VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

INTERNATIONAL UNIVERSITY
SCHOOL OF BIOMEDICAL ENGINEERING

Practice 2: Animal Cells and Microbiologies

BM067IU

REPORT
LAB 2: MICROBIOLOGICAL CULTURE

Submitted by
Bùi Thị Nhật Linh-BEBEIU22246

Date Submitted: 21/12/2023

Date Performed: 14/12/2023
Lab Section: Thursday morning
Course Instructor: Ph.D. Truong Phuoc Long
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Table of Contents

I. 1
1. Objectives 1
2. Background information 1
2.1 Aseptic Technique 1
2.2 Biological Safety Cabinets 3
2.3 Culturing Techniques 4
2.4 Culturing Conditions 4
2.5 Colony Morphology 5
II. Error! Bookmark not defined.
1. 5
2. Equipment 5
3. 5
3.1 Broth Culturing 5
3.2 Agar Slant 6
3.3 Streak Plate 7
III. Result and Discussion 8
4. Result 8
4.1 Broth Culturing 8
4.2 Agar Slant 9
4.3 Streak Plate 10
5. Discussion 10
5.1 Broth Culturing 10
5.2 Agar Slant 10
5.3 Streak Plate 10
IV. 11
V. 12

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List of figures
Figure 1. Growth E. Coli in nutrient Broth……………………………………………..……..8
Figure 2. Growth (A) E. Coli and (B) Bacillus Clausii in nutrient agar slant from an agar
plate………………………………………………………………………………………..……9
Figure 3. Growth (C) Bacillus Clausii and (D) E. Coli in nutrient agar from a streak plate exhibiting
isolated colonies………………………………………………………………………….……..10

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 I. Introduction

1. Objectives
In this laboratory, we are handling microbial cultures and media, working in a biosafety
cabinet, performing culturing techniques, and studying colony morphology. Moreover, we
also use sterile equipment and media to transfer and culture microorganisms.

2. Background information
2.1 Aseptic technique
To prevent the contamination and transmission of microorganisms, aseptic technique
encompasses a set of safety measures that should be followed. It is crucial to ensure the
sterilization of all materials used and to be cautious to avoid introducing new
microorganisms from the surroundings into cultures. When working in the laboratory, it is
important to consistently adhere to standard aseptic techniques.

Personal Protection and Cleanliness

• No guests are allowed in the lab, including children or minors.

• Know emergency procedures, use and location of emergency equipment (emergency
exits, fire extinguishers, fire blanket, eye wash station, first aid kit, and broken glass
container).
• In case of fire, evacuate the room and assemble outside the building.
• Report all accidents, no matter how insignificant they appear, to a laboratory
supervisor.
• Be aware of your surroundings and potential dangers created by others.
• Do not smoke, eat, drink, chew gum, or apply cosmetics in a laboratory.
• Wear protective clothing such as long pants, closed-toe shoes, a lab coat, and
goggles.
• Tie long hair up or behind the shoulders. Do not wear long, dangling jewelry or
scarves.
• Dispose of gloves in the laboratory trash. Do not wear lab coats and gloves in public
areas. You will need to dispose of gloves and take off your lab coat before leaving
the lab.
• Cover cuts or scrapes with a sterile, waterproof bandage before entering a lab.
• Wash skin immediately and thoroughly if contaminated by chemicals or
microorganisms.
• Wash your hands regularly, with soap and water, especially after working with
bacteria.
• If you are pregnant or ill, please let your lab instructor know immediately.
• Let your lab instructor know before leaving the classroom.[1]

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 Handling Microbial Cultures and Media
When working with microbial cultures, it is crucial to prevent cross-contamination by
other microorganisms. Here are some tips for maintaining a sterile work area and handling
microbial cultures and media effectively:

Maintain a Sterile Work Area

• Before and after use, disinfect all work surfaces with 70% ethanol or an appropriate
disinfectant. This is especially important after any spills.
• Keep your workspace uncluttered and only have the necessary equipment for your
experiment on the work surfaces.
• Ensure that you have all the necessary supplies before starting an experiment to
reduce the likelihood of careless contamination.
• If available, work in a biosafety cabinet that has been sterilized using ultraviolet light
and an appropriate disinfectant.
• Avoid opening windows or using fans that circulate outside air. If possible, work in
a laboratory setting with air vents covered with filters to prevent contamination by
airborne particles.
• Clean water baths are used for thawing or warming media or solutions regularly.
• Routinely sterilize incubators used for microbial propagation.

