SCHOOL OF SCIENCE AND TECHNOLOGY
Master of Science in Medical Laboratory Science
SCI8007SEF Medical Microbiology & Virology I
Laboratory Manual
Practical 1
2023 Autumn Term
Name ___________________________
Student ID _______________________
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 1
THE MICROBIOLOGY LABORATORY SAFETY RULES
Laboratory Safety in a microbiology laboratory is important. Infection might be caused by
the microorganisms being studied. Virulent human pathogens are used in this laboratory
course, these pathogens can cause disease in an immunocompetent host. Students need to
strictly follow the safety instructions to prevent potential infection. In addition to
microorganisms, some potentially harmful chemicals are used in this laboratory course.
The laboratory procedures involve use of glassware, open flames, and sharp objects, it can
cause damage if the laboratory apparatus is not properly used. To avoid biological hazard,
chemical hazard and laboratory accident, the following precautions should be strictly
followed:
1.
The laboratory procedures must be read before attending the laboratory session. By
being knowledgeable about the procedures, you will assure the safety of yourself and
of your fellow laboratory students.
2.
Laboratory coats are required. Wearing old clothing is also desirable, since many
reagents can produce permanent stains on clothes. Anyone wearing shorts must wear
a mid-calf length laboratory coat.
3.
Except using microscope or using computer or at other times as designated by the
laboratory technician or instructor, safety glasses must be worn in this laboratory
course.
4.
Students must not wear sandals or open-toed or canvas shoes because of the
possibility of chemical spills and the risk of cuts and infection from broken glass.
Students wearing inappropriate footwear will not be allowed to work in the
laboratory.
5.
Long hair must be tied back to minimize the chance of contamination and fire hazard during
the experiments.
6.
Smoking, eating, and drinking in the laboratory are strictly prohibited.
7.
Before entering the laboratory, coats, books, and other paraphernalia (except
laboratory manual) should be placed in designated locations. These items shall never
be placed on bench tops.
8.
At the beginning and the end of each laboratory session, bench tops shall be wiped
with a disinfectant.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 2
9.
Observe strict personal hygiene. Wash your hands with soap at the start of the
laboratory session before performing any laboratory procedures and wash your hands
again before leaving the laboratory at the end of the session.
10.
All cultures are potentially pathogenic and the precautions below should be followed
at all times:
a.
Broth cultures must always be carried in a test tube rack when moving around
the laboratory.
b.
Broth cultures must be kept in a test tube rack on the bench tops when not in
use.
c.
Broth cultures must never be pipetted by mouth. Always use a suction aid
(never use your mouth) when filling a pipet or use a pipette with a biological
or chemical reagent.
d.
Spilled cultures should be covered with paper towels and then saturated with
disinfectant solution. Following 15 minutes of reaction time, the towels should
be removed and disposed of in the manner indicated by the instructor.
11.
Spills, cuts and other accidents should be immediately reported to the instructor or
laboratory technician. Further treatment shall be considered if necessary.
12.
To avoid aerosol generation, proper techniques for flaming the inoculating loops
shall be used. Cultures and reagents shall also be mixed in a way to avoid splashing.
Proper techniques of these procedures will be demonstrated by the instructor or
laboratory technician.
13.
Removal of media, equipment, and cultures from the laboratory is prohibited unless
it is directed by the instructor or laboratory technician.
14.
During and at the end of each laboratory period, used glassware and plasticware are
disposed to different trays, namely ‘infectious plasticware’, and ‘non-infectious
plasticware’, ‘non-infectious glassware’. Infectious glassware and broken glassware
are disposed to sharp box.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 3
Part 1 Streaking for Isolation and Interpretation of
Primary Culture Results
Learning objectives:
✓ Basic laboratory safety in medical microbiology laboratory
✓ Basic laboratory technique of clinical microbiology laboratory:
➢ Inoculate bacterial cultures on agar plates using streaking
technique that shall separate the individual bacterial cells to
obtain well-isolated discrete colonies on primary culture
media
➢ Observe key colony morphology
➢ Interpret primary culture results with colony morphology and
gram stains results.
✓ Students shall be able to explain the principle and intended use of
various types of media and classify a given medium as a nonselective, selective, enrichment or differential medium.
✓ Students shall be able to discuss the various incubation conditions
used in a clinical microbiology laboratory.
Materials:
•
•
•
•
•
Inoculating loop
Bacti-Incinerator/Bunsen burner
Broth cultures of
o Staphylococcus aureus
o Escherichia coli
o Enterococcus faecalis / Streptococcus pneumoniae
o Salmonella species
o Pseudomonas aeruginosa
Agar plates of
o Tryptic Soy Agar (TSA)
o MacConkey agar (MAC)
o Chocolate agar (CHOC)
Sterile swabs
References:
Mahon and Lehman, Textbook of Diagnostic Microbiology, Sixth Edition, Chapter 6
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 4
Principles:
Culture remains the diagnostic gold standard for many bacterial infections. Most bacteria
can be cultivated in vitro (outside of a living organism) using artificial culture media
(plural; singular is “medium”). A bacterial culture media is a preparation in which bacteria
can use the nutrients of culture media as their food and multiply.
Clinical microbiology laboratories use a wide variety of growth media for isolation of
commonly encountered bacterial pathogens. Culture media vary in their form. Liquid
culture medium is called broth. It can be solidified by adding a solidifying agent, agar. Agar
consists of a mixture of polysaccharides extracted from algae. Except acting as a solidifying
agent, it also provides nutrients to grow microorganisms. Other compositions of a media is
determined by the species to be cultivated. Enriched media contain the nutrients required
to support the growth of a wide variety of organisms, (including some of the more fastidious
ones), they are commonly used to harvest as many different types of microbes as are present
in the specimen. Selective media generally selects for the growth of a desired organism,
suppressing the growth of non-desired organisms. Differential media takes advantage of
biochemical properties of target organisms, often leading to a visible change when growth
of target organisms are present. The selection of primary media shall enable the growth of
suspected causative bacteria from a particular clinical sample.
The streak-plate procedure is commonly used to isolate pure cultures of bacteria, or
colonies, from mixed populations by simple mechanical separation. Primary inoculation is
often made with a loop, swab, or other suitable devices. Generally, 1-2 drops of liquid
specimens, swab dipped into fecal or sputum specimen or direct swab specimens are used
as primary inoculum. As the bacteria in the primary inoculum is diluted by streaking it over
successive quadrants, the number of bacteria decreases. Usually, by the third or fourth
quadrant, only a few bacteria are transferred which shall give discrete colony. Isolation of the
bacterial colonies is essential to obtain pure cultures prior to characterizing the colony
morphology and biochemical properties of isolated bacterial organisms.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 5
Procedure:
1. Obtain a paper template of a petri dish to practice streaking using a pen. Perform
successful streaks on the paper template at least twice PRIOR to continuing to step
#2.
2. Working in groups, label the bottom of agar plates with your initials, date, and the
organism. The bottom is the section with the agar. Label according to the following:
•
Staphylococcus aureus – TSA / MAC / CHOC
•
Escherichia coli – TSA / MAC
•
Enterococcus faecalis / Streptococcus pneumoniae – TSA / MAC / CHOC
•
Salmonella species – TSA / MAC
•
Pseudomonas aeruginosa – TSA / MAC
3. Turn on the Bunsen burner/ Bacti-Incinerator.
4. For each broth culture, remove a sterile swab from its wrapper, holding it between
the thumb and forefinger of the right hand.
5. Next hold one of the broth tubes in the left hand and remove and hold the cap with
the little finger and palm of the right hand.
6. Heat the neck of the broth tube by holding against the opening of incinerator. Dip
the swab into the broth culture tube, saturating it well. Press and swirl the swab
against the inside of the tube above the broth to remove excess. Withdraw the swab,
heat the neck of the broth tube again, and replace the cap. Replace the broth tube in
a rack.
7. Remove the agar plate from its lid with the left hand. While holding the plate,
inoculate the agar heavily near the periphery of the plate spanning to approximately
¼ of the agar. This area is referred to as the first quadrant (See Figure 1 step #1.) You may
use the same swab for each broth culture to inoculate the first quadrant on several plates,
as long as the swab is used to inoculate the plates in this order: TSA, CHOC, and MAC.
This order is important since it starts from non-selective media to selective media. If a
selective agar plate is inoculated first, the reagents such as antimicrobials and/or other
chemicals from the selective plate may be carried over to the next agar plate.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 6
8. Replace the plate into its lid and discard the swab into the appropriate biohazard
container.
9. Flame the wire loop and let it cool for 2-3 seconds.
10. Holding the agar plate with the left hand, streak the original inoculum at a 90°
clockwise angle (Figure 1 step #2.) Hold the bacteriological loop loosely between
the thumb and index finger. Allow the weight of the loop to exert its own pressure.
There is no need to exert additional pressure with the hand.
11. Replace the plate into its lid and flame the loop. Let it cool again and streak again
clockwise to 90 degrees from the second streak (Figure 1 step #3).
