Download Isolation and Identification of Gram Positive Cocci

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
 The mannitol fermentation test.
 The Coagulase Test
 The DNase Test
 Novobiocin (NB) disc
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The mannitol fermentation test.
Inoculate the bacteria into a mannitol microtube,incubate at 370C for 18h.S.aureus will
ferment mannitol to produce acid,which causes
the medium to turn yellow.
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Test
Staphylococcus aureus
Staphylococcus
epidermidis
Mannitol fermentation
Positive
Negative
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Staphylococcus
saprophyticus
Usually positive
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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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
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Identification of Gram-positive cocci
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types Of Streptococcus
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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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(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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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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Isolation and Identification of group A beta hemolytic (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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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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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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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
 S.salivarius
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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(ethylhydrocupreine hydrochloride).
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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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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End of lecture
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