B.sc. Microbiology II Bacteriology Unit 4.1 Bacterial Growth

Bacterial Growth

Course: B.Sc. MicrobiologySem II

Sub: BacteriologyUnit 4.1

• Bacterial growth is the asexual reproduction, or celldivision, of a bacterium into two daughter cells, in aprocess called binary fission. Providing no mutationalevent occurs the resulting daughter cells aregenetically identical to the original cell. Hence, "localdoubling" of the bacterial population occurs.

Cell Growth and Binary Fission

• In microbiology, growth is defined as an increase in the number ofcells. Microbial cells have a finite life span, and a species ismaintained only as a result of continued growth of its population.

• There are many reasons why understanding how microbial cellsgrow is important. For example, many practical situations call forthe control of microbial growth, in particular, bacterial growth.

• Knowledge of how microbial populations can rapidly expand isuseful for designing methods to control microbial growth,whether the methods are used to treat a life-threateninginfectious disease or simply to disinfect a surface. Knowledge ofthe events surrounding bacterial growth also allows us to see howthese processes are related to cell division in higher organisms.

• In a growing rod-shaped cell,elongation continues until the celldivides into two new cells. Thisprocess is called binary fission(“binary” to express the fact thattwo cells have arisen from one).

• In a growing culture of a rod-shapedbacterium such as Escherichia coli,cells elongate to approximatelytwice their original length and thenform a partition that constricts thecell into two daughter cells.

Cell Growth and Binary Fission

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• This partition is called a septum and results from the inwardgrowth of the cytoplasmic membrane and cell wall fromopposing directions; septum formation continues until thetwo daughter cells are pinched off.

• There are variations in this general pattern. In some bacteria,such as Bacillus subtilis, a septum forms without cell wallconstriction, while in the budding bacterium Caulobacter,constriction occurs but no septum is formed.

• But in all cases, when one cell eventually separates to formtwo cells, we say that one generation has occurred, and thetime required for this process is called the generation time.

Growth curve

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Lag Phase

• When a microbial culture is inoculated into a freshmedium, growth usually begins only after a period of timecalled the lag phase.

• This interval may be brief or extended, depending on thehistory of the inoculum and the growth conditions.

• If an exponentially growing culture is transferred into thesame medium under the same conditions of growth(temperature, aeration, and the like), there is no lag andexponential growth begins immediately. However, if theinoculum is taken from an old (stationary phase) cultureand transferred into the same medium, there is usually alag even if all the cells in the inoculum are alive. This isbecause the cells are depleted of various essentialconstituents and time is required for their biosynthesis.

• A lag also ensues when the inoculum consists of cells thathave been damaged (but not killed) by significanttemperature shifts, radiation, or toxic chemicals because ofthe time required for the cells to repair the damage.

• A lag is also observed when a microbial population istransferred from a rich culture medium to a poorer one; forexample, from a complex medium to a defined medium. Togrow in any culture medium the cells must have a completecomplement of enzymes for synthesis of the essentialmetabolites not present in that medium. Hence, upontransfer to a medium where essential metabolites must bebiosynthesized, time is needed for production of the newenzymes that will carry out these reactions.

Exponential Phase• during the exponential phase of growth each cell divides to form two

cells, each of which also divides to form two more cells, and so on, fora brief or extended period, depending on the available resources andother factors.

• Cells in exponential growth are typically in their healthiest state andhence are most desirable for studies of their enzymes or other cellcomponents.

• Rates of exponential growth vary greatly. The rate of exponentialgrowth is influenced by environmental conditions (temperature,composition of the culture medium), as well as by genetic

• characteristics of the organism itself. In general, prokaryotes growfaster than eukaryotic microorganisms, and small eukaryotes growfaster than large ones. This should remind us of the previouslydiscussed concept of surface-to-volume ratio. Recall that small cellshave an increased capacity for nutrient and waste exchangecompared with larger cells, and this metabolic advantage can greatlyaffect their growth and other properties

Stationary Phase• In a batch culture (tube, flask bottle, Petri dish), exponential

growth is limited. Consider the fact that a single cell of abacterium with a 20-min generation time would produce, ifallowed to grow exponentially in a batch culture for 48 h, apopulation of cells that weighed 4000 times the weight of Earth!This is particularly impressive when it is considered that a singlebacterial cell weighs only about one-trillionth (10-12) of a gram.

• Obviously, this scenario is impossible. Something must happen tolimit the growth of the population. Typically, either one or both oftwo situations limit growth: (1) an essential nutrient of theculture medium is used up, or (2) a waste product of the organismaccumulates in the medium and inhibits growth. Either way,exponential growth ceases and the population reaches thestationary phase.

