Most Probable Number (MPN) Test: Principle, Procedure, Results • Microbe Online (2022)

By Nisha Rijal•Updated: 05/04/22•7 min read

Bacteriology

Most Probable Number (MPN) is used to estimate the concentration of viable microorganisms in a sample by means of replicating liquid broth growth in ten-fold dilutions. It is commonly used in estimating microbial populations in soils, waters, and agricultural products. MPN test is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods.

MPN is most commonly applied for quality testing of water i.e to ensure whether the water is safe or not in terms of bacteria present in it. A group of bacteria commonly referred to as fecal coliforms act as an indicator of fecal contamination of water. The presence of very few fecal coliform bacteria would indicate that water probably contains no disease‑causing organisms, while the presence of large numbers of fecal coliform bacteria would indicate a very high probability that the water could contain disease‑producing organisms making the water unsafe for consumption.

(Video) Most Probable Number (MPN) - Microbiology

Table of Contents

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.

MPN test is performed in 3 steps

  1. Presumptive test
  2. Confirmatory test
  3. Completed test

Presumptive test

The presumptive test is a screening test to sample water for the presence of coliform organisms.

If the presumptive test is negative, no further testing is performed, and the water source is considered microbiologically safe.

(Video) MPN Most Probable Number Water Quality Testing: Microbiology

If the presumptive test is negative, no further testing is performed, and the water source is considered microbiologically safe. If, however, any tube in the series shows acid and gas, the water is considered unsafe and the confirmed test is performed on the tube displaying a positive reaction.

The method of the presumptive test varies for treated and untreated water.

Requirements

  • Medium: Lactose broth or MacConkey broth or Lauryl tryptose (lactose) broth
  • Glasswares: Test tubes of various capacities (20ml, 10ml, 5ml), Durham tube
  • Others: Sterile pipettes

Preparation of the Medium

  • Prepare medium (either MacConkey broth or lactose broth) in single and double strength concentrations.
  • For untreated or polluted water :
    • Dispense the double strength medium in 10 tubes (10mL in each tube) and single strength medium in 5 tubes (10 mL in each tube)and add a Durham tube in an inverted position.
  • For treated water:
    • Dispense the double strength medium in 5 tubes (10mL in each tube) and 50 mL single strength medium in 1 bottle and add a Durham tube in an inverted position.
  • Examine the tubes to make sure that the inner vial is full of liquid with no air bubbles.
  • Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Procedure of MPN test

For untreated (polluted) water
  1. Take 5 tubes of doublestrength and 10 tubes of singlestrength for each water sample to be tested.
  2. Using a sterile pipette add 10 mL of water to 5 tubes containing 10 mL double strength medium.
  3. Similarly, add 1 mL of water to 5 tubes containing 10 mL single strength medium and 0.1 mL water to the remaining 5 tubes containing 10 mL single strength medium.
  4. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs.
  5. Compare the number of tubes giving a positive reaction to a standard chart and record the number of bacteria present in it.
    For example, a water sample tested shows a result of 3–2–1 (3 × 10 mL positive, 2 × 1 mL positive, 1 × 0.1 mL positive) gives an MPN value of 17, i.e. the water sample contains an estimated 17 coliforms per 100 ml

To view the full table download the PDF file from the link given in the reference.

For treated (unpolluted) water
  1. Take 1 tube of single strength (50mL) and 5 tubes of double strength (10mL) for each water sample to be tested.
  2. Using a sterile pipette add 50 mL of water to the tubes containing 50 mL single strength medium.
  3. Similarly, add 10 mL of water to 5 tubes containing 10 ml double strength medium.
  4. Incubate the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs.
  5. Compare the number of tubes giving a positive reaction to a standard chart and record the number of bacteria present in it.
    For example, a water sample tested shows a result of 1-4 (1 × 50 mL positive, 4 × 10 mL positive) gives an MPN value of 16, i.e. the water sample contains an estimated 16 coliforms per 100 mL.

Confirmatory Test

Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to confirm the presence of coliform, a confirmatory test is done.

(Video) Most Probable Number (MPN) Method

From each of the fermentation tubes with positive results transfer one loopful of medium to:

  1. 3 mL lactose-broth or brilliant green lactose fermentation tube,
  2. to an agar slant and
  3. 3 mL tryptone water.

