Most probable number (MPN) technique Lecture Video :https://youtu.be/vOB9AQo54vU

1. Most probable number
(MPN) Technique
A.R. Deborah
I M.Sc. Applied Microbiology
Scared heart college (Autonomous)
Tirupattur

2. Water
Water is the essential part of life. There are
thousands of microorganisms which live in water and
transported through it.
People affected due to bacterial infectious diseases
transmitted through water (eg:cholera, typhoid fever
and bacillary dysentery).
Coliform bacteria (Escherichia coli) strain is the
indicator organism used to indicate faecal
contamination and water analysis is mainly based on
the concept of faecal indicator bacteria.

3. Most Probable Number
 Most Probable Number (MPN) is a method used to estimate the
concentration of viable microorganisms in a water sample.
 MPN is most commonly applied for quality testing of water i.e to
ensure whether the water is safe or not.
 In drinking water, the presence of very few faecal coliform bacteria
would indicate that a water probably contains no disease- causing
organisms.
 while the presence of large numbers of faecal coliform bacteria
would indicate a very high probability that the water could contain
disease -producing organisms making the water unsafe for
consumption.

4. Principle
 The most probable number (MPN) is a statistical method used to estimate
the viable numbers of bacteria in a sample by inoculating broth in 10-fold
dilutions and is based on the principle of extinction dilution.
 The coliform groups comprises in all aerobic and for cultivating anaerobic
Gram negative non sporulating, rod shaped bacteria that ferment lactose
with gas formulating within 48 hours at 35 °C coliform includes bacteria of
most common internal organism.
 Coliform bacterial densities can be determined by either Multiple tube
fermentation technique (MPN Test) by Membrane filter (MF) procedure.
The Multiple tube fermentation technique providing the Most portable
number (MPN) is an Indirect count technique relying on statistical
interpretation of growth. The test was conducted in three steps such as
Presumptive test, Confirmatory test and Complete test.

5. Materials Required
a) Brilliant Green Lactose Broth
b) Eosin Methylene Blue (EMB) agar
c) Gram staining kit
d) Erlenmeyer flask
e) Pipettes
f) Test tubes
g) Durham’s tube
h) Petridishes
i) Other lab wares

6. Presumptive test - procedure
 Distribute 10 ml Single strength lactose broth (SSLB) each to 10 test tubes
and Double distilled strength Lactose broth (DSLB) to 5 test tubes.
 Add the Durham's tube in inverted position and sterile them at a time.
 After autoclave, Inoculate the 0.1 ml of water sample to each 5 tubes of
SSLB, 1 ml of water sample to each 5 tubes of SSLB tubes and 10 ml of
water sample in 5 tubes of DSLB correspondingly.
 Incubate the tubes at 37 °C and examine the gas formation in Durham’s
tubes at 24 hours (positive test) or not 48 hours (doubt full tests) proceed
to confirmed.
 Compare the number of tubes giving positive reaction to a standard chart
and record the number of bacteria present in it.

7. For example: A water sample tested shows a result of 4–2–0 (4× 10
ml positive, 2 × 1 ml positive, 0 × 0.1 ml positive) gives an MPN value
is 22,
i.e. the water sample contains an estimated 22 Coliforms per 100 ml.

9. Confirmative test - Procedure
Take a loopful culture from the Lactose broth tube from
the highest dilution that still showed positive test and
streaks it on EMB agar plate.
Incubate the plates at 37 °C for 24 hours.
Observe for the typical Escherichia coli colonies showing
greenish metallic sheen.

10. Complete test
 Pick up a suspicious colony (Escherichia coli) from EMB agar plate, make a smear and
conduct Gram staining.