1. Isolation and Identification of Gram Positive Cocci

2. Identified method for Staphylococci
Gram-stain
Isolation and culture
Pure culture
Direct identification
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3. Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters
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5. Direct identification
The mannitol fermentation test.
The Coagulase Test
The DNase Test
Novobiocin (NB) disc
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6. The mannitol fermentation test.
Inoculate the bacteria into a mannitol micro-tube,incubate at 370C for 18h.S.aureus will ferment mannitol to produce acid,which causes the medium to turn yellow.
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7. Staphylococcus aureus Staphylococcus epidermidis
Test
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Mannitol fermentation
Positive
Negative
Usually positive
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8. The Coagulase Test
Coagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting.
It might be a virulence factor with the coagulated blood around the bacteria protecting them from the immune system.
Coagulase-negative strains are often as pathogenic as coagulase-positive strains.
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9. The DNase Test
Inoculate DNase agar plates with a loop so that the growth is in plaques about 1 cm in diameter.I
ncubate at 370C overnight.
Flood the plate with 1 N hydrochloric acid.Clearing around the colonies indicates DNase activity.
The hydrochloric acid reacts with unchanged deoxyribonucleic acid to give a cloudy precipitate.
A few other bacteria,e.g. Serratia,may give a positive reaction.
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10. Staphylococcus aureus Growing on DNase Agar
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11. Blood agar with a novobiocin (NB) disc
Staphylococcus aureus Growing on Blood Agar
Staphylococcus saprophyticus Growing on Blood Agar
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12. Identification of Gram-positive cocci
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13. types Of Streptococcus
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14. β haemolytic steps: Lancefield groups
Historically
A, B, C, F and G
group specific antigens
cell wall polysaccharide
group D and Enterococci
lipoteichoic acid moiety
Not always β haemolytic
Now: 18 different groups of strep: from
A-H, and from K-T
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15. (β) Haemolytic Streptococcus groups
S. pyogenes
Group A
S. agalactiae
Group B (occasionally α or none)
Streptococcus equismilis
Group C (occasionally α or none)
Streptococcus bovis
Group D (α or none, occasionally β)
Streptococcus anginosus
Group F & G
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16. Gp A strep: S. pyogenes Laboratory ID
Gram +ve
Catalase
Haemolysis
small, round, grey colonies
β haemolysis
zone is large
Gp A antigen
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17. Sensitivity to the antibiotic bacitracin
Isolation and Identification of group A beta hemolytic (Streptococcus pyogenes)
on Blood agar. Streptococcus pyogenes produces a zone of beta hemolysis around 2-3mm in diameter surrounding each colony
Sensitivity to the antibiotic bacitracin
Group A Strep is senstive, other b haemolytic streps are resistant.
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18. Bacitracin sensitivity
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19. Group B: S. agalactiae S. agalactiae Identification Catalase negative
Appearance on BA
Larger colonies than Gp A
Small zone of hemolysis
Latex agglutination Gp B
Bacitracin resistant
CAMP positive
Growth on Mac (weak)
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20. Presumptive ID Tests - CAMP
The “CAMP” test (acronym for developers) is used to differentiate S. agalactiae (GBS), which is positive, from other beta hemolytic strep, all of which are negative
“CAMP factor” is a soluble hemolysin produced by GBS that combines in a synergistic way with a similar hemolysin of S. aureus to form an arrowhead zone of clearing. See image
The staph is streaked perpendicularly to the unknown strep. After appropriate incubation time an “arrowhead-shaped” clear zone of hemolysis will appear.
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21. S. agalactiae Synergistic haemolysis observed between betahemolytic
Staphylococcus
aureus and group B
streptococci.
􀁺 Positive reaction: arrowhead haemolysis pattern
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22. α – Haemolytic streps: Viridans streps
Not groupable by Lancefield groups
No group specific CHO
Streptococcus pneumoniae
Primary human pathogen
S.sanguis
S.mitis
S.mutans
S.salivarius
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24. The Pneumococcus (Streptococcus pneumoniae)
1: Isolation on Blood agar
Pneumococci frequently require enriched media and increased CO2 tension for initial isolation.
They are usually isolated on Blood agar and incubated in a candle jar (a closed container in which a lit candle is placed to remove O2 and increase CO2 ) at 37C. On Blood agar, colonies appear small, shiny, and translucent.
They are surrounded by a zone of alpha hemolysis
2: Optochin sensitivity
Pneumococci are the only streptococci that are sensitive to the drug optochin(ethylhydrocupreine hydrochloride).
3: Gram stain: gram-positive, diplococci
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26. The Genus Enterococcus
Enterococci are gram-positive streptococci, typically occurring in pairs and short chains, that are normal flora of the intestinal tract. Enterococci responsible for a variety of opportunistic infections in humans, and serologically belong to Lancefield group D streptococci.
On Bile Esculin agar
Unlike most bacteria, the enterococci will grow in the presence of the bile salts in the medium.
They hydrolyze the esculin, producing esculetin which reacts with the iron salts in the medium turning the agar black
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28. Enterococcus
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29. Laboratory ID: Enterococcus
white colonies
alpha and no haemolysis (rarely beta)
Also grow on MacConkey
Resembles S. pneumoniae in Gram stains
Bile esculin positive
Grows on Mac
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30. End of lecture
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