The membrane filtration method and multiple tube method are described for testing water samples for indicator organisms like coliforms and E. coli. The membrane filtration method uses a vacuum to pull the water sample through a membrane filter which retains bacteria. The filter is then placed on a culture medium and incubated. Colonies are counted and reported as CFU/100ml. The multiple tube method involves inoculating different volumes of the water sample into lactose broth tubes, incubating, and observing for acid/gas production to determine the most probable number of coliforms present in the original sample using statistical tables.

2. 1-Membrane filtration method
2-Pour plate method
3-Spread plate method
4-Multiple tube method
5-ATP testing method

3. Requirements:
 Filtration assembly
 Membrane filter(nominal pore size 0.2 or 0.45mm).
 Vacuum pump
 Sample

4.  In the membrane-filtration (MF) method, a minimum
volume of 10 ml of the sample (or dilution of the
sample) is taken.
 Introduced aseptically into a sterile or properly
disinfected filtration assembly containing a sterile
membrane filter (nominal pore size 0.2 or 0.45mm).
 A vacuum is applied and the sample is drawn through
the membrane filter.

5.  All indicator organisms are retained on or within the
filter, which is then transferred to a suitable selective
culture medium in a Petri dish.
 Petri dish is transferred to an incubator at the
appropriate selective temperature where it is
incubated for a suitable time to allow the replication of
the indicator organisms.

7.  Visually identifiable colonies are formed and counted,
and the results are expressed in numbers of “colony
forming units” (CFU) per 100 ml of original sample.

9.  Both methods are same but with few differences.
 In both the methods serial dilutions of the sample is used.

10.  From each serial dilution culture is prepared but in
pour plate method two types of colonies are obtained
surface and sub-surface as compared to spread plate
method.

11.  In pour plate method both agar and sample are poured
together and are mixed thoroughly.
 While in spread plate method sample is spread on
agar.
 After all the pouring and spreading petri dishes are
incubated at 37°c for 24 hrs.

12.  After incubation colonies are obtained and counted with
naked eye.

13.  The multiple-tube method is also referred to as the most
probable number(MPN) method.
Aim:
 Total or presumptive coliform count.
 Confirmed coliform and ecoli count.
Requirements:
 Sterile bottle
 MacConkey’s broth with bromocresol purple.
 Brilliant green bile lactose broth.
 Tryptone water.
 Test tubes.

14. MacConkey’s broth:
 Make 50ml double strength M.B
 Pour 10ml double strength M.B in 5 test tubes.
 Pour 5ml single strength M.B in 5 test tubes.
 Each tube has durham’s tube.
 Autoclave the prepared meida.

15.  Take sterilized bottle of 100 ml with 0.1ml of 1.8%
sodium thiosulphate.
 Flame the mouth of tape and bottle before sample
collection.
 Let the water running for2-3 minutes then collect
sampl.
 cap the bottle and thoroughly mix the water.

16.  Pour 50ml sample in 50ml double strength M.B
 5ml sample in each 5 tubes with double strength M.B.
 1ml sample to each 5 test tubes with single strength
M.B.
 Then incubate all the tubes at 37°c for 18-24 hrs.

17.  After incubation observe those tubes with acid and gas
production.
 These tubes will give presumptive coliform count.
 it is based on an indirect assessment of microbial
density in the water sample by reference to statistical
tables to determine the most probable number of
microorganisms present in the original sample.

18.  The concentration of microorganisms in the original
sample can then be estimated from the pattern of
positive results (the number of tubes showing growth
in each volume series) by means of statistical tables
that give the “most probable number” per 100 ml of the
original sample.