CENTRES OF DIPLOMA STUDIES

GROUP MEMBERS
1
2
3
4
5

UMMI SURAYA BINTI MD YATIM MUSTAFA

HALIMI NURUL ATIQAH BINTI HONAN
WAN FATIN AFIFAH BT WAN MOHD FAUZI
ANISHA ADZIHAN BINTI BAKRI
AINATUL NAIMAH BINTI ZAILAN SHUKRI

MATRIC
NO.
AA120108
AA120212
AA120695
AA121458
AA121403

AMALAN TEKNOLOGI SALIRAN DAN MESRA

ALAM
BACTERIAL COUNT

1.0 OBJECTIVE

Students will be able to measure the bacteriological quality of water sample by

performing total plate count.

2.0 LEARNING OUTCOMES

At the end of the laboratory courses, students will be able
1. Become proficient at dilutions.
2. Become proficient at performing a standard plate count and determining bacterial
counts in a sample.
3.0 THEORY
Bacteria are remarkably adaptable to diverse environmental conditions: they are found
in the bodies of all living organisms and on all parts of the earthin land terrains and
ocean depths, in arctic ice and glaciers, in hot springs, and even in the stratosphere. Our
understanding of bacteria and their metabolic processes has been expanded by the
discovery of species that can live only deep below the earth's surface and by species that
thrive without sunlight or in the high temperature and pressure near hydrothermal vents
on the ocean floor. There are more bacteria, as separate individuals, than any other type of
organism; there can be as many as 2.5 billion bacteria in one gram of fertile soil.
Many studies require the quantitative determination of bacterial populations. The two
most widely used methods for determining bacterial numbers are the standard, or viable,
plate count method and spectrophotometric (turbidimetric) analysis. Although the two
methods are somewhat similar in the results they yield, there are distinct differences. For
example, the standard plate count method is an indirect measurement of cell density and
reveals information related only to live bacteria. The spectrophotometric analysis is based
on turbidity and indirectly measures all bacteria (cell biomass), dead and alive. The
standard plate count method consists of diluting a sample with sterile saline or phosphate
buffer diluent until the bacteria are dilute enough to count accurately. That is, the final
plates in the series should have between 30 and 300 colonies. Fewer than 30 colonies are
not acceptable for statistical reasons (too few may not be representative of the sample),

and more than 300 colonies on a plate are likely to produce colonies too close to each
other to be distinguished as distinct colony-forming units (CFUs). The assumption is that
each viable bacterial cell is separate from all others and will develop into a single discrete
colony (CFU). Thus, the number of colonies should give the number of bacteria that can
grow under the incubation conditions employed. A wide series of dilutions (e.g., 10 -4 to
10-10) is normally plated because the exact number of bacteria is usually unknown.
Greater accuracy is achieved by plating duplicates or triplicates of each dilution.
Increased turbidity in a culture is another index of bacterial growth and cell numbers
(biomass). By using a spectrophotometer, the amount of transmitted light decreases as the
cell population increases. The transmitted light is converted to electrical energy, and this
is indicated on a galvanometer. The reading, called absorbance or optical density,
indirectly reflects the number of bacteria. This method is faster than the standard plate
count but is limited because sensitivity is restricted to bacterial suspensions of 10 7 cells or
greater.

4.0 EQUIPMENTS & MATERIALS

1. Petri plate
2. Pipette
3. Test tube
4. Glass rod
5. Bunsen burner
6. Incubator
7. Ethanol 95% @ methanol
8. Sterilizer
9. Microscope
10. Bacteria medium: Peptone = 5g, Beef Extract=3g, Agar=15g, Distilled water=600
mL

5.0 PROCEDURES

Procedures of preparing nutrient media

1. Peptone, beef extract, agar and distilled water mixed in 600mL beaker and boiled.
2. The agar cooled up to 45-50oC.
3. For the Spread Plate test, the nutrient media poured into half of the six petri plates.
Note: All the agar preparation procedures should be performed under laminar flow to keep
the samples sterile. Use gloves to prevent contamination of the samples
Dilution procedures
1. A clean, sterile, dry pipette used to remove 0.1mL from the bacteria sample and
blew into the 9.9mL of dilution fluid (normally deionizer/distilled water) in tube#1
and mixed thoroughly by blowing lots of bubbles with the pipette for a couple
seconds. The pipette discharged into the used jar for later cleaning. Notice tube#1
now contains 1/100 the concentration of bacteria in the original sample because
0.1mL is 1/100 of 10mL. Since nearly 0.1mL of liquid may cling to the outside of
the pipette, you must wipe the pipette with Kleenex or toilet paper before inserting
the pipette into tube#1.
2. Another clean, sterile, dry pipette used to remove 0.1mL from tube#1, wipe pipette,
blow contents of pipette into tube#2, and continue blowing bubbles for a second or
two for good mixing.
3. Using another clean, sterile, dry pipette remove 0.1mL from tube#2, wipe, blow
contents of pipette into tube#3, continue blowing bubbles for a second or two for
mixing.
4. The same procedures repeated until tube#6. The diagram below referred for better
0.1 mL

understanding.

