Lab.2.

Practical Microbial Physiology

Bacterial Growth Curve

Growth is an orderly increase in the quantity of cellular constituents.

In most bacteria, growth involves increase in cell mass and number of ribosome, 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.

Four phases of the bacterial growth cycle are

recognized:

1. Lag Phase.

2. Log phase.

3. Stationary phase.

4. Decline (death) phase.

Typical growth curve for a bacterial population

1. Lag phase: The length of the lag phase is dependent on some factors:

§ Age of culture inoculum

 old culture -> long lag

 young culture-> short lag

§ Size of inoculum

 few cells -> long lag

 many cells -> short lag

§ Environment

 pH, temp, gases and salinity

 sub optimum -> long lag

 optimum-> short lag

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth
wherein all the cells are dividing regularly by binary fission.
3. Stationary and Decline Phase. Exponential growth cannot be continued forever in a batch
culture (e.g. a closed system such as a test tube or flask). 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".

Growth rate: it means cell number/time

1
or Growth rate (min-1) = mean generation time

Generation time: Time taken for a cell or population to double in numbers called Generation
Time.

t G = generation time

G= t = time of growth
3.3 log b/B B = number of bacteria at the beginning of a time interval

b = number of bacteria at the end of the time interval

Generation times for some common bacteria under optimal conditions of growth:

Bacterium Medium Generation Time (min.)

Escherichia coli Glucose-salts 17

Bacillus megaterium Sucrose-salts 25

Streptococcus lactis Milk 26

Streptococcus lactis Lactose broth 48

Staphylococcus 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

Methods for measuring bacterial numbers:

1. Direct microscopic counts: by using special slides known as counting chambers.

2. Indirect viable cell counts, also called plate counts: 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 is
related to the viable number of bacteria in the sample.

3. Turbidity method

Protocol for growth curve and detecting generation time:

1. Your flask will be inoculated by the instructor prior to class and allowed to incubate at 37° C in
incubator during lecture.

2. Starting at time 0, you will do the following every 30 minutes for a total of 4 time points:

3. Each time you must perform serial dilution, and from each dilution inoculate a plate of
Trypticase soy agar by spreading methods.

4. After 24 hr incubation at 37oC, choose appropriate plate with 25-250 colonies, determine the
number of cfu/ml of the original culture for each of the time sets and record the data in the table
below.

5. Convert the cfu/ml to log of cfu/ml, draw growth curve then calculate the generation time.

 Remember that B and b can be any two time points.

Incubation Time (min) Bacterial concentration (cfu/ml) Log of cfu/ml

Calculation of the generation time

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?
G= t_____
3.3 log b/B

G= 240 minutes
3.3 log 107/104

G = 240 minutes
3.3 x 3

G = 24 minutes