LABORATORY # 3
Differentiation of Staphylococcus sp.
Laboratory #3
Differentiation of Staphylococcus
Skills= 20 Points
Objective:
At the completion of this activity, the student will be able to:
1.Correctly differentiate Staphylococcus aureus, Staphylococcus epidermidis and
Staphylococcus saprophyticus through the use of colony morphology, catalase test,
hemolytic properties, gram stain, coagulase tests, Staphyloslide and
Novobiocin sensitivity.
Materials:
Blood agar cultures of:
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
3% hydrogen peroxide (Catalase reagent)
2-Microscope slides
Microscope
Immersion oil
Coagulase plasma
Gram stain materials
1-Staphyloslide
2-Novobiocin disks
1-TSA
References:
1.
Mahon and Manuselis, Textbook of Diagnostic Microbiology, Fourth Edition,
Chapter 14
2.
BD BBL Coagulase Plasma package insert
3.
BD BBL Staphyloslide package insert
Principles:
Bacteria in the genus Staphylococcus are gram positive spherical cells that occur singly,
occasionally in pairs, but most frequently in irregular clumps. The appearance of a gram
stained 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, S.aureus
being catalase positive and Streptococcus catalase negative.
MLAB 2534 – Laboratory 3 – Page 1
LABORATORY # 3
Differentiation of Staphylococcus sp.
Staphylococci are normal flora on the skin and mucous membranes, but can cause
infection under certain circumstances. S.aureus is more pathogenic than the other
common members of the genus, S.epidermidis and S.saprophyticus. Disease processes
with S.aureus are numerous. The portal of entry is variable, since they gain access to
the body via the skin, the respiratory tract or the genito-urinary tract. Some of the
infections of humans in which S.aureus is the etiological agent are boils, carbuncles,
impetigo, meningitis, osteomyelitis, urinary infections, and food poisoning.
S.epidermidis has been known to cause various hospital-acquired infections (such as
prosthetic or indwelling devices), whereas S.saprophyticus is mainly associated with
urinary tract infections in young females who are sexually active. S.aureus can be
differentiated from S.epidermidis and other gram positive cocci by the following S.
aureus characteristics:
Test(s)
Pigment
Hemolysis
Catalase
Coagulase
Novobiocin
Mannitol
S = Sensitive
R = Resistant
S. aureus
Gold-white
+
+
+
S
+
S. epidermidis
White-yellow
+
S
-
S. saprophyticus
White-yellow
+
R
-
Procedure:
Working in Pairs:
1. Colony Morphology
Observe individual colonies of S.aureus, S.epidermidis and S.saprophyticus. Record
the colony morphology and color of each on the chart at the end of this exercise.
2. Hemolytic Properties
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 medium, there will be a round, clear zone surrounding the
colony because the red blood cells in that area have been lysed (zone of hemolysis).
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.
a. Observe the blood agar plates of S.aureus, S.saprophyticus and
S.epidermidis; note any zone of hemolysis around well-isolated colonies.
Report results as hemolytic, or non-hemolytic.
b. Record your observations in the appropriate columns of the chart.
MLAB 2534 – Laboratory 3 – Page 2
LABORATORY # 3
Differentiation of Staphylococcus sp.
3. Gram Stain
As previously mentioned, species of Staphylococcus and gram positive cocci may
RARELY occur singly, occasionally in pairs, but most often in clusters.
a. Prepare smears of S.epidermidis and S.saprophyticus. Use your smear from Lab
#2 for S.aureus.
b. Carefully gram stain each slide, EXCEPT the S.aureus smear, 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 organism.
2. Record your observations.
4. Catalase Test
Staphylococcus species contain the enzyme catalase, whereas most species of
Streptococcus (another gram positive coccus) do not. Catalase will break down
hydrogen peroxide. When mixed with 3% hydrogen peroxide, catalase positive
organisms will generate bubbles of oxygen which are visible to the naked eye.
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.
H2O2
→
H2O + O2 (gas bubbles)
a. Test S. aureus, S.epidermidis and S.saprophyticus 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.
b. Add 1 or 2 drops of 3% hydrogen peroxide.
a. Rapid appearance and sustained production of gas bubbles or effervescence
is 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 will be reported as
“positive,” whereas as negative result (no bubbles) will be reported as
“negative.”
5. Coagulase Test
Coagulase is an exoenzyme that causes fibrin of blood plasma to clot. Pathogenic
S.aureus produces coagulase, while non-pathogenic strains are coagulase negative.
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. Free Coagulase is an intracellular enzyme produced when the organism is
MLAB 2534 – Laboratory 3 – Page 3
LABORATORY # 3
Differentiation of Staphylococcus sp.
cultured in broth.
The slide test is simple to perform and rapid, but detects bound coagulase only.
Therefore, all negative slide coagulase must be followed by a tube test, which will
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 acts on prothrombin in coagulase plasma.
