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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
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