International University School of Biomedical Engineering: Semester 1. 2019 - 2020
International University School of Biomedical Engineering: Semester 1. 2019 - 2020
International University School of Biomedical Engineering: Semester 1. 2019 - 2020
2019 – 2020
INTERNATIONAL UNIVERSITY
SCHOOL OF BIOMEDICAL ENGINEERING
BIOLOGY FOR BME LABORATORY
Lab 2 – Media And Bacterial Species
1. ABSTRACT
The experiment is about observing the cultures for signs of growth and how to inoculates streak plate.
Aseptic technique is fundamental to the success and safety of an experiment. It’s avoid health risk and
hazardous or sterile materials on lab.
2. CULTURING TECHNIQUE
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting
them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial
cultures are foundational and basic diagnostic methods used as a research tool in molecular biology.
Microbial cultures are used to determine the type of organism, its abundance in the sample being tested,
or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the
cause of infectious disease by letting the agent multiply in a predetermined medium. For example, a
throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into
a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative
agent of strep throat. Furthermore, the term culture is more generally used informally to refer to
"selectively growing" a specific kind of microorganism in the lab.
It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population
of cells or multicellular organisms growing in the absence of other species or types. A pure culture may
originate from a single cell or single organism, in which case the cells are genetic clones of one another.
For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a
gelatinous substance derived from seaweed. A cheap substitute for agar is guar gum, which can be used
for the isolation and maintenance of thermophiles.
Broth Culture
A broth culture is a liquid nutritional medium used to culture live microorganisms in either a test tube or
an Erlenmeyer flask. This form of culture allows for the rapid growth of many microorganisms, and is
often used to prepare specimens for cryopreservation or to propagate large volumes of microbial cultures.
Below we describe an aseptic protocol for the inoculation of a broth culture.
Materials
Bunsen burner
Sterile broth media
Slant or broth culture
Wire inoculation loop
NOTE 1: Sterile, disposable loops can be used to inoculate cultures. These loops are ready-to-use
and do not require flame sterilization.
Procedure
1. 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.
2. 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.
3. Pass the opening of the culture tube through the Bunsen burner flame. This will kill any
microorganisms on the lip of the tube.
4. 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.
5. Flame the mouth of the culture tube again, replace the cap, and set it aside.
6. With your free hand, pick up a tube of sterile media and remove the cap with the fourth and fifth
fingers of the hand that is holding the loop.
7. Pass the opening of the media tube through the Bunsen burner flame. This will kill any
microorganisms on the lip of the tube.
8. Gently insert the inoculation loop into the media and move the loop back and forth several times
to inoculate the media.
9. Remove the inoculation loop and flame the mouth of the test tube again, replacing the cap. Set the
tube aside.
10. Sterilize the inoculating loop as described in step 1.
11. Incubate the broth culture under the appropriate growth conditions.
Agar Slant
Agar slants are a form of solid media generated from the addition of a gelling agent, such as agar, to a
broth culture. To prepare an agar slant, agar is added to the broth culture then heated within an autoclave
to dissolve 5 the agar and sterilize the media. This medium will then solidify at 42°C. Agar slants provide
a large surface area on which to grow microorganisms and are frequently used as a method to temporarily
store actively growing cultures. Below we describe an aseptic protocol for the inoculation of an agar
slant.
Materials
Slant or broth culture
Wire inoculation loop
Bunsen burner
Sterile agar slant
Slant or broth culture
Procedure
1. 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 (NOTE 1).
2. 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.
3. Pass the opening of the culture tube through the Bunsen burner flame. This will kill any
microorganisms on the lip of the tube.
4. 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.
5. Flame the mouth of the culture tube again, replace the cap, and set it aside.
6. 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 2).
7. Pass the opening of the media tube through the Bunsen burner flame. This will kill any
microorganisms on the lip of the tube.
8. 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.
9. Remove the inoculation loop and flame the mouth of the test tube again, replacing the cap. Set the
tube aside.
10. Sterilize the inoculating loop as described in step 1.
11. Incubate the agar slant under the appropriate growth conditions.
Streak Plate
Similar to the agar slant, streak plates are a form of solid media generated from the addition of a gelling
agent, such as agar, to a broth culture. This medium is then added to a petri dish, rather than a test tube,
and allowed to solidify. The term “streak plate” refers to how the culture is inoculated on the agar surface.
This particular inoculation technique is used to isolate a pure colony from a single microbial species.
Samples obtained from the resulting pure colony can then be grown on a new plate so that the organism
can be identified, studied, or the colony morphology can be analyzed. For more information on the
analysis of colony morphology, see the section entitled “Colony Morpholgy.” Below we describe an
aseptic protocol for the inoculation of a streak plate.
