Most Probable Number (MPN) Test: Principle, Procedure, Results

 

Purpose:

To enumerate the number of bacteria present in the water by the MPN method.

To identify the bacteria present in the drinking water sample.

Principle:

Water to be tested is diluted serially and inoculated in lactose broth, coliforms if present in water utilizes the lactose present in the medium to produce acid and gas. The presence of acid is indicated by the color change of the medium and the presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium. The number of total coliforms is determined by counting the number of tubes giving positive reaction (i.e both color change and gas production) and comparing the pattern of positive results (the number of tubes showing growth at each dilution) with standard statistical tables.

Type of container and additives

Sterile container

Requirements:

Equipment: Test tubes, Durham tube, sterile plates, laminar airflow, Incubator.

Reagents/ Media: MacConkey broth, Stains, Biochemical reagents

Sample collection:

After flaming the tap and allowing the water to run for five minutes to clear out the stagnant water in the line, Specimen must be collected aseptically into sterile 500 ml ground-glass-stoppered bottles containing sufficient sodium thiosulfate (about 0.3 ml of a 10% solution to 300 ml. of water) to reduce the chlorine in a treated water supply at the moment of collecting the specimen. Place immediately in a portable ice chest or a refrigerator and hold at 5°C until the examination can be started - always within a few hours of collection.

Culture procedure:

Test for the presence of thiosulfate in the specimen to be certain that the free chlorine has been neutralized. Add one drop of N/10 iodine solution to 3-5 ml of the specimen. If the solution remains iodine-colored, thiosulfate is absent. If the iodine color disappears upon shaking, then thiosulfate is present and free chlorine is absent, and the specimen is satisfactory for testing.

Presumptive Test for Coliform Bacilli:

1. One 50 ml quantity of water added to 50 ml double strength MacConkey broth.

2.   Five 10 ml quantities each to 10 ml double strength MacConkey broth.

3.     Five 1 ml quantities each to 5 ml single strength MacConkey broth.

4.     Five 0.1 ml quantities each to 5 ml single strength MacConkey broth and Incubate at 35-37°C.

5.     Examine after 24 and 48 hours look for changes in color of broth and for gas formation.

6.     NOTE: Do not confuse air bubbles with actual gas production. True gas formation is indicated by turbidity (showing bacterial growth) and the evolution of small bubbles throughout the medium when the tube is shaken gently, and by accumulation in the Durham tube to at least 10 percent of the capacity of the Durham tube.

7.     Absence of broth color change and gas formation at the end of 48 hours indicates a negative test. Presence of gas and color changes of broth is presumptive evidence of the presence of coliforms and indicates the necessity of further procedures

Confirmatory Test:

Subculture from the tubes showing gas to plates of MacConkey Agar and Gram staining preparation made from MacConkey agar should be examines microscopically.

Result interpretation:

Coliform Bacilli

Report the probable number of coliform bacilli per 100 ml of specimen according to McCrady's table and classify as follows on the basis of Presumptive Test results.

NOTE: Regardless of the count, if typical faecal E.coli is isolated, report the water as unsatisfactory. If Ps. aeruginosa or other Gram negative bacilli are found in great numbers, this also should be reported.

Reference intervals:

McCredie’s Table:

S.No	Most Probable Number (Mpn)/100 ml	QUALITY OF WATER
1	0	Highly satisfactory
2	1-2	Satisfactory
3	3-10	Suspicious
4	More than 10	Unsatisfactory

Reporting of test results:

      Negative Report:

        Result: No coliform grown in culture.

        Interpretation: Satisfactory.

Positive Report:

       Result: Coliform grown in culture (mpn/100 ml)

    Interpretation: Unsatisfactory.  Take necessary remedial action and repeat the investigation.

Completed Test

The positive results from the confirmatory test may be false; it is desirable to do completed tests. For this inoculum from each positive tube of the confirmatory test is streaked on a plate of Eosin Methylene Blue agar is incubated at 37°C for 24 hours.

After incubation, plates are examined for the presence of typical colonies. Coliforms can produce greenish metallic sheen colony which is differentiates it from non-coliform colonies (show no greenish metallic sheen) on EMB agar. 

Quality control procedures:

Check the reliability of media, with known bacteria/ ATCC strains of bacteria and whenever new batches are used.

Check the reliability of stains with known bacteria/ ATCC strains with both positive and negative bacteria and whenever new batches are used.

Reference:

1. Myer’s and Koshi’s., Manual of diagnostic procedures in medical microbiology and immunology/serology – CMC, Vellore -2001(page: 101).
2. Ananthanarayan and Paniker's, Textbook of Microbiology, Eleventh Edition-2013; Page:-640.