changes in cell numbers and in metabolic activities. Techniques of microbial cell growth measurement categorizes into following types Techniques of growth

measurement Enumeration of Cell number

Direct Microscopic count

Electronic counting chamber

Breed method

Indirect viable count

Coulter counter

Cell mass measureme nt

Cell activity measureme nt

Nitrogen content

Turbidometry

Dry weight

Plate count

Haemocytometer

Membrane filter technique

Proportional counter

Tube count (MPN)


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Cell number counting or measuring techniques involve direct counts, (visually or instrumentally) and indirect viable cell counts. 1. DIRECT MICROSCOPIC COUNTS:  Direct microscopic counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>10 7 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.  A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In order to detect and prove that thermophilic bacteria were growing in boiling hot springs, T.D. Brock immersed microscope slides in the springs and withdrew them periodically for microscopic observation. The bacteria in the boiling water attached to the glass slides naturally and grew as micro-colonies on the surface.  Enumeration of microorganisms is especially important in dairy microbiology, food microbiology, and water microbiology.

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Enumeration of Cell number


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


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PROPORTIONAL COUNT  Standard suspension of particles( for example plastic beads where the number of particles per volume is known) is mixed with equal amount of cell suspension  This mixed suspension is spread on the slide.  Heat fixed and stained using suitable stain.  The particles and cells in each microscopic field is counted.  Average count of particles and cells is taken from the Number of fields.  Example:  Average count for particle is 5 and average count for cell is 30 per field if number of partilcles in 1 ml of standard suspension is 10,0000, then number of cells per 1ml of cell suspension is 30  ---------x10,000= 60,000 cells/ ml 5

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Counting chambers, consisting of a ruled slide and a cover slip. It is constructed in such a manner that the cover slip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted.  Different techniques of direct cell count includes  Dry smear count: Breed method  Proportional count  Haemocytometer BREED METHOD  This method is also called as dried smear cell count.  A known volume of microbial cell suspension (0.01 ml) is spread uniformly over a glass slide covering a specific area (1 sq. cm).  The smear is then fixed by heating and stained  For milk first treat the slide with some fat solvents to remove the fat and stained . For milk Newman’s stain serves both the purposes.  For bacterial broth stain the smear with appropriate stain.  Wash in gentle stream of water and air dry.  Examined under oil immersion lens and count the number of cells at least in 20-30 microscopic fields.  The counting of total number of cells is determined by calculating the total number of microscopic fields per one square cm. area of the smear.  The total number of cells can be counted with the help of following calculations: 1. Area of microscopic field = πr2 r (oil immersion lens) = 0.08 mm. Area of the microscopic field under the oil immersion lens= πr2 = 3.14 x (0.08 mm)2 = 0.02 sq. mm. 2. Area of the smear one sq. cm. = 100 sq. mm. 3. Then, the no. of microscopic fields = 100 / 0.02= 5000 4. No. of cells 1 sq. cm. (or per 0.01 ml microbial cell suspension) = Average no. of microbes per microscopic field x 5000 Application: Use for enumeration of milk bacteria


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Figure: Direct count of bacteria with a Petroff-Hausser cell counter.


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Neubauer counting chamber

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============================================================================ DIRECT COUNTING IN LIQUID BROTH By Haemocytometer (Neubauer Chamber) Or Petroff-Hausser Cell Counter  Another quick and precise method of counting total number of cell in abroth is by using Neubauer counting chamber (Haemocytometer) or by use of Petroff Hausser Counting Chamber.  Counting chambers, consisting of a ruled slide and a cover slip. It is constructed in such a manner that the cover slip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted.  Among two counting chambers, Neubauer counting chamber is suitable for large sized bacterial cells like Azotobacter  Petroff-Hausser counting chamber, suitable for counting small sized bacterial cell.  Procedure 1. Dip the chamber and cover glass in mild detergent solution for few minutes and rinse thoroughly with distilled water, drain excess water and air dry. 2. Place the cover glass over the counting chamber in appropriate manner. 3. With the help of a Pasteur pipette draw small quantity of bacterial broth. 4. Fill the chamber 5. Observe under light microscope (40 x) 6. Count the number of cells in each large square. 7. Make count in at least 10-15 large squares. Note observations. 8. Calculate the total population as follows. Total cell count (cells/ml) = Average No. of cells per large square x Dilution factor x Factor for large square


