BAIRD-PARKER RPF AGAR – ready-to-use

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 presence 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 68883, NF V0 057-1 and ISO 5944 – IDF 60. The pre-poured media can also be used for the enumeration
and confirmation of pathogenic staphylococci according to the standard XP T90-412 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 black 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.
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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 90-412, 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 black 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 .........................................................................................9.47 g
- Meat extract ...................................................................................4.74 g
- Yeast extract ..................................................................................0.95 g
- Sodium pyruvate ............................................................................9.47 g
- Glycine .........................................................................................11.36 g
- Lithium chloride ..............................................................................4.73 g
- Bacteriological agar .......................................................................14.2 g
- Rabbit plasma, EDTA..................................................................25.0 mL
- Bovine fibrinogen ...........................................................................5.00 g
- Trypsin inhibitor...........................................................................25.0 mg
- Potassium tellurite.......................................................................25.0 mg
pH of the complete medium at 25°C: 7.2 ± 0.2.
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QUALITY CONTROL
-
- Prepared medium : amber agar.
Typical culture response after 48 hours of incubation at 37°C
36°C (2) :
(1)
or after 44 hours of incubation at
Characteristics
Growth
(Productivity Ratio : PR)
Microorganisms
Colony
color
Fibrin halo
Staphylococcus aureus
DSM 799
Staphylococcus aureus
ATCC® 25923
(1)
Staphylococcus epidermidis ATCC 12228
(1)
Escherichia coli
ATCC 25922
PR ≥ 50%
PR ≥ 50%
limited, score 0-1
inhibited, score 0
black
black
black
presence
presence
absence
(2)
66% ≤ R3 ≤ 150%
inhibited
black
presence
(1)
(1)
(2)
(1)
(2)
Staphylococcus aureus
Enterococcus faecalis
CIP 53.154
CCM 2541
Productivity ratio PR according to XP CEN ISO/TS 11133-2
Productivity ratio R3 according to XP T90-412 ; NF T 90-461
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
BM06708
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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com/
PHOTO SUPPORT
Product Reference : BM06708
Media used for : Detection / enumeration / confirmation of coagulase positive Staphylococci.
Staphylococcus aureus
Characteristic colonies
Gray to black 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 black surrounded by a halo of fibrin.
(reduction of tellurite and plasma coagulation)
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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com/
BIBLIOGRAPHY
Loeb, L. 1903. The influence of certain bacteria on the coagulation of blood. Jour. Med. Res., 10: 407.
Duthie, E.S. 1954. Evidence of Two Forms of Staphylococcal Coagulase. J. gen. Microbiol., 10: 427-436.
Baird-Parker, A.C. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. Jour. of
Appl. Bact., 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 Staphylococcus aureus (PPSA). Can. Jour. of Microbiol., 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. Neth. Milk Dairy J., 30: 222-229.
Hauschild, A.H.W., Park, C.E., and Hilsheimer, R. 1979. A modified pork plasma agar for the enumeration of Staphylococcus
aureus in foods. Can. J. of Microbiol., 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. Can. Jour. of Microb., 30: 470-474.
Sawhney, D. 1986. The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar. Journ. of
Applied Bacteriology. 61: 149-155.
de Buyser, M.L., Audinet, N., Delbart, M. O., Maire, M., and Françoise, F. 1998. Comparison of selective culture media to
enumerate coagulase-positive staphylococci in cheeses made from raw milk. Food microbiol. 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.
ISO 5944 / FIL 60. Décembre 2001. Lait et produits à base de lait. Détection des staphylocoques à coagulase positive.
Technique du nombre le plus probable.
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.
NF V 08-057-1. Janvier 2004 (2e 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 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 EN ISO 6888-3 (V 08-014-3). Juin 2003 (3e 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 90-461 (juillet 2001), NF T 90-461/A1 (juin 2005) et NF T 90-461/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 2010-01-15.
They are susceptible to modification at any time, without warning.
Code document : BM067/A/2003-01 : 7.
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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tel : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com/