LAB S20 Flashcards
hemolysis and Lancefield classification of S. pyogenes
group A beta hemolytic
hemolysis and lancefield classification of S. agalactiae
group B beta hemolytic
hemolysis of S. pyogenes
alpha hemolytic
morphology of streptococcus and enterococcus in microscope
G+ cocci in chains
morphology of S. pneumoniae in microscope
cocci in pairs
slightly elongated lanceolate
surrounded by halo (polysaccharide capsule)
three culture media used for streptococci species
5% sheep BAM
Columbia CNA agar
PEA agar
inoculation technique for streptococcus
streak stab technique
purpose of streak swab technique in streptococcus inoculation
creates a relatuvely anaerobic environment
streptococcal growth is enhanced by
5-10% CO2
most streptococci will grow on agar media at incubation of:
35-37 C within 48 hours
About 0.5 mm in diameter, grayish white, convex, entire, transparent to translucent, matte or glossy; large zone of β-hemolysis, 2x-4x the diameter of the colony.
Group A
Larger than group A streptococci; translucent to opaque; flat, glossy;
narrow zone of β-hemolysis
Group B
Grayish white, glistening; wide zone of β-hemolysis
Group C
Grayish white, small, matte; narrow to wide zone of β-hemolysis
Group F
Grayish white, matte; wide zone of β-hemolysis
Group G
Small, gray, glistening
young are dome-shaped, later umbilicated
S. pneumoniae
Minute to small, gray, domed, smooth or matte; α- or ɣ-hemolysis
Viridans streptococci
Small, cream or white, smooth, entire; α–, β-, or ɣ-hemolysis
Enterococcus spp.
oxygen labile hemolysin
streptolysin O
oxygen stable hemolysis
streptolysin S
causes beta hemolysis in the surface of agar
streptolysin S
causes beta hemolysis im the subsurface of the agar
streptolysin O
results in the greening of agar
alpha hemolysis
alpha hemolytic streptococci
S. lneumoniase, viridans group, Enterococcus
hemolysis of Entterococcus species cultivated 24 hours in aerobic atmosphere at 37C
gamma hemolysis
other gamma hemolytic species
group D streptococci viridans group (sometimes)
two characteristics for presumptive identification of S. pneumoniae
a-hemolytic dome shaped mucoiddue to large amounts of capsular polysaccharide
checker-piece/ nail head colonies due to organism autolysis
4 tests for identification of Beta hemolytic streptococci
Bacitracin (Taxo A) test
SXT (trimethoprim-sulfamethoxazole) test
CAMP test
sodium hippurate hydrolysis test
Used for presumptive identification of group A β-hemolytic streptococci
bacitracin (taxo A) test
concentration of bacitracin in taxo A test
0.04 u
Used for presumptive identification of groups C, F, or G β-hemolytic streptococci
SXT (trimethoprim- sulfamethoxazole test)
concentration of trimethoprim in SXT test
1.25 g
concentration of sulfamethoxazole in SXT test
23.75 ug
bacitracin susceptible, SXT resistant
group A
bacitracin resistant, SXT resistant
group B
bacitracin resistant or susceptible, SXT susceptible
Group C, F, G
CAMP test meaning
Christie, Atkins, Munch- Petersen (1944)
Used for presumptive identification of bacitracin-resistant Group B β-hemolytic streptococci
CAMP test
extracellular substance enhancing hemolytic activity of S. aureus
CAMP factor
when group B streptococci enhances hemolytic effect of S. aureus, it is termed as
synergistic hemolysis
synergistic hemolysis is observed as
arrow head shaped zone where the two organisms intersect
positive camp test (arrow head zone) is characteristic of
Group B B-hemolytic streptococcus
CAMP negative
Group A streptococcus
also used for presumptive identification of bacitracin-resistant Group B β-hemolytic streptococci
sodium hippurate hydrolysis test
Group B streptococcus hydrolyzes hipourate into:
benzoate and glycine
mixed with benzoic acid for precipitation to determine hippurate hydrolysis
ferric chloride
how to determine positive benzoic acid test
wait for precipitate to persist for 10 minutes
mixed with glycine to determine hippurate hydrolysis
ninhydrin
ninhydrin mechanism of action on glycine
deaminate alpha amink compounds which releases ammonia and carbon dioxide, forming a purple or deep blue color
positive result for sodium hippurate hydrolysis
10 mins precipitate for benzoic acid
deep blue or purple color for glycine
hippurate positive streptococcus
Group B
hippurate negative streptococcus
Group A
2 tests for identification of alpha hemolytic streptococci
Optochin Taxo P susceptibility test
Bile solubility test
other name for optochin
ethylhydrocuprein hydrochloride
principle of optochin susceptibility
inhibits growth of S. pneumoniae
more than 14 mm ZOI for optochin
S. pneumoniae
less than 14 mm ZOI for optochin
viridans or group D
S. pneumoniae produces an autolytic intracellular amidase enzyme known as
desoxycholate
desoxycholate is activated by
bile
In bile solubility test, S. pneumoniae undergoes:
rapid autolysis
bile solubility test positive result
disappearance of colonies = S. pneumoniae
bile solubility negative result
intact colonies = viridans and group D
Three other methods to distinguish between S. pneumoniae and viridans group
- Quellung or Neufeld reaction
- inulin fermentation test
- mouse virulence test
principle of Quellung reaction
anticapsular antibodies react with polysaccharide capsule of S. pneumoniae
Positive quellung reaction:
clumping, halo effect, light blue outline = S. pneumoniae
negative quellung reaction:
viridans streptococci
principle of inulin fermentation test
S. pneumoniae can ferment inulin with acid and gas
positive inulin fermentation test
S. pneumoniae
negative inulin fermentation
viridans group
based on sensitivity of white mouse to S. pneumoniae
mouse virulence test
inoculation method of white mouse
intraperitoneal injection
positive mouse virulence test
death of mouse within 1-2 days = S. pneumoniae
Tests for identification of Group D streptococci and Enterococcus species
- Bile esculin test
2. Salt tolerance test
principle of bile-esculin test
ability of Group D and Enterococcus species to grow in 40% bile
hydrolyze esculin into glucose and esculetin
esculetin reacts with ferric ions (from ferric citrate)
results into black diffusible complex
bile esculin test positive result:
blackening of medium: Group D/ Enterococcus
bile esculin test
no blackening = viridans
salt tolerance test principle
ability of enterococcus to grow in the presence of 6.5% NaCl
positive result of salt tolerance
visible turbidity/ change of color from purple to yellow = enterococcus
negative result of salt tolerance
no turbidity and color change = Group D
Other methods to distinguish between Group D and enterococcus
- pyrrolidonyl arylamidase (PYR) test
2. susceptibility to penicillin
principle of pyrrolidonyl arylamidase test
pyrrolidonyl arylamidase hydrolyzes PYR, which releases free B-naphthylamid + addition of diazo dye coupler forming a red Schiff base
PYR positive
Enterococcus
PYR negative
Group D streptococci
penicillin resistant
Enterococcus
penicillin susceptible
Group D
