STAPHYLOCICCI BASMAH ALMAARIK Lecture 3 Morphology Gram positive

STAPHYLOCICCI BASMAH ALMAARIK Lecture # 3

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

� Coagulase +ve: � Staphylococcus aureus. � Coagulase –ve: � Staphylococcus saprophyticus. � Staphylococcus epidermidis. All Staphylococci are CATALASE +VE

� Coagulase +ve � Staphylococcus aureus � Blood Agar: � Creamy colonies 1 -2 mm � Beta-heamolysis � Coagulase -ve � Staphylococci � Blood Agar: � Grayish white colonies � No heamolysis

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

� Coagulase +ve � Staphylococcus aureus � � Coagulase -ve � Staphylococci Mannitol Salt Agar: � Growth � Change color from Red to Yellow (Mannitol fermenting) Differential: Staph aureus can ferment mannitol and give diffused yellow color � Mannitol Salt Agar: � Growth � No yellow color no fermentation. Selective: Containing very high salt conc only staph can grow

� Coagulase +ve � Staphylococcus aureus � Mac. Conkey �Pink Agar: colonies (lactose fermenting) � Coagulase -ve � Staphylococci � Mac. Conkey �Only Agar: some give Pink colonies (lactose fermenting)

Biochemical Tests are: 1 - Catalase 2 - Coagulase 3 -DNase 1. Catalse: � 2 H 2 O 2 + 2 H 2 O � Streptococci vs. Staphylococci

1. 2. 3. one drop of H 2 O 2 on a clean slide using a cover slip, transfer 1 colony of organism to H 2 O 2 Observe air bubbles formation Never use the loop to transfer organism

2. Coagulase test: Fibrinogen in plasma Fibrin 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 B. Free (tube) coagulase test: 1. 2. 3. 4. 1 ml pepton + 1 ml EDTA plasma Emulsify loop full of organism Incubate 30 min x 37ºC If negative incubate at RTº overnight

(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.

3. DNase Test: 1. 2. 3. 4. 5. 6. DNase enzyme hydrolyze DNA Spot-inoculate organism on a DNA containing media Incubate 37ºC x 24 hr Flood plate with 1 mol/ml HCL sol HCL will precipitate unhydrolyzed DNA Tip off the excess acid Clearing around colonies DNase positive strain No Clearing around colonies DNase negative strain

DNASE TEST: DNA agar precipitat ed by HCL Opec zone due to lack of DNA Staph. aureus

(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.

� Novobiocin Disk: � Staphylococcus saprophyticus Resistant epidermidis Sensitive

� 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: Ø Blood agar plate. Ø Mannitol Salt Agar. o For coagulase –ve staph do Novobicin sensitivity testing