0.1 mL 0.1 mL 0.1 mL 0.1 mL

0.1 mL

5. Our tubes labeled with the dilution factor as to notice the bacteria content in the
test tubes contain 9.9 mL dilution fluid
test tubes contain 9.0 mL dilution fluid
water
water
sample
sample
1/106
1/10
1/100
1/104
1/105to use
1/103
Note: There are many
types of
pipettes,
and you
are advised
blow out pipette, that is
1.0 mL
1.0 mL
indicated by a frosted ring on the pipette at the top end.
1.0 mL
1.0 mL
1.0 mL
1.0 mL
1/106
1/104
1/105
1/103
water
sample

tubes.

Spread Plate test method

1. A clean, dry, sterile pipette used to remove 0.1mL of diluted sample from each test
tube into six different petri plates contain sterile agar.
2. The petri plate closed.
3. All the petri plates placed inside the incubator for 24 hours with a temperature of
37oC.

Pour Plate test method

1. A clean, dry, sterile pipette used to remove 0.1mL of diluted sample from each test
tube into six different petri plates.
2. The agar poured into the plates, and waited until the agar to solidify.
3. The petri plate closed.
4. All the petri plates placed inside the incubator for 24 hours with a temperature of
37oC.
Methods of counting bacteria
1. After being incubate for 1 day, the petri plates took out.
2. The petri plate placed on the counting chamber.
3. The bacteria colonies on the culture were counted.

6.0 RESULTS & CALCULATIONS / ANALYSIS

Plating

Average

Method

Colony/

Pour

plate
119

Plate
Spread

Dilution
1/102

1/10
190

125

175

200

180

1/103
155
160

1/104
85
90

Total
1/105
75
80

1/106

bacteria

35

/ mL
1.19

40

1023
7.5

1023

Plate

7.0 DATA ANALYSIS

1. Show the calculation for each of the plating method and fill in the above table.
Calculation for Pour Plate Method
Average

= (190+175+155+85+75+35)
6
=

119

Total Bacteria = 119 x 101 x 102 x103 x 104 x 105 x 106

= 1.19x 1023

Calculation for Spread Plate Method

Average

= (200+180+160+90+80+40)
6
= 750

Total Bacteria = 750 x 101 x 102 x103 x 104 x 105 x 106

= 7.5 x 1023
2. Analyze the results by using the appropriate method. Explain your findings.
Based on our findings, we choose Spread Method as the appropriate to
calculate the amount of bacteria. This is because it is a little quicker than the pour
plate method.
3. State the systematic bias error that could occur during this experiment.

Talking during doing the experiment.

When doing the experiment, we should not have to talk out of the
experiment topic because it can cause an error to our experiment.
The temperature need to be in between 40oC-50oC.
The temperature mixing of agar, beef and peptone should be less than
50oC. If the temperature is more than 50oC the result of our experiment is
not the exactly one. Plates with fewer than 20 or more than 200 total
colonies usually give statistically invalid results.
Error cause by equipment
During the experiment, the equipment use must be sterilized if not
it will affect the result. But due to the not functional sterilized
pipette, we use unsterilized pipette, luckily the result is not
ridiculous.
Besides that, all the preparation of procedure must be done under
the laminar flow to avoid the bacteria from entering into the

sample.
Sterilizer door and Incubator.
We have to limit when opening the sterilizer door. There is a marking on
the door that remain us to be very alert with it. If we open the sterilizer door

more than the limit, the sample will have a fungus. The sample that we put
in the incubator need to be for 24 - 48 hours. On our experiment, we done it
for 24 hours which is not give a good result.

4. Usually, the result shows different reading for both methods. However, in some
cases, both methods produce the same result. Explain why the results are
indistinguishable.
Normally we measured volume of each dilution on the surface of an agar
plate and spread it around. Apart from that, not all of the cells in the sample are
placed on the agar surface and well separated from each other as we spread them
around. Some cells will adhere to the "spreader" and you seldom get full separation
of all of the cells the result is being overlapping colonies that might not be
distinguishable from each other.

8.0 QUESTIONS
1. Explain the meaning of phrases two times ten to the eight cells per mL in your
own convenient terminology.

