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Potable (drinking) water has great importance. Water is polluted by several waste materials and are responsible for infections like dysentry, typhoid, cholera etc. Analysis of water samples on a routine basis is not possible for each pathogen. Hence water is examined to detect Escherichia coli which indicates faecal contamination since this bacterium is always present in human intestines.
There are three basic tests to detect coliform bacteria in water: 1. Presumptive Test 2. Confirmed Test 3. Completed Test.
These tests are sequentially performed on each sample of water. They detect the presence of coliforms (faecal contaminants), the Gram negative, non-spore forming bacilli that ferment lactose and produce gas and acid when the samples are incubated for 24-48 hours at 37°C.
Part 1: Presumptive Test:
Principle:
Coliforms (not other enteric organisms) use lactose as carbon and energy source and produce acid and gas.
Requirements:
1. Water samples from sewage plant, pond and tap.
2. Lactose fermentation broth 1 x (and 2x) (PH 69).
Beef extract 3.0 g
Peptone 5-0 g
Lactose 5-0 g
Distilled water 1000.0 ml
For 2x broth use double the concentration of ingredients.
Double strength lactose fermentation broth (LB 2x)-15 tubes.
Single strength lactose fermentation broth (LB 1x)-30 tubes both with Durhams tubes.
3. Test tubes 45.
4. Test tube rack.
5. 10 ml pipettes.
6. 1 ml pipettes.
7. 0.1 ml pipettes.
8. Mechanical pipetting device.
9. Bunsen Flame.
10. Glass marking pencil.
11. Incubator.
12. Laminar clean air flow hood.
Procedure:
1. Set up three series of tubes each having three groups. Label them with water source and volume of sample inoculated.
2. Collect water samples and keep them ready:
3. Sewage, pond and tap water samples are transferred into the series of labelled tubes as shown above with separate sterile pipettes and incubate all tubes for 48 hours at 37°C.
Production of yellow colour and gas in Durhams tubes is indicative of lactose fermentation and is presumptive evidence of the presence of coliforms in the sample. The most probable number (MPN) of coliforms can be estimated by determining the number of tubes in each group that show gas formation.
Part 2: Confirmed Test:
In water samples where presumptive test was positive, confirmation of the presence of coliforms is done. The positive presumptive test suggests that the water sample is nonpotable. Confirmation test is necessary since positive presumptive tests can also be due to the presence of some non-coliforms that are not recognised as faecal contaminants.
Principle:
The dye methylene blue inhibits growth of gram-positive bacteria. In an acidic environment, eosin methylene blue in EMB agar forms a complex that precipitates out, on to the coliform colonies producing dark centers with a green metallic sheen, characteristic of E.coli, the indicator of faecal contamination.
Requirements:
1. Eosin-Methylene Blue Agar (pH. 7.2) EMB. (Levine):
2. 24 hour old positive cultures in lactose broth from each of the three series from presumptive test.
3. EMB agar plates.
4. Bunsen flame.
5. Glass marking pencil.
6. Laminar clean air flow hood.
7. Inoculating loop.
8. Lactose broth.
9. Incubator.
Procedure:
1. Label EMB agar plates with the source of the sample of water: sewage-pond- tap.
2. Streak EMB agar plates with positive 24 hour lactose broth cultures from sewage water series.
3. Repeat with pond and tap water and incubate all plates at 37°C for 24 hours. E.coli colonies appear with dark centers and green metallic sheen.
Part 3: Completed Test:
This test is the final test for analysis of water sample where the colonies that appeared on EMB agar plates are used.
Principle:
Confirmation of colonies that appear on EMB agar is done by Gram staining and lactose fermentation tests.
Requirements:
1. One 24 hour old coliform-positive EMB agar culture.
2. Nutrient agar slant and lactose fermentation broth.
3. Gram staining set.
4. Bunsen flame.
5. Inoculation loop.
6. Microscope.
Procedure:
Label tubes with source of water and label one nutrient broth and lactose broth with same colony of each sample and incubate at 37°C for 24 hours. Tubes with acid and gas and Gram-negative bacilli confirms presence of E.coli.