Transcript The mannitol fermentation test.

General Microbiology Laboratory
Isolation and Identification of Gram
Positive Cocci
Identified method for Staphylococci
Gram-stain
 Isolation and culture
 Pure culture
 Direct identification
Staphylococci are Gram-positive cocci, typically
arranged in clumps or Grape-like clusters
Direct identification
 The mannitol fermentation test.
 The Coagulase Test
 The DNase Test
 Novobiocin (NB) disc
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.
Test
Staphylococcus aureus
Staphylococcus
epidermidis
Mannitol fermentation
Positive
Negative
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.
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.
Staphylococcus aureus Growing on DNase Agar
Blood agar with a novobiocin (NB) disc
Staphylococcus aureus
Growing on Blood Agar
Staphylococcus saprophyticus
Growing on Blood Agar
Identification of Gram-positive cocci
types Of Streptococcus
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
(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
Gp A strep: S. pyogenes Laboratory ID
Gram +ve
 Catalase
 Haemolysis
 small, round, grey colonies
 β haemolysis
 zone is large
 Gp A antigen
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.
Bacitracin sensitivity
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)
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.
S. agalactiae
Synergistic haemolysis
observed between
betahemolytic
Staphylococcus
aureus and group B
streptococci.
􀁺 Positive reaction:
arrowhead haemolysis
pattern
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
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
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
Enterococcus
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
End of lecture