Water Quality Analysis by Most Probable Number (MPN) technique

Aim

To determine the potability of the given water sample

Principle

The major source of human pathogens which are transmitted mainly through oral fecal route, such as those belonging to Salmonella species, Shigella species, pathogenic Escherichia coli, Vibrio cholerae, Yersinia enterocolitica, Campylobacter species, various viruses such as Hepatitis A, Hepatitis E, Rota virus and parasites such as Entamoeba histolytica and Giardia species are drinking water.  To determine the potability of drinking water it is necessary to screen the water sample for the presence of these microorganisms.  Since it is tedious to monitor for these pathogens on a regular basis, possible fecal contamination in the water body is determined by screening for the presence of indicator microorganisms. Among indicators, the coliform group of bacteria is routinely tested.  These are rod shaped, Gram-negative, non-spore forming, motile bacteria which can ferment lactose with the production of acid and gas when incubated at 35–37°C.  Escherichia coli, Enterobacter aerogenes, etc. are coliforms. Escherichia coli is the most common coliform and its presence is principally associated with fecal contamination.

Most probable number (MPN) analysis is a statistical method based on the random dispersion of microorganisms per volume in a given sample to detect fecal coliforms in a water sample.

MPN test is completed in three steps:

1.      Presumptive test

2.      Confirmed test

3.      Completed test

 

Presumptive Test

Principle

In this test, water samples are inoculated into fermentation tubes filled with a selective growth medium (lactose broth), which contain inverted Durham tubes for detection of fermentation gas. Three sets of three lactose broth tubes are inoculated with different measured amounts of the water sample and incubated at 37°C for overnight and observed for gas formation.  Presence of gas indicates a positive presumptive test and indicates the presence of coliforms in the water sample since coliforms are capable of utilizing lactose as carbon source.

Procedure

1.      Double strength and single strength lactose broth were prepared and dispersed into test tubes with Durham’s tube and sterilized.

2.    3 tubes containing 10 ml double strength lactose broth and 3 tubes containing 9 ml single strength lactose broth and 3 tubes containing 9.9 ml single strength lactose broth were set up for each water sample.

3.      Using sterile pipette 10 ml of water sample was transferred to a set of 3 tubes containing 10 ml double strength lactose broth and 1 ml water sample was transferred to a set of 3 tubes containing 9 ml single strength lactose broth and 0.1 ml water sample was transferred to set of 3 tubes containing 9.9 ml single strength lactose broth.

4.      All the tubes were incubated at 37°C for overnight

5.      After incubation the tubes were observed for gas production.

Observation and Result

The production of gas after 24-hour incubation indicates a positive presumptive test for coliform bacteria. 

MPN of bacteria present in 100 ml water can be obtained from Mc Crady's probability table or MPN table by comparing the number of positive and negative tubes observed from the 9 inoculated lactose fermentation tubes.  The MPN index of the water sample obtained by referring the standard table is tabulated in table 1.  The tubes showing positive result are retained and used for confirmed test.

MPN index for samples A, B, C and D were noted as 4, 9, 460 and 7 respectively. This shows the presence of E.coli or Enterobacter respectively. This was confirmed by further test.

 

Presumptive test (Left hand side)

Sl. No.	Tubes showing gas production			MPN Index (per 100 ml)
	Lactose broth (2x) 10 ml	Lactose broth (1x) 1 ml	Lactose broth (1x) 0.1 ml
1	1	0	0	4
2	2	0	0	9
3	3	3	1	460
4	1	1	0	7

Confirmed Test

Principle

In the presumptive test, gas formation in lactose fermentation tubes indicates that the water is unsafe to drink.  However, gas formation may also be due to some non-coliform organisms such as clostridium. Confirmed test serves to confirm the presence of coliform bacteria after either a positive or doubtful presumptive test. Eosin methylene blue agar plates are used which contains methylene blue that inhibit gram positive bacteria.  Gram negative lactose fermenters (coliforms) such as E coli produce dark centered small colonies having a green metallic sheen while Enterobacter aerogenes form large colonies and lack metallic sheen.

 A loopful of growth from a presumptive tube is also transferred into a tube of brilliant green lactose (BGLB) and incubated at 37°C for 24 hours. This is a selective medium for detecting coliform bacteria and contains a Durham tube to detect gas production.

Procedure

1.   Eosin methylene blue agar plates and BGLB tubes containing inverted Durham’s tubes were prepared.

2.  From the positive  presumptive test tubes of each water sample, a loopful of medium was transferred to BGLB tubes and also streaked into EMB agar plates.

3.      The plates and tubes were incubated 37°C for 24 hours.

4.      After incubation, tubes were observed for acid and gas production and the plates were observed for the presence of typical coliform colonies.

Observation and Result

The BGLB tubes inoculated with presumptive tubes of sample A, B, C  and D showed colour change from green to yellow which indicated acid production and presence of gas in Durham’s tubes.  These samples also showed dark centered small colonies having a green metallic sheen in EMB plates which indicated a positive confirmed test.

 

Confirmed test (Left hand side) 

Sl. No.	Colony morphology in EMB plate	Acid/Gas production in BGLB tube	Presence / absence of coliforms	Result
1	Dark centred colour colonies with green metallic sheen	Yes	Presence of coliforms	Non potable
2	Dark centred colonies with green metallic sheen	Yes	Presence of coliforms	Non potable
3	Dark centred colonies with green metallic sheen	Yes	Presence of coliforms	Non potable
4	Dark centred colonies with green metallic sheen	Yes	Presence of coliforms	Non potable

Completed Test

Principle

This is the final step in the bacteriological examination of drinking water and helps to further confirm doubtful and, if desired, positive confirmed test results. 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 acid and 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.

Procedure

1.    Single strength brilliant green lactose broth containing inverted Durham’s tubes and nutrient agar slants were prepared.

2.      The slants and tubes were inoculated with colonies obtained from confirmed test.

3.      The tubes and slants were incubated at 37°C for 24 hours.

4.      After incubation the tubes were examined for gas formation and the colonies from slants were Gram stained and observed under microscope.

Observation and Result

Gas production in Brilliant green lactose broth inoculated with colonies form EMB plates of sample A, B, C  and D  confirmed the presence of coliforms.  The presence of gram negative bacilli upon gram staining further confirmed the presence of E.coli in the given water sample.  The water sample A, B, C  and D  are not potable. 

  

Completed Test (Left hand side)

Water sample	Gas production in lactose broth	Gram staining	Result
A	Positive	Negative	Non potable
B	Positive	Negative	Non potable
C	Positive	Negative	Non potable
D	Positive	Negative	Non potable