Measurement of Microbial Growth by Total Viable Count Method

Objective:

To find the generation time of microorganisms by total viable count method.

Principle:

The number of cells capable of dividing and forming the colonies on solid medium are called viable number cells. Total viable count method is more convenience because there is virtually no other reliable and virtual way to count living organisms. In this method, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium. The suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then incubated under conditions that permit microbial reproduction so that colonies develop that can be seen without the aid of a microscope. It is assumed that each bacterial colony arises from an individual cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor, the number of bacteria in the original sample can be determined.

Materials required:

-Nutrient agar

- 10- 12 hrs old broth culture

-Sterile pipette  

-L –rod

-Turn table

-Petri dishes

Procedure:

-  The bacterial sample is serially diluting upto 10⁴ dilutions (add 0.5 ml of sample in 4.5 ml of saline).     

-Transfer the 0.1 ml from each dilution tubes to the corresponding agar plates by spread plate technique.

-  Incubate the plates  at 37 ͦ c  for 24 hours

- following the incubation number of cells will be counted.

- Repeat this process from initial time of inoculation (0^th hour) to 7-8 times for every 30 min.  

- Calculate the CFU each time and the results will be tabulated and plotted the graph.

- To determine the generation time by using the formula Gt = t2 - t1  (CFU2 -CFU1).

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

CFU calculation:

No. of CFU / volume plated (ml) x total dilution used    to     No. of CFU/mL

Nutrient Agar Composition:

Peptone   5g

Beef extract   2g

Yeast extract    3g

Sodium chloride   5g

Agar   15 g

Distilled water   1000ml

Saline  Preparation: 

0.9 gm of Nacl in 100ml of water.