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RPF SUPPLEMENT
Code: SR0122
A selective and diagnostic supplement for the isolation, enumeration and confirmation of Staphylococcus aureus from food and other specimens. When used with Baird-Parker Agar Base (RPF) CM0961
Vial contents (each vial is sufficient for 100ml of medium) |
|
Fibrinogen |
0.375 g |
Rabbit plasma |
2.5 ml |
Trypsin inhibitor |
2.5 mg |
Potassium tellurite |
2.5 mg |
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differs from Baird-Parker Medium in that the egg yolk emulsion has been replaced
by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 laboratories6,7. 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.