BAIRD-PARKER RPF AGAR INTENDED USE Baird Parker RPF (RPF = Rabbit Plasma Fibrinogen) Agar is used for the direct detection and enumeration of coagulase positive staphylococci. The medium has the advantage of considerably reducing the number of confirmation tests for the presence of coagulase positive staphylococci particularly when atypical colonies are observed on other selective media. The medium allows the simultaneous enumeration and confirmation to be performed in a single operation. HISTORY The production of free coagulase considered to be the principal characteristic for recognizing the pathogenicity of staphylococci, in particular Staphylococcus aureus, was at the origins of the development of Baird Parker with Rabbit Plasma Fibrinogen. Initial formulations of the incorporation of plasma in solid culture media revealed inconsistencies between the results obtained by the coagulase tube test and the formation of a characteristic halo of fibrin around colonies. The differences arose from the fact that certain strains possessed a coagulase that also activated the plasminogen-plasmin system as well, resulting in fibrinolysis and the disappearance of the fibrin halo. In order to overcome this difficulty, it was recommended to add soybean trypsin inhibitor. In order to favor the detection of coagulase produced by Staphylococcus aureus, Devoyod et al. studied in 1976, the incorporation of pork plasma into Baird-Parker medium. Hauschild subsequently improved the performance of Devoyod s medium through the inclusion of bovine fibrinogen and a trypsin inhibitor, with a correlative decrease in the amount of plasma. In 1983, Beckers et al. modified Hauschild's medium, replacing the pork plasma with rabbit plasma, classically used in the coagulase tube test. These authors also inoculated the medium in depth, rather than the previously used double layer technique of Devoyod. Finally, the formulation was improved in 1986 by Sawhney, after completing studies relative to the toxicity of potassium tellurite towards Staphylococcus aureus in a rabbit plasma fibrinogen medium. PRINCIPLES - The growth of staphylococci is favored by sodium pyruvate and glycine. - Accompanying microflora is inhibited by lithium chloride and potassium tellurite (added extemporaneously), as well as a high concentration of glycine. - Rabbit plasma was chosen for its excellent specificity towards staphylococcal coagulase and by its aptitude to rapidly produce clot formation by forming staphylothrombin from prothrombin. The rabbit plasma is reinforced with bovine fibrinogen. Staphylothrombin acts by cutting the A and B fibrinopeptides of fibrinogen, thereby initiating the polymerization process that results in the appearance of fibrin halos surrounding the colonies. - Soybean trypsin inhibitor prevents fibrinolysis. - The coloration of staphylococcal colonies is due to the reduction of potassium tellurite to telluride. In addition, the presence of tellurite favors the inhibition of contaminating Gram-positive microflora. 1/6
PREPARATION OF BASE MEDIUM - Suspend 54.9 g of dehydrated base medium (BK055) in 900 ml of distilled or deionized water. - Slowly bring to boiling, stirring with constant agitation until complete dissolution. - Dispense in flasks by adding 90 ml per flask, if to be used with Rabbit Plasma Fibrinogen Supplement for 100 ml, BS034 (it is also possible to use the RPF Supplement BS038). Or For large volume preparation, it is also possible to dispense into 1000 ml capacity flasks by adding 450 ml of medium per flask, if the RPF Supplement for 500 ml is chosen (BS038). - Sterilize in an autoclave at 121 C for 15 minutes. NOTE : Incomplete agar melting during preparation will invariably lead to significant inconsistency in the gel strength of the solidified agar, after sterilization and cooling. RECONSTITUTION OF THE SUPPLEMENTS Rabbit Plasma Fibrinogen Supplement for 90 ml base: BS034 - Using aseptic techniques, add 10 ml of sterile distilled water at room temperature. The dissolution may be facilitated by using sterile distilled water preheated to at least 37 C but no more than 44 C. - Turn end-over-end to dissolve. Avoid frothing the solution. Immediate dissolution is not always obtained. To accelerate the process, a mechanical agitator (vortex) for tubes can be used to fully dissolve the lyophilisate. The supplement should be completely dissolved before adding to Baird- Parker Agar base. Rabbit Plasma Fibrinogen Supplement for 450 ml base: BS038 - Using aseptic techniques, fill the vial of lyophilisate with 50 ml of sterile distilled water, preheated to 37 C but not more than 44 C. - Turn end-over-end to dissolve. Avoid frothing the solution. The supplement should be completely dissolved before adding to Baird-Parker Agar base. INSTRUCTIONS FOR USE - Melt the medium for the minimum amount of time necessary in order to achieve total liquefaction Cool and maintain at 44-47 C. - For 90 ml of base media, add 10 ml of reconstituted Rabbit Plasma Fibrinogen Supplement (BS034). - For 450 ml of base media, add 50 ml of the reconstituted Rabbit Plasma Fibrinogen Supplement (BS038). - Mix well to insure a proper and complete homogenization. Use immediately. Enumeration of coagulase positive staphylococci in foods and animal feeding stuffs: - Transfer 1 ml of the sample to analyze and its tenfold dilution series into sterile Petri dishes. - Pour 10 to 15 ml of complete medium. - Homogenize by swirling. - Let solidify on a cool surface. - It should be noted that the instructions for certain standards (NF EN ISO 6888-3, NF V 08-057-1, notably) authorize an alternative surface inoculation. - Incubate at 37 C for 24 and 48 hours. 2/6
Enumeration of pathogenic staphylococci for the bacteriological examination of waters, using membrane filtration method without confirmation: - Pour 10 to 15 ml of complete medium. - Let solidify on a cool surface. - Place the membrane, filtered side up, on the surface of the agar, insuring a complete contact. - Return the plate and incubate at 36 ± 2 C for 21 ± 3 hours and 44 ± 4 hours. The membrane quality should be validated prior to initiating this application. Confirmation of pathogenic staphylococci for the bacteriological examination of waters. - Pour 18 ml of complete medium. - Let solidify on a cool surface. - Re-isolate the number of characteristic colonies according to the recommendations of the standard XP T 90-412, or transfer the membrane from Baird Parker Egg Yolk Tellurite medium [(BK055 + BS060), BM018 or BM091]. - Incubate at 36 ± 2 C for 21 ± 3 hours. RESULTS Coagulase positive staphylococci are characterized by the formation of gray or colonies surrounded by an opaque halo of fibrin that is clearcut, stable and well visible. Count only those plates containing less than 100 characteristic colonies. Use of the RPF technique eliminates the need for confirming the results by the coagulase tube test. For water testing controls, it is necessary to gently raise the membrane in order to verify the presence (or not) of fibrin halos at 21 ± 3 hours of incubation. TYPICAL COMPOSITIONS For 950 ml of base medium : (can be adjusted to obtained optimal performance) - Tryptone...10.0 g - Meat extract...5.0 g - Yeast extract...1.0 g - Sodium pyruvate...10.0 g - Glycine...12.0 g - Lithium chloride...5.0 g - Bacteriological agar...15.0 g For a supplement vial to be added to 90 ml 1 or 450 ml 2 of base media - Rabbit plasma, EDTA... 2.5 ml 1... 12.5 ml 2 - Bovine fibrinogen... 0.5 g 1... 2.5 g 2 - Trypsin inhibitor... 2.5 mg 1... 12.5 mg 2 - Potassium tellurite... 2.5 mg 1... 12.5 mg 2 1 Supplement BS03408 2 Supplement BS03808 ph of the complete medium at 25 C: 7.2 ± 0.2. 3/6
QUALITY CONTROL - Dehydrated base medium: cream-white powder, free-flowing and homogeneous. - Appearance : white to pinkish lyophilisate, giving an amber and opalescent solution after reconstitution. - Complete prepared medium : amber agar. - Typical culture response after 48 hours of incubation at 37 C : Microorganisms Growth (Productivity Ratio : P R ) Characteristics Colony color Fibrin halo Staphylococcus aureus DSM 799 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 12228 Escherichia coli ATCC 25922 P R 50% P R 50% limited, score 0-1 inhibited, score 0 presence presence absence - Typical cultural response after 44 hours of incubation at 36 C (XP T 90-412 ; NF T 90-461) Microorganisms Characteristics Growth (Productivity Ratio : R 3 ) Colony color Fibrin halo Staphylococcus aureus CIP 53.154 Enterococcus faecalis CCM 2541 66% R 3 150% inhibited presence STORAGE / SHELF LIFE Dehydrated base medium : 2-30 C. - The expiration date is indicated on the label. Prepared medium from dehydrated base (benchmark value*) : - Base in flasks : 6 months at 2-8 C. - Complete media in plates : 15 days at 2-8 C. Rabbit Plasma Fibrinogen Supplement : - Store between 2-8 C, shielded from light. - The expiration dates are indicated on the labels. - Once reconstituted, the product must not be stored for more than 8 days at 2-8 C. Do not freeze. PACKAGING Code Dehydrated base medium : - 500 g bottle BK055HA - 5 kg drum BK055GC RPF Supplement : - 8 vial pack for 100 ml complete media / vial BS03408-1 vial pack for 500 ml complete media / vial BS03808 4/6
