BAIRD-PARKER RPF AGAR

Transcription

1 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

2 RECONSTITUTION OF THE SUPPLEMENTS Rabbit Plasma Fibrinogen Supplement for 90 ml base: reagent R2 from RPF kit BT005 - 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 not 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 190 ml base: reagent R2 from RPF kit BT010 - Using aseptic techniques, add 10 ml of sterile distilled water preheated to 37 C at least (44 C max). - 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. INSTRUCTIONS FOR USE - Melt the medium for the minimum amount of time necessary in order to achieve total liquefaction RPF kit BT005 or BT010, reagent R1). - Cool and maintain at C. - For 90 ml of base media (reagent R1 from RPF kit BT005), add 10 ml of reconstituted Rabbit Plasma Fibrinogen Supplement (reagent R2 from RPF kit BT005). - For 190 ml of base media (reagent R1 from RPF kit BT010), add 10 ml of reconstituted Rabbit Plasma Fibrinogen Supplement (reagent R2 from RPF kit BT010). - 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 , NF V , notably) authorize an alternative surface inoculation. - Incubate at 37 C for 24 and 48 hours. 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 10 to 15 ml of complete medium. - Let solidify on a cool surface. - Transfer the number of characteristic colonies according to the recommendations of the standard XP T , or transfer the membrane from Mannitol Salt agar (BK030 or BM085) or from Baird Parker + Egg Yolk Tellurite [(BK055 + BS060), BM018 or BM091]. - Incubate at 36 ± 2 C for 21 ± 3 hours. 2/6

3 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 1 L of complete medium : (can be adjusted to obtained optimal performance) - Tryptone g - Meat extract g - Yeast extract g - Sodium pyruvate g - Glycine g - Lithium chloride g - Bacteriological agar g - Rabbit plasma, EDTA ml - Bovine fibrinogen g - Trypsin inhibitor mg - Potassium tellurite mg ph of the complete medium at 25 C: 7.2 ± 0.2. QUALITY CONTROL - 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 Staphylococcus epidermidis ATCC Escherichia coli ATCC P R 50% P R 50% limited, score 0-1 inhibited, score 0 presence presence absence 3/6

4 - Typical cultural response after 44 hours of incubation at 36 C (XP T ; NF T ) Microorganisms Growth Characteristics (Productivity Ratio : R3) Colony color Fibrin halo Staphylococcus aureus CIP Enterococcus faecalis CCM % R 3 150% inhibited presence STORAGE / SHELF LIFE Ready-to-melt media in vials : - Store between 2-8 C, shielded from light. - The expiration date is indicated on the label. PACKAGING Code RPF kit : - Composed of 6 x 90 ml vials of base media (R1) and BT freeze-dried RPF supplement vials (R2) - Composed of 6 x 190 ml vials of base media (R1) and BT freeze-dried RPF supplement vials (R2) 4/6

5 PHOTO SUPPORT Product Reference : BT00508, BT01008 Media used for : Detection / enumeration / confirmation of coagulase positive Staphylococci. Staphylococcus aureus Baird Parker RPF agar Ref : BT00508 Incubation : 24 hours / 37 C Characteristics : Coagulase positive staphylococci are gray to surrounded by a halo of fibrin. 5/6

6 BIBLIOGRAPHY LOEB, L The influence of certain bacteria on the coagulation of blood. Journal of Medical Research, 10 : DUTHIE, E.S Evidence of two forms of staphylococcal coagulase. Journal of General Microbiology, 10 : BAIRD-PARKER, A.C An improved diagnostic and selective medium for isolating coagulase positive staphylococci. Journal of Applied Bacteriology, 25 : DEVOYOD, J.J., MILLET, L. et MOCQUOT G 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) : STADHOUDERS, J., HASSING, F. and van AALST-van MAREN, N.O 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 : HAUSCHILD, A.H., PARK, C.E. and HILSHEIMER, R A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods. Canadian Journal of Microbiology, 25 : BECKERS, H.J., van LEUSDEN, F.M., BINDSCHEDLER, O. and GUERRAZ, D 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 : SAWHNEY, D The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar. Journal of Applied Bacteriology, 149 : FIL provisoire 145A. Novembre 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 Comparison of selective culture media to enumerate coagulase-positive staphylococci in cheeses made from raw milk. Food microbiology, 15 : NF EN ISO (V ). Octobre 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 /A1 (V /A1). Décembre 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 (V ). Janvier 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 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 (V ). 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 Juin Qualité de l eau. Recherche et dénombrement des staphylocoques pathogènes. Méthode par filtration sur membrane. NF T Août 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 They are susceptible to modification at any time, without warning. Code document : BT005 RPF/E/ : 1. 6/6