Table of Contents Page

1.0 OBJECTIVES ........................................................................................................................................................... 3

2.0 APPARATUS/ MATERIALS .................................................................................................................................. 3
3.0 THEORY/INTRODUCTION .................................................................................................................................. 6
4.0 PROCEDURE ........................................................................................................................................................... 7
4.1 Tap water ..................................................................................................................................................................... 7
4.2 Surface Water (Goma lakes) ...................................................................................................................................... 8
5.0 DATA COLLECTION ............................................................................................................................................. 9
6.0 DATA ANALYSIS .................................................................................................................................................... 9
7.0 DISCUSSION .......................................................................................................................................................... 11
8.0 RECOMMENDATIONS ........................................................................................................................................ 12
9.0 CONCLUSION........................................................................................................................................................ 12
10.0 REFERENCES ........................................................................................................................................................ 12

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List of Figures
Figure 1; Preparation of turbid water…………………………………………………………………………...…3
Figure 2; sampling beakers ....................................................................................Error! Bookmark not defined.
Figure 3; Drum containing water and clay………………………………………………………………………..4
Figure 4; student collecting samples from a sedimentation column ......................Error! Bookmark not defined.
Figure 5; Turbidimeter…………………………………………………………………………………………….4
Figure 6; Student Stirring turbid water ................................................................. Error! Bookmark not defined.
Figure 7; Plot of turbidity against time for sampling point 1. ...............................Error! Bookmark not defined.
Figure 8; Plot of turbidity against time for sampling point 3. ...............................Error! Bookmark not defined.
Figure 9; Plot of turbidity against time for sampling point 5. ...............................Error! Bookmark not defined.
Figure 10; Plot of turbidity against time for sampling point 7. .............................Error! Bookmark not defined.

List of Tables

Table 1; Turbidity for discrete settling ..................................................................... Error! Bookmark not defined.
Table 2; Turbidity for flocculent settling.................................................................. Error! Bookmark not defined.
Table 3; 100 x (𝐶𝑡/𝐶𝑜) vs time for discrete settling................................................ Error! Bookmark not defined.
Table 4; 100 x (𝐶𝑡/𝐶𝑜) vs time for flocculent settling ............................................ Error! Bookmark not defined.

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1.0 OBJECTIVES

To get introduced to sampling methods and microbiological examination methods used in water
supply and sanitation.
To determine the concentration of microbiological parameters (i.e. the number of total coliforms
or faecal coliforms per 100 ml) in laboratory tap water and water from the Goma lakes so as to
assess if these water sources have been contaminated by pathogens.

2.0 APPARATUS/ MATERIALS

Colony counter with a Magnifying Lens

Sterile Sartorius funnel.
Alcohol burner.
2 litre suction flask.
Electrical vacuum pump.
Sterile membrane filters 50mm diameter with a pore size of 0.45 microns.
Glass petri dishes with culture medium.
Pair of forceps
Sterile pipettes.
An incubator
Sterilized sampling bottles

Figure 1; Sterilized sampling bottles in a tray

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Figure 2; Electrical vacuum pump Figure 3; Tap water from a tap

Figure 4; Colony counter with a Magnifying Lens Figure 5; Total coliforms incubator

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Figure 6; Total coliform colony on filter medium Figure 7; Feacal coliform colony on filter medium

Figure 8; Feacal coliform incubator

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3.0 THEORY/INTRODUCTION

The most importance of water borne diseases has long been recognised. The main causes of human enteric
diseases are pathogenic microorganisms. Contamination of drinking water by human or animal excrement
constitutes the most common mechanism for the transmission of these organisms to humans, not only
directly, but also indirectly, through food preparation.
The discharge of wastes from municipal sewers is one of the most important water quality issues world-
wide. It is of particular significance to sources of drinking-water. Municipal sewage contains human faeces
and water contaminated with these effluents may contain pathogenic (disease-causing) organisms and,
consequently, may be hazardous to human health if used as drinking-water or in food preparation. Faecal
contamination of water is routinely detected by microbiological analysis.
The primary objective of the bacteriological examination of water is thus the detection of faecal
contamination. Although it is possible to detect the presence of the various pathogens in water, the
isolation and identification of many of them are often extremely complicated and seldom yield qualitative
results. The approach that has been adopted is to analyse for indicator organisms that inhabit the gut in
large numbers and are excreted in human faeces. The presence of these indicator organisms in water is
evidence of faecal contamination and, therefore, of a risk that pathogens are present. If indicator organisms
are present in large numbers, the contamination is considered to be recent and/or severe.
The indicator organisms used are often a group of bacteria which are not pathogenic themselves but
indicate the possible presence of pathogens. These organisms are known as coliform organisms. There
are two types of coliforms; these are total coliforms and faecal coliforms.
Total coliforms refer to a large group of Gram-negative, rod-shaped bacteria that share several
characteristics. The group includes thermo tolerant coliforms and bacteria of faecal origin, as well as some
bacteria that may be isolated from environmental sources. Thus the presence of total coliforms may or
may not indicate faecal contamination. Faecal coliform has been used in water microbiology to denote
coliform organisms which grow at 44.5 C and ferment lactose to produce acid and gas. In practice, some
organisms with these characteristics may not be of faecal origin and the term “thermotolerant coliform”
is, therefore, more correct and is becoming more commonly used. Nevertheless, the presence of
thermotolerant coliforms nearly always indicates faecal contamination.
The advantages of using the coliform organisms include the fact that they are normal inhabitants of the
intestinal tract of humans and animals. They are also not pathogenic except the one of a particular strain
called the Escherichia Coli (E. coli). They are usually excreted in large numbers by both healthy and
carriers, hence easy to identify if the coliform organisms are not present, then the conclusion is that the

