University of Santo Tomas

Faculty of Pharmacy
Department of Medical Technology

LIMITED FACE-TO-FACE MODULE

Enriched Virtual Mode During the Quarantine Period

Section: Microbiology

Topic Title: Culture Media Preparation, Streak Plating Method, and Gram Staining

Duration: 2 hours 30 minutes

Intended Learning Outcomes

At the end of the topic session, the interns should be able to:
1. Determine the proper procedure in the preparation, dispensing and storage of
commonly used culture media
2. Demonstrate the proper steps of preparing an agar petri plate and broth medium
3. To provide rationale for aseptic technique
4. To transfer bacteria aseptically from one form of culture medium to another
5. To explain the rationale and procedure for the Gram Stain
6. To interpret the prepared slide stained using Gram Stain procedure

Materials/Equipment
▪ Student handouts
▪ Reference books

Topic Outline
● Post-laboratory Conference

Topic Presentation
Each student will have to undergo one-on-one conference with the faculty
preceptor regarding the tactual performance of Culture Media Preparation, Streak Plating
Method, and Gram Staining. Before the conference, the student must be able to introspect
and have a self-reflection on the experiential learning that happened during the face-to-
face session with the preceptor. The student must evaluate himself if the procedures
listed below are successfully performed. During the presentation, the significance of
procedures listed down below must be well explained.
 University of Santo Tomas
Faculty of Pharmacy
Department of Medical Technology
Activities

A. Self-Reflection on Experiential Learning

The student must be able to Satisfactory Unsatisfactory Comment/

done done Problems
encountered
Wash hands with antiseptic and put on ✓
gloves and face protection
Assemble materials and equipment ✓

Select an agar plate to be inoculated and ✓

label the bottom of the plate
Pick up tube containing a culture in one ✓
hand; use fourth and fifth fingers of the
other hand holding the inoculating loop to
remove the cap from the tube (hold cap
with fingers for entire procedure; do not
lay cap down)

Insert tip of inoculating loop into culture ✓

and pick up a small amount of bacterial
culture

Sterilize the mouth of the tube, replace ✓

cap, and set tube
in test tube rack; do not allow anything to
touch the loop

Lift the agar plate lid just enough to insert ✓

the loop and gently spread the inoculum
over the surface of one quadrant of the
agar plate
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Faculty of Pharmacy
Department of Medical Technology
Able to make at least 3 quadrants of ✓
streak and sterilizing the loop on a flame
in between each streak
Place the agar plate upside down in the ✓
35° to 37° C incubator to incubate
overnight (18 to 24 hours)
Discard contaminated disposables into ✓
appropriate sharps or biohazard
containers and clean work area with
surface disinfectant
Able to prepare a bacterial smear and air ✓
dried completely
Heat fix the smear and allow the slides to ✓
cool
Flood the slides with crystal violet for the ✓
manufacturer’s
recommended time, usually 1 minute
Rinse the stain off the slides with a gentle ✓
stream of water
from a beaker, faucet, or plastic squeeze
bottle and
tilt the slides to remove excess water
Flood the slides with Gram’s iodine for the ✓
recommended
time
Decolorize and rinse smears one at a ✓
time:
a. Hold slide using forceps or clothespin
and add the decolorizer until no more
purple color runs off the slide
NOTE: Decolorize no longer than a few
seconds to prevent over-decolorization
b. Rinse the slide with water immediately
to remove the decolorizer and stop the
reaction; tilt the slide to remove
excess water and return slide to staining
rack
Counterstain the smears by flooding the ✓
slides with safranin
for the recommended time
Rinse the slides, tilt to remove excess ✓
water; wipe the
back of the slide with paper towel to
remove stain; stand
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Faculty of Pharmacy
Department of Medical Technology
slides on end or blot between sheets of
bibulous paper
to dry
Observe that the organisms either as ✓
gram-positive or gram negative; describe
their morphology (color, shape,
arrangement)

B. Post Laboratory General Questions

Culture Media Preparation

1. Give the components of a simple culture medium and their corresponding
purposes
The following are the characteristics of a simple culture media:
Hydrogen and oxygen are found in water.
Carbohydrates: The most common ingredient added to culture
media for energy.
Agar is a solidifying or gelling agent.
Peptone/Meat Extract: Nutrients; Carbon and Nitrogen Source
Calcium, magnesium, iron, trace metals: phosphates, sulphates,
and other key inorganic components include calcium, magnesium,
iron, and trace metals such as phosphates and sulphates.

