Laboratory Tests

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

To evaluate the presence of microorganisms in qualitative and quantitative water, the most probable number approach is utilised (mainly coliforms). The abbreviation...

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This article writter by MN Editors on October 11, 2022

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Most Probable Number (MPN) Test: Principle, Procedure, Results
Most Probable Number (MPN) Test: Principle, Procedure, Results
  • To evaluate the presence of microorganisms in qualitative and quantitative water, the most probable number approach is utilised (mainly coliforms). The abbreviation “MPN” stands for most likely number.
  • Multiple tube fermentation test or MPN method quantifies the amount of coliforms in 100 ml of water sample to measure the water’s quality.
  • The MPN test consists of a series of test tubes containing fermentative broth to which water samples are added in a predetermined ratio.
  • We can detect coliform in the water sample by examining the generation of acid and gas in the fermentation tube.
  • A shift in colour (from red to yellow) in the fermenting broth indicates the generation of acid.
  • When gas bubbles form in an upside-down Durham tube, it means that gas is being made in the fermentation tube. When estimating or determining the number of microorganisms in large samples of water, soil, etc., the most probable number is frequently employed.
  • The MPN test identifies the presence or absence of faecal coliforms, which can contaminate water with faeces and render it unfit for human consumption.
  • Determining the concentration of microorganisms or water quality, i.e. whether water is potable or not, necessitates water quality testing.
  • This lesson will cover the most probable number test’s definition, principles, techniques, advantages, and downsides.

What is MPN?

  • MPN refers to the most likely number. It refers to the qualitative and quantitative investigation of water that can identify faecal coliforms.
  • E.coli is a frequent faecal 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 Most Probable Number

The water to be tested is serially diluted and inoculated in lactose broth. If coliforms are present in the water, they consume the lactose in the medium to produce acid and gas. The presence of acid is shown by a change in the medium’s colour, whilst the presence of gas is indicated by the collection of gas bubbles in an inverted Durham tube present in the media. Total coliforms are determined by counting the number of tubes exhibiting a positive reaction (i.e., both colour change and gas production) and comparing the pattern of positive results (the number of tubes exhibiting growth at each dilution) with conventional statistical tables. The MPN test is conducted in three phases;

  • Presumptive test.
  • Confirmatory test.
  • Completed test.

Objectives of Most Probable Number (MPN)

  • Using the MPN method, count the amount of bacteria in the drinking water.
  • Identify the microorganisms present in the sample of drinking water.

Presumptive Test

  • This test, which is a particular enrichment process for coliform bacteria, is carried out in fermentation tubes containing a selected growth medium (MacConkey lactose broth) and inverted Durham tubes for the detection of fermentation gas.
  • A series of lactose broth tubes are infected with measured quantities of the test water sample.
  • There may be three or four groups of three, five, or more tubes in the series of tubes.
  • Lactose, sometimes a surfactant such as Na-lauryl sulphate or Na-taurocholate (bile salt), and frequently a pH indicator colour, such as bromcresol purple or brilliant green, are the primary selection variables present in the medium.
  • Lactose has a selective effect because many bacteria cannot ferment this sugar, whereas coliform bacteria and several other species of bacteria can.
  • The surfactant and dye do not inhibit coliform bacteria, but they do inhibit numerous other bacteria, such as spore formers.
Presumptive Test
MPN test

Requirements

  • Lactose broth, MacConkey broth, or Lauryl tryptose (lactose) broth are all acceptable options.
  • Glasswares: Different-sized test tubes (20ml, 10ml, 5ml), Durham tubes
  • In addition: sterile pipettes

Preparation of the Medium

  • Prepare both single and double concentrations of the medium (either MacConkey broth or lactose broth).
  • For untreated or polluted water : Dispense the double strength media in 10 tubes (10 mL per tube) and the single strength medium in 5 tubes (10 mL per tube) and add an inverted Durham tube.
  • For treated water: Dispense the double strength medium in 5 tubes (10 mL per tube), the single strength medium in 1 bottle containing 50 mL, and add an inverted Durham tube.
  • Check the tubes to ensure that the inner vial is filled with liquid and has no air bubbles.
  • Autoclave at 15 pounds of pressure (121 degrees Celsius) for 15 minutes.

Procedure of Presumptive Test

1. For untreated (polluted) water

  1. Take 5 tubes of double concentration and 10 tubes of single concentration for each sample of water to be examined.
  2. Using a sterile pipette, add 10 mL of water to five tubes containing 10 mL of double-concentration medium.
  3. Add 1 mL of water to five tubes containing 10 mL of single-strength medium and 0.1 mL of water to five tubes containing 10 mL of single-strength media.
  4. All tubes should be incubated at 37°C for 24 hours. If no positive tubes develop, re-incubate for up to 48 hours.
  5. Compare the number of tubes exhibiting a positive reaction against a standard chart and note the quantity of bacteria present in the sample.
  6. For instance, a water sample with a result of 3–2–1 (3 10 mL positive, 2 1 mL positive, and 1 0.1 mL positive) has an MPN value of 17, indicating that the sample contains around 17 coliforms per 100 ml.

