1. STAPHYLOCICCI Lecture # 3

2. Staphylococcus sp.  Morphology:  Gram positive cocci.  In clusters  Culture:  Facultative anaerobes  Incubation 37ºC x 24 hr

3. Staphylococcus sp.  Coagulase +ve:  Staphylococcus aureus.  Coagulase –ve:  Staphylococcus saprophyticus.  Staphylococcus epidermidis. All Staphylococci are CATALASE +VE

4. CULTURE  Blood Agar :  Creamy colonies 1-2 mm  Beta-heamolysis  Blood Agar:  Grayish white colonies  No heamolysis Coagulase +ve Staphylococcus aureus Coagulase -ve Staphylococci

5. CULTURE  MacConkey Agar:  Pink colonies (lactose fermenting)  MacConkey Agar:  Only some are give Pink colonies (lactose fermenting) Coagulase +ve Staphylococcus aureus Coagulase -ve Staphylococci

6. 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 aureus Coagulase -ve Staphylococci Differential: Staph aureus can frement mannitol and give diffused yellow color Selective: Containing very high salt conc only staph can grow

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

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

9. Coagulase Test Coagulase PositiveCoagulase Negative

10. Coagulase Test B. Bound (slide) coagulase: 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

11. 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.

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

13. 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.

14. Coagulase Negative Staphylococci  Novobiocin Disk:  Staphylococcus saprophyticus  Resistant  Staphylococcus epidermidis  Sensitive

15. Micrococcus  Gram Positive Cocci in pairs and tetrads.  Catalase +ve.  Generally harmless, but can cause disease in immunocompromised host.

16. Micrococcus  Blood agar or chocolate agar: Small, non-hemolytic, variably pigmented (white, orange, yellow pink) colonies.  MacConkey: no growth.

17. 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.