BAIRD-PARKER RPF AGAR ready-to-use

Transcription

1 BAIRD-PARKER RPF AGAR ready-to-use 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 of coagulase positive staphylococci particularly when atypical colonies are observed on other selective media. The ready-to-use plates allow the confirmation of coagulase positive staphylococci as described in standards NF EN ISO and NF V The pre-poured media can also be used for the enumeration and confirmation of pathogenic staphylococci according to the standard XP T for water analysis. 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 of tellurite favors the inhibition of contaminating Gram-positive microflora. 1/5

2 INSTRUCTIONS FOR USE Confirmation of coagulase positive staphylococci in the context of samples destined for human or animal consumption, dry dairy products and environmental samples : - From tubes of Giolitti and Cantoni broth with Tween 80 (BK159, BM110/111) incubated under anaerobic conditions at 37 C, inoculate a loop (10 µl) of the growth culture onto a RPF plate. Enumeration of pathogenic staphylococci in water analysis, by membrane filtration : - Aseptically place the membrane on the surface of the agar, filtered side up and making sure that the membrane and agar are in close contact. - Incubate at 36 ± 2 C for a total of 44 ± 4 h. Due to the rare absorption of halos after 48 hours of incubation, it is necessary to perform a primary reading after 21 ± 3 h and to then extend the incubation to 44 ± 4 h. Confirmation of pathogenic staphylococci in the context of water controls : - 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 h. RESULTS Coagulase positive staphylococci are characterized by the formation of gray or colonies surrounded by an opaque halo of fibrin that is clear-cut, 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. TYPICAL COMPOSITION (can be adjusted to obtained optimal performance) For 1 liter of medium : - 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 ± /5

3 QUALITY CONTROL - Prepared medium : amber agar. - Typical culture response after 48 hours of incubation at 37 C: Microorganisms Growth Characteristics Colony color Fibrin halo Staphylococcus aureus DSM 799 Staphylococcus aureus ATCC Staphylococcus epidermidis ATCC Escherichia coli ATCC good, score 2 good, score 2 limited, score 0-2 inhibited, score 0 absence - Typical culture response after 44 hours of incubation at 36 C according to XP T and NF T Microorganisms Growth Characteristics Colony color Fibrin halo Staphylococcus aureus CIP Enterococcus faecalis CCM % R 3 150% inhibited STORAGE / SHELF LIFE Pre-poured (complete) media in Petri dishes : - Store between 2-14 C, shielded from light. - The expiration date is indicated on the label. PACKAGING Code Pre-poured plates (Ø 90 mm) : - 20 plates BM /5

4 PHOTO SUPPORT Product Reference : BM06708 Media used for : Detection / enumeration / confirmation of coagulase positive Staphylococci. Staphylococcus aureus Characteristic colonies Gray to colony surrounded by an opaque halo of fibrin Baird-Parker RPF agar Ref : BM06708 Incubation 24 hours at 37 C (surface) Characteristics : Coagulase positive staphylococci are gray to surrounded by a halo of fibrin. (reduction of tellurite and plasma coagulation) 4/5

5 BIBLIOGRAPHY Loeb, L The influence of certain bacteria on the coagulation of blood. Jour. Med. Res., 10: 407. Duthie, E.S Evidence of Two Forms of Staphylococcal Coagulase. J. gen. Microbiol., 10: Baird-Parker, A.C An improved diagnostic and selective medium for isolating coagulase positive staphylococci. Jour. of Appl. Bact., 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 Staphylococcus aureus (PPSA). Can. Jour. of Microbiol., 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. Neth. Milk Dairy J., 30: Hauschild, A.H.W., Park, C.E., and Hilsheimer, R A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods. Can. J. of Microbiol., 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. Can. Jour. of Microb., 30: Sawhney, D The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar. Journ. of Applied Bacteriology. 61: de Buyser, M.L., Audinet, N., Delbart, M. O., Maire, M., and Françoise, F Comparison of selective culture media to enumerate coagulase-positive staphylococci in cheeses made from raw milk. Food microbiol. 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. 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. 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 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 (juillet 2001), NF T /A1 (juin 2005) et NF T /A2 (mai 2007). Qualité de l eau. Contrôle qualité des milieux de culture. 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 : BM067/A/ : 8. 5/5