12. Repeat step 9, streak the fourth quadrant (Figure 1 step #4).
13. Flame the loop to remove all organisms.
14. Repeat steps 3-12 with the other broth cultures.
15. Have the instructor review and initial your streaking technique.
16. Incubate at 35° C for 24 hours.
NOTE: If cultures cannot be examined after 24 hours (i.e., weekend), the agar
plates shall be refrigerated after incubation.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 7
Figure 1
NOTE: Different methods exist for removing organisms from the loop between quadrants.
Generally, the metal loop is sterilized between each quadrant by incinerating the loop.
Some clinical microbiologists flame once after the initial quadrant and then rotate the loop
to streak sequential quadrants using unused side of the loop. When using plastic loops,
some may stab the loop several times into the peripheral region of the agar to clear the loop
between quadrants. Please be aware that plastic loop shall not be flamed!
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 8
Other Inoculation Techniques:
The quadrant (4 corner) method is used in this course as we are working primarily with
broth cultures with numerous colony forming units (CFU). However, when working with
primary specimens obtained directly from a patient, such as on a swab, a three (3) corner
inoculation or streaking technique is frequently used. It is because the number of organisms
or CFU found in patient primary specimens is most often much fewer than those grown in
broth cultures.
Evaluation of Isolation Technique
Examine the agar plates prepared by the instructors, theses plates were incubated for 24
hours before the lab session. There should be sufficient growth of each organism on the
plates and the isolation should be such that in the last corner or quadrant of growth there
should be well-isolated colonies. To determine the amount of growth of each colony-type,
the culture should also be evaluated according to the following semi-quantitative measures:
Growth in 1st quadrant only = Scanty growth
Growth in 1st, 2nd, and 3rd quadrants = Moderate growth
Growth in all quadrants = Heavy growth
Procedure:
1. Using the Colony Morphology handout from the instructor, evaluate the colony
morphology of each culture and include results on the worksheet below.
2. If there is no growth on the plate, indicate that on the worksheet by using “NG”
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 9
Practical 1 Part 1
Results Form
Name:
Student ID:
Marks:
/ 24
1. Examine agar plates prepared by the instructors and note the colony morphology.
(1 mark @)
Gram +/-, cell morphology [Rod/cocci]
Amount of
Growth
Plate
Colony Morphology
E. coli on TSA
Moderate growth, smooth, white colur colony
E. coli on MAC
Moderate growth, medium, pink colony
Pseudomonas on TSA
Moderate
Pseudomonas on MAC
Moderate
Large, spreading
Salmonella on TSA
Moderate
White, large, smooth
white colony, large, swarming
Large,
Salmonella on MAC
S. aureus on TSA+SB
17
Hemolysis, Alpha
S. aureus on TSA
12
Circula, Medium, white, smooth
S. aureus on MAC
No growth
Circula, Bile salt & to inhibit growth
S. aureus on CHOC
24
Circula
Enterococcus faecalis /
Streptococcus pneumoniae on TSA
17
Circula
Enterococcus faecalis /
Streptococcus pneumoniae on MAC
Inhibited, 4
Circula
Enterococcus faecalis /
Streptococcus pneumoniae on CHOC
9
Circula
S. Pneumoniae on MAC
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 10
PRE-LAB STUDY QUESTIONS
Practical 1 Part 1 Streaking for Isolation and Interpreting Primary Culture Results
Name:
Student ID: 13094812
Marks:
/8
1. Define the following types of media and give an example. (4 marks)
a. Selective media
Selective media is a type of media that allows the growth of specific microorganisms while
inhibiting the growth of others. MacConkey agar, which interfere with the growth of many
gram-positive bacteria and favor the growth of gram-negative bacteria
b. Enriched media
Enriched media is a type of media that contains supplements such as blood, serum to enhance the
growth of fastidious bacteria. An example of enriched media is blood agar.
c. Differential media
Differential media is a type of media that distinguishes one microorganism type from another growing
on the same media. An example of differential media is Mannitol Salt Agar.
d. Enrichment media
Enrichment media is a type of media that selects for certain microorganisms by including a nutrient
that the desired microorganism can use but its competitors cannot. Luria broth is an example of
enrichment media, it is used to culture fast-growing bacteria such as E. coli.
2. Why is it important to have isolated bacterial colonies on a culture plate? (1 mark)
Identification of unknown microorganisms: To identify an unknown microorganism, it is necessary to
separate a mixture of microbial species
3. The agar in a nutrient agar serves as a
a surface for bacterial growth . (1 mark)
4. Why is a loop flamed before it is placed into a specimen? (1 mark)
It is an effective method to prevent contamination.
5. A four quadrant technique is more preferred than a three quadrant technique when
working with broth cultures. Why? (1 mark)
Since adding one more quadrant can have a greater dilution of the inoculum, allowing operators to
obtain single colonies
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 11
Part 2 Preparation of Smears and Gram Staining
Learning objectives:
At the end of this activity, the student shall be able to:
✓ Describe the Gram stain procedure.
✓ Prepare and evaluate gram stain smears.
✓ Correctly interpret and record Gram stains on primary specimens,
as well assingle-organism slides.
✓ Evaluate specimen quality based on specimen types and Gramstained smear results.
✓ Discuss the clinical applications of Gram-stained smears.
✓ Identify the characteristics of a quality Gram stain.
✓ Recognize key microscopic characteristics of bacteria on Gram stains.
✓ Distinguish significant findings from artifact on a Gram-stained smear.
✓ Identify special bacterial structures: capsules, flagella, and endospores.
✓ Explain the significance of these bacterial structures in
diagnosis and identification of disease.
✓ Perform or describe the techniques that identify these special structures.
✓ Identify acid-fast bacilli on a stained preparation.
✓ Explain the significance of acid-fast bacilli in a specimen.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 12
Materials:
•
24-hour streaked plates of:
o Staphylococcus aureus
o Escherichia coli
o Enterococcus faecalis / Streptococcus pneumoniae
o Salmonella sp.
o Pseudomonas aeruginosa
• microscope slides
• Inoculating equipment:
• Bunsen burner / Bacti-Incinerator
• Inoculating loop
• Staining equipment:
• Forceps
• Wash bottle
• Gram Stain Reagents:
• Crystal violet
• Gram's iodine
• Acetone-alcohol
• Gram's safranin
• Microscope
• Immersion oil
• Demonstration slides of capsules, flagella, endospores, and acid-fast
bacilli
References:
Mahon and Lehman, Textbook of Diagnostic Microbiology, Sixth Edition, Chapter 7
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 13
Principle:
Gram staining is the most crucial staining technique in clinical microbiology for
identification of routine cultures. Gram stain is a differential stain requiring a primary stain
(crystal violet) and a counter-stain (safranin or basic fuchsin).
The first step in gram staining is the application of the primary stain, crystal violet for an
initial staining of a slide with heat-fixed smear. The next step is fixation of primary stain,
it involves using a mordant i.e. iodine to form an insoluble complex with crystal violet in
the bacterial cell. Subsequently, a decolorizer, often a solvent of ethanol or acetone-alcohol,
is applied. After decolorization, the smear is stained with a counter-stain.
Microorganisms that resist decolorization and retain the crystal violet-iodine complex
appear deep purple microscopically and are called Gram positive (G+). Microorganisms
that decolorize and counter-stained by safranin (or basic fuchsin) appear red and are called
Gram-negative (G -).
Before decolorization, both Gram positive (G+) and Gram-negative (G -) bacteria are
stained deep purple with crystal violet. The cell wall of G+ bacteria is thick, consisting
many layers of peptidoglycan interspersed with teichoic, and the lipid content is low. The
decolorizer causes the thick cell wall of G+ bacteria cell to dehydrate and shrink, which
closes the pores in the cell wall and prevents the crystal violet- iodine complex from
washing away during decolorization. The Crystal Violet-Iodine complex remains bound to
the cell of G+ cells after decolorization and thus G+ cells appear deep purple after Gram
staining. In contrast, the decolorizer dissolves the outer membrane of G – organisms, which
is formed of lipids. The cell wall of G – cell is composed of thin layer of peptidoglycan.
During decolorization, it becomes porous and does not retain the crystal violet stain. Then,
when G – cell is again stained with safranin, the G – cell is stained red in color.
Note: If the decolorization step is omitted, all bacteria shall appear Gram-positive.
Direct Gram stains from clinical specimens can provide the physician with valuable
information to begin treating patients with antibiotics long before culture results are
obtained. In addition, the results of the Gram stain can confirm specimen acceptability,
identify specific infectious agents, and determine the probability of infection by observing
indicators of inflammation.
Of note, clinical Gram stain results are preliminary only, and the physician should evaluate
antibiotic therapy when culture and antibiotic sensitivity results are available. Also, due to
the presence of normal flora, it is uncommon to perform routine Gram stains on nonsterile specimens such as stool and sputum.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 14
Procedure:
1. Preparation of Smears: Prepare smears of organisms from the plates: S. aureus from
TSA, S. aureus from CHOC, E. coli from TSA, E.coli from MAC, Streptococcus from
TSA, Streptococcus from CHOC, Salmonella from TSA, Salmonella from MAC,
Pseudomonas from TSA and Pseudomonas from MAC.
