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Lab 8
Identification of Staphylococci and Streptococci
In this lab we will examine methods for identifying unknown pathogenic cultures.
Organisms from the genus Staphylococcus and Streptococcus cause a number of
diseases. Sometimes both are capable of causing the same disease. It is important to
distinguish which organism you are dealing with, as the most effective treatment may
vary depending on which one is causing the infection.
Exercise 1: Catalase Test
The Catalase test is used to differentiate between Staphylococci and
Streptococci. This simple, and immediate, test identifies organisms that possess the
enzyme catalase, which breaks down hydrogen peroxide into water and gaseous
oxygen. When hydrogen peroxide is added to Staphylococci, which is catalase-positive,
bubble form. Streptococci do not form bubbles since they do not have catalase.
Note that this is just one way that you can differentiate staphylococci from
streptococci. Another simple way that you have already learned is by Gram staining.
Both are Gram-positive cocci, but staphylococci form clusters while streptococci are
found in chains.
Objective: To differentiate between Staphylococcus and Streptococcus by identifying
the presence of the enzyme catalase.
Materials:
 Staphylococcus aureus slant
 Streptococcus pyogenes slant
 Clean microscope slides
 Hydrogen peroxide
 Pipet
 Sharpie
Procedure:
1. Label a microscope slide with the name of the organism. You can test both
organisms on one slide by putting one culture on each end of the slide
2. Place a drop of hydrogen peroxide on each side of the slide
3. Aseptically transfer a small amount of bacteria to one of the hydrogen peroxide
drops
4. Watch for the production of bubbles. This will happen very quickly
5. Aseptically transfer a small amount of the second bacteria to the other drop of
hydrogen peroxide
6. Watch for the production of bubbles
Results:
1. What does the catalase test differentiate?
2. How is the presence of catalase detected?
3. Give the formula for this reaction.
4. Which genus is catalase positive?
5. What are some other ways you can differentiate Staphylococcus from
Steptococcus?
Tests to determine the species of Staphylococci
There are three major species of pathogenic Staphylococci; Staphylococcus
aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis. While S.
aureus is the main cause of infection among the Staphylococci, S. saprophyticus and S.
epidermidis are the source of several infections of clinical importance. S. aureus
infections include gastroenteritis, toxic shock syndrome (TSS), pneumonia, meningitis,
osteomylelitis, bacteremia, urinary tract infections (UTI). S. saprophyticus is one of the
leading causes of UTI’s in sexually active women. S. epidermidis is associated with
infections of catheters, IV lines, and prosthetic devices. We will be looking at a number
of ways of differentiating between these organisms in this lab.
Exercise 2: Coagulase
Coagulase is an enzyme used by S. aureus to cause the formation of blood
plasma clots around themselves. This clots acts as a mechanism of evading the hosts’
immune defense. Because S. aureus produces coagulase and the other species of
staphylococcus do not, inoculation of a tube containing rabbit plasma will give a positive
indication of S. aureus.
Objective: To identify a Staphylococcus aureus by identifying the presence of the
enzyme coagulase.
Materials:
 Staphylococcus aureus broth
 Staphylococcus epidermidis broth
 2 tubes of rabbit plasma per group of 4 students
Procedure:
1. Label the rabbit plasma tubes
2. Inoculate rabbit plasma with a heavy amount of each Staphylococcus
3. Place in your labs incubation bin for incubation at 37°C overnight
Results:
1. What is coagulase?
2. What benefit might a bacterium gain by forming a plasma clot around itself?
3. What species of Staphylococcus produces coagulase?
4. What does a positive test for coagulase look like?
Exercise 3: Mannitol Salt Agar (MSA)
MSA contains mannitol (carbohydrate), 7.5% sodium chloride (salt), and phenol red (pH
indicator). Because most bacteria cannot survive in a high salt environment, this media
is selective for staphylococci. While all staphylococci can grow on this media, only the
pathogenic forms (S. aureus) can ferment mannitol. Fermentation of mannitol produces
acid, which causes the pH indicator phenol red to turn yellow (note: phenol red is yellow
below pH 6.8, red at pH 7.4 to 8.4, and pink above pH 8.4)
Objective:

To determine if a bacterium is from the genus Staphylococcus (Selective
property).

