Microbial Analysis of Water

Laboratory Report No. 11

Tharrah Anne O. Banogon

Bio 120.1 Section 3 W(1:00-7:00pm)

November 3, 2017
I. Introduction

Water contains sufficient nutrients to support growth of various organism (Patil,

Chaudhari, Kulkarani, & Chincholkar, 2008). In developing countries, natural
sources of water used in settlements are very prone to fecal contamination
(Franchem, 1980; Evison & James, 1977). It is also known that the majority of the
rural population of these countries is dependent on such sources for its domestic
water requirement (WHO, 1973). With this case, periodic bacteriological
examination is performed to detect sewage pollution of water to eliminate the
possibility that disease that can be transmitted by water (Patil, Chaudhari,
Kulkarani, & Chincholkar, 2008).

One of the traditional and easiest way in microbiology to be able to analyze

bacteriological water quality is through most probable number (MPN)
(Pommerville, 2010). The MPN method is a statistics-based test which estimates
the number of fecal coliforms in a water sample based on the degree of lactose
fermentation by organisms in the sample (CollinCountyCommunityCollegeDistrict,
2001). In this laboratory, the sample water to be tested with MPN method. In the
analysis, the detection of coliform bacteria (gram-negative enteric bacteria)
involves three sets of tests, called the presumptive, confirmed, and completed test
(Pommerville, 2010). The goal of this experiment is to determine if the water
sample is contaminated by fecal coliforms and if the water sample is safe for
drinking or not.

II. Materials and Methods

The working area is cleaned and necessary materials are prepared before
starting the experiment. All the material mentioned used in determining the most
viable plate count technique is provided by provided by the Microbiology
Laboratory of the Department of Biological Sciences of the University of the
Philippines Visayas. The water sample to be tested is obtained from a well of
household in Barangay Laguna, Guimbal, Iloilo that is used through an electric
water pump. It is obtained by overflowing an unopened plastic bottle and storing it
in the refrigerator until the day it is used.
A. Presumptive Test

The lactose broth tubes used in this experiment have Durham tubes inside.
Three tubes of double -strength lactose (or lauryl tryptose) broth (DSLB) and six
tube of single-strength lactose broth are obtained and are labelled with the date,
assigned amount of water sample to be transferred, and the name of the
experimenter. Beforehand, a 10mL pipette is sterilized using an autoclave. The
bottle with the water sample is shaken multiple times. Using the sterile 10mL
pipette, 10mL of the water sample is aseptically transferred into the three tube of
double-strength lactose broth (Figure 1A) and the tubes are mixed carefully.

Then 1mL of the water sample is aseptically transferred to three tubes of single-
strength lactose broth (SSL) (Figure 1B). The three tubes are mixed carefully.
Another three tubes of single-strength lactose broth are mixed with 0.1mL of the
water sample (Figure 1C). All the lactose tubes are then incubated for 24 hours at
a temperature of 350C.

A B C

Figure 1. Presumptive test, (A) 10mL water sample to 3 DSL tubes, (B) 1mL
water sample to 3 SSL tubes and (C) 0.1mL water sample to 3 SSL tube.

The tubes are observed after 24 hours and the tubes that have shown any
presence of gas (10% or more) are positive for the presumptive test. After
observing the tubes, they are incubated again for another 24 hours at 350C. After
the given incubation period and the tubes are observed again. The tubes that
produce gas after 48 hours is indicated as a doubtful test and the tubes that haven’t
shown any sign of gas production are negative for the presumptive test.
 B. Confirmed Test

Positive tubes with different dilution of samples are selected to be used in this
test. One Eosin-Methylene Blue (EMB) plate is labelled and is divided into three
parts for the 3 dilution of water samples from the tubes selected. An inoculating
loop is sterilized by passing it through the flame produced by an alcohol lamp. The
tubes are inoculated into the EMB plate and is incubated for 48 ± 3 hours at a
temperature of 350C. after the incubation period coliform colonies are then
identified and results are recorded.

C. Completed Test

The plate from the confirmed test is used in this test. The culture from the three
section of the plate that shows a green metallic sheen characterization is gram-
stained. The smear of the culture is then evaluated if there is demonstration of
gram-negative non-sporing rods bacteria then the culture is a satisfactory
completed test.