Handling Media

• Before and after use, sterilize the outside container of all media and reagents with
70% ethanol. Also, do not leave containers of media open longer than necessary.
• Aliquot sterile solutions into smaller volumes whenever possible. If you are unsure
of the sterility of your media, it is best to discard it immediately.
• Avoid pouring sterile liquids from one container to another, this increases the
likelihood of media contamination. Rather, use sterile pipettes for the aseptic transfer
of media.
• Never mouth pipette. This poses a health risk for personnel as well as increases the
risk of contamination.
• Always use sterile glass or disposable plastic pipettes to work with liquid media. Use
each pipette only once to avoid cross contamination.
• Do not open sterile media, petri dishes, or pipette containers until you are ready to
use them.
Handling Microbial Cultures
• Obtain microbial strains from an authenticated, trusted source. Research
laboratories run the risk of misidentified or contaminated strains when using
microbial cultures obtained from colleagues.
• Before working with media and microbial cultures, sanitize your hands and
work area with an appropriate disinfectant. Disinfectants are commonly

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 applied to laboratory surfaces to destroy any present microorganism by
denaturing proteins or disrupting the cell membrane.
• Ensure you are wearing appropriate sterile, protective clothing.
• To aid in the prevention of culture contamination, all glassware, equipment,
media, and reagents should be sterilized prior to use by either filter-
sterilization or use of an autoclave.
• Handle only one microbial culture at a time.
• Avoid passaging and subculturing microbial strains too many times.
• Inspect cultures daily for signs of cross-contamination.
• Strains should be preserved as low-passaged cultures grown under optimal
growth conditions.[2]

2.2 Biological Safety Cabinets

A biological safety cabinet (BSC) is a primary engineering control used to protect
personnel against biohazardous or infectious agents and to help maintain quality control of
the material being worked with as it filters both the inflow and exhaust air. It is sometimes
referred to as a laminar flow or tissue culture hood.[3]

Biological Safety Cabinet Classes

Class I: A Class I cabinet Class I biosafety cabinet is defined as a ventilated cabinet
that provides personnel and environmental protection. Class I BSCs are designed with an
open front with inward airflow (personnel protection) and HEPA-filtered exhaust air
(environmental protection). They pull room air through the front of the cabinet and across
the worksurface, away from the operator (similar to a fume hood) and use a HEPA filter at
the exhaust outlet. They commonly recirculate air back to the laboratory but can be
externally exhausted if needed.

Class II: A Class II cabinet is defined as a ventilated cabinet for personnel, product and
environmental protection, often used for microbiological work or sterile pharmacy
compounding. In some labs, these containment hoods are referred to as cell culture or tissue
culture hoods. In pharmacy settings, these hoods are referred to as chemo hoods. Class II
BSCs are designed with an open front with inward airflow (personnel protection), downward
HEPA-filtered laminar airflow (product protection) and HEPA-filtered exhaust air
(environmental protection). These cabinets are further differentiated by types based on
construction, airflow and how they interface with exhaust systems — A1, A2, B1, B2 and
C1.

Class III: A Class III cabinet is defined as a totally enclosed, ventilated cabinet with
leak-tight construction and attached rubber gloves for performing operations in the cabinet.
These cabinets have a transfer chamber with interlocked doors that allow for sterilization of
materials before entering/exiting the glove box. Materials can also be taken in and out

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 through a dunk tank filled with a disinfecting solution. The cabinet is maintained under
negative pressure and supply air is drawn in through HEPA filters. The exhaust air is treated
with either double HEPA filtration or single HEPA filtration followed by air incineration
and then exhausted outside.

