PALCAM AGAR INTENDED USE

PALCAM AGAR INTENDED USE PALCAM Agar is a selective medium used for the differentiation and isolation of Listeria monocytogenes from milk and cheese, as well as in other food products, even highly contaminated. PALCAM Agar is also used in the context of the rapid alternative method for the detection of species belonging to the genus Listeria (Listeria spp) (COMPASS Listeria Agar), in human food products and environmental samples. It is officially AFNOR-certified NF VALIDATION, under the reference number BKR 23/02 11/02, of which the validity runs until November 28, 2014. HISTORY The medium was formulated by van Netten et al. in 1989 to overcome the insufficient selectivity of media used in the past for the detection and enumeration of Listeria. The studies were based on the work of Rodriguez (1984), who was the first to use esculin and iron salts to visualize Listeria monocytogenes by its esculinase-positive character. Many selective media for Listeria containing esculin, however, also enable the growth of several group D streptococci so that the use of esculin was only of limited value. Based on the work of Rocourt (1987), van Netten supplemented the medium with D-mannitol in order to differentiate mannitol-positive enterococci from mannitolnegative Listeria. Based on these two principles, and using the ecometric evaluation method, the authors showed that the combined action of ceftazidim and lithium chloride was more effective in terms of selectivity than that obtained by using 2-phenylethanol. PALCAM Agar is a combination of ALPAMY medium (van Netten et al., 1988 b) and Oxford Medium (Curtis et al., 1989). The authors showed that among the 13 selective media tested, PALCAM Agar gave satisfactory results, producing highly typical Listeria colonies at the same time as inhibiting almost all other contaminating bacteria. PRINCIPLES - Peptones and yeast extract favor the excellent growth of Listeria. - Glucose and starch are the energy sources for microbial development. - Sodium chloride maintains osmotic balance. - Listeria hydrolyze esculin to glucose and esculetin, the latter compound forming a black complex with ferric ions supplied by ferric citrate. - Accompanying microflora are inhibited by lithium chloride, ceftazidim, polymyxin and acriflavin. - The fermentation of mannitol by contaminating bacteria that may grow causes phenol red to turn yellow, thereby orienting the diagnosis. 1/5

PREPARATION - Suspend 68.9 g of dehydrated base medium (BK145) in 1 liter of distilled or deionized water. - Slowly bring to boiling, stirring with constant agitation until complete dissolution. - Dispense 100 ml in 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. INSTRUCTIONS FOR USE - Melt the medium (if it was prepared in advance). - Cool and maintain the medium at 44-47 C. - Per 100 ml of base, aseptically add 1 ml of reconstituted PALCAM Selective Supplement (BS004 or BS049). - Mix well. - Pour into sterile Petri dishes. - Let solidify on a cold surface. - Dry in an incubator with the covers partially removed. - For enumeration, transfer 0.1 ml of the sample to analyze (or of a selective enrichment broth) and its serial tenfold dilutions to the plates prepared as above or to ready-to-use plates (BM020) that have been brought to room temperature. Spread the inoculum on the surface of the agar with a sterile triangle. - For detection, isolate individual colonies by inoculating a loop of selective enrichment broth. - Incubate at 37 C for 24 and 48 hours. When using COMPASS Listeria Agar in the AFNOR-certified procedure, take an isolated characteristic colony on the surface of COMPASS Listeria Agar (blue-green colonies with or without an opaque halo) and re-isolate by stabbing onto the surface of PALCAM Agar (up to 15 stabbings per agar plate). Incubate at (37 ± 1) C for (24 ± 3) hours. The presence of a characteristic colony confirms the affiliation to the genus Listeria. RESULTS After 24 hours of incubation, Listeria monocytogenes forms olive-green colonies with a hollow black center and surrounded by black zones. When the colonies reach confluence, the medium becomes brown-black. PALCAM Agar is highly selective, but it is sometimes possible to observe colonies of staphylococci or enterococci (which ferment mannitol and produce yellow colonies with a yellow halo, thereby being easily distinguished from Listeria). Suspected colonies are subjected to biochemical identification tests. 2/5

