Differentiation of Staphylococcus from Micrococcus

Staphylococcus Micrococcus
Catalase Test + +
Aerobic Growth + +
Anaerobic Growth + -
Glucose utilization in (OF media) Fermentative Oxidative or
Nonsaccharolytic
Modified Oxidase Test - +
Benzidine - +
Reaction to lysostaphin (2000ug/ml) Susceptible Resistant
Reaction to Furazolidone Susceptible Resistant
Reaction to bacitracin (0.04 U) Resistant Susceptible

IDENTIFICATION AND DIFFERENTIATION OF STAPHYLOCOCCI

1. Bacitracin Susceptibility Test

Principle:

 Bacitracin (0.04U) inhibits growth of Micrococcus &

Stomatococcus while having no effect on Staphylococcus

Purpose:

 To differentiate Staphylococcus from Micrococcus & Stomatococcus

RESULT:

 Susceptible
- zone of inhibition greater than 10mm;
- Micrococcus & Stomatococcus

 Resistant
- No zone of inhibition
- Staphylococcus

2. Modified Oxidase Test or Microdase Test

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 Principle:

 Micrococcus possess cytochrome C in the cytoplasm oxidase

system whereas most staphylococci do not

 Bacteria w/ cytochrome C produces a dark-blue and product w/

modified oxidase reagent (6% tetramethylphenylenediaminde HCl
in dimethyl sulfoxide)

Purpose:

 To differentiate Staphylococcus (-) species from Micrococcus (+) spp.

RESULT:

 Positive
- Dark blue color
- Micrococcus

 Negative
- No color change
- Staphyolococcus

3. Coagulase test

Principle:

 Coagulase from S. aureus binds fibrinogen that causes plasma to clot

Purpose:

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  To differentiate S. aureus (+) from CoNS (-)

2 TYPES OF COAGULASE

1. Bound Coagulase/Clumping factor

o Clumping factor directly to converts fibrinogen to fibrin
o Detected by rapid slide coagulase test

 Positive Result:

 agglutination/clumping
 S. lugdunensis & S. schleiferi are also positive

2. Free Coagulase/Staphylocoagulase

o Staphylocoagulase reacts with CFR from coagulase CFR

complex that resembles thrombin and indirectly converts
fibrinogen to fibrin

o Detected by tube coagulase test

o Clot formed may undergo autolysis due to fibrinolysin & will

result false-negative

o Inoculate with plasma and incubate @ 35 C for 1-4 hours

 Positive Result:

 Clot formation within 1-4 hours of incubation

4. DNase Test

Principle:

 Test to determine ability if aerobes to hydrolyze DNA

 DNase agar is pale green due to DNA-methyl green complex

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  If the organism hydrolyses DNA, the green color fades & the colony is
surrounded by COLORLESS

Purpose:

 To differentiate S. aureus (+) from S. epidermitis (-)

RESULT:

 Positive
- Hydrolosis of surrounding medium or clear zone
- S. aureus

 Negative
- No clearing is observed
- Staphylococcus epidemitis

5. Novobiocin Screening Test

Principle:

 Of the clinically significant coagulase negative staphylococci (CoNS),

Staphylococcus saprophyticus is resistant to 5 ug/ of Novobiocin

Purpose:

 Test to screen coagulase negative staphylococci from urine specimens

 Detect S. saprophyticus

RESULT:

 Sensitive
- S. aureus & S. epidermitis

 Negative
- S. saprophyticus

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 OTHER TEST

Test Purpose Principle Result

PYR test Differentiates  PYR is hydrolyzed to Negative:

Pyrrolidinyl S. aureus L-pyrrolidone & beta- o S. aureus
naphthylamine when acted by
from: enzyme pyrrolidonyl Positive:
arylamidase o S. lugdunensis
S. lugdunensis o S. intermedius
S. intermedius  Beta-naphthylamine combines o S. schleiferi
S. schleiferi w/ p-
dimethylaminocinnamaldehyd
e to from a red compound

VP Test Differentiates  Determines if an organism Negative:

Voges-Proskauer S. aureus uses butanediol o S. lugdunensis
Test fermentation/butylene
from: glycol pathway. Positive:
o S. aureus
S. lugdunensis  End product is acetoin (pink-
red color)

ANTIMICROBIAL SUSCEPTIBILITY TESTING

When is AST done among staphylococci?

 Serious infections with S. aureus require AST

 They produce beta-lactamase/penicillinases which make them
resistant to penicillin

Some staphylococci produce the mecA gene which codes for an altered penicillin-
binding protein (PBP) making them resistant to methicillin, nafcillin, & oxacillin

 MRSA – methicillin resistant Staphylococcus aureus

 MRSE – methicillin resistant Staphylococcus epidermitis
 BORSA – borderline oxacillin-resistant Staphylococcus aureus
 VRSA – vancomycin resistant Staphylococcus aureus
 VISA – vancomycin-intermediate Staphylococcus aureus

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 DIAGNOSTIC TESTS FOR STREPTOCOCCI

Group Species Hemolysis Bacitracin CAMP Bile Esculin SXT

Sensitivity Test sensitivity

Group A Beta Sensitive - - Resistant

S. pyogenes
Group B Beta Resistant + - Resistant
S. agalactiae
Group C Beta Resistant - - Sensitive
S. equi
Group D Alpha, Resistant
E. fecalis Beta, or Resistant - + (some are
S. equinis none sensitive)
Viridans Resistant
S. salivarus Alpha or (some are - - Sensitive
S. mitis none sensitive)
S. mutans