2.3 Culturing Techniques

Culture media are solutions of nutrients that are used to support the growth of
microorganisms. They can be categorized into several groups based on their composition
and purpose. Here are the different types of culture media:
Defined Media: Defined media have an exact chemical composition, with all the
nutrients and their quantities known.
Complex Media: Complex media have an unknown chemical composition. They
contain reagents of biological origin, such as yeast extract and peptone, where the exact
chemical composition is not known.
Selective Media: Selective media are formulated to inhibit the growth of certain
bacterial species while promoting the growth of specific species.
Enrichment Media: Enrichment media also allow for the growth of specific bacterial
species.
2.4 Culturing Conditions
Culturing conditions play a crucial role in the successful growth and propagation of
microorganisms. The specific conditions required for culturing can vary depending on the
type of microorganism and the purpose of the culture.
Different microorganisms have different temperature requirements for optimal growth.
Some microorganisms thrive at room temperature, while others require higher temperatures,
such as those found in incubators set at 37°C.
The pH level of the culture medium can impact microbial growth. Some
microorganisms prefer acidic conditions, while others thrive in alkaline environments.
Adjusting the pH of the culture medium to the optimal range for a specific microorganism
is important for successful culturing.
The presence or absence of oxygen can determine whether a microorganism is
classified as aerobic or anaerobic. Aerobic microorganisms require oxygen for growth,
while anaerobic microorganisms cannot tolerate oxygen and may even be harmed by its
presence. The choice of aerobic or anaerobic culture conditions depends on the specific
microorganism being cultured.
Microorganisms require specific nutrients for growth, including carbon, nitrogen,
phosphorus, and various trace elements. The composition and concentration of these
nutrients in the culture medium must be optimized to support the growth of the desired
microorganism.
Agitation of the culture medium can be important for certain microorganisms. It helps
to distribute nutrients evenly and prevent the accumulation of waste products. Agitation can
be achieved through shaking, stirring, or using specialized bioreactors.

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 Maintaining a sterile environment is crucial to prevent contamination of the culture. All
equipment, media, and reagents must be sterilized before use. Proper aseptic techniques,
such as working in a laminar flow hood and using sterile techniques, should be followed to
minimize the risk of contamination.

2.5. Colony Morphology

Colony morphology refers to the characteristics and appearance of bacterial colonies
grown on solid media. It is an important tool for describing and identifying different
bacterial species. Here are some key aspects of colony morphology:
Form: The form refers to the shape of the colony. Common forms include circular,
irregular, filamentous, and punctiform (tiny).
Size: The size of the colony can vary and is often measured in millimeters. Tiny
colonies are referred to as punctiform.
Surface: The surface of the colony can have different appearances, such as shiny,
smooth, veined, rough, dull, wrinkled, or glistening.
Elevation: Elevation describes the side view of a colony. It can be flat, convex,
umbonate, raised, or pulvinate (very convex).
Margin/Border: The margin or border refers to the edge of the colony. It can be entire
(smooth), undulate (wavy), lobate, or filamentous.
Color: The color of a bacterial colony can vary and may be influenced by
environmental factors. It is important to note that color alone is not sufficient for
identification, but it can provide additional information.

II. Materials and equipment

1. Materials
Distilled water
Ethanol 700
Slant and broth bacterial culture: Escherichia Coli (EC) and Bacillus Clausii (BC)
Sterile agar slant, petri dish with agar, and broth media
2. Equipment
Bunsen burner
Wire inoculating loop
3. Experimental procedures
3.1 Broth Culturing
Step 1: Sanitize the experimental area using ethanol 700 and cotton wool for safety
precautions.
Step 2: Sterilize a wire inoculating loop by heating it to a red glow using an alcohol
burner. Make sure to sterilize both the loop and the lower portion of the handle. Allow the
loop to cool before using it to obtain a culture.

