FACULTY OF CIVIL AND ENVIRONMENTAL ENGINEERING

DEPARTMENT OF WATER RESOURCES AND

ENVIRONMENTAL ENGINEERING

ENVIRONMENTAL ENGINEERING LABORATORY

LABORATORY INSTRUCTION SHEETS

SUBJECT CODE BFC 3121

EXPERIMENT CODE MA2
EXPERIMENT TITTLE BACTERIA COUNT
COURSE CODE
 KOD ETIKA PELAJAR
(KEP)
JAB. KEJ. SUMBER AIR & ALAM SEKITAR
FAKULTI KEJURUTERAAN AWAM & ALAM SEKITAR
KUiTTHO

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usaha saya sendiri. Saya juga mengaku tidak menerima atau memberi sebarang bantuan
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apa yang tersebut di dalamnya adalah benar.

___________________________
Tandatangan Pelajar

Nama : _______________________________

No. Matrik :____________________________

Tarikh :________________________________

FACULTY OF CIVIL & ENVIRONMENTAL ENGINEERING

 DEPARTMENT OF WATER RESOURCES AND
ENVIRONMENTAL ENGINEERING

ENVIRONMENTAL ENGINEERING
LABORATORY

SHORT REPORT

SUBJECT CODE
CODE & EXPERIMENT
TITLE
COURSE CODE
EXPERIMENT DATE
NAME OF STUDENT
NO.OF GROUP
GROUP MEMBER 1.
2.
3.
4.
5.
NAME OF LECTURER/
INSTRUCTOR/TUTOR
DATE OF SUBMISSION
MARKS ATTENDANCE & /10%
DISCIPLINE
INTRODUCTION /5%
RESULTS /15%
DATA ANALYSIS /15%
DISCUSSION /25%
CONCLUSION /5%
REFERENCES /5%
TOTAL /80%
EXAMINERS COMMENT APPROVAL RECEIVE

FACULTY : CIVIL & ENVIRONMENTAL

PAGE NO.: 1/5
ENGINEERING
DEPT : WATER RESOURCE &
EDITION: MA2
ENVIRONMENTAL ENGINEERING

LAB : ENVIRONMENTAL ENGINEERING REVISION NO.: 03

EFFECTIVE DATE: 01/12/2007

EXPERIMENT : BACTERIA COUNT AMMEND. DATE: 20/11/2007
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 to :

1. Be more proficient at dilutions.
2. Be more 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 billions of 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 diluted
enough to be counted accurately. Hence, 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 it has limitation where
sensitivity is restricted to bacterial suspensions of 107 cells or greater.

PREPARED BY: BALKIS A. TALIP

SIGNATURE:
DATE: 20 NOVEMBER 2007

FACULTY : CIVIL & ENVIRONMENTAL

PAGE NO.: 2/5
ENGINEERING
DEPT : WATER RESOURCE &
EDITION: MA2
ENVIRONMENTAL ENGINEERING
REVISION NO.: 03
LAB : ENVIRONMENTAL ENGINEERING
EFFECTIVE DATE: 01/12/2007
EXPERIMENT : BACTERIA COUNT AMMEND. DATE: 20/11/2007
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

5.1 Media preparation: Please prepare the nutrient media using the microbiology standard method.

5.2 Sample preparation: Prepare the serial dilution of the water sample using the appropriate
dilution factor.

5.3 Plating procedures: Using the pour plate and spread plate method.

FACULTY : CIVIL & ENVIRONMENTAL

ENGINEERING PAGE NO.: 4/5
DEPT : WATER RESOURCE &
ENVIRONMENTAL ENGINEERING EDITION: MA2

REVISION NO.: 03
LAB : ENVIRONMENTAL ENGINEERING
EFFECTIVE DATE: 01/12/2005
EXPERIMENT : BACTERIA COUNT AMMEND. DATE: 20/12/2005
 6.0 RESULTS & CALCULATIONS / ANALYSIS

Plating Average Dilution Total

Method Colony bacteria
1/10 1/100 1/1000 1/104 1/105 1/106
/plate / mL
Pour 119 190 175 155 85 75 35
Plate
Spread 125 200 180 160 90 80 40
Plate

7.0 DATA ANALYSIS

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

2. Analyze the results by using appropriate method. Explain your findings.

3. State the systematic bias error that could occur during this experiment.

4. Usually, the results give different readings for both methods. However, in some cases,
both methods produce the same results. Explain why the results are indistinguishable.

8.0 QUESTIONS

1. Explain the meaning of a phrase two times ten to the eight cells per mL in your own
convenient terminology.

2. What the meaning of TNTC and the significant amount due to the TNTC?
Give the formula for determining bacteria count.

3. Design an experiment to compare the bacteria counts in different water samples (tapwater,
lake water, swimming pool water and rainbarrel water). Explain the difference of bacteria
count for each type of water sample?

4. In many experiments there are 2 types of control used which are positive and negative
control. Based on this experiment what is the suitable control? How will the control affect
1/100
1/10 your findings?