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For example In Neubauer counting chamber  Average cell count per large square was 25  Dilution factor is 102  Factor for large square is 10 4 Therefore Total cell count (cells/ml) =25 x 102 x104 = 2.5 x107 cells/ml In Petroff-Hausser counting chamber  Average cell count per large square was 25  Dilution factor is 102  Factor for large square is : 2.5 x10 5 Therefore Total cell count (cells/ml) =25 x 102 x2.5x105 = 6.5 x108 cells/ml Limitations: o Motile bacteria are difficult to count by this method, and as happen with other microscopic methods, dead cells are about as likely to be counted as live ones. o A rather high concentration of cells is required to be countable-about 10 million bacteria per milliliter. Advantages o No incubation time is required, and they are usually reserved for applications in which time is the primary consideration. ======================================================================================= Advantage of Direct Microscopic count  Rapid, Simple and easy method requiring minimum equipment.  Morphology of the bacteria can be observed as they counted.  Very dense suspensions can be counted if they are diluted appropriately. Limitations of Direct Microscopic count  Dead cells are not distinguished from living cells.  Small cells are difficult to see under the microscope, and some cells are probably missed.  Precision is difficult to achieve  A phase contrast microscope is required when the sample is not stained.  The method is not usually suitable for cell suspensions of low density i.e. < 10 7 Cells per ml, but samples can be concentrated by centrifugation or filtration to increase sensitivity. =================================================================================== 2. ELECTRONIC COUNTING CHAMBERS Electronic counting chambers count numbers and measure size distribution of cells. For cells the size of bacteria the suspending medium must be very clean. Such electronic devices are more often used to count eucaryotic cells such as blood cells. Coulter Counter Coulter counter is an electronic used to count number of microbes preferably protozoa microalgae and yeasts. In This method, the sample of microbes is forced through a small orifice (small hole). On the both sides of the orifice, electrodes are present measure the electric resistance or conductivity when electric current is ed through the orifice. Every time a microorganism es through the orifice, electrical resistance increases or the conductivity drops and the cell is counted. The Coulter counter gives accurate results with larger cells. The precaution to be taken in this method is that the suspension of samples should be free of any cell debris or other extraneous matter.



1. Electrode 5. Electrode

2. Flow of Suspending Fluid 6. Particle Location

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3. Bacterial Cell 4. Orifice 7. Measurement of Voltage


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PLATE COUNT  Technique of Viable Count.  The bacterial culture need not contain all living cells.  There might be few dead as well.  Only living cells will form colony when grown in proper solid medium and under standard set or growth conditions.  This fact is used to estimate number of living or dead bacterial cells (viable count) in the given culture.  Estimates thus obtained are expressed as a colony forming unit (CFU).  Viable count technique is very much useful in the dairy industry and the food industry for quantitative analysis of milk and spoilage of food products.  There are two ways of performing a plate count: the spread plate method and the pour plate method.  Pour plate method.  Spread plate method

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3. INDIRECT VIABLE CELL COUNTS: Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating. If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample. Methods of indirect count includes  Plate count  Membrane filter count  MPN


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Pour plate method.

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 A pour plate is a method of melted agar inoculation followed by Petri dish incubation.  A known volume (usually 0.1-1.0 ml) of culture is pipette into a sterile Petri plate; melted agar medium is then added and mixed well by gently swirling the plate on the table top.  Because the sample is mixed with the molten agar medium, a larger volume can be used than with the spread plate.  However, with the pour plate method the organism to be counted must be able to briefly withstand the temperature of melted agar, 45oC  The cultures are inoculated into melted agar that has been cooled to 45oC.  The liquid medium is well mixed then poured into a Petri dish (or vice versa).  Plates incubated at optimum conditions for desired incubation time.  Colonies form within the agar matrix rather than on top as they do when streaking a plate.  Pour plates are useful for quantifying microorganisms that grow in solid medium.  Because the "pour plate" embeds colonies in agar it can supply a sufficiently oxygen deficient environment that it can allow the growth and quantification of microaerophiles.  After incubation number of colonies were counted and  Calculate the total viable count as follows. Viable cell count (cells/ml) = Average No. colonies x Dilution factor ---------------------------------------------------------ml of sample taken  For example, if a plate containing a 1/1,000,000 dilution of the original sample  Shows 150 colonies  1 ml sample taken  The number of CFUs per ml of sample =  In the case of the example above 150 x 1,000,000 = 150,000,000 CFUs per ml Application:  This method is used to count only live (viable) cells.  This method is used to enumerate bacteria in milk, water, foods, soils; cultures etc and the number of bacteria are expressed as colony-forming units (CFU) per ml.