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 Step 3: Hold a culture tube with one hand and remove the cap using the fourth and fifth
fingers of the other hand. Avoid placing the cap on the lab bench to prevent contamination.
Keep the open tube at an angle to prevent dust from falling into the tube.
Step 4: Pass the opening of the culture tube through the alcohol burner flame to kill any
microorganisms on the lip of the tube.
Step 5: Insert the sterile wire loop into the culture tube. If there is a culture growing on
an agar slant, touch the inoculation loop onto the culture surface and gather a small amount
of the microorganism. If it is a broth culture, submerge the inoculating loop within the
culture.
Step 6: Flame the mouth of the culture tube again, replace the cap, and set it aside.
Step 7: With your free hand, take a tube of sterile media and remove the cap using the
fourth and fifth fingers of the hand holding the loop.
Step 8: Pass the opening of the media tube through the alcohol burner flame to kill any
microorganisms on the lip of the tube.
Step 9: Gently insert the inoculation loop into the media and move it back and forth
several times to inoculate the media.
Step 10: Remove the inoculation loop, flame the mouth of the test tube again, replace
the cap, and set the tube aside.
Step 11: Sterilize the inoculating loop as described in step 2.
Step 12: Incubate the broth culture under the appropriate growth conditions.

3.2 Agar Slant

Step 1: Use ethanol 700 and cotton wool to sanitize the experimental area and ensure
safety cautions.
Step 2: Flame a wire inoculating loop to sterilize it. The inoculating loop should be
heated to a red glow using an alcohol burner. Ensure that both the loop and the lower portion
of the handle have been sterilized. Allow the loop to cool before obtaining a culture. If the
loop is too hot, it will cause the cells to burst.
Step 3: With your other hand, pick up a culture tube and remove the cap with the fourth
and fifth fingers of the hand that is holding the loop. Never lay a cap down on the lab bench,
this will result in contamination. Always hold the open tube at an angle so that dust does not
fall into the tube and contaminate the culture.
Step 4: Pass the opening of the culture tube through the alcohol burner flame. This will
kill any microorganisms on the lip of the tube.
Step 5: Insert the sterile wire loop into the culture tube. If you have a culture growing
on an agar slant, touch the inoculation loop onto the culture surface and gently gather a small
amount of the microorganism. If you have a broth culture, the inoculating loop can be
submerged within
the culture.
Step 6: Flame the mouth of the culture tube again, replace the cap, and set it aside.

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 Step 7: With your free hand, pick up the sterile agar slant and remove the cap with the
fourth and fifth fingers of the hand that is holding the loop (See step 3).
Step 8: Pass the opening of the media tube through the alcohol burner flame. This will
kill any microorganisms on the lip of the tube.
Step 9: Gently insert the inoculation loop into the test tube and gently streak the
microbial culture onto the surface of the agar slant. Do not puncture the agar.
Step 10: Remove the inoculation loop and flame the mouth of the test tube again,
replacing the cap. Set the tube aside.
Step 11: Sterilize the inoculating loop as described in step 2.
Step 12: Incubate the agar slant under the appropriate growth conditions.