Materials
Slant or broth culture
Wire inoculation loop
Bunsen burner
Sterile petri dish with agar
Procedure
1. 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..
2. 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.
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.
4. Pass the opening of the culture tube through the Bunsen burner flame. This will kill any
microorganisms on the lip of the tube.
5. 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.
6. Flame the mouth of the culture tube again, replace the cap, and set it aside.
7. With your free hand, pick up the agar-half of the petri dish, leaving the lid face up on your bench.
8. Streak out the culture onto section 1 of your plate. Return the petri dish to its lid.
9. Sterilize the inoculating loop as described in step 1.
10. 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.
11. Sterilize the inoculating loop as described in step 1.
12. 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.
13. Sterilize the inoculating loop as described in step 1.
14. Incubate the petri dish under the appropriate growth conditions. Be sure to incubate the plate
agar-sideup, 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.
Streak plate set-up How to inoculate a streak plate Example of a streak plate
exhibiting isolated colonies following incubation
isolated colonies following incubation
Spread Plate
Similar to the streak plate technique, a spread plate is another form of inoculation that used to isolate pure
colonies from a single microbial species. This particular technique is primarily used to quantify the
number of microorganisms within an actively growing broth culture. For more information on the
enumeration of a microorganism, view the section entitled “Bacterial Enumeration”. Below we describe
an aseptic protocol for the inoculation of a spread plate.
Materials
Broth culture
Sterile 1 mL pipette
Pipette bulb
Glass or metal streaking “hockey” stick
70% Ethanol
Bunsen burner
Sterile petri dish with agar
NOTE 2: Pre-drying the plates will aid in spreading the culture media evenly and quickly
Procedure
1. Place a petri dish agar-side-down onto the bench top.
2. Select a sterile 1 mL pipette and gently attach it to the pipette bulb. Do not touch the tip of the
pipette, this will contaminate it.
3. With your other hand, pick up the culture tube and remove the cap with the fourth and fifth
fingers of your opposite hand. 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.
4. Pass the opening of the culture tube through the Bunsen burner flame. This will kill any
microorganisms on the lip of the tube.
5. Insert the sterile pipette tip into the culture tube and aspirate 0.1 mL of growing culture.
6. Remove the pipette from the culture tube, flame the mouth of the culture tube again, replace the
cap, and set it aside. Carefully hold the pipette, do not touch the tip or lay the pipette on the bench
top.
7. With your free hand, pick up the lid to the petri dish and hold it at an angle over the plate. This
will prevent dust from falling into your culture while you inoculate.
8. Slowly dispense the culture media from the pipette onto the center of the plate. Replace the petri
dish lid, and properly dispose of the pipette.
9. Sterilize the hockey stick by submerging the short, flat portion of the hockey stick into the 70%
ethanol. Pass the hockey stick quickly through the Bunsen burner flame to light it on fire. Do not
leave the hockey stick in the flame. Once the flame has burned out, allow the hockey stick to cool
for a few seconds.
10. With your free hand, lift up the lid of the petri dish. Place the sterile portion of your hockey stick
onto the agar to dissipate the heat. Then spread the inoculum over the plate, evenly spreading the
culture media on the surface of the agar. Gently turn the plate while spreading the media to ensure
the surface of the agar is evenly covered. Do not puncture the agar (See: NOTE 2).
11. Continue to spread the culture media until it has dried onto surface of the plate. Replace the petri
dish lid and re-sterilize the hockey stick.
12. 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
spread plate technique, you will obtain isolated colonies on your plate.
3. CULTURING CONDITION
Because bacteria can grow and thrive in a variety of environments, optimal growth temperatures may
vary significantly between species. In general, most pathogenic or commensal bacterial strains grow well
at body temperature (37°C). In contrast, many environmental strains thrive at lower temperatures, often
within a range of 25°C to 30°C.
Bacterial species can be categorized based on their growth temperature; these include psychrophiles (0°C
to 20°C), mesophiles (25°C to 40°C), and thermophiles (45°C to 122°C). Strains can withstand
considerable drops in temperature and survive several days at 4°C. In lower temperatures, bacterial
growth and metabolism are significantly diminished.
To varying requirements for optimal growth temperatures, bacteria also differ in their use of oxygen for
respiration. Aerobic organisms, such as Bacillus species, use oxygen as a terminal electron acceptor
during respiration. Helicobacter pylori, also require the use of oxygen, but at lower levels than naturally
occurring in the environment. In contrast, anaerobic organisms use electron acceptors such as nitrate or
sulfate, among other inorganic acceptors.