Your group shall make a total of 10 smears, putting two (2) smears on each slide.
a. Use clean slides. If necessary, rinse each in alcohol to remove any grease and airdry.
b. Label each slide appropriately, handling the slides by the edges only. Use a wax
pen. DO NOT uses an ink marker. Writing using an ink marker dissolves during
the staining process.
c. A circle is marked one side of the slide using a wax pencil. Marking the slide
makes it convenient to identify the surface of the slide that contains the smear. On
the opposite side, place a small drop of sterile saline on the center of circle. Using
a sterilized inoculating loop, obtain a small amount of the colony to be stained and
emulsify in the drop of water on the slide. Spread over an area of about the size of
a nickel.An adequate smear is one that, when held up to the light, a thin film is
faintly visible. Do not make the smear too thick, the smear should be one cell
thick.
d. Allow the smear to air dry and then heat fix by holding the slide, smear side out,
against the opening of the Bacti-Incinerator for about 3 seconds or passing the slide
through the flame 2-3 times. Hold the slide with a wooden slide holder to prevent
burning of your fingers. Check to make sure the slide has been heated sufficiently
by gently placing the slide, smeared surface up, against the back of the hand; the
slide should feel moderately warm. After cooling, the slide is ready for staining.
2. Gram Stain
a. Place the slides, smear surface up, on the staining rack over the sink or a staining
station.
b. Flood the slide with crystal violet, and allow to react for 30 seconds.
c. Handling the slide with forcep/slide holder, tilt it to about 45ºangle to drain the dye off.
d. Continue to hold the slide at an angle and immediately rinse it thoroughly with a
gentle stream of water from a wash bottle.
e. Place the slide on the rack and flood it with iodine. Allow it to react for thirty
30 seconds.
f. With forceps/slide holder, tilt the slide and allow to drain.
g. Immediately rinse the slide thoroughly with water from a wash bottle.
h. Replace the slide on the rack and flood it with decolorizer (acetone alcohol) until
the run-off becomes clear. Adjust decolonization time according to the thickness
of the smear.
i. Rinse immediately with water. This stops the decolorizing process.
j. Replace the slide on the staining rack and flood it with safranin. Allow to stand
for 30-45 seconds.
k. Drain the slide and wash thoroughly with water.
l. Allow the slide to dry in a rack or blot (not rub) carefully with bibulous paper.
m. When slides are dried, observe g ra m s t a i ni ng r esul t s using oil immersion
microscope and report results in report sheet.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 15
3. Quality Control
a. Daily
i. Check the appearance of reagents for precipitate or crystal sediment.
ii. Prepare a smear of Escherichia coli (ATCC 25922) and Staphylococcus
epidermidis (ATCC 12228) or Staphylococcus aureus (ATCC 25923).
Fix and stain. Expected results:
1. Escherichia coli: pink, Gram negative rods
2. Staphylococcus epidermidis or aureus: deep purple, Grampositive cocci
b. Gram stain quality
Many factors can affect the Gram reaction. Some of these factors include
whether the smear is too thick or thin, excessive heat fixing, improper
decolonization, antibiotic therapy, age of the colony, precipitated stain, length
of counterstain, and the presence of mucus or other proteinaceous material.
c. Quality assurance
i.Compare final culture results with Gram stain reports. Morphologies seen
on the Gram stain should be recovered in the culture.
ii. Annual review of reference slides to assess competency.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 16
Special Staining Techniques: Acid-fast Bacilli Stain, Capsule Stain, Endospore
Stain, and Flagella Stain
Some bacteria possess cell walls and other structures that are best demonstrated
by methods other than the Gram Stain. This exercise deals with a differential stain for the
special type of waxy cell walls possessed by Mycobacterium and with methods used to
demonstrate endospores, capsules, and flagella. In addition to their value in identification
of certain bacteria, demonstration of these structures is important for your understanding
of the basic structure and function of bacterial cells in disease processes.
Bacterial Capsules
The capsule is a gelatinous, slimy material surrounding the bacterial cell. In many
cases the capsule helps protect the cell against phagocytosis. Thus potential pathogens are
protected from the body's natural defenses and are more likely to cause disease than noncapsulated strains. The capsule also allows bacteria to adhere to surfaces, such as mucous
membranes and teeth. Other functions of a capsule include protection from dehydration
and loss of nutrients. In this exercise, capsules are demonstrated by the negative stain, in
which the capsule shows up as a clear area or halo surrounding the cell against the dark
background of nigrosin stain (India ink).
Bacterial Flagella
Flagella are structures that enable bacteria to be motile. They may occur singly at one end, in
tufts at one or both ends, or arranged all around the cell.
monotrichous = a single flagellum
amphitrichous = a single flagellum at both ends of the cell
lophotrichous = two or more flagella at one or both ends of the cell
peritrichous = flagella distributed over the entire cell
The number and arrangement of flagella can be used to help identify bacteria.
Flagella are demonstrated by special stains using mordants that increase the width of the
flagella and are then stained with carbol-fuchsin so that they may be seen with the
microscope. NOTE: The pink color of the microbes is due to the color of the primary
carbol-fuchsin stain, and is NOT an indication of a Gram reaction, as in the Gram stain
procedure.
Bacterial Endospores
Endospores are very resistant structures that are formed by certain bacteria under adverse
conditions. Two genera of Gram-positive bacilli (rods) are endospore-formers: Bacillus
and Clostridium. Endospores enable the organism to survive drying and lack of nutrients,
so they can exist in dust and soil for many years. Their presence in dust accounts for much
of the laboratory contaminants. The very thick spore wall does not stain easily, so the
endospores will appear in Gram stains as unstained areas inside the cell. To stain the
spores themselves, (1) carbol-fuchsin or (2) malachite green stain is heated so that it
will be absorbed by the wall of the endospore so that they appear (1) red or (2) green.
The vegetative part of the cell will decolorize upon rinsing with 95% ethanol and can then
be counterstained with (1) methylene blue or (2) safranin for contrast.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 17
Acid-Fast Bacilli
The cell walls of the genus Mycobacterium, which includes the pathogens of
tuberculosis and leprosy, are different from most other types of bacterial cell walls
because they are waxy and stain poorly, if at all. However, they will take up the acid-fast
stain. This stain uses carbol-fuchsin to which phenol has been added. The cell wall then
resists decolonization with acid-alcohol. (alcohol plus hydrochloric acid; thus the name
"acid-fast") The end result is an organism that retains the carbol-fuchsin color. Other
organisms will decolorize with the acid-alcohol and will take up the counterstain brilliant
green or methylene blue. Mycobacterium species are therefore often called "acid-fast
bacilli" (AFB).
Materials:
Prepared demonstration slides of capsules, flagella, endospores, and acid-fast bacilli
Procedures:
I.
Capsule stain by the negative method
Examine the demonstration slides with oil immersion for the presence of
capsules. They should appear as tiny, unstained, “halos” around the bacteria
cells.
II.
Flagella stain: Examine the demonstration slides under oil-immersion for the
presence of flagella. They should appear as thin, whip-like "tails". Remember,
this is not a gram stain, and the color does not designate a Gram reaction.
III.
Endospores stain
Examine the demonstration slides under oil-immersion for bacterial endospores.
They will appear as small (1) pink or (2) green or colorless circles or ovals
inside the bacilli.
IV.
Acid-fast stain
Examine the demonstration slides with oil-immersion for the presence of the acidfast organisms (“AFB” = acid-fast bacilli). They should appear as clumps
(“cords”) of tiny, fuschia-colored bacilli. Other, non-acid-fast bacteria will appear
blue.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 18
Practical 1 Part 2 Results Form
Name:
Student ID:
Marks:
/ 12
1. Gram Stains
The student shall record Gram stain results in the table below.
If noorganisms are seen, note “No organism seen or NOS”
Plate
Gram Stain Result
Note down Gram reaction and cell morphology
(1 mark @)
E. coli on TSA
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
E. coli on MAC
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Pseudomonas on TSA
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Pseudomonas on MAC
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Salmonella on TSA
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Salmonella on MAC
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
S. aureus on TSA
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
S. aureus on MAC
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
S. aureus on CHOC
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Enterococcus faecalis /
Streptococcus pneumoniae
on TSA
Enterococcus faecalis /
Streptococcus pneumoniae
on MAC
Enterococcus faecalis /
Streptococcus pneumoniae
on CHOC
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
Gram positive cocci / Gram positive rod / Gram negative cocci /
Gram negative rod / NOS / Other: ____________________
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 19
Name:
Practical 1 Part 2 Results Form
Student ID: 13094812
Marks:
/8
2. Special stains
Examine the slides with special stains provided by the instructor.
Draw the appearance of the structures and annotate them. (2 marks per slide)
A. Capsules
Special stain: ___________
Objective lens: _____X
C. Endospores
Special stain: ___________
Objective: _____X
B. flagellum / flagella
Special stain: ___________
Objective: _____X
D. Acid-fast bacilli
Special stain: ___________
Objective: _____X
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 20
PRE-LAB STUDY QUESTIONS
Practical 1 Part 2 Preparation of Smears and Gram Staining
Name:
1.
Student ID:
Marks:
/ 35
Why are direct Gram stains ordered on clinical specimens? (1 mark)
Gram staining allows for the identification of bacterial morphology, which can be used to narrow down the
possible bacterial species
2.
