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MPN Test

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27 views

MPN Test

Uploaded by

TJ guitar
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© © All Rights Reserved
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Name of the experiment: Determination of plate counts and MPN test for a given

sample.

Aim of the experiment: To determine the plate counts and MPN test of a given
sample.

Apparatus required:-
1. Medium: Lactorebrom or Mac Conkey broth or Laurytryplore( lactose) broth.

2. Glass ware: Test tubes of various capacities (20ml, 10 ml, 5ml)

3. Sterile pipettes.

Theory:-

Most probable number (MPN) is a method used to estimate the number of coli forms in
water. It involves inoculating different dilutions of water sample into lactose broth and
observing gas production, which indicates the presence of coliforms.

MPN test is performed in 3 steps:

1. Presumptive test.

2. Confirmatory test.

3. Completed test.

Presumptive test

The method of presumptive test varies for treated and untreated water.
Preparation of medium :-

1. Prepare medium in single and double strength concentration.

2. For untreated or polluted water.

• Dispense the double strength medium into10 tubes (10 ml in each tube) and single
strength medium in 5 tubes (10ml in each tube) and add a durham tube in an
inverted position.

3. For treated water-

• Dispense the double strength medium in 5 tubes (10 ml each tube) and 50ml single
strength medium in 1 bottle and a durham tube in an inverted position.

4. Sterilize by autoclaving at 15 lbs pressure (121°c) for 15 minutes.

Procedure of MPNtest

(i) For untreated (polluted water).

1. Take 5 tubes of double strength and 10 tubes of single strength for each water
sample to be tested.
2. Using a sterile pipet add 10 ml of water to 5 tubes of double strength.

3. Similarly add 1ml of water to 5 tubes of 10 ml singe strength and 0.1 ml of water to
the remaining 5 tubes.

4. Incubate all the tubes at 37°C for 24 hrs if no tubes appear positive re-incubate
for upto 48 hrs

5. Compare the number of tubes giving a positive reaction to standard chart and
record the number of bacteria present in it.

(i)For treated (unpolluted) water:-

1. Take 1ml of single strength (50ml) and States of double strength (10 ml for each)
water sample to be treated.

2. Using a sterile pipets add some of 50 ml of water to the 5 tubes containing some
of single strength medium.

3. Similarly add 10 ml of water to 5 tubes containing 10 ml of double Strength


medium.

4. Incubate it for 24 hrs at 37°C and if it does not show re-incubate upto48 hrs.

5. Compare the number of tubes giving positive reaction to a standard chart and
record the number of bacteria present in it.

Confirmatory test:-

Some microorganism other than coliforms also produce acid and gas form Lactose
fermentation. In order to confirm the presence of coliform, a confirmatory test is
done. For each of the fermentation tubes with positive result transfer one loopful of
medium to: 1) 3ml lactose broth or brilliant green lactose fermentation tube.

2) To an agar slant and

3) 3ml tryptone water

Incubate the inoculated lactose-broth fermentation tube at 37°C and inspect gas
formation after 24± 2 hours. If no gas production in seen, further incubate, up to a
maximumof 48±3 hours to check gas produced, The agar slant should be incubated
at 37°C for 24±2 hours and gram-stained preparation made from the slant, should
be examined microscopically.

The formation of gas in lactose broth and the demonstration of gram-negative, non-
spore forming bacilli in the corresponding agar indicates the presence of a member
of the coliform form group in sample examined.

The absence of gas formation in lactose broth or the failure to demonstrate gram
negative, non spore forming bacilli in the corresponding agar slant constitutes a
negative test.
Tryptone water test:- 1. Incubate, the tryptone water at (44.5± 0.2°C) for 18-24 hrs.

2. Following incubation, add approximately 0.1ml of Kovacs reagent and mix gently.

3. The presence of indole is indicated by a red color in the kovacs reagent, forming
a film the aqueous phase of the medium.

a. Confirmatory tests positive for indole, growth and gas production show the
presence of thermotolerant E. col.

b. Growth and gas production in the indole confirm thermotolerant coliforms.

Completed test:-

Since some of the positive results from the confirmatory test may be false, it is
desirable to do completed test. For this inoculum from each positive tube of the
confirmatory test is streaked on a plate of EMB or Endo agar.

In this process a loopful of a sample from each positive BGLB tube is streaked onto
selective medium like Eosinmethylene blue agar or Endo's medium. One plate each
is incubated at 37°c and another al 44.5±0.2°C for 24 hrs.

Following incubation, all plates are examined for the presence of typical colonies.

Coliforms produces colonies with a greenish metallic & sheen which is differentiated
it from non-coliform colonies (show no sheer).The presence of typical colonies on
high temperature (44.5±0.2) indicates the presence of thermotolerant E.coli.

IS Code name and limits:-

IS 1622: 1981 Methods of Sampling and Microbiological examinations of water.


For drinking water MPN should be zero per 100 ml as IS guidelines.

IS 5402: 2012- Microbiology of food and animal feeding (or) Stuffs

For drinking water, the total plate count should be less than 100 CFU/ml at 37°C
within 24 hours.

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