1. Microbial Growth Growth in Batch Culture
Mean Generation Time and Growth Rate
Measurement of Microbial Growth
Continuous Culture
Factors Influencing Growth
Growth in Natural Environments

2. Growth in Batch Culture
“Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction
A “batch culture” is a closed system in broth medium in which no additional nutrient is added after inoculation of the broth.

3. Growth in Batch Culture
Typically, a batch culture passes through four distinct stages:
Lag stage
Logarithmic (exponential) growth
Stationary stage
Death stage

4. Growth in Batch Culture

5. Mean Generation Time and Growth Rate
The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes.
Growth rate (min-1) =
Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation

6. Mean Generation Time and Growth Rate

7. Mean Generation Time and Growth Rate

8. Measurement of Microbial Growth
Microscopic cell counts
Calibrated “Petroff-Hausser counting chamber,” similar to hemacytometer, can be used
Generally very difficult for bacteria since cells tend to move in and out of counting field
Can be useful for organisms that can’t be cultured
Special stains (e.g. serological stains or stains for viable cells) can be used for specific purposes
Serial dilution and colony counting
Also know as “viable cell counts”
Concentrated samples are diluted by serial dilution

9. Measurement of Microbial Growth
Serial dilution and colony counting
Also know as “viable cell counts”
Concentrated samples are diluted by serial dilution
The diluted samples can be either plated by spread plating or by pour plating

11. Measurement of Microbial Growth
Serial dilution (cont.)
Diluted samples are spread onto media in petri dishes and incubated
Colonies are counted. The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual).
A simple calculation, with a single plate falling into the statistically valid range, is given below:

12. Measurement of Microbial Growth
Serial dilution (cont.)
So you’ve seen all this before, huh?
What if there is more than one plate in the statistically valid range of 25 – 250 colonies?
For details on this standard method, see: FDA Bacteriological Analytical Manual
and specifically the section on: Aerobic Plate Counts
Really.
heh, heh.

13. Measurement of Microbial Growth
Where: C = Sum of all colonies on all plates between n1= number of plates counted at dilution (least diluted plate counted) n2= number of plates counted at dilution (dilution 2 = 0.1 of dilution 1) d1= dilution factor of dilution 1 V= Volume plated per plate

14. Measurement of Microbial Growth
Membrane filtration
Used for samples with low microbial concentration
A measured volume (usually 1 to 100 ml) of sample is filtered through a membrane filter (typically with a μm pore size)
The filter is placed on a nutrient agar medium and incubated
Colonies grow on the filter and can be counted

15. Measurement of Microbial Growth
Turbidity
Based on the diffraction or “scattering” of light by bacteria in a broth culture
Light scattering is measured as optical absorbance in a spectrophotometer
Optical absorbance is directly proportional to the concentration of bacteria in the suspension

16. Measurement of Microbial Growth
Mass determination
Cells are removed from a broth culture by centrifugation and weighed to determine the “wet mass.”
The cells can be dried out and weighed to determine the “dry mass.”
Measurement of enzymatic activity or other cell components

17. Growth in Continuous Culture
A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate.
This method is accomplished in a device called a chemostat.
Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained.

18. Lab Ex. 19: Enumeration of Bacteria – Pour Plate Technique
Bacterial growth may be determined based on increase in the
number of cells.
Reproduction and thus growth leads to increase in cell number and
thus the population of the bacterial culture.
Bacterial growth follows specific patterns as seen in the growth
curve patterns for bacteria.
The time it takes for a population of bacterial cells to double in
number is called the generation time.
Bacterial growth may be determined by using either
Cell Mass methods or Cell Number methods.

19. Cell Mass methods:
Turbidity measurements by Spectrophotometry
Total Protein
Total DNA
Cell Number methods:
Direct Microscopic Counts (hemocytometer)
Pour Plate
Most Probable Number
Membrane Filter
Enumeration methods may yield either total counts or viable counts.
Total count is a count of cells including dead and live cells.
Viable count is a count of only those cells that are alive in the sample.

20. Pour Plate method:
This is the most commonly used method for enumeration of
bacteria in a wide variety of samples including milk, food,
meat, soil etc.
Pour plate methods yield a count of only the living cells in the
sample and thus are a viable count.
There are two steps to the process: dilution of the sample so
that various dilutions of the sample may be inoculated
onto plates and a count of the colonies that grow made;
the second step is the plating of the dilutions so that each
cell in the diluted sample may then grow and form colonies
that will in turn become visible to the naked eye and can be
counted.

21. Dilution is important since the colonies will have to be counted and
with a concentrated sample there may be too many colonies
than can be accurately counted.
Plating is important since a count of only the living cells is
required in this procedure (only living cells will be able to
multiply and form colonies)
Samples of milk, meat and soil will be used in the exercise.
Rules to keep in mind:
- only living cells are counted. Why?
- only plates with colony numbers between are useable
(<30 or >300 are not statistically valid)
- use aseptic technique
- dilutions to be done accurately

23. Procedure:
- aseptically dilute sample with sterile water blanks
- make 1:10 dilutions as instructed, based on sample
- aseptically transfer 1ml volume of dilutions that are to be
plated onto sterile Petri plates
- pour molten agar onto the sample in Petri dish and mix
thoroughly
- incubate as instructed for growth of colonies
- during next lab period, count colonies on plates
- use the formula:
cfu/ml = # of colonies x 1
dilution