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Lab Exercise 19 Gram Positive Cocci
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Lab Exercise 19 Gram Positive
Cocci
Staphylococci © Gloria Delisle,
author. Licensed
for use, ASM MicrobeLibrary (linked to
http://www.microbelibrary.org)
Objectives:
1. Perform, interpret and define the
relevance of the catalase test.
2. Perform, interpret and define the
relevance of the coagulase test.
3. Perform, interpret & define the relevance
of the test for hemolysis on BAP.
4. Describe the Gram stain & arrangement
of major GPC families.
5. Determine the identification of an unknown
GPC organism using the above tests.
6. Relate the medical significance of each of the GPC covered in the lab.
Medically significant Gram Positive cocci are represented by two main families:
Micrococcaceae ( including the genera Staphylococcus and Micrococcus) and
Streptococcaceae (including the genera Streptococcus and Enterococcus).
I. Micrococcaceae - Catalase +, Gram positive cocci in clusters
A. Genus Micrococcus – These bacteria are rarely associated with disease and are
common environmental contaminants. They Gram stain as GPC in tetrads and
produce yellow (Micrococcus luteus) or rose (M. roseus) colored pigments on
enriched media.
B. Genus Staphylococci - Salt tolerant GPC
1. Staph. aureus - These are pathogenic bacteria causing wound
infections, abscesses, carbuncles, bacteremia, septicemia, osteomyelitis.
Associated with purulent discharges and capable of producing a wide
range of exotoxins (including hemolysins and DNAse), characterized as
highly invasive. Causes food poisoning by the production of a heat
resistant toxin. Important in nosocomial infections especially MRSA
(Methicillin or Multiple Resistant Staph. aureus). Nasal carriers are
important.
2. Staph. epidermidis - Normal skin flora, nonhemolytic, coagulase negative.
Opportunistic pathogen isolated from catheters and I.V. lines and
associated with transplant and immunosuppressed patients.
3. Staph hemolyticus - Normal skin inhabitant, beta hemolytic but coagulase
negative. Opportunistic pathogen associated with UTI's.
Lab Exercise 19 Gram Positive Cocci
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II. Streptococcaceae - GPC in chain and pairs, easily over decolorized. This family
produces a large number of exotoxins including hemolysins, erythrogenic toxins,
nephrotoxins, and cardiohepatic toxins. Pathogenesis depends on species, strain,
portal of entry and immune response. The Streptococcaceae are fastidious requiring
blood agar for growth and producing typical characteristics as the blood cells are
destroyed (hemolysis) for nutrients.
A. Streptococci - chains and pairs, some
encapsulated, bile esculin and salt negative
1. Strep. pyogenes - Group A strep, beta
hemolytic, causing strep throat, scarlet
fever, peurperal fever (post natal sepsis),
skin infections such as impetigo, and
pneumonia. Post infection sequela such
as glomerulonephritis and rheumatic fever
represent serious syndromes if infections
are not treated immediately.
Streptococci © Gloria Delisle,
author. Licensed for use,
ASM MicrobeLibrary (linked to
http://www.microbelibrary.org)
2. Strep. agalactiae - Group B beta
hemolytic strep causing neonatal
meningitis thought to be associated with
asympotomatic vaginal carriers,. Recently
reported in AIDS patients.
3. Strep. pneumoniae -Alpha hemolytic,
mucoid, and lancet- shaped. Virulent strains are encapsulated. Causes classis
pneumonia, ear and eye infections.
4. Alpha strep - variety of nonpathogenic normal flora found on the skin
and in the mouth. Occasionally associated with bacterial endocarditis.
Many of these alpha strep are found on the respiratory tract as normal
flora. Strep mutans is one of these strep associated with dental caries.
Strep. sanguinis and Strep. parasanguinis are normal oral flora.
B. Enterococci - Salt tolerant bile esculin positive strep. Normal flora of the G.I. tract.
Opportunistic pathogens infecting decubiti (bedsores), causing UTI'S and associated with IV
contamination. The enterococci are medically significant due to growing antibiotic
resistance, they are referred to as VRE (Vancomycin Resistant Enterococci). Many species
make up this group of strep including Enterococcus faecalis and Enterococcus faecium.
More information can be found at http://www.cdc.gov/ncidod/eid/vol4no2/adobe/huy.pdf
Lab Exercise 19 Gram Positive Cocci
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The table below represents a simple differentiation between these genera of bacteria. Refer
to the Atlas for sample test results.
Family
Micrococcaceae
Streptococcaceae
Test results
Staphylococcus
Micrococcus
Streptococcus
Enterococcus
Arrangement in
broth
grape-like clusters
tetrads
sarcina
chains (some
pairs)
paired
(occasional
chains)
hemolysis on
Blood agar
Staph. epidermidis
negative
alpha, beta,
gamma
alpha or
gamma
Staph. aureus
+
growth on
nutrient agar
good
good
poor or none
good
Catalase
positive
positive
negative
negative
MSA - Mannitol
Salt Agar NaCl tolerance
growth
Staph. epidermidis
negative
negative
growth
negative
negative
negative
Staph. aureus
Coagulase
+
Staph. epidermidis
Staph. aureus
Materials:
Hydrogen peroxide
dropper
slide
Coagulase tube
MSA plate
Blood Agar plate
+
Stock cultures:
Labeled 1-4
Staphylococcus aureus
Staphylococcus epidermidis
Micrococcus luteus
Streptococcus sanguinis or Strep.
parasanguinis
Lab Exercise 19 Gram Positive Cocci
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Procedure:
1. Gram stain each of the stock cultures and
record the results.
2. Divide a Blood Agar plate into 4 sections and
label them. Inoculate each section with a streak
from each stock culture.
3. Perform a catalase test as described in the
Atlas, by transferring a loopful of bacteria from
each stock culture to a glass slide. Using a
dropper apply a drop of hydrogen peroxide to
each bacterial smear and record the results.
Bubbling = positive
4. Divide an MSA plate into quadrants and inoculate it in the same fashion that the BAP
was inoculated.
5. Perform a coagulase test, using the pre-made tubes and inoculating a tube with each
of the stock cultures. Cover the tube with parafilm and incubate for 24-48 hours.
6. Incubate the BAP plate, MSA plate and coagulase at 37C. During the next lab period
read and record the results.
7. Determine from the identification chart on page 1 of this lab, the identity of each
stock culture.
Lab Exercise 19 Gram Positive Cocci
Lab Report Exercise 19 Bacterial Identification of Gram Positive Cocci
Name____________________
1. Use your book and the atlas as a reference. Define the following terms and describe
a positive test.
beta hemolysis -
alpha hemolysis -
gamma hemolysis -
2. Describe a positive coagulase test and identify the species of organism that would
produce a positive test.
3. What advantage might coagulase give to a pathogen as it invades the human body?
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Lab Exercise 19 Gram Positive Cocci
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4. Fill in the chart below with your results and determine the identity of each of your
stock cultures as a lab team.
stock #
Gram
stain
hemolysis
- BAP
MSA plate
catalase
test
1
2
3
4
Last Updated 6/30/2017
©Janet Fulks
coagulase Identification