Experiment:-

Aim: Testing for microbiological quality of potable water (Coli form test).
Principle: The number of total coliforms (Enterobacter, Klebsiella,Citrobacter, Escherichia) in
a water sample can be determined by a statistical estimation called the most probable number
(MPN) test. This test involves a multiple series of Durham fermentation tubes and is divided
into three parts: the presumptive, confirmed, and completed tests. In the presumptive test,
dilutions from the water sample are added to lactose or lauryl tryptose broth fermentation tubes.
After 24 to 48 hours of incubation at 35°C, one looks for bacteria capable of fermenting lactose
with gas production, presumably coliforms. (The lauryl tryptose broth is selective for gram-
negative bacteria due to the presence of lauryl sulfate.) In the confirmed test, one transfers
material from the highest dilution of those lactose broth tubes that showed growth and gas
production into brilliant green lactose bile broth, which is selective and differential for coliforms.
The tube is incubated for 48 ± 3 hours at 35°C. Gas formation in the Durham tube is a confirmed
test for total coliforms.
In the completed test, a sample from the positive green lactose bile broth is streaked onto
Levine’s EMB or LES Endo agar and incubated for 18 to 24 hours at 35°C. On EMB agar,
coliforms produce small colonies with dark centers. On LES Endo agar, coliforms produce
reddish colonies. Samples are then inoculated into brilliant green lactose bile broth and onto a
nutrient agar slant. These tubes are incubated for 24 hours at 35°C. If gas is produced in the
lactose broth, and the isolated bacterium is a gram-negative (based on a Gram stain) nonsporing
rod, the completed test is positive.
An estimate of the number of coliforms (most probable number) can also be done in the
presumptive test. In this procedure, 15 lactose broth tubes are inoculated with the water sample.
Five tubes receive 10 ml of water, 5 tubes receive 1 ml of water, and 5 tubes receive 0.1 ml of
water. A count of the number of tubes showing gas production is then made, and the figure is
compared to a table (table 1) developed by the American Public Health Association. The number
is the most probable number (MPN) of coliforms per 100 ml of the water sample. (It should be
noted that the MPN index usually comes from the presumptive test if raw sewage is being tested
and comes from confirmed or completed tests for other types of samples.)
 Fig
: MPN procedure for bacteriological examination of water for the presence of Coliforms by the
presumptive, confirmed and completed tests.
Table1: Most probable number (MPN) of coliform bacteria (sourced from Harley-Prescott,
Laboratory exercises in Microbiology).

In this experiment, a simplified form of the MPN test is followed. The samples will be incubated
in lactose broth followed by evaluation of gas production and streaking on EMB agar plates for
confirmation.
Reagents: Lactose broth (1x and 2x), water samples (A and B), durham tubes, boiling tubes,
EMB agar plates, dilution series, pipettes.
Procedure:
1. Prepare two sets of 2x lactose broth (each set contains 5 boiling tubes with inverted durham
tubes with 10ml lactose broth each). Prepares 4 sets of 1x lactose broth (each set contains 5
boiling tubes with inverted durham tubes with 5ml lactose broth each).
2. Inoculate the 2x sets with 10 ml water samples (5 tubes for sample A and 5 for sample B).
3. Inoculate 5 tubes with 1x lactose broth with 1ml sample A and 5 others with 1ml sample B.
4. Inoculate 5 tubes with 1x lactose broth with 0.1 ml sample A and 5 others with 0.1 ml sample
B.
5. Incubate for 24-48 hr at 35-37°C and observe for gas production.
6. For confirmation, prepare suitable dilutions of the tubes giving positive readings and streak
on EMB agar plates. Incubate for 24-48 hr at 35-37°C and observe for growth on E.coli, the
most common faecal contaminant and indicator organism for water.
Observation Table:
 Sample A Sample B
10ml 1ml 0.1ml 10ml 1ml 0.1ml
Time 2x 2x 2x 2x 1x 1x 1x 1x 1x 1x 1x 1x 2x 2x 2x 2x 1x 1x 1x 1x 1x 1x 1x 1x
24h
48h
Gas production:- Presence: + ; Absence: -
Table: Identification of bacterial growth on EMB agar plate (sourced from HIMEDIA)
Growth on EMB Probable bacteria
Pink, without sheen Enterobacter aerogenes
purple with black centre and green metallic Escherichia coli
sheen
Pink, mucoid Klebsiella pneumoniae
Colourless Proteus mirabilis/ Salmonella typhimurium

Result:
The MPN count of sample A is --- and B is ---. On EMB, coliforms from pink to red colonies
and some may have green metallic sheen.
No. of coliforms per 100 ml of water = colony count x 100
Volume of sample used
Water is considered safe for drinking if it contains fewer than 4 coliforms per 100 ml water.
Precautions:
1. If sewage or pond water sample is used for the test, it should be mixed thoroughly by shaking
prior to inoculation of lactose fermentation tubes.
2. After sterilization, the durham tubes in the lactose fermentation tubes should always be free
from air bubbles.