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 Spread plate technique is an additional method of quantifying microorganisms on solid medium.  With the spread plate method, a volume of an appropriately diluted culture usually no greater than 0.1 ml is spread over the surface of an agar plate using a sterile glass spreader.  The plate is than incubated until the colonies appear, and the number of colonies counted.  Instead of embedding microorganisms into agar, as is done with the pour plate method, liquid cultures are spread on the agar surface.  After incubation number of colonies were counted and  Calculate the total viable count as follows. Viable cell count (cells/ml) = Average No. colonies x Dilution factor ---------------------------------------------------------ml of sample taken  For example, if a plate containing a 1/1,000,000 dilution of the original sample  Shows 150 colonies  0.1 ml sample taken  The number of CFUs per ml of sample =  In the case of the example above 150 x 1,000,000 1500,000,000 CFUs per ml --------------------- = ---------------------------------------0.1 An advantage of spreading a plate over the pour plate method is that cultures are never exposed to 45 oC (i.e. melted agar temperatures). Note: Surface of the plate must be dry, so that the liquid that is spread soaks in. volume greater than 0.1ml are rarely used because the excess liquid does not soak in and may cause the colonies to coalesce as they from, making them difficult to count. ===========================================================================  Advantage of plate count method  This method is used routinely and with satisfactory results for the estimation of bacterial populations in milk, water, foods, and many other materials.  Its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted.  It is easy to perform and can be adapted to the measurement of populations of any magnitude.  Limitation of plate count technique  Only living cells develop colonies that are counted;  Clumps or chains of cells develop into a single colony;  Colonies develop only from those organisms for which the cultural conditions are suitable for growth.

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Spread plate method


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MEMBRANE FILTER COUNT  When the quantity of bacteria is very small, as in lakes or relatively pure streams, bacteria may be counted by filtration methods.  In this technique, at least 100 ml of water ed through a thin membrane filter( bacteriological filter) whose pores are too small to allow bacteria to .  Microbial cell numbers are frequently determined using special membrane filters possessing millipores small enough to trap bacteria.  Technique  In this technique a sample containing microbial cells ed through the membrane filter.  The filter is then placed on solid agar medium or on a pad soaked with nutrient broth (liquid medium) and incubated until each cell develops into a separate colony.  Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes also vary with medium and the microorganism.  A colony count gives the number of microorganisms in the filtered sample, and specific media can be used to select for specific microorganisms.  Calculate the total viable count as follows. Viable cell count (cells/ml) = Average No. colonies x Dilution factor ---------------------------------------------------------ml of sample filtered  For example, if a plate containing a 1/1,000,000 dilution of the original sample  Shows 150 colonies  Volume of sample filtered is 100  The number of CFUs per ml of sample =  In the case of the example above 150 x 1,000,000 = --------------------- = 15, 00,000 CFUs per ml 100



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Application:  This technique is especially useful in analyzing aquatic samples  This method is applied frequently for detection and enumeration of coliform bacteria, which are indicators of faecal pollution of food and water.  Filtration of very dilute (low cell number) samples, such as ocean water or groundwater. Advantages  Technique is highly sensitive (theoretically, a single cell can be detected)  It allows for inspection and positive identification of the organism counted. Limitations:  Only living cells develop colonies that are counted;  Clumps or chains of cells develop into a single colony  Colonies develop only from those organisms for which the cultural conditions are suitable for growth.  The latter makes the technique virtually useless to characterize or count the total number of bacteria in complex microbial ecosystems such as soil or the animal rumen or gastrointestinal tract. MOST PROBABLE NUMBER METHOD (MPN METHOD)  Organisms that cannot grow on solid media can still be enumerated using the most probable number method  Once again, cultures are diluted, here into a suitable broth medium  "The more tubes that show growth, especially at greater dilutions, the more organisms were present in the sample."  Particularly, at some greater dilution there will be on average no organisms added per tube of broth, while at lesser dilutions there will be organisms in every tube; the middle dilution at which the transition is made from all tubes inoculated with organism to few or none represents approximately the inverse of the concentration of organisms in the original culture  The basis of the test is that multiple tubes of culture medium are inoculated with various

