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8. DETERMINATION OF MPN


10.1 Introduction

The quality of drinking water can sometimes becomes suspect because of the presence of
microorganisms such as Escherichia Coli (E-coli), faecal streptococci and anaerobic spore
forming clostridium perfringenes, excreted from the intestines of warm blooded animals
including man and also birds. Water supplied for human consumption must be free from
these organisms. These organisms by themselves are generally harmless. (Certain types of
Escheria Coli are known to cause gastroenteritis in new born babies). Their presence in
Water is indicative of sewage contamination and is presumptive evidence of possible
presence, especially during epidemics, of pathogenic organisms such as -Salmonella typhi
(causing typhoid fever), vibreo cholerae (causing cholera), Shigella dysenteriae (causing
bacillery dysentery), Entamoeba histolytica (causing amoebic dysentery), Giardia lambliae
(causing gastrointestinal infection), Hepatitis viruses A, B, Non -B (causing infective
Jaundice), polioviruses, types 1, 2, and 3 (causing Poliomyelitis), parasitic flukes or flat
worms (causing Schistosomiasis), clostridium tetani (causing tetanus infection), eggs of
worms (causing ascaritis and other infections).

As a routine procedure, it is neither practicable nor essential to isolate and identify specific
pathogenic organisms in samples of drinking water. It is enough to evaluate the probability
of their presence in a domestic supply. To achieve this end, the organisms most commonly
used as indicators of pollution are E-coli and coliform group as a whole. As organisms of
the coliform group are foreign to water, their presence in water samples is regarded as an
evidence of sewage contamination. Some part of the sewage could have come from carriers
and persons afflicted by one or more of the above waterborne diseases

When pathogens are present in sewage, they are always outnumbered by excremental
organism-coli and other coliforms, which are hardier, more resistant and longer lasting.
These coliforms organisms are easier to detect in water .If they are not found in water
samples, it can be inferred that, pathogens are also absent.

10.2 Bacteriological standards for drinking water
BIS-
1. Water in Distribution System including Consumer Premises-

a) Throughout any year 95% of samples should not contain any coliform organisms in 100ml.
b) No sample should contain E-coli in 100 ml.
c) No sample should contain more than 10 coliform organisms per 100 ml,
d) Coliform organisms should not be detectable in 100 ml of any two consecutive samples.

2. Unpiped Water Supplies (such as wells, bore holes and springs)

a) No sample should contain more than 10 coliform organisms per 100 ml and
b) No sample should contain E-coli in 100 ml.
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10.3 Methods of Evaluation of Bacteriological Quality of Water.

10.3.1 Identification of Specific Pathogenic Organisms.
This requires very large quantities of sample for analysis, a wide variety of culture media and
detailed methods to isolate different pathogens for specific identification. This method is
uneconomical, time consuming and assumes special significance, only when characteristics of a
particular pathogen is to be studied to formulate a defense programme or develop specific drugs
to inhibit the growth of the disease causing organism. This method is rarely adopted as a routine
test for water.

10.3.2 Membrane Filtration Method for Detection of Faecal Streptococci

This method involves counting of coliform organisms in water by filtering a measured volume
(10 ml or 100 ml) through membrane of cellulose esters. All the bacteria are retained on the
membrane. The membrane is placed on a well dried plate of glucose -azide agar. This is then
incubated at 37 C for 4 hours and then at 44C for 44 hours.
All reddish colonies are counted as faecal streptococci.

10.3.3 MPN

The most probable number of coliform organisms in a water sample is a statistical estimate of the
density of bacteria most likely to produce a particular result of some significance, associated
with the bacterial quality of water.


a) Principle-
Coliform bacteria (which are gram negative, non -spore forming and rod shaped) are bile
Tolerant and capable of fermenting lactose in 48 hours at 37 C, with production of acids and
gas. Production of gas is evidence of presence of coliform organisms.

b) Equipment -

1) Biological incubator
2) Autoclave
3) pH meter

c) Glass-ware - (All of borosil)

1) Fermentation tubes with glass stoppers or screw caps-
Alternatively, the following serves the purpose -
50 ml graduated cylinders (without spout) with close fitting stoppers -5 nos,
25 ml graduated cylinders (without spout) with close fitting stoppers -5nos,
10 ml graduated cylinders (without spout) with close fitting stoppers -5nos,
100 ml graduated cylinders (normal type) with close fitting stoppers -2nos,
2) Shell vials or Durham tubes-
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5 mm dia, 3.0 cm long (or similar size) - 15 nos.
3) Graduated pipettes: 10 ml -1 no, 5 ml -1 no.
4) Conical flask, 500 (or beaker, 500 ml) -1no.

d) Chemicals - (All of best quality - analar grade)
1) Lactose- Food for bacteria. Lactose is broken down to produce acids (chiefly CH
3
COOH) and
gases (CO
2
and H
2
).
2) Peptone- Nutrient essential for bacterial growth.
3) Bile salt - Inhibits growth of non-intestinal organisms present in the water sample.
4) Sodium Chloride - preservative, prevents vegetative growth.
5) Neutral Red - Indicator, indicates production of acids turning from red to rosy pink and with
more production of acids to yellow.

6) Dilution Water -

Stock buffer -

Dissolve 34.0 g KH
2
PO
4
in 500 ml of distilled water, adjust pH to 7.4 -7.5 on a pH meter
using 1N NaOH and dilute to one liter with distilled water and mix thoroughly.