The use of oxygen and inorganic compounds by anaerobic organisms can differ greatly between species.
Clostridium species, can only survive and reproduce in the absence of oxygen; these organisms are often
killed by the presence of oxygen. Similarly Aerotolerant anaerobes, such as Lactobacillus species, cannot
use oxygen during respiration; however, unlike strict anaerobes, these microorganisms can tolerate
oxygen 8 for short periods of time.
When working with anaerobic cultures, it is important to avoid unnecessary exposure to oxygen.
Anaerobic conditions may be obtained for either transfer or incubation by the methods listed below.
Unless specifically mentioned, the standard anaerobic gas mixture is 80% N₂, 10% CO₂, and 10% H₂.
Anaerobic conditions for transfer may be obtained by either of the following: 1. Use of an anaerobic gas
chamber. 2. Placement of test tubes under a gassing cannula system hooked to anaerobic gas. During
incubation, anaerobic conditions may be maintained by any of the following:
1. Loose screw-caps on test tubes in an anaerobic chamber.
2. Loose screw-caps on test tubes in an activated anaerobic GasPak™ jar.
3. Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.
Below, we describe a general procedure to analyze the temperature and oxygen requirements of a
microorganism.
MATERIALS
Broth culture.
Sterile agar plates per culture.
Sterile pre-reduced agar plates per culture.
Wire inoculation loop Bunsen burner.
PROCEDURE
Inoculate the six sterile agar plates using the streak plate culturing technique.
Under anaerobic conditions, inoculate the three pre-reduced agar plates using the streak plate
culturing technique.
Incubate the plates agar-side-up at either 25°C, 30°C, or 37°C under the following conditions:
o Aerobic – incubate plates in a standard incubator.
o Microaerophilic – incubate plates in a candle jar. This can be any jar large enough to hold
petri dishes and a candle. Place your plate and a lit candle into the jar and seal the lid.
The candle flame will consume most of the oxygen in the jar, producing elevated levels
of carbon dioxide.
o Anaerobic – incubate pre-reduced plates in a sealed anaerobic jar, such as the GasPak™
anaerobic system (Becton Dickinson). This system consists of a polycarbonate jar, a lid
with a gasket to prevent air flow, an indicator strip for the detection of oxygen, and a
ready-to-use chemical sachet that creates an anaerobic atmosphere.
Observe the cultures for signs of growth.
4. ASEPTIC TECHNIQUE
Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and
sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be
opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest
on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab
period.
Personal Protection and Cleanliness
In a Microbiology laboratory, it is important to maintain the health and safety of all personnel. To avoid
health risks, laboratory scientists should wear protective gear and be aware of any potential hazards
nearby. Generally, protective gear serves two purposes; to protect the scientist from laboratory hazards
and to shield the experiment from unintentional contamination. Below, we describe several mechanisms
to maintain laboratory safety.
Upon entering and exiting a lab, be sure to thoroughly wash your hands with antimicrobial soap.
While in the lab, avoid touching your face or eyes. Further, do not handle make-up or contacts as they
may become contaminated.
Only enter the lab wearing closed-foot shoes. Open-toed shoes should never be worn in a laboratory as
it leaves you susceptible to potential cuts or infection from broken glassware or sharp equipment.
While performing an experiment, wear a clean, fitted laboratory coat. For additional protection, use a
closed-front laboratory coat. Laboratory coats should never leave the lab, and should be cleaned
frequently. Additionally, ensure that the sleeves of your coat are fitted so that they will not catch fire near
an open flame, nor become entangled.
When working with microorganisms, wear fitted, disposable gloves. Flakes of dry skin harbor bacteria,
which may provide a source of contamination; wearing gloves will mitigate the risk. Additionally,
disposable gloves decrease the risk of infecting any hand wounds.
Pull back long hair or use a hair cover. Long hair is notorious for attracting dust, a potential source of
contamination. Additionally, long hair could be a health hazard when working near an open flame.
Use protective eye-wear or a face shield when working with hazardous materials or cultures. If your
eyes come in contact with microorganisms or harmful chemicals, wash your eyes for 15 minutes in an
eyewash and seek medical attention immediately.
Handling Microbial Cultures and Media
When working with any microbial strain, propagation success depends heavily on the prevention of
crosscontamination by other microorganisms. Sources of contamination can include non-sterile supplies,
media, reagents, unclean work surfaces and incubators, airborne particles, and unclean gloves. Below, we
describe some tips for handling microbial cultures and media.
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.
Maintain an uncluttered work space; all work surfaces should only contain equipment that is required
for your experiment.
Ensure that you have all necessary supplies before beginning an experiment. Being prepared will reduce
the likelihood of careless contamination.