  

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

dilutions of a water sample and incubated at a constant temperature for a given period of time. If coliformic bacteria are present in a tube this is detected by growth within the tube and the production of gas. Any gas produced is collected in an inverted gas collection tube placed within a larger test tube containing the culture medium. The result of the analysis, in of the most probable number of coliforms, depends upon the number of tubes which show a positive reaction. Typically, the MPN value is determined from the number of positive tests in a series of 5 replicates made from 3 different dilutions or inoculation amounts (15 samples altogether). For example, sample inoculation amounts may be 10, 1 and 0.1 ml per test tube. The test method can be described as follows:



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When using the above equation, that the count of positive tubes starts with the highest dilution in which at least one negative result has occurred. When more than three test dilutions are incubated, the following rules are used in determining MPN value:  Choose the highest dilution that gives positive results in all five portions tested or the largest number of positives and the two next higher dilutions.  Where positive results occur in dilutions higher than the three chosen according to the above rule, they are incorporated in the results of the highest chosen dilution up to a total of five.  If only one dilution gives a positive result, two dilutions immediately lower and higher giving zero positives should be chosen so as to keep the positive result in the middle of the series. Table : MPN Index and 95% Confidence Limits for Various Combinations of Positive and Negative Results when Ten 10 mL Portions are used

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Some examples for estimation of MPN for various cases are given in Table 3.11. The selected combinations are shaded. Calculations are explained below:


Table. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results with Five Tubes per Dilution (10 mL, 1.0 mL, 0.1 mL) Department of Microbiology, D.B. Science College, Gondia.

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Table: Examples for reading and calculating MPN values.



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CELL MASS MEASUREMENT 1. Dry weight technique: The cell mass of a very dense cell suspension can be determined by this technique. In this technique, the microorganisms are removed from the medium by filtration and the microorganisms on filters are washed to remove all extraneous matter, and dried in desiccators by putting in weighing bottle (previously weighted). The dried microbial content is then weighted accurately. This technique is especially useful for measuring the growth of micro fungi. It is time consuming and not very sensitive. Since bacteria weigh so little, it becomes necessary to centrifuge several hundred millions of culture to find out a sufficient quantity to weigh. 2. Measurement of nitrogen content As the microbes (bacteria) grow, there is an increase in the protein concentration (i.e. nitrogen concentration) in the cell. Thus, cell mass can be subjected to quantitative chemical analysis methods to determine total nitrogen that can be correlated with growth. This method is useful in determining the effect of nutrients or anti-metabolites upon the protein synthesis of growing culture. Nitrogen estimation is carried out by Kjeldahl method 3. Measurement of Turbidity (Turbidometry) Rapid cell mass determination is possible using turbidometry method. Turbidometry is based on the fact that microbial cells scatter light striking them. Since the microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. One visible characteristic of growing bacterial culture is the increase in cloudiness of the medium (turbidity). When the concentration of bacteria reaches about 10 million cells (107) per ml, the medium appears slightly cloudy or turbid. Further increase in concentration results in greater turbidity. When a beam of light is ed through a turbid culture, the amount of light transmitted is measured, Greater the turbidity, lesser would be the transmission of light through medium. Thus, light will be transmitted in inverse proportion to the number of bacteria. Turbidity can be measured using instruments like spectrophotometer and nephlometer Determination of cell mass using turbidometry method

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1. Source of Light of a single wave-length (monochromatic) 2. Filter 3. Tube with cell Free medium 4. Tube with suspension of microorganisms 5. Photocell or Detector



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McFarland standards In microbiology, McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range. Original McFarland standards were made by mixing specified amounts of barium chloride and sulfuric acid together. Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in the solution. A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4). Now there are McFarland standards prepared from suspensions of latex particles, which lengthens the shelf life and stability of the suspensions. The standard can be compared visually to a suspension of bacteria in sterile saline or nutrient broth. If the bacterial suspension is too turbid, it can be diluted with more diluent. If the suspension is not turbid enough, more bacteria can be added. McFarland Nephelometer Standards: McFarland Standard No.

