Contents

ABSTRACT............................................................................................................................................ 2
RESULTS ............................................................................................................................................... 4
CALCULATION .................................................................................................................................... 4
DISCUSSION ......................................................................................................................................... 6
CONCLUSION ....................................................................................................................................... 7
RECOMMENDATIONS ........................................................................................................................ 8
APPENDICES ........................................................................................................................................ 9
REFERENCES ..................................................................................................................................... 10

Figures and Table

Figure 1: Methodology of Membrane Filtration Technique ................................................................... 3
Figure 2 : Parafilm M used ................................................................................................................... 9
Figure 3 : Effluent sample …………………………………………………………………………………………………………………9
Figure 4 : Influent sample ………………………………………………………………………………………………………….……..9
Figure 5 : Incubated samples for 18hr ……………………………………………………………………………………………….9
Table 1 : Result obtained ……………………………………………………………………………………………………………..….4

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ABSTRACT

The Membrane Filter (MF) Technique was introduced in the late 1950s as an
alternative to the Most Probable Number (MPN) procedure for microbiological analysis of
water samples. The MF Technique offers the advantage of isolating discrete colonies of
bacteria, whereas the MPN procedure only indicates the presence or absence of an
approximate number or organisms (indicated by turbidity in test tubes).

The membrane filter (MF) technique is highly reproducible, can be used to test
relatively large sample volumes, and usually yields numerical results more rapidly than the
multiple-tube fermentation procedure. The MF technique is extremely useful in monitoring
drinking water and a variety of natural waters. However, the MF technique has limitations,
particularly when testing waters with high turbidity or large numbers of non-coliform
bacteria. When the MF technique has not been used previously, it is desirable to conduct
parallel tests with the method the laboratory is using currently to demonstrate applicability
and comparability.

As related to the MF technique, the coliform group is defined as those facultative

anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that develop red colonies
with a metallic (golden) sheen within 24 h at 35°C on an Endo-type medium containing
lactose. Some members of the total coliform group may produce dark red, mucoid, or
nucleated colonies without a metallic sheen. When verified these are classified as atypical
coliform colonies. When purified cultures of coliform bacteria are tested, they produce
negative cytochrome oxidase and positive -galactosidase test reactions. Generally, pink
(non-mucoid), blue, white, or colourless colonies lacking sheen are considered non-coliforms
by this technique.

Membrane filters have a known uniform porosity of predetermined size

(generally 0.45 µm) sufficiently small to trap microorganisms. Using the membrane
filter technique, sample is passed through the membrane using a filter funnel and vacuum
system. Any organisms in the sample are concentrated on the surface of the membrane. The
membrane, with its trapped bacteria, is then placed in a special plate containing a pad
saturated with the appropriate medium. The passage of nutrients through the filter during
incubation facilitates the growth of organisms in the form of colonies, on the upper surface of

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the membrane. Discrete colonies thus formed can be easily transferred to confirmation
media.

There are several benefits of the Membrane Filtration Technique which is Membrane
filter technique is an effective, accepted technique for testing fluid samples for
microbiological contamination. It involves less preparation than many traditional methods,
and is one of a few methods that will allow the isolation and enumeration of
microorganisms. Membrane filters are used extensively in the laboratory and in industry to
sterilize fluid materials. It also permits testing of large sample volumes and provides presence
or absence information within 24 hours.

Figure 1: Methodology of Membrane Filtration Technique

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RESULTS

Table 1 : Result obtained

Influent Effluent
Dilution Factor No. of colony No. of colony
(CFU/100ml) (CFU/100ml)

6 6000 10 10000
RAW
95 95000 6 6000

10^4 113 1.13*10^9 54 5.4*10^8

10^6 1 1*10^9 1 1*10^9

10^7 18 1.8*10^11 48 4.8*10^11

10^8 19 1.9*10^12 76 7.6*10^12

CALCULATION

Sample calculation:

