1.

0 INTRODUCTION

Traditionally, indicator bacteria have been used to determine any possible presence of
fecal contamination and to estimate the amount of contamination in water and foods. Present
standards for the sanitary quality of the materials such as water and foods, with respect to
fecal contamination which based on the concentrations of indicator bacteria. Total Coliform
Fermentation Technique is the most widely used indicator bacteria which define as “All of
the aerobic acid and facultative anaerobic, Gram-negative, non-spore forming rod-shaped
bacteria which ferment lactose with gas formation within 48 hours at 35°C” (In, 2008). This
technique can be done by either the multiple-tube fermentation or by using the membrane
filter.

The SOP of this technique involves a series of preliminary and confirmatory tests in
which gas bubbles are formed in small glass vials as it is the prove of the action of coliforms
bacteria in broth medium. Basically, this experiment was about to determine the Most
Probable Number (MPN) of bacteria (per 100mL sample) which is one of the most used
technique, to distinguish total coliforms from other bacteria and to understand the method of
dilution to extinction. During the present work, the method that been involved was total
coliforms which defined as bacteria and its been composed various type of bacteria that
belong to Enterobactecteriaceae’s group. This group of bacteria can be mostly found in
certain location in general human, animal waste, soil and water in which influenced by
surface water (Coliform Bacteria in Drinking Water Supplies, 2007).

The existence of total coliforms may or may not indicate contaminate of fecal. The
total coliforms count commonly give a general indication of the sanitary condition of a water
supply. On the other hand, for the extreme cases, a low or even zero count for thermotolerant
coliforms in a high count for a total coliform group. It is not important to indicate the
existence of fecal contamination. The growth of the types of coliform can be suitable with the
presence of soil or organic matter in the water or by conditions its suitable for.

The experiment was usually tested by carrying out the multiple-tube fermentation
technique. The method results in imprecise quantification. The growth of bacteria is able
determine by increasing the media turbidity and the changes in individual color. Meanwhile,
by placing a small inverted Durham tube in the media to trap any gas produced, the gas
production is can be determined.
OBJECTIVES

Objectives that can be obtained from this experiment were:

1) To distinguish total coliforms from other bacteria,

2) To understand the method of dilution to extinction, and
3) To determine the MPN (Most Probable Number) of bacteria per 100mL sample
2.0 PROCEDURE

Preparation of McConkey’s Broth :

Lactose, peptone and

sodium chloride was
placed in 500 mL
beaker and mixed with
250 mL distilled water
(A).
A and B was mixed in
The 2.5g bilesalt was
500 mL volumetric
dissolved with 100 mL
flask and added with
diatilled water in 500
100 mL of distilled
mL beaker (B).
water.

The 1% Bromocresol Distilled water was

The pH was adjusted Purple was added in added until the final
to 7.4. ethanol (10 mL + 1L volume became 500
Broth) mL.

Preparation of 1% Bromocresol purple :

The 1g Bromocresol Purple salt (C4H16Br2O5S) was

placed in 250 mL beaker.

The 100 mL ethanol was added and mixed thoroughly.

The 1% of Bromocresol Purple solution was transfer into

25 fermentation tubes by using burette.

The 4 dilution bottles containing 9 mL Ringers solution,

pipette in metallic container, 25 fermentation tubes
containing 12 mL McConkeys Broth and Durham tubes
were autoclave for 1 hour 45 minutes at 121 ℃ to kill
microorganism and prevented contamination happen.
Dilution and incubate steps :

By using sterile pipette,

The fermentation
inoculated the McConkeys
The sample was tubes were
Broth with starting from the
diluted in Ringers arranged in rows
highest dilution ( 5x 10-4 mL
solution. of five each in a
sample dilution until 5x 100
test tube rack.
mL original water sample.

The results for absence

The inoculated The water sample
of heavy growth, gas
tubes were was mixed with
and acidic production
incubated at 35 ℃ McConkeys'
was recorded after 48
for 48 hours. Broth gently.
hours.
3.0 RESULTS AND DISCUSSION

This experiment is all about the microbiological analysis of standard presumptive

total coliform fermentation technique. The sample that has been used is from MICET sewage.
Basically, this experiment runs 5 sets which contain different dilution such as 100, 101, 102,
103, and 104. Each of the set have 5 tubes which means 25 tubes has been used in order to
determine the present of the bacteria.

