Most Probable Number Test Definition, Principle & Method

To evaluate the presence of microorganisms in qualitative and quantitative water, the most probable number model is employed (mainly coliforms). The abbreviation “MPN” stands for most probable number test. Multiple tube fermentation test or MPN method quantifies the amount of coliforms in 100 ml of water sample to measure the water’s quality.

Definition of MPN

MPN refers to the most probable number. It refers to the qualitative and quantitative investigation of water that can identify faeces coliforms. E.coli is a frequent faeces contamination found in water, and ingesting it can result in severe disease. Thus, in the most probable number method, E.coli is employed as a “Pollution indicator” to determine the quality of the water. The most probable number consists of three sets of techniques, including presumptive, confirmatory, and completed-test procedures.

Principle of MPN

A most probable number is a statistical procedure that compares the results to conventional statistical tables. It requires three dilutions of fermentative broth and water sample. The production of acid and gas suggests a positive outcome. The quantitative analysis of coliform is determined by counting the number of tubes giving a positive result and comparing the pattern of positive results with the statistical data.

Procedure of MPN

MPN is a method, which involves three consecutive tests, namely presumptive, confirmatory and completed-test.

i) Presumptive Test

The preliminary or screening test employs a series of fermentation tubes containing known concentrations of lactose broth. The negative presumptive analysis indicates that the water is microbiologically safe and that further testing is not required. However, if the results are positive, the presence of coliforms must be confirmed using the confirmed MPN-test.

According to the Chambers, 40-390 million per millilitre of coliforms are required for gas generation. The generation of gas depends on the ratio of coliform to non-coliform bacteria. If the ratio of non-coliform bacteria is large, gas production will also decrease. The presumptive test consists of the subsequent steps:

Figure 1: Presumptive Test. Image Source: Microbe Online

  1. Prepare and autoclave MacConkey purple media of single and double strength in test tubes using Durham’s tube and autoclave it.
  2. Take three sets of five test tubes each, one with 10 ml of double strength (DS) and the other two with 10 ml of single strength (SS).
  3. Transfer 10 ml of water to each of the DS broth tubes using sterile pipettes. Transfer 1 ml of water sample to each of the five tubes of one set of SS broth and 0.1 ml of water to each of the five tubes of the last set of SS broth. Incubate the tubes for 24 hours at 37°C.
  4. Observe the colour change and gas production in Durham’s tube following incubation.
  5. Record the number of positive results from each set and compare it to the standard chart to determine the probable coliform count per 100 ml of water sample.

ii) Confirmed Test

By examining the positive tubes from the presumptive test, the confirmatory test verifies the presence of coliform. The presumptive test’s gas output does not prove that the water sample contains coliform. Water contains a variety of other bacteria that may provide a false presumptive test result.

The water contains yeasts and Clostridium species that can ferment lactose by releasing gas and acid. Verifying the presence of coliform in water is therefore necessary. A verified test can often be carried out in one of two ways: Brilliant green lactose bile broth (BGLB), Eosin methylene blue agar medium (EMB)

Confirmed Test

Figure 2: Confirmed Test. Image Source: Biologyreader

i) Lactose-broth

Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2 hours. If no gas production is seen, further incubate up to a maximum of 48 ±3 hours to check gas production.

ii) Media slants

Take a loopful of suspension from a positive tube and inoculate it on the agar surface. The agar slants should be incubated at 37°C for 24± 2 hours. Colonies must be examined macroscopically.

iii) Completed Test

It is the final test to ascertain the presence of coliforms. A completed test involves the following steps:

Figure 3: Completed Test. Image Source: Biologyreader

  1. Transform a typical coliform colony from an agar plate into a tube of brilliant green bile broth with Durham’s tube and a nutrient agar slant on the surface.
  2. Incubate for 24 hours at 35°C.
  3. After 24 hours, check the broth for gas production and perform Gram staining on the nutrient agar slant for organisms.

Results of MPN

i) Presumptive Test

Positive: The development of 10 percent or more gas in the Durham tube within 24 to 48 hours, along with turbidity in the growth media and colour change, indicates a positive presumptive test for coliform bacteria and, thus, for the likelihood of faecal contamination. The test is just presumptive because various other species of bacteria can yield identical results under these conditions.

Negative: No growth or formation of gas in Durham’s tube.

i) Presumptive Test

Positive: Formation of gas in lactose broth and the formation of a coliform-like colony on EMB agar suggest the existence of a coliform member in the analysed sample. Coliforms create colonies with a metallic sheen that is distinguishable from non-coliform colonies. The appearance of typical colonies at high temperatures implies that thermoresistant E. coli is present.

Negative: The absence of gas formation in lactose broth or the failure to demonstrate coliform-like colonies on the EMB agar.

iii) Completed Test

Positive: The presence of gas in the brilliant green bile broth tube and Gram-negative, non-spore-forming rods on NA slant constitutes a positive completed test for the presence of coliform bacteria, which, in turn, infers possible contamination of the water sample with fecal matter.

Negative: Absence of growth and gas formation in the broth. Absence of gram-negative, non-sporing rods on Gram staining.

Uses of MPN Test

  1. It is widely utilised in the estimation of microbial populations in a variety of environments, including soils, waterways, and agricultural products.
  2. Plate count enumeration procedures can be complicated by the presence of particulate material in samples.
  3. In addition to that, it has been proposed as a potential alternative approach to trend studies conducted in the field of environmental monitoring.
  4. It is also useful for counting bacteria that form colonies reluctantly on agar plates or membrane filters but readily grow in liquid media.

Advantages of MPN

  1. The MPN method can be used to analyse turbid water that contains sediments, sludge, mud, and other materials.
  2. It is simple to interpret the results by looking for gas production or gram-negative rod-shaped coliform growth.
  3. The Most Probable Number test has the benefit over membrane filtration in that it may be used with all types of water.

Limitations of MPN

  1. Most probable number test consists of three tests that take a while to return findings in order to confirm the presence of coliforms.
  2. A very sensitive approach like most probable number test occasionally produces inaccurate results.
  3. The most probable number approach necessitates the use of numerous test tubes and glasses for the preparation of the media.