Water Quality Analysis - 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 ideally completed in three steps:

1.      Presumptive test

2.      Confirmed test

3.      Completed test

 

Presumptive & Confirmed 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.

The gas formation in lactose fermentation tubes n the presumptive test, 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.

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.   A loopful of water sample was also streaked into Eosin methylene blue (EMB) agar plates.  The tubes and plates were incubated 37°C for 24 hours. 

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

Observation 

The production of gas in lactose broth 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 0 respectively. This shows the presence of E.coli or Enterobacter respectively. The presence of E.coli was confirmed by the appearance of dark centered small colonies having a green metallic sheen in EMB plates.

Result

The presence of gas in lactose broth and dark centered small colonies with metallic sheen in EMB plates indicates that the water sample is non-potable.  

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	0	0	0	0

 Confirmed test (Left hand side) 

Sl. No.	Colony morphology in EMB plate	Presence / absence of coliforms	Result
1	Dark centred colour colonies with green metallic sheen	Presence of coliforms	Non potable
2	Dark centred colonies with green metallic sheen	Presence of coliforms	Non potable
3	Dark centred colonies with green metallic sheen	Presence of coliforms	Non potable
4	No dark centred colonies with green metallic sheen	Absence of coliforms	Potable

 

Nitrification of organic compounds in Soil

 

Aim

To demonstrate nitrification in the given soil sample

Principle

Nitrification is the process by which ammonia (NH₃) or ammonium (NH4⁺) is converted to nitrate (NO₃⁻). Nitrification is the  result of oxidation of ammonium to nitrite (NO₂⁻) by nitrosifying or ammonia-oxidizing bacteria and oxidation of nitrite (NO₂⁻) to nitrate (NO₃⁻) by the nitrite-oxidizing bacteria. Nitrification is an important step in the nitrogen cycle in soil. The nitrate released into the soil is highly soluble and easily assimilated by photoautotrophic bacteria, algae, and plants that convert it into the amino acids needed for their  enzyme and protoplasm construction.

The chemoautotrophic bacteria such as Nitrobacter, Nitrococcus, Nitrosococcus, and Nitrosomonas  use the energy of reduced nitrogen compounds, such as ammonium and nitrite, as an energy source for the autotrophic production of organic compounds. Russian soil microbiologist Winogradsky showed that Nitrosomonas species and Nitrobacter species take part in nitrification, which is one of the essential phases of nitrogen cycle.

There are several factors influencing the growth of nitrifying bacteria in soil. Presence of good amount of organic matter in soil is important for the growth and proliferation of the nitrifying bacteria. In acid soils, nitrification is poor due to decrease in the population of nitrifying bacteria. Water logged soil deficient in oxygen are not congenial for nitrification. Similarly either too low (below 5⁰C) and too high soil temperature (above 40⁰C) is not conducive for the optimum functioning of nitrifying microorganisms. 

Materials required

Ammonium Sulfate Broth (Nitrite Forming Broth), Nitrite Broth (Nitrate Forming Broth),Trommsdorf's reagent, diphenyl amine reagent, soil sample.

Methodology

1. Tubes containing ammonium medium and nitrite medium were prepared and sterilized.

2. A loopful of soil was inoculated into the tubes. The tubes were then incubated at room temperature for 3-7 days.

3. Following incubation, test for nitrite was carried out by adding 2-3 drops of Trommsdorf's reagent to the incubated tubes and observed for colour change.

4. Similarly, the test for nitrate was carried out by adding 2-3 drops of diphenyl amine to incubated tubes and observing the colour change.

5. The results were compared with the uninoculated control and recorded.

Observation  

Ammonium sulfate broth (nitrite forming broth) was observed to see if ammonium has been converted to nitrite (NH₄⁺  --->     NO₂^-). Nitrate forming broth will be observed to see if nitrite has been converted to nitrate (NO₂^-  ---> NO₃^-).

Result

When Trommsdorf’s reagent was added to the tubes containing the ammonium medium, a blue black coloration was obtained. Similarly, when diphenyl amine reagent was added to the tubes containing nitrite medium, a blue black coloration was obtained which showed the presence of nitrifiers in the soil sample.

Detection of nitrification in soil sample   (left hand side)

Colour development	Intensity	Inference
No color	-	No nitrite/nitrate
Pale blue	+	Small amount of nitrite/nitrate
Blue	++	More nitrite/nitrate
Blue/black	+++	Large amount of nitrite/nitrate