STAPHYLOCICCI Basmah almaarik Lecture # 3

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

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

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

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

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

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)

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

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

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

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

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 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

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

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. 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