Why a Gram stain is performed on all CSF specimens? (1 mark)
Gram staining is a rapid diagnostic tool that can provide preliminary results within minutes
3. Why is Gram stain not ordinarily done on stool? (1 mark)
Stool contains a large number of normal flora, which can make it difficult to identify pathogenic bacteria
using Gram staining.
4.
Discuss how cell wall structure determines the Gram stain reaction of a bacterium. (2
marks)
The Gram-positive cell wall is thick and consists of many layers of peptidoglycan, the nature of their cell wall,
Gram-positive bacteria stain purple after Gram staining. The Gram-negative cell wall is thinner and the outer
membrane contains lipopolysaccharides, which are responsible for the endotoxic properties of Gram-negative
bacteria, leading to a red color after counterstaining
5.
State the four reagents used in the Gram stain procedure, including their function. (4
marks) Crystal violet: Primary stain that stains both Gram-positive and Gram-negative bacteria purple by binding to
negatively charged cell wall structures
Iodine: Mordant
Acetone:Decolourizer
Safranin: A counterstain that counterstains the Gram-negative bacteria pink, it is a positively charged molecule
that binds to negative bacterial cell wall structures
6.
List four factors that can affect the Gram reaction. (2 marks)
1.) The lifespan of the culture
2.) Thickness of the smear
3.) Type of organism
4.) Excessive heat fixation
7. In a Gram-stained smear, bacteria are enumerated with the
mark)
100x
objective. (1
8. In a Gram-stained smear from a sputum sample, epithelial cells are enumerated with the
40x
objective. (1 mark)
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 21
9. What is the importance of performing these special stains? What information do they
give you? (4 marks)
Acid-fast Bacilli Stain: The acid-fast bacilli stain is used to identify acid-fast bacteria that contain mycolic acid in their cell walls. The
information provided by the acid-fast stain helps to assess the adequacy of preliminary diagnosis and antimicrobial therapy selected
after collecting culture specimens and before final identification of the microorganism.
Capsule Stain: The capsule stain is used to identify the presence of capsules surrounding bacterial cells. Capsules are protective
structures that help bacteria evade the host immune system. The information provided by the capsule stain helps to assess the
virulence of the bacteria and the potential for causing disease.
Endospore Stain: The endospore stain is used to visualize bacterial endospores, which are highly resistant structures that allow
bacteria to survive in harsh environments. The information provided by the endospore stain helps to identify the presence of
endospores and the potential for bacterial survival in the environment.
Flagella Stain: The flagella stain is used to visualize bacterial flagella, which are whip-like structures that allow bacteria to move. The
information provided by the flagella stain helps to identify the motility of the bacteria and the potential for spreading infection.
10.
a. Is a bacterium that possesses a capsule more virulent? (1 mark)
Yes, as they have more protection from the capsule acting as a sheild.
b. What are the functions of a capsule? (1 mark)
Protection mainly.
11.
a. Why are endospores important to a bacterial cell? Under what conditions are they
formed? (2 marks)
Endospores are important to a bacterial cell as they allow them to survive in harsh environments.
Granting resistant to ultraviolet radiation, high temperature, extreme freezing, and chemical disinfectants
Formation is usually triggered by a lack of nutrients.
b. What genera of bacteria can produce endospores? (2 marks)
Bacillus and Clostridium
c. Give an example of the genus and species of four pathogenic bacteria that
produce bacterial endospores. (4 marks)
Bacillus anthracis, Clostridium botulinum, Clostridium tetani, and Bacillus cereus
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 22
12.
a. What are the genera that the acid-fast stain is used to identify? (1 mark)
The acid-fast stain is used to identify acid-fast organisms such as members of the genus Mycobacterium
b. Name two diseases that can be diagnosed with the aid of the acid-fast stain. (2
marks)
Two diseases that can be diagnosed are tuberculosis and leprosy.
c. What is the function of flagella? (1 mark)
Movement of cells.
13.
Write a brief explanation of why each one of the following bacterial structures
requires a "special" staining technique in order to be observed. (Explain why they
cannot be demonstrated using a Gram Stain.)
a.
Capsule (1 mark)
Difficult to stain using the Gram stain because they are composed of polysaccharides or other materials that are not
easily penetrated by the crystal violet-iodine complex.
b.
Flagella (1 mark)
Too thin to be seen using the Gram stain
c.
Endospore (1 mark)
Endospores are highly resistant structures that are not easily penetrated by the crystal violet-iodine complex.
d.
Acid-fast bacilli (1 mark)
Acid-fast bacteria have a unique cell wall that is resistant to most compounds, including the crystal violet-iodine complex
from gram stain.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 23
Part 3 Presumptive Identification of unknown Bacteria
Learning Objective:
At the completion of this activity, the student shall be able to presumptively identify and
differentiate Escherichia coli, Pseudomonas aeruginosa, Salmonella species, Staphylococcus
aureus and Streptococcus pneumoniae through 1) observing colony morphology, hemolytic
properties and Optochin sensitivity, and 2) using Gram stain, oxidase test, catalase test,
coagulase tests (Staphaurex/slide test and tube test), indole test and oxidase test.
Materials:
• Blood agar cultures of:
o Escherichia coli
o Pseudomonas aeruginosa
o Salmonella species
o Staphylococcus aureus
o Streptococcus pneumoniae
• Trypticase soy agar (TSA) cultures of:
o Escherichia coli
o Pseudomonas aeruginosa
o Salmonella species
o Staphylococcus aureus
o Streptococcus pneumonia
• MacConkey agar cultures of:
o Escherichia coli
o Pseudomonas aeruginosa
o Salmonella species
o Staphylococcus aureus
o Streptococcus pneumoniae
• Gram stain materials
• 3% hydrogen peroxide (Catalase reagent)
• Staphaurex™ Latex Agglutination Test / Rabbit coagulase plasma (Coagulase test)
• Bile salt (sodium deoxycholate)
• Kovac's reagent (Indole test)
• Oxidase reagent
• Sterile cotton swab, wooden stick, dropper, test tube and filter paper
• Microscope slides
• Microscope and Immersion oil
• Optochin disks
References:
Mahon and Lehman, Textbook of Diagnostic Microbiology, Sixth Edition, Chapter 6 -10, 14,
15, 19 and 21
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 24
Principles
Bacteria in the genus Staphylococcus are Gram-positive spherical cells that occur singly,
occasionally in pairs, but most frequently in irregular clusters. The appearance of a Gramstained smear is usually sufficient to distinguish staphylococci from the streptococci
because of the characteristic cell grouping (grape-like clusters) of staphylococci. These two
groups may also be distinguished by the presence of the enzyme catalase, in which
staphylococci are catalase-positive and streptococci are catalase-negative.
Staphylococcus species contain the enzyme catalase, whereas most species of
Streptococcus (another Gram-positive coccus) do not. Catalase break down hydrogen
peroxide. When mixed with 3% hydrogen peroxide, catalase positive organisms generate
bubbles of oxygen which are visible to the naked eye whereas catalase negative organisms
do not. It is preferable to test colonies from media without blood since erythrocytes possess
catalase activities. In addition, be careful not to gouge agar when picking up colonies for
this same reason.
catalase
H2O2
→
H2O + O2 (gas bubbles)
Some bacteria synthesize the enzyme hemolysin. Hemolysin is an exoenzyme that lyses
red blood cells. If a colony of bacterial cells is producing hemolysin and secreting it into the
blood agar medium, there shall be a round, clear zone surrounding the colony because the
red blood cells in that area have been lysed (zone of hemolysis). This type of grouping is
referred to as Brown’s classification. The three types of hemolysis are alpha, beta and
gamma:
Alpha hemolysis is caused by oxidization of hemoglobin to green methemoglobin. It is
characterized by an indistinct zone around the colony with a greenish discolorization.
Beta hemolysis is caused by complete lysis of the red cells characterized by a clear, colorless
zone around the colony.
Gamma hemolysis is actually a misnomer in that there is no apparent hemolysis or
discoloration around the colony of blood agar.
S. aureus is usually hemolytic, but sometimes it is not. The presence or absence of
hemolytic properties, therefore, cannot be used as a definitive identification of
Staphylococcus species. S. aureus usually produces coagulase, while most coagulasenegative staphylococci do not produce coagulase. Coagulase is an exoenzyme that causes
fibrin of blood plasma to clot. Two forms of coagulase may be produced by S. aureus: free
and/or bound. Bound coagulase, also known as clumping factor, is attached to the cell wall
of the organism, whereas free coagulase is an extracellular enzyme.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 25
Staphaurex is a latex slide agglutination test for the differentiation of staphylococci that
posses clumping factor and/or Protein A (found in S. aureus). This kit consists of yellow
latex particles coated with human fibrinogen and IgG. Once the latex reagent is mixed with
colonies, staphylococci that have protein A or clumping factor crosslink occur giving a
visible agglutination. The slide test is simple to perform and rapid, but detects bound
coagulase only. Therefore, all negative slide coagulase results must be followed by a tube
test, which shall detect both bound and free coagulase. The test tube test is more sensitive
because it can pick up smaller quantities of coagulase. In the tube test procedure, free
coagulase is liberated from the cell which activates prothrombin in plasma, thereby
converting fibrinogen to fibrin and promoting the clotting of plasma or blood.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 26
Streptococci may instead be presumptively classified according to the type of hemolysis
produced on blood agar.
