MICROBIOLOGICAL ASSAY OF WATER, SOIL AND AIR

SAMPLING

The sample should be representative of the bacterial quality; hence extreme care should be taken
to avoid contamination. Pre-sterilised (at 121oC for 15 minutes) and paper wrapped glass bottles
are used. If the water is known to have residual chlorine, 0.2 ml of 3% sodium thiosulphate
solution should be added prior to sterilisation. While sampling in reservoirs, the bottle is lowered
in water (at a depth of 15 to 30 cm). The bottle is held there by the base in one hand, while with
the other hand the stopper and cover are removed. These should be retained in hand while the
bottle is filled, stopper is then replaced. The filled bottle is finally pulled up. Do not fill the bottle
completely, but allow an air space of about 3 cm. While sampling from the taps, the external
fittings on the tap are removed and the tap is sterilised by flame on a piece of cotton, soaked in
methylated spirit until it is quite hot. The water is then allowed to flow for 2 minutes, the bottle
is opened near the tap. Water is collected and the bottle is immediately closed. Avoid any
contamination by hand.

TRANSPORT, PRESERVATION AND STORAGE

Samples after collection should be immediately taken to the laboratory for examination. If the
processing is not possible within one hour, the samples should be transported in ice. In
laboratory, if immediate analysis is not possible, the samples can be preserved at 4°C up to 6
hours, but in no case more than 24 hrs.

Most probable number (MPN) method for counting E. coli

(coliform)
APPARATUS AND MATERIALS

 Test tubes, 25 ml 50 ml
 Durham tubes
 Water bath with a stable 44.5±0.2°C
 Autoclave
PRINCIPLE
For the waters suspected of having high density, several dilutions are used. As a routine one 50
ml, five 10 ml and five 1 ml volumes of the water sample are inoculated. MacConkey’s broth is
suitable for this test.
Procedure:

Step I: Presumptive test:

 At first three set of 5 test tubes are taken.
 10 ml of double strengthen liquid media (MacConkey broth) is placed in each test tubes of
1st set
 Similarly, 10 ml of single strengthen liquid media is placed in each test tubes of 2 nd set and
3rd
 ** double strengthen broth refers to broth made up using twice the normal amount of broth
powder.
 Lactose broth or lauryl sulphate broth or tryptose lauryl broth is used as liquid media for
the test.
 Then, Durham’s tube is inserted in inverted position in each test tube of all sets.
 All the test tubes are then cotton plugged and sterilized using autoclave for 15 minutes at
15 lb/inc pressure at 121 °
 After cooling water sample is added in each test tubes as follows;
 Add 10 ml water sample in each test tubes of 1st set
 Add 1 ml water sample in each test tubes of 2nd set
 Add 0.1ml water sample in each test tubes of 3rd
 Then incubate all test tubes at 35.5 °C for 24 hours. After incubation gas production in
Durham’s tubes is observed.
 Tubes in which gas production is 10% or more is recorded as positive tube and tubes in
which gas production is less than 10% is recorded as doubtful.
 Doubtful test tubes are further incubated for 24 hours and again gas production is noted. If
gas production is still less than 10%, then tube is recorded as negative and are discarded
  ** doubtful result is given by other gas producing lactose fermenting bacteria other than
coliforms such as Lactobacillus, Streptococci, Bacillus, Clostridium Clostridium produces
more than 10% gas but only after 48 hours of incubation.
 All the positive test tubes are taken for confirmatory test.

Step II: Confirmatory test for MPN method:

 Positive tubes obtained from presumptive test are now confirmed for coliform.
 For confirmation of coliform, brilliant green lactose bile (BGLB) broth is used as culture
media, because BGLB broth inhibits growth of gram positive bacteria such as
lactobacillus, Streptococci, Bacillus and Clostridium
 Coliforms can grow in this BGLB medium
 For confirmation, one loopful of sample from each positive tubes obtained from
presumptive test is inoculated in respective tubes containing Brilliant green lactose bile
broth and incubated for 24 hours at 35.5 °
 Gas production 10% or more are recorded as positive while less than 10% is recorded as
doubtful. Doubtful tubes are again incubated and the result is recorded.
 All the positive test tubes are now confirmed for presence of coliforms.
 Finally the number of bacteria present in water sample is determined from previous MPN
chart. Alternatively number of coliforms can also be calculated by the formula;
 Coliforms/100ml = numbers of positive tubes
_/ volume of samples in negative tubes * volume of samples in whole experiments

Interpretation:
 Three types of colonies are obtained in culture media;
 Typical colony: they are pink colored with greenish metallic appearance or
nucleated colony. Coliforms gives typical colony
 Atypical colony: they are pink and non-nucleated colony. Coliforms as well as other
lactose fermenting organisms gives atypical colony
 Non-typical colony: they are non-pink colony given by non-coliforms