0.5

1.0% Barium chloride (ml) 1.0% Sulfuric acid (ml)

2

3

4

0.05 0.1

0.2

0.3

0.4

9.95 9.9

9.8

9.7

9.6

6.0

9.0

12.0

Approx. cell density (1X10^8 CFU/mL) 1.5

1

3.0

% Transmittance*

74.3 55.6 35.6 26.4 21.5

Absorbance*

0.132 0.257 0.451 0.582 0.669

*at wavelength of 600 nm

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 More than a million cells per milliliter must be present for the first traces of turbidity to be visible. Therefore, about 10 to 100 million cells per milliliter are needed to make a suspension turbid enough to be read on a spectrophotometer. Therefore, turbidity is not a useful measure of contamination of liquids by relatively small numbers of bacteria.



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CELL ACTIVITY (BIOCHEMICAL ACTIVITY) MEASUREMENT The activities of living cells are measured. All measurements are indirect, and all measurements are based upon the activities of live (viable) cells only. The methods includes A. Chemical methods or assays:  Any chemical changes in the media will be proportional to the activity of the cells.  One can monitor the rate at which glucose amounts in the media decline.  The faster glucose amounts decline, the more active the organisms.  The same can be said of oxygen usage and decline.  Carbon dioxide (waste product of metabolism) will increase at a greater rate if the organisms are active.  Rates of increases in acidity (shown by a decline in pH) are also used to determine cell activity. B. Physical methods or assays:  these are sensitive and fast methods  Microcalorimetry: Minute temperature changes over time can be monitored to assess cell activity using sensitive thermometers called thermistors. Antibiotic sensitivity can be tested using microcalorimetry.  Radioactive isotopes: Radioactively labeled carbon released from glucose during metabolic activities in the form of carbon dioxide, can be detected and counted using a scintillation counter. The greater the count, the more active the culture. Antibiotic sensitivity can also be measured using radioactive isotopes. 1) Advantages:  Giving counting of metabolically active cells

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2) Disadvantages  Not giving count on metabolically in active cells.  Nonspecific techniques 3) Applications:  For elucidating microbial growth kinetics.  Useful for determining kinetics of fermentation



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Table 1. Some Methods used to measure bacterial growth Method

Application

Direct microscopic count

Enumeration of bacteria in milk Cannot distinguish living from or cellular vaccines nonliving cells

Viable cell count (colony counts)

Enumeration of bacteria in milk, Very sensitive if plating conditions foods, soil, water, laboratory are optimal cultures, etc.

Turbidity measurement

Estimations of large numbers of Fast and nondestructive, but bacteria in clear liquid media cannot detect cell densities less and broths than 107 cells per ml

Measurement of total N or protein

Measurement of total cell yield only practical application is in the from very dense cultures research laboratory

Measurement of Biochemical activity e.g. O2 uptake CO2 Microbiological assays production, ATP production, etc.

Comments

Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers

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Measurement of dry weight or wet Measurement of total cell yield probably more sensitive than total weight of cells or volume of cells in cultures N or total protein measurements after centrifugation



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B. PURE CULTURE AND CULTURAL CHARACTERISTICS CONCEPT OF PURE A population of microbial cells of only one species which were derived from a single cell is called as pure culture. Process of isolation of one kind of microbial population from mixture of many different kinds is called as pure culture technique. MIXED CULTURE A population of microbial cells of only different types which were derived from a different cells is called as mixed culture. Example: Milk is a mixed culture of bacteria

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In biology, axenic describes a culture of an organism that is entirely free of all other "contaminating" organisms. The earliest axenic cultures were of bacteria or unicellular eukaryotes, but axenic cultures of many multicellular organisms are also possible.[1] Axenic culture is an important tool for the study of symbiotic and parasitic organisms in a controlled manner. Preparation Axenic cultures of microorganisms are typically prepared using a dilution series of an existing mixed culture. This culture is successively diluted to the point where subsamples of it contain only a few individual organisms, ideally only a single individual (in the case of an asexual species). These subcultures are allowed to grow until the identity of their constituent organisms can be ascertained. Selection of those cultures consisting solely of the desired organism produces the axenic culture. Axenic cultures are usually checked routinely to ensure that they remain axenic. One standard approach with microorganisms is to spread a sample of the culture onto an agar plate, and to incubate this for a fixed period of time. The agar should be an enriched medium that will the growth of common "contaminating" organisms. Such "contaminating" organisms will grow on the plate during this period, identifying cultures that are no longer axenic. Experimental use As axenic cultures are derived from very few organisms, or even a single individual, they are useful because the organisms present within them share a relatively narrow gene pool. In the case of an asexual species derived from a single individual, the resulting culture should consist of identical organisms (though processes such as mutation and horizontal gene transfer may introduce a degree of variability). Consequently, they will generally respond in a more uniform and reproducible fashion, simplifying the interpretation of experiments. Problems The axenic culture of some pathogens is complicated because they normally thrive within host tissues which exhibit properties that are difficult to replicate in vitro. This is especially true in the case of intracellular pathogens. However, careful replication of key features of the host environment can resolve these difficulties (e.g. host metabolites, dissolved oxygen), such as with the Q fever pathogen, Coxiella burnetii.