The meaning of the phrases two times ten to the eight cells per m refer to
the experiment is 100000000 bacteria per m is a difficult number to write
down or comprehend. 100,000,000 are not much better. 10 8 mean the same
thing. 108 = 100,000,000 = 100 million. The 8 is referring to the number of
zeros. 103 = 10 x 10 x 10 = 1000.1 x 108 is the scientific notation for 100
million. 2 x 108 is the scientific notation for 200 million. So, thats mean
scientific notation, is easy. "2 times ten to the eighth" means 2 * 10 * 10 * 10 *
10 * 10 * 10 * 10 * 10. Or in simple word two times ten to the eight cells per
mL is there were 200 million bacteria per milliliter. 10 to the 8 are very
familiar to any bacteriologist as a bacterial culture having that titer is just
barely turbid.

2. What the meaning of TNTC and the significant amount due to the TNTC?
Give the formula for determining bacteria count
The meaning of TNTC is To Numerous To Count and significant amount due
to TNTC when sample that we counting are more than 300.
Formula :
Number of bacteria = number of colonies/dilution factor

3. Design the experiment for comparing the bacteria counts of water sample (tap
water, lake water, swimming pool water and rain barrel water). Explain the
different of bacteria count for each type of water sample?
Experiment designed to compare the bacteria counts of water sample like tap
water, lake water, swimming pool water and rain barrel water can be summarized
as follow.
Before that, remember to use sterile tools at all stages of the study.
a. Put a drop water of well water on a sterile plate of TGY or other agar.
b. Spread it uniformly over the surface of the agar with any non-absorbent
sterile tool. A glass rod bent into an L-Shape is ideal. The bottom of a
teaspoon will also work.
c. Incubate the plate at 30oC or at room temperature.

d. Examine the plate frequently. At room temperature, it will probably take a

day or two for single cells to grow into colonies large enough for us to see.
Different species of bacteria grow at different speeds and some species
will take many days.
e. Count the colonies. There are about 18 drops per mL, the size of drop
depends on the orifice. Small tips make small drops and it can take 30+
drops to make one milliliter.
Example calculation of bacteria titer:
If the pipette gives 18 drops per mL and you counted 10 colonies, then the
bacterial count for your sample is 18 x 10 = 180 bacteria per milliliter. Keep in
mind that on TGY agar most common bacteria will grow and the colonies you find
will be mostly harmless bacteria. There are agars which permit the growth of
coliforms or the coliforms give colored colonies.
4. In many experiment there are 2 types of control used which are positive and
negative control. Due to this experiment what is the suitable control? How the
control will effect to your finding?
From the results obtain from this experiment, we found that the positive control
is not the suitable control. This is because both of the control Colonies probably
indicates contamination by water, dust and the most is affect by human error.

9.0 DISCUSSION
Bacteria are found everywhere in our environment. Bacteria reproduce by a process
called fission, or the splitting of an organism into two separate organisms. This process can
occur every 15 minutes under favorable conditions.The plate count method relies on bacteria

growing a colony on a nutrient medium so that the colony becomes visible to the naked eye
and the number of colonies on a plate can be counted. To be effective, the dilution of the
original sample must be arranged so that on average between 10 and 100 colonies of the
target bacterium is grown.
There are two types of Plate method that can be used which is Spread Plate Method
and Pour Plate Method. From the experiment that we have conducted there are small different
that we can see from the value of spread plate method compared to pour plate method.
Theoretically, the result between spread plate method and pour plate method should be same
because they use the same material and quantity of mixture. However, from our result there is
small difference from total of the bacteria and it may be cause by the error while conducting
the experiment.

10.0 CONCLUSION
The objective of the bacteria count is to measure the amount of bacteriological of
water sample by performing total plate count. Bacteria are kind of dangerous microorganisms
and can affect human life through consumption of water and foods. While the vast majority

of bacteria pose no threat to us, in some bacteria can offer bad effect and may produce serious
disease. In order to reduce the incidence of such disease, the bacteria count is very important
to conduct into water supply so that further inspection and treatments.
In this analysis, there are two method has been used to count the number of bacteria
exist in water samples. These are Pour Plate method and Spread Plate method. By using the
Pour Plate method, the average number of colony resulted is 119 which is 1.19 X 10 23
bacteria per ml and by using the Spread Plate method, the average number of colony is 750
which is 7.5 X 1023 bacteria per ml.
The Environmental Protection Agency (EPA) through Water Quality Standard
imposed that for the recreational waters (freshwater) the geometric mean of the indicated
bacterial densities should not exceed one or the other of the following two selected bacteria
that is E. coli 126 per 100 ml or Enterococci 33 per 100 ml. The experiment result revealed
that the water is very unsuitable for human consumes and give bad effect to consumers.
In conclusion, based on this analysis, both method has a different result of bacteria
colony and numbers which is the percentage of different between two results is 85%.
Regarding to this experiment result, the objectives of this experiment was reached and
fulfilled as to measure the bacteria content as well as the water quality.