• In the stationary phase, there is no net increase or decrease incell number and thus the growth rate of the population iszero. Although the population may not grow during thestationary phase, many cell functions can continue, includingenergy metabolism and biosynthetic processes.

• Some cells may even divide during the stationary phase butno net increase in cell number occurs. This is because somecells in the population grow, whereas others die, the twoprocesses balancing each other out. This is a phenomenoncalled cryptic growth.

Death Phase• If incubation continues after a population reaches the stationary phase,

the cells may remain alive and continue to metabolize, but they willeventually die. When this occurs, the population enters the deathphase of the growth cycle. In some cases death is accompanied byactual cell lysis. Figure 5.10 indicates that the death phase of thegrowth cycle is also an exponential function. Typically, however, therate of cell death is much slower than the rate of exponential growth.

• The phases of bacterial growth are reflections of the events in apopulation of cells, not in individual cells. Thus the terms lag phase,exponential phase, stationary phase, and death phase have nomeaning with respect to individual cells but only to cell populations.Growth of an individual cell is a necessary prerequisite for populationgrowth. But it is population growth that is most relevant to the ecologyof microorganisms, because measurable microbial activities requiremicrobial populations, not just an individual microbial cell.

Measurement of Microbial growth

• Population growth is measured by tracking changes in the number of cells or changes in the level of some cellular component. The latter could be protein, nucleic acids, or the dry weight of the cells themselves. We consider here two common measures of cell growth: cell counts and turbidity, the latter of which is a measure of cell mass.

Microscopic Counts• A total count of microbial numbers can be achieved using a

microscope to observe and enumerate the cells present in a culture ornatural sample. The method is simple, but the results can beunreliable.

• The most common total count method is the microscopic cell count.Microscopic counts can be done on either samples dried on slides oron samples in liquid. Dried samples can be stained to increase contrastbetween cells and their background. With liquid samples, speciallydesigned counting chambers are used. In such a counting chamber, agrid with squares of known area is marked on the surface of a glassslide.

• When the coverslip is placed on the chamber, each square on the gridhas a precisely measured volume. The number of cells per unit area ofgrid can be counted under the microscope, giving a measure of thenumber of cells per small chamber volume. The number of cells permilliliter of suspension is calculated by employing a conversion factorbased on the volume of the chamber sample.

Microscopic Counts

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• A second method of enumerating cells in liquid samples iswith a flow cytometer. This is a machine that employs a laserbeam and complex electronics to count individual cells. Flowcytometry is rarely used for the routine counting of microbialcells, but has applications in the medical field for counting anddifferentiating blood cells and other cell types from clinicalsamples. It has also been used in microbial ecology toseparate different types of cells for isolation purposes.

Microscopic Counts

• Microscopic counting is a quick and easy way of estimating microbial cellnumber. However, it has several limitations.

• Limitations:

1. Without special staining techniques, dead cells cannot be distinguishedfrom live cells.

2. Small cells are difficult to see under the microscope, and some cells areinevitably missed.

3. Precision is difficult to achieve.

4. A phase-contrast microscope is required if the sample is not stained.

5. Cell suspensions of low density (less than about 106 cells/milliliter) havefew if any bacteria in the microscope field unless a sample is firstconcentrated and re-suspended in a small volume.

6. Motile cells must be immobilized before counting.

7. Debris in the sample may be mistaken for microbial cells.

Microscopic Counts

Viable Counts• A viable cell is one that is able to divide and form offspring,

and in most cell-counting situations, these are the cells we aremost interested in.

• For these purposes, we can use a viable counting method.

• To do this, we typically determine the number of cells in asample capable of forming colonies on a suitable agarmedium.

• For this reason, the viable count is also called a plate count.

• The assumption made in the viable counting procedure is thateach viable cell can grow and divide to yield one colony.

• Thus, colony numbers are a reflection of cell numbers.

• There are at least two ways of performing a plate count: thespread-plate method and the pour-plate method.

spread-plate method• In the spread-plate method, a volume (usually 0.1 ml or less) of

an appropriately diluted culture is spread over the surface of anagar plate using a sterile glass spreader.

• The plate is then incubated until colonies appear, and thenumber of colonies is counted.

• The surface of the plate must not be too moist because theadded liquid must soak in so the cells remain stationary.

• Volumes greater than about 0.1 ml are avoided in this methodbecause the excess liquid does not soak in and may cause thecolonies to coalesce as they form, making them difficult to count.

pour-plate method

• In the pour-plate method, a known volume (usually 0.1–1.0 ml) of cultureis pipetted into a sterile Petri plate.

