Application

Chromocult Coliform Agar

Selective agar for the simultaneous detection of total coliforms and E. coli in drinking water and processed food samples.

Intended Use
Chromocult Coliform Agar is a selective and differential chromogenic culture medium intended for use in microbiology laboratories analyzing food and water. Within 24 hours this medium enables the detection, differentiation and enumeration of E. coli and coliforms from drinking water and processed food matrices such as frankfurters, cooked chicken and non-fat dried milk. The manufacturing process for food and water often result in metabolic injury to the microorganisms and/or reduction of microbial members. Sub lethal microbial injury is caused by chemical or heat treatment as well as storage under frozen, dried or chilled conditions. Traditional culture media used for the detection of coliforms/ E. coli often contain ox bile or bile salts to inhibit Gram-positive bacteria. These strong inhibitors however may limit the growth of the targeted microorganisms if they are already sub lethally damaged. A careful selection of inhibitors used in the selective media is required to ensure the growth and recovery of these damaged microorganisms. Chromocult Coliform Agar contains Tergitol7 as an inhibitor of Gram-positive bacteria which has no negative effect on the growth of the targeted coliforms/ E. coli. Chromocult Coliform Agar is therefore the ideal medium for the detection of coliforms/ E. coli in drinking water and processed foods.

Validation Studies
Water testing Chromocult Coliform Agar membrane filter method has been approved and accepted by the U.S. Environmental Protection Agency (EPA) for monitoring total coliforms and E. coli under the Total Coliform Rule (40 CFR 141.21). In validation studies for the detection of total coliforms and E. coli in drinking water the Chromocult Coliform Agar method was compared to the EPA-approved reference method (mENDO LES Agar). The Chromocult Coliform Agar method offered precise and accurate identification and measurement of total coliforms and E. coli. In Spain Chromocult Coliform Agar membrane filter method has been approved and accepted as an alternative method according to the European Union Directive on Drinking Water. In validation studies for testing the equivalence of the quantitative detection of E. coli and coliforms, the Chromocult Coliform Agar method was compared to the reference method of the ISO 9308-1:2000 "Detection and enumeration of Escherichia coli and coliform bacteria, Part 1: Membrane filtration method." The equivalence study was performed according to the recommendations of ISO 17994 "Water quality - Criteria for the establishment of equivalence between microbiological methods." It was clearly demonstrated that Chromocult Coliform Agar is "equivalent or better" when compared to the reference method (Lactose TTC Agar with Tergitol 7). Food testing

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Application

Chromocult Coliform Agar has been validated by the AOAC Research Institute under the Performance Tested MethodsSM program for the analysis of frankfurters, cooked chicken and non-fat dried milk. The most probable number (MPN) method for coliform bacteria and E. coli (AOAC official method 966.24) was used for method comparison testing. The Chromocult Coliform Agar method was found to be equivalent to the AOAC official method.

Mode of Action
The interaction of selected peptones, pyruvate, sorbitol and phosphate buffer promotes rapid colony growth, even for sub lethally injured coliforms. The growth of Gram-positive bacteria as well as certain Gram-negative bacteria is inhibited by the presence of Tergitol7, which has no negative effect on the growth of the coliform bacteria. Further, the combination of two chromogenic substrates permits the simultaneous detection of total coliforms and E. coli. The addition of antibiotic supplements is recommended if sample material is known to have high level of background flora (especially if Aeromonas species are expected). Coliform identification The substrate Salmon-GAL is used for the identification of -D-galactosidase, which is characteristic for coliforms. This interaction results in a salmon to red color of the coliform colonies. E. coli identification The substrate X-glucuronide is used for the identification of -D-glucuronidase, which is characteristic for E. coli. E. coli cleaves both Salmon-GAL and X-glucuronide, so that positive colonies take on a dark-blue to violet color. These are easily distinguished from other coliform colonies, which have a salmon to red color.

Typical Composition (g/litre)

Peptone 3.0; sodium chloride 5.0; sodium dihydrogen phosphate 2.2; di-sodium hydrogen phosphate 2.7; sodium pyruvate 1.0; tryptophan 1.0; agar-agar 10.0; Sorbitol 1.0; Tergitol 7 0.15; 6-chloro-3-indoxyl-beta-Dgalactopyranoside 0.2; 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid 0.1; isopropyl-beta-Dthiogalactopyranoside 0.1.

Preparation
Preparation of agar medium Suspend 26.5 g in 1000 ml of purified water and heat to boiling with frequent agitation until completely dissolved (approx. 35 min). Do not autoclave! Do not overheat! Immediately cool the medium in the water bath at 45-50 C. pH: 6.8 0.2 at 25C. Preparation of optional antibiotic supplements (for water testing) Cefsulodin solution: Dissolve 10 mg of cefsulodin in 5 ml of purified water and sterilize by membrane filtration (0.2 m nominal pore size). Aseptically add the solution (5 ml per liter of medium) to one liter of liquefied medium.

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E. coli/Coliform Selective Supplement (EMD Chemicals cat. no. 1.00898.0001): The supplement is a mixture of vancomycin (2.5 mg/vial) and cefsulodin (2.5 mg/vial) in lyophilized form. To prepare the supplement, suspend the lyophilisate in the original vial by adding 2 ml of sterile purified water. Shake vigorously until the solution is clear. Aseptically add the contents of 2 vials (4 ml per liter of medium) to one liter of liquefied medium. Antibiotic solutions must be mixed evenly into sterilized Chromocult Coliform Agar while the medium is still liquefied, but only after the medium has been cooled to 45-50C. The antibiotic solutions are sensitive to heat. Pour plates directly after adding the antibiotic solution. Preparation of plates Dispense in Petri dishes to a depth of at least 4 mm (approximately 18 ml in a 90 mm plate). The medium is slightly opalescent and yellowish. The agar plates must be dry before surface inoculation. In case of visible water dry them before use. Prepared plates can be stored for up to 6 months (or up to 1 month if antibiotic supplements have been added) at 2 - 8C and protected from light. To prevent plates from becoming dry seal them in plastic pouches or bags.

