Schematic diagram for the presumptive identification of gram-positive cocci.

CR, resistant;
S, susceptible.
 STREPTOCOCCUS TEST PRINCIPLE AND POSITIVE NEGATIVE RESULTS AND CORESPONDING ORGANISMS

TEST PRINCIPLE
CATALASE TEST To perform a catalase test, a plastic, disposable Postive: STAPHYLOCOCCCUS Negative: STREPTOCUCCUS
inoculating loop or wooden applicator stick is
used to place some of the colony onto a small
area of a glass microscope slide. A drop of 15%
hydrogen peroxide is added; the production of
bubbles of oxygen gas is a positive result
-USED TO DIFFERENTIATE STAPHYLOCOCCUS
AND STREPTOCOCCUS
HEMOLYSIS PATTERN hemolysis on sheep RBCs on agar Beta hemolysis: Beta ,alapha and none:
Alpha (α)-Partial lysis of RBCs around colony Group A (s. pyogenes) Eneterococus
Greenish discoloration of area around colony Group B
Beta Gorup c Alpha and none:
(β) Complete lysis of RBCs around colony Clear Viridans
area around colony Alpha hymolysis: Aerococcus
Nonhemolytic-No lysis of RBCs around colony S. Pneumoniae Pediococcos
No change in agar s.viridans Leuconostoc
Alpha-prime (α′) or wide zone Small area of Some group B
intact RBCs around colony surrounded by a
wider zone of complete hemolysis Gamma hemolysis :
Most Group B

Lance field Group antigen Most streptococci, except for many of the A: S. pyogenes Anginosus group, mutans group, mitis
viridans group, have a group or common C B: S. agalactiae group, salivarius group belongs to A, C, F, G,
carbohydrate (polysaccharide), which can be C:S. dysgalactiae, S. equi N,
used to serologically classify an isolate D; S. bovis group,E. faecalis, E. faecium
S.pneumoniae

L-pyrrolidonyl α-naphthylamide (PYR) PYR-impregnated disks serve as the substrate Positive: Negative
to produce α-naphthylamine, which is detected s. Pyogenes (group a) Group D
in the presence of D- Enterococcus spp. s. agalactiae
dimethylaminocinnamaldehyde (DMCA) by the Aerococcus Other beta hemolytic spp
production of a red color S.pnemoniae
Viridans steptococci
prediococcus
optochin test In the optochin test, a filter paper disk Resistant: Sensetive:
containing optochin (ethylhydrocuprein s. Pyogenes s. pneumoniae
hydrochloride) is added to the surface of an S. Agalactase
SBA plate that has just been inoculated with an Other B hemolytic spp
α-hemolytic Streptococcus. The plate is Enterococcus
 incubated overnight at 35° C in a CO2 Group D
incubator. A zone of inhibition greater than 14 Viridans
mm with a 6-mm disk or a zone of inhibition
greater than 16 mm with a 10-mm disk is
considered susceptible
BIle esculiunn solubility test est for bile solubility takes advantage of the S. Positive Negative
pneumoniae autocatalytic enzyme amidase. Enterococcus Other spp not specified
Under the influence of a bile salt or detergent, s.pneumoniae
the organism’s cell wall lyses during cell prediococcus
division. A suspension of S. pneumoniae in a
solution of sodium deoxycholate lyses, and the
solution becomes clear. Other α-hemolytic
organisms do not undergo autolysis, and the
solution remains cloudy. Suspensions of
bacteria made in saline serve as negative
controls.
CAMP test The CAMP test can be performed in three Postive Negative
ways. One is with the use of a β-lysin– s. Agalactiae (group B) others not specififed spp
producing strain of S. aureus; another is with
the use of a disk impregnated with the β-lysin.
Both methods take advantage of the enhanced
hemolysis occurring when the β-lysin and the
hemolysin produced
by GBS are in contact. The result is a
characteristic arrowhead-shaped hemolytic
pattern
A third method, the rapid CAMP test (or spot
CAMP test), involves placing a drop of
extracted β-lysin on the area of confluent
growth of the suspected GBS. After incubation
at 35° C for at least 20 minutes, enhanced
hemolysis is observed
Hippurate Hydrolysis A useful test for differentiating S. agalactiae Positive:
from other β-hemolytic streptococci is the
hippurate hydrolysis test. S. agalactiae S.agalactiae
possesses the enzyme hippuricase (also called Areococcus
hippurate hydrolase), which hydrolyzes sodium Pediococcus
hippurate to form sodium benzoate and glycine
Leucine aminopeptidase (LAP) is a peptidase that hydrolyzes peptide bonds Postive Negative
adjacent to a free amino group. Because LAP S.pyogenes Aerococcus
reacts most quickly with leucine, it is called S.agalactiae Leuconoctoc
leucine aminopeptidase. The substrate, Other bhemolytic spp
leucine-βnaphthylamide, is hydrolyzed to β- Group D
naphthylamine. After the addition of p- S.Pneumoniae
dimethylaminocinnamaldehyde reagent, a red Viridans
color develops pediococcus

Voges-Proskauer Test The VP test detects acetoin production from Positive

glucose. In the modified VP test, a heavy anginosus group
suspension of bacteria is made in 2 mL of VP
broth. After about 6 hours of incubation at 35°
C, a few drops of 5% α-naphthol and 40%
potassium hydroxide are added. The tube is
shaken vigorously to increase the
concentration of dissolved oxygen, and the
broth is incubated at room temperature for 30
minutes. The formation of a red or pink color is
a positive reaction.
Other Susceptibility test
Vancomycin SMZ
Resistant : Greater percentage susceptible:
Prediococcus Enterococcus Resistant:
Leuconostoc S.pyogenes
S.galactiae
Enterococcus
Sensetive:
S.pyogenes
S.agalactiae Sensetive:
Other B hemolytic species Other Beta hemolytic spp
Group D S.pneumoniae
S.peumoniae Viridans
Viridans
Aerococcus
 Organisms positive for bile esculin are Positve :
separated into group D streptococci or Enterococcus
Salt tolerance test Enterococcus by the salt tolerance test. Growth Aerococcus
in 6.5% NaCl broth
Variables:
Pediococcus
Leuconostoc