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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 a water sample
2. To describe the significance of counting bacteria

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3.0

THEORY

Microorganism in the water sample can exist in the form of bacteria, protozoa,
fungi and virus.
Microorganism is an organism that is microscopic or submicroscopic, which is too
small to be seen under naked eyes. However, the numbers of microorganisms in a
given sample are required to know in certain aspect such as dairy industries,
diseases investigation, and pollution control. Bacteria is one of the most common
microorganism normally was checked in analyzing water.
A variety of methods have been developed for the enumeration of bacteria like
direct microscopic counts, filtration and viable plate counts. Among the methods
of enumeration, viable plate counts are being used most frequently to measure
bacterial populations.
In viable plate counts, we are measuring the number of viable cells, unlike the
microscopic counts which cannot distinguish live from dead cells. However, it
takes some time for the visible colonies to grow. Before doing plate counts, serial
dilutions are required. This is because it is hard to count more than 300 colonies
on an agar plate if we inoculated directly from the original bacterial suspension or
sample without serial dilutions.
Plate counts could be performed either using pour plate method or spread plate
method and each of them have their advantages and limitations. All the visible
colonies are calculated and represented as colony forming units (CFU). Then, the
CFU is multiplied with the corresponding dilution factor. As a result, the population
of original sample is known.

FACULTY : CIVIL &


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

EDITION:
REVISION
03
NO:
EFFECTIVE
Feb 2016
DATE:
AMENDMENT
DATE:

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4.0

EQUIPMENTS AND MATERIALS


1)
2)
3)
4)

Conical Flask (1000ml)


Test tube rack
Parafilm
Pipette

5) Spatula
6) Magnetic bar
7) Glass rod
8) Test Tube
9) Autoclave/Sterilizer
10)

Incubator

11)

Colony counter

12)

Petri dish

13)

Agar : Peptone = 5g, Beef Extract = 3g, Agar = 15g, Distilled

water = 600ml

All equipments used in microbiuology work should be sterilized in an autoclave


at 121 C for 15 minutes

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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
5.2

Dilution procedures
0.1 mL

1.

2.

0.1 mL 0.1 mL

0.1 mL 0.1 mL

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
1.0water
mL
mL (sterile
containing bacteria and blow it into the 9.9mL of dilution 1.0
fluid
1.0 mL
deionized/distilled water) in tube#1 and mix thoroughly by
blowing lots of
1.0 mL
bubbles with the pipette for a couple seconds. Discard the
into the
1.0pipette
mL
used jar for later cleaning. Notice tube#1 now contains 1/1
.
Since
nearly
1.0 mL
6
1/10
0.1mL of liquid may cling to the outside of the pipette, you
must wipe the
1/104
pipette with Kleenex or toilet paper before inserting the pipette
into
5
1/10
tube#1.
3
1/10
Using another clean, sterile, dry pipette remove 0.1mL from
tube#1, wipe
water
pipette, blow contents of pipette into tube#2, continue blowing
sample 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.

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FACULTY : CIVIL &


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

5.3

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

Spread Plate test method

One of the techniques for performing a standard plate count is the spread plate
method. As the name implies, serial dilutions of a sample are spread onto the
surfaces of agar plates.
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

The pour plate, like other viable plate count methods, involves adding a sample to
a solid medium that will support microbial growth incubating the plates so that
each bacterial cell multiplies to form a colony, and counting the number of
colonies that develop.
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 using colony counter.

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6.0

RESULTS AND CALCULATIONS

Sample of Table for Spread plate and Pour Plate Method


Dilution
10-1
10-2
10-3
10-4
10-5
10-6
10-7

Count 1

Count 2

Average

Concentration of bacteria in original sample = Number of bacteria X dilution


factor (in all dilution)
Average = Concentration of bacteria in original sample / Number of dilution that
the colony appear or could be counted
Unit to be used CFU/ml (colony forming unit/ml sample)
7.0

QUESTIONS

(7.1) Can you use un-sterilize water as dilution water in serial dilution?
(7.2) Which method you can observe more clearer and high number of bacteria
colony (spread plate or pour plate method)?
(7.3) List three (3) types of water sample with high bacteria concentration.
(7.4) Which method is more flexible to be used as enumeration procedure for
various type of bacteria (spread plate or pour plate method) and Why?
(7.5) Suggest two (2) methods in achieving 1/100 dilution other than by adding
1mL of sample in 99 mL of dilution water

8.0 DISCUSSION

PREPARED BY :

SIGNATURE :

POSITION :
DATE :

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