FACULTY OF ENGINEERING TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY

CELL AND
TISSUE
ENG. TECH.
LABORATORY
KOD
ETIKA
PELAJAR
LABORATORY INSTRUCTION SHEETS

(KEP)

COURSE CODE

BNN 30104

EXPERIMENT CODE

JABATAN TEKNOLOGI KEJURUTERAAN KIMIA

EXPERIMENT TITLE
DATE

Bacterial Cells Counting : Pour Plate Method

FAKULTI TEKNOLOGI KEJURUTERAAN

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FACULTY: ENNGINEERING

EDITION:

TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY

REVISION NO:

EXPERIMENT: Bacterial Cells

EFFECTIVE DATE:

Counting : Pour Plate Method

AMENDMENT DATE:

18/2/2014

1.0 OBJECTIVE
The objectives of this experiment are to:
a. perform and describe cultarable enumeration technique
b.
perform and explain the growth of culture for quantification
2.0 LEARNING OUTCOMES
i)
The student will be able to learn and practice sterile techniques used to culture
bacteria
ii)
The student will be able to understand the phases of microbial growth

3.0 INTRODUCTION / THEORY

Bacterial cultivation technique involves the technique of transferring inoculums. Inoculums is
a small amount of microorganisms used in cultivation to produce more microorganisms and
product. Inoculum transfer is performed aseptically using sterile loop. In this experiment,
student will be exposed to several standard techniques of bacteria cultivation.
Bacteria cell growth can be determined by counting the number of the bacteria cells. The
counting of bacteria is performed to determine the total number of bacteria in a population.
Bacteria may be counted in such a way as to obtain an estimate either of the total number of
organisms alive or dead, or of the number of living organisms only. The first is referred to as the
TOTAL COUNT (direct method), the second as the VIABLE COUNT ( indirect metod).
There are a few method used to determine number of living cells, which can grow in an
optimum condition for the particular bacteria. An accurate picture of the living cells in a
microbial population can be observed if the cells grow in an optimum condition. The methods
used are:
a. Pour plate method
b. Spread plate method
c. Bacteria counting using membrane filtration, etc.
Other method that can be determine the growth is by measuring the increment of particular
parameter with time, i.e. protein, dry cells weight, RNA, etc. Cells turbidity also shows the
growth and this can be performed using spectrophotometer with a certain wavelength.

FACULTY: ENNGINEERING

EDITION:

TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY

REVISION NO:

EXPERIMENT: Bacterial Cells

EFFECTIVE DATE:

Counting : Pour Plate Method

AMENDMENT DATE:

18/2/2014

To obtain an accurate results, a few factor must be controlled namely temperature, pH,
medium content and dilution of bacteria suspension. In this exercise, you will be introduced to
several techniques from the bacteria counting methods.
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 diluents 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.

FACULTY: ENNGINEERING

EDITION:

TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY

REVISION NO:

EXPERIMENT: Bacterial Cells

EFFECTIVE DATE:

Counting : Pour Plate Method

AMENDMENT DATE:

18/2/2014

Pour plate method is one of the most convenient techniques commonly used to count bacteria.
It has the advantages of not requiring previously plates, and is often used to assay bacterial
contamination of foodstuffs. This methods is also used for isolation of mixed bacteria cultures.
4.0 APPARATUS & MATERIALS
1 bacteria culture
100ml sterile salt solution for serial dilution
7 test tube/sterile container
2 sterile pipettes (5 ml or 10 ml)
8 sterile petri dish

5.0 PROCEDURES
Dilution procedure
1. Please prepare the nutrient media using the microbiology standard method.
2. Dilution method. Prepare the serial dilution of the water sample using the
appropriate dilution factor:
a. Use a clean, sterile, dry pipette to remove 0.1mL from the bacteria sample and
blow it into the 9.9mL of dilution fluid (normally deionized/distilled water) in
tube#1 and mix thoroughly by blowing lots of bubbles with the pipette for a
couple seconds. Discard the pipette 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.
b. Using another clean, sterile, dry pipette remove 0.1mL from tube#1, wipe
pipette, blow contents of pipette into tube#2, continue blowing bubbles for a
second or two for good mixing.
c. 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.