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 water did not come in contact with faecal matter, hence no pathogenic bacteria present in water. Since
the number of coliforms is usually much larger than the possible pathogens, there is a great margin of
safety. The use of the coliform bacteria does not require specialised bacteriologists and analysis is not
time consuming. They can easily be detected by relatively simple analytical procedures though the
results can only be obtained after a period of 24 hours after culturing in an oven.
The membrane filtration method can only support the growth of limited number of colonies. The
optimum number being 20 to 80 colonies, the choice of the volume of water to be filtered depends on
the type of water source. The following serves as a guide:-
 Good quality treated water 100 ml
 Untreated drinking water 10 – 50 ml
 Surface water 0.1 – 10 ml
 Raw sewage 0.1 – 0.0001 m

4.0 PROCEDURE
4.1 Tap water

1. The tap mouth was cleaned with cotton wool which was dipped in methylated spirit
2. The tap was opened for one minute to clear the water stored within the pipe so that the water collected is
got from the storage pipes.
3. Then, the tap mouth was sterilized using a cotton wool swab dipped in methylated spirit.
4. The tap was again opened for one minute.
5. A sampling bottle with its lid wrapped in aluminum foil was used to collect the water from the tap and
was labeled
6. The work place was sterilized using methylated spirit.
7. The filter bed and funnel were sterilized using a flame on a cotton wool swab and distilled water was
poured into the funnel.
8. The distilled water was then sucked by the suction pump.
9. The funnel was removed and the membrane filter was put centrally on the funnel bed using sterilized
forceps.
10. The funnel was put back in position.
11. The sample was poured into the funnel until it reached 100 ml level marked on inside of the funnel.
12. The suction pump was turned, after water filtered, the funnel was removed and the filter membrane was
also removed from the filter bed using sterilized forceps.

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 13. The filter membrane is then placed in Petri dish containing MFC Agar media for faecal coliform analysis
making sure bubbles are not trapped in-between.
14. The petri dishes was labeled and put in the incubators for 24 hours.
15. The number of the colonies were counted by putting the petri dishes on the electronic machine to count
the colonies.
4.2 Surface Water (Goma lakes)

1. The sampling bottle was put upside down to a depth of 30 cm turning with the mouth up but at a tilted
orientation away from the hand (creating a current)
2. The sample was then taken to the laboratory.
3. The work area was then sterilized.
4. Six sterilized test tubes were each filled with 9ml of distilled water placed on a rack.
5. Then using a sterilized micro pipette, 1 ml of the sample was transferred from the water sample and
added to 9ml of distilled water in the first test tube to dilute the test sample.
6. The distilled water and the specimen were mixed thoroughly using a mechanical vibrating machine.
7. After thoroughly diluting the sample a 1 ml sample was transferred into the next test tube for further
dilution. A 1ml of this sample was then transferred to the next test tube for further dilution and the same
process was continued and gave a dilution factor of 1:100 (0.01 ml) of the sample.
8. The funnel was mounted on a funnel bed connected to a suction pump and distilled water was added to
the funnel to a certain distance.
9. Then the pump was switched on to suck the water on the funnel.
10. The funnel was then sterilized with a flame and distilled water was poured to the funnel to rinse it.
11. The funnel was removed and the sterile membrane filter was put centrally using sterilized forceps and
the funnel was put back in position.
12. Then the sample in the last test tube was poured in the filter and the pump was switched on until all the
water had gone through the filter.
13. After filtration the filter membrane was removed from the funnel.
14. Then the filter membrane placed nicely in the Petri dish with ensuring that no bubbles are formed.
15. The same procedure was repeated for the same sample but the filter membrane was put in a different
growing medium this time.
16. The two Petri dishes with filter membranes; one for faecal coliforms containing different sample
volumes or dilutions and another one containing two different sample volumes or dilutions for total
coliform determination were put into the incubators upside down.
17. These were incubated for a period of 24 hours before the colonies could be counted.
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5.0 DATA COLLECTION
The results that were obtained from both the laboratory tap water sample and the Goma lakes water
sample are as follows below:

