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Code: SR0122

Directions
Reconstitute one vial  as directed, aseptically add the contents to 90 ml of sterile Baird-Parker Agar Base (RPF) CM0961 cooled to 48°C. Mix well and use immediately.

Description
Rabbit Plasma Fibrinogen Agar (RPF Agar) is based on the formulation described by Beckers et al¹. This medium is a modification of Baird-Parker Medium and is recommended for the selective isolation, enumeration and confirmation of Staphylococcus aureus from food and other specimens².
The RPF Agar formulation retains the Baird-Parker Agar Base which has been specifically formulated to resuscitate injured cells³. This medium differs from Baird-Parker Medium in that the egg yolk emulsion has been replaced by fibrinogen, rabbit plasma and trypsin inhibitor. The fibrinogen was added to enhance the coagulase reaction in the RPF Agar⁴. Rabbit plasma was selected and it was found to be more specific for the coagulase activity when compared to other sources of plasma¹. Trypsin inhibitor was added to prevent fibrinolysis.
RPF supplement is a modification of the original formulation where potassium tellurite content has been reduced four-fold, i.e. from 0.01% to 0.0025% w/v. This reduction was necessary as it was discovered in the Oxoid laboratories that some strains of S. aureus were sensitive to potassium tellurite when used at 0.01% w/v in RPF Agar⁵. This modification of RPF Agar was found to give comparable growth and selectivity to that achieved on Baird-Parker Medium. The improved productivity of RPF Agar has also been confirmed by other laboratories^6,7. The reduction in potassium tellurite concentration in RPF Agar results in S. aureus forming white or grey or black colonies, surrounded by an opaque halo of precipitation, i.e. the coagulase reaction.

Technique
Surface Inoculation Method
1. Prepare the RPF Agar plates as directed.
2. Process the food sample in a stomacher or Waring blender using the recommended sample size and diluent.
3. Separate plates are inoculated with 0.1 ml of the prepared samples and subsequent dilutions as required.
4. Incubate at 35°C and examine after 24 and 48 hours incubation.
5. Count all the colonies that have an opaque halo of precipitation around them. Do not limit the count to black colonies.
6. Report as number of coagulase positive staphylococci isolated per gram of food.

Pour Plate Method
1. Prepare the RPF Agar as directed and hold at 48°C.
2. Process the food sample in a stomacher or Waring blender using the recommended sample size and diluent.
3. Add 1ml of the prepared sample (initial suspension and subsequent decimal dilution) into each sterile Petri dish.
4. Aseptically add 20 ml of sterile RPF Agar and mix gently.
5. Incubate at 35°C and examine after 24 to 48 hours.
6. Count all the colonies surrounded by an opaque halo of precipitation.
7. Report as number of coagulase positive staphylococci isolated per gram of food.

Precautions
Colonies of some contaminating organisms growing in close proximity to the coagulase positive colonies may partially digest the coagulase halo reaction.

References
1. Beckers H. J., van Leusden F. M., Hogeboom W. M. and Delfgon-van Asch E. H. M. (1980) (English summary) De Ware(n)-Chemicals 10. 125-130.
2. Beckers H. J., van Leusden F. M., Bindshedler O. and Guerraz D. (1984) Can. J. Microbiol. 30. 470-474.
3. Baird-Parker A. C. (1962) J. Appl. Bacteriol. 25. 12-19.
4. Hauschild A. H. W., Park C. E. and Hilsheimer R. (1979) Can. J. Microbiol. 25. 1052-1057.
5. Sawhney D. (1986) J. Appl. Bact. 61. 149-155.
6. Beckers H. J. (1985) Personal Communication.
7. van Schothorst M. (1985) Personal Communication.