1. STAPHYLOCICCI Basmah almaarik
Lecture # 3

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

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

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

5. Hemolysis 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
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

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

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

8. Catalse: 2H2O2  O2 + 2H2O Streptococci vs. Staphylococci
Biochemical Tests
Biochemical Tests are:
1- Catalase
2- Coagulase
3-DNase
Catalse:
2H2O2  O2 + 2H2O
Streptococci vs. Staphylococci

9. Never use the loop to transfer organism
Catalase test
one drop of H2O2 on a clean slide
using a cover slip, transfer 1 colony of organism to H2O2
Observe air bubbles formation
Never use the loop to transfer organism

10. Coagulase Test A. Bound (slide) coagulase test:
Fibrinogen in plasma  Fibrin
A. Bound (slide) coagulase test:
One drop of saline on clean slide
Emulsify loopful of organism in saline
Add one drop of plasma
Mix rocking for 2 min

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

12. 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.

13. Biochemical Tests DNase Test: 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

14. DNase Test: DNA agar precipitated by HCL Staph. aureus
Opec zone due to lack of DNA

15. 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.

16. Coagulase Negative Staphylococci
Novobiocin Disk:
Staphylococcus saprophyticus  Resistant
Staphylococcus epidermidis  Sensitive

17. Your Work For Today.
For staphylococcus aureus Prepare Gram Stain, show it to the instructor.
Perform Catalase test.
Perform slide Coagulase test.
Perform DNA’s test.
Do subculture of the organism to:
Blood agar plate.
Mannitol Salt Agar.
For coagulase –ve staph do Novobicin sensitivity testing