To determine if a Staphylococcus is the mannitol fermenting Staphylococcus
aureus (Differential property).
Materials:

Staphylococcus aureus broth

Staphyloccus epidermidis broth

Escherichia coli broth

One MSA plate per group of 4

Inoculating loop

Bunsen burner
Procedure:
1. With a marker, divide the plate in thirds and thoroughly label the bottom of the
plate
2. Inoculate one-third with a straight-line streak of Staphylococcus aureus, one-third
with a straight-line streak of Staphylococcus epidermidis, and one-third with a
straight-line streak of Escherichia coli.
3. Place upside down in your lab sections incubation bin
4. Incubate at 37°C overnight
Results:
1. What makes MSA a selective media?
2. What does MSA select for?
3. How is a positive result for the selective property determined?
4. What makes MSA a differential media?
5. What does MSA differentiate between?
6. How is a positive result for the differential property determined?
7. Which bacteria did MSA select for?
8. Which bacteria fermented mannitol?
Exercise 4: Novobiocin Sensitivity Test
The novobiocin sensitivity test uses paper disks impregnated with the antibiotic
novobiocin. A small amount of broth is spread over a portion of the plate and the
novobiocin disk is place on this area. The antibiotic diffuses a known distance into the
media. Bacteria that are susceptible to the antibiotic will not grow well, leaving an area
known as a zone of inhibition around the antibiotic disk. Those that are resistant grow
despite the presence of the antibiotic. S. saprophyticus is resistant to novobiocin, while
S. aureus and S. epidermidis are susceptible.
Objective: To differentiate Staphylococcus saprophyticus from the other Staphylococci
based on its resistance to the antibiotic novobiocin.
Materials:
 Staphylococcus aureus broth
 Staphylococcus epidermidis broth
 Staphylococcus saprophyticus broth
 3 Sterile cotton swab
 1 Blood agar plate per group of 4
 Novobiocin disks
 Forceps
Procedure:
1. Label and divide the blood agar plate in thirds with a sharpie
2. Dip the sterile cotton swab into one of the broths
3. Spread the culture in the appropriate section to create a square
4. Using the forceps, gently press a novobiocin disk in the center of your bacterial
spread
5. Repeat steps 2 through 4 for the other two broth cultures
6. Place your plate upside down in your labs incubation bin to be incubated at 37°C
overnight
Results:
1. What is novobiocin?
2. Which species of Staphylococcus is resistant to novobiocin?
3. Should you see growth of this species in close proximity to the novobiocin disk?
Why or why not?
4. Should you see growth of the susceptible species in close proximity to the
novobiocin disk? Why or why not?
Tests to determine the species of Streptococci
We will be using several different organisms in this section; Streptococcus pyogenes,
Streptococcus agalactiae, Enterococcus faecalis (note that E. faecalis was previously
named Streptococcus faecalis) and Streptococcus pneumoniae. Streptococci can be
classified in a couple of ways. One way is by the type of hemolysin, if any, it produces.
Beta-hemolytic streptococci completely hydrolyze red blood cells. Alpha-hemolytic
streptococci partially hydrolyze red blood cells. Non-hemolytic, or sometimes called
gamma-hemolytic, are not hemolytic at all and do not affect the red blood cells. Another
way to classify streptococci is based on a carbohydrate antigen present on the bacterial
cell wall called the Lancefield antigen. Today’s organisms come from groups A
(Streptococcus pyogenes), B (Streptococcus agalactiae), and D (Enterococcus
faecalis). Streptococcus pneumoniae does not have surface carbohydrate antigens that
fall into one group.
Like the staphylococci, streptococci are the cause of many infections. Among
the infections that Streptococcus pyogenes causes are cellulitis, pharyngitis, necrotizing
fasciitis, scarlet fever, TSS, rheumatic fever, and post-streptococcal glomerulonephritis.
Streptococcus agalactiae is responsible for several infections in newborns. S.
agalactiae in the birth canal may be transferred to the fetus during a vaginal delivery
and cause meningitis, pneumonia, or sepsis. Enterococcus faecalis is responsible for
cases of UTI, biliary tract infections, and subacute bacterial endocarditis. Streptococcus
pneumoniae causes pneumonia, otitis media, and meningitis.
Exercise 5: Hemolysis
Streptococci may produce an enzyme called hemolysin, which destroys or
partially destroys red blood cells. We will use a blood agar plate to determine the type