Work may be performed in a thoroughly sterilized biosafety cabinet. Biosafety cabinets can be
sterilized via ultraviolet light in conjunction with 70% ethanol or an appropriate disinfectant.
Do not open windows or use fans that circulate outside air. If possible, work in laboratory settings that
have air vents covered with filters. This will prevent the contamination of cultures by airborne particles.
Frequently clean water baths used for thawing or warming media or solutions.
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
Before working with media and microbial cultures, wipe your work area with 70% ethanol or an
appropriate disinfectant.
Ensure you are wearing appropriate protective clothing. This will protect you from the culture as well as
reduce accidental culture contamination.
Only use sterile glassware, equipment, media, and reagents. Check media for contamination by
observing for turbidity.
Handle only one microbial culture at a time. The risk of cross contamination or misidentification
increases when more than one strain is handled at a time.
When working with test tube cultures, hold cultures at an angle after you remove the lid to avoid
airborne particles from falling into the culture. Sterilize the outside of the culture tube using a Bunsen
burner flame.
When working with plated cultures, hold the petri dish lid at an angle after you remove the lid to avoid
airborne particles falling into the culture dish.
When handling a microbial culture, work quickly and carefully in an environment that has minimal
distractions. Do not leave the lid off your culture for extended periods of time.
Ensure that all biosafety cabinets are routinely recertified Biosafety Cabinet Sanitation Clean work
surfaces with an appropriate disinfectant before and after use.
If the biosafety cabinet is equipped with germicidal UV lights, decontaminate work surfaces before and
after use by turning on the UV light for at least 15 minutes. Never use the UV light while the biosafety
cabinet is in use.
Using an appropriate disinfectant, wipe down the outer surface of all pipettes, pipette tip boxes, media,
materials, etc. prior to placing them in the biosafety cabinet.
Always wear a clean lab coat and sterile gloves when working in a biosafety cabinet. Proper use of a
Biosafety Cabinet
Only raise the biosafety cabinet sash to the recommended level, this will reduce disruption to the air
flow as well as assist in the prevention of airborne contaminant entry. When using a biosafety cabinet,
limit the amount of movement in the cabinet and do not remove your arms. Additionally, limit the access
to the area around the biosafety cabinet. This will reduce disruption to the airflow.
Do not use open flames within a biosafety cabinet. The resulting heat from the flame can disrupt the air
flow provided by the biosafety cabinet, increasing the risk for contamination. Additionally, gas leaks
associated with Bunsen burners or the use of an alcohol-based disinfectant near an open flame can result
in fire or injury.
5. COLONY MORPHOLOGY
A colony is defined as a visible mass of microorganisms all originating from a single mother
cell. ... Colony morphology can sometimes be useful in bacterial identification. Colonies are described as
to such properties as size, shape, texture, elevation, pigmentation, effect on growth medium
Shape – This refers to the form of the colony, or its overall appearance. Common shapes include circular,
rhizoid, irregular, filamentous, and spindle.
Margin – The margin refers to the edge of a colony, and may be an important characteristic in organism
identification. Common margins include entire, undulate (resembling waves), lobate (lobed structure),
curled, rhizoid, filamentous, or erose (irregularly notched).
Elevation – Elevation describes the side-view of the colony. The most common are flat, raised, convex
(curved surface), pulvinate (cushion-shaped), and umbonate (having a knobby protuberance).
Size – The size of a colony is often a useful characteristic for identification, and can be measured. Sizes
are often described as punctiform (shaped like a point), small, moderate, or large.
Appearance – The outwards aspects of the colony surface, often depicting if the colony is glistening
(glossy, shiny) or dull (cloudy).
Optical Property – This describes the opacity of the colonies. Colonies are frequently described as
opaque (impervious to light), translucent (lets light through diffusely), or transparent (allows light to pass
through without disruption).
Texture – Describes the consistency of a colony. Rough colonies have a granular, flattened surface and
are often associated with loss of virulence. Smooth colonies have a glistening, rounded surface. Other
terms used to describe colony texture include mucoid (gummy, viscous),butyrous (buttery texture), or dry
(brittle or powdery colonies).
Pigmentation – Some bacterial species produce pigments which can be either water soluble or soluble in
fat. The production of pigments may vary depending on the environmental conditions or age of the
colony.
lactobacillus acidophilus
bacillus subtilis
streptococcus faecalis
escherichia coli
Staphylococcus aureus
6. CONCLUSION
colony morphology of different species can be observed easily. We have observed the cultures for signs
of growth and how to inoculates streak plate.
Knowing about Aseptic technique is fundamental to the success and safety of an experiment. It’s avoid
health risk and hazardous or sterile materials on lab.