For Influent Sample

 No Dilution

E.coli in term of CFU/100ml = 6 x 1 x

= 6 x 103CFU/100ml

E.coli in term of CFU/100ml = 95 x 1 x

= 95 x 103CFU/100ml

 Dilution 104

E.coli in term of CFU/100ml = 113 x 104 x

= 1.13 x 109 CFU/100ml

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  Dilution 106

E.coli in term of CFU/100ml = 1 x 106 x

=1 x 109 CFU/100ml

 Dilution 107

E.coli in term of CFU/100ml = 18 x 107 x

= 1.8 x 1011 CFU/100ml

 Dilution 108

E.coli in term of CFU/100ml = 19 x 108 x

= 1.9 x 1012 CFU/100ml

For Effluent Sample

 No Dilution

E.coli in term of CFU/100ml = 10 x 1 x

= 10000 CFU/100ml

E.coli in term of CFU/100ml = 6x 1 x

= 6000 CFU/100ml

 Dilution 104

E.coli in term of CFU/100ml = 54 x 104 x

= 5.4x 108 CFU/100ml

 Dilution 106

E.coli in term of CFU/100ml = 1 x 106 x

=1 x 109 CFU/100ml

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  Dilution 107

E.coli in term of CFU/100ml = 48 x 107 x

=4.8 x 1011 CFU/100ml

 Dilution 108

E.coli in term of CFU/100ml = 76x 108 x

= 7.6 x 1012 CFU/100ml

DISCUSSION

The experiment was conducted by using three types of samples which is tab water and
lake (Influent) and lake (Effluent) water. The experiment was conducted by using differences
dilution factor in each sample. There were about five different dilution factor taken to
conduct this experiment which is 100 , 104 , 106 , 107 𝑎𝑛𝑑 108 . There were four group in the
class so each group done different dilution factor and will combined the data, as for group 1,
the dilution factor that been carried out is dilution factor of 100 𝑎𝑛𝑑 108 . While conducting
the experiment, the dilution factor for 108 is been done correctly and carefully to avoid any
mistakes occur and can avoid error in data.

The term “faecal coliform” has been used in water microbiology to denote coliform
organisms which grow at 44 or 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. Usually, more than 95 per cent of thermotolerant coliforms isolated from
water are the gut organism Escherichia coli, the presence of which is definitive proof of
faecal contamination. As a result, it is often unnecessary to undertake further testing to
confirm the specific presence of E. coli. In the laboratory thermotolerant coliforms are grown
on media containing lactose, at a temperature of 44 or 44.5 °C. They are provisionally
identified by the production of acid and gas from the fermentation of lactose.

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 From the result data, the highest colony data in influent is 19 colonies in lake influent
for 108 dilution factor at 1.9*10^12 (CFU/ 100ml) while the lowest 6 colonies in raw influent
for 100 dilution factor at 6000 (CFU/ 100ml). This is shown that the raw water have less total
coliforms and faecal coliforms present. The highest colony in effluent data is 76 colonies in
lake effluent for 108 dilution factor at 7.6*10^12 (CFU/ 100ml) while the lowest colony form
is 6 colonies in raw influent for 100 dilution factor at 6000 (CFU/ 100ml). This is shown that
the raw water have less total coliforms and faecal coliforms present.In overall the test shown
that, the is result of the colony is unproportionable colony counts that do not correlate to
dilution factors. This occur because of there were group were done the test is not shaking or
mixing well the dilutions before plating. It’s need to mix well in between dilutions and do not
let dilutions set very long before plating.

CONCLUSION

As conclusion the Microbiology Filtration Technique is an effective, accepted

technique for testing fluid samples for microbiological contamination. It involves less
preparation than many traditional methods and is one of a few methods that will allow the
isolation and enumeration of microorganisms. The Microbiology Filtration Technique also
provides presence or absence information within 24 hours.

Thus, conclusive the test shown that, the is result of the colony is unproportionable
colony counts that do not correlate to dilution factors. This are may due to certain error when
handling water samples and when conducting the test. There are several precaution steps that
can be taken before conducting the test. Firstly, theoretically almost any volumes of non-
turbid water could be filtered through the disk, the organisms from any given volume being
deposited in the disk. Therefore, we should be careful when handling the filter paper, the
forceps need to be cleaned and the filter paper must be placed correctly on top of the funnel
to vacuum.

Next, when transfer the membrane to one medium to another should be fast enough
and correctly placed to ensure full contact between samples and buffered water. This is for
purposes of selection or differentiation of organisms thus allowing isolation and enumeration

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of discrete colonies of bacteria. Thus, when conducting the experiment, the dilution factor for
108 need to be done correctly and carefully to avoid any mistakes occur and can avoid error
in data.

RECOMMENDATIONS

There are some improvement that can be made to get a more accurate result:

i) Sample selection
Samples that contain a high level of bacteria must be diluted, so that the bacteria that
grows on the filter is at a density that can be measured. The ideal sample volume for non-
potable water or wastewater yields 20–80 coliform colonies per filter. When the sample
volume is less than 20 mL (diluted or undiluted), add 10 mL of sterile dilution water to
the filter funnel before vacuum is applied. The dilution water will help to distribute the
bacteria uniformly across the membrane filter.

ii) Sample dilution

Non-potable water samples must be diluted to a level at which the bacteria can be
measured. The ideal sample volume for total coliform testing yields approximately 20 to
80 coliform colonies and not more than 200 colonies of all types per filter. Ideal sample
volumes for fecal coliform testing yield approximately 20 to 60 coliform colonies per
filter. Analyze three different sample volumes when the coliform number is uncertain.

iii) Accuracy check

Positive and negative controls are important. Pseudomonas aeruginosa is recommended
as negative control, while Escherichia coli is recommended as positive control for total
and fecal coliforms. Potable water samples from municipal treatment facilities should be
negative for total coliforms and fecal coliforms.

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APPENDICES

Figure 2: Parafilm M used Figure 3 : Effluent sample

Figure 4 : Influent sample Figure 5 : Incubated samples for 18hr

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REFERENCES

 https://microbeonline.com/membrane-filter-technique/
 https://laboratory.pall.com/content/dam/pall/laboratory/literature-library/non-gated/id-
7290.pdf

 Hay J1, K. W. (n.d.). Membrane filtration method for bacteriological testing of water:
enhanced colony visualization and stability on purification of phenol red indicator.
Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/7764596

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