D. Determination of E. coli

After the MPN method further confirmation of the presence of E. coli is

performed if the coliform contaminant from the results is E. coli (the EMB plate
cultures would show a metallic green sheen). If this is present, the sample is
inoculated from these plates on the following media: SIM agar butt, two MRVP
broth tubes, and citrate slant. Then the biochemical reactions in these media are
determined after 48 hours of incubation. A typical E. coli would be positive for indole
and methyl red production and negative for the rest.

III. Results and Discussion

All the lactose broth tubes in the following tables below are incubated for 24
hours at a temperature of 350C when it was observed. A following column is added
id the tube produced any gas after 48 hours of incubation at 350C. The sample
teste is from a well of household in Barangay Laguna, Guimbal, Iloilo that is
obtained and used through an electric water pump. It is obtained by overflowing an
unopened plastic bottle. It was taken on October 10, 2017 and is tested on October
11, 2017. The sample is stored on a refrigerator for the night of October 10, and
was brought to the microbiology lab at 1:00pm. Before the sample was tested, it
was shaken for about 25 times.
A. Presumptive Test

Table 1. Double-Strength Lactose (DSL) with 10mL water sample.

After 24 After 48
Tube hours Description (24 hours) hours Remarks
Number
+ - + -
1 Broth turns into darker color of brown, very
Positive
turbid, forms sediments and it produces gas
test.
(bubbles >10%) on the Durham tube.
2 Broth turns into darker color of brown, very
Positive
turbid, forms sediments and it produces gas
test.
(bubbles >10%) on the Durham tube.
3 Broth turns into darker color of brown, very
Positive
turbid, forms sediments and it produces gas
test.
(bubbles >10%) on the Durham tube.
*The check mark indicates if there is gas production more than 10 % on a tube.

The Double-Strength lactose broth tubes after observed for 24 hours

shows the production of gas by the indication of great number of bubble in the
Durham tubes. After 48hours the tubes condition is still the same. The result is
all the three tubes are positive test.

Table 2. Single-Strength Lactose (SSL) with 1mL of water sample.

After 24 After 48
Tube hours Description (24 hours) hours Remarks
Number
+ - + -
1 Broth turns into darker color of brown,
Positive
turbid, forms sediments and it produces gas
test.
(bubbles >10%) on the Durham tube.
2 Broth is in the same state and no changes, Negative
it is clear and bubble formation is negative. test
3 Broth turns into darker color of brown,
Doubtful
turbid, forms sediments and it produces gas
test.
(bubbles <10%) on the Durham tube.
*The check mark indicates if there is gas production more than 10 % on a tube.

Only one the SSL tubes with 1mL of the water sample is positive for the
presumptive test. The tube number 2 is negative for the result and tube number
 3 is a doubtful result since the significant amount of bubbles (bubbles >10%)
only appears after it is incubated for 48 hours.

Table 3. Single- Strength Lactose with 0.1mL water sample.

After 24 After 48
Tube hours Description (24 hours) hours Remarks
Number
+ - + -
1 Broth turns into darker color of brown,
Positive
turbid, forms sediments and it produces gas
test.
(bubbles >10%) on the Durham tube.
2 Broth turns into darker color of brown,
Positive
turbid, forms sediments and it produces gas
test
(bubbles >10%) on the Durham tube.
3 Broth turns into darker color of brown,
Doubtful
turbid, forms sediments and it produces gas
test.
(bubbles <10%) on the Durham tube.
*The check mark indicates if there is gas production more than 10 % on a tube.

Two of the SSL tubes with 0.1mL water sample are positive for the presumptive
test and tube number 3 is a doubtful result since the significant amount of bubbles
(bubbles >10%) only appears after it is incubated for 48 hours.

The results of the lactose broth tubes give an MPN index number of 3-1-2. A
3-1-2 test results indicates that 120 coliforms are present in 100 mL of sample
based on the table found in the Appendices (Most Probable Number (MPN) Index
for Various Combinations of Positive and Negative Results When Three 10-ml
Portions, Three 1-ml Portions and Three 0.1-ml Portions Are Used ). The doubtful
tubes listed on the following tables above are said so because they didn’t produce
significant amount of gas which is the form of bubbles in the Durham tubes. The
organisms in the doubtful tubes are non-fecal coliforms and are said to cause
doubtful sanitary significance (Mara, 1978).

