VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

INTERNATIONAL UNIVERSITY
SCHOOL OF BIOMEDICAL ENGINEERING

Practice 2: Animal Cells and Microbiologies

BM067IU

REPORT
LAB 1- Microbiology Lab Equipment,
media preparation and aseptic technique

Submitted by
Huỳnh Nhật Minh - BEBEIU23062

Date Submitted: 23/09/2024

Date Performed: 20/09/2024
Lab Section: Monday afternoon
Course Instructor: Ph.D. Truong Phuoc Long
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Table of Contents
I. Introduction.............................................................................................................................1
1. Equipment in Microbial Culture..............................................................................................1
2. Necessary Nutritional and Environmental Factors for Microorganism Culture......................1
3. Sterilization Methods...............................................................................................................2
II. Experimental............................................................................................................................2
1. Materials and equipment......................................................................................................2
a. Materials...........................................................................................................................2
b. Equipment........................................................................................................................4
2. Experimental procedures.....................................................................................................7
III. Results and discussion.........................................................................................................9
1. Results..................................................................................................................................9
2. Discussion................................................................................................................................9
IV. Conclusion...........................................................................................................................9
V. References.............................................................................................................................10

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List of Figures

Figure 1. Autoclave machine (LC.104 room from BME-HCMIU)......................................1

Figure 2. (A) Nutrient Broth; (B) Nutrient Agar................................................................10

List of Tables
Table 1. Chemicals using in this lab....................................................................................4
Table 2. Equipments using in this lab....................................................................................................
Table 3. Steps for preparing microbial culture media using nutrient agar (NA)...................................

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I. Introduction

1. Equipment in Microbial Culture

Microbial culture laboratories often utilize the following equipment to enable

precise and safe culturing processes:
 Autoclave: Autoclaves, often known as steam sterilizers, are
commonly utilized in healthcare and industrial purposes. An autoclave
is a machine that uses steam under pressure to destroy hazardous
bacteria, viruses, fungus, and spores on objects placed within a
pressure vessel. The products are heated to the necessary sterilizing
temperature for a certain period of time. The moisture in the steam
effectively transmits heat to the objects, destroying the protein
structure of the bacteria and spores.
 Shaker: A laboratory incubator shaker is a type of laboratory
equipment that incubates and shakes samples at a specified
temperature and speed. It is widely used in biotechnology, micro
biology, and molecular biology to promote the growth and
development of cells, bacteria, and enzymes.
 Incubator: An incubator is an insulated box that can adjust
temperature, humidity, and other environmental factors to promote
development, hatching, and reproduction.
 Centrifuge: A centrifuge is a laboratory device that separates fluids,
gasses, and liquids based on their density. This separation is
accomplished by quickly spinning a container filled with the material.
The centrifugal forces created during this process drive the denser
material to the bottom of the container.
 Petri dishes and test tubes: contain culture medium for microbial
growth, either liquid or solid.

Figure 1. Autoclave machine (LC.104 room from BME-HCMIU)

 2. Necessary Nutritional and Environmental Factors for Microorganism Culture

Each kind of microbe has unique requirements for its life habitat. The
following are significant aspects that affect their growth:
 Temperature: Microorganisms are classified into three types based on
their optimal temperature requirements: mesophilic, psychrophilic, and
thermophilic. Mesophilic microorganisms typically thrive at
temperatures ranging from 15°C to 43°C. However, psychrophilic
microbes have been seen to thrive and proliferate at temperatures as
low as 0°C. Thermophilic organisms can grow in temperatures higher
than 80°C. Pathogenic organisms require a temperature of roughly
37°C (body temperature), whereas saprophytes have a considerably
greater latitude range.
 pH level: The acidity or alkalinity of the environment is crucial for
microbial growth. Most bacteria prefer neutral pH levels (around 6-8),
but some thrive in extreme acidic or alkaline environments.
 Light: Light exposure can impact certain microorganisms, particularly
photosynthetic microbes, which require light to generate energy.
Others may be sensitive to light and grow best in dark conditions.
 Nutrients: Microorganisms require a variety of nutrients such as
carbon, nitrogen, and trace elements for energy and growth. The
specific nutrient needs vary depending on the type of microbe and its
metabolic pathways.
 Oxygen: Microorganisms have varying oxygen requirements. Aerobic
microbes need oxygen for survival and energy production, while
anaerobes grow in the absence of oxygen. Facultative anaerobes can
switch between aerobic and anaerobic conditions, adapting to either
environment.

3. Sterilization Methods

Sterilization is critical in microbiology for preventing contamination and

maintaining clean cultures. Typical sterilizing procedures include:
 Physical Sterilization:
 Autoclaving: Autoclaving involves the use of high pressure
saturated steam at about 121 degrees centigrade to sterilize
medical instruments and other related articles.
 Steam Sterilization: Steam sterilization is divided into two
main types: intermittent sterilization and single sterilization.
Intermittent sterilization is done at medium temperatures over
multiple cycles, while single sterilization is performed at high
temperatures (similar to autoclaving).
 Dry Heat Sterilization: Dry heat sterilization involves heating
the air at high temperatures (between 160-180 degree Celsius)
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 to eliminate microorganisms suitable for materials with poor
heat resistance (need to be wrapped carefully in paper to avoid
contamination).
 Radiation Sterilization: High-energy rays such as UV and
Gamma rays are used for sterilization.
 Chemical Sterilization:
 Ethanol: Ethanol is effective for disinfection but not suitable
for sterilization, as it cannot reliably kill bacterial spores (90%
alcohol is not used as much as 70% alcohol because 90%
alcohol evaporates too quickly and its high osmotic pressure is
not suitable for disinfection like 70% alcohol.)
 Ozone (O3): Ozone gas attacks the cellular constituents by
oxidation. It is environmentally friendly, but is very reactive
and can corrode stuff.
 Hydrogen Peroxide (H2O2): Vaporized hydrogen peroxide,
which is used on medical instruments. It is very efficient as a
means of transportation and does not harm the environment, but
it is not good for items that are easily affected by moisture.

