IDENTIFICATION OF STAPHYLOCOCCUS AUREUS

OBJECTIVE
The test for Staphylococcus aureus is provided to determine compliance with the requirements given in individual monograph/specifications,.
PRINCIPLE
Identification of Staphylococcus aureus is the detection of a pathogenic bacteria which causes infection in human body. It can be identified by using specific, differential media which support growth of only Staphylococcus aureus.
REAGENT AND MEDIA
Fluid Soybean - Casein Digest Medium
Pancreatic digest of Casein 17.0 g
Papai 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 of between 7.1 and 7.5. Filter, if necessary, to clarify. Distribute into suitable containers and sterilize in an autoclave at 121 OC for about 15 min.
Mannitol - Salt Agar Medium
Pancreatic digest of Casein 5.0 g
Peptic digest of animal tissue 5.0 g
Beef Extract 1.0 g
D-Mannitol 10.0 g
Sodium Chloride 75.0 g
Agar 15.0 g
Phenol Red 0.025 g
Water 1000 mL
Final pH after sterilization 7.4 ± 0.2
Mix, then heat with frequent agitation, and boil for 1 min. to effect solution.
Vogel - Johnson Agar Medium
Pancreatic Digest Casein 10.0 g
Yeast extract 5.0 g
Mannitol 10.0 g
Dibasic Potassium Phosphate 5.0 g
Lithium Chloride 5.0 g
Glycine 10.0 g
Agar 16.0 g
Phenol Red 25.0 mg
Water 1000 mL
Boil the solution of solids for 1 min. sterilize, cool to between 45 - 50 OC and add 20 mL of sterile Potassium tellurite solution (1 in 100)
Baird - Parker Agar Medium
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 OC for 15 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 24 h. at 37 OC before use. Any contaminated media should be discarded Instead of preparing media, one can also use dehydrated media of Hi media / Difco and rehydrate the required quantity as per instruction son the bottle label, dispense in required quantities and sterilize at 15 psi at 121 OC for 15 min.
PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
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
Non-Fatty Products Insoluble in Water:
1. Suspend 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 dilute to 100 mL with the same medium.
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3. A suitable surface-active agent such as 0.1 % w/v of Polysorbate 80 may be added to assist the suspension of poorly wettable substances.
4. If necessary, adjust the pH of the suspension to about 7.
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80.
2. If necessary, heat to not more than 40 OC
3. Mix carefully while maintaining the temperature in a water bath or in an oven.
4. Add 85 mL of Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not more than 40 OC, if necessary,
5. Maintain this temperature for the shortest necessary for formation of an emulsion and in any case for not more than 30 min.
6 If necessary, adjust the pH of the emulsion to about 7.0
For Fluid Specimen in Aerosol Form:
1. Chill the container in an Alcohol-dry ice mixture for approx. 1 h., cut open the container allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible and transfer the quantity of test material required for the procedures specified in one of the two proceeding paragraphs, as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residues.
PROCEDURE
As per IP’95
Primary Test
1. Place the prescribed quantity in a sterile screw-capped jar containing g 100 mL of fluid soybean-casein digest medium and incubate at 35 - 37 OC for 24 - 48 h.
2. Subculture on a plate containing a layer of Mannitol salt agar medium or Vogen-Johnson agar medium and incubate at 35 - 37 OC for 18 - 24 h.
3. Examine the resulting growth by Gram’s staining and apply the coagulase test. Gram-positive cocci (in cluster) in yellow colonies (in Mannitol salt agar medium) and in colonies, black surrounded by yellow zones (in Vogen - Johnson agar medium), and giving an positive coagulase test indicate the presence of Staphylococcus aureus.
Confirmatory Test :
Coagulase Test:
1. Transfer representative colonies from the agar surfaces of the Mannitol salt agar medium or Vogel-Johnson agar medium to individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37 OC examine the tubes after 24 h.
2. If coagulation in any degree is observed, the test is positive.
As per BP
Primary Test:
1. Inoculate 100 mL of casein digest broth with a quantity of the solution, suspension or emulsions thus obtained containing 1 g or 1 mL of the preparation being examined, after pretreatment as described above.
2. Mix and incubate at 35 - 37 OC for 24 - 48 h.
3. Subculture on a suitable medium such as Baird-Parker agar.
4. Incubate at 35 - 37 OC for 24 - 48 h.
5. If no growth of micro-organisms is detected, the preparation being examined passes the test.
6. Black colonies of Gram-positive cocci often surrounded by clean zones may indicate the presence of Staphylococcus aureus.
Confirmatory Test:
1. For catalase-positive cocci, confirmation may be obtained, for example, by coagulase and deoxyribonuclease tests.
2. The preparation being examined passes the test if cultures of the type described do not appear or if the Confirmatory biochemical tests are negative.
As per USP
Primary Test
1. To the specimen ad Fluid Soybean-Casein digest medium to make 100 mL mix, and incubate.
2. Examine the medium for growth and if growth is present, 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.
3. Cover and invert the dishes and incubate.
4. 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.
Confirmatory Test
Coagulase Test
1 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 OC, examining the tubes after 3 h. and subsequently at suitable intervals up to 24 h.
3. Test positive e 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.
TABLE I
Morphological Characteristics of Staphylococcus aureus on Selective Agar Media


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 screw-capped jar containing 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.
3. Use the method as per the pharmacopoeial status (grade) of the material. In case of In-house specifications, follow the method as per IP or as specified.