(Presumptive) Identification of streptococci
Test
Streptococcus
pneumoniae
Group A
streptococci
β
+
-
Group B
streptococci
β,’
-*
+*
-*
Group D,
Enterococcus
Group D,
non-Enterococcus
α
±
-
Viridans
streptococci
α
+*
-*
-*
Hemolysis
Bacitracin
CAMP^
BileEsculin^
6.5%
NaCl^
Optochin#
Bile
Solubility
α,β,’,γ
-*
-*
+*
α,γ
-*
-*
+*
-
-*
-
-*
+*
-*
+
+
-*
-*
-
-*
-*
-*
-*
-*
-*
Catalase
-*
-*
-*
-*
PYR
-*
+
-*
+*
-*
CAMP test: Christie–Atkins–Munch-Peterson test; PYR test: Pyrrolidonyl Arylamidase test
* Occasional exceptions occur.
Remark: Most clinical laboratories do not use all of tests listed above to identify members of the
genus Streptococcus. In the consideration of time, instead of using tests that requires an incubation
time ≥24 hour ^, rapid tests, such as bile solubility test, the Lancefield grouping or PYR test is used
#
Optochin (ethylhydroxycupreine hydrochloride), a quinine derivative, selectively inhibits the
growth of S. pneumoniae in very low concentration. Optochin may inhibit other alpha-hemolytic
streptococci, but only at higher concentrations. S. pseudopneuomiae is also susceptible to optochin,
but usually marginal (e.g. 14 – 15 mm). In addition, optochin-resistant S. pneumoniae does exist.
Additional test, such as bile solubility, may be required to identify S. pneumoniae.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 27
Technical Notes
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 28
Principles (Gram negative bacteria):
Gram-negative bacilli belonging to the family Enterobacteriaceae are commonly
encountered microorganisms in the clinical microbiology laboratory. Many members of
this group are indigenous to the gastrointestinal tract. Members of the Enterobacteriaceae
may be recovered from infections of virtually every anatomical site.
Gram stain morphology is neither helpful in separating members of the Enterobacteriaceae
from other Gram-negative bacilli nor in making species identifications. The morphology
of colonies growing on blood agar is also of limited diagnostic usefulness because most
species appear as dull gray, dry to mucoid colonies. Some species of Proteus may grow
diffusely over the agar surface as a thin film, a phenomenon called swarming.
MacConkey agar is a differential medium for the selection and recovery of the
Enterobacteriaceae and related enteric Gram-negative bacilli. The bile salts and crystal
violet inhibit the growth of Gram-positive bacteria and some fastidious Gram-negative
bacteria. Lactose is the sole carbohydrate. Lactose-fermenting bacteria produce colonies
that are varying shades of red, due to the conversion of the neutral red indicator dye (red
below pH 6.8) from the production of mixed acids. Colonies of non-lactose-fermenting
bacteria appear colorless or transparent.
Indole is one of the metabolic degradation products of the amino acid tryptophan. Bacteria
that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating
tryptophan with the production of indole, pyruvic acid, and ammonia. Indole production is
an important characteristic in the identification of many species of micro-organisms, being
particularly useful in separating E. coli (positive) from members of the KlebsiellaEnterobacter group (mostly negative). The indole test is based on the formation of a red
color complex when indole reacts with the aldehyde group of p-paraminobenzaldehyde
(Kovac’s reagent).
The cytochrome system is usually only present in aerobic organisms which are capable of
utilising oxygen as the final hydrogen receptor. Cytochrome containing organisms produce
an intracellular oxidase enzyme that catalyzes the oxidation of cytochrome c. The end
product of this metabolism is either water or hydrogen peroxide (broken down by
catalase).The oxidase test detects the presence of a cytochrome oxidase system that
catalyse the transport of electrons between electron donors in the bacteria and a redox dye
reagent tetramethyl-p-phenylene-diamine. The dye is reduced to deep purple color.
Organisms which produce cytochrome oxidase or indophenol oxidase as part of their
respiratory chain are oxidase-positive and turn the dye reagent blue/purple. Organisms
lacking cytochrome oxidase do not oxidize the reagent, leaving it colorless within the limits
of the test, and are oxidase-negative. The test reagent, N, N, N’, N’-tetramethyl-pphenylenediamine dihydrochloride acts as an artificial electron acceptor for the enzyme
oxidase. The oxidised reagent forms the colored compound indophenol blue. This test is
used to assist in the identification of Pseudomonas, Neisseria, Aeromonas, Campylobacter,
Vibrio, Brucella and Pasteurella, all of which produce the enzyme cytochrome oxidase.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 29
Procedure:
Working in groups: Each pair is provided with 5 unknown culture plates.
Exercise 1. Colony Morphology
Observe individual colonies of 5 unknown culture plates. Record the colony
morphology and color of each on different agar medium on the Practical 1 Part 3
Results Form.
Exercise 2. Hemolytic Properties
a. Observe the blood agar plates of 5 unknown culture plates; note any zone of
hemolysis around well-isolated colonies.
b. Report results as hemolytic, or non-hemolytic. Record your observations in the
appropriate columns of the Results Form.
Exercise 3. Gram Stain
a. Prepare smears of the 5 unknown culture plates.
b. Carefully Gram stain each slide, and allow to air dry or blot gently with bibulous
paper.
c. Under oil immersion, observe the Gram reaction, morphology and arrangement of
cells for each unknown organism.
d. Record your observations.
Exercise 4. Catalase Test
a. Test the 5 unknown organisms for catalase activity. For each, use a clean slide.
With a loop or wood applicator stick, transfer cells from the center of a well-isolated
colony to the surface of the slide (Do NOT to take any blood agar with the colonies!
This can result in a false positive. It is preferable to test colonies from media without
blood since erythrocytes possess catalase activities).
b. Add 1 or 2 drops of 3% hydrogen peroxide.
▪ Rapid appearance and sustained production of gas bubbles or effervescence are
indicative of a positive test. Since some bacteria may possess enzymes other
than catalase that can decompose hydrogen peroxide, a few tiny bubbles
forming after 20 to 30 seconds is not considered a positive test.
c. Record results of each organism in the chart. A positive result shall be reported as
“positive,” whereas as a negative result (no bubbles) shall be reported as “negative.”
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 30
Exercise 5. Coagulase Test
In this exercise perform a slide and tube coagulase test on all organisms, regardless of
the results of the slide test.
▪
Slide test (to detect bound coagulase)
i. Place a drop of physiological saline on each end of a slide, or on two
separate slides.
ii. With the loop or a wodden stick, emulsify a portion of the isolated colony
in each drops to make two thick suspensions.
iii. Add a drop of rabbit plasma to one of the suspensions, and mix gently.
iv. Look for clumping of the organisms within 10 seconds.
Control v. No plasma is added to the second suspension to differentiate any granular
appearance of the organism from true coagulase clumping.
▪
Tube Test
i. For each organism which had a negative rapid slide test, perform
the tube test.
ii. For each organism, label a small test tube with patient name or
organism name. Place approximately 0.2 mL of coagulase plasma
into each tube.
iii. Inoculate the plasma with a large loopful of the colonies to be tested.
iv. Incubate the tubes in a 35°C incubator or water bath. Coagulase
positive organisms usually produce a visible clot within 1-4 hours.
Examine the tubes after 4 hours of incubation. DO NOT shake or
agitate the tube which would cause dissolution of the clot. A positive
result is notated as a “positive,” no grading is required.
v. After the 4 hour incubation time, if the specimen is still negative,
add a parafilm and reincubate the tubes.
vi. After 24 hours, if the absence of a clot is observed, report as
‘negative”. A negative result would also be interpreted as coagulasenegative staphylococci.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 31
▪
Staphaurex
i. Screen suspect staphylococcus colonies using a Gram stain before
proceeding to the latex test procedure.
ii. Bring reagents to room temperature before use.
iii. Just prior to use, shake the latex to obtain an even suspension.
iv. Dispense a drop of the latex suspension on the reaction card for each
culture to be tested.
v. Cultures may be tested direct from the primary plate, or a subculture
can be made on blood or nutrient agar for subsequent testing. Best
results are obtained from enriched media such as blood agar or
nutrient agar. The use of fresh cultures grown overnight is
recommended. Select 2-5 isolated colonies of the suspect
staphylococci from the primary medium.
vi. Take a mixing stick and pick up some of the culture by touching it
with the flat end of the stick.
vii. Emulsify the sample of culture in a drop of latex by rubbing with
the flat end of the stick. Rub thoroughly, but not too vigorously or
the surface of the card may be damaged. Spread the latex over
approximately half of the area of the circle. Discard the mixing stick.
viii.Rotate the card gently for up to 20 seconds and examine for
agglutination, holding the card at normal reading distance from the
eyes.
ix. Dispose of the card – Do not reuse.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory Manual
Page | 32
Exercise 6. Bile solubility test
The bile solubility test is used to identify and distinguish S. pneumoniae from alpha-hemolytic
Streptococcus spp. The test may be performed using a cell suspension on a slide or in a tube or
by adding the reagent directly to the colony. The principle of bile solubility test the lysis of
pneumococcal cells when sodium deoxycholate (bile salts) is applied to the colony under specific
conditions of time and temperature, but other streptococci do not lyse.
