Aim
To determine the potability of the
given water sample
Principle
The
major source of human pathogens which are transmitted mainly through oral fecal
route, such as those belonging to Salmonella
species, Shigella species, pathogenic
Escherichia coli, Vibrio cholerae, Yersinia enterocolitica,
Campylobacter species, various viruses such as Hepatitis A, Hepatitis E,
Rota virus and parasites such as Entamoeba
histolytica and Giardia species
are drinking water. To determine the
potability of drinking water it is necessary to screen the water sample for the
presence of these microorganisms. Since
it is tedious to monitor for these pathogens on a regular basis, possible fecal
contamination in the water body is determined by screening for the presence of indicator microorganisms. Among indicators, the coliform group
of bacteria is routinely tested. These are rod shaped, Gram-negative, non-spore forming, motile bacteria which can
ferment lactose with the production of acid and gas when incubated at 35–37°C. Escherichia
coli, Enterobacter aerogenes, etc. are
coliforms. Escherichia coli is the most common coliform and its presence is principally
associated with fecal contamination.
Most
probable number (MPN) analysis is a statistical method based on the random
dispersion of microorganisms per volume in a given sample to detect fecal
coliforms in a water sample.
MPN
test is completed in three steps:
1.
Presumptive test
2.
Confirmed test
3. Completed test
Presumptive
Test
Principle
In this test, water samples are
inoculated into fermentation tubes filled with a selective growth medium
(lactose broth), which contain inverted Durham tubes for detection of
fermentation gas. Three sets of three lactose broth tubes are inoculated with different
measured amounts of the water sample and incubated at 37°C
for overnight and observed for gas formation.
Presence of gas indicates a positive presumptive test and indicates the
presence of coliforms in the water sample since coliforms are capable of
utilizing lactose as carbon source.
Procedure
1.
Double strength and single strength
lactose broth were prepared and dispersed into test tubes with Durham’s tube
and sterilized.
2. 3 tubes containing 10 ml double
strength lactose broth and 3 tubes containing 9 ml single strength lactose
broth and 3 tubes containing 9.9 ml single strength lactose broth were set up
for each water sample.
3.
Using sterile pipette 10 ml of water
sample was transferred to a set of 3 tubes containing 10 ml double strength
lactose broth and 1 ml water sample was transferred to a set of 3 tubes
containing 9 ml single strength lactose broth and 0.1 ml water sample was
transferred to set of 3 tubes containing 9.9 ml single strength lactose broth.
4.
All the tubes were incubated at 37°C
for overnight
5.
After incubation the tubes were
observed for gas production.
Observation and Result
The production of gas after 24-hour
incubation indicates a positive presumptive test for coliform bacteria.
MPN of bacteria present in 100 ml water can be obtained from Mc Crady's probability table or MPN table by comparing the number of positive and negative tubes observed from the 9 inoculated lactose fermentation tubes. The MPN index of the water sample obtained by referring the standard table is tabulated in table 1. The tubes showing positive result are retained and used for confirmed test.
MPN index for samples A, B, C and D were noted as 4, 9, 460 and 7 respectively. This shows the presence of E.coli or Enterobacter respectively. This was confirmed by further test.
Presumptive test (Left hand side)
|
Sl. No. |
Tubes showing gas
production |
MPN Index (per 100 ml) |
||
|
Lactose broth (2x)
10 ml |
Lactose broth (1x)
1 ml |
Lactose broth (1x)
0.1 ml |
||
|
1 |
1 |
0 |
0 |
4 |
|
2 |
2 |
0 |
0 |
9 |
|
3 |
3 |
3 |
1 |
460 |
|
4 |
1 |
1 |
0 |
7 |
Confirmed
Test
Principle
In the presumptive test, gas formation in lactose
fermentation tubes indicates that the water is unsafe to drink. However, gas formation may also be due to
some non-coliform organisms such as clostridium.
Confirmed test serves
to confirm the presence of coliform bacteria after either a positive or
doubtful presumptive test. Eosin methylene blue agar plates are used which
contains methylene blue that inhibit gram positive bacteria. Gram negative lactose fermenters (coliforms)
such as E coli produce dark centered
small colonies having a green metallic sheen while Enterobacter aerogenes form large colonies and lack metallic sheen.
A loopful of growth from a presumptive tube is also transferred
into a tube of brilliant green lactose (BGLB) and incubated at 37°C for 24
hours. This is a selective medium for detecting coliform bacteria and contains
a Durham tube to detect gas production.
Procedure
1. Eosin methylene blue agar plates and BGLB tubes containing
inverted Durham’s tubes were prepared.
2. From the positive presumptive test tubes of each water sample, a loopful of medium was transferred to BGLB tubes and also streaked into EMB agar plates.
3. The
plates and tubes were incubated 37°C for 24 hours.
4.
After incubation, tubes were
observed for acid and gas production and the plates were observed for the
presence of typical coliform colonies.
Observation and Result
The BGLB tubes inoculated with presumptive tubes of sample A, B, C and D showed colour change from green to yellow which indicated acid
production and presence of gas in Durham’s tubes. These samples also showed dark centered small
colonies having a green metallic sheen in EMB plates which indicated a positive
confirmed test.
Confirmed test (Left hand side)
|
Sl. No. |
Colony morphology
in EMB plate |
Acid/Gas production in BGLB tube |
Presence / absence of coliforms |
Result |
|
1 |
Dark centred colour colonies with green metallic
sheen |
Yes |
Presence of coliforms |
Non potable |
|
2 |
Dark centred colonies with green metallic
sheen |
Yes |
Presence of coliforms |
Non potable |
|
3 |
Dark centred colonies with green metallic
sheen |
Yes |
Presence of coliforms |
Non potable |
|
4 |
Dark centred colonies with green metallic
sheen |
Yes |
Presence of coliforms |
Non potable |
Completed Test
Principle
This is the final step in the
bacteriological examination of drinking water and helps to further confirm
doubtful and, if desired, positive confirmed test results. A typical coliform
colony from an EMB agar plate is inoculated into a tube of brilliant green bile
broth and on the surface of a nutrient agar slant. They are then incubated at
37°C for 24 hours. After 24 hours, the broth is checked for the production of
acid and gas, and a Gram stain is made from organisms on the nutrient agar slant.
If the organism is a Gram-negative, non spore-forming rod and produces gas in
the lactose tube, then it is positive that coliforms are present in the water
sample.
Procedure
1. Single strength brilliant green lactose broth
containing inverted Durham’s tubes and nutrient agar slants were prepared.
2.
The slants and tubes were inoculated
with colonies obtained from confirmed test.
3. The
tubes and slants were incubated at 37°C for 24 hours.
4.
After incubation the tubes were
examined for gas formation and the colonies from slants were Gram stained and
observed under microscope.
Observation and Result
Gas production in Brilliant green lactose broth inoculated
with colonies form EMB plates of sample A, B, C and D confirmed the presence of coliforms. The presence of gram negative bacilli upon
gram staining further confirmed the presence of E.coli in the given water sample.
The water sample A, B, C and D are not potable.
Completed
Test (Left
hand side)
|
Water sample |
Gas production in lactose broth |
Gram staining |
Result |
|
A |
Positive |
Negative |
Non potable |
|
B |
Positive |
Negative |
Non potable |
|
C |
Positive |
Negative |
Non potable |
|
D |
Positive |
Negative |
Non potable |
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