Download A. GRAM'S POSITIVE COCCI

A. GRAM'S POSITIVE COCCI
2. STREPTOCOCCUS SPP.
Microscopic examination
Microscopic examination
Morphological Description
Type of stain:
 Gram reaction:
 Color:
 Shape:
 Size:
 Arrangment:

gram stain
gm +ve
violet
cocci
small
chains or pairs
Classification of streptococci
1. hemolysis
on blood agar
α hemolysis
(Partial hemolysis )
β hemolysis
(Complete hemolysis)
γ hemolysis
(non hemolysis
*S. pyogenes
*S. pneumonia
*Viridans
streptococci
*S. faecalis
Cultivation
Culture Media
1. Blood agar
2. Chocolate agar
Culture Media

Blood agar

Type: enriched, differential
medium
Constituents: nutrient agar +510% defibrinated blood
Principle: species of streptococci give various types of
hemolysis on blood agar
Use: * for isolation of
pathogenic bacteria
* differentiate Streptococcus spp. according to
their hemolytic activity




Chocolate agar

Type: enriched
Constituents: blood agar heated at
56oC
Principle: hemoglobin releases
hemin (X-factor), required
by fastidious organisms
Use : permits growth of fastidious
bacteria & to help in the
recovery of Streptococcus spp



Tests
• Blood hemolysis:
α, β, γ
• Biochemical tests: 1. Bile solubility
2. Bacitracin sensitivity
3. Optochin sensitivity
• Serological test:
Antistreptolysin O
test (ASO)
Blood hemolysis
Hemolysis on blood agar
(α) Alpha haemolysis
(β) Beta-haemolysis
(γ) Gamma-haemolysis
Principle
Use
Incomplete blood
haemolysis
green zone around the
growth due to H2O2
production, hemoglobin
Met hemoglobin
Complete blood
haemolysis
producing clear zone
around the growth
To differentiate m.o.
according to their
haemolytic activity
S. pneumoniae(α)
S. viridans (α)
Absence of blood
haemolysis around
the growth
Streptococcus faecalis
(γ)
Staphylococcus
epidermidis (γ)
To differentiate m.o.
according to their
haemolytic activity
e.g. Staph. aureus (β)
Strept. pyogenes(β)
Bile solubility
Medium: Nutrient broth + bile salt.
 Principle: Lysis of m.o. sensitive to bile salts

as Streptococcus pneumoniae
 Use: To differentiate between α-haemolytic
streptococci
S. pneumoniae +ve
& S. viridans -ve
Bacitracin sensitivity


Medium: Blood agar
Reagent: 0.04 unit bacitracin disc is
applied to the surface
of the agar plat
 Principle: S. pyogenes (β-hemolytic) is
sensitive to bacitracin
Bacitracin Test +ve
α & γ hemolytic streptococci are
resistant to bacitracin
 Use: To differentiate between Strept. pyogenes (S)
from other α & γ hemolytic streptococci (R)
Optochin sensitivity
Medium: Blood agar
 Reagent: Optochin (ethyl hydrocuprein HCl)
 Principle: Optochin inhibits the growth of some

m. o.

Use: To differentiate between α-haemolytic
streptococci S. pneumoniae +ve
& S. viridans -ve
+ve
-ve
-ve S. viridans
+ve S. pneumoniae
Optochin Sensitivity Test
Antistreptolysin O test (ASO)
Serologic Agglutination Test




Medium: Polystyrene latex particles coated with
Streptolysin O Antigen
Reagent: Streptolysin O Antigen
Principle: Reagent + Serum → agglutination
(+ ve Ag-Ab reaction)
Use: Detection of anti-streptolysin O in
patient’s serum is an indication for
streptococcal infection.
ASO test
+ve
-ve
Day
1
Mixed culture
Gram stain and streak for isolation
MAcConkey
Day
2
TSA
Gram stain and select a Gram negative colony
Oxidase (-)
Lac negative on
MAC
Oxidase (+)
Lac positive on
MAC
Lac negative
on MAC
Day
3
Day
4
Urea (+)
Green sheen
on EMB
Dark purple
on EMB
Glucose
fermentation (-)
H2S (+)
on TSI
Citrate (-)
Citrate (+)
Lipase (+) on
tributyrin
Proteus vulgaris
Escherichia coli
E. aerogenes
Ps. aeruginosa
Day
1
Mixed culture
Gram stain and streak for isolation
MSA
Day
2
TSA
Gram stain and select a Gram positive colony
Catalase (+)
Day
3
Day
4
Negative for
mannitol on MSA
Positive for
mannitol on MSA
Trehalose
(-)
Nitrate
Nitrate
(+)
(-)
Novobiocin
sensitive
Novobiocin
sensitive
Novobiocin
resistant
Staphylococcus
epidermidis
Staphylococcus
aureus
Staphylococcus
saprophyticus