PRACTICAL 5

Biological parameters

INTRODUCTION

The heterotrophic plate count, formerly known as the standard plate count is a method of assessing the
total number of several kinds of bacteria in the water sample (water, wastewater, changes during
wastewater treatment). Colonies may arise from pairs, chains, cluster or single cells, all of which
included in the term of colony-forming units (CFU). Three different methods can be used to determine
CFU; pour plate method, spread plate method, and membrane filter method.

Coliform bacteria are found in the intestines of humans and other animals. Therefore, the presence of
these kinds of bacteria is an indication of fecal contamination from human or animal waste products.
The coliform group consists of several genera of bacteria belonging to the family Enterobacteriaceae.
The historical definition of this group has been based on the method used for detection (lactose
fermentation). When the fermentation technique is used, this group is defined as all facultative
anaerobic, gram-negative, nonspore forming bacteria that ferment lactose with gas and acid formation
within 48 h at 35oC. The standard test for the coliform group can be carried out by multiple-tube
fermentation technique or presence-absence procedure. In this experiment, the multiple-tube
fermentation technique is described. Results of the examination of replicate tubes and dilutions are
reported in terms of the Most Probable Number (MPN) of organisms present. This number, based on
certain probability formulas, is an estimate of the mean density of coliforms in the sample. This
experiment will focus on biological parameter testing through spread plate and standard total coliform
fermentation techniques.

OBJECTIVE

After conducting this experiment, students should be able to:

1. Analyse the presence of common microorganisms in water samples.

2. Analyse the presence of fecal microorganisms in water samples.
3. Evaluate the water quality based on its bacteriological characteristics.

MATERIALS AND EQUIPMENTS

Standard plate count:

 Sterile water
 Tryptone glucose extract agar
 Sterile bottle with stopper
 Sterile flask for water blank
 Five sterile pipettes
 Four sterile Petri dishes
 Bent glass rod
 Incubator
Coliform bacteria:

 Lactose Broth
 Sterile bottle with stopper
 Nine fermentation tubes containing lactose broth and a small inverted vial
 Four sterile pipettes
 Incubator

1. Heterotrophic plate count (Spread plate technique)

a) A series of dilutions has been prepared from original samples as follows:

i. A sterile water blank containing about 100 ml of sterile has been obtained water
and ten sterile test tubes.
ii. Each tube is labelled from 101 to1010 .
iii. A sterile pipette has been used to transfer 9 ml of sterile water into each tube.
iv. A sterile pipette has been used to transfer 1 ml of water sample to the first test
tube. There has been Mix well and continued to transfer 1 ml of sample from
the first tube to the second tube. The procedure has been repeated until all
dilutions complete.
b) 0.5 ml of diluted sample has been pipette onto the surface of a pre-dried agar plate (tryptone
glucose yeast extract agar).
c) A sterile bent glass rod has been use to distribute inoculums over the surface of the medium by
rotating the dish using hand or on a turntable.
d) The inoculums have been entirely absorbed into the medium before incubating.
e) The Petri dishes has been incubate for 48 hours at 35oC
f) The number of bacterial colonies growing on each plate has been count after 48 hours. Since the
size of the original sample has been know, the number of bacteria should been determine with
per millilitre of the original sample.
g) Number of bacteria/ml (CFU/ml) = No of colonies/volume of sample x dilution factor.

2. Standard total coliform fermentation technique

a) A series of dilutions has been prepared from original samples as described in spread plate
method (1a).
b) Fermentation tubes containing 10 ml of sterile lactose broth and Durham tube has been
obtained.
c) Dilution should contain in to three tubes. For example, three should be labelled with 101 of
dilution; three has been labelled with 102 of dilution and three has been labelled with 103 of
dilution and so on.
d) A sterile pipette has been used to place 1 ml of the water sample of each dilution into each of
the fermentation tubes labelled with sample dilution accordingly.
 e) The solutions have been Mix well and incubate at 35oC for 24 hours.
f) The tube has been examined after 24 hours for the presence of gas in the small inverted vial.
g) At the end of 48 hours the solution has been Examine again.
h) All tubes that show a positive test has been recorded. A test is positive if the gas has collected in
the vial and the culture is cloudy.
i) After incubation, the number of tubes showing growth (marked positive) and no growth
(marked negative) has been recorded.

RESULTS

Heterotrophic plate count

Dilution 101 102 103 104 105 106 107 108 109 1010

Number of colonies

CFU/ml

Show calculations:

Total coliform

Number of positive tubes

MPN / ml Show calculations:

DISCUSSIONS

The experiment that we ran is a failure as no results were gained throughout the experiment.
Neither the Heterotrophic Plate Count nor Total Coliform. There are reasons that might lead to the
failure in getting results for this experiment.

Firstly, the correct time taken for the incubation usually will take from few hours and up to 7
days to get better results. But in our experiment, we only did the experiment for two hours and waiting
for the results 24 hours after that. So, during that time, the bacteria growth might not take place as it
can be considered still early for them to form colonies of theirs. Next, while we’re about to look for the
results, the Petri dish containing agar and the bacteria itself was melted after being incubated, and
considered to be broken and cannot be recycled again. This occur due to the overheating of the
incubator and the temperature might increase whilst the Petri dishes were placed inside it. The melting
of the dishes ruined the whole process of the forming of the colonies of bacteria as the temperature is
too high in the incubator. Even though it will not cause heat shock to the bacteria but the change in
shape of the Petri dishes might influences their formation. Supposedly, this heterotrophic plate count is
actually a screening test to any presence of bacteria, and the bacteria should form in colonies by their
types. Each colony comprise of different kind of bacteria.

Next, the second experiment was uncompleted during this experiment was hand on. This is
because the data that we obtained was not very accurate and hard to make the conclusion. Our group
was given the high dilution for drain water which were 107 ,108 ,109 dan 101 ,102 ,103 for the tap
water. Every dilution, we prepared 3 sample and the we result we get was negative for all the three
solution because there no bubble present in the test tube. Besides, sterile process was not been done in
correct way and it cause the microorganism outside the test tube a reaction together with the sample.

Conclusion

We have studied the interaction between one coliform (E.coli) and several common waterborne
organisms. Clearly, more research with other coliform genera and other aquatic bacteria is needed. The
results from this report and elsewhere indicate that coliform injury and suppression result from complex
biological and physiochemical factors. An understanding of these factors and their control will lead to
greater accuracy in the determination of portable water quality.
Appendix