PHOTO SUPPORT Product Reference : BK055HA + BS03408 or BS03808 Media used for : Detection / enumeration / confirmation of coagulase positive Staphylococci. Staphylococcus aureus Baird Parker RPF agar Ref : BK055HA + BS03408 Incubation : 24 hours / 37 C Characteristics : Coagulase positive staphylococci are gray to surrounded by a halo of fibrin. 5/6
BIBLIOGRAPHY LOEB, L.. 1903. The influence of certain bacteria on the coagulation of blood. Journal of Medical Research, 10 : 407-419. DUTHIE, E.S.. 1954. Evidence of two forms of staphylococcal coagulase. Journal of General Microbiology, 10 : 427-436. BAIRD-PARKER, A.C.. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. Journal of Applied Bacteriology, 25 : 12-19. DEVOYOD, J.J., MILLET, L. et MOCQUOT G.. 1976. Un milieu gélosé pour le dénombrement direct de Staphylococcus aureus : milieu au plasma de porc pour S. aureus (PPSA). Canadian Journal of Microbiology, 22 (11) : 1603-1611. STADHOUDERS, J., HASSING, F. and van AALST-van MAREN, N.O.. 1976. A pour-plate method for the detection and enumeration of coagulase-positive Staphylococcus aureus in the Baird-Parker medium without egg yolk. Netherland Milk Dairy Journal, 30 : 222-229. HAUSCHILD, A.H., PARK, C.E. and HILSHEIMER, R.. 1979. A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods. Canadian Journal of Microbiology, 25 : 1052-1057. BECKERS, H.J., van LEUSDEN, F.M., BINDSCHEDLER, O. and GUERRAZ, D.. 1984. Evaluation of a pour-plate system with a rabbit plasma-bovine fibrinogen agar for the enumeration of Staphylococcus aureus in food. Canadian Journal of Microbiology, 30 : 470-474. SAWHNEY, D.. 1986. The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar. Journal of Applied Bacteriology, 149 : 149-155. FIL provisoire 145A. Novembre 1997. Lait et produits à base de lait. Dénombrement des staphylocoques coagulase-positifs. Techniques de comptage des colonies. De BUYSER, M.L., AUDINET, N., DELBART, M.O., MAIRE, M. and FRANCOISE, F.. 1998. Comparison of selective culture media to enumerate coagulase-positive staphylococci in cheeses made from raw milk. Food microbiology, 15 : 339-346. NF EN ISO 6888-2 (V 08-014-2). Octobre 1999. Microbiologie des aliments. Méthode horizontale pour le dénombrement des staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 2 : Technique utilisant le milieu gélosé au plasma de lapin et au fibrinogène. NF EN ISO 6888-2/A1 (V 08-014-2/A1). Décembre 2003. Microbiologie des aliments. Méthode horizontale pour le dénombrement des staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 2 : Technique utilisant le milieu gélosé au plasma de lapin et au fibrinogène. Amendement 1 : Inclusion des données de fidélité. XP CEN ISO/TS 11133-2 (V 08-104-2). Janvier 2004. Microbiologie des aliments. Guide pour la préparation et la production des milieux de culture. Partie 2 : Guide général pour les essais de performance des milieux de culture. NF V 08-057-1. Janvier 2004 (2 e tirage de Décembre 2004). Microbiologie des aliments. Méthode de routine pour le dénombrement des staphylocoques à coagulase positive par comptage des colonies à 37 C. Partie 1 : Technique avec confirmation des colonies. NF EN ISO 6888-3 (V 08-014-3). Juin 2003 (3 e tirage d Avril 2005). Microbiologie des aliments. Méthode horizontale pour le dénombrement des staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 3 : Recherche et méthode NPP pour les faibles nombres. XP T 90-412. Juin 2006. Qualité de l eau. Recherche et dénombrement des staphylocoques pathogènes. Méthode par filtration sur membrane. NF T 90-421. Août 2006. Essais des eaux. Examens bactériologiques des eaux de piscines. *Benchmark value refers to the expected shelf life when prepared under standard laboratory conditions following manufacturer s instructions. It is provided as a guide only and no warranty, implied or otherwise is associated with this information. The information provided on the package take precedence over the formulations or instructions described in this document. The information and specifications contained in this technical data sheet date from 2011-04-18. They are susceptible to modification at any time, without warning. Code document : BK055 RPF/E/2003-01 : 10. 6/6