MOST PROBABLE NUMBER (MPN) TEST FOR WATER QUALITY
AIM: To enumerate the number of
bacteria present in the water sample by the MPN method and to identify the
bacteria present in the drinking water sample.
Principle:
The most probable number (MPN) test is a statistical
method test based on the random dispersion of microorganisms per volume in a
given sample. In this
method, measured volumes of water are added to a series of tubes containing a
liquid indicator growth medium. The media receiving one or more indicator
bacteria show growth and a characteristic color change. The color change is
absent in those receiving only an inoculum of water without indicator bacteria.
From the number and distribution of positive and negative reactions, the MPN of
indicator organisms in the sample may be estimated by reference to statistical
tables.
MPN test is completed in three
steps:
1. Presumptive test
2. Confirmed test
3. Completed test
Presumptive test: This test, a specific
enrichment procedure for coliform bacteria, is conducted in fermentation tubes
filled with a selective growth medium (lactose broth), which contains inverted
Durham tubes for the detection of fermentation gas. A series of lactose broth
tubes are inoculated with measured amounts of the water sample to be tested.
The series of tubes may consist of three or four groups of three, five, or more
tubes.
The main selective factors found in the medium are lactose,
sometimes a surfactant such as Na-lauryl sulfate or Na-taurocholate (bile salt),
and often a pH indicator dye for facilitating detection of acid production,
such as bromcresol purple or brilliant green. The selective action of lactose
occurs because many bacteria cannot ferment this sugar, whereas coliform
bacteria and several other bacterial types can ferment it. The surfactant and
dye do not inhibit coliform bacteria, whereas many other bacteria, such as the
spore formers, are inhibited.
Confirmed test: This test serves to confirm
the presence of coliform bacteria when either a positive or doubtful
presumptive test is obtained.
1. A loopful of growth from such a
presumptive tube is transferred into a tube of brilliant green lactose bile
(BGLB) 2% broth (or other lactose broth) and incubated at 35°C for 48 hours.
This is a selective medium for detecting coliform bacteria in water, dairy, and
other food products. A selective agent in the medium is lactose. The broth tube
also contains a Durham tube to detect gas production.
2. A plate of Endo agar (or EMB
agar) is streaked with a loopful of growth from a positive tube and incubated
at 35°C for 18–24 hours. Typical coliform bacteria (E. coli and Enterobacter aerogenes) exhibit good growth on this
medium and form red to black colonies with dark centers or sheen. Salmonella typhi exhibits good growth but
the colonies are colorless. S. aureus growth is inhibited altogether.
Completed Test: A typical coliform colony from
an Endo 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 35°C for 24
hours. After 24 hours, the broth is checked for the production of 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.
Materials required: Test tubes, Durham tubes,
Conical flask, Petriplates, Lactose broth, EMB Agar, Endo agar, BGLB Broth,
Gram’s staining kit, Glass slide, Inoculation loop, Microscope.
Procedure for Presumptive test:
1. Prepare Lactose broth of single
and double strength in test tubes with Durham’s tube and autoclave it.
2. Take three sets of test tubes
containing five tubes in each set; one set with 10 ml of double strength (DS)
other two containing 10 ml of single strength (SS) .
3. Using sterile pipettes,
transfer 10 ml of water to each of the DS broth tubes. Transfer 1 ml of water
sample to each of 5 tubes of one set of SS broth and transfer 0.1 ml water to
five tubes of remaining last set of SS broth tubes.
4. Incubate the tubes at 37°C for
24 hours.
5. After incubation, observe the
gas production in Durham’s tube and the color change of the media.
6. Record the number of positive
results from each set and compare with the standard chart to give presumptive
coliform count per 100 ml water sample.
Procedure for Confirmed test:
Some microorganisms other than coliforms also produce acid and gas
from lactose fermentation. In order to confirm the presence of coliform, a
confirmatory test is done. For this, a loopful of suspension from a positive
tube is inoculated into a 3 ml lactose-broth or brilliant green lactose
fermentation tube and to an agar plate (EMB agar or Endo Agar) or slant.
Gas formation
BGLB Broth
A.
Inoculation of the lactose-broth
1. Incubate the inoculated
lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2
hours.
2. If no gas production is seen,
further incubate up to a maximum of 48 ±3 hours to check gas production.
B.
Inoculation in media slants
1. Take a loopful of suspension
from a positive tube and inoculate it on the agar surface.
2. The agar slants should be
incubated at 37°C for 24± 2 hours.
3. Colonies must be examined
macroscopically.
Procedure
for Completed test:
1. Transform a typical coliform
colony from the agar plate into a tube of brilliant green bile broth with
placed Durham’s tube and on the surface of a nutrient agar slant.
2. Incubate at 35°C for 24 hours.
3. After 24 hours, check the broth
for the production of gas, and perform Gram staining for organisms on the
nutrient agar slant.
Result
for Presumptive test:
Positive: The formation of 10% gas or
more in the Durham tube within 24 to 48 hours, together with turbidity in the
growth medium and the color change in the medium constitutes a positive
presumptive test for coliform bacteria, and hence for the possibility of fecal
pollution.
·
The
test is presumptive only because under these conditions several other types of
bacteria can produce similar results.
Negative: No growth or formation of gas in
Durham’s tube.
Result
for Confirmed test:
Positive: Formation of gas in
lactose broth and the demonstration of a coliform-like colony on the EMB agar
indicate the presence of a member of the coliform group in the
sample examined.
Coliforms
produce colonies with a greenish metallic sheen which differentiates them from
non-coliform colonies (show no sheen). The presence of typical colonies at high
temperatures (44.5 ±0.2) indicates the presence of thermo tolerant E.coli.
Negative: The absence of gas
formation in lactose broth or the failure to demonstrate coliform-like colonies
on the EMB agar.
Result
for Completed test:
Positive: The presence of gas in
the brilliant green bile broth tube and Gram-negative, non-spore-forming rods on
NA slant constitutes a positive completed test for the presence of coliform
bacteria, which, in turn, infers possible contamination of the water sample
with fecal matter.
Negative: Absence of growth and gas
formation in the broth. Absence of gram-negative, non-sporing rods on Gram’s
staining.