2. In a tabulated form, give an example of each differential and selective culture

media and state the purpose of each incorporated substance
Differential
Seller’s Agar For differentiating nonfermentative bacili like
Pseudomonas, Acinetobacter, and Alkaligenes
PYR Agar Differentiate beta hemolytic Streptococci and
Enterococci based on their ability to produce enzyme,
pyrrolinodyl Arylamidase or pyroglutamyl
Aminopeptidase
Selective
Phenylethanol Agar For the selective isolation of Gram-positive cocci and
anaerobic Gram-negative bacilli
New York City Agar For the isolation of pathogenic species of Neisseria like
Neisseria gonorrhoeae and Mycoplasma hominis, and
Ureaplasma urealyticum
3. Give examples of culture media classified according to its: physical state (liquid,
semi-solid, solid), manner or formation (butt, butt and slant, slant, plated)
• Physical State
▪ Liquid
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Faculty of Pharmacy
Department of Medical Technology
• Thioglycolate broth: For the recovery of aerobes, anaerobes,
microaerophilic, and fastidious microorganisms
• Gram negative broth: increases the number of pathogens in
a stool sample
▪ Semi-Solid
• SIM (Sulfide Indole Motility): for motility testing
• Stuart’s Medium: for maintenance of viability of
microorganism
▪ Solid
• Salmonella Shigella Agar (SSA): for the isolation and
differentiation of Salmonella and Shigella
• Manner of Formation
▪ Butt
• Schaedler Agar: for the recovery of aerobes and anaerobes
• SIM (Sulfide Indole Motility): for motility testing
• Amie’s Medium: for maintaining the viability of the organisms
from source to the laboratory
▪ Butt and Slant
• Triple sugar iron agar (TSI): for the identification of Gram-
negative enteric bacilli based on their ability to ferment
glucose, lactose, and sucrose.
• Lysine iron agar (LIA): for the identification of Gram-negative
enteric bacilli based on their ability to decarboxylate or
deaminate lysine and to produce hydrogen sulfide or not.
▪ Slant
• Loeffler’s coagulated serum slants: for the isolation of
Clostridium diphtheriae
▪ Plated
• MacConkey Agar (MAC): For isolation and differentiation of
lactose and nonlactose fermenting gram-negative bacilli
• Mannitol Salt Agar (MSA): For isolation and differentiation

Streak plating Method

4. Explain why the use of aseptic technique is important in the bacteriology
laboratory.
• The term aseptic procedure refers to the absence of bacteria, viruses, and
other microbes. In bacteriology, this technique is critical for preventing
contamination while moving organisms from one culture medium to another.
Contaminants may proliferate in the form of colonies if aseptic method is not
followed. It also protects workers from becoming infected.
5. When is a loop preferable for transferring bacteria? How about an inoculating
needle?
A solid or dense media is retrieved with an inoculating needle, whereas
liquid media is retrieved with an inoculating loop. In contrast to an
 University of Santo Tomas
Faculty of Pharmacy
Department of Medical Technology
inoculating needle, which is a straight wire, an inoculating loop has a loop
at the end of the wire that can be calibrated to 1 uL or 10 uL.
6. What is the purpose of flaming the loop before and after each use?
It is critical to disinfect the loop/needle before and after each usage in
order to avoid contamination of the organism to be isolated as well as the
culture media or agar.
7. Why must the loop be cooled before using it in a culture? Should you set it down
to let it cool? How do you determine when it has already cooled?
After making the inoculating loop red hot, it's critical to cool it down since a
red-hot loop or needle could kill the organism that needs to be isolated.
The inoculating loop or needle cannot be set down to cool because it may
become contaminated.

