Most Probable Number (MPN) Test for Water (Viable Cells)

Theory

The routine microbial examination of water to determine its portability is not based on the isolation and identification of pathogenic microorganisms but based on finding a microorganism whose presence indicates that water has been contaminated with an intestinal origin, and therefore the possibility of the presence of pathogenic microorganisms.

The fecal contamination of drinking water is evaluated by using coliform bacteria as an indicator. An indicator organism is an organism that can be used as an indication of pollutions.

Coliforms ferment lactose within 48 hours and produce lactic acid and carbon dioxide.

Lactose -------->  Lactic acid  + carbon dioxide

In the coliform test, determining the sanitary quality of water involves 4 stages such as;

• Presumptive test
• Confirmed test
• Completed test
• Positive completed test

In this lab presumptive test is carried out. In the presumptive test, the indicator is phenol red. The color changes from red to yellow. In the basic medium, phenol red is red whereas in the acidic medium it is yellow.

Procedure

First two serial dilutions 10^-1, 10^-2 of water samples provided, were prepared. Next 1ml aliquots were transferred (each from 10⁰, 10^-1, 10^-2 tubes) into the lactose broth in Durham fermentation tubes. This was done in duplicates. Finally, the tubes were incubated at 35⁰C for 48 hours.

Observation

Sample	No of positive tubes						CFU/ml	CFU/100ml
	100		10-1		10-2
	1	2	1	2	1	2
Tap water	0	0	0	0	0	0	0	0
Tubewell water	0	1	0	1	0	1	2	2*100 = 200
Rain water	1	1	1	1	0	1	70	70*100 =7000
Bottled water	0	0	0	0	0	0	0	0
Well water	1	1	1	1	0	1	70	70*100 =7000
River water	1	1	1	1	1	0	70	70*100 =7000
Drain water	1	1	1	1	1	1	100	100*100 =10000
Stream water	1	1	1	1	1	1	100	100*100 =10000

Discussion

The presence of specific indicator organism signals that a given water sample is contaminated with pathogens. The most widely used indicator for microbial water contamination is the coliform group. Coliforms are used as indicators of water contaminations because many of them inhibit the intestine of humans and other animals in large numbers. Thus their presence in water indicates fecal contamination. Therefore most probable number method can be used to detect and enumerate coliforms in water.

Lactose fermentation after 48 hours at 35⁰C indicates a positive presumptive test for the presence of coliforms in the sample. In this practical, the indicator is phenol red. In a neutral medium, phenol red gives an orange color while it gives a yellow color in acidic medium. Hence the color changes from orange to yellow shows that the medium has changed from neutral to acidic. That indicates the formation of lactic acid due to lactose fermentation. The gas bubble in the fermentation tube also indicates the lactose fermentation. Gas bubble is carbon dioxide. By referring to a statistical table no of coliforms per 100ml can be obtained (MPN Table).

According to the above results, the highest number of colony forming unit (CFU)/100ml was shown by drain water and stream water. Stream water flows through various areas and also polluted water from industries which are coming from drainages used to flow into streams. Hence both drain and stream water are highly polluted. Thus the coliform contaminations may be higher, due to the exposure to various situations. Next highest is well water, river water, and rainwater. Well water does not flow. River water flows but does not expose to various areas as the stream water. This is why CFU of river water does not exceed the CFU of stream water. Although rainwater is exposed it does not contaminate with pollutants. But CFU value of rainwater has a high value. It may be due to instrumental or experimental errors. This can occur due to unclean containers or when counting a deformed gas bubble may be counted. Because when the gas bubble is very large the bottom of the gas bubble is difficult to identify. Both tap and bottled water have zero values, which indicates these two samples are not contaminated with coliforms. Since tap water is purified before distributing to houses it is already checked for coliforms. Only if it is free from coliforms the distribution is done. Bottled water is also purified and sterilized.

For the success of the results, it is important to use sterile containers to collect water samples. And a wide-mouthed, sterile, glass-stoppered bottles can be used. When collecting tap water, let the water to flow initially and then take the sample, whereas, in a stream, water is collected below the water surface. If transportation is a necessary water sample is packed in ice. Otherwise, due to environmental conditions, the activity of the organism may change. To inhibit further activities of coliforms, after 48 hours the samples should place in a refrigerator.

According to the national water and drainage board, total coliforms equal to 10 while fecal coliforms equal to 0, are acceptable values in drinking water /100ml. These values can vary depending on the country.

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