ENVIRONMENTAL BIOTECHNOLOGY

Experiment 9

Aim of the Experiment : Determination of Most Probable Number (MPN)

Principle

It is a statistical method based on the probability theory. In this technique, the sample serially diluted till the number of organisms reach the point of extinsion. From each of the dilutions several multiple tubes of a specific medium are inoculated. Presence of organism is indicated by acid or gas in the medium. Pattern of positive and negative test results are then used to estimate the number of bacteria in the original sample. Since the test gives the most probable number of organisms present in the sample it is also known as MPN test.

Materials required

1. 1 MLBB tube having 50 ml double strength (2x) medium.

2. 5 MLBB tubes each having 10 ml double strength (28) medium.

3. 11. MLBB tubes each having 5 ml single strength (X) medium.

4. Sterile 10 ml and 1 ml pipettes. 5. Water sample to be tested.

Procedure:

1. Shake the water sample vigorously to ensure uniform distribution of organisms.

2. Dilute the sample (if necessary) by transferring 10 ml of water sample by sterite pipette to 90ml of sterile distilled water. This makes 10^-1 dilution. Make more dilution if needed.

3. With the sterile graduated pipettes inoculate the water sample (diluted sample, if the dilution is done) as follow.

• 1 tube of MLBB having 50 ml (2X) medium with 50 ml sample.

• 5 tubes of MLBB having 10 ml (2X) medium with 10 ml of sample each.

• 5 tubes of MLBB having 5 ml (X) medium with 1 ml of sample each.

• 5 tubes of MLBB having 5 ml (X) medium with 0.1 ml of sample each.

4. One tube of MLBB having 5 ml (X) medium is left uninoculated, which serves as control.

5. Incubate all tubes at 37°C for 24 hours.

6. Examine tubes for acid and gas after 24 hours.

7. If no tube shows acid and gas reintubate all tubes for another 24 hours

8. At the end of the incubation period, record the number of positive tubes in each of three sets (i.e. 10.ml, 1 ml and0.1ml), and interprete results as follows.

Interpretation

McCrady in 1918 computed tables (refer Appendix-III, page no. 205) regarding the most et probable number of organisms present in 100 ml of water, on the basis of various combinations of positive and negative results in the amounts used for tests. Number of organisms per 100 ml is read from the McCrady's table, and the number is multiplied by the dilution factor (if any), to come to the final number.