Working Dilution Water

Add 1.25 ml stock buffer solution to one liter of distilled water and mix. This water should be
used for preparing the broth.

e) Preparation of Medium (MacConekey Broth), per 100 ml of sample to be Tested.
1) Take 250 ml of working dilution water in a 500 ml conical flask (or beaker)-(A)
2) Add peptone (10 g), bile salt (2.5 g) Sodium chloride (2.5 g).
3) Boil the mixture for two minutes.
4) Add lactose (5 g) and allow the broth to cool to room temperature.
5) Add 1% neutral red till the broth becomes dark red. This broth is of double strength.
6) Take out 50 ml of double strength broth in sterile beaker (B) and add an equal volume of
boiled and cooled working dilution water. This broth is of single strength.

f) Sterilization of Glass Ware -
All glass-ware used for bacteriological tests (including sampling bottles) should be thoroughly
washed using a good detergent and hot water, rinsed with hot water to remove detergent residues
and finally rinsed with distilled water. All glass-ware, washed as above, should be sterilized in an
autoclave at 160 C (not less than 121 C) and at a pressure of 1.05 kg/cm
2
for not less than 15
minutes. (sterilization for 2 hours is recommended as a standard practice).

g) Procedure for presumptive test
The MPN test consists of the following four steps.
1) Sampling

Collect bacteriological samples in sterile glass bottles with ground glass stoppers protected with
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a piece of linen, aluminum foil or paper. 300 ml capacity BOD bottles are ideal for sampling.
Start MPN test within one hour of sampling. If the samples are to be transported from a distance,
hold the sample below 10 C during a transportation period not exceeding 6 hrs. Keep such
samples in a refrigerator on receipt in the laboratory and start the test within 2 hrs. (Under
unavoidable circumstances however the duration of time between the collection of samples and
their examination may be extended up to a maximum of 30 hrs.)


2) Inoculation

a) Arrange the sterilized fermentation tubes to form 3 sets-
50 ml. tubes forming set A, 25 ml.
tubes forming set B, 10 ml. tubes forming
set C.

Set A Set B Set C










10ml sample 1ml sample 0.1ml sample

b) Pour 25 ml. double strength MacConekey broth into each 50 ml tube in set A.
Pour 12 ml double strength MacConekey broth into each 25 ml tube in set B.
Pour 5 ml double strength MacConekey broth into each 10 ml tube in set C.

c) Shake the sample bottle to distribute the microorganisms (if any), most evenly. Extract
100ml of the sample into a sterile graduated jar.

d) Using 10 ml pipette, inoculate 10ml of the sample into each of the tubes in set A. Using
5 ml pipette, inoculate 1ml of the sample into each of the tubes in set B. Using 1 ml
pipette, inoculate 0.1ml of the sample into each of the tubes in set C.

e) Insert a sterile Durham tube, upside down into each fermentation tube. Firmly stopper
each fermentation tube. Turn the tube upside down shake and hold it till the Durham tube
is completely filled with the broth with no air bubble trapped inside. The entire work of
inoculation should be done under sterile conditions.

3) Incubation-

a) Place the inoculated fermentation tubes at 37 C in a thermostatically controlled biological air
incubator.

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b) Examine after 24 hours, for production of gas and also for change in color of the broth.
Any gas produced is seen trapped in inverted Durham tubes. Fermentation results in a drop in
Ph of the broth, which is indicated by a rosy pink or, in a more advanced stage, yellow
coloration.

c) Continue incubation for 24 hours more. Again examine all the tubes for production of gas and
also for change in color of the broth.

4) Observation-
After 48 hours of incubation, count the number of positive results i.e. the number of tubes
producing gas in each set, irrespective of the amount of gas produced. A negative result is
indicated by no gas and no change in color of the medium.

Record the result.

Refer to MPN statistical tables and read out MPN index / 100 ml of sample.

Observation Table-

Sample no Description
of Sample
Number of tubes giving positive
result out of
MPN
index per
100 ml
Remarks
5 tubes of
10 ml
5 tubes of
1ml
5 tubes of
0.1ml






Conclusion:
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MPN Reference Table (Need Not Be Written I n The J ournal)

Number of tubes
Giving positive result out of
MPN
index
per
100ml
Number of tubes
Giving positive result out of
MPN
index
per
100ml
5 of 10
ml each
5 of 1ml
each
5 of 0.1
ml each
5 of 10ml
each
5 of 1
ml each
5 of
0.1ml
each

0 0 0 < 2 2 0 0 5
0 0 1 2 2 0 1 7
0 1 0 2 2 1 0 7
0 2 0 4 2 1 1 9
1 0 0 2 2 2 0 9
1 0 1 4 2 3 0 12
1 1 0 4 3 0 0 8
1 1 1 6 3 0 1 11
1 2 0 6 3 1 0 11
3 1 1 14 5 1 2 63
3 2 0 14 5 2 0 49
3 2 1 17 5 2 1 70
3 3 0 17 5 2 2 94
4 0 0 13 5 3 0 79
4 0 1 17 5 3 1 110
4 1 0 17 5 3 2 140
4 1 1 21 5 3 3 180
4 1 2 26 5 4 0 130
4 2 0 22 5 4 1 170
4 2 1 26 5 4 2 220
4 3 0 27 5 4 3 280
4 3 1 33 5 4 4 350
4 4 0 34 5 5 0 240
5 0 0 23 5 5 1 350
5 0 1 31 5 5 2 540
5 0 2 43 5 5 3 920
5 1 0 33 5 5 4 1066
5 1 1 46 5 5 5 > 2400