ENVIRONMENTAL BIOTECHNOLOGY

Experiment 10

Aim of the Experiment : Presumptive test for coliform group of bacteria.

Principle

It is based on the principle that coliforms if present in water, will ferment lactose to produce acid and gas within 24 48 hours. Production of acid is indicated by pH indicator and gas is collected in Durham's vial, both of which are present in the medium.

Media used are highly selective for coliforms, which inhibit growth of gram-positive organisms. MacConkey’s lactose bile broth (MLBB) or formate lactose glutamate medium or laurel tryptose broth or brilliant green lactose bile broth (BGLB) can be used for presumptive test.

Different volumes of water are inoculated in MLBB or BGLB which permits growth of coliforms only. Coliforms will ferment lactose to produce acid and gas within 24-48 hours and test is considered as positive. Hence, it may be presumed that coliforms may be present in water hence the name presumptive test.

Materials required

1. Water sample.

2. Sterile 1 ml and 10 ml pipettes.

3. 5 MLBB tubes, each with 10 ml double strength medium (2X).

4. 3 MLBB tubes, each with 5 ml single strength medium (X).

Procedure:

1. Shake the water sample vigorously to ensure uniform distribution of organisms.

2. With sterile graduated pipettes inoculate the water sample as follows. MLBB double strength tubes with 10 ml water.

1 MLBB single strength tubes with 1 ml water. 1 MLBB single strength tubes with 0.1 ml water.

3. One tube of MLBB single strength is not inoculated and hence, serves as control.

4. Incubate all tubes at 37 °C for 24 hours.

5. Examine tubes for the presence of acid and gas after 24 hours.

6. If no gas has formed, reincubate all the tubes for another 24 hours (total 48 hours).

7. Record the presence or absence of acid & gas at each examination, & interprete as followes.

Interpretations:

1. Absence of gas even after 48 hours indicate a negative test and the presumptive test is terminated. The water sample is assumed to be potable.

2. Presumptive test is considered positive if any one or more of tubes show acid and gas. Positive tubes are retained for confirmed test.

Note:

1. Double strength broth: (contains double the concentration of ingredients except water) is used when large volumes of water are to be inoculated, because the medium would otherwise be too diluted and may not support the growth of bacteria.

2. False positive presumptive test may be produced due to:

a) Presence of lactose fermenting organisms other than coliforms.

b) A synergistic association where the joint action of two organisms on a carbohydrate results into production of gas which will not be formed by either species when grown sperately. Synergism is frequently caused by a joint action of gram-positive and gram-negative organism following togather, e.g

Staphylococcus aureus + Proteus vulgaris Streptococcus faecalis + Salmonella paratyphi A Staphylococcus aureus + Salmonella schotimulleri

False positive presumptive test can be overcome by adding bile salts and triphenylmethane dyes which inhibit growth of gram-positive bactria and thereby eliminate synergistic effect.

3. Overfermentation: this kind of results are obtained when ratio of number of organisms to the amount sugar is comparatively very high. Due to less amount of sugar, acid produced is not sufficient enough to lower the pH at which growth of organism is inhibited. Thus, growth continues and organisms switch over to peptones when sugars are depleted. Since end prod ucts of protein metabolism are alkaline in nature the basicity of medium increases. Hence, MLBB appears yellow even though the test is positive.