Most probable number

(MPN)
Most probable number (MPN)
► Most probable number (MPN) analysis is a statistical method based on the
random dispersion of microorganisms per volume in a given sample.
► It is commonly used in estimating microbial populations in soils, waters,
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
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 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.
Procedure
1. Prepare medium (either MacConkey broth or lactose broth) in single and double strength
concentration.
2. Take three sets of test tubes containing five tubes in each set ;one set with 10 ml of double
strength (DS) other two containing 10 ml of single strength (SS) and add Durham tube in
inverted position
3. Make sure that the Durham tube are full of liquid and no air bubble
4. Using a sterile pipette add 10 mL of water to 5 tubes containing 10 mL double strength
medium.
5. 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.
6. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs.
7. After incubation , observe the gas production in Durham’s tube and color change of the media.
8. Compare the number of tubes giving a positive reaction to a standard chart and record the
number of bacteria present in it.
► MPN index, which is the estimated number of coliforms per
100mL of water. An MPN index below 2 is considered to be
drinkable. Using the numbers from before, 5, 2, and 0, this
water sample has an MPN index of 50. This sample isn't
safe to drink.
Double strength Single strength Single strength
Result
► Positive: The formation of 10% gas or more in the Durham tube
within 24 to 48 hours, together with turbidity in the growth medium
and the colour change (red to yellow) and hence for the possibility
of coliform bacteria and fecal pollution.
• The test is presumptive only, because under these conditions several
other types of bacteria can produce similar results.
► Negative: No growth or formation of gas in the Durham’s tube.
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:
1. 3 mL brilliant green lactose fermentation tube, add and add Durham tube in inverted
position
► Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas
formation after 24 hours. If no gas production is seen, further incubate up to a
maximum of 48 ±3 hours to check gas production
2. A plate of Eosin Methylene Blue (EMB) agar is streaked with a
loopful of growth from a positive tube, and incubated at 37°C for 24
hours. Typical coliform bacteria (E. coli and Enterobacter aerogenes)
exhibit good growth on this medium and form red to black colonies with
with dark centers or a sheen
Result
► Positive: Formation of gas in lactose broth
► the demonstration of a coliform-like colony on the EMB agar
► Coliforms produce colonies with greenish metallic sheen which
differentiates it from non-coliform colonies (show no sheen.
► Negative: The absence of gas formation in lactose broth or the
failure to demonstrate coliform-like colonies on the EMB agar.
Completed Test
► Since some of the positive results from the confirmatory test may be false, it is desirable
to do completed tests.
► A typical coliform colony from an EMB 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 37°C for 24 hours.
► After 24 hours, the broth is checked for the production of gas in
lactose tube
► Gram stain is made from organisms on the nutrient agar slant.
► Result
► If the organism is a Gram-negative, nonspore-forming rod and
produces gas in the lactose tube, then it is positive that coliforms are
present in the water sample
• ADV
• 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 analysed by membrane filtration.
• LIMITATIONS
• Poor accuracy and precision associated with MPN counts usually
means 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.