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Culture Media Quality control Positive control: Expected results Escherichia coli ATCC® 26922" Good growth; purple-pink colonies with flourescence Negative control: ‘Staphylococcus aureus ATCC* 25928" No growth “This orgenism is avaliable as a Cult-Loop® References 1. ‘Association of Official Analytical Chemists’ ED.A, Bacteriological Analytical Manual 8th Edition (Revision ‘A/1998) AOAC, Arlington Va. VIOLET RED BILE GLUCOSE AGAR Code: CMo485 4 glucose-containing selective medium for the detection and enumeration of Enterobacteriaceae in food products. Formula gmilitre Yeast extract, 3.0 Peptone 7.0 Sodium chloride 5.0 Bile Salts No. 3 16 Glucose 10.0 Neutral red 0.03 Crystal violet 0.002 Agar . 12.0 pH7.4 £02 Directions ‘Suspend 38.5 g in 1 litre of distilled water. Bring to the boil. Continue to boil for 2 minutes or for the minimum time necessary to dissolve completely and ensure that there are no remaining flecks of unmelted agar. No further sterilisation is necessary or desirable. Mix well and dispense into tubes or dishes. Description Results from tests that may be applied to water to detect coli-aerogenes organisms as possible indicators of faecal contamination possess far less significance when applied to raw foods. In the examination of foodstuffs, detection of a more defined group of organisms, the Enterobacteriaceae, that ferment glucose to produce acid and/or gas has been recommended". in addition to coliforms this group includes salmoneliae and shigellae, which do not ferment lactose, and enterotoxigenic Escherichia coli. It also contains organisms, Such as Klebsiella and Citrobacter, which are more resistant to heat than coliforms and are therefore better indicators of failure of processes that use minimal heat. The difficulties of measuring the total Enterobacteriaceae content of foodstuffs have been studied by Mosse! of al, who showed that the addition of glucose to an existing medium for the detection of coliforms improves the performance. They added 10 g per litre of glucose to crystal violet neutral red bile lactose agar Wiolet Red Bile Agar C0107), and named the modified formulation MacConkey Glucose Agar. Further work by Mossel et 21? showed that the lactose could be omitted resulting in the formulation of Violet Red Bile Glucose Agar. The continued inclusion of lactose would not provide test results leading to more accurate identification. Exclusion of lactose renders the medium more economical to make as less weight is required per litre - ‘Media that contain bie salts have an intrinsic toxicity for Enterobact been under stress™*", Considerable cifferences have been observed among six commercial preparations of Violet Red Bile Agar'* with regard to productivity for Enterobacteriaceae', and the intensity of their metabolism. In conjunction with Oxoid the components of the medium were examined and Mossel drafted a specification as follows: \ceae, even for cells that have not 2374 2008 ce Culture Media 1. Approved media have to be clear and yield colonies of satisfactory size. They have to give reproducible counts of typical colonies of Enterobacteriaceae. 2. When challenged for intrinsic toxicity by an anaerobic metabolic test using a strain of Yersinia enterocolitica (Serotype 03) as a sensitive indicator, media must promote adequate growth, acid formation and, where required, adequate gas formation. 3 Media have to satisfy the confirmation rate of typical colonies, ie. the number of colonies confirmed as Enterobacteriaceae divided by the number of colonies tested. Violet Red Bile Glucose Agar has been developed to satisfy all of these criteria and complies with the recommendations of ISO" Technique Prepare a series of dilutions of the samples so that at least one will be included that will yield 100-200 colonies from a 1 mi aliquot. Transfer 1 mi aliquots of each dilution to 9 cm Petri dishes using 2 plates for each dilution. Add 15 ml of medium, cool to 47°C. Gently swirl the piates 3 times clockwise and 3 times ant: Clockwise. After the medium has solidified overlay with 10 mi of the same medium and leave to solidly. Invert the dishes and incubate at >42°C for 18 hours, 32°C for 24-48 hours or 4°C for 10 days depending on the groups of Enterobacteriaceae to be recovered’. The agar overlay ensures anaerobic conditions which suppress the growth of non-fermentative Gram- negative bacteria. It also encourages the fermentation of glucose which favours the formation of clearly visible purple colonies, surrounded by a purple helo, Characteristic appearance of colonies Round, purple 1-2 mm diameter surrounded by purple haloes. Although colony size is generally 1-2 mm, size can be affected by a number of influences and all red colonies should be counted. Confirmation of the identity of these colonies must be made by further tests. Storage conditions and Shelf life Store the dehydrated medium at 10-30°C and use before the expiry date on the label. Store the prepared medium at 2-8°C and use as frestily as possible. Appearance Dehydrated medium: Straw-pink coloured, free-flowing powder. Prepared medium: Purple coloured gel. Quality Control Positive control: Expected results’ ~ Escherichia coll ATCC® 25922* Good growth; purple/pink colonies with halo Negative control: ‘Staphylococcus aureus ATCC® 6538" No growth “This ceganiom is avaiable as a Cult-Loop® Precautions This medium is not completely specific for Enterobacterlaceae, other organisms may grow e.g. Aeromonas and Yersinia species. The selective activity of this medium diminishes after 24 hours incubation and organisms previously suppressed may exhibit growth. Medium for the poured plate procedure should be freshly prepared, cooled to 47°C and used within 3 hours. References 4. WHO Technical Report Series N.596 (1976) Geneva, p.51. 2. Mosse! D. A. A. (1958) Zbl. Bakt. |. Ref. 166. 421-432. 3. Mosse! D. A. A., Mengerink W. H. J. and Scholts H. H. (1962) J. Bacteriol. 84. 381. 4, Mosse! D. A. A, Eelderink I, Koopmans M. and van Rossem F, (1978) Lab. Practice 27. No.12. 1049- 1050. 5. Mossel D. A. A., Eelderink |., Koopmans M. and van Rossem F. (1979) J. Food Protect. 42. 470-475. 6. Mossel D. A. A. (1978) Food Technol, Austral. 30. 212-219. 7. Kroninger D. L. and Banwart G. J. (1978) J. Food Sci. 43. 1328-1929. 8. Bridson E. Y, (1978-79) in Van Monster tot Resultaat Nederland Society for Microbiology. Wageningen, pp.58-67. 2006 2.375

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