Biochemical Tests for differentiating Staphylococcus, Micrococcus and Streptococcus compiled by JTC2019

I. Staphylococcus and Micrococcus

Biochemical Reagent / culture media Positive result Differentiates Principle Precautions/
test ( + and S organism in Notes
bold )
Catalase 3%H2O2 Bubbling/ Staphylococcus (and Detection of enzyme Do not get
Effervescence Micrococcus) from catalase colonies from
Streptococcus BAP
(False +)

Modified 6% Dark Blue Micrococcus from Detection of Do not use

oxidase tetramethylphenylenediamine coloration Staphylococcus cytochrome c from iron loop
–HCl (TMPD-HCl) in dimethyl Exceptions: cytochrome oxidase (False +)
sulfoxide (DMSO) S. sciuri
S. lentus
S. vitulus
Coagulase test
Slide Rabbit plasma ( 1-2 drops ) Clumping S. aureus and S. Screening test that Do not get
coagulase ( add colonies first before intermedius (also detects bound colonies from
adding the rgt) S.lugdunensis ) coagulase/clumping culture
from S. epidermidis factor ; Test for media with
and S. saprophyticus pathogenicity ; Proceed high salt
to tube coagulase test if content
negative (MSA)

Tube Rabbit plasma ( 1 ml ) Coagulum / S. aureus and S. Confirmatory test that Do not
coagulase (add the rgt first before adding clump ( not intermedius from S. detects free coagulase incubate
the colonies ) ; clumping) epidermidis and S. beyond 4hrs ;
Incubate at 37C for 4hrs ; check saprophyticus organism
every 30 min forms
staphylokin-
ase that
breaks down
coagulum
( False - )

Oxidation OF medium ;pH indicator Change in color Differentiates Differentiates

Fermentation from Red to Staphylococcus and organisms in their
-test yellow Micrococcus ability to oxidize and
(if no color ferment sugars
change =
asaccharolytic )

a. Closed tube Layered with mineral oil Yellow coloration Staphylococcus Differentiates
Oxidation Use of OF medium and pH (break down of + closed tube organisms in their
Fermentati-on indicator sugar) + open tube ability to oxidize and
test + = fermenter ferment sugars
b. Open tube Tube is open Yellow coloration Micrococcus Differentiates
Oxidation Use of OF medium and pH (break down of + open tube organisms in their
Fermentati-on indicator sugar) - closed tube ability to oxidize and
test += oxidizer ferment sugars
DNAse Test DNA agar with toluidine blue Clear zone / DNAse producing Detection of DNAse Also (+) for
and methyl green as pH hydrolysis of Staphylococcus enzyme activity ; Grp B
indicators ; ( binding of pH surrounding aureus breakdown of DNA Streptococcus
indicator to DNA ) medium

Gelatinase 12 % gelatin Liquefied or Staphylococcus Identification of Make sure to

test hydrolyzed aureus from other organisms capable of refrigerate the
gelatin Staphylococcus liquefying/ gelatin
[Enterococcus(+)] hydrolyzing gelatin medium
Differentiation for coagulase positive Staphylococcus spp.
Voges MRVP broth Red coloration Differentiates Determines if Can also
Proskauer (Methyl Red Voges Proskauer coagulase positive organism utilizes the differentiate
test broth ) Staphylococcus spp. butylene glycol viridans
Rgt: + = S. aureus ( also pathway ( formation streptococcus
40% KOH w/ 0.3% creatine S. schleiferi and of acetoin from from one
5% α naphthol in 100% S. lugdunensis ) glucose ) another
methanol - = S. intermedius
PYR test Pink to cherry red Differentiates Detects Can also
( Pyrrolidonyl coloration coagulase positive pyroglutamylaminope differentiate
Arilamadase Staphylococcus spp. ptidase or Grp D strep
test or + = S. intermedius pyrrolidonylamylamid and Can
Pyrrolidonyl ( also S. ase or presumptively
aminopeptid- lugdunensis ) pyrrolidonylaminopep identify Grp A
ase test ) - = S. aureus tidase Strep.
Differentiation for coagulase negative Staphylococcus spp. (CONS)
Novobiocin 5 ug Novobiocin S – Has obvious Differentiates Antibiotic resistance
test zone of inhibition coagulase negative and susceptibility of
Staphylococcus spp. the Staphyloccocus
R- Has minimal S= S. epidermidis sp. To Novobiocin
zone of inhibition ( also S.
haemolyticus )
R= S. saprophyticus

