Explain the Procedure Estimation of the Amount of Bacteria?

Now carry out the exercise following the steps enumerated herewith.

1. Prepare ten fold dilutions of E. coli culture in diluent till 10^-7 dilution is obtained. For making dilution, label 7 tubes of sterile water blanks as 10^-1 through 10^-7. Look at the Figure for details. Mix the E. coli culture by rolling the tube between the palms for uniform distribution of cells. Aseptically transfer 1 ml of culture with a sterile pipette to a tube marked 10^-1. This is 1:10 dilution (10 times diluted culture). Discard the pipette in a beaker of disinfectant.

2. Thoroughly mix the 1:10 dilution and with a fresh sterile pipette, transfer 1 ml of 10^-5 dilution to a tube marked 10^-2 to achieve 1:100 or 100 times dilution.

3. Repeat the step 2 till get 10^-7 dilution, i.e., 1 ml of 10^-2 is transferred to a tube labeled 10^-3  (1000 times dilution) and 1 ml from 10^-3 to a tube marked 10^-4 (10,000 times dilution) and so on. Each time a fresh sterile pipette has to be taken.

4. Liquefy the nutrient agar medium by boiling and maintain in a liquid state at a temperature of 45°C by keeping in a water bath.

5. Label the sterile petri plates as 10^-5, 10^-6 and 10^-7 in duplicate. Add 1 ml of each 10^-5, 10^-6 and 10^-7 to the labeled petri plates.

6. Aseptically pour 20-25 ml of molten nutrient agar at temperature of 45°C to each petri plate having dilution. Gently rotate the plate to ensure uniform distribution of cells in the medium.

7. After solidification of agar, incubate the plates at 37°C for 24 hours in an inverted position. Following incubation, cell counts are made both for surface and subsurface colonies. For manual counting, each colony is counted and marked with a marker on the bottom of the plate.