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AXENIC CULTURE



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ISOLATION OF PURE CULTURE  Microorganisms are generally found in nature (air, soil and water) as mixed populations.  Even the diseased parts of plants and animals contain a great number of microorganisms, which differ markedly from the microorganisms of other environments.  To study the specific role played by a specific microorganism in its environment, one must isolate the same in pure culture.  However, it is not easy to isolate the individual microorganisms from natural habitats and grow them under imposed laboratory conditions.  If inoculums from any natural habitat is taken and allowed to grow in a culture medium, a large number of diverse colonies may develop that, due to crowdedness, may run together and, thereby, may lose individuality.  Therefore, it is necessary to make the colonies well-isolated from each other so that each appears distinct, large and shows characteristic growth forms.  Such colonies may be picked up easily and grown separately for detailed study.  Several methods for obtaining pure cultures are in use.  Some common methods are in everyday-use by a majority of microbiologists, while the others are methods used for special purposes. Common Methods of isolation of pure culture Pure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria, many other microfungi, and unicellular microalgae, may be most commonly obtained by plating methods such as streak plate method, pour plate method and spread plate method. But, the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method. 1. Streak Plate Method:  This technique is developed in a laboratory of Robert Koch. By Friedrich Loeffler and George Graffky.  This method is used most commonly to isolate pure cultures of bacteria.  A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. The successive streaks "thin out" the inoculums sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited Various methods of streaking



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 Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/ needle and restreaked onto fresh media to ensure purity. 2. Pour Plate Method:  This technique is developed in a laboratory of Robert Koch.  This method involves plating of diluted samples mixed with melted agar medium.  The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium.  Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C).  The bacteria and the melted medium are mixed well.  The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated.  When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies).  These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity. Pour plate method has certain disadvantages as follows: 1. The picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, 2. The microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms. However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture. The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium



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4. Serial Dilution Method  As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions.  The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid medium. Serial dilution method  If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ ml.  If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism.  If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism

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3. Spread Plate Method  In this method the mixed culture of microorganisms is not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline. A drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod.  The medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate. Spread plate method


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5. Enrichment culture:  Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture.  The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation.  The medium of known composition and specific condition of incubation favors the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.  Example: 1. A selectively enriched culture of Nitrobacter spp. was obtained by inoculating soil in a salt containing NaNo2 at pH 8.5 and incubating it in a dark incubator at 250-300C. 2. Enrichment of Lactobacillus spp. from milk is carried out by subjecting inoculation ok milk in Lactobacillus MRS Media followed by incubation at 370C.


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Selective media will permit the growth of one type of bacteria while preventing the growth of other types. This will facilitate the isolation of a desired species. Using selective media pure culture of desired strain can be isolated. A specimen with mixed microbial population when streaked on the surface of selective media for desired strain, this medium due to selective components only allow the growth of desired strain and inhibit the growth of rest. Due to which the colonies appeared on such medium were only of desired strain. Each colony of desired strain denotes pure culture. For example: MacConkey’s medium is selective. It contains bile salts and crystal violet; these will inhibit the growth of Gram-positive organisms and favoring growth of gram negative bacteria. Mannitol salt agar which inhibits the growth of salt intolerant organisms and favour growth of salt tolerant Bacteria ( S. aureus). Differential media will allow visual differentiation between two or more species of bacteria. Using selective media pure culture of desired strain can be isolated. A specimen with mixed microbial population when streaked on the surface of differential media for desired strain, this medium due to differential ingredients shows different colony characteristics of different bacterial strain. Colonies with desired characteristics denotes desired strain. Each colony with desired characteristics denotes pure culture. Examples include blood agar, MacConkey’s medium and Mannitol Salt agar  Colonies growing on blood agar are differentiated by hemolysis patterns (greening: alpha hemolysis, clearing - beta hemolysis and no hemolysis -gamma).  Using MacConkey’s medium, lactose fermenting and lactose non-fermenting bacteria can be distinguished. Organisms that are able to ferment lactose will produce an acid end-product that causes a change in the pH of the surrounding media. A pH indicator (neutral red), is present in the media and changes from

