Detection of Staphylococcus aureus

1. APPARATUS

1. Autoclave
2. BOD - Incubator
3. Bacteriological Incubator
4. pH – meter
5. Hot air Oven
6. Laminar Air flow
7. Analytical Balance
8. Spirit Lamp / Bunsen Burner
9. Water Bath
10. Micropipette
11. Petri dishes
12. Test tubes
13. Media bottles   

2.   REAGENTS   AND  MEDIAS

1. Soyabean Casein Digest Medium
2. Mannitol - Salt Agar Medium
3. Vogel - Johnson Agar Medium
4. Baird - Parker Agar Medium

Prepare media (2.1 – 2.4 )as per the current version of Respective GTP.

All the above media should be incubated for 48 h at 32.5⁰C ± 2.5⁰C before use.  Any   contaminated media should be discarded.

1. PROCEDURE    
   1. Pre-Treatment Of The Preparation Being Examined
      1. Water - Soluble Products :
         1. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 ml with the same medium
         2. If necessary, adjust the pH to about 7.0 ± 0.2.
   2.  Enrichment
      1. Take 10 ml or 10 g, the quantity corresponding to 1.0 g or 1.0 ml (Else as per the specification) of the pretreated sample in a suitable vessel, add a volume of Fluid Soyabean-Casein digest medium to make 100 ml and incubate at 32.5⁰C±2.5⁰C for 24 to 48 hours.
      2. Examine the medium for growth, and if growth is present, mix by gently shaking.
      3. Use an inoculating loop to streak a portion of the medium on the surface of Vogel-Johnson agar medium (or Baird-Parker agar medium, or Mannitol-Salt agar medium), on petridishes.
      4. Cover and invert the dishes and incubate at 32.5⁰C ± 2.5⁰C for 24 to 48 hours.
      5. If, upon examination, none of the plates contains colonies having the characteristics listed in Table I for the media used, the test specimen meets the requirements for freedom from Staphylococcus aureus.

Table –I

               Morphological Characteristics of Staphylococcus aureus on Selective Agar Media

1. Do the gram staining identification of suspected colonies and with the aid of an inoculation loop, transfer representative suspected colonies from agar surfaces of Vogel-Johnson agar medium (or Baird-Parker agar medium, or Mannitol-Salt agar medium) to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without suitable additives.
2. Incubate in a water bath at 37±1^OC, examining the tubes after 3 hrs and subsequently at suitable intervals up to 24 hrs.
3. Test positive and negative controls simultaneously with the unknown specimens.
4. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus.
5. Note :
   1. Carry out a control test by repeating the test, adding the prescribed quantity  and a volume of broth containing 10 to 50 cells / colonies of Staphylococcus aureus (ATCC 6538; NCTC 10788), prepared from a 24 h. culture in fluid soybean-casein digest medium, to a sterile 100 ml of fluid soybean-casein digest medium.
   2. The test is invalid if the results do not indicate that the control contains Staphylococcus aureus.         

Staphylococcus aureus, bacteriological incubator, analytical balance