3.3 Streak Plate

Step 1: Use ethanol 700 and cotton wool to sanitize the experimental area and ensure
safety cautions.
Step 2: Place a petri dish agar-side-up onto the bench top. Using a permanent marker,
draw a “T” on your plate to separate your plate into 3 sections.
Step 3: Flame a wire inoculating loop to sterilize it. The inoculating loop should be
heated to a red glow using a Bunsen burner. Ensure that both the loop and the lower portion
of the handle have been sterilized. Allow the loop to cool before obtaining a culture. If the
loop is too hot, it will cause the cells to burst.
Step 4: With your other hand, pick up a culture tube and remove the cap with the fourth
and fifth fingers of the hand that is holding the loop. Never lay a cap down on the lab bench,
this will result in contamination. Always hold the open tube at an angle so that dust does not
fall into the tube and contaminate the culture.
Step 5: Pass the opening of the culture tube through the Bunsen burner flame. This will
kill any microorganisms on the lip of the tube.
Step 6: Insert the sterile wire loop into the culture tube. If you have an agar slant, touch
the inoculation loop onto the culture surface and gently gather a small amount of the
microorganism. If you have a broth culture, the inoculating loop can be submerged within
the culture.
Step 7: Flame the mouth of the culture tube again, replace the cap, and set it aside.
Step 8: With your free hand, pick up the agar-half of the petri dish, leaving the lid face
up on your bench.
Step 9: Streak out the culture onto section 1 of your plate. Return the petri dish to its
lid.
Step 10: Sterilize the inoculating loop as described in step 2.
Step 11: Once cool, pick up the agar-half of the petri dish and pass the inoculation loop
1-3 times through section 1, dragging the culture through section 2 multiple times. Be sure
your streaks do not overlap. Return the petri dish to its lid.
Step 12: Sterilize the inoculating loop as described in step 2.

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 Step 13: Once cool, pick up the agar-half of the petri dish and pass the inoculation loop
1-3 times through section 2, dragging the culture through section 3 multiple times. Be sure
your streaks do not overlap. Return the petri dish to its lid.
Step 14: Sterilize the inoculating loop as described in step 2.
Step 15: Incubate the petri dish under the appropriate growth conditions. Be sure to
incubate the plate agar-side-up, this will prevent condensation from dropping into the
culture. With appropriate streak plate technique, you will obtain isolated colonies in section
3 of your plate.

III. Result and Discussion

4. Result
4.1 Broth Culturing

Figure 1. Growth E. Coli in nutrient Broth

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 4.2 Agar Slant

A B

Figure 2. Growth (A) E. Coli and (B) Bacillus Clausii in nutrient agar slant
from an agar plate

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 4.3 Streak Plate

C D

Figure 3. Growth (C) Bacillus Clausii and (D) E. Coli in nutrient agar from a streak
plate exhibiting isolated colonies

5. Discussion
5.1 Broth Culturing
During the process of growing bacteria in gravy, there are several reasons that can cloud the
gravy. One of them is that bacteria can reproduce and produce mucus in the gravy environment.
This mucus can cause the meat to become cloudy and lose its transparency. In addition, bacteria can
also produce other metabolites and by-products during growth and decomposition of organic matter
in meat juice. These substances can cause cloudiness and loss of transparency in gravy.
5.2 Agar Slant
Off-white or pale straw-colored colonies with a moist and smooth texture appear in agar slant.
For E. Coli, colonies may appear creamy or mucoid, non-spore-forming, so no spores are observed.
For Bacillus Clausii, colonies may appear irregular, dry, and raised, spore formation can be
observed under appropriate conditions.
5.3 Streak Plate
E. coli colonies on a streak plate are typically small, round, and may appear creamy or mucoid.
The streaking pattern helps to separate individual bacterial cells, leading to well-isolated colonies.
Bacillus Clausii colonies on a streak plate may be irregular, dry, and raised. The spore-forming
ability may result in distinctive features in the colonies.

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IV. Conclusion
The conclusion of the biological culture in a laboratory setting is biological cultures allow for
the isolation of individual microorganisms from complex samples. Pure cultures are essential for
the accurate identification and characterization of specific bacteria, viruses, fungi, or other
microorganisms.

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V. References
[1] Lab Safety. (2022, January 19).
[2] Ph.D., C. W. (2012, October 10). 8 Tips on Handling Microbial Cultures.
[3] Biological Safety Cabinets | Environment, Health & Safety. (n.d.).

[4] What are two main types of culture media? | AAT Bioquest. (n.d.).

- End -

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