Test tube method
About 0.5 ml of sterile saline is dispensed into a small test tube. A heavy suspension of the
organism is prepared in the saline (equivalent to 1 McFarland standard). The suspension is
divided into two tubes, one labeled “TEST” and the other labeled “CONTROL.” Two to five
drops of bile reagent (10% sodium deoxycholate) are dispensed into both the tubes marked
“TEST” and “CONTROL.” Both the tubes are gently mixed. The tubes are then incubated for
three hours at 35°C, checking hourly for clearing, or each tube can be examined by Gram stain
or methylene blue wet mount for lysis of cells at an interval of 15 minutes.
Exercise 7. Indole Test
1. Inoculate a cotton swab with one colony from the agar if E. coli is suspected. Add one drop
of indole reagent to the cotton swab and observe for a pink-red color within two (2) minutes.
2. Record results based on interpretation below.
3. The development of a pink-red color is indicative of a positive result. No color change or a yellow
color indicates a negative result. Record as either “Positive” or “Negative” in report form.
Exercise 8. Oxidase Test
1.
2.
3.
4.
Filter Paper Spot Method
Use a loop and pick a well-isolated colony from a fresh (18- to 24- hour culture) bacterial
plate and rub onto a small piece of filter paper.
Place 1 or 2 drops of 1% oxidase reagent on the organism smear.
Observe for color changes.
Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10
seconds. Microorganisms are delayed oxidase positive when the color changes to purple
within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change
or it takes longer than 2 minutes.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 33
Quality Control
Quality control ensures that the information generated by the laboratory is accurate, reliable,
and reproducible. This is accomplished by assessing the quality of the specimens,
monitoring the performance of test procedures, reagents, media, and personnel. For the
reagents used in this laboratory session, QC should be performed weekly and on each new
lot or shipment of the product using standard laboratory strains, such as American Type
Culture Collection (ATCC). Results should be reported in the QC log. Components of kit
tests should not be interchanged with those from another kit.
Technical Notes
➢ Hydrogen peroxide (catalase reagent) is subject to degradation, especially
if exposed to light. The reagent should be kept in an amber-colored
container and checked daily against a known positive and negative control.
➢ It should never be assumed that an unknown organism is a Gram positive
coccus without performing a Gram stain in addition to a catalase test.
➢ Do not perform differentiation tests on a mixed culture, wait for isolated
colonies.
➢ For the purpose of this laboratory session, the student is exposed to multiple
methods to identify whether the organism produces coagulase. However, in
the clinical setting, usually only one method, such as a kit test is used.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 34
Name: ________________________Student ID:_____________________Marks:_________ / 55
Practical 1 Part 3 Results Form
Test (5 mark @ row)
Colony Morphology
▪ Trypticase soy agar (TSA)
▪ Blood agar (BA)
▪ MacConkey agar (MAC)
Plate A
Plate B
Plate C
TSA:
TSA:
TSA:
TSA:
BA:
BA:
BA:
BA:
White, smooth, large
MAC:
White, glistening,
medium
MAC:
White, rough, large
MAC:
-ve, rod
-ve, rod
-ve, cocci
Plate E
TSA:
BA:
Greenish, Mucoid
White, circula, small
glistening
MAC:
Complete hemolysis,
Beta
only for +ve
Hemolytic Properties
Gram Stain & Morphology
Plate D
+ve, cocci
MAC:
Alpha
+ve, cocci
Optochin sensitivity on BA only for +ve, cocci
test for Strap
Catalase +ve, cocci, for beta , for Stap
Slide coagulase test
Tube coagulase
Bile solubility test For +ve, alpha, for strep.
Indole test For -ve, rod, E.Coli
Oxidase test
+ve
-ve
-ve
+ve
-ve
For -ve
Presumptive identification
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 35
PRE-LAB STUDY QUESTIONS
Practical 1 Part 3 Presumptive Identification of unknown Bacteria
Name:
Student ID:
Marks:
/ 16
1. Describe the principle and function(s) of the tests listed below:
Test Principle and Function(s) (2 marks @)
Test
It is used to differentiate Streptococcus pneumoniae (optochin sensitive) from
Optochin sensitivity test
other alpha-hemolytic streptococci (optochin resistant). The test involves placing
a Taxos P disc (impregnated with optochin) on a smear of the bacteria and
incubating the plate.
Bacitracin sensitivity test
Catalase test
Staphaurex /
Slide coagulase test
Tube coagulase test
Bile solubility test
Indole test
Oxidase test
It is used to differentiate beta-hemolytic streptococci (Streptococcus pyogenes
and Streptococcus agalactiae) from other beta-hemolytic streptococci. The test
involves placing a Taxos A disc (impregnated with bacitracin) on a smear of
the bacteria and incubating the plate.
Used to differentiate bacteria that produce catalase from those that do not.
Catalase is an enzyme that breaks down hydrogen peroxide into water and
oxygen. The test involves adding hydrogen peroxide to a bacterial culture and
observing for the production of oxygen bubbles. Bacteria that produce catalase
will produce oxygen bubbles, while bacteria that do not produce catalase will not
produce oxygen bubbles.
Used to differentiate Staphylococcus aureus (positive) from other
Staphylococcus species (negative). The test involves adding a drop of
Staphaurex reagent to a bacterial culture and observing for the formation of a
clot. S. aureus will form a clot, while other Staphylococcus species will not
form a clot.
Used to differentiate S. aureus (positive) from other Staphylococcus species
(negative). The test involves adding a bacterial culture to a tube of rabbit
plasma and observing for the formation of a clot. S. aureus will form a clot,
while other Staphylococcus species will not form a clot.
Used to differentiate Streptococcus pneumoniae (soluble) from other
alpha-hemolytic streptococci (insoluble). The test involves adding a bacterial
culture to a tube of bile and observing for the dissolution of the bacterial
cells. S. pneumoniae will dissolve in bile, while other alpha-hemolytic
streptococci will not dissolve in bile.
Used to differentiate bacteria that produce indole from those that do not. Indole
is a byproduct of the breakdown of tryptophan by bacteria. The test involves
adding Kovac's reagent to a bacterial culture and observing for the production
of a red color. Bacteria that produce indole will produce a red color, while
bacteria that do not produce indole will not produce a red color.
Used to differentiate bacteria that produce cytochrome c oxidase from those that
do not. Cytochrome c oxidase is an enzyme that is involved in the electron
transport chain in bacteria. The test involves adding a drop of oxidase reagent to
a bacterial culture and observing for the production of a blue color. Bacteria that
produce cytochrome c oxidase will produce a blue color, while bacteria that do
not produce cytochrome c oxidase will not produce a blue color.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 36
PRE-LAB STUDY QUESTIONS
Practical 1 Part 3 Presumptive Identification of unknown Bacteria
Name:
1.
Student ID: 13094812
Marks:
/6
What test differentiates streptococci from staphylococci? (1 mark)
Coagulase test
2.
Describe what α, β, and gamma hemolysis look like on blood agar (3 marks). Why is hemolysis
important in the identification of streptococci? (1 mark)
Alpha: Partial hemolysis that produces a greenish discoloration around the bacterial colony.
Beta: Complete hemolysis that produces a clear zone around the bacterial colony.
Gamma: No hemolysis of red blood cells. There are no notable zones around the colonies
Hemolysis is important in the identification of streptococci because different types of streptococci
produce different types of hemolysis.
3.
The optochin disk test is incubated at 25 / 35 ± 2°C for 18-24 hours in 5-10% CO2 enriched
environment / ambient air. Delete the inappropriate answers; 0.5 mark each.
The optochin disk test is incubated at 35 ± 2°C for 18-24 hours in a 5-10% CO2 enriched environment.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 37
PRE-LAB STUDY QUESTIONS
Practical 1 Part 3 Presumptive Identification of unknown Bacteria
Name:
1.
Student ID:
13094812
Marks:
/7
What two processes do MacConkey reactions determine? (2 marks)
MacConkey reactions determine lactose fermentation and gram-negative bacterial growth.
2.
Define fermentation. (1 mark)
Fermentation is an anaerobic process that is performed by a cell to generate chemical energy
such as ATP from pyruvate, but without going through the citric acid cycle and the electron
transport chain system.
3.
Using your textbook and lecture notes, list two medically important Gram-negative
bacilli which arelactose positive (ferment lactose). List other two medically
important Gram-negative bacilli which are lactose negative.
Lactose positive (ferment lactose):
Lactose negative:
Escherichia coli, Klebsiella pneumoniae
Pseudomonas aeruginosa, Salmonella enterica
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
(2 marks)
(2 marks)
Page | 38
PRE-LAB STUDY QUESTIONS
Practical 1 Part 3 Presumptive Identification of unknown Bacteria
13094812
Name: ____________________Student ID: _______________Marks:
__________/ 5
4.