of hemolysin produced by the Streptococci. Both Streptococcus pyogenes and
Streptococcus agalactiae are beta-hemolytic, which means they completely destroy red
blood cells. Inoculation of a blood agar plate with either of these organisms will result in
a clearing around the colonies. Enterococcus faecalis is either alpha- or gammahemolytic, depending on the strain. Alpha-hemolysis results in partial breakdown of the
red blood cells and appears as a green coloration around the colonies due to a
hemoglobin metabolite of the unlysed RBC’s. Non-hemolysis (gamma-hemolysis) will
not cause a color change in the media, just growth of colonies. Streptococcus
pneumoniae is alpha-hemolytic.
Objective: To determine the type of hemolysis performed by different species of
Streptococcus.
Materials:
 Streptococcus pyogenes broth
 Streptococcus mitis broth
 Enterococcus faecalis broth
 1 Blood agar plate per group of 4
Procedure:
1. Label and divide the blood agar plate in thirds with a sharpie.
2. Straight-line streak each section with the appropriate organism
3. Put your plate upside down in your labs incubation bin for incubation at 37°C
overnight
Results:
1. What appearance does an alpha-hemolytic bacterium produce?
2. What appearance does a gamma-hemolytic bacterium produce?
3. What species of Streptococcus are beta-hemolytic?
Exercise 6: Bacitracin Sensitivity
In the bacitracin sensitivity test, a paper disk impregnated with the antibiotic
bacitracin is placed on a blood agar plate in an area in which you have spread a
bacterial culture. If an organism is susceptible to bacitracin, a clear zone will form
around the disc. Organisms resistant to bacitracin will grow around the disc. The test is
useful in differentiating the beta-hemolytic group A strep (Streptococcus pyogenes is
bacitracin susceptible) from other beta-hemolytic streptococci.
Objective: To identify Streptococcus pyogenes based on its susceptibility to the
antibiotic bacitracin.
Materials:
 Streptococcus pyogene broth
 Streptococcus agalactiae broth
 1 Blood agar plate per group of 4
 2 Sterile cotton swabs
 Bacitracin discs
 Forceps
Procedure:
1. Label and divide the blood agar plate in half with a sharpie.
2. Dip the sterile cotton swab into one of the broths
3. Spread the culture in the appropriate section to create a square
4. Using the forceps, gently press a bacitracin disk in the center of your bacterial
spread
5. Repeat steps 2 through 4 for the other broth culture
6. Place your plate upside down in your labs incubation bin to be incubated at 37°C
overnight
Results:
1. What is bacitracin?
2. Which species of Streptococcus is susceptible to bacitracin?
3. Should you see growth of this species in close proximity to the bacitracin disk?
Why or why not?
4. Should you see growth of the resistant species in close proximity to the bacitracin
disk? Why or why not?
Exercise 7: Bile Esculin Test
Bile esculin agar is a media used to detect Group D streptococci and
enterococci. This media contains bile, which inhibits the growth of organisms except
Group D streptococci and enterococci. These organisms can hydrolyze esculin into
glucose and esculetin. The glucose is used for energy metabolism and the esculetin
reacts with iron salts in the media to form a black precipitate. This dark coloration
indicates that you have a Group D strep or enterococci.
Objective: To identify Enterococcus faecalis based on its ability to grow in the
presence of bile and utilize esculetin as an energy source.
Materials:
 Enterococcus faecalis broth
 Streptococcus pyogenes broth
 1 bile esculin plate per group of 4
Procedure:
1. Label and divide the bile esculin agar plate in half with a sharpie.
2. Straight-line streak the surface of each half with the appropriate organism.
3. Put in your labs incubation rack to be incubated at 37°C overnight
Results:
1. What makes this a selective media?
2. Explain the differential property of this media?
3. What types of bacteria will grow on the bile esculin media?
4. What does a positive test look like?
These charts outline the “expected” results for each test. Make sure you know the expected
results for each organism on each test for your practical (i.e. memorize these charts!)
Catalase
Coagulase
DNase
MSA
Novobiocin
S. aureus
+
+
+
beta
susceptible
S. saprophyticus
+
resistant
Catalase
Hemolysis
S. pyogenes
beta
S. agalactiae
beta
S.
Alpha
Bacitracin
Bile esculin
sensitive
-
resistant
-
resistant
-
S. epidermidis
+
susceptible
E. faecalis
Alpha or
gamma
resistant
+