HALF-FRASER BROTH INTENDED USE Half-Fraser Broth is used for the selective and differential enrichment of Listeria monocytogenes in milk and dairy products, as well as in other food products which may contain it. HISTORY The medium studied by Fraser et al. in 1988 is a modification of the formulation of Donnelly and Baigent. The composition of the base is identical to that of UVM Broth and was modified by the addition of lithium chloride as selective agent and of ferric ammonium citrate to visualize cultures that hydrolyze esculin, by the resultant blackening of the medium. PRINCIPLES - The very good recovery of Listeria monocytogenes is assured by the concentration differences in nalidixic acid and acriflavine between Half-Fraser and Fraser, as well as the two enrichment steps themselves. Half-Fraser Broth allows the primary enrichment step, with secondary enrichment being performed in Fraser Broth. - Polypeptone, yeast extract and meat extract furnish the nutrients required for the growth of Listeria. - The high sodium chloride content increases the selectivity of the medium. - Phosphates buffer the pH of the medium. - Esculin is hydrolyzed by Listeria to glucose and esculetin, the latter compound forming a black complex with ferric ions supplied by ferric citrate, added just before use, which also favors the growth of Listeria. - Lithium chloride inhibits the growth of most enterococci which can also hydrolyze esculin. - Nalidixic acid blocks the DNA replication of bacteria sensitive to this antibacterial agent. - The growth of secondary Gram-positive microflora is inhibited by acriflavine. PREPARATION - Dissolve 55.0 g of dehydrated Fraser base II medium (BK133) or Half-Fraser base (BK173) in 1 liter of distilled or deionized water. - Stir slowly until complete dissolution. - Dispense in flasks at 225 mL per flask. - Sterilize in an autoclave at 121°C for 15 minutes. - Cool the medium to 25°C. Fraser base II medium (BK133) : - Aseptically add 2.25 mL of reconstituted Half Fraser Selective Supplement BS030 or 2.0 mL of reconstituted Selective Supplement BS032. - Mix well. 1/5 Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com Half-Fraser base (BK173) : - Aseptically add 2.25 mL of a Ferric ammonium citrate solution, 5% (BS059 or BS062) - Mix well. INSTRUCTIONS FOR USE Primary enrichment - To media prepared as above, or to ready-to-use vials BM016, aseptically add 25 g of the product to test. - Mix well. - Incubate at 30°C. - After 24 hours of incubation, carry out isolation on the appropriate selective media : Oxford Agar (BK110 + BS003 or BM019), PALCAM Agar (BK145 + BS004/BS049 or BM020), COMPASS Listeria Agar (BM123 or BM124). - In addition, transfer 0.1 mL of the resulting culture to 10 mL of Fraser Broth (BK133 + BS031, BK115+BS059 or BS062, BM013). Please refer to the Fraser broth datasheet. The ready-to-use 3 liter (BM133) or 5 liter (BM134) flexible bags allow for the use of an automated system in the preparation of stock solutions for primary enrichment (refer to instruction manuals of the systems in question for more information). Secondary enrichment - After inoculation, incubate Fraser Broth at 37°C. - After 24 and 48 hours of incubation, conduct selective isolations in selective solid media using a platinum loop. NOTE : Base medium BK173 can not be used for the secondary enrichment. RESULTS Identify suspected colonies and subject them to identification tests. NOTE : All the tubes, with or without blackening, must be transferred to selective media. A minimum period of 24 hours is necessary to permit the visualization of the black color. Suspected samples must systematically be transferred to selective isolation media, even in the absence of a color change. 2/5 Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com TYPICAL COMPOSITION of complete media (can be adjusted to obtain optimal performance) For 1 liter of medium : - Polypeptone .................................................................................10.00 g - Yeast extract ..................................................................................5.00 g - Meat extract ...................................................................................5.00 g - Sodium chloride ...........................................................................20.00 g - Disodium phosphate, anhydrous....................................................9.60 g - Monopotassium phosphate ............................................................1.35 g - Esculin ...........................................................................................1.00 g - Lithium chloride ..............................................................................3.00 g - Nalidixic acid ...............................................................................10.0 mg - Acriflavin (chlorhydrate) ..............................................................12.5 mg - Ferric ammonium citrate ................................................................0.50 g pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. QUALITY CONTROL - Dehydrated medium : yellowish powder, free-flowing and homogeneous. - Prepared medium (complete) : brownish solution with a bluish reflection, can present a precipitate. - Typical culture response after 24 hours of incubation at 30°C, followed by subculture on Oxford Agar: Microorganisms Growth on Oxford agar Listeria monocytogenes + Enterococcus faecalis + Escherichia coli CIP 59.53 ATCC® 29212 ATCC 25922  10 characteristic colonies Listeria monocytogenes + Enterococcus faecalis + Escherichia coli CIP 78.31 ATCC 29212 ATCC 8739  10 characteristic colonies Enterococcus faecalis Escherichia coli ATCC 29212 ATCC 25922 inhibited inhibited STORAGE / SHELF LIFE Dehydrated base medium : 2-30°C, shielded from light. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Base media in vials : 6 months at 2-8°C, shielded from light. - Complete medium in vials : 8 days at 2-8°C, shielded from light. Ready-to-use media in vials or flexible bags, Selective supplements for Half-Fraser Broth Ferric ammonium citrate, 5% sterile solution : - Store between 2-8°C, shielded from light. - The expiration dates are indicated on the labels. 3/5 Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com PACKAGING Code Ready-to-use media in vials : - 10 x 225 mL BM01608 Ready-to-use media in flexible bags : - 3 x 3 liters - 2 x 5 liters BM13308 BM13408 Dehydrated Fraser base II medium (without ferric ammonium citrate, nalidixic acid or acriflavine) : - 500 g bottle - 5 kg drum BK133HA BK133GC Half-Fraser Selective Supplements (for base BK133) : - 10 vial pack (for 500 mL) - 8 vial pack (for 2.25 liters) Dehydrated Half-Fraser base (without ferric ammonium citrate) : - 500 g bottle - 5 kg drum Ferric ammonium citrate, 5% sterile solution Supplement : - 10 x 90 mL vials - 7 x 10 mL tubes BS03008 BS03208 BK173HA BK173GC BS05908 BS06208 BIBLIOGRAPHY Donnelly, C.W., and Baigent, G.J. 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. App. Environ. Microbiol., 52: 689-695. Fraser, J.A., and Sperber, W.H. 1988. Rapid detection of Listeria spp. in food and environmental samples by esculin hydrolysis. J. Food. Prot., 51: 762-765. 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 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. 4/5 Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com PHOTO SUPPORT Product reference : [BK133HA, GC + BS03008 or BS03208], [BK173HA, GC + BS05908 or BS06208] ; BM01608, BM13308 or BM13408 Media used for : Selective and differential enrichment of Listeria monocytogenes in foods Presumptive positive sample Negative control Listeria monocytogenes Fraser broth Ref : BM01308 Incubation : 24 hours / 37°C Characteristics : blackened tube (at right) a presumptive indication of Listeria monocytogenes growth (reduction of esculin), however confirmation is still required. Note : Half Fraser will demonstrate the same type of blackening, but in 225 mL vials. *Benchmark value refers to the expected value 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 2009-07-31. They are susceptible to modification at any time, without warning. Code document : BK133/A/2003-01 : 11. 5/5 Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com