Sample Preparation
Prepare test samples using standard laboratory techniques such as those described in the Bacteriological Analytical Manual, Standard Methods for the Examination of Water and Wastewater or the ISO standard specific for the product concerned. To minimize possible interference between the coloration of coliforms/ E. coli and the sample (e.g. low pH), it is advisable to dilute the sample 1:10 in a buffered solution (e.g. add 450 ml Butterfield's phosphate buffer, buffered peptone water, or buffered sodium chloride peptone broth to blender jar containing 50 g of sample and blend 2 min) and then to perform further dilutions as needed.

Application
Water testing For water testing the agar medium base should be used with the addition of antibiotics. In the USEPA study the agar medium base was supplemented with 10 mg of cefsulodin per liter. The European study in Spain was performed with the agar medium base supplemented with the E. coli/Coliform Selective Supplement. Chromocult Coliform Agar is usually inoculated by the membrane filter method. Filter an appropriate volume of sample (e.g., 100 ml for municipal drinking water, 250 ml for bottled water, or 1 ml for surface water) using a membrane filter. Place the filter on Chromocult Coliform Agar ensuring that no air is trapped underneath. Incubate the inoculated dishes aerobically at 35-37C in an inverted position for 24 hours. After incubation, examine the plates for the presence of typical colonies of E. coli and other coliforms. NOTE: The type of membrane filter affects the size and coloration of colonies. The best results were obtained using membrane filters of cellulose mixed-ester material, e.g. Pall Gelman GN-6 (OSSMER et al., 1999). Food testing

Merck KGaA, 64271 Darmstadt, Germany, Tel. +49(0)6151 72-2440 EMD Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA, +1-978-715-1335

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Application

For processed food samples the agar medium base can be used without the addition of antibiotic supplements (as in the AOAC study). For fresh food samples with a higher microbial load, Chromocult Coliform Agar ES (EMD cat. no. 1.00850) is recommended. Chromocult Coliform Agar is usually inoculated by the pour plate method. Using a sterile pipette, transfer 1 ml of the liquid test sample (or 1 ml from the appropriate dilution) to a sterile Petri dish. Pour into each Petri dish approximately 15 ml of the Chromocult Coliform Agar while the medium is still liquefied, but only after the medium has been cooled to 45-50C in a water bath. Carefully swirl the plate until the inoculum is thoroughly mixed with the medium. Allow the mixture to solidify on a cool horizontal surface. Incubate the inoculated dishes aerobically at 35-37C in an inverted position (agar side up) for 24 hours. After incubation, examine the plates for the presence of typical colonies of E. coli and other coliforms.

Results
Count the dark blue to violet colonies as E. coli and the salmon to red colonies as other coliforms. The total of all red and blue colonies is the total coliform count. Some E. coli (3-4%) are -glucuronidase-negative and appear as salmon-red colonies, e.g. E. coli O157 strains. Accompanying flora appear as colorless colonies, except for some organisms, which possess -D-glucuronidase activity. These colonies appear light blue to turquoise in color. The total coliform count should not exceed 150 typical CFU and 300 total CFU (total coliforms and accompanying bacteria) per plate and 100 typical CFU and 200 total CFU per membrane filter, respectively. Above these levels, the colonies cannot be counted accurately. Samples, which are expected to exceed these maximum levels, should be diluted prior to inoculation. Calculate the number of E. coli and other coliforms per milliliter or per gram of sample from the number of characteristic colonies in the plates.

Limitations
Chromocult Coliform Agar is designed for rapid colony growth, even for sub lethally injured coliforms. The selectivity of the agar medium base is limited. Samples with a high microbial load can lead to overgrowth of the whole plate. For samples with high microbial load use the agar medium base with antibiotics (approved for water testing) or the Chromocult Coliform Agar ES (approved for food testing). Some strains of Aeromonas spp. can grow as salmon to red colonies (similar to coliforms) on Chromocult Coliform Agar when using the agar medium base without cefsoludin. Aeromonads can be easily differentiated from coliforms by the oxidase reaction. Coliforms are oxidase-negative whereas aeromonads are oxidase-positive. Plates inoculated with food samples using the spread plate method might produce poorly distinguished, uncharacteristic colonies that are difficult or impossible to enumerate. For food samples use the pour plate method.

Merck KGaA, 64271 Darmstadt, Germany, Tel. +49(0)6151 72-2440 EMD Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA, +1-978-715-1335

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Application

Performance Characteristics
Water Testing USEPA study: The specificity of Chromocult Coliform Agar was assessed for recovery and identification of total coliforms and E. coli. Both positive and negative results were evaluated according to the EPA's ATP format for the presence or absence of total coliforms and E. coli as compared to the reference methods. Based on the verifications for total coliforms, Chromocult Coliform Agar was found to have a false positive error of 2.0 % while the false negative rate (undetected target error) was 0 %. For E. coli, the false positive and negative rates were 0 %. EU Equivalence Study in Spain: The Chromocult Coliform Agar method was compared to the ISO 9308-1:2000 reference method (Lactose TTC Agar with Tergitol 7). The coliform enumeration was 43% higher with the Chromocult method compared to the TTC method. The E. coli enumeration was strongly dependent on the confirmation method. When confirmation was based only on indole at 44C, the number of confirmed E. coli colonies with the Chromocult method was between