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MATERIALS

1. Media used: Baird Parker Medium, Blood Agar, Mannitol Salt Agar, Cooked Meat
Medium (+10% salt), Gelatine Medium, Nutrient broth.
2. Rabbit Plasma
3. Food samples: milk, burger.

METHODS
1. Isolate and identified S.aureus from meat product.
(a) Enrichment technique
A pitch of burger or minced beef were transferred to a bottle of Cooked Meat Medium
(10% salt as enrichment to suppress other organisms). Incubated at 37C for 24 to 48
hours.
(b) Plating
After 24 to 48 hours of incubation, a loopful from the medium was transferred and
streak on Mannitol Salt Agar to obtain isolated colonies. Incubated at 37C for 24 to
48 hour and typical staphylococcal colonies were selected (positive colonies are
surrounded by yellow halos as against negative colonies which are surrounded by red
or purple zones).
(c) Confirmation
The positive colonies were picked out from the Mannitol Salt Agar and carried out the
following inoculations:
i. To B-hemolysis: Streaked on Blood Agar plates. Incubated at 37C for 24 to 48
hours.
ii. To detect the presence of gelatinolytic enzymes: Inoculated into Gelatine
Medium and incubated at 37C for 24 to 48 hours. After incubation, keep the
gelatin tube in a refrigerator for up to 4 hours. If the gelatine does not set by
that time it may indicate that the organism has produces the enzyme to
hydrolyse the gelatin and hence the gelatin would not solidify. On the other
hand, if the gelatin had set, this indicates that no enzymes was produced.
iii. To detect the presence of coagulase: positive colonies were inoculated into
Nutrient broth and incubated at 37C for 24 to 48 hours. After 24 to 48 hours,
0.2 mL of bacteria suspension was taken and added to 0.5mL of coagulase
plasma with EDTA tubes. Incubated in a 37C water bath and examined
periodically for clot formation. S. aureus should clot the plasma within 4 hours.
2. Enumeration of S.aureus from milk sample.
(a) 1 mL of milk sample was pipette and serial dilutions were made up to 1:10.
(b) From each diluent above, 0.1 mL was delivered into respective Baird Parker plates.
Spread out evenly on the agar surface with a glass rod. Allowed to settle for about 15
minutes and incubated at 37C for 24 to 48 hours.
(c) After incubation, the development of jet black circular convex colonies of 1.0 to 1.5
mM diameter surrounded by a zone of precipitation (or zone of opacity) was observed.
Count the colony forming units of S. aureus.