B. Confirmed Test
Positive lactose tubes of different dilution of samples are inoculated on an EMV
plate. The illustration below shows the cultures after incubating the plate for 48
hours at 350C.
 Figure 2. EMB inoculation of 3 positive tubes from the presumptive test. (1)
DSL with 10mL of water sample, (2) SSL with 1mL of water sample and (3) SSL
with 0.1mL of water sample

The first section shows growth of a dark purple culture, which indicates that
there is a presence of a gram-negative bacteria that is a lactose fermenter
(DalynnBiologicals, 2004). The second section shows a green metallic sheen
culture. According to Dalynn Biologicals, this kind of culture have bacteria that are
fast-lactose fermenters and with all the characterization of the culture in quadrant
2, it pertains to one species of bacteria, Escherichia coli (2004). Then lastly the
third section which was inoculated with a SSL mixed with 0.1ml of the water
sample, shows a pink colored culture. This type of color differentiation pertains that
there is presence of gram-negative bacteria that are non-lactose fermenter
(DalynnBiologicals, 2004).

C. Completed Test
The green metallic sheen culture in section 2 of Figure 2, is further tested for
the presence of Escherichia coli. The test to confirmed the presence of the said
bacteria is done by gram staining.
 The microorganism observed on
the stained smear is rod-like in shape and
retained red dye (safranin) which
indicates that it is a gram-negative
bacterium.

Figure 3. Gram Stain of an EMB culture from section 2.

This result from the gram staining further confirmed that the culture in section
2 of the EMB plate has the presence of Escherichia coli. From the results of the
presumptive test, it can be concluded that the water sample tested is not safe for
drinking or non-potable and contains fecal coliforms.

D. Determination of E. coli

Multiple tests are needed to really detect and further confirmed E. coli. The
culture used for the several tests is from section 2 of the EMB plate (Presumptive
Test). The colonies are inoculated into 2 MR-VP broth tubes, a SIM stab, and a
citrate slant.

Figure 4. Determination of E. coli

The tubes are incubated for 72 hours on room temperature (280C). Based on
the results gathered no changes are seen in the SIM stab and citrate slant. For
MR-VP, it is positive for methyl-red, negative for indole and positive acetyl-carbinol,
indicating that other bacteria were present in the latter observations.
IV. Literature Cited

• Aneja, K. R. (2003). Experiments in Microbiology, Plant Pathology and

Biotechnology. New Delhi: New Age International Publishers.

• CollinCountyCommunityCollegeDistrict. (2001). Most Probale Number (MPN)

Presumptive Test. Retrieved from Collin County Community College District Net:
http://iws2.collin.edu/dcain/CCCCD%20Micro/most_probable_number_presumptive.h
tml

• DalynnBiologicals. (2004). EOSIN METHYLENE BLUE AGAR. Dalynn Biologicals

Inc.

• Evison, L., & James, A. (1977). Microbial criteria for tropical water quality . Wastes
and Health in Hot Climates (ed. R. G. Feachem, M. G. McGarry, & D. D. Mara), ch.3.
Chichester: Wiley.

• Franchem, R. (1980). Bacterial standards for drinking water quality in developing

countries. Lancet ii, 255-256.

• Mara, D. D. (1978). Bacterial Analysis of Drinking Water. In Small Water Supplies,

Ross Institute Bulletin No.10 (ed. S. Cairncross and R. Feachem), appendix A.
London: School.

• Patil, U., Chaudhari, A., Kulkarani, J., & Chincholkar, S. (2008). Foundations in
Microbiology; Fifth Edition. Abhyudaya Pragati: NIRALI PRAKASHAN.

• Pommerville, J. C. (2010). Alcamo's Fundamentals of Microbiology:Body Systems .

Sudbury, Massachusetts: Jones and Barltett Pubishers .

• WHO. (1973). Community water supply and sewage disposal in developing

countries. World Health Statistics Report 26 (11), 720-783.
V. Appendices

Table (1): Most Probable Number (MPN) Index for Various Combinations of
Positive and Negative Results When Three 10-ml Portions,Three 1-ml
Portions and Three 0.1-ml Portions are used.