Staphylococcus aureus (S. aureus)

Staphylococcus aureus (S. aureus)

Gram Positive Bacteria
Media BPA

Outline:
Presumption by BPA
Confirmation by BHI, MSA, coagulase test and thermonulease test


Weigh 11g sample to 99ml of peptone water
Put into stomacher for 2 min
Do serial Dilution to achieve 10E-2 and 10E-3
Do spread plate on BPA
3 duplicate for each dilution
Incubate 35˚C for 48 hrs
Observe the colonies
Classify them into Typical, suspect, non typical
Do a colony count for typical and suspect
Pick 4 representative colony of typical colony
Divide BHI agar and MSA agar into 4 sections
Streak 4 colonies into the each of the section
Inoculate 2 colonies into 2 BHI broth (One colony for each broth)
Incubate BHI aerobically 35˚C for 24hr for BHI and MSA anaerobically
Pick 2 colony from BHI for coagulase test


Colonies in BPA will be black shinny convex colon with clear zone
Colonies in BHI will be Yellow round colony
Colonies in MSA will be yellow colonies with yellow zone


Positive coagulase test will show clotting
Thermonuclease test
From Step 13 , after incubation, put the BHI broth in water bath 100˚C for 15min
Divide Toludine Blue DNAase agar into half
Streak each tube (BHI broth)into the respective half
Positive colony will be pink colour in Thermonuclease test

Do the same for suspect colonies

If BHI, MSA , coagulase test and Thermonuclease test is positive, the colony count for typical is confirmed in BPA

If BHI, MSA , coagulase test and Thermonuclease test is positive, the colony count for suspect is confirmed in BPA.

Media Explanation

BPA (Baird-Parker Agar)
· Selective differential media
· LiCl and Potassium Tellurite suppresses non-osmophile
· Egg Yolk contains lecithin
· S.aureus produces lecithinase, therefore it will produce colony with clear zone

Blood-Brain-Infusion (BHI)
· Contains may micronutrients which will enriched S .aureus

MSA (Mannitol Salt Agar)
· Suppresses non-osmophile
· Differentiate non mannitol fermenters
· S. aureus ferments manitol carbohydrate to acid
· When pH decreases phenol red will turn yellow

Toludine Blue DNA agar
· Differentiate heat resistant DNAase producers (S.aureus) from heat resistant DNAase non producers
· Heating will isolates heat stable enzymes activity
· DNAase is produce, DNA hydrolysed into nucleotide
· Nucleotide reacts with toludine, a pdt which is blue colour