1.

OBJECTIVE
The bacteriological quality of water sample is measured by performing total plate
count.

2.0

THEORY
Bacteria are the highest population of microorganisma in a wastewater
treatment plant will belong to the bacteria. They are single-celled organisms
which use soluble food. Conditions in the treatment plant are adjusted so that
chemoherotrophs predominate. Bacteria are highly adaptable to diverse
environmental conditions.
Understanding of bacteria and their metabolic processes has been
expanded vastly. 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. In bacterial colony assays, the patterns are formed within culture
media that has been inoculated with bacterial cells. This allows the cells to
reproduce and form bacterial colonies within and/or on the surface of the medium.
When the colonies are sufficiently large, they are usually visible to the naked eye,
which allows researchers to determine the number of colonies formed. In
addition, various visual characteristics of the colonies, such as shape, size,
pigmentation, and opacity, can be used to help determine the type of bacterium
present.
Most experiment requires the quantitative determination of bacterial
populations. The two typical methods for determining bacterial numbers are the
standard, or viable, plate count method and spectrophotometric (turbidimetric)
analysis. Although the two methods yield the similar results, there are distinct
differences. 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. The assumption is that each viable bacterial cell is separate from
all others and will develop into a single discrete colony (CFU).

3.0

MATERIAL
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 = 600mL

4.0

PROCEDURE

Procedures of preparing nutrient media

1. 5g of peptone, 3g of beef extract and 15g of agar are mixed in 600mL distilled
water and boiled.
2. After boiling, the agar is left cooled to 45-50oC.

Dilution
1. 0.1mL of the bacteria sample is blown into a test tube (Tube #1) contained of
9.9mL of dilution fluid (distilled water) by using a cleaned, sterile, dried pipette.
Continue blowing bubbles for a second or two for good mixing
2. 0.1mL sample from Tube #1 is blown into Tube #2, continue blowing bubbles for
a second or two for good mixing.
3. Another 0.1mL from of sample from Tube #2 is pipette and blown into Tube #3,
continue blowing bubbles for a second or two for mixing.
4. The same procedures are repeated until Tube #6.
5. Label your tubes with the dilution factor as to notice the bacteria content in the
tubes.

Spread Plate test method

1. For the Spread Plate test, the nutrient media is poured into half of the six petri
plates.
2. 0.1mL of diluted sample from each test tube is sprinkled into six different petri
plates contain sterile agar using a stick.
3. The petri plates are then closed and labeled.
4. All the petri plates are placed inside the incubator for 24 hours with a
temperature of 37oC.

Pour Plate test method

1. 0.1mL of diluted sample from each test tube is sprinkled into six different petri
plates contain sterile agar using a stick.
2. The agar is poured into the plates and till the agar solidified.
3. The petri plates are then closed and labeled.
4. All the petri plates are placed inside the incubator for 24 hours with a
temperature of 37oC.

Methods of counting bacteria

1. The petri plates are taken out after being incubated for 24 hours.
2. The selected petri plate is placed on the counting chamber.
3. The bacteria colonies on the culture is counted and recorded.

5.0

RESULTS

Plating
Average
Method Colony/plate
Pour
Plate
Spread
Plate

Dilution

Total
bacteria
/ mL

1/10

1/100

1/1000

1/104

1/105

1/106

13.33

14

17

13

23

800

17.83

10

15

17

30

15

1070

20

8.0

DATA ANALYSIS

(1)

Show the calculation for the plating method and fill in the above table.

POUR PLATE
Average Colony/plate = 9 + 14 + 17 + 13 + 23 + 4 = 13.33
6
Total bacteria / mL =
9 + 14 + 17 + 13 + 23 + 4 = 80
80 bacteria = 0.1 mL
? = 1 mL
1 X 80 = 800 bacteria
0.1
1mL = 800 bacteria

SPREAD PLATE
Average Colony/plate = 10 + 15 + 20 + 17 + 30 + 15 = 17.83
6
Total bacteria / mL =
10 + 15 + 20 + 17 + 30 + 15 = 107
26 bacteria = 0.1 mL
? = 1 mL
1 X 107 = 1070bacteria
0.1
1mL = 1070 bacteria