Susceptibility 300 units Polymyxin B S – Has obvious Differentiates Antibiotic resistance

to Polymyxin B zone of inhibition coagulase negative and susceptibility of
test Staphylococcus spp. the Staphyloccocus
R- Has minimal S= S. saprophyticus sp. To Polymyxin B
zone of inhibition (also S.
haemolyticus )
R= S. epidermidis

Ornithine Ornithine decarboxylase tube Violet coloration Identify and confirm Enterics
decarboxylase (ODC tube) presence of S. E.cloaceae
test lugdunensis E.aerogenes
Urease Urease S. epidermidis and Detection of Urease S. epidermidis
production S.saprophyticus ; enzyme and
S. aureus and S.saprophytic-
S.lugdunensis are us are both
Variable (depends) CONS
 II. Streptococcus
Biochemical test / Reagent / Culture Positive Differentiates Principle Precaution / Note
Differentiating test media result (+ and S organism in
bold)
Growth in BAP 5% defibrinated α – partial Organisms based on Hemolytic patterns Do not overheat
sheep blood /BAP hemolysis their hemolytic ( CAP instead of
β- complete patterns BAP )
hemolysis α- Viridans
γ- no Streptococcus, S. All Streptococcus
hemolysis pneumoniae are pinpoint
(mucoid=capsulated), colonies except
Grp D and for S. pneumoniae
Enterococcus ( capsule )
β- Grp A: S. pyogenes
( mucoid = Leuconostoc,
capsulated ) Aerococcus and
- Grp B : S. Pediococcus
agalactiae (small resemble Viridans
zone/ narrow zone) S.
- Grp C : S. (α hemolytic ; can
dysgalactiae and also be γ
S. equi hemolytic )
- Grp F and G : S.
anginosus
γ- Grp D and
Enterococcus
( Viridans )
Lancefield’s Lancefield’s Reaction to Grp A = antisera A Grouped based on presence Streptococcus
classification ( Slide antisera Lancefield’s Grp B = antisera B of Common C Carbohydrate pneumoniae and
agglutination test ) antisera Grp C = antisera C antigen and Teichoic acid Viridans
Grp D = antisera D Streptococcus are
Grp F = antisera F not classified
Grp G= antisera G under the
Rebecca
Lancefield’s
classification
Differentiation for β-hemolytic Streptococcus spp.
Bacitracin Bacitracin S- Has Differentiates Grp A Antibiotic susceptibility to Among all the
susceptibility test obvious Streptococcus from Bacitracin Streptococcus
(Taxo A) zone of Grp B,C,F,G spp. , only grp A
inhibition Streptococcus / S.
S- Grp A pyogenes is
R- has R- Grp B,C,G,F Bacitracin
minimal susceptible
zone of
inhibition
Sulfamethoxazole Sulfamethoxazole S- Has Differentiates Grp A Antibiotic susceptibility to SMX
susceptibility test ( SXT or SMX ) obvious and Grp B
zone of Streptococcus from
inhibition Grp C,F,G

R- has S- Grp C,F,G

minimal R- Grp A and B
zone of
inhibition
CAMP test A heavy vertical Arrow head Identifies Grp B Enhanced hemolysis (contact Staphylococcus
( Christie, Atkins, streak of hemolysis hemolytic of hemolysin and β-lysin) aureus is β-
Munch-Petersen Staphylococcus pattern streptococcus from hemolytic
test ) aureus Grp A
+ = Grp B
streptococcus
( Streptococcus
agalactiae )
PYR test Pink to Presumptive test Detects Can be used to
cherry red that identifies β- pyroglutamylaminopeptidase differentiate
coloration hemolytic or pyrrolidonylamylamidase or coagulase positive
Streptococcus pyrrolidonylaminopeptidase staphylococcus ;
Can be used to
+ = Grp A differentiate Grp
D Streptococcus

