Most Probable Number (MPN) Test

• 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 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
• Presumptive test
• Confirmatory test
• 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, 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.
• 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.
Take 5 tubes of double strength and 10
tubes of single strength for each water
sample to be tested.

Using a sterile pipette add 10 mL of

water to 5 tubes containing 10 mL
double strength medium.

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.
Incubate all the tubes at 37°C for 24 hrs.
If no tubes appear positive re-incubate
up to 48 hrs.
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
 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.
• From each of the fermentation tubes with positive results transfer
one loopful of medium to:
• 3 mL lactose-broth or brilliant green lactose fermentation tube,
• to an agar slant and
• 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.
• A plate of LES Endo agar (or EMB agar) is streaked
with a loopful of growth from a positive tube and
incubated at 35°C for 18–24 hours.
• Typical coliform bacteria (E. coli and Enterobacter
aerogenes) exhibit good growth on this medium and
form red to black colonies with dark centers or a
sheen.
• Salmonella typhi exhibits good growth but the
colonies are colorless.
• S. aureus growth is inhibited altogether.
 Completed Test

• This test helps to further confirm doubtful and, if desired,

positive confirmed test results.
• A typical coliform colony from an LES Endo agar plate is
inoculated into a tube of brilliant green bile broth and on the
surface of a nutrient agar slant. They are then incubated at
35°C for 24 hours.
• After 24 hours, the broth is checked for the production of gas,
and a Gram stain is made from organisms on the nutrient agar
slant.
• If the organism is a Gram-negative, non-spore-forming rod and
produces gas in the lactose tube, then it is positive that
coliforms are present in the water sample.
• Objectives of Most Probable Number (MPN)
• To enumerate the number of bacteria present
in the drinking water by the MPN method.
• To identify the bacteria present in the drinking
water sample.
• Advantages of Most Probable Number (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.
• Permits samples that cannot be analyzed by
membrane filtration.
• Limitations of Most Probable Number (MPN)
• Poor accuracy and precision associated with
MPN count usually mean that the method is
one of last resort — to be considered only
when other counting methods are
inappropriate.
• Laborious and expensive in terms of materials,
glassware, and incubator space.
• It has relatively a large margin of error.