SUMMARY OF RXNS TO BACITRACIN AND SXT

ORGANISM BACITRACIN SXT

GAS Susceptible Resistant
GBS Resistant Resistant
GROUP C, F, G Resistant Susceptible

DIAGNOSTIC TESTS FOR BETA HEMOLYTIC STREPTOCOCCI

1. Bacitracin Susceptibility Test

Principle:
 Determines the effect of 0.04 U bacitracin on an organism
 Streptococcus pyogenes is inhibited by bacitracin while other beta-
hemolytic streptococci are resistant

Purpose:

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  Differentiates GAS from other beta-hemolytic streptococci

RESLUTS:

 Susceptible: GAS
 Resistant: GBS, Group C, F, G (non-group A Strep)

2. SXT Sensitivity Test

SXT 2 antibiotics:

 1.25 ug trimethoprim
 27.75 ug sulfamethoxazole

RESLUTS:

 Susceptible:
 Zone of inihibition
 Viridans & Group C

 Resistant:
 No zone of inhibition
 GAS

3. Hippurate Test

Principle:

 Hydrolysis of hippuric acid by hippuricase

→ produces glycine & benzoic acid

 Glycine is deaminated by ninhydrin (oxidizing agent)

 This is indicated by production of purple color

Hippuric acid → glycine + benzoic acid

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 Glycine + ninhydrin → purple/ dark blue color

Purpose:

 Differentiates GAS from GBS & other beta-hemolytic streptococci

Test Organism Reaction

Streptococcus agalactiae Positive: Dark blue color changes
Streptococcus pyogenes Negative: No color change or faint blue

4. CAMP Test

Principle:

 GBS can be distinguished from other B-hemolytic streptococci by

their production of CAMP factor

 CAMP (Christie, Atkins, Munch-Peterson)

→ Peptide that acts together with beta-hemolysin of S.

aureus, enhancing effect of hemolysis

Purpose:

 Differentiates GBS from other beta-hemolytic streptococci

RESLUTS:

 Positive
- Arrow zone head of hemolysis
- Indication presence of GBS
 Negative
- No increased hemolysis
- Other beta-hemolytic streptococci (GAS, C, F, G)

DIAGNOSTIC TESTS FOR GROUP D STREPTOCOCCI

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 1. Bile Esculin Agar Test

Principle:

 Group D streptococci (GDS) can grow in the presence of 40% bile &
hydrolyze esculin

 They will turn the indicator (ferric ammonium citrate)

→ dark brown color.

 Dark brown color results from the combination of esculetin (end

product of esculin hydrolysis) w/ ferric ions form a phenolic iron
complex

Purpose:

 Differentiates GDS from all other streptococci

RESLUTS:

 Positive
- Blackening of slant
- GDS grow readily on bile esculin agar
- Imparting dark brown to the medium
 Negative
- No blackening of slant
- Other streptococci are inhibited by bile salts

NOTE.

(+) Enterococcus faecalis

(-) Streptococcus mitis

2. PYR (pyrrolidonyl napththylamide)

Principle:

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  Presence of L-pyrroglutamylaminopeptidase detected in presence N

 To detect the presence of N-methylaminocinnamaldehyde reagent by

production of bright red colored product

Purpose:

 Differentiates Group D Enterococci from other Group D non-enterococci

RESLUTS:

 Positive
- Bright red color
- Entreococcus

 Negative
- No color change (orange)
- Non-enterococcus

3. PYR (pyrrolidonyl napththylamide)

Principle:

 Used to determine the ability of an organism to grow in high

concentrations of salt.

 A heat infusion broth containing 6.5% NaCl is used as test medium.

→ this broth also contains a small amount of glucose &

bromcresol purple as pH indicator

Purpose:

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  Differentiates Group D Enterococci (+) from other Group D non-
enterococci (-)

RESLUTS:

 Positive
- Turbidity in broth w/ or w/o color change from purple to yellow
- Enterococci

 Negative
- No turbidity & no color change
- Group D Non-enterococcus

4. Pyruvate Broth

Principle:

 Used to determine the ability of an organism to utilize pyruvate

Purpose:

 Differentiates Enterococcus faecalis (+) from Enterococcus faecium (-)

RESLUTS:

 Positive
- Color change from Green to yellow
- Enterococcus faecalis

 Negative
- No color change
- Yellow-green indicates weak rxn & should be regarded as negative
- Enterococcus faecium

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MISCELLANEOUS TEST STREPTOCOCCI

1. MRS Broth

Principle:

 MRS (Mann, Rogosa, Sharpe) broth test

 Used to determine whether organism forms gas during glucose

fermentation.

 Some Lactobacillus spp. & Leuconostoc spp.

Purpose:

 Differentiates Leuconostoc spp from all other genera except

Lactobacilli

RESLUTS:

 Positive
- Gas product or bubble in Durham tube
- Leuconostoc spp

 Negative
- No gas production or bubble in Durham tube

2. LAP disk

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 Principle:

 LAP (leucine aminopeptidase) disk

 Is a rapid test for detection of enzyme leucine aminopeptidase

 LAP disk contains leucine-beta-naphthylamide which serves as

substrate for the detection of LAP

 The resulting beta-naphthylamine produces RED color upon the

addition of cinnamaldehyde reagent

Purpose:

 Used to identify catalase negative gram + cocci

 Rapid test to identify Streptococci, Enterococci, & Pediococcus
 Which differentiate them from Aerococcus & Leuconostoc

RESLUTS:

 Positive
- Red color w/n 1 minute after adding cinnamaldehyde
- Gram (+), catalase (-), cocci

 Negative
- No color change
- Development of slight yellow color

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