Determination of bacterial generation time - Direct microscopic method and turbidity method

 

Determination of bacterial generation time - Direct microscopic method and turbidity method

 

AIM: To determine the generation time of given bacterial culture by Direct microscopic method and turbidity method.

PRINCIPLE: Bacterial population growth studies require inoculation of viable cells into a sterile broth medium and incubation of the culture under optimum temperature, pH and gaseous conditions. Under these conditions, the cells will reproduce rapidly and the dynamics of the microbial growth can be charted by means of a population growth curve, which is constructed by plotting the increase in cell numbers versus time of incubation. The curve can be used to delineate stages of the growth cycles. It also facilitates measurement of cell number and the rate of growth of a particular organism under standardized conditions as expressed by its generation time, the time required for a microbial population to double. The stages of a typical growth curve are: Lag, Log, Stationary and Death phase. Construction of a complete bacterial growth curve requires that aliquots of a 24-h shake flask culture be measured for population size at intervals during the incubation period. Spectrophotometric measurement of developing turbidity at regular intervals can be used as an index of increasing cellular mass. The generation time can be determined by simple extrapolation from the log phase. Instead of cell number, it is often more convenient to use dry cell weight per volume X as a measure of cell biomass concentration. During the exponential phase in batch we can write:

dX/dt =X

Where is the specific growth rate of the cells.

A. DIRECT MICROSCOPIC COUNTS

MATERIALS REQUIRED:

Petroff-Hausser counting chamber, Cover slips, Sterile diluent (nutrient broth or sterile saline), Pasteur pipets

PROCEDURE:

1. Clean Petroff-Hausser counting chamber with 70% alcohol and let air dry.

2. Mix culture well and apply a single drop to counting chamber with Pasteur pipet. Examine the counting chamber using high power, oil immersion objective.

3. Make a preliminary estimation of the concentration of cells from the overnight culture using the following formula:

Total cells counted x 2.0x107 x dilution factor = cells/ml

# Small squares counted

Therefore, if you counted an average of 15 cells per small square, then you would have a final concentration of 3.0 x 108 cells/ml. 37 4.

You may have to adjust downward using one of your initial serial dilutions so that the counts per Small Square are in the 5 to 15 cell range.

5. Once this is done, make sure to allow time for cells to settle and move focus through the suspension (i.e., up and down) so as to count all cells within the small square “box”.

6. Count the number of bacterial cells in at least 10 small squares.

B. TURBIDITY METHOD

MATERIALS REQUIRED: Culture: 12-18 h nutrient broth culture of E. coli  

Medium: Nutrient Broth

Equipment & Accessories: Laminar hood, Orbital incubator shaker, 250 ml conical flasks, 15 ml test tubes, Glassware marker, 1.0 and 0.2 ml sterile disposable tips, Micropipettes

PROCEDURE:

1. An over-night culture of E. coli is used to inoculate 100 ml of nutrient broth in a 250 ml conical flask. 2. The flask containing culture was incubated in an orbital shaker at 37°C, 180 rpm.

3. Aliquots of the culture were taken aseptically at regular intervals and the turbidity was measured in a spectrophotometer at 600 nm using nutrient broth as blank.

4. Optical density of the samples at 600 nm was recorded till 24 h of growth.

 5. The O.D600 values as a function of time were plotted in a semi-log paper to generate the growth curve.

6. The generation time of the bacteria can be determined by extrapolation from the growth curve.

 7. Plot the growth curve and calculate the generation time from the curve.

8.  A graph is plotted between biomass concentrations vs. time.

9. Linear part of the graph, which is exponential phase of growth, is taken for specific growth calculation.

RESULT:

Tabulate the result based on the dilution factor and respective method of determining generation time calculation method

Direct microscopic count

S. No.	Incubation time (h)	Number of cells/cu.mm
1.
2.
3.
4.
5.
6.
7.

 

Determination of generation time by turbidity method

S. No.	Incubation time (h)	Optical Density (600 nm)
1.	0
2.	1.0
3.	2.0
4.	3.0
5.	4.0
6.	5.0
7.	6.0