1. APPARATUS
1.1. Autoclave
1.2. BOD - Incubator
1.3. Bacteriological Incubator
1.4. pH – meter
1.5. Hot air Oven
1.6. Laminar Air flow
1.7. Analytical Balance
1.8. Spirit Lamp / Bunsen Burner
1.9. Water Bath
1.10. Micropipette
1.11. Petri dishes
1.12. Test tubes
1.13. Media bottles
2. REAGENTS AND MEDIAS
2.1 Soyabean Casein Digest Medium
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Distilled Water 1000 ml
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after Sterilization between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121–124OC for about 30 min.
2.2 Baird - Parker Agar Medium
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic digest of Casein 10.0 g
Beef Extract 5.0 g
Yeast Extract 1.0 g
Lithium Chloride 5.0 g
Agar 20.0 g
Glycine 12.0 g
Sodium Pyruvate 10.0 g
Water 950 ml
pH after sterilization 7.2 ± 0.2
Heat to boiling for 1 min., shaking frequently. Adjust the pH so that after sterilization it is 6.6 - 7.0. Sterilize by heating in an autoclave at 121–124OC for 30 min., cool to 45 - 50 OC and add 10 ml of sterile 1 % w/v solution Potassium tellurite and 50 ml of egg yolk emulsion.
All the above media should be incubated for 48 h at 32.50C ? 2.50C before use. Any contaminated media should be discarded.
3. PROCEDURE
3.1. PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
3.1.1. Water - Soluble Products :
3.1.1.1. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Soyabean casein digest medium 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
3.1.1.2. If necessary, adjust the pH to about 7.0 ± 0.2.
3.2. Enrichment
3.2.1. 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 300C to 350C for 18 to 48 hours.
3.2.2. Examine the medium for growth, and if growth is present, mix by gently shaking.
3.3. Primary Test
3.3.1. Use an inoculating loop to streak a portion of the medium on the surface of Baird-Parker agar medium petridishes.
3.3.2. Cover and invert the dishes and incubate at 300C to 350C for 18 to 72 hours.
3.3.3. 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 Baired parker Agar
Characteristic colonial Morphology Gram Stain
Black, Shiny surrounded by clear zones 2-5 mm
Positive Cocci
(in clusters)
3.4. Confirmatory Test (Coagulase test )
3.4.1. With the aid of an inoculation loop, transfer representative suspected colonies from agar surfaces of Baird-Parker agar medium to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without suitable additives.
3.4.2. Incubate in a water bath at 37±1OC, examining the tubes after 3 hrs and subsequently at suitable intervals up to 24 hrs.
3.4.3. Test positive and negative controls simultaneously with the unknown specimens.
3.4.4. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus.
3.5. Note :
3.5.1. 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.
3.5.2. The test is invalid if the results do not indicate that the control contains Staphylococcus aureus.
Autoclave, general testing procedure, laminar air flow, bod - incubator