Complete the table of Key Biochemical Tests for Differentiating Enterobacterales below (5 marks):
Indole Test
Methyl Red
Voges-Proskauer
Citrate
Urease
Phenylalanine
deamination
Motility test
ornithineC
decarboxylase
lysine
decarboxylase
Glucose
Lactose
Sucrose
Mannitol
Sugar Fermentation
Hydrogen sulfide
production
IMViC test
Triple Sugar
Iron
TSI slant
K or A/AG
+
-
+
-
+
+
-
+
+
-
AG
AG
AG
AG
A/AG
-
-
-
+
+
-
-
+
+
+
AG
AG
AG
AG
Morganella morganii
K/AG
-
+
+
-
-
+
+
+
+
-
AG
-
-
-
Proteus mirabilis
K/AG
+
-
+
+
+
+
+
+
+
-
AG
-
-
-
Providencia rettgeri
K/A
-
+
+
-
+
+
+
+
-
-
A
-
-
-
-
-
+
+
-
-
+
+
+
AG
-
A
A
A
A
ORGANISM
Citrobacter freundii
Enterobacter aerogenes
Escherichia coli
Klebsiella pneumoniae
w
Salmonella typhi
Serratia marcesens
K/AG
-
+Late
Shigella sonnei
Yersinia enterocolitica
A/A
Pseudomonas
aeruginosaNE
K/NC
-
+
+
-
-
+
-
-(35oC);
+(25oC)
+
-
A
-
A = acid; K = alkaline; NC = no change; G = gas; +Late = late fermentation; w = weak production;
NE
= Non-Enterobacteriaceae (nonfermentative aerobes).
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 39
Practical 1 Part 4 Urine culture and microscopic urinalysis
Learning objectives:
At the end of this activity, the student shall be able to:
✓ Understand the workflow of urine bench in medical microbiology lab.
✓ Perform microscopic urinalysis
Materials:
•
•
•
•
•
•
Microscope slides
Inverted microscope
Urine specimens (from students) (50ml falcon)
Urine specimens for microscopic examination (prepared by instructors)
CHROMID® CPS® ELITE agar (CPSE)
Cystine lactose electrolyte deficient agar (CLED)
Reference:
Mahon and Lehman, Textbook of Diagnostic Microbiology, Sixth Edition, Chapter 37
Principles:
Urinary tract infection (UTI) is a very common infection in out-patient settings.
Majority of the cases are caused by Escherichia coli. Staphylococcus
saprophyticus usually causes UTI in sexually active women. Other causative
agents of UTI include Enterobacteriaceae, pseudomonads and Staphylococcus
aureus. Less commonly, Enterococcus species, Lancefield group B streptococci,
urease-positive corynebacteria and Candida species are involved. In patients with
urogenital abnormalities or dysfunction, more than one organism may be
responsible for the infection. All urine specimens shall undergo a dipstick test
and/or microscopy to look for the presence of white blood cells, red blood cells,
nitrites and bacteria.
Remark: All children under 3 years of age should have a microscopy performed
since dipsticks can be unreliable in this age group due to frequent voiding.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 40
Types of specimen
Urine (midstream urine), suprapubic aspiration, catheterized urine, bag specimen
Criteria of specimen rejection
•
•
•
•
•
Un-refrigerated specimen older than 2 hours may be subject to overgrowth
and may not yield valid results;
unlabeled specimen;
mislabeled specimen;
specimen in expired transport container;
24 hours urine specimens.
Storage
At room temperature unless delay is inevitable; it must be refrigerated
Note:
• First morning specimens yield highest bacterial counts from overnight
incubation in the bladder, and are the best specimens.
• Colony count interpretation standards are based on controlled studies from
first early morning collections.
• Forced fluids or randomspecimens dilute the urine and may cause reduced
colony counts.
• Hair from perineum will contaminate the specimen. The stream from a
male may be contaminated by bacteriafrom beneath the prepuce.
• Bacteria from vaginal secretions, vulva or distal urethra may contaminate
transport.
• Organisms from hands or clothing might contaminate. Receptaclemust be
sterile.
Specimen processing
1. Urine culture
2. Urine dipstick (Macroscopic urinalysis)
3. Urine Microscopy (Microscopic urinalysis)
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 41
Urine culture
A known volume of urine is cultured in order to allow quantification of the number of
organisms in the original urine, although because of imprecisions in the method this is
usually referred to as ‘semi-quantitative’ culture.
Bacterial quantification
Determining a colony-forming unit (CFU):
A calibrated loop holding a defined volume (e.g. 1 microliter or L) may be used to
quantify bacteria in specimens, such as urine. If 1-L loop is used, the number of
CFUis multiplied by 1000 to calculate the CFU per milliliter (mL) of urine.
Procedure:
1. Dip a sterile 10 L calibrated loop into a well-mixed midstream urine specimen
(collected by students on their own using a 50mL falcon before the laboratory
session).
2. With the calibrated loop, streak the CPSE/CLED plate from top to middle, as
shown on the nextpage.
3. With the same loop, cross streak the primary inoculation at right angles.
4. Repeat for additional plates, as directed.
5. Incubate aerobically at 35 oC O2 incubator for 24 hours.
6. To obtain the number of CFUs per mL of urine, multiply the number of colonies
on the plate by the appropriate dilution factor. If a 10 L loop was used, the
dilution factor is 100. If a 1 L loop was used the dilution factor is 1000. For
example, if a 1 L calibrated loop was used, and 300 colonies grew on the plate,
the colony countwould be 300 X 1000= 300,000 or 3 x 105 CFU/mL.
7. The final calculated result is reported as CFU/mL.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 42
Streaking pattern used in this practical.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 43
Identification on CHROMID® CPS® ELITE
CPSE contains specific chromogenic substrates for the identification of common urine pathogens:
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 44
CLED agar (cysteine lactose electrolyte deficient medium) is a valuable noninhibitory growth medium used in the isolation and differentiation of urinary organisms.
Being electrolyte deficient, it prevents the swarming of Proteus species. Cystine promotes the
formation of cystine-dependent dwarf colonies. Bromothymol blue is the indicator used inthe
agar, it changes to yellow in case of acid production during fermentation of lactose or changes
to deep blue in case of alkalinization. Lactose-positive bacteria build yellow colonies. Bacteria
which decarboxylate L-Cystine cause an alkaline reaction and build deep blue colonies.
Presumptive identification can be made from the CLED agar plate:
Escherichia coli
Klebsiella species
Proteus species
Salmonella species
Pseudomonas
aeruginosa
Enterococcus faecalis
Staphylococcus
aureus
yellow, opaque colonies with a slightly deeper
coloured centreabout 1.25 mm diameter
(Non-lactose fermenting strains - blue colonies)
extremely mucoid colonies varying in color from
yellow towhitish-blue
translucent blue colonies usually smaller than
Escherichia coli
flat blue colonies
green colonies with typical matt surface and rough
periphery
yellow colonies about 0.5 mm diameter
Coagulase negative
staphylococci
deep yellow colonies about 0.75 mm diameter, uniform
in colour
pale yellow or white, more opaque than
Enterococcus faecalis,often with paler periphery
Corynebacteria
very small grey colonies
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 45
Confirmatory testing (Urine culture)
Confirmatory testing should be done on all organisms, if necessary make purity plates on blood agar
first.
Table 3. Confirmatory identification tests for urinary tract isolates
Organism
Identification methods
Staphylococci
1.
2.
3.
Catalase
Coagulase / Staphaurex
Novobiocin disc if coagulase negative (S. saprophyticus is
resistant)
Streptococci/Enterococcus
1.
2.
3.
Catalase
Enterococcus spp.:group and sub onto bile-esculin agar
Other streptococci: sub on blood agar (+optochin disc) and
identify using conventional tests (haemolysis, Lancefield
grouping, etc.)
Gram negative bacilli
(GNB)
1.
E. coli: confirm with indole only (inoculate Motility
Indole Lysine medium)
Other coliforms: ID using biochemistry tests
(sugars set)+/- API 20E
Pseudomonas spp.: confirm as Pseudomonas
aeruginosa with oxidase and growth of green
colonies on Columbia agar after incubation at
42C. If not P. aeruginosa report as Pseudomonas
sp. (ID with API 20NE only if clinically indicated)
2.
3.
Other organisms
(e.g. yeasts)
ID only if clinically significant
Interfering factors:
1. Patient on antibiotic therapy.
2. Improper sample collection.
Page | 46
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Pathogens and commensals of Urine specimen
Common pathogens
E. coli and other Enterobacteriaceae
Enterococcus spp
Staphylococcus aureus
Staphylococcus saprophyticus
Acinetobacter spp
Pseudomonas spp
β-haemolytic streptococci
Salmonella spp (early stage of infection)
Corynebacterium jeikeium
Neisseria gonorrhoeae
Gardnerella vaginalis
Parasites
Schistosoma haemetobium
Trichomonas vaginalis
Commensal flora
the urine is sterile except for the
urethral mucosa which support the
growth of microflora as:
Diphtheroid bacilli
Lactobacillus spp
Coagulase negative Staphylococci
α Haemolytic Streptococci
Bacillus spp
Non pathogenic Neisseria spp.
