ChromoCult Coliform Agar

1 ChromoCult Coliform AgarFor detection of coliforms in drinkingwater and processed food of actionPrimarily the interaction of carefully selected peptones inaddition to pyruvate, sorbitol and a phosphate bufferguarantees rapid colony growth, even for the sublethallyinjured coliforms. The growth of accompanying Gram-positive bacteria flora aswell as some Gram-negative bacteria flora is largely inhibitedby the use of Tergitol 7 which has no negative effect on thegrowth of the Coliform , Merck has developed a new combination of two chromogenic substrates which allow for the simultaneousdetection of total coliforms and identificationThe characteristic enzyme for coliforms, -D-galactosidase.

2 Cleaves the Salmon-GAL substrate and resulting in a salmonto red colouration of the Coliform identificationThe substrate X-glucuronide is used for the identification of -D-glucuronidase which is characteristic for cleaves both Salmon-GAL and X-glucuronide, so thatpositive colonies take on a dark-blue to violet colour. These are easily distinguished from the other Coliform colonieswhich have a salmon to red colour. The inclusion of trypto-phane improves the indole reaction, thereby increasingdetection reliability when it is used in combination with theSalmon-GAL and X-glucuronide reaction as part of anadditional confirmation for composition (g/litre)Peptones ; sodium chloride ; sodium dihydrogenphosphate ; disodium hydrogen phosphate ; sodium pyruvate ; tryptophan ; agar - agar ; Sorbitol ; Tergitol 7 ; chromogenic mixture g in 1 litre of demin.

3 Water by heating in aboiling water bath or in a flowing stream of steam. Stir thecontents regularly to assist dissolution (approx. 35 min.).Some turbidity may occur but this does not effect the performance!Do not autoclave! Do not overheat!pH: at 25 :If the sample material contains high Gram-positivebacteria resp. Pseudomonas and Aeromonas spp. Supplement to the medium cooled to 45 - 50 plates are opalescent and yellowish. Store at 4 2 C and protect from light. To prevent plates from becoming dryseal in plastic-pouches or bags. Shelf-life under theseconditions: 6 procedureInoculate the medium by the pour-plate method or surfacespreading.

4 In addition the membrane-filter technique can alsobe used. Incubation: 24 hours at 35 - 37 :dark-blue to violet colonies (Salmon-GAL and X-glucuronide reaction) Total coliforms:salmon to red colonies (Salmon-GAL reaction)and dark-blue to violet colonies ( ).Other Gram-negatives:colourless colonies, except for some organisms which possess -D-glucuronidase activity. These colonies appear light-blue to turquoise. In order toconfirm , overlay the dark-blue to violet colonies with a drop of Kovacs Indole reagent. If the reagent turns to acherry-red colour after a few seconds the reaction is positiveand confirms the presence of method:The simultaneous detection of total coliforms and usingChromoCult Coliform agar (CCA) relies on the specificcolouration of target colonies.

5 Ossmer et. al (1999) reportedthe effect of the type and brand of membrane filters on thegrowth and colour formation of coliforms and on best performance was obtained using filters of cellu-lose-mixed-ester material, Pall GN 6 or Schleicher andSch ll ME 25. It is advised to use one of these filters as refe-rence for the validation of membrane Coliform AgarSelective agar for the simultaneous detection of total coliforms and in drinking water and processed food approved, the approval by AOAC is (500 g)Literature US-EPA approved methodFederal Register / Vol.

6 67, No. 209,29. October 2002, Rules and RegulationsFRAMPTON, E. W., RESTAINO; L. and BLASZKO, L. : Evaluation of the -glucuronidase substrate 5-bromo-4-chloro-3-indolyl- -D-glucuronide (X-GLUC) in a 24 hour direct platingmethod for Escherichia coli. J. Food Protection 51: 402 404 (1988)KILIAN, M. and B LOW, P.: Rapid diagnosis of Enterobacteriaceae. I. Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. B 84: 245 251 (1976)LE MINOR, L. and BEN HAMIDA, F.: Advantages de la recherche de la -galactosidase sur celle de la fermentation du lactose en milieu complexe dans le diagnosticbact riologique, en particulier des Enterobacteriaceae.

7 Ann. Inst. Pasteur (Paris) 102:267 277 (1962)MANAFI, M. and KNEIFEL, W. A:combined chromogenic-fluorogenic medium for the simultaneousdetection of total coliforms and in water. Zentralbl. Hyg. 189: 225-234 (1989)OSSMER, R.; SCHMIDT, W.; MENDE, U.: ChromoCult Coliform agar Influence of Membrane FilterQuality on Performance Poster Presentation Congresso de laSociedad. Espanola de Microbiologia, Granada, Spain (1999)New Zealand Dairy Industry; Microbiological Methods Manual,Section 48: Product Test Methods Enteric Indicator Organisms. NZTM 2; (1998)AdditivesCat.

8 Indole reagent 100 Indole30 16 Selective 20 platesColiform AgarPall 66278 Cellulose-mixed-ester-GN6 membrane filtersSchleicher & Cellulose-mixed-Sch ll 406870ester-ME 25/21 Escherichia coli dark-blue to violet+++ATCC 11775 Escherichia coli dark-blue to violet+++DSMZ 502 Citrobacter freundiisalmon to red+--ATCC 8090 Salmonella enteritidiscolourless---ATCC 13076 Enterococcus faecalisinhibitedATCC 19433 Quality controlTest strainsColony colourSalmon-GAL X-Glucuronide makes the KGaA64271 Darmstadt, GermanyFax+ 49 (0) 61 51 / 72 33 80E-mail: provide information and advice to our customers to the best of our knowledge and ability, but without obligation or liability.

9 Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged is a registered trademark of Merck KGaA, Darmstadt, 28610807/04