This product then acts on fibrinogen to form a clot. In this exercise perform a slide
and tube coagulase test on both organisms, regardless of the results of the slide
test.
a. Rapid Slide Test
i. For each organism, use a clean glass slide. Place one drop of
coagulase plasma on each. Emulsify a loopful of the colonies
(approximately 2-4) to be tested in the drop on the appropriate
slide. Colonies should be from an 18-24 hour old culture.
ii. Mix with the loop or wood applicator stick to obtain a smooth
suspension. Gently rock the slide.
iii. If the test is coagulase positive, visible clumps will appear within 1
to 2 minutes. It may be necessary to observe the mixture over a
lamp to see the clumping appearance. A positive result is notated
as a “positive,” no grading is required. A positive result would also
be interpreted as S.aureus on the patient report form because S.
aureus is the only Staphylococcus that is coagulase positive.
iv. If no agglutination is observed, it will be reported as “negative.”
v. Discard slides in a biohazard waste container and record results for
each organism.
b. 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 and ID
number. Place approximately 0.5 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 37°C incubator or waterbath. Coagulase
positive organisms usually produce a visible clot within 1-4 hours.
Examine the tubes periodically by gently tipping the tube. 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. A
positive result would also be interpreted as S.aureus on the patient
report form because S.aureus is the only Staphylococcus that is
coagulase positive.
v. After the 1-4 hour incubation time, if the specimen is still negative,
parafilm the tubes and reincubate for 24-48 hours and observe
MLAB 2534 – Laboratory 3 – Page 4
LABORATORY # 3
Differentiation of Staphylococcus sp.
again for clot formation.
vi. After 24 hours, if the absence of a clot is observed, report as
‘negative”. A negative result would also be interpreted as
Coagulase-negative Staphylococcus on the patient report.
6. Staphyloslide
This procedure is a latex slide agglutination test for the differentiation of
staphylococci that possess clumping factor and/or Protein A (found in S. aureus).
This kit consists of blue latex particles coated with human fibrinogen and IgG. Once
the latex reagent is mixed with colonies, Staph that have protein A or clumping
factor will cross-link giving a visible agglutination.
a. Mix the latex reagent by gentle inversion.
b. Label the test circle with patient or organism name.
c. Dispense 1 drop of the test latex onto one circle on the reaction card and one
drop of Control latex onto another circle.
d. Using 3-5 isolated colonies of S.aureus from a nonselective media, such as blood
agar, combine with the test latex reagent, spreading the solution over the entire
test circle.
e. Using 3-5 isolated colonies of S.aureus from a nonselective media, such as blood
agar, combine with the control reagent, spreading the solution over the entire
control circle.
f. Hand rock the card for 20 seconds. Observe for agglutination by reading
macroscopically.
i. A positive result is regarded as agglutination within 20 seconds with no
agglutination in the control circle. This indicates the presence of S.
aureus. A negative result is obtained if no agglutination occurs and a
smooth suspension remains at 20 seconds in the test circle. A negative
reaction would be reported on the patient chart as Coagulase-negative
Staphylococci.
g. Dispose of reaction circle in appropriate biohazard container.
7. Novobiocin Susceptibility
a. Label the blood agar plate with organism or patient name, student name, and
date.
b. Streak S. epidermidis and S. saprophyticus on a split blood agar plate to obtain
a good lawn of growth.
c. Place a 5 µg novobiocin disk in the center of each inoculum.
d. Incubate at 37°C for 18 to 24 hours.
e. Examine the plate for inhibition around the disk. Measure the zone diameter in
millimeters and record results. The diameter should be read at the place of
complete inhibition, disregarding colonies that are only detected by close
scrutiny or by using transmitted light.
a. Susceptible zone: > 12 mm
b. Resistance: equal to or lesser than 12 mm.
MLAB 2534 – Laboratory 3 – Page 5
LABORATORY # 3
Differentiation of Staphylococcus sp.
f. Interpret whether the organism is susceptible or resistant using the zone sizes,
but for reporting purposes use either “S” (susceptible) or “R” (resistant) in the
report form.
a. S. saprophyticus is resistant; S. epidermidis is sensitive.
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 lab, QC should be performed weekly and on
each new lot or shipment of the product using ATCC control organisms. 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) 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.
Other catalase-positive bacteria can resemble staphylococci, in particular
yeasts.
 Do not perform differentiation tests on a mixed culture, wait for isolated
colonies.
 For the purpose of this lab, 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.
MLAB 2534 – Laboratory 3 – Page 6
LABORATORY # 3
Differentiation of Staphylococcus sp.
Report Form: Staphylococcus Differentiation
Points= 20
Name ___________________________________ Date _______________________
Test
S. aureus
S. epidermidis
S. saprophyticus
Colony Morphology
Hemolytic
Properties
Gram Stain &
Morphology
Catalase
Rapid
Slide Coagulase
Tube Coagulase
Staphyloslide
Novobiocin
Sensitivity
MLAB 2534 – Laboratory 3 – Page 7
LABORATORY # 3
Differentiation of Staphylococcus sp.
Lab #3: Staphylococci Biochemical Tests and Media
Study Questions
Points: 12
Name:__________________________
Biochemical Test or Test Principle
Media
Date:_____________________
Explain how this test is used to identify the
Staphylococci
Gram Stain
Catalase
Production
MLAB 2534 – Laboratory 3 – Page 8
LABORATORY # 3
Differentiation of Staphylococcus sp.
Biochemical Test or Test Principle
Media
Coagulase slide
test
Explain how this test is used to identify the
Staphylococci
Coagulase tube
test
Staphyloslide
Novobiocin
resistance
MLAB 2534 – Laboratory 3 – Page 9