FACULTY: ENNGINEERING

EDITION:

TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY

REVISION NO:

EXPERIMENT: Bacterial Cells

EFFECTIVE DATE:

Counting : Pour Plate Method

AMENDMENT DATE:

18/2/2014

d. Keep applying the same procedures until tube#6. Refer the below diagram for
better understanding.
e. Label your tubes with the dilution factor as to notice the bacteria content in
the tubes
Notes:
# All the agar preparation procedures should be performed under laminar flow to
keep the samples sterile. Use gloves to prevent contamination of the samples
# 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
0.1 mL

0.1 mL

0.1 mL

0.1 mL 0.1 mL

0.1 mL

test tubes
contain 9.9 mL
dilution fluid

water
sample

1/10

1/100

1/103

Pour plate procedure

1.
2.
3.
4.
5.
6.
7.

1/104

1/105

1/106

test tubes
contain 9.0 mL
dilution fluid
water

sample
Using clean, dry, sterile pipette to remove 0.1mL of diluted sample from each test tube
1.0 mL
into six different petri plates
Pour the agar into the plates. Wait until the agar to solidify.
1.0 mL
Close the petri plates.
Place all the petri plates inside the incubator for 18-24 hours with a temperature
of
1.0 mL
37oC
After being incubate for 1 day, take out the petri plates.
1.0 mL
Place the petri plate on the counting chamber.
1.0 mL
Count the bacteria colonies on the culture cells in 1 ml suspension.
1.0 mL

6.0 RESULTS & CALCULATIONS / ANALYSIS

1/106
1/104
1/105
1/103
water

sample

FACULTY: ENNGINEERING

EDITION:

TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY

REVISION NO:

EXPERIMENT: Bacterial Cells

EFFECTIVE DATE:

Counting : Pour Plate Method

AMENDMENT DATE:

Plating

18/2/2014

Dilution

Method

1/10

1/100

1/1000

1/104

1/105

1/106

Pour
Plate

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.

7.0 DISCUSSION AND CONCLUSION

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. 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?

FACULTY: ENNGINEERING

EDITION:

TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY

REVISION NO:

EXPERIMENT: Bacterial Cells

EFFECTIVE DATE:

Counting : Pour Plate Method

AMENDMENT DATE:

18/2/2014

5. 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
your findings?
6. You may have difficulties on counting bacterial colonies in some of the methods above.
There is an automated way to count bacteria in a liquid culture. Give one example.

1.0 REFERENCE
Cid, A. G., and V. B. Rajal. 2011. New teaching strategies to improve student performance in
fundamentals of biotechnology. J. Microbiol. Biol. Educ. 12:4647.
Marintcheva, B. 2012. Motivating students to learn biology vocabulary with Wikipedia. J.
Microbiol. Biol. Educ. 13:6566.
Rosalie J. Cot, 1998. Aseptic Technique for Cell Culture, Current Protocols in Cell Biology,
Current Protocols in Cell Biology. Part 1.3.1-1.3.10 by John Wiley & Sons, Inc. Becton
Dickinson Microbiology Systems Sparks, Maryland,
Chatigny, M.A. 1986. Primary barriers. In Laboratory Safety: Principles and Practices (B.M.
Miller, D.H.M. Grschel, J.H. Richardson, D.Vesley, J.R. Songer, R.D. Housewright, and W.E.
Barkley, eds.) pp. 144-163. American Society for Microbiology, Washington, D.C. Freshney, R.I.
1994. Culture of Animal Cells: A Manual of Basic Technique, 3rd ed., pp. 51-69. Wiley-Liss,
New York.
Prepared by / Disahkan oleh:

Signature/Tandatangan:
Name/Nama: Dr Norshuhaila Mohamed Sunar
Date/Tarikh :

Approved by / Disahkan oleh :

Signature / Tandatangan :
Name / Nama : Prof. Madya Dr. Ishak Baba
Date / Tarikh :