Most Probable Number (MPN) Test: Principle, Procedure, Results
Most Probable Number (MPN) is a method used to estimate the concentration of viable
microorganisms in a sample by means of replicate liquid broth growth in ten-fold dilutions. It
is commonly used in estimating microbial populations in soils, waters, agricultural products
and 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.
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.
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 concentration.
▪ 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
MPN Water Testing
1. Take 5 tubes of double strength and 10 tubes of single strength 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
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.
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 positive
