Streptococci Biochemical reactions
StreptococciBiochemical reactionsByDr. Nabil El AilaAssistant
Professor of Molecular MicrobiologyMedical Technology DepartmentAl
-Aqsa University
Dr. Nabil El AilaDiagnostic Microbiology1StreptococciCharacters
of StreptococciGram positive cocci1m in diameterChains or
pairsUsually capsulatedNon motile
Non spore formingFacultative anaerobesFastidiousCatalase
negative (Staphylococci are catalase positive)
Dr. Nabil El Aila Diagnostic MicrobiologyHemolysis on Blood
agar
-hemolysis-hemolysis-hemolysis Dr. Nabil El AilaDiagnostic
Microbiology18.05.094ClassificationBased on
O2AerobesAnaerobesPeptostreptococciGrowth on BA hemolysis hemolysis
hemolysisIncomplete hemolysis (green color)Complete hemolysis / /
no hemolysis Strep. viridans Strep. pneumoniaeEnterococcus
fecalisLancefield grouping specific C carbohydrate Ag on cell wall
Group A U (21 groups)Group AS. pyogenesGroup BS. agalactiaeGroup
CS. equisimitisGroup DEnterococcusOther groups (E-U)Differentiation
between -hemolytic streptococciThe following tests can be used to
differentiate between -hemolytic streptococciLanciefield
ClassificationBacitracin susceptibility TestSpecific for S.
pyogenes (Group A)CAMP testSpecific for S. agalactiae (Group B)
Dr. Nabil El Aila Diagnostic MicrobiologyLaboratory Diagnosis:
Group A StreptococcusColony morphologyTransparent, smooth, and
well-defined zone of complete or b- hemolysis
Dr. Nabil El Aila Diagnostic MicrobiologyLaboratory Diagnosis:
Group A
StreptococcusIdentificationCatalase-negativeBacitracin-susceptiblePYR-positiveBile-esculinnegative6.5%
NaCl-negative
Group A streptococci is susceptible to Bacitracin disk (left);
The right shows resistance Dr. Nabil El Aila Diagnostic
MicrobiologyBacitracin sensitivityPrinciple:Bacitracin test is used
for presumptive identification of group ATo distinguish between S.
pyogenes (susceptible to B) & non group A such as S. agalactiae
(Resistant to B)Bacitracin will inhibit the growth of gp A Strep.
pyogenes giving zone of inhibition around the
diskProcedure:Inoculate BAP with heavy suspension of tested
organismBacitracin disk (0.04 U) is applied to inoculated BAPAfter
incubation, any zone of inhibition around the disk is considered as
susceptible
8Pyrrolidonyl arylamidase Test (PYR) I.Principle Some bacteria
like group A streptococci and Enterococcu s species produce
pyrrolidonyl arylamidase which hydrolyzes the substrate L-
pyrrolidonyl --naphthylamide to form -naphthylamine. A pink to red
color forms when p-dimethylaminocinnam-aldehyde (PYR reagent) is
added to -naphthylamine. II. Inoculum Strains are grown on blood
agar plates overnight at 35C in CO2 . More than 1 day of incubation
may be necessary for more fastidious genera such as the gemellae,
alloiococci, and helcococci. The strains to be tested are grown on
a blood agar plate until sufficient growth is seen to heavily
inoculate the disks. III. Reagents and Materials PYR disk (Remel) -
PYR reagent Loops - Deionized Sterile water Dr. Nabil El Aila
Diagnostic MicrobiologyIV. Procedure The procedure that is used in
the Streptococcus laboratory is modified from the package insert.
The LAP test is usually done simultaneously. 1. Place the disks on
blood agar plate in an area of little or no growth or on a slide.
The moisture from the plate is usually sufficient to rehydrate the
disk. If the disk is placed on a slide, then a tiny drop of sterile
deionized water is added. (DO NOT OVERSATURATE THE DISK). 2. Using
a loop or wooden stick, inoculate the disks heavily. Using two or
more loop-fulls of culture is necessary for satisfactory results.
3. Leave the plates with the disks on the bench at room temperature
for 10 minutes. 4. Add the detection reagent and read after 3
minutes. V. Reading and Interpretation The development of a red
color within 3 minutes is positive. No change in color or a yellow
color is negative. The color develops immediately. Discard the test
after 10 minutes. VI. Limitations False negative reactions may
result if too little inoculum is used.
PYR positivePYR Negative Dr. Nabil El Aila Diagnostic
Microbiology
Laboratory Diagnosis: Group B b-Hemolytic Streptococcus Colony
morphologyGrayish-white, mucoid, creamy, narrow zone of
b-hemolysisPresumptive Identification
testsCatalase-negativeBacitracin-resistant Dr. Nabil El Aila
Diagnostic MicrobiologyLaboratory Diagnosis: Group B b-Hemolytic
StreptococcusPresumptive identification
testsBile-esculin-hydrolysisnegativeDoes not grow in 6.5%
NaClCAMP-testpositiveHipppurate Hydrolysis
S. agalactiae shows the arrow-shaped hemolysis near the
staphylococcus streak, showing a positive test for CAMP factor Dr.