Gram Staining

8. Why is it important to gently roll the swab across the slide when preparing a
smear?
Because the morphology and arrangement of bacteria are critical in the
identification of the organism, gently rolling the swab across the slide is
necessary to avoid loss of cellular elements and disruption of bacterial
arrangements.
9. Why must the smear for Gram stain be heat-fixed before it is stained?
It's critical to heat-fix the smear in order to eliminate the bacteria and avoid
infection. It also secures the smear in the slide and makes it easier for the
sample to absorb stains.
10. Why will old (more than 48 hours) cultures of Gram positive bacteria stain as
Gram Negative?
The reason for this is that bacteria in old cultures lose their peptidoglycan
cell walls with time, causing gram positive bacteria to resemble gram
negative bacteria.
11. Give the possible implications of the following improper procedures
The smear is applied in is too thick of a layer
Uneven staining and/or decolorization would ensue from a thick
smear. Gram positive bacteria would appear to be gram negative
bacteria because the crystal violet left would cause cell stacking,
making it impossible to tell which bacteria is which. Stacking
compromises bacterial form, and a large number of cells on a slide
will render the slip opaque, blocking light penetration and making
interpretation difficult.
bacteria are over-heated during fixation
The microbe would be harmed by overheating, which would result
in cell breakage, resulting in cell distortion following staining If left
over the flame for too long or kept stationary, it might scorch the
slide. Aerosolization can occur if heat fixing is done before the
 University of Santo Tomas
Faculty of Pharmacy
Department of Medical Technology
smear has entirely dried. When heat-fixed, the smear should also
face upward to avoid killing the microbe.
Use of expired culture smears or too old cultures
It could result in a gram varied consequence. Gram-positive
bacteria's cell walls may break down, making them unable to retain
the Crystal Violet stain.
Under-decolorization: alcohol (decolorizer) is washed away before it had
any effect on the cell wall
It could result in a gram varied outcome. Under-decolorized
organisms appear somewhat violet when they should be pink. It
may also generate crystal stains, necessitating decolorization and
restaining. If the alcohol was rinsed away rapidly, the backdrop of
the slide may be hazy.
Over-decolorization: Leaving alcohol (decolorizer) on the slide for too long
It could result in a gram varied consequence. Because the crystal
violet was totally washed away and only safranin was visible, over-
decolorized organisms seem pink when they should be violet.

C. Critical Thinking Scenarios

Case 1: Jose’s work assignments in bacteriology included wiping down work surfaces
and disposing of all contaminated materials and cultures appropriately. He used a
surface disinfectant to wipe the counters every hour during the work day. Several
times during each shift he gathered all the used culture plates and other contaminated
supplies into biohazard bags. After closing the bags securely, he placed them
alongside the other trash to be picked up by housekeeping services.

Questions for Case 1:

1. Should Jose be commended for ensuring the safety of everyone working in the
laboratory?
• Yes, Jose should be commended for this.
2. Explain your answer.
• Jose deserves praise for making sure that everyone is secure. He wiped the
surface of the table. According to the example above, work surfaces must be
followed religiously. He also disposed of potentially infectious waste,
contaminated items, and cultures in the appropriate bags, which he secured
snugly and securely.
 University of Santo Tomas
Faculty of Pharmacy
Department of Medical Technology

Case 2: Marion made a smear for a Gram stain and left it to dry while she went to
lunch. While she was gone Christine had extra time, so she stained it. When she
examined it under the microscope bacteria were scarce and large clear areas were
present on the slide. Christine prepared another smear, let it dry, heat-fixed it, and
stained it. She had no problem observing the morphology of the bacteria present on
the second slide
Question for Case 2:
1. Give the possible reason/s why Christine encountered the problem with the
Gram stain.
• The sample was exposed to lengthy air drying, which resulted in huge clear
areas in the smear that looked like holes under the microscope and may
have caused the bacteria to precipitate, making them scarce under the
microscope.

Rubrics
 University of Santo Tomas
Faculty of Pharmacy
Department of Medical Technology

References
Lehman, D. C. and Mahon, C. R. (2019). Textbook on Diagnostic Microbiology, 6th ed. USA:
Elsevier.
 University of Santo Tomas
Faculty of Pharmacy
Department of Medical Technology
Cardona, C. and Tiburcio, J. Laboratory Manual in Bacteriology. Philippines. C and E.

Prepared by

Name of Faculty Preceptors Signature

Miguel Carlos G. Arada, RMT, MSMT
Elner H. Del Rosario, RMT, MSMT

Reviewed by:

Mr. Benjie M. Clemente, MPH Ms. Ruth R. Bangaoil, MSMT

Member Member
Limited Face-to-Face Limited Face-to-Face

Ms. Mary Rose V. Pingol, MSMT Ms. Laarni E. Gloriani, MSMT

Member Asst. Coordinator
Limited Face-to-Face Limited Face-to-Face

Assoc. Prof. Ma. Frieda Z. Hapan, PhD

Chair, Teaching and Learning Committee

Endorsed by:

Mr. Joemarie T. Malana, MSMT Prof. Edilberto P. Manahan, PhD

Coordinator Chair
Medical Technology Clinical Internship Program Department of Medical Technology
Committee

Approved by

Prof. Aleth Therese L. Dacanay, PhD

Dean