TYPICAL COMPOSITION of the complete medium (can be adjusted to obtain optimal performance) For 1 liter of medium : - Peptones...23.00 g - Yeast extract...3.00 g - Glucose...0.50 g - Starch...1.00 g - D-mannitol...10.00 g - Esculin...0.80 g - Ferric ammonium citrate...0.50 g - Sodium chloride...5.00 g - Lithium chloride...15.00 g - Polymyxin B (sulfate)...10.00 mg - Ceftazidime...20.00 mg - Acriflavin...5.00 mg - Phenol red...0.08 g - Bacteriological agar...10.00 g ph of the ready-to-use medium at 25 C : 7.2 ± 0.2. QUALITY CONTROL - Dehydrated medium : pinkish powder, free-flowing and homogeneous. - Prepared (complete) media : red agar. - Typical culture response after 48 hours of incubation at 37 C : Microorganisms Listeria monocytogenes CIP 59.53 Listeria monocytogenes CIP 78.31 Escherichia coli ATCC 25922 Enterococcus faecalis ATCC 29212 Growth (Productivity ratio: P R ) P R 50% P R 50% inhibited, score 0 inhibited, score 0 Characteristics olive-green colonies surrounded by a black halo olive-green colonies surrounded by a black halo STORAGE / SHELF LIFE Dehydrated medium : 2-30 C. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Base media in vials : 6 months at 2-8 C. - Complete media in plates : 1 month at 2-8 C, shielded from light. Pre-poured (complete) media in plates, PALCAM Selective Supplement : - Store between 2-8 C, shielded from light. - The expiration dates are indicated on the labels. 3/5

PHOTO SUPPORT Product reference : [BK145HA, GC + BS00408 or BS04908], BM02008 Media used for : Selective isolation and enumeration of Listeria monocytogenes in foods. Listeria monocytogenes PALCAM agar Ref : BM02008 Incubation : 48 hours / 37 C Characteristics : olive-green colonies with distinctive concave center, surrounded by a black halo (indicative of esculin hydrolysis). 4/5

PACKAGING Code Pre-poured media in Petri plates (Ø 90 mm) : - 20 plates BM02008 Dehydrated base medium (without Polymyxin, Ceftazidime, Acriflavin) : - 500 g bottle BK145HA PALCAM Selective Supplement (qs. 500 ml) : - 10 vial pack BS00408 PALCAM Selective Supplement (qs. 2.5 liters) : - 8 vial pack BS04908 BIBLIOGRAPHY van Netten, P., van de Ven, A., Perales, I., and Mossel D.A.A. 1988. A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods. Int. Jour. of Food Microb., 6: 187-198. van Netten, P., Perales, I., and Mossel, D.A.A. 1988. An improved selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods. Letters in Applied Microbiology, 7: 17-21. van Netten, P., Perales, I., van de Moosdijk., Curtis G.D.W., and Mossel D.A.A. 1989. Liquid and solid selective differential media for the detection and enumeration of Listeria monocytogenes and other Listeria spp. International Journal of Food Microbiology, 8: 299-316. Bind, J.L. 1991. Mise en évidence et dénombrement des Listeria à partir de produits laitiers. Lait, 71: 99-105. Journal Officiel du 7 Avril 1992. Contrôle microbiologique des produits végétaux ou d origine végétale, 5144. (arrêté du 13 mars 1992). NF EN ISO 11290-1 (V 08-028-1). Février 1997. Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes. Partie 1 : Méthode de recherche. XP V 08-062. Octobre 2000. Microbiologie des aliments. Méthode de dénombrement de Listeria monocytogenes. Méthode de routine. 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 11290-1/A1 (V 08-028-1/A1). Février 2005. Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes. Partie 1 : Méthode de recherche. Amendement 1 : Modification des milieux d isolement, de la recherche de l hémolyse et introduction de données de fidélité. BKR 23/2-11/02 ALTERNATIVE ANALYTICAL METHODS FOR AGRIBUSINESS Certified by AFNOR Certification www.afnor-validation.org *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 2011-07-01. They are susceptible to modification at any time, without warning. Code document : BK145/A/2003-01 : 6. 5/5