Download Isolation and Identification of

General Microbiology Laboratory
Isolation and Identification of Gram
Positive Cocci
Identified method for Staphylococci
Gram-stain
 Isolation and culture
 Pure culture
 Direct identification
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Staphylococci are Gram-positive cocci, typically
arranged in clumps or Grape-like clusters
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Direct identification
Coagulase Test
Mannitol fermentation test.
DNase Test
 Novobiocin (NB) disc
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Test
Staphylococcus aureus
Staphylococcus
epidermidis
Mannitol fermentation
Positive
Negative
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Staphylococcus
saprophyticus
Usually positive
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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Staphylococcus aureus Growing on DNase Agar
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Blood agar with a novobiocin (NB) disc
Staphylococcus aureus
Growing on Blood Agar
Staphylococcus saprophyticus
Growing on Blood Agar
S
R
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Types Of Streptococcus
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(β) Haemolytic Streptococcus groups
S. pyogenes
• Group A
 S. agalactiae
• Group B (occasionally α or none)
Streptococcus bovis
• Group D (α or none, occasionally β)
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Group A strep: S. pyogenes
Gram +ve cocci
 Catalase (+ve)
Small, round, grey colonies
 β-haemolysis
 zone is large
 Group A antigen
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Isolation and Identification of (Streptococcus pyogenes)
1. on Blood agar. Streptococcus pyogenes produces a
zone of beta hemolysis around 2-3mm in diameter
surrounding each colony
2. Sensitivity to the antibiotic bacitracin
3. Group A Strep is senstive, other b haemolytic streps
are resistant.
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Bacitracin sensitivity
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Identification of Streptococci
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Group B: S. agalactiae
S. agalactiae
Identification
 Catalase negative
Bacitracin resistant
 CAMP positive
 Growth on Mac (weak)
Appearance on BA
 Larger colonies than Group A
 Small zone of hemolysis
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CAMP test
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.
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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S. agalactiae
Synergistic haemolysis
observed between
betahemolytic
Staphylococcus
aureus and group B
streptococci.
􀁺 Positive reaction:
arrowhead haemolysis
pattern
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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
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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.
3: Gram stain: gram-positive, diplococci
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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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Enterococcus
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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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End of lecture
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