Figure 3.0: Fermentation tubes containing 12mL McConkey’s after autoclave

Figure 3.1: Solution of water sample and McConkeys' Broth after incubate for 2 days
Based on the observation in Figure 3.0, the colour of the solution is purple after
autoclave and the colour of the solution remain after adding the dilution sample and Durham
tubes but, in the Figure 3.1, the colour of the solution change to milky yellow after 2 days
incubate at 35 ºC. The changes of the solution colour mean there are presence of the total
coliform of e-coli because the total coliform is grown in or on a medium containing lactose at
temperature 35 ºC (Bartram J. and Pedley S., 1996). The presence of the total coliform also
can be determined by the production of gas from the fermentation of lactose where there was
presence of gas in the tubes when the cover of the tube is being removed.

Table 3.0: Present of total coliform in the tube

Dilution 100 101 102 103 104
Sample
Source No of
4/5 3/5 2/5 3/5 5/5
Positives

Combination
4-3-2
of positives

1
Factor  m 2
vi ni  2
SElog   MPN 2  
applied
 i 1 exp vi MPN   1 


MPN/100mL 39

The Most Probable Number known as MPN is most commonly applied for quality
testing of water to ensure whether the water is safe or not in terms of bacteria present in
it that have stated by Nisha Rijal (2017) and Robert Blodgett (2010). The presence of the total
coliform also has been proved with the value of the Most Probable Number (MPN) that has
been taken from the Table 7.0 in the appendix by de Man, J. C. (1983). In order to determine
the MPN number, each set of the dilution tubes for 100, 101, 102, 103, and 104 that contain
bubble in the Durham tubes is being calculated as shown in Table 3.0. the result shows that
the combination of the positives was 4-3-2 so the MPN/100mL that get from the Table 7.0
was 39 and it lower than 100. However, it has been proved by Peeler, J. T., (1992) stated that
the MPN is particularly useful for low concentrations of organisms that less than 100/g,
especially in milk and water or untreated water, and for those foods whose particulate matter
may interfere with accurate colony counts. Hence, the result for this experiment was
achieved.
 Moreover, there might be some error during run the experiment it may occurred
during weigh the McConkey’s broth and during measure the broth solution may not accurate.
Besides, the result also can be affected during inoculate the duplicate solutions that can cause
contamination happened.
4.0 CONCLUSION AND RECOMMENDATIONS

As a conclusion, this experiment have been conduct to identified production of e-coli

in water sample. The presence of the total coliform of e-coli were detected from water sample
that has been collected from UniKL MICET plant. Besides, the value of the MPN/100mL
was 4-3-2. Which we can get the value from Table 7.0 was 39 and it lower than 100.

While conducting this experiment, there were few recommendations can be done to
get an excellent experiment’s results. Firstly, the pipette should be replaced with the latest
technology which is by using micropipette. Micropipette is more accurate and parallax error
can be avoided. Secondly, the beakers use for solution preparation must be cleaned to remove
any unwanted foreign object from contaminating the solutions. Thirdly, the dilution must be
done correctly and it shall not exceed the calibration mark at the Erlenmeyer flask or ideal
expected results will not be achieved.
5.0 REFERENCES

Bartram J. and Pedley S., (1996). United Nations Environment Programme and the
World Health Organization: Microbiological Analyses.

de Man, J. C. (1983). MPN tables, corrected. Eur. J. Appl. Biotechnol. 17:301-305.

Nisha Rijal (2017). Microbe Online: Most Probable Number (MPN) Test: Principle,
Procedure and Results. Retrieved February 26, 2018, from
https://microbeonline.com/probable-number-mpn-test-principle-procedure-results/

Peeler, J. T., G. A. Houghtby, and A. P. Rainosek. (1992). The most probable number
technique, Compendium of Methods for the Microbiological Examination of Foods, 3rd Ed.,
105-120

Robert Blodgett (2010). Center for Food Safety and Applied Nutrition. (n.d.).
Laboratory Methods - BAM Appendix 2: Most Probable Number from Serial Dilutions.
Retrieved February 25, 2018, from
https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm109656.htm

Coliform Bacteria in Drinking Water Supplies (2007). [Online]. [Accessed 24-02-2018].

Available from world wide web;
https://www.health.ny.gov/environmental/water/drinking/coliform_bacteria.htm.

In, E. (2008). Techniques In Environmental Helath Sciences, Fall 2008. Indicator Bacteria -
Total and Fecal Coliforms, E.coli, 1–10.
J. Bartram and S. Pedley. (1996). Chapter 10 - Microbiological Analyses. [Online].
[Accessed 24-02-2018]. Available from world wide web:
http://www.who.int/water_sanitation_health/resourcesquality/wqmchap10.pdf.
6.0 APPENDICES

Table 7.0: Reference to determine the MPN of the sample (de Man, J. C. 1983)