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6. Use of selective and differential media:


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yellow to red in an acidic environment. Lactose fermenting bacteria growing on MacConkey’s media will appear pink whereas non-lactose fermenters will be white. Mannitol salt agar allows discrimination among salt tolerant organisms. Those that ferment mannitol will produce acid turning the pH indicator (phenol red) to a yellow color. Those that cannot ferment mannitol will leave the media the red color of phenol red at nuetral pH.

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7. Specific isolation technique:



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 Micromanipulator method: Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down. A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette. Now a hanging drop is searched, which contains only a single microorganism cell. This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism. The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialized studies. Proof of Purity of Cultures Assuming that one has isolated a pure culture, how does one establish that it is pure? A pure culture is one in which the cells are all of one kind, i.e., demonstrate "likeness". Hence, the proof of purity of cultures consists of demonstrating the "likeness" of microorganisms in the culture. It is based on certain criteria as follows: 1.The microorganisms look alike microscopically and stain in the same fashion. 2. When plated, all the colonies formed look alike. 3. Streaks, stabs, etc. are uniform. 4. Several isolated colonies perform identically, i.e., ferment the same sugars, and so on.

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8. Single Cell Isolation methods:  An individual cell of the required kind is picked out by this method from the mixed culture and is permitted to grow.  The following two methods are in use.  Capillary pipette method: Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.


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Need of Preservation of Pure Culture  Most microbiological laboratories maintain large collection of strains, frequently referred to as stock culture collection. These organisms are needed for laboratory classes and research work, as test agent for particular procedures, or as reference strains for taxonomic studies.  Most biological companies maintain large culture collections. The strains are used for screening of new, potentially effective chemotherapeutic agents; as assay for vitamins and amino acids; as agent for production of vaccines, antisera, antitumor agents, enzymes, and organic chemicals  Such reference cultures are cited in company patents  For these and other purposes it is extremely important to have properly identified and catalogued strains of bacteria available. Methods of preservation 1. Periodic Transfer to Fresh Media:  Strains can be maintained by periodically preparing a fresh stock culture from the previous stock culture.  The culture medium, the storage temperature, and the time interval at which the transfers are made vary with the species and must be ascertained beforehand.  The temperature and the type of medium chosen should a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible.  The transfer method has the disadvantage of failing to prevent changes in the characteristics of a strain due to the development of variants and mutants 2. Refrigeration:  Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms.  This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped.  Thus their growth continues slowly, nutrients are utilized and waste products released in medium.  This results in, finally, the death of the microbes after sometime.

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MAINTENANCE AND PRESERVATION OF PURE CULTURES OF BACTERIA  Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination.  Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (sub culturing) to allow continuous growth and viability of microorganisms.  The transfer is always subject to aseptic conditions to avoid contamination.  Since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time.  In addition, there is a risk of genetic changes as well as contamination.  Therefore, it is now being replaced by some modern methods that do not need frequent sub culturing.  These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying).

3. Paraffin Method/ preservation by overlaying cultures with mineral oil :  This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature.  The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium.  This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture can be preserved form months to years (varies with species).  The advantage of this method is that we can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the original culture.  The simplicity of the method makes it attractive, but changes in the characteristics of a strain can still occur. 4. Cryopreservation:  Cryopreservation (i.e., freezing in liquid nitrogen at -196°C or in the gas phase above the liquid nitrogen at -150°C) helps survival of pure cultures for long storage times.  In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol or Dimethyl Sulfoxide (DMSO) that prevent the cell damage due to formation of ice crystals and promote cell survival.  This liquid nitrogen method has been successful with many species that cannot be preserved by lyophilization and most species can remain viable under these conditions for 10 to 30 years without undergoing change in their characteristics, however this method is expensive. 5. Lyophilization (Freeze-Drying)  Most bacteria die if cultures are allowed to become dry, although spore- and cyst-formers can remain viable for many years.  However, freeze-drying can satisfactorily preserve many kinds of bacteria that would be killed by ordinary drying.  In this process, a dense cell suspension is placed in small vials and frozen at -60 to -78°C. The vials are then connected to a high-vacuum line. The ice present in the frozen suspension sublimes under the vacuum, i.e. evaporates without first going through a liquid water phase. This results in dehydration of the bacteria with a minimum of damage to delicate cell structures  The vials are then sealed off under vacuum and stored in a refrigerator. Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.  Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small area. Furthermore, the small vials can be sent conveniently through the mail to other microbiology laboratories when packaged in a special sealed mailing containers.  Lyophilized cultures are revived by opening the vials, adding liquid medium, and transferring the rehydrated culture to suitable growth medium.  Advantage of Lyophilization  Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small area  Small vials can be sent conveniently through the mail to other microbiology laboratories when packaged in a special sealed mailing containers  Lyophilized cultures can be revived by opening the vials, adding liquid medium, and transferring the rehydrated culture to a suitable growth medium.


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Glycerol at -20°C  Grow a pure culture on an appropriate solid medium.  When the culture is fully developed, scrap it off with a loop.  Suspend small clumps of the culture in sterile neutral glycerol.  Distribute in quantities of 1-2 ml in screw-capped tubes or vials.  Store at -20°C.  Avoid repeated freezing and thawing.  Transfer after 12-18 months.

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6.

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STOCK CULTURE COLLECTION CENTERS CULTURE COLLECTION  The ultimate source of all strains of industrial microorganisms is the natural environment.  But through the years, a number of industrial strains have been deposited in Culture Collections.  There are a number of culture collections which serve as the repositories of microbial cultures, for example ATCC (American Type Culture Collection), NCTC (National Collection of Type Cultures) of UK etc.

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THE ROLE OF CULTURE COLLECTIONS  Since the early days of microbiology, astronomical numbers of microorganisms were isolated from a wide variety of natural sources, and used for the scientific research and for the industrial fermentation.  However, large numbers of microorganisms had lost in the past, and they are no longer available. Microbiologists often lose microbial cultures that they studied because of the change of their interests and difficulties in keeping the cultures.  This is due to the absence of reliable culture collections in which microorganisms are maintained properly and supply them promptly on demand.  Through the study of microbial cultures maintained in the culture collection, potential properties of microorganisms have been developed, and the future perspective of microbiology will be presumed.  Therefore, reliable and well-organized culture collections are needed as the depository and for the promotion of research and application of the strains.  In fact, culture collections play a role of the depository of the type strains in bacteriology, and the study of bacterial systematics will not be completed without culture collections consequently.  Major tasks of culture collections are the collection and maintenance of important and useful microbial cultures, supply of the cultures on demand, and preparation of their informative documents.  Social needs for culture collections are increasing year by year, and effective and smooth management is required for better services of culture collections. In this point of view, exchange of information and cooperation among culture collections are the most important matter to achieve the purpose of the culture collection.  According to the data of World Data Center for Microorganisms (WDCM) (14), culture collections are distributed in 58 countries in the world.  Thus improvement of culture collections is critical and crucial for the further development of microbiology, microbial industry, and biotechnology. In addition, good operation and management of culture collections are in great part due to the activity of personnel in the culture collections.



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NAMES OF SOME INTERNATIONAL CENTERS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

American Type Culture Collection (ATCC): Maryland USA. Central Bureau For Scimmel Cultures: Baarn, Netherlands Centre de Collections de Types bocrobeins: Lausanne, Switzerland. Commonwealth Mycological institute: Surrey, England. Colture collection of algae and protozoa: Cambridge,England. Culture collection Unit: Illnois, USA. Pasteur institure: Paris, Microbiological culture collections: Osaka, Japan. National Collection of Type cultures: London, England. National collection of Yeast cultures: Surrey, England National Collection Of industrial Bacteria: Aberdeen, Scotland. USSR Antibiotic research Institute: Moscow, USSR

NAMES OF SOME NATIONAL CENTERS

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1. National chemical laboratory Pune, India. 2. Microbial type culture collection (MTCC), Chandigarh, India 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.


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