FACULTY : CIVIL &

ENVIRONMENTAL ENG.
DEPART : WATER &
ENVIRONMENTAL ENG.
LAB : ENVIRONMENTAL
ENGINEERING
EXPERIMENT : BACTERIAL
COUNT
1.0

REVISION
02
NO:
EFFECTIVE 28/12/20
DATE: 15
AMENDMENT
DATE:

OBJECTIVE

2.0

EDITION:

Students will be able measure the bacteriological quality of water sample

by performing total plate count on agar at 37C

LEARNING OUTCOMES

At the end of the laboratory courses, students will be able

1. To count bacteria in water sample
2. To describe the significance of counting bacteria

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3.0

THEORY

There are few microbiology parameters that can be used in determining the
water quality of sample. However, the most common method used to determine
the quality of water pollution is bacteria count.
Bacteria were the only form of life on earth for 2 billion years. They were first
observed by Antony Van Leeuwenhoek in the 17th century: bacteriology ad an
applied science began to develop in the late 19th century as a result of research in
medicine and in fermentation processes, especially by Lois Pasteur and Robert
Koch. Bacteria are remarkably adaptable to diverse environmental conditions;
they are found in the bodies of all living organisms and on all parts of the earth-in
land terrains and ocean depths, in arctic ice and glaciers, on 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 earths 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.
Bacteria are often maligned as the causes of human and animal disease (e.g.
Leptospira, which causes serious disease in livestock). However, certain bacteria,
the actinomycetes, produce antibiotics such as streptomycin and nocardicin;
others live symbiotically in the guts of animals (including humans) or elsewhere
in their bodies, or on the roots of certain plants, converting nitrogen into a usable
form. There are five methods of counting bacteria; Dilution and Plating, Counting
Chambers, Most Probable Number, Membrane Filters, Photometer and
Spectrometers. In dilution and plating method, two types of plating are commonly
used (i.e Pour and Spread Plate).
Bacteria are grouped in a number of different ways. Most bacteria are of one of
three typical shapes-rod-shaped (bacillus), round (coccus, e.g.; streptococcus),
and spiral (spirillum). An additional group, vibrios, appears as in complete spirals.
The cytoplasm and plasma membrane of most bacterial cells are surrounded by a
cell wall; further classification of bacteria is based on cell wall characteristics.
They can also be characterized by their patterns of growth, such as the chains
formed by streptococci of flagella; other bacteria have rigid rod like protuberances
called pili that serve as tethers.
FACULTY : CIVIL &
ENVIRONMENTAL ENG.
DEPART : WATER &
ENVIRONMENTAL ENG.
LAB : ENVIRONMENTAL
ENGINEERING
EXPERIMENT : BACTERIAL
COUNT

EDITION:
REVISION
02
NO:
EFFECTIVE 28/12/20
DATE: 15
AMENDMENT
DATE:

2|Page

4.0

EQUIPMENTS AND MATERIALS

1) Cornical Flask (1000ml)

2) Test tube rack

3) Spatula

4) Parafilm

5) Magnetic bar

6) Pipette

7) Glass rod

8) Autoclave

9) Test Tube

10)

Incubator

11)

Magnetic bar

12)

Sterilizer

13)

Petri dish

14)

Microscope

15) Agar : Peptone = 5g, Beef Extract = 3g, Agar = 15g, Distilled
water = 600ml
5.0

PROCEDURES

5.1
1)
2)
3)
4)

Procedures of preparing Nutrient Media agar

Prepare distilled water in 600ml beaker and boil it.
Mix peptone, beef extract and agar
Cool the agar up to 45-50oC.
For the Spread Plate test, pour the nutrient media 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

FACULTY : CIVIL &

ENVIRONMENTAL ENG.
DEPART : WATER &
ENVIRONMENTAL ENG.
LAB : ENVIRONMENTAL
ENGINEERING

EDITION:
REVISION
02
NO:
EFFECTIVE 28/12/20
DATE:
15
3|Page

5.2

EXPERIMENT : BACTERIAL
COUNT
Dilution procedures
0.1 mL

1.

2.

0.1 mL 0.1 mL

0.1 mL 0.1 mL

AMENDMENT
DATE:

0.1 mL

test tubes
contain 9.9 mL
dilution fluid
water
test tubes
sample
6
1/10
1/100
1/103
1/104
1/105
1/10
contain 9.0 mL
dilution fluid
water
sample
Use a clean, sterile, dry pipette to remove 0.1mL from the
sample
1.0bacteria
mL
1.0
mL
and blow it into the 9.9mL of dilution fluid (normally deionized/distilled
1.0 mL
water) in tube#1 and mix thoroughly by blowing lots of bubbles
with the
1.0 mL
pipette for a couple seconds. Discard the pipette into the1.0
used
mL jar for later
cleaning. Notice tube#1 now contains 1/100 the concentration
1.0 mL of bacteria
1/106 nearly
in the original sample because 0.1mL is 1/100 of 10mL. Since
4
1/10must
0.1mL of liquid may cling to the outside of the pipette, you
wipe the
5
1/10
pipette with Kleenex or toilet paper before inserting the pipette
into
3
1/10
tube#1.
water
Using another clean, sterile, dry pipette remove 0.1mL from
tube#1, wipe
sample

pipette, blow contents of pipette into tube#2, 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. Keep applying the same procedures until tube#6. Refer the below
diagram for better understanding.
5. Label your tubes with the dilution factor as to notice the bacteria content
in the tubes.
Note: There are many types of pipettes, and you are advised to use blow out
pipette, that is indicated by a frosted ring on the pipette at the top end.

FACULTY : CIVIL &

ENVIRONMENTAL ENG.
DEPART : WATER &
ENVIRONMENTAL ENG.

EDITION:
REVISION 02
NO:
4|Page

LAB : ENVIRONMENTAL
ENGINEERING
EXPERIMENT : BACTERIAL
COUNT

EFFECTIVE 28/12/20
DATE: 15
AMENDMENT
DATE:

5.3 Spread Plate test method

1. Using clean, dry, sterile pipette to remove 0.1mL of diluted sample from
each test tube into six different petri plates contain sterile agar.
2. Close the petri plate.
3. Place all the petri plates inside the incubator for 18-24 hours with a
temperature of 37oC.
5.4 Pour Plate test method
1. Using clean, dry, sterile pipette to remove 0.1mL of diluted sample from
each test tube into six different petri plates.
2. Pour the agar into the plates. Wait until the agar to solidify.
3. Close the petri plates.
4. Place all the petri plates inside the incubator for 18-24 hours with a
temperature of 37oC.
5.5 Methods of counting bacteria
1. After being incubate for 1 day, take out the petri plates.
2. Place the petri plate on the counting chamber.
3. Count the bacteria colonies on the culture.
6.0

RESULTS AND CALCULATIONS

PREPARED BY :

SIGNATURE :

POSITION :
DATE :

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