Differentiation of Grp B β-hemolytic Streptococcus from other β-hemolytic Streptococcus

Hippurate hydrolysis Ninhydrin Violet /Blue Differentiates of Grp Detection of the enzyme Can be used to
reagent coloration B Streptococcus from Hippuricase ( enzyme that differentiate
with other hydrolyses hippurate to viridans
hydrolysis β-hemolytic benzoic acid and glycine ) Streptococcus
of Streptococcus
hippurate
Presumptive identification of Grp D Streptococcus
Bile Esculin hydrolysis 40% bile and Brown- Presumptive test that Ability to break down Esculin
esculin black identifies Grp D and grow in 40% Bile
precipitate Streptococcus from
and other grps
hydrolysis
of esculin All Grp D are positive
Differentiates Group D Streptococcus ( Enterococcus vs Non-Enterococcus )
PYR test Pink to Differentiates Grp D Detects
cherry red Enterococcus from pyroglutamylaminopeptidase
coloration non-Enterococcus or pyrrolidonylamylamidase
or
pyrrolidonylaminopeptidase

Salt tolerance test 6.5% NaCl broth Differentiates Grp D Ability to grow in 6.5% NaCl
Enterococcus from
non-Enterococcus
Differentiates Viridans Streptococcus
Voges Proskauer MRVP broth Red Differentiates Determines if organism Can be used to
test (Methyl Red Voges coloration Viridans utilizes the butylene glycol differentiate
Proskauer broth ) Streptococcus from pathway ( formation of coagulase positive
one another acetoin from glucose ) Staphylococcus
- = S. mitis
Sugar fermentation ``
Hippurate Ninhydrin reagent Violet Differentiates Detection of the enzyme Can Presumptively
hydrolysis coloration Viridans Hippuricase ( enzyme that identify Grp B
and Streptococcus from hydrolyses hippurate to Streptococcus from
hydrolysis one another benzoic acid and glycine ) other β-hemolytic
of Streptococcus
hippurate
Differentiation of Streptococcus-like organisms from other Streptococcus
Leucine Red Aerococcus and Differentiates This test can
Aminopeptidase coloration Leuconostoc are Streptococcus, separate
test (LAP) LAP- Enterococcus and Aerococcus and
Pediococcus from Leuconostoc from
Aerococcus and other
Leuconostoc Streptococcus
( Aerococcus and
Leuconostoc are LAP - )

Differentiation of other α-hemolytic Streptococcus

Optochin test Optochin disk (main S- Has S- S. pneumoniae Presumptive identification of Capsular swelling
(Taxo B or P) component : obvious R- other S. pneumoniae ; Antibiotic test, mouse
Ethylhydrocuprein zone of α-hemolytic susceptibility and resistance virulence test and
HCl ) inhibition Streptococcus to optochin inulin fermentation
test can be other
R- has presumptive ways
minimal to identify S.
zone of pneumoniae
inhibition
Bile solubility test Sodium Clear Presumptive ID of Lysis of the cell wall due to The autocatalytic
desoxycholate solution S. pneumoniae presence of bile salts; amidase enzyme of
Presence of autocatalytic S. pneumoniae is
amidase enzyme of S. responsible for the
pneumoniae bile solubility
activity and the
lysis activity.
Serologic tests for S. pyogenes ( Scarlet fever )
Dick’s test Scarlatinal toxin Redness of The presence or Susceptibility test for the Determines risk of
skin absence of the scarlatinal toxin getting the
antitoxin/ ( intradermal injection ) infection
antibodies against + = high risk of
the scarlatinal toxin getting infected
Schultz Charlton Scarlatinal toxin Blanching/ Detects the presence Diagnostic or confirmatory Neutralized since
test neutralized of the antitoxin/ test for scarlet fever the body has the
phenomenon antibody against the (intradermal injection) antitoxin to
scarlatinal toxin neutralize the
scarlatinal toxin