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.

The agar slants should be incubated at 37°C for 24± 2 hours and Gram-stained preparations made from the slants 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 group in the 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 (absence of coliforms in the tested sample).

Tryptone Water Test

  1. Incubate the tryptone water at (44.5 ±0.2°C) for 18-24 hours
  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 over the aqueous phase of the medium.

a. Confirmatory tests positive for indole, growth, and gas production show the presence of thermotolerant E. coli.
b. Growth and gas production in the absence of indole confirm thermotolerant coliforms.

(Video) Most Probable Number: How to perform the test for microbial analysis

Completed Test

Since some of 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 EMB or Endo agar.

In this process, a loopful of a sample from each positive BGLB tube is streaked onto selective medium like Eosin Methylene Blue agar or Endo’s medium. One plate each is incubated at 37°C and another at 44.5± 0.2°C for 24 hours.

High temperature incubation (44.5 ±0.2) is for detection of thermotolerant E.coli.

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

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

Advantages of MPN

  • Ease of interpretation, either by observation or gas emission
  • Sample toxins are diluted
  • Effective method of analyzing highly turbid samples such as sediments, sludge, mud, etc.
  • that cannot be analyzed by membrane filtration.

Disadvantages of MPN

  • It takes a long time to get the results
  • Results are not very accurate
  • Requires more hardware (glassware) and media
  • Probability of false positives

References and further reading

  1. WHO:Multiple Tube Method for Thermotolerant (Faecal Coliform)

Most Probable Number (MPN) Test: Principle, Procedure, Results • Microbe Online (5)

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

(Video) Most Probable Number MPN Method for Coliform Detection in Water and Food samples

FAQs

What is the result of the MPN test? ›

The MPN confirmation procedure is carried out by transferring positive presumptive tubes (gas production within 24–48 h) to 2% brilliant green bile lactose broth to test for gas production within 48 h at 35 °C for total coliforms and at 44.5 °C for fecal coliforms. Positive tubes are used to calculate the MPN.

What is the MPN test explain where it is used? ›

Most Probable Number (MPN) is used to estimate the concentration of viable microorganisms in a sample by means of replicating liquid broth growth in ten-fold dilutions. It is commonly used in estimating microbial populations in soils, waters, and agricultural products.

What Is the Most Probable Number test MPN meaning and in which test we are using it? ›

The Most Probable Number (MPN) or multi-tube method (McCrady)1 is a statistical method used to estimate the number of viable cells of a particular microorganism in a sample, most commonly food or water. It is used when samples contain too few bacteria to provide reliable viable cell numbers by classical plate count.

What does most probable number MPN mean? ›

The most probable number (MPN) is the number of organisms that are most likely to have produced laboratory results in a particular test.

What is an acceptable MPN value? ›

According to the World Health Organization, a zero count of E. coli per 100 ml of water is considered safe for drinking. A count of 1–10 MPN/100 ml is regarded as low risk; 11–100 MPN/100 ml is medium risk.

What indicates a positive tube in MPN coliform test? ›

The inoculated tubes are incubated for 48 + 3 hr at 35 + 0.5EC. Formation of gas at any time in the tube indicates a positive confirmed test. 2.1. 3 Completed Test: The completed test is performed on all samples showing a positive result in the confirmed test.

What do you mean by MPN in microbiology? ›

A statistical estimate of the number of coliform-group organisms per unit volume of sample water. Expressed as a density or population of organisms per 100 mL of sample water.

What is MPN analysis? ›

The most probable number (MPN) analysis is a statistical method based on the random dispersion of microorganisms per volume in a given sample. In this method, measured volumes of water are added to a series of tubes containing a liquid indicator growth medium.

How is the number of coliforms determined in the MPN method? ›

All the positive test tubes are now confirmed for presence of coliforms. Finally the number of bacteria present in water sample is determined from previous MPN chart. Alternatively number of coliforms can also be calculated by the formula; Coliforms/100ml = numbers of positive tubes.

What is the acceptable level of coliform bacteria per 100 ml of drinking water? ›

The Maximum Contaminant Level (MCL) for bacteria in drinking water is zero total coliform colonies per 100 milliliters of water as established by the EPA.