II. Experimental
1. Materials and equipment
a. Materials

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 Name of
Quantity Function Illustration
Chemicals
Provides
essential
nutrients,
vitamins, and
growth factors
for
Meat Extract, microorganisms.
HiMedia
1 1g
Laboratories Pvt.
Ltf, India.

A source of
nitrogen and
amino acids,
promoting
growth and
Peptone, HiMedia metabolism of
2 Laboratories Pvt. 2g microorganisms.
Ltd, India.

Maintains
osmotic balance
and enhances
Sodium Chloride microbial growth
(NaCl), by providing
Guangdong necessary ions.
3 1g
Guanghua Sci-
Tech Co. Ltd.,
China

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 4 Agar, Vietnam 4g Agar is used to
solidify
solutions. With
two properties—
heat resistance
(from 40°C to
100°C) and the
fact that
microorganisms
do not absorb
nutrients from
agar (preventing
liquefaction)—
agar is widely
used to create
media for
microbial
culture.
5 Distilled Water 200ml Acts as a solvent
to dissolve the
ingredients and
create a suitable
liquid medium
for culturing
microbes.
Table 1. Chemicals using in this lab
b. Equipment
Name of
Illustration
Equipments

1 Stirring rod

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 2 Measuring cup

3 Erlenmeyer flask

4 Analytical balance

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 5 Microwave

6 Medical cotton

7 Lab wrapping paper

8 Rubber band

Table 2. Equipments using in this lab

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 2. Experimental procedures
Ste
Description of steps Illustration
p
All the equipment was washed and
dried with laboratory quality paper to
eliminate interference with microbial
growth.
1

200 ml of distilled water was measured

into a plastic container and warmed in
a microwave oven at medium power
for roughly 2 minutes to get warm
2 water (up to 60°C but not boiling).

Chemicals were measured using a

microbalance. Consequently, 1g of
meat extract, 2g of peptone, 1g of
sodium chloride and 4g of agar were
3 weighed and each chemical was placed
in a different beaker.

Distilled water at a temperature of 60

degrees centigrade was added to each
of the beakers with the chemicals in it,
and the chemicals were dissolved in the
4 water by stirring. The two solutions
were then mixed together in a flask and
the flask was rolled slowly.

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 The flask was put into the microwave
and observed very carefully. When
bubbling occurred, the microwave was
turned off and boiling was continued
on a stove top until all the water had
boiled. Precautions were made to avoid
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carryover, particularly from the
bubbles formed by the meat extract and
peptone. This process was continued
until the meat extract as well as the
peptone dissolved in the solution.

The flask was plugged using sterile

cotton wool. Enough of the cotton was
pulled off to stuff into the opening of
the flask and compacted with the
6 thumb before it was placed into the
mouth of the flask. If cotton wool was
long, any extra was put back into the
flask or torn off if necessary.

The opening of the flask was covered

with paper and tied firmly with rubber
bands.

The flask was placed in an autoclave

(used only for preparing culture media
not the autoclave used for sterilizing
the equipments). The autoclave was set
8 to operate for 15 min at 121°C (time
was started after the temperature had
reached 121°C).

The culture medium for microbial

9 growth was successfully prepared.
Then clean the laboratory.

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 Table 3. Steps for preparing microbial culture media using nutrient agar
(NA)

III. Results and discussion

1. Results
After removing the media from the autoclave, we have 2 types of media

Figure 2. Nutrient Broth and Agar

2. Discussion
When producing bacterial culture medium, it's vital to remember the following:
- Wipe your hands and working surfaces often to reduce the risk of bacterial introduction to
the culture media.
- Cultivate bacteria with specialized instruments and supplies.
-Make sure all instruments are cleaned before they are used on the baby.
-Read and follow the instructions provided in the literature regarding the culture media
pertinent to the specific culture being conducted.
-Be careful to follow correct procedure and proportions in mixing the parts.
-Make sure that the culture medium has the right pH for the microorganisms that you are
using in your experiment.
-ensure that the culture media remains at the best temperature and in a place where it is not
exposed to light.
-Regularly as advised look out for contamination.
IV. Conclusion
In conclusion, we learn about the environmental and nutritional needs for microbe culture in
a laboratory setting in this lab. In addition, we know how an autoclave for sterilization or
disinfection operates. Moreover, we learn how to make the media needed for the culture of
microorganisms for preparing the broths, slants, and plates that will be used in the next lab
sessions.

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V. References
[1] Lab Manuals - Practice 2: Animal Cells and Microbiologies.

[2] 4 oz Online Vietnam | Ubuy. (n.d.). Ubuy Vietnam.

[3] Bruslind, L. (2019, August 1). Microbial Nutrition. Pressbooks.

[4] JoVE Science Education Database. Lab Safety. Proper Use of Autoclaves. JoVE, Cambridge,
MA, (2023).
[5] Tankeshwar, A. (2023, December 6). Bacterial Culture Media: Classification, Types, Uses.
Microbe Online.
[6] H. (2018, February 16). Difference Between Nutrient Agar and Nutrient Broth. Pediaa.Com.
- End -

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