MANNITOL SALT AGAR INTENDED USE Mannitol Salt Agar is used for the selective isolation, detection and enumeration of pathogenic staphylococci in milk, meat products, fish and seafood, other food products, pharmaceuticals, cosmetics, and biological samples of animal origin. It is also used in the bacteriological examination of swimming pool water, spas and drinking water using membrane filtration. HISTORY The experiments of Koch showed that staphylococci could tolerate hypersalted media at 7.5%. Chapman confirmed these initial results and observed that staphylococci which coagulated rabbit plasma formed yellow colonies on this medium, while most other bacteria were inhibited. PRINCIPLES - The high sodium chloride concentration inhibits the growth of most bacteria other than staphylococci. - Mannitol fermentation, shown by the color change of the ph indicator (phenol red) to yellow orients the diagnosis. - The demonstration of pathogenic staphylococci is confirmed by a coagulase test and, optionally, deoxyribonuclease and phosphatase tests. PREPARATION - Suspend 111.0 g of dehydrated medium (BK030) in 1 liter of distilled or deionized water. - Slowly bring to boiling, stirring with constant agitation until complete dissolution. - Dispense in tubes or flasks - 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. 1/5
INSTRUCTIONS FOR USE General use : - Melt the medium (if prepared in advance). - Cool and maintain at 44-47 C. - Pour into sterile Petri dishes. - Let solidify on a cold surface. - Dry in an incubator with the covers partially removed. - Transfer 0.1 ml of the product to analyze and its serial tenfold dilutions to the Petri dishes. - Spread the inoculum on the surface of the medium with a sterile triangle. - Incubate at 37 C for 24 and 48 hours. Water testing : - Melt the medium (if prepared in advance). - Cool and maintain at 44-47 C. - Pour into sterile Petri dishes (the thickness of the agar should be at least 5 mm). - Let solidify on a cold surface. - Aseptically filter the appropriate volume of water through a nitrocellulose membrane. - Place the membrane, filtered side up, on the surface of the agar prepared as described (or use ready-to-use plates BM148), insuring a complete contact. The agar plates should be brought to room temperature before manipulation. - Incubate at (36 ± 2) C for (44 ± 4) hours. RESULTS Pathogenic staphylococci form luxuriant pigmented colonies, surrounded by a yellow ring due to the fermentation of mannitol. Non-pathogenic staphylococci generally give rise to small red colonies which do not change the color of the medium. Several strains of Staphylococcus epidermidis can ferment mannitol. After 48 hours of incubation, several strains of enterococci, Bacillus, Micrococcus and Serratia may grow. TYPICAL COMPOSITION (can be adjusted to obtain optimal performance) For 1 liter of medium : - Tryptone...5.0 g - Peptic digest of meat...5.0 g - Meat extract...1.0 g - Mannitol...10.0 g - Sodium chloride...75.0 g - Phenol red...25.0 mg - Bacteriological agar...15.0 g ph of the ready-to-use medium at 25 C : 7.4 ± 0.2. 2/5
QUALITY CONTROL - Dehydrated medium : pinkish powder, free-flowing and homogeneous. - Prepared medium : red agar. - Typical culture response after 24-48 hours of incubation at 37 C (XP CEN ISO/TS 11133-2) : Microorganisms Growth (Productivity Ratio : P R ) Characteristics Staphylococcus aureus DSM 799 Staphylococcus aureus ATCC 25923 (1) Staphylococcus aureus CIP 53.154 Staphylococcus epidermidis ATCC 12228 Escherichia coli ATCC 25922 (1) Enterococcus faecalis CCM 2541 P R 50% P R 50% 66% R 3 150% good, score 2 inhibited, score 0 inhibited (1) 48 hours incubation according to NF T90-461 ; Productivity ratio : R 3 ) pink colonies surrounded by a red zone STORAGE / SHELF LIFE Dehydrated medium : 2-30 C. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Media in vials : 6 months at 2-8 C. - Media in plates : 1 month at 2-8 C. Pre-poured media in plates : - Store between 2 8 C, shielded from light. - The expiration date is indicated on the label. PACKAGING Code Dehydrated medium : - 500 g bottle BK030HA Pre-poured media in Petri plates (Ø 55 mm) : - 20 plates BM14808 3/5
BIBLIOGRAPHY Chapman, G.H. 1945. The significance of sodium chloride in studies of staphylococci. J. Bacteriol., 50: 201. Chapman, G.H. 1948. An improved Stone medium for the isolation and testing of food poisonning staphylococci. Food Research, 13: 100-105. Journal Officiel du 21 septembre 1968. Méthodes officielles de prélèvement et d'analyse bactériologiques des glaces et crèmes glacées. (arrêté du 30 août 1968). 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 T 90-461. Juillet 2001 et NF T 90-461/A1. Juin 2005. Qualité de l eau. Microbiologie. Contrôle qualité des milieux de culture. United States Pharmacopeia 29. 2006. Microbial Limit Tests, 2503-2508. ISO 22718. Février 2006. Cosmétiques. Microbiologie. Détection de Staphylococcus aureus. 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. NF EN ISO 22718 (T 75-605). Septembre 2009. Cosmétiques. Microbiologie. Détection de Staphylococcus aureus. 4/5
PHOTO SUPPORT Product reference : BK030HA; BM14808 Media used for : Selective isolation, detection and enumeration of pathogenic Staphylococci. Left : Staphylococcus aureus Right : Staphylococcus epidermidis Mannitol Salt (Chapman) agar Ref : BK030HA Incubation : 24 hours / 37 C Characteristics : Staphylococcus aureus (upper left) : Yellow colonies surrounded (indicating mannitol fermentation) ; Staphylococcus epidermidis (bottom right) : White colonies surrounded by a red halo (no mannitol fermentation) *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 2010-01-12. They are susceptible to modification at any time, without warning. Code document : BK030/A/2000-09 : 9. 5/5