2. Analyze the results by using the appropriate method. Explain your findings.
The method for determining bacterial numbers is the plate count method. The
standard plate count method consists of diluting a sample until the bacteria are
dilute enough to count accurately. For both Spread Plate and Pour Plate method,
the bacteria colonies decrease as the sample are more diluted. This is because the
diluted sample from Tube # 6 containing lesser bacteria compared to Tube # 1.
From the findings, the bacteria colonies from Pour Plate method is more than the
bacteria colonies from Spread Plate method. This might be caused by the
inactiveness of bacteria sample incubated under the agar in the petri plate.
3. State the systematic bias error that could occur during this experiment.
-

A calibration error in a measuring instrument.

Scale errors while counting bacteria using the counting chamber.

Bias error due to different individual in judging colony size.

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 indistinguishasble.
The difference in results may because of the different procedures carried out to
get the bacteria count. For Spread Plate, the nutrient is poured into the petri plate
and then sprinkler the sample. For Pour Plate method, it is the other way round.
The quantity of agar and sprinkled on are not accurately measured and place in all
the petri plate, therefore this different in quantity of agar and sample may
influence the bacteria result. To produce indistinguishable results, the amount of
agar and sample poured must be exactly the same.

7.0

DISCUSSION AND QUESTION

1. Explain the meaning of phrases two times ten to the eight cells per mL in
your own convenient terminology.
2108 cells/mL, is a terminology used to describe the scientific notation for
bacterial count. 2108 cells/mL = 200,000,000 cells/mL.
2. What the meaning of TNTC and significant amount due to the TNTC
Too Numerous to Count (CFU per plate more than 250 colonies)
3. Give the formula for determining bacteria count.
CFU/ mL = CFU/plate x dilution factor x 1/aliquot
4. 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 kind of water sample.
a. Pour the nutrient media is into half of four petri plates.
b. Sprinkle the diluted sample from each water source into four different
petri plates contain sterile agar using a stick.
c. Close the petri plates and label it.
d. Place all the petri plates into the incubator for 24 hours with a
temperature of 37oC.
e. After 24 hours, remove the petri plates from the incubator and count the
bacteria colony in the counting chamber.
The bacteria colonies consist in tap water probably will have the least bacteria
since tap water is treated to reach a safe condition for human consumption. Water
sample from the swimming pool will have minimum bacteria colonies too since
pool water is chlorinated. Lake water and rain barrel water might consist of large

numbers of bacteria colony since the water is untreated and might be

contaminated with various contamination source.
5. 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
far the control will effect to your finding.
Positive control. The positive control confirms that the basic conditions of the
experiment were able to produce a positive result, even if none of the actual
experimental samples produce a positive result.

8.0

CONCLUSION

This experiment is designed to count the bacteria in the selected water sample.
Two method has been used for the counting purpose which is Pour Plate method and
Spread Plate method. By using the Pour Plate method, the average number of colony is
13.33 which is 800 bacteria per mL and the by using the Spread Plate method, the
average number of colony is 17.83 which is 1070 bacteria per mL.
Bacteria are dangerous microorganisms and can affect human health through
consumption of water and foods. However, not all bacteria post threat to human, but there
are 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.
The US 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. By referring to the foreign standard, the collected water
sample result is unsuitable for recreational use and human consumption.
The analysis shows that the percentage difference of the results between two
methods is approximately 25%. The objectives of this experiment was reached and
fulfilled as to measure the bacteria content as well as the water quality. The involved
group members are well understand the concept of the experiment for bacteria counting.

9.0

REFERENCE
1. Hammer, Mark J. (2001)Water and Waste water Technology Frouth Edition
New Terzey: Prentice Hall
2. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition.
Prentice Hall. Upper Saddle River, New Jersey. pp. 140-144.
3. Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction.
Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA,
pp. 147-154, 158-166.
4. Davis, Mackenzie L. and David A. Cornell, Introduction to Environmental
Engineering, 2nd Ed., McGraw-Hill Publishing Company, New York, 1991.
5. Hammer, Mark J., Water and Wastewater Technology, 2nd Ed., John Wiley & Sons,
New York, 1986.