General Microbiology LaboratoryGeneral Microbiology Laboratory

Bacterial Generation TimeBacterial Generation Time

Microbial GrowthMicrobial Growth

Growth:Growth: an increase in the number of cells, not an increase in size

Bacterial species only maintained if population continues to grow

Generation:Generation: growth by binary fissionGrowth rate:Growth rate: cell number/time or cell mass/timeGeneration time:Generation time: time it takes for a cell to divide and

the population to double; most are 1-3 hours.

Generation times vary markedly with the species of microorganism and environmental conditions; they can range from 10 minutes for a few bacteria to several days with some eucaryotic microorganisms.

Generation TimesGeneration Times

Bacterium MediumGeneration Time (minutes)

Escherichia coli Glucose-salts 17

Bacillus  megaterium Sucrose-salts 25

Streptococcus lactis Milk 26

Streptococcus lactis Lactose broth 48Staphylococcus aureus

Heart infusion broth 27-30

Lactobacillus acidophilus

Milk 66-87

Rhizobium japonicum

Mannitol-salts-yeast extract

344-461

Mycobacterium tuberculosis

Synthetic 792-932

Treponema pallidum Rabbit testes 1980

What occurs during binary fission?What occurs during binary fission?

The Growth CycleThe Growth Cycle

The population growth is studied by analyzing the growth curve of a microbial culture.

The standard bacterial growth curve describes various stages of growth a pure culture of bacteria will go through, beginning with the addition of cells to sterile media and ending with the death of all of the cells present.

Typical growth curve for a bacterial populationTypical growth curve for a bacterial population

Phases of GrowthPhases of Growth

Lag phaseLag phase

• The period of apparent inactivity in which the cells are adapting to a new environment and preparing for reproductive growth.

• Cells are usually synthesizing new components.

• In practice, bacteria from one medium to another, where there are 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.

• Cells may increase in size during this time, but simply do not divide (by binary fission).

• Lag phase varies considerably in length depending upon the condition of the microorganisms and the nature of the medium.

Log (exponential) phaseLog (exponential) phase

• The period in which the organisms are growing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing.

• Generation time can be easily obtained from the exponential phase of a growth curve

• The population is most uniform in terms of chemical and physical properties during this period.

Stationary phaseStationary phase

• Eventually population growth decrease, and the growth curve becomes horizontal.

• rate of cell growth = rate of cell deathrate of cell growth = rate of cell death

• Cell death may result from Nutrient limitation & Toxic waste accumulation (e.g. acid buildup from fermentation); as well as O2 depletion, critical population level reached.

Death phaseDeath phase

• Stationary phase, in a standard bacterial growth curve, is followed by a die-off of cells, called Death phase.

• It is the period in which the cells are dying at an exponential rate.

• Some of the reasons are: continued accumulation of wastes, loss of cell's ability to detoxify toxins, etc.

Balanced and Unbalanced GrowthBalanced and Unbalanced Growth

• Balanced (exponential) growth occurs when all cellular components are synthesized at constant rates relative to one another.

• Unbalanced growth occurs when the rates of synthesis of some components change relative to the rates of synthesis of other components. This usually occurs when the environmental conditions or nutrient levels change.

Measurement of Bacterial GrowthMeasurement of Bacterial Growth

Cell numbersCell numbers

Metabolic rateMetabolic rate

Cell numbersCell numbers

1- Direct microscopic count1- Direct microscopic count• accomplished by direct microscopic

observation on specially etched slides (such as Petroff-Hausser chambers or hemocytometers) or by using electronic counters.

2- Viable count (colony forming units)2- Viable count (colony forming units)

Cell mass / numbersCell mass / numbers

Turbidity Turbidity or or optical densityoptical density • it is one of the optical methods for counting cells; can estimate

cell numbers accurately by measuring visible turbidity. • Light scattered is proportional to number of cells.

• Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm.

• Accurate measure of cells when concentration not too high. Easy and quick to measure (can measure a sample in less than a minute).

• This technique measures the total mass of organisms and does not distinguish between dead and viable organisms.

Metabolic rateMetabolic rate

002 2 uptake, C0 uptake, C022 and ATP production and ATP production

Turbidity measurements of microbial growthTurbidity measurements of microbial growth

Optical Density (Counting by Spectrophotometer)

End of lecture