Anaerobic cocci
Commensal Mycobacterium
Commensal Mycoplasma spp.
Page | 47
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Urine Microscopy
1.
2.
3.
4.
5.
6.
After testing urine, if necessary, with dipstick, place about 10 mL in plastic conical
tube labeled with patient's name and cover tube with tight fitting cover.
Place tube in centrifuge and place a second tube containing equal amount of water
(with tight fitting cover on) directly opposite tube with urine, to act as counter
weight.
Centrifuge urine specimen according to manufacturer's directions at a RCF
(relative centrifugal force) of 400 x g for 5-10 minutes. After centrifuge has
stopped, remove tube and pour off the supernatant, leaving any sediment in the
bottom of the tube.
With a plastic pipette, mix the remaining liquid and sediment and remove a few
drops of the mixture. If there is no obvious sediment present, remove a few drops
of urine from bottom of tube.
Place one drop of the sediment solution on a glass slide and cover with a coverslip.
Using the microscope, examine the sediment using phase contrast or bright light
under low (10x) and high (40x) power, scanning several fields to obtain average
numbers of formed elements
Interpretation of Results
1.
Report the presence of WBC's, RBC's, and casts using a high powered field
(H.P.F.; 400x magnification). The latter need to be identified as hyaline, WBC,
RBC, epithelial casts. Samples containing crystals should be forwarded to the
clinical laboratory for evaluation.
2.
Report the presence of epithelial cells, bacteria, crystals, and trichomonas.
Describe the abundance of epithelial cells, bacteria, crystals, and trichomonas
in a H.P.F. with a rating i.e. "few”, “moderate”, or “many".
3.
Identify presence of yeast (mycelial forms or hyphae).
.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 48
Technical note:
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 49
Useful images for urine microscopy
Red cells, White cells, and bacteria
Red Blood Cell
White Blood Cell
Bacteria
Epithelial cells (indicate that the urine is not a clean catch)
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 50
Useful images for urine microscopy
White cell casts (found when there is inflammation of the kidney pelvis or tubules)
Red cell casts (indicate haemorrhage into the renal tubules or glomerular bleeding; orange red colour)
Hyaline casts (associated with damage to the glomerular filter membrane)
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 51
Useful images for urine microscopy
Crystals
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 52
Technical note
Urine bench Result reporting in a clinical laboratory:
• Report urine microscopy as an initial report.
Results
Microscopic examination
WBC/ RBC/ Epithelial cell
Casts
Crystals
Bacteria / Yeast /
Trichomonas species /
Other parasites
Reporting format
No. of [WBC / RBC /
Epithelial cell] per field (20X)*
Corresponding no. of cells
per μL
<12^
<10 cells/μL^
- (<10 cells/μL)
12-120^
10-100 cells/μL^
+ (10-100 cells/μL)
>120 or packed field^
>100 cells/μL^
++ (>100 cells/μL)
Hyaline cast / Granular cast / Cellular cast / Leukocyte cast /
RBC cast / Epithelial cell cast / Fatty cast present
(Hyaline cast / Granular cast / Cellular cast /
Leukocyte cast / RBC cast / Epithelial cell cast / Fatty cast) present.
No cast seen.
No cast seen.
Abnormal crystal present
[Name of crystal] present.
No crystal or normal crystal seen
No abnormal crystal seen.
Bacteria / Yeast / Trichomonas species/ Other parasites present
Bacteria / Yeast / Trichomonas species / [Name of parasite] present.
* Microscopy Result obtained using routine test procedures in a clinical microbiology laboratory:
1. Dispense 60 μL of well-mixed urine into a well of a flat-bottomed microtitre plate. Make sure that the specimen covers the whole
bottom surface.
2. After all specimens have been dispensed, allow specimens to settle for 10-15 mins.
3. Examine the microtitre plate under inverted microscope with a 20X objective. Scan several fields in each well to check for even
distribution of cells.
4. Count the number of WBCs, RBCs and ECs per representative field.
5. Examine the presence and type of casts, presence of bacteria, yeasts, Trichomonas species or other parasites.
^Calculation for conversion of number of cells per 20X objective field to corresponding number of cells per μL is as follows:
Radius of well (r) = internal diameter of well/2 = 6.5 mm/2 = 3.25 mm; Surface area = πr2 = 33.19 mm2;
Fluid depth = volume (μL) of dispensed urine /surface area (mm2) = 60 mm3 / 33.19 mm2= 1.807 mm
Field of view (FOV) diameter = Field number (marked on eyepiece)/objective magnification = 18 (in this example) /20 = 0.9 mm
The volume that is observed in one field can be calculated knowing the well depth and the field of view as: π x (radius of FOV)2 x fluid
depth = π × (0.9/2)2 × 1.807 = 1.2 mm3 (i.e. 1.2 μL) Thus 120 cells in a field corresponds to approximately 100 cells /μL.
• Report the isolated pathogen and its sensitivity pattern as a final report.
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 53
Turn around time:
• Urine microscopy results should be available 1 hour after
specimen receipt.
• Isolation of a possible pathogen can be expected after 2-3 days.
• Negative culture will be reported out 1-2 days after the receipt of the
specimen.
Additional information
• A single culture is about 80% accurate in the female; two containing the
same organism with count of 105 or more represents 95% chance of true
bacteriuria; three such specimens mean virtual certainty of true bacteriuria.
• Urinary tract infection is significantly higher in women who use diaphragmspermicide contraception, perhaps secondary to increased vaginal pH and a
higher frequency of vaginal colonization with E.coli.
• A single clean voided specimen from an adult male may be considered
diagnostic with proper preparation and care in specimen collection.
• If the patient is receiving antimicrobial therapy at the time the specimen is
collected, any level of bacteriuria may be significant.
• When more than two organisms are recovered, the likelihood of
contamination is high;thus, the significance of definitive identification of the
organisms and susceptibility testing in this situation is severely limited. A
repeat culture with proper specimen collection including patient preparation
is often indicated.
• Periodic evaluation of diabetics and pregnant women for asymptomatic
bacteriuria has been recommended.
• Institutionalized patients, especially elderly individuals, are prone to urinary
tract infections, which can be severe.
• Cultures of specimens from Foley catheters yielding multiple organisms with
high colony counts usually represents colonization of the catheter and not
true significant bacteriuria.
• Most laboratories limit the number of organisms which will be identified
when recovered from urine to two. Similarly, most do not routinely perform
susceptibility tests on isolates from presumably contaminated specimens.
• Failure to recover aerobic organisms from patients with pyuria or positive
Gram's stains of urinary sediment may indicate the presence of mycobacteria
or anaerobes.
• As the number of patients who are chronically catheterized increases, so does
the controversy on what constitutes a diagnostic specimen.
• Few clinical studies have been performed to support the identification of
more than two organisms or implicate usual site flora (eg, diptheroids, alpha
or gamma streptococci, and coagulase-negative staphylococci other than S.
saprophyticus).
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 54
Practical 1 Part 4
Results Form
Name:_________________________Student ID:_____________________Marks: _________/ 21
1. Colony Counts on urine specimens
a. CPSE / CLED agar plate prepared by instructor for your group:
Group:
Sample A:
Sample B:
Sample C:
Sample D:
CFU/mL (1 mark)
CFU/mL (1 mark)
CFU/mL (1 mark)
CFU/mL (1 mark)
b. Midstream urine specimen of your own on CPSE / CLED:
Number of bacterial colony:
Bacterial semi-quantification: <104 / 104-105 / >105 CFU/mL* (1 mark)
*Delete the incorrect answer
2. Microscopic examination (2 marks per field)
Urine sample
Presence of RBC
(Yes / No)
Presence of WBC
(Yes / No)
Others
A
B
Y
N
N
Y
Crystal
Bacteria
Calcium osolate,
Observed in
kidney stone
causing internal
bleeding
Urinary tract
infection
C
N
D
N
Y
Yeast,
budding features
WBC first
indication of
UTI
N
Bacteria
No WBC
observed in high
bacteria counts.
Thus it is a
contamination
case.
Could be some
pre-analytic
error such as
over-incubation
at Room Temp.
Urine color
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 55
Biochem analyse to
Macro,micro
Practical 1 Part 4
PRE-LAB STUDY QUESTIONS
Name:
Student ID:
Marks: ____________/3
Instructions:
The student may use the course textbook, course lecture notes, or
Internet to answer the following question.
1. What laboratory tests are commonly performed in urine bench of medical
microbiology lab? (3 marks)
Urine culture is a very common laboratory test that checks urine for the presence of
microorganisms which causes infections. A urine sample is placed into a container
with a growth medium that the germs can grow on, and the urine culture is then
placed in an incubator for up to one day. If there are bacteria or fungi in the urine,
visible colonies can grow. Urine culture is an important diagnostic tool used to
identify the type of bacteria causing the infection and to determine the appropriate
antibiotic treatment.
Special Stain
1.) Capsule stain, staining the background, Black
2.)
SCI 8007SEF Medical Microbiology & Virology I – Laboratory
Manual
Page | 56