Most Probable Number (mpn): Principle, Procedure, and Results

Who discovers the most probable number?

    Understanding and Teaching the Most Probable Number Technique, published in 1975 .J. L. Oblinger and J. A. Koburger 540-545 in J. Milk Food Technol., 38(9).

Define Most Probable Number

Using ten-fold dilutions of liquid broth growth, the Most Probable Number (MPN) technique, is used to calculate the concentration of live microorganisms in a sample. It is frequently applied to the estimation of microbial populations in agricultural products, waterways, and soils. When samples contain particulate matter that obstructs plate count enumeration techniques, MPN tests are particularly helpful.

      To determine whether the water is safe to drink in terms of the number of bacteria present, MPN is most frequently used to assess the quality of water. Fecal contamination of water is indicated by a kind of bacteria known as fecal coliform. In contrast, the presence of large numbers of fecal coliform bacteria would indicate an extremely high possibility that the water could be contaminated with pathogens, making it dangerous to drink. Very few fecal coliform bacteria would suggest that water is likely to be free of pathogenic microorganisms.

Most probable numbers Principle

      A statistical technique called a most probable number compares the outcomes to the normative statistical tables. It involves three sets of dilutions containing the water sample and fermentative broth. A successful outcome is indicated by the production of gas and acid. The quantitative analysis of coliform is calculated by counting the number of tubes yielding a positive result and contrasting the pattern of positive results with the statistical data.

Which methods are used for the Most Probable Number?

Three tests are conducted back-to-back using the MPN technique, including;

  • Presumptive test
  • Confirmatory test

Presumptive test

In the preliminary or screening test, lactose broth in various fermentation tubes is utilized at known concentrations. The negative presumptive analysis shows that the water is safe from a microbiologic standpoint and does not call for additional testing. However, if the findings are positive, the validated MPN test must be used to verify the presence of coliforms.

       According to Chambers, 40–390 million coliforms are needed for every milliliter of gas to be produced. The ratio of coliforms to non-coliforms affects gas formation. The production of gas will also be reduced if the non-coliform bacteria ratio is high. The following steps make up the presumptive test:

Requirements of Presumptive test

culture medium:

           Lauryl tryptose (lactose) broth, MacConkey broth, or lactose broth


           Durham tube, test tubes of varying sizes (20ml, 10ml, and 5ml).

In addition,

          sterile pipettes.

The procedure of the Presumptive test

  1. To make a single-strength medium, Add 40 g of the Lactose broth powder to 1000 ml of distilled water in a sterile conical flask. To create a double strength medium, combine 80 g of the Lactose broth with 1000 ml of distilled water in the other flask.
  2. The flasks should be sterilized for 15 minutes at 121 degrees Celsius in an autoclave.
  3. Water samples should be collected in a series of test tubes after autoclaving. In the three sets, add water with the known concentration in the following amounts: 10 ml in the first five tubes, 1 ml in the next five tubes, and 0.1 ml in the last five tubes. 10 ml of double-strength Lactose medium should be added to 10 ml of water to be tested. In contrast, apply 10 ml of lactose medium at single strength to a water sample to assess 1 ml and 0.1 ml.
  4. The tubes should be incubated for 24 to 48 hours at 35 degrees Celsius.
  5. Count the number of positive tubes of each set and check the number from the table mentioned below


        Results are determined after incubation based on any color change or gas production in the Lactose broth.

Presumptive test results

      The reddish-yellow coloration of Lactose broth and the formation of gas inside the Durham tubes will show a positive outcome for the presumptive test. A negative result for the presumptive test will be obtained if the color of the broth stays the same, i.e., red, and no gas forms.

Confirmation Test

         It is the confirmatory test that verifies the presence of coliform by examining the presumptive test’s positive test tubes. The presumptive test’s gas output does not prove the presence of coliform in the water sample. The presence of numerous more bacteria in water can result in a misleading presumptive test.

         There are some yeasts and Clostridium species in the water, and they can ferment lactose to produce gas and acid. The detection of coliform in water must therefore be confirmed. There are typically two ways to conduct a verified test:

In a culture medium containing;

  • Eosin methylene blue (EMB)
  • Brilliant green lactose bile broth (BGLB)


  1. Make a solution of the following ingredients to create a brilliant green lactose bile broth medium first:
  2. 10 g of peptone
  3. 10 g of lactose
  4. 20 g of bile salt
  5. Brilliant Green: 0.0133 g
  6. 1 L of distilled water
  7. The BGLB medium should be autoclaved for 15 minutes at 121 degrees to sterilize it.
  8. Gently shake the tubes with positive presumptions.
  9. Place a loop’s worth of culture in the BGLB fermentation tube after that. Gram positive bacteria cannot thrive in the BGLB medium because of the vivid green dye.
  10. The test tubes should be incubated for 48 hours at 35 degrees Celsius.

Confirmed Test Observations

        Keep an eye on the test tubes with the BGLB medium and the inoculum of the presumed positive test to see if any gas is being produced in the inverted Durham tube.


        The presence of coliforms is shown by the gas generation in the BGLB medium.

The Durham tube of the BGLB fermentation tube producing gas indicates the presence of coliform bacteria. The development of gram negative, non-sporing rods in the agar slants is proof that coliforms are present.

Presumptive positive testing in EMB medium


  1. First, combine the required ingredients to create a solution of eosin methylene blue agar medium:
  2. 10 g of peptone
  3. Agar: 15 g
  4. 10 g of lactose
  5. 0.4 g of eosin Y
  6. 0.065 g Methylene blue
  7. 2 grams of dipotassium hydrogen phosphate
  8. 1L of distilled water
  9. EMB agar should be autoclaved for 15 minutes at 121 degrees Celsius and sterilized it.
  10. On the sterile Petri plates, pour the EMB medium and let it set up.
  11. Gently shake the tubes with positive presumptions.
  12. A loopful of culture should then be streaked onto the EMB agar media.
  13. The test tubes should be incubated for 48 hours at 35 degrees Celsius.


     Check the plates for bacterial colonies developing on the EMB media. Typically, three different types of colonies form:

  • Typical Colonies
  • Atypical Colonies
  • Negative Colonies

Typical Colonies:

        It is verified by the development of a nucleated colony and may take the shape of a metallic shine.

Atypical Colonies:

       Colonies that are non-nucleated, opaque, and mucoid are produced.

Negative Colonies:

     Different colony types are produced by it.


     The development of a typical colony is a sign that coliform bacteria are present.

Advantages and disadvantages of the most probable method


  • Ease of interpretation using either gas emissions or observation
  • Toxin samples are diluted.
  • An effective way to examine materials that are too extremely turbid to be tested is by membrane filtration, such as sediments, sludge, mud, etc.


  • Results don’t appear for a very long time.
  • The results aren’t particularly precise.
  • Extra media and hardware (glassware) are needed.
  • Chance of erroneous positive results.


Rimsha Bashir
Rimsha Bashir

Rimsha Saith is a highly knowledgeable microbiologist with a keen interest in the field. Her expertise and passion are in her writing for Microbiology. As a writer, Rimsha has authored numerous articles that have been well-received by both health and medical students and industries.

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