Duterte, Jan Rommel T.

Exercise No. 13
STANDARD MOST PROBABLE NUMBER (MPN) COLIFORM TEST
I.

II.

III.

Introduction
Microorganisms can be found everywhere, whether in air, land, or water.
Some of these microorganisms are harmless to humans, while some can
cause disease. One of the microorganisms that can be found in these
environments is the coliform bacteria.
Coliform bacteria are a commonly used indicator of the sanitary quality
of food and water. They are defined as rod-shaped Gram-negative non-spore
forming and motile or non-motile bacteria which can ferment lactose with the
production of acid and gas when incubated at 3537C. Coliforms can be
found in the aquatic environment, in soil and on vegetation; they are
universally present in large numbers in the feces of warm-blooded animals.
While coliforms themselves are not normally causes of serious illness, they
are easy to culture, and their presence is used to indicate that
other pathogenic organisms of fecal origin may be present.
Two techniques are commonly used to detect the presence of coliforms
in water. The first of these is called the multiple fermentation tube or most
probable number technique. In this method measured portions of a water
sample are placed in test-tubes containing a culture medium. The tubes are
then incubated for a standard time at a standard temperature.
Objectives
a) Determine the presence of coliform group of bacteria in a water sample
b) List and explain each steps in the multiple-tube technique for determining
coliforms in the water sample
c) Obtain some index as to possible number of coliform bacteria present in
the water sample being tested
Methodology
A. Materials and Apparatus
100 mL water sample in a
sterile container
9-20 mL test tubes
9-Durham tubes
Lauryl Sulfate Tryptose
broth (LST)

Brilliant Green Lactose

broth (BGLB)
Eosin Methylene Blue
(EMB) agar plates
Gram-staining reagents
Sterile pipettes 1mL
Alcohol
Alcohol lamp

B. Experimental Procedure
B.1 Presumptive MPN
A water sample to be analyzed for coliform bacteria was collected,
placed in a sterile container, and set aside. Nine Durham tubes were
submerged upside down in test tubes containing lactose broth. These test
tubes were then inoculated with 1, 0.1 and 0.01 mL of the water sample.
The test tubes were incubated at 35C for 2 days. The positive result for
coliform bacteria was gas production and this was recorded for each
dilution.
B.2 Confirmatory MPN
The lactose broth tubes that had gas production were placed into
brilliant green lactose bile broth in Durham tubes and streaked onto eosin
methylene blue agar. The tubes and plates were incubated for 2 days at
35C. Positive result was the gas production and a green streak on the
agar plate and this was recorded.
B.3 Completed Test for the Presence of Coliforms
A small amount of broth was transferred from the positive brilliant
green lactose bile broth into a Durham tube and incubated at 35C for 2
days. Positive eosin methylene blue plates were transferred to a nutrient
agar slope. This was also incubated at 35C for 2 days. Gas formation is
positive and this was recorded.
IV.

Results and Discussion

Figure 1. Presumptive Test for the Water Sample after addition of 1.0mL, 0.1 mL and 0.01 mL water.

Table 1. Presumptive Test Result

Time

Volume of water sample

use
1.0 mL 0.1 mL
0.01

MPN per 100

mL water
sample

mL
0

Total test tubes positive at 24

3
0
230
hrs
Total test tubes positive at 48
3
0
0
230
hrs
As seen in the picture above only the lactose broth inoculated with
1mL of water sample showed gas production. Following the calculations in
the lab manual, the MPN per 100mL of sample is 230.

A sample was taken from the tubes and transferred to brilliant green
lactose bile broth and the same results are observed, only the 1mL water
sample showed positive results.
Two kinds of bacteria were observed on the EMB plates: Enterobacter
and E. coli. As with the test tubes, the same results were found: only 1 mL
addition gave positive tests for E.coli and Enterobacter. E. coli is seen as
metallic green colonies on the EMB plates, while Enterobacter forms a
shiny brown metallic colonies. The same with the results of E.coli, it only
gives positive to 1 mL addition for 24 hrs and 48 hrs.

Figure2. Addition of 1.0 mL water sample on EMB

Figure 3. Addition of 0.1 mL of water sample on EMB

Figure 4.Addition of 0.01 mL of water sample on EMB

Table 2. Confirmatory Test result
Time

24 hrs

48 hrs

BGLB
Gas form
Gas
formation on
the 1.0mL
addition
Gas
formation on
the 1.0mL
addition

Time

24 hrs

48 hrs

EMB
Microorganism
E.coli
Enterobacter
E. coli
Enterobacter
present in the present in the
1.0mL
1.0mL
inoculation
inoculation
E. coli
Enterobacter
present in the present in the
1.0mL
1.0mL
inoculation
inoculation

V.

VI.

Conclusion
The presence of coliform in water was detected using lactose broth,
brilliant green lactose bile broth, and eosin methylene blue agar. The
standard MPN of coliform bacteria was then calculated. E. coli and
Enterobacter was observed on all the techniques done, and these bacteria
are part of the coliform group of bacteria. The multiple-tube technique was
done in order to validate the presence of coliform in the chosen water
sample.
References
American Public Health Association (APHA), Standard Methods for the
Examination of Water and Wastewater (19th ed.), APHA, Washington,
DC (1995).