STAPHYLOCICCI

Lecture # 3

Staphylococcus sp.

Morphology: Gram positive cocci. In clusters

Culture: Facultative anaerobes Incubation 37ºC x 24 hr

Staphylococcus sp.

Coagulase +ve: Staphylococcus aureus.

Coagulase –ve: Staphylococcus saprophyticus. Staphylococcus epidermidis.

All Staphylococci are CATALASE +VE

CULTURE

Blood Agar: Creamy colonies 1-2 mm Beta-heamolysis

Blood Agar: Grayish white colonies No heamolysis

Coagulase +ve

Staphylococcus aureus

Coagulase -ve Staphylococci

CULTURE

MacConkey Agar: Pink colonies (lactose

fermenting)

MacConkey Agar: Only some are give Pink

colonies (lactose fermenting)

Coagulase +ve

Staphylococcus aureus

Coagulase -ve Staphylococci

CULTURE

Mannitol Salt Agar: Growth Change color from Red to

Yellow (Mannitol fermenting)

Mannitol Salt Agar: Growth No yellow color no

fermentation.

Coagulase +ve

Staphylococcus aureusCoagulase -ve Staphylococci

Differential: Staph

aureus can frement mannitol and give diffused

yellow color

Selective: Containing very high salt conc only staph can grow

Biochemical Tests

Biochemical Tests are:

1- Catalase

2- Coagulase

3-DNase

1. Catalse: 2H2O2 O2 + 2H2O

Streptococci vs. Staphylococci

Coagulase Test

2. Coagulase test: Fibrinogen in plasma Fibrin

A. Free (tube) coagulase:1. 1 ml pepton + 1 ml EDTA plasma2. Emulsify loop full of organism3. Incubate 30 min x 37ºC4. If negative incubate at RTº overnight

Coagulase Test

Coagulase Positive Coagulase Negative

Coagulase Test

B. Bound (slide) coagulase: 1. One drop of saline on clean slide2. Emulsify loopful of organism in saline3. Add one drop of plasma4. Mix rocking for 2 min

Coagulase Test(Latex agglutination test)

• Several latex agglutination test kits are available to identify Staphylococcus aureus.

• Latex particles coated with fibrinogen.• Rapid clumping of latex particles occurs

when mixed with colony material of S. aureus.

Biochemical Tests

3. DNase Test:DNase enzyme hydrolyze DNA

4. Spot-inoculate organism on a DNA containing media

5. Incubate 37ºC x 24 hr6. Flood plate with 1 mol/ml HCL sol

HCL will precipitate unhydrolyzed DNA4. Tip off the excess acid5. Clearing around colonies DNase positive strain6. No Clearing around colonies DNase negative

strain

MRSA (methicillin resistant Staphylococcus aureus)

• These strains are resistant to methicillin and related penicillins and are particularly difficult to treat because they are also resistant to most other common antibiotics.

• MRSA strains cause hospital infections, particularly wound infection and septicaemia.

• Vancomycin is often needed to treat MRSA infections.

Coagulase Negative Staphylococci

Novobiocin Disk:

Staphylococcus saprophyticus Resistant Staphylococcus epidermidis Sensitive

Micrococcus

Gram Positive Cocci in pairs and tetrads. Catalase +ve. Generally harmless, but can cause

disease in immunocompromised host.

Micrococcus

Blood agar or chocolate agar:

Small, non-hemolytic, variably pigmented (white, orange, yellow pink) colonies.

MacConkey:

no growth.

Your Work For Today.

1. Prepare Gram Stain, show it to the instructor.

2. Perform Catalase test.

3. Perform Coagulase test.

4. Perform DNA’s test.

5. Do subculture of the organism to:

Blood agar plate.

Mannitol Salt Agar.