VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

INTERNATIONAL UNIVERSITY
SCHOOL OF BIOTECHNOLOGY

REPORT 5
BACTERIAL POPULATION COUNTS
INTRODUCTION TO BACTERIAL IDENTIFICATION
PROCESS
CULTURING UNKNOWN BACTERIUM FOR
SUBSEQUENT ANALYSIS
Course: Practice in Microbiology – BTBT22IU11 group 4
Instructor: MSc. Hoang Thi Lan Xuan
Group number: 01

Name Student ID Contribution

1 Đặng Hoàng Trâm Anh BTBCIU22097 33.3%

2 Phan Vũ Gia Hân BTBTIU22201 33.3%

3 Vũ Thị Thanh Thuỷ BTBCIU22093 33.3%

Score: /100
Date of submission: June 3rd, 2024

Academic year: 2023 – 2024

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TABLE OF CONTENTS

I. Introduction………………………………………………….. 2

II. Procedure…………………………………………...……….. 2

III. Results………………………………………………….….... 4

IV. Discussion…………………………………………………... 6

V. Conclusion……………………………………………............. 9

VI. References………………………………………………........ 9
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I. INTRODUCTION
Bacteria, microscopic organisms found abundantly throughout our world, play essential roles
in various fields. To understand their influence, we need to determine not only how many
bacteria are present but also who they are. Bacterial population counts, achieved through
techniques like the standard plate count, estimate the number of viable cells in a sample.
Identification processes, which may involve microscopy, staining, and biochemical tests, then
help distinguish between different species based on their unique characteristics. Finally,
culturing unknown bacteria allows us to isolate and grow them in a controlled environment.
This culturing provides enough material for further analysis, enabling detailed identification
and exploration of the bacteria's properties and functions. Through these combined methods,
we gain a deeper understanding of the fascinating world of bacteria and their impact on our
lives.

II. PROCEDURE
1. Materials
Broth culture of E.Coli Alcohol burner
Sterile LB medium Tissue paper and marking pen

Nutrient agar plates Soil, Ethanol

Micropipettes and Micro-tips Distilled water and glass stick

Eppendorf tubes Beaker

Spreader Quebec colony counter

2. Procedure
For dilution steps
To dilute the culture broth, add 900 µL of sterile LB in 9 Eppendorf tubes. Add 100 µL of
culture broth to the first tube and gently dispense until homogenous. Transfer 100 µL to the
second tube and repeat the procedures above. Continue performing serial dilutions until
generating the final dilution 10!" .

Plating E.Coli into nutrient agar plates

1. After finishing dilution, use the spread-plate technique to transfer 100µL from each
diluted sample to a sterile LB agar plate.
2. Incubate the plates at 37° C for 16 hours in an inverted position.
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Plating soil into nutrient agar plates

1. Mix the soil with distilled water and heat that mixture (based on the chosen method).
2. Use the substance on the surface without solid materials and dilute until the dilution of
10!# .
3. Use the spread-plate technique to transfer 100 µL from a diluted sample to an agar plate.
4. Incubate the plates at 37° C for 16 hours in an inverted position.

Steps for counting and calculations

1. Lay out the plates on the table in order of dilution.
2. Place the plate on the Quebec colony counter.
3. Start counting every colony, regardless of how small or insignificant until finishing that
plate. And then compare to see which plate should be chosen to estimate the number of
bacteria in the original culture.
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III. RESULTS
1. Quantitative plating methods

Figure 1. Colonies observation on Petri dishes

(Dilution factor from 10-5 to 10-9)

Enumeration of bacteria:

NUMBER OF COLONIES

Group 3 Group 2 Group 1 Group 4 Group 5

Plate
(DF:10-5) (DF:10-6) (DF:10-7) (DF:10-8) (DF:10-9)

190 colonies TNTC TNTC 33 colonies TFTC

* TNTC stands for too numerous to count

* TFTC stands for too few to count
* DF stands for dilution factor
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2. Identify of microorganisms in soils (by serial dilution- agar plate

technique)

Procedure Result Observation

Soil plates with heat treatment

show a great diversity of
organisms. As we can see
Heat
bacteria, yeast, fungi.
treatment

Figure 2.1. soil plate with heat treatment

(the result of group 3)

Soil plate with no heat treatment

Without heat
appeared with clumps and they
treatment
might be yeast.

Figure 2.2. Soil plate without heat

treatment
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IV. DISCUSSIONS
1. Why is it necessary to perform a plate count in conjunction with the turbidity
measurement?
Turbidity is quick, but it does not indicate whether or not cells are alive. A plate count indicates
the presence of live cells, but it is time-consuming. Together, they provide a clearer picture: a
rapid check for total cells (turbidity) and confirmation of live cells (plate count).

2. What is a Colony-forming Unit (CFU)?

A Colony-forming unit (CFU) is a unit used in microbiology to estimate the number of viable
bacteria or fungi in a sample (viable here means the cells are alive and able to multiply). One
CFU is considered as one colony.