• Melted agar medium, tempered to just about gelling temperature, is thenadded and mixed well by gently swirling the plate on the bench top.

• Because the sample is mixed with the molten agar medium, a largervolume can be used than with the spread plate.

• However, with this method the organism to be counted must be able towithstand brief exposure to the temperature of molten agar (45–50 oC).

• Here, colonies form throughout the medium and not just on the agarsurface as in the spread-plate method.

• The plate must therefore be examined closely to make sure all colonies arecounted.

• If the pour-plate method is used to enumerate cells from a natural sample,another problem may arise; any debris in the sample must bedistinguishable from actual bacterial colonies or the count will beerroneous.

Turbidimetric Methods• During exponential growth, all cellular components increase in

proportion to the increase in cell numbers. • Thus, instead of measuring changes in cell number over time, one

could instead measure the increase in protein, DNA, or dry weight of a culture as a barometer of growth.

• However, since cells are actual objects instead of dissolved substances, cells scatter light, and a rapid and quite useful method of estimating cell numbers based on this property is turbidity.

• A suspension of cells looks cloudy (turbid) to the eye because cells scatter light passing through the suspension.

• The more cells that are present, the more light is scattered, and hence the more turbid the suspension.

• What is actually assessed in a turbidimetric measurement is total cell mass.

• However, because cell mass is proportional to cell number, turbidity can be used as a measure of cell numbers and can also be used to follow an increase in cell numbers of a growing culture.

Nutritional Requirements

Introduction

• Bacterial growth involves

– An increase in the size of the organisms

– An inctrease in the number of organisms

an increase in the total mass (biomass)

– Requirements for growth

– Nutrients

– Enviromental conditions

– Sourse of energy

The Common Nutrient Requirements

• Macroelements (macronutrients)– H, O, C, N, S, P

– required in relatively large amounts

• Micronutrients (trace elements)– K, Ca, Mg, Fe, Cu, Mn, Zn, Co, Mo and Ni

– required in trace amounts

– often supplied in water or in media components

Carbon

• Classification of microorganisms on the basis of carbon source

1. Photoautotrophs

2. Photoorganotrophs

3. Chemoautotrophs (Lithotrophs)

4. Heterotrophs

Classification of microorganisms on the basis of carbon source

1. Photoautotrophs

– Use CO2 as the principal carbon source– Eg. Photosynthatic bacteria (cyanobacteria) ,

Algae

Cyanobacteria can be helpful in agricultureas they have the capability to fixatmospheric nitrogen to soil.

2. Photoorganotrophs

» Use light as energy sourse but need some energy compound – acetate as a sourse of carbon

» Eg. Certain photosynthatic eubacteria

[Eubacteria- A large group of bacteria with simple cells and rigid cell walls]

CLASSIFICATION OF MICROORGANISMS ON THE BASIS OF CARBON SOURCE

3. Chemoautotrophs (Lithotrophs)• Use CO2 as the sourse of carbon • Obtain energy by the oxidation of reduced

organic substances (ammonia reduced form of sulphur and ferrous iron)

• Certain eubacteria (can be cultured in strictly mineral media)

4. Heterotrophs• use organic molecules as their source of carbon• versatile in their ability to use diverse sources of

carbon. Burkholderia cepacia can use over 100 different carbon compounds.

CLASSIFICATION OF MICROORGANISMS ON THE BASIS OF CARBON SOURCE

Nitrogen

• Major component of protein and nucleic acid

• Needed for synthesis of important molecules(e.g., amino acids, nucleic acids)

• Most organisms obtain N in the oxidized form of nitrate

• Use N through assimilation of reduce nitrate (NO3

-) and Nitrite (NO2-) → form ammonium

ion (NH4+) as end product

Sources of nitrogen

• Organic molecules (amino acids)

• Ammonia (NH3)

• Nitrate via assimilatory nitrate reduction

• Nitrogen gas via nitrogen fixation

Sulphur

• Sulphate is the principal source of sulphur

• Sulfur forms part of the structure of coenzymes, and found in cysteinyl and methionyl side chains of proteins.

• Reduced forms of sulphur present in organic compounds (S containing amino acids) are also utilized

• Microorganisms use sulphate (SO42-) as

sulfure source → end product is hydrogen sulfide (H2S).

Other elements

• Inorganic compounds present in theenvironment and those released indecomposition of organic substrates areprincipal sources of other major nutrientelements and micronutrients.

• Phosphorus that are bound in organiccompounds is releases as phosphoric acidduring decomposition.

