Most probable number (MPN) method

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 (e g: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 Brilliant Green Lactose Broth Eosin Methylene Blue (EMB) agar Gram staining kit Erlenmeyer flask Pipettes Test tubes Durham’s tube Petridishes 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 C oliforms per 100 ml.

8. 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.

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