2. For treated (unpolluted) water

  1. Take 1 vial of single concentration (50 mL) and 5 vials of double concentration (10 mL) for each water sample to be examined.
  2. Add 50 mL of water to the tubes containing 50 mL of single-strength medium using a sterile pipette.
  3. Add 10 mL of water to 5 tubes holding 10 mL of double-concentration medium.
  4. At 37°C, incubate the tubes for 24 hours. If no positive tubes develop, re-incubate for up to 48 hours.
  5. Compare the number of tubes exhibiting a positive reaction against a standard chart and note the quantity of bacteria present in the sample.
  6. For instance, a water sample with a result of 1–4 (1 50 mL positive, 4 10 mL positive) has an MPN value of 16, indicating that the sample contains around 16 coliforms per 100 mL.

Presumptive Test Result

Positive

  • The development of 10% 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

  • There is no gas expansion or creation in the tube of Durham.

Confirmatory Test

It is the test that confirms the presence of coliform by analysing the positive tubes from the preliminary test. The presence of gas in the presumptive test does not necessarily indicate the presence of coliform in the water sample. Numerous additional bacteria are present in water, which can result in a false positive during a presumptive test.

The water contains yeasts and Clostridium species that can ferment lactose by creating both acid and gas. Therefore, confirming the presence of coliform in water becomes required. A confirmed test can often be conducted in two ways:

  • In an emerald-green lactose-bile broth (BGLB)
  • In eosin methylene blue agar media, bacteria are cultured (EMB)
Confirmatory Test
Confirmatory Test

Procedure 

  1. Prepare a vivid green lactose bile broth medium by combining the following ingredients:
    1. Peptone: 10 g
    2. Lactose: 10 g
    3. Bile salt: 20 g
    4. Brilliant green: 0.0133 g
    5. Distilled water: 1 L
  2. Autoclave the BGLB medium for fifteen minutes at 121 degrees Celsius.
  3. Gently shake the positive presumptive tubes.
  4. Transfer then a loopful of culture to the BGLB fermentation tube. The BGLB medium’s vivid green dye inhibits the development of gram-positive bacteria.
  5. Incubate the test tubes at 35 degrees Celsius for 48 hours.

Confirmed Test Result

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 (show no sheen). The appearance of typical colonies at high temperatures (44.5 0.2) implies that thermoresistant E. coli is present.

Negative

  • Lack of gas production in lactose broth or absence of coliform-like colonies on EMB agar.

Tryptone Water Test 

  • At (44.5 0.2°C), incubate the tryptone water for 18 to 24 hours.
  • After incubation, add approximately 0.1 mL of Kovacs reagent and thoroughly combine.
  • The presence of indole is indicated by a red hue in the Kovacs reagent, which forms a film over the medium’s aqueous phase.

a. Positive confirmatory indole, growth, and gas generation tests indicate the existence of thermotolerant E. coli.

b. In the absence of indole, growth and gas production confirm thermotolerant coliforms.

Completed Test

This test helps confirm dubious and, if required, confirmed positive test results. A typical coliform colony from an LES Endo agar plate is injected into a tube of vivid green bile broth and a nutrition agar slant. They are then incubated for 24 hours at 35°C. After 24 hours, the broth is examined for the formation of gas, and the organisms on the nutrient agar slant are stained with a Gram stain. If the organism is a Gram-negative, non-spore-forming rod that creates gas in the lactose tube, then the presence of coliforms in the water sample is confirmed.

  1. Transform a typical coliform colony from the nutrient agar slant into a tube of vivid green bile broth using Durham’s tube and a nutrient agar plate.
  2. Incubate for 24 hours at 35°C.
  3. After 24 hours, evaluate the broth for the formation of gas and Gram stain the nutrient agar slant for organisms.
Completed Test
Completed Test

Completed Test Result

Positive

  • The presence of gas in the vivid green bile broth tube and Gram-negative, non-spore-forming rods on the NA slant indicates that the test for the presence of coliform bacteria has been successfully performed, which suggests that the water sample may be contaminated with faeces.

Negative

  • No growth and no gas production in the soup. Gram staining reveals the absence of gram-negative, nonspore-producing rods.

Uses

  • It is frequently employed in the estimation of microbial populations in soils, waterways, and agricultural products.
  • The technique is especially useful for samples containing particles that interfere with plate count enumeration techniques.
  • It has also been recommended as an alternative approach to environmental monitoring trend studies.
  • It is especially useful for counting bacteria that form colonies only hesitantly on agar plates or membrane filters, but thrive in liquid media.

Advantages of  MPN

  • Easy interpretation by observation or gas emission
  • Toxin samples are diluted.
  • Method for analysing many samples, including sediments, sludge, muck, etc.

Disadvantages of MPN

  • It requires some time to achieve results.
  • Results are not particularly precise.
  • More hardware (glassware) and media are required.
  • Probability of erroneous positive results.

References

  • Williams, M. G., & Busta, F. F. (1999). TOTAL VIABLE COUNTS | Most Probable Number (MPN). Encyclopedia of Food Microbiology, 2166–2168. doi:10.1006/rwfm.1999.4000
  • Rowe R, Todd R, Waide J. Microtechnique for most-probable-number analysis. Appl Environ Microbiol. 1977 Mar;33(3):675-80. doi: 10.1128/aem.33.3.675-680.1977. PMID: 16345226; PMCID: PMC170744.
  • Sui Sien, Leong & Ismail, Johan & Denil, N.A. & Sarbini, Shahrul & Wasli, Wafri & Lingoh, Arlene. (2018). Microbiological and Physicochemical Water Quality Assessments of River Water in an Industrial Region of the Northwest Coast of Borneo. Water. 10. 1648. 10.3390/w10111648. 
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