This document describes laboratory exercises for identifying different gram positive cocci, including Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae. Identification is based on gram stain morphology, growth characteristics like hemolytic patterns on blood agar, and biochemical tests for catalase, coagulase, optochin sensitivity, and bile solubility. The goal is to differentiate clinically relevant gram positive cocci like S. aureus, S. pyogenes, and S. pneumoniae.

1. Practical Microbiology
ENV 341
Dr Mohamed El Zowalaty
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2. Laboratory exercise # 1
Gram positive cocci
Catalase Positive
1 – Staphylococcus aureus
2. Staphylococcus epidermidis
3. Micrococcus luteus
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3. 1. Microscopical examination
Staphylococcus aureus Sputum smear
Gram positive cocci arranged in clusters or bunches
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4. 2. Growth on nutrient agar medium
Golden yellow colonies
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Catalase test
Negative and positive catalase (O2 is evolved from H2O2) test results. The absence of
bubbling from a transferred colony indicates a negative test.
3. Catalase test
Positive formation of bubbles or effervescence

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Due to production of
coagulase enzyme which
produces clot or
coagulation of plasma.
4. Coagulase test
Positive formation of clot or coagulation of plasma

7. Due to production of gelatinase
enzyme which liquefy gelatin
5. Gelatinase test
Positive
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8. Positive , with fermentation of mannitol and production of acid,
Red Yellow color
6. Growth on Mannitol Salt agar
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9. Beta hemolytic, clear zone
of hemolysis around
colonies
7. Growth on Blood agar
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2. Staphylococcus epidermidis 3. Micrococcus luteus
Gelatinase, coagulase negative
Non hemolytic
Catalase positive

11. Laboratory exercise # 2
Gram positive cocci
Catalase Negative
Streptococci
1. Streptococcus pyogenes (GAS)
2. Streptococcus agalactiae (GBS)
3. Streptococcus pneumoniae
4. Streptococcus viridans
5. Streptococcus faecalis
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13. 1. Gram stain
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Gram positive cocci arranged in chains

14. A. Beta hemolytic, clear
zone of hemolysis around
colonies
2. Growth on Blood agar
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15. Streptococcus pyogenes (GAS)
Streptococcus agalactae (GBS)
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Beta
hemolytic=
clear zone
on blood
agar plate

16. 2. Bacitracin test
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Beta hemolytic=
clear zone on
blood agar plate
Zone of growth
inhibition
Zone of growth
inhibition
Streptococcus pyogenes (GAS) Streptococcus agalactae (GBS)

17. B. Alpha hemolytic,
greenish zone of
hemolysis around colonies
2. Growth on Blood agar
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Proper streaking and growth of Alpha hemolytic
streptococci on blood agar medium with a surrounding
green zone of alpha-hemolysis

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2. Optochin test
Streptococcus pneumoniae
Streptococcus pneumoniae strain on blood agar showing alpha hemolysis (green zone
surrounding colonies). Note the zone of inhibition around a filter paper disc
impregnated with optochin. (sensitive to optochin)

20. • The quellung reaction (swelling
reaction) forms the basis of
serotyping and depends on the
swelling of the capsule upon
binding of homologous
antibody.
• The test consists of mixing a
loopful of colony with equal
quantity of specific antiserum
and then examining
microscopically at 1000X for
capsular swelling.
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3. Capsule swelling test

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Optochin test for S. pneumoniae using optochin disks. The
strain on the left is S. viridans and is resistant to optochin
with no zone of inhibition. The strain on the right is
susceptible to optochin and is S. pneumoniae.

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23. Optochin test
Streptococcus viridans
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Streptococcus viridans strain on
blood agar showing alpha hemolysis
(green zone surrounding colonies).
No zone of growth inhibition (
Resistant) around a filter paper disc
impregnated with optochin.

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Results of the bile solubility test are shown for two different strains of bacteria. For
strain 1, a slight decrease in turbidity is observed in the tube containing the bile salts (2nd
from left), but the contents are almost as turbid as the control tube (far left); therefore,
strain 1 is not S. pneumoniae. For strain 2, all turbidity in the tube containing the bile salts
(far right) has cleared, indicating that the cells have lysed, in contrast to the control tube
(2nd from right), which remains turbid; therefore, strain 2 is S. pneumoniae
4. Bile solubility test
S. Pneumoniae
No Turbidity
( No growth)
Clear
S. viridans
Turbidity
(growth)

25. Enterococcus faecalis
(Streptococcus faecalis)
• C. Gamma
Nonhemolytic
streptococci,
• Group D
streptococci,
• vancomycin-
resistant
enterococcus
(VRE).
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26. Acknowledgment
• I, Dr Mohamed El Zowalaty, would like to
express special thankfulness to the US CDC
Public Health Image Library, NIH/NIAID, Dr
Kenneth Toda, and others for providing the
photomicrographs available in the public
domain.
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