Table 1; water Sample from the tap

Amount of sample per 100ml Total coliforms per 100ml Faecal coliforms per 100ml

100 0 0

Table 2; water sample from the Goma lakes

Amount of sample per 100ml Total coliforms Faecal coliforms

(Dilution factor)
1.0 60 34
0.1 27 9
0.01 0 0

6.0 DATA ANALYSIS

The concentration of microbiological parameters is expressed in units of number of coliforms per 100ml (No.
CFU/100ml). When other smaller volumes are used; the concentration is computed from the formula:

𝑁𝑜.𝑜𝑓 𝐶𝑜𝑙𝑖𝑓𝑜𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

Coliforms/100ml = (
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑(𝑚𝑙)
) × 100

Sample calculation 1( tap water):

0
Total Coliforms/100ml = (100 𝑚𝑙) × 100

= 0 CFU/100ml

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 0
Faecal Coliforms/100ml = (100 𝑚𝑙) × 100
= 0 CFU/100ml

Sample calculation 2( Goma lakes):

 For 1ml dilution factor;

60
Total Coliforms/100ml = (1𝑚𝑙) × 100

= 6000 CFU/100ml

34
Faecal Coliforms/100ml = (1𝑚𝑙) × 100

= 3400 CFU/100ml

 For 0.1ml dilution factor;

27
Total Coliforms/100ml = (0.1𝑚𝑙) × 100

= 27000 CFU/100ml

9
Faecal Coliforms/100ml = (0.1𝑚𝑙) × 100

= 9000 CFU/100ml

 For 0.01ml dilution factor;

0
Total Coliforms/100ml = (0.01𝑚𝑙) × 100

= 0 CFU/100ml

0
Faecal Coliforms/100ml = (0.01𝑚𝑙) × 100

= 0 CFU/100ml

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7.0 DISCUSSION
Coliform bacteria are described and grouped based on their common origin or characteristics, as either
Total or Faecal coliform. The Total coliform group includes Faecal coliform bacteria such as
Escherichia Coli (E.coli), as well as other types of coliform bacteria that are naturally found in the soil.
Faecal coliforms exist in the intestines of warm blood animals and humans, and are found in bodily
waste, animal droppings, and naturally in soil. 95% of the Faecal coliform bacteria is comprised of
E.Coli which is excreted on feacal matter (faeces).

Total coliform do not necessarily indicate recent water contamination by faecal waste. However, the
presence or absence of these bacteria in treated water is often used to determine whether water
disinfection is working properly. The presence of Faecal coliform in well water may indicate recent
contamination of the groundwater by human sewage or animal droppings which could contain
pathogens. This is why the coliform bacteria are considered “indicator micro-organism”; their presence
warns a potential presence of pathogens and should alert the sanitary engineer to take necessary actions.

Two methods for the examination of coliform bacteria in water are available and these are; Membrane
filter method and multiple fermentation tube method. In this experiment the membrane filter method
was used to determine the number of faecal and total coliforms in the two samples from the laboratory
tap and also from the Goma lakes.

It can clearly be seen from the calculated and graphically presented results that the number of Total
coliforms from Goma lakes was more than the number of Faecal coliforms as expected since the Faecal
coliform group is part of the Total coliform bacteria. The presence of both Total coliforms and Faecal
coliforms in the Goma lakes indicate the presence of pathogens in the lake.

Water from both tap had zero coliforms per 100ml and this is because of the chlorine disinfection in the
distribution system. Therefore, using the standards of the World Health Organisation and the Zambia
Bureau of Standards, the water from the taps is fit for human consumption.

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8.0 RECOMMENDATIONS

The experiment should be performed in smaller groups so that everyone can participate and it
would help in maintaining a sterile environment because the laboratory would be less crowded.
A better method of collecting the sample from the Goma lakes such as tying the collection bottle
to a wire and immersing it in the water should be employed. This is because the method we used
of wearing gloves and dipping the hand in the lake was not sufficient considering that the other
part of the hand was in contact with the water during the collection.

9.0 CONCLUSION
It was a great privileged to be exposed to the sampling and bacteriological examination methods used in water
supply and sanitation. Both samples, that is, tap water and water from the Goma lakes had their levels of
contamination found with tap water containing no coliforms and Goma lakes water containing both faecal and
total coliforms. Here it was also learnt that for best results, use of standard plate count is not such an ideal
method. Since the number of total coliforms is more than the number of faecal coliforms it can therefore be
ascertained that these results Reliable and the experiment was correctly done. It can finally be concluded that
the water in the Goma lakes is contaminated by pathogens while that from the laboratory tap is not
contaminated by pathogens.

10.0 REFERENCES
1. Dr. Wolfram Shaefer, (1992) Public Health Engineering, UNZA.
2. Dr. Tembo (2019) CEE 4412 Environmental Engineering Water Quality Lecture Notes, UNZA.
3. J. Bartram and S. Pedley,(1996) chapter 10 - microbiological analyses, World Health Organization.

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