The document discusses the Most Probable Number (MPN) technique used to check water quality by testing for the presence of fecal coliform bacteria. The MPN technique involves a three step process: 1) A presumptive test where water samples are added to lactose broth tubes in different volumes and observed for gas formation, 2) A confirmatory test where cultures are streaked on EMB agar plates and observed for E. coli colonies, and 3) A complete test involving Gram staining of colonies to confirm the presence of E. coli. The number of positive lactose broth tubes corresponds to an MPN value estimate of the number of coliform bacteria per 100ml of water.
3. WATER
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Water is an essential part of life. There are thousands of
microorganisms that live in water and are transported through it.
People are affected due to bacterial infectious diseases
transmitted through contaminated 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.
4. MOST PROBABLE NUMBER
Most Probable Number (MPN) is a method used to check
the water.
MPN is most commonly applied for quality testing of water.
In drinking water, the presence of very few faecal coliform
bacteria would indicate that 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.
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5. PRINCIPLE
The most probable number (MPN) is a statistical method.
The coliform groups comprise all aerobic and cultivating anaerobic
bacteria.
Coliform bacterial densities can be determined by either the Multiple
tube fermentation technique (MPN Test). 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 Presumptive test,
Confirmatory test, and Complete test.
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6. MATERIALS REQUIRED
Brilliant Green Lactose Broth
Eosin Methylene Blue (EMB) agar
Gram staining kit
Erlenmeyer flask
Pipettes
Test tubes
Durham’s tube
Petri dishes
Other lab wares
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7. 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.
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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) giving an MPN value is 22,
i.e. the water sample contains an estimated 22 Coliforms per 100 ml.
11. CONFIRMATIVE TEST - PROCEDURE
Take a loopful culture from the Lactose broth tube from the highest
dilution that still showed a positive test and streaks it on the EMB agar
plate.
Incubate the plates at 37 °C for 24 hours.
Observe the typical Escherichia coli colonies showing a greenish
metallic sheen.
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