3. For the Standard Plate Count method, if your culture has been diluted up to
1,000,000 times yet you are still unable to count the CFU (due to clumping),
suggest two ways that you can overcome this problem next time.

- Way 1: Verify that the culture and diluent were put in the correct amounts, as even minor
errors might have serious implications. Also, ensure that the material is thoroughly mixed and
homogenized before making dilutions. This can help to break up cell clumps or aggregates.

- Way 2: Try different dilution techniques, such as using a vortex mixer or a mechanical
homogenizer, to further disperse the cells and prevent clumping.

4. For bacterial enumeration, list at least 2 problems might arise from

pipetting/working with very small volume of culture

- Inaccuracy: Pipettes have a minimum accurate volume they can dispense. Working with very
small volumes that may fall outside this range. Inaccuracies in dispensed volume can
significantly skew your final bacterial count, leading to underestimates or overestimates of the
actual population.

- Cell Loss: Loss can occur during transfers from place to place. When working with small
volumes, even minor losses during pipetting or transfers can significantly impact your final
count. This loss of cells further reduces the accuracy of your bacterial enumeration.
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5. Discussion for Lab 5 results:

a. Give comments about the class Standard Plate Count result regarding (i)
contamination, (ii) spreading technique and (iii) the logic of the change in
colony number over different diluting concentrations:

Plate Comments

Group 1 (DF: 10-7) (ii), TNTC

Group 2 (DF:10-6) (ii),(iii),TNTC

Group 3 (DF: 10-5) No comment

Group 4 (DF: 10-8) No comment

Group 5 (DF: 10-9) TFTC

b. Which plate (i.e. which Dilution Factor) should you use to estimate the number
of living E.coli cells in the original culture? Explain your choice.

As we know, statistically valid plate counts are between 30 to 300 colonies, too few colonies
may not accurately reflect the concentration of the plated culture, more than a few hundred
would be difficult to count due to overcrowding, and we got 190 colonies in 10-5 dilution tube
and 33 colonies in 10-8 dilution tube. Therefore, we will choose a plate with 10-5 dilution factor
or a plate with 10-8 dilution factor to estimate the number of living E.coli cells in the original
culture.

c. Use that plate (Q.5b) to show calculation for:

● Estimating the Number of living cells per ml of the original culture:

!"# ((
Cell/mL = = = 33 x 10+ cell/mL
$ & '" )** & )*!"

● Estimating the Number of living cells per µl of the original culture:

,-../01 (( & )*$

Cell/µL = = = 33 x 10( cell/µL
)*# )*#
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d. Give comments about the culturing results from your soil sample regarding
kinds of microbes (i.e. Bacteria, fungus, etc.) and in relation to with/without
heat treatment. Pictures of the culture should be provided.

The two images show soil samples in Petri dishes, the 2.1 was heated at 70oC for 10 minutes,
and the 2.2 was without heat treatment. The soil sample that underwent heat treatment shows
numerous colonies, which indicate the presence of microorganisms such as bacteria, yeast, or
fungi.

In contrast, the soil sample without heat treatment appears to have a very small quantity of
visible colonies or growth. The major part of this Petri dish shows some giant clumps that do
not have any obvious microorganisms.

The difference in the appearance of the two soil samples suggests that the heat treatment has
influenced the microbial population or growth dynamics within the heated soil sample. Heat
can kill or inactivate some microorganisms while promoting the growth of others that are more
heat-resistant. The distinct colonies in the heated sample could represent heat-tolerant or heat-
activated microbes that have proliferated after the heat treatment. While some microorganisms
that are sensitive to high temperatures and some other matters, such as organic compounds or
nutrients, were killed. Heat reduced the quantity of microorganisms in the sample, and
eventually, only those microorganisms that survived at this temperature could grow and appear
as colonies on the agar surface.

Whereas in the sample that was not treated with heat, the number of microorganisms
completely remained and kept growing. In the end, because of the huge amount of them, they
appeared as clumps on the agar surface instead of clearly formed colonies, which indicate the
presence of specific microorganisms.
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V. CONCLUSION
In conclusion, the combined approach of bacterial population counts, identification processes,
and culturing provides a powerful toolkit for unraveling the secrets of the microbial world. By
quantifying bacterial abundance, differentiating between species, and isolating unknown
bacteria for further study, we gain a comprehensive picture of their presence and influence.
This knowledge is essential in various fields, from ensuring food safety and developing
antibiotics to understanding the complex ecosystems bacteria inhabit. As we continue to refine
these techniques, we unlock new possibilities for harnessing the power of bacteria and
mitigating their potential threats.

VI. REFERENCES
a. 23-24 S2 Practice in Microbiology. MSc. Hoang Thi Lan Xuan, School of
Biotechnology, IU (International University)