Growth Factors

• Apart from macro- and micro-nutrients, somemicroorganisms require additional organiccompounds in very small quantities) which areessential for their metabolism,These accessory compounds are growth Factor.

• Includes– Vitamins– Amino acids– Purines & pyrimidines (for synthesis of nucleic acid)– Sterols etc.

• Bases of nucleic acids

• Adenine and guanine are purines

• Cytosine, thymine, and uracil are pyrimidines

• Also found in energy triphosphates (ATP and GTP)

Growth factors and their functions

Practical importance of growth factors

• development of quantitative growth-response assays for measuring concentrations of growth factors in a preparation

• industrial production of growth factors by microorganisms

Uptake of Nutrients by the Cell

• Some nutrients enter by passive diffusion

• Most nutrients enter by:

– facilitated diffusion

– active transport

– group translocation

Factors affecting Microbial Growth

Introduction

• Microbial growth is greatly affected by chemical andphysical nature of their surroundings instead ofvariations in nutrient levels and particularly the nutrientlimitation.

• For successful cultivation of microorganisms it is not onlyessential to supply proper and balanced nutrients butalso it is necessary to maintain proper environmentalconditions.

• As bacteria shows divers food habits, it also exhibitsdiverse response to the environmental conditions.

• Growth and death rates of microorganisms are greatlyinfluenced by number of environmental factors such aswater acidity, temperature, oxygen requirement and pH.

Water Acidity

• Water is one of the most essentialrequirements for life.

• Thus, its availability becomes most importantfactor for the growth of microorganisms.

• The availability of water depends on twofactors - the water content of the surroundingenvironment and the concentration of solutes(salts, sugars etc.) dissolved in the water.

• In most cases, the cell cytoplasm possesseshigher solute concentration in comparison to itsenvironment.

• Thus, water always diffuses from a region of itshigher concentration to a region of the lowerconcentration. This process is called osmosis.

• When a microbial cell is placed in hypertonicsolution (or, solution of low water activity), itloses water and shrinkage of membrane takesplace.

• This phenomenon is called plasmolysis.

• Microorganisms show variability in their ability to adapt the habitats of low water activity.

• Microorganisms like S. aureus can survive over a wide range of water activity and are called as osmotolerant (as water activity is inversely related to osmotic pressure).

• Most microorganisms grow well only near pure water activity (i.e. around 0.98-1).

• halophiles require high concentration of salts

Temperature

• As temperature influences enzymic reactionsit has an important role in promoting orpreventing microbial growth.

• Four groups depending on their optimumgrowth temperature and the temperaturerange at which they will grow.

• Thermophiles have optimum growth at 55 °Cand a growth range of 30 - 75 °C

• Mesophiles have optimum growth at 35 °Cand a growth range of 10 - 45 °C

• Psychrotrophs have optimum growth at 20 -30 °C and a growth range of 0 - 40 °C

• Psychrophiles have optimum growth at 15 °Cand a growth range of -5 - 20 °C

Oxygen• The atmosphere of earth contains about 20% (v/v) of oxygen.

Microorganisms capable of growing in the presence of atmosphericoxygen are called aerobes whereas those that grow in the absence ofatmospheric oxygen are called as anaerobes.

• The micro-organisms that are completely dependent on atmosphericoxygen for growth are called obligate aerobes whereas those that donot require oxygen for growth but grow well in its presence are calledas facultative anaerobes.

• Aerotolerants (e.g. Enterococcus faecalis) ignore O2 and can grow inits presence or absence.

• In contrast, obligate anaerobes (e.g., Bacteroids, Clostridiumpastewianum, Furobacterium) do not tolerate the presence of oxygenat all and ultimately die.

• Few microorganisms (e.g., Campylobacter) require oxygen at very lowlevel (2-10%) of concentration and are called as microaerophiles. Andthey are damaged by the normal atmospheric level of oxygen (20%).

pH• The intracellular pH of any organism must be

maintained above the pH limit that is critical forthat organism.

• The control of intracellular pH is required in orderto prevent the denaturation of intracellularproteins.

• Each organism has a specific requirement and pHtolerance range.

• Most micro-organisms grow best at neutral pH(7.0).

• Yeasts and moulds are typically tolerant of moreacidic conditions than bacteria.

Reference

Books:

1. Microbiology by pelczar

2. Introduction to Microbiology, By A. S. Rao

3. A Text Book of Microbiology, By P. Chakraborty

4. Biology of microorganisms By M. T. Madigan, J. M. Martinko, D. A. Stahl and D. P. Clark

Images:

1 to 3 Biology of microorganisms By M. T. Madigan, J. M. Martinko, D. A. Stahl and D. P. Clark