Lecture # 3. Morphology: Gram positive cocci. In clusters Culture: Facultative anaerobes Incubation 37ºC x 24 hr.

STAPHYLOCICCI BASMAH ALMAARIK

Lecture # 3

STAPHYLOCOCCUS SP.

Morphology: Gram positive cocci.In clusters

Culture:Facultative anaerobesIncubation 37ºC x 24 hr

STAPHYLOCOCCUS SP.

Coagulase +ve: Staphylococcus aureus.

Coagulase –ve: Staphylococcus saprophyticus. Staphylococcus epidermidis.

All Staphylococci are CATALASE +VE

Blood Agar: Creamy colonies 1-2

mm Beta-heamolysis

Coagulase -ve Staphylococci

Blood Agar: Grayish white colonies No heamolysis

CULTURE Coagulase +ve Staphylococcus aureus

HEMOLYSIS

α-hemolysis:

the agar under the colony is dark and greenish

γ-hemolysis: the agar under and around the colony is unchanged, and the organism is called non-hemolytic

β-hemolysis:  complete hemolysis- is a complete lysis of RBCs in the media around and under the colonies: the area appears lightened (yellow) and transparent

Mannitol Salt Agar: Growth Change color from Red

to Yellow (Mannitol fermenting)

Coagulase -ve Staphylococci

Mannitol Salt Agar: Growth No yellow color no

fermentation.

CULTURE Coagulase +ve Staphylococcus aureus

Differential: Staph

aureus can ferment mannitol and give diffused

yellow color

Selective: Containing very high salt conc only staph can grow

CULTURE

Coagulase +ve Staphylococcus aureus

MacConkey Agar:Pink colonies

(lactose fermenting)

Coagulase -ve Staphylococci

MacConkey Agar:Only some give

Pink colonies (lactose fermenting)

BIOCHEMICAL TESTS

1. Catalse: 2H2O2 O2 + 2H2O

Streptococci vs. Staphylococci

Biochemical Tests are:

1- Catalase

2- Coagulase

3-DNase

CATALASE TEST

1. one drop of H2O2 on a clean slide2. using a cover slip, transfer 1 colony of

organism to H2O23. Observe air bubbles formation

Never use the loop

to transfer organism

COAGULASE TEST

A. Bound (slide) coagulase test:

1. One drop of saline on clean slide

2. Emulsify loopful of organism in saline

3. Add one drop of plasma

4. Mix rocking for 2 min

2. Coagulase test:Fibrinogen in plasma Fibrin

COAGULASE TEST

B. Free (tube) coagulase test:1. 1 ml pepton + 1 ml EDTA

plasma2. Emulsify loop full of organism3. Incubate 30 min x 37ºC4. If negative incubate at RTº overnight

2. Coagulase test:Fibrinogen in plasma Fibrin

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

DNASE TEST:

Staph. aureus

Opec zone

due to lack of DNA

DNA agar

precipitated by HCL

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

YOUR WORK FOR TODAY.

For staphylococcus aureus Prepare Gram Stain, show it

to the instructor.

1. Perform Catalase test.

2. Perform slide Coagulase test.

3. Perform DNA’s test.

4. Do subculture of the organism to:

Blood agar plate.

Mannitol Salt Agar.

o For coagulase –ve staph do Novobicin sensitivity

testing