Nabil El Aila Diagnostic MicrobiologyCAMP Test I. Principle Some
bacteria produce CAMP factor (a diffusible extracelluar protein)
that synergistically acts with the beta-lysin of Staphylococcus
aureus and enhances the lysis of red blood cells. The purpose of
the CAMP test is to aid in the identification of nonhemolytic group
B streptococci and other -hemolytic streptococci. II. Specimen
Growth from a blood agar plate or any solid media. III. Reagents
and Materials TSA-sheep blood agar Dr. Nabil El Aila Diagnostic
MicrobiologyIV. Procedure 1. The CAMP test is performed on
TSA-sheep blood agar. A single streak of -lysin producing S. aureus
made across the center of the plate. Strain SS-695 (Strep. Lab
number is a -lysin producing strain of S. aureus. 2. A single
colony of the unknown strain (beta hemolytic streptococci) is
picked up with an inoculating loop and used to make a single streak
perpendicular but not touching the S. aureus streak. A 2-3 mm space
should remain between the streaks. 3. Incubate the inoculated plate
normal atmosphere overnight at 35C. Group B streptococci and a few
other beta-streptococci produce an enhancement of the -lysin
activity of the S. aureus strain. V. Reading and Interpretation
This enhanced activity is in the shape of an arrowhead at the
juncture of the two streaks, with the widest portion of the
arrowhead on the group B side. VI. Limitations Do not incubate in
an anaerobic environment or under CO2. Some S. pyogenes strains
will give a positive reaction when incubated in CO2. CAMP test
Dr. Nabil El Aila Diagnostic Microbiology
Hipppurate Hydrolysis Test
I. Principle Some bacteria like group B streptococci produce the
enzyme hippurate hydrolase which hydrolyzes sodium hippurate to
form benzoic acid and glycine. The addition of ferric chloride to
benzoic acid forms an insoluble brown ferric benzoate precipitate.
II. Inoculum An overnight culture in Todd Hewitt broth incubated at
35 C or a fresh bacterial suspension in Todd Hewitt broth may be
used as the inoculum. An inoculating loopful of culture from a
blood agar plate may also be used. III. Reagents and Materials 1.
Hippurate broth commercial suppliers. 2. Ferric choride (FeCl3)
commercial supplies. Labeled as TDA if purchased for bioMereiuxIV.
Procedure 1. The hippurate broth is inoculated with one drop of a
fresh (16-20 h) Todd-Hewitt broth culture. 2. The broth is
incubated for up to 7 days or until turbid growth is seen at 35C.
3. The tube of broth is then centrifuged to sediment the bacteria.
4. Pipette 0.8 ml the clear supernatant to a small clear tube (13 x
100). 5. Add 0.2 ml of ferric chloride reagent to the supernatant.
Mix well. V. Results and Interpretation A heavy precipitate that
does not clear within 10 minutes indicates a positive test. A clear
golden-brown liquid indicates a negative test. VI. Limitations
Growth should be turbid before testing. Some fastidious organisms
may show poor growth Dr. Nabil El Aila Diagnostic
MicrobiologyLaboratory Diagnosis: Streptococcus Group D and
Enterococcus SpeciesIdentification testsCatalase: may produce a
weak catalase reactionHydrolyze bile esculinDifferentiate Group D
from Enterococcus sp. with 6.5% NaCl or PYR test Dr. Nabil El Aila
Diagnostic MicrobiologyLaboratory Diagnosis: Streptococcus Group D
and Enterococcus SpeciesMicroscopic morphologyCells tend to
elongate Colony morphologyMost are non-hemolytic, although some may
show a- or, rarely, b-hemolysisPossess Group D antigen
Dr. Nabil El Aila Diagnostic Microbiology
Bile Esculin Test
I.Principle A selective and differential medium used in the
identification of catalase-negative bacteria. The selective agent
bile, inhibits most gram positive bacteria. The enterococci and
Streptococcus bovis will grow. Esculin in the medium is hydrolyzed
to esculetin and dextrose. The esculetin reacts with ferric
chloride in the media to form a black-brown color. II. Inoculum An
overnight culture in Todd Hewitt broth incubated over night at 35 C
or a fresh bacterial suspension in Todd Hewitt broth may be used as
the inoculum. An inoculating loopful of culture may also be used.
III. Reagents and Materials 1. Bile esculin slant (Remel) Dr. Nabil
El Aila Diagnostic MicrobiologyIV. Procedure 1. Inoculate tube with
1 drop of inoculum allowing drop to run down slant. Alternatively,
the slant may be inoculated with a loopful of growth from a blood
agar plate. 2. The slant is then incubated at 35C for 2 days in
ambient air. Fastidious organisms may be held up to 14d. V. Reading
and Interpretation The bile esculin test is positive when a black
color forms over one-half or more of the slant. If no blackening
occurs the test is negative. VI. Limitations Do not incubate medium
in a carbon dioxide atmosphere. The increase in C02 will cause the
viridans streptococci to grow better and increase the likelihood of
a positive BE reaction. Streptococcus bovis and enterococci do not
require C02 for good growth.
Dr. Nabil El Aila Diagnostic MicrobiologyIdentification Schema
Schema to differentiate Group A and B from other b-hemolytic
streptococci
Identification SchemaSchema to differentiate Enterococcus and
Group D streptococci from other nonhemolytic streptococci
Laboratory Diagnosis:Streptococcus pneumoniaeColony
morphologySmooth, glistening, wet-looking,
mucoida-HemolyticCO2enhances growth
Dr. Nabil El Aila Diagnostic MicrobiologyLaboratory Diagnosis:
Streptococcus pneumoniaeIdentificationCatalase
negativeOptochin-susceptibility-testsusceptibleBile-solubility-testpositive
Dr. Nabil El Aila Diagnostic MicrobiologyDifferentiation between
-hemolytic streptococciThe following definitive tests used to
differentiate between S. pneumoniae & viridans streptococci
Optochin TestBile Solubility TestInulin Fermentation Dr. Nabil
El Aila Diagnostic MicrobiologyOptochin Susceptibility
TestPrinciple:Optochin (OP) test is presumptive test that is used
to identify S. pneumoniaeS. pneumoniae is inhibited by Optochin
reagent (