How do you read coliform results? ›

Interpreting Your Well Water Test Results
  1. Total Coliform: 0–5. E.coli: 0. ...
  2. Total Coliform: > or = 6. E.coli: 0. ...
  3. E.coli: 1 or higher. Interpretation: Your water is unsafe for drinking unless boiled.
  4. Total Coliform: est (estimate) E.coli: est (estimate) ...
  5. Total Coliform: o/g (overgrown) E.coli: o/g (overgrown)

Is MPN test is used to detect E. coli? ›

MPN - Completed test for E. coli. To perform the completed test for E. coli, gently agitate each gassing EC tube, remove a loopful of broth and streak for isolation on a L-EMB agar plate and incubate for 18-24 h at 35°C ± 0.5°C .

Why is an MPN an estimate rather than an accurate number? ›

Because it is not practical (nor meaningful) to determine the relative size of cells in a microbial population. If a bacterium has a generation time of 30 minutes, and you start with 100 cells at time 0, how many cells will you have in 30, 60, 90, and 120 minutes?

Is the MPN procedure qualitative or quantitative? ›

It is actually a qualitative test rather than quantitative indicating only the presence of coliforms, not their numerical presentation. This test is carried out in three continuous stages: presumptive test, confirmed test and completed test through which the presence of indicator organism E.

Can coliform bacteria make you sick? ›

Most coliform bacteria are not harmful. However, some can make you sick. A person that has been exposed to these bacteria may have an upset stomach, vomiting, fever, or diarrhea. Children and the elderly are more at risk from these bacteria.

What is a high coliform count? ›

They are a group of closely related, mostly harmless bacteria that live in soil and water as well as the gut of animals. Coliforms count is a hygienic indicator and high level of coliform counts generally indicates unsanitary condition or poor hygiene practices during or after food production.

How do you convert MPN to CFU? ›

Popular Answers (1) The simple answer is that they are equivalent- one MPN is equal to one CFU. Both units measure the estimated number of bacteria in a water sample.

What is the difference between E. coli and coliform bacteria? ›

The main difference between E coli and coliform is that the E. coli are a type of bacteria; that is, a fecal coliform whereas the coliform is a bacterium involved in the fermentation of lactose when incubated at 35–37°C. The other type of coliform bacteria is non-fecal coliforms that are Enterobacter and Klebsiella.

What is the acceptable level of coliform in food? ›

coli. It appeared that a limit of 10 coliform organisms per g as a suggested standard could be met with several types of foods.

What is the MPN index? ›

MPN Index means the most probable number of coliform organisms in a given volume of sewage as determined by statistical estimation; Sample 1Sample 2. MPN Index means the most probable number of coliform organisms in a given volume of wastewater which, in. Sample 1.

What is MPN on a product? ›

Use the MPN [mpn] attribute to submit your product's Manufacturer Part Number (MPN). MPNs are used to uniquely identify a specific product among all products from the same manufacturer.

Which is the dye is used in the 1st step of MPN test? ›

Shake the positive presumptive tubes gently. Then transfer a loopful of culture into the BGLB fermentation tube. The brilliant green dye in the BGLB medium inhibits the growth of gram-positive bacteria. Incubate the test tubes for 48 hours at 35 degrees Celsius.

What is the temperature at which MPN test is performed? ›

Explanation: MPN test is performed at 37oC where different samples of water are mixed with lactose broth and incubated in test tubes for 48 hours.

Is MPN equal to CFU? ›

MPN (most probable number) is equivalent to CFU (colony forming units). The use of either MPN or CFU is based on the method used to detect bacteria, and both are valid measures of bacteria density.

How can we check the purity of a pure culture? ›

1: be sure that one colony has been selected when streaking on the appropriate media. 2: perform a Gram stain. 3: perform other tests required to classify the organism in the corresponding category.

What is the necessity of confirmatory test during MPN test? ›

The purpose of confirmatory test is to confirm the presence of coliform bacteria in the water sample. Brilliant green lactose bile broth fermentation tubes are used for the confirmed phase.

What is the normal MPN count for potable water? ›

This EPA recreational water quality standard requires less than 126 MPN (or CFU) of generic E. coli per 100 ml water. For well water, some guidelines use 2.2 MPN total coliform per 100 ml water as a threshold above which the well should be treated.

What is the microbial limit for drinking water? ›

The most common allowable bacterial numbers used by health departments, water-supply agencies, and local jurisdictions vary from 100/ml to 500/ml of colony-forming units.

What is a high E. coli count in urine? ›

For that reason, up to 10,000 colonies of bacteria/ml are considered normal. Greater than 100,000 colonies/ml represents urinary tract infection. For counts between 10,000 and 100,000, the culutre is indeterminate. Sensitivity refers to the antibiotics tested to be effective in stopping the bacteria.

What happens if your well tests positive coliform? ›

Although total coliforms can come from sources other than fecal matter, a positive total coliform sample should be considered an indication of pollution in your well. Positive fecal coliform results, especially positive E. Coli results, should be considered indication of fecal pollution in your well.

What to do if well tests positive for bacteria? ›

What to do if Your Water Tests Positive for Coliform Bacteria
  1. Use boiled or bottled water or water from a safe alternative source for cooking and drinking until you address the issue. ...
  2. Disinfect your well with a chlorine solution. ...
  3. Test your well water again after disinfection to confirm there are no coliform bacteria.

What are good water test results? ›

ACCEPTABLE RESULTS: Hard water is beneficial to health. However, values near 150 mg/l are ideal from an aesthetic viewpoint, if the corrosivity index is satisfactory.

How do you test for E. coli Microbiology? ›

To diagnose illness caused by E. coli infection, your doctor sends a sample of your stool to a laboratory to test for the presence of E. coli bacteria. The bacteria may be cultured to confirm the diagnosis and identify specific toxins, such as those produced by E.

Which test is used to detect E. coli? ›

In the present work, for the first time, we have developed a litmus test using Whatman blotting paper that can be used to detect the E. coli bacteria in water samples. This kind of litmus paper strips are very useful in water quality testing to determine whether the water being tested is safe to drink or not.

How do you identify coliform bacteria? ›

Depending on the media used, the color of the agar plate will help indicate whether coliform are present in the sample: MacConkey agar will turns pink and cloudy indicating the presence of coliforms that ferment lactose. Eosin methylene blue agar will show a metallic green sheen in the presence of coliforms.

Is most probable number accurate? ›

7.4.

The MPN is only effective of the examination of bacteria, as it does not provide reliable results for the enumeration of fungi. To determine the accuracy and sensitivity of the test methods used for microbial limit testing, 10 g or 10 mL samples of the test material are examined.

How will you determine positive result in MPN test? ›

The MPN confirmation procedure is carried out by transferring positive presumptive tubes (gas production within 24–48 h) to 2% brilliant green bile lactose broth to test for gas production within 48 h at 35 °C for total coliforms and at 44.5 °C for fecal coliforms. Positive tubes are used to calculate the MPN.

What is the importance of MPN test in the determination of water potability? ›

Usually MPN method is used to detect coliform bacteria in water or food. MPN method for coliforms is a three-step process and all the media used are liquid media. The MPN of total coliforms in potable (drinking) water is based on the World Health Organization definition of total coliforms.

Why is double strength broth used in MPN? ›

Double-strength broth used in some tubes of the mpn series because in single strength 10 ml of sample is mixed with 10 ml of broth which will get diluted due to which growth will not be accurate but in double strength, due to high concentration all the cell will get proper nutrient and thus,give positive and accurate ...

What does MPN per 100ml mean? ›

A statistical estimate of the number of coliform-group organisms per unit volume of sample water. Expressed as a density or population of organisms per 100 mL of sample water.

What is the normal MPN count for potable water? ›

This EPA recreational water quality standard requires less than 126 MPN (or CFU) of generic E. coli per 100 ml water. For well water, some guidelines use 2.2 MPN total coliform per 100 ml water as a threshold above which the well should be treated.

How do you read coliform results? ›

Interpreting Your Well Water Test Results
  1. Total Coliform: 0–5. E.coli: 0. ...
  2. Total Coliform: > or = 6. E.coli: 0. ...
  3. E.coli: 1 or higher. Interpretation: Your water is unsafe for drinking unless boiled.
  4. Total Coliform: est (estimate) E.coli: est (estimate) ...
  5. Total Coliform: o/g (overgrown) E.coli: o/g (overgrown)

What is the necessity of confirmatory test during MPN test? ›

The purpose of confirmatory test is to confirm the presence of coliform bacteria in the water sample. Brilliant green lactose bile broth fermentation tubes are used for the confirmed phase.

What is a safe coliform level? ›

The Maximum Contaminant Level (MCL) for bacteria in drinking water is zero total coliform colonies per 100 milliliters of water as established by the EPA. The total coliform test is the basic yardstick for determining the biological quality in a water supply.

What is the acceptable level of coliform bacteria per 100 mL of waste water? ›

coli) is none detectable per 100 mL. Maximum Acceptable Concentration for Drinking Water = none detectable per 100 mL This means that in order to conform to the guideline: • For every 100 mL of drinking water tested, no total coliforms or E. coli should be detected.

What is the acceptable level of coliform bacteria per 100 mL of recreational water? ›

Recreational waters that are not designated beaches should not have more than 406 E. coli/100 mL in any one sample, or more than 126/100 mL in a 60-day, three-sample geometric mean average. Occasional higher numbers are not unusual, particularly after storm events and where urban or agricultural runoff occurs.

How do you convert MPN to CFU? ›

Popular Answers (1) The simple answer is that they are equivalent- one MPN is equal to one CFU. Both units measure the estimated number of bacteria in a water sample.

What is the MPN index? ›

MPN Index means the most probable number of coliform organisms in a given volume of sewage as determined by statistical estimation; Sample 1Sample 2. MPN Index means the most probable number of coliform organisms in a given volume of wastewater which, in. Sample 1.

What happens if you drink water with coliform bacteria? ›

Most coliform bacteria are not harmful. However, some can make you sick. A person that has been exposed to these bacteria may have an upset stomach, vomiting, fever, or diarrhea. Children and the elderly are more at risk from these bacteria.

What does a positive result for coliform mean? ›

What does a positive coliform test result mean? A positive coliform test means possible contamination and a risk of waterborne disease. A positive test for total coliforms always requires more tests for fecal coliforms or E. coli.

What happens if your well tests positive coliform? ›

Although total coliforms can come from sources other than fecal matter, a positive total coliform sample should be considered an indication of pollution in your well. Positive fecal coliform results, especially positive E. Coli results, should be considered indication of fecal pollution in your well.

What is considered a high total coliform count? ›

Sometimes, coliform bacteria results are reported as "TNTC" (too numerous to count) or "confluent." TNTC means that the bacteria concentration was so high that it could not be counted (generally higher than 200 colonies per 100 mL).

Which media is used in completed test for MPN analysis? ›

It is a final test in which a loopful of sample from positive confirmatory tubes is streaked on Eosin methylene blue agar or M-endo agar and incubated for 24 hours. Three types of colonies are obtained in culture media; Typical colony: they are pink colored with greenish metallic appearance or nucleated colony.

What is MPN analysis? ›

The most probable number (MPN) analysis is a statistical method based on the random dispersion of microorganisms per volume in a given sample. In this method, measured volumes of water are added to a series of tubes containing a liquid indicator growth medium.

What is the temperature at which MPN test is performed? ›

Explanation: MPN test is performed at 37oC where different samples of water are mixed with lactose broth and incubated in test tubes for 48 hours.

Videos

1. Microbiological Examination of Water -MPN
(Sharrel Rebello)
2. Presence Absence and MPN testing for Coliform Bacteria
(TheWaterSifu)
3. Asst.Prof.P.K.Gakkhad: Most Probable Number(MPN)- Presumptive Test (SYBSc Microbiology MB222) Lec:29
(KJS UG MICROBIOLOGY)
4. MPN test for Water in detail by IES Vishnu Maurya
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5. Total Coliforms
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6. BACTERIOLOGICAL TEST | MPN TEST| E COLI | COLIFORM BACTERIA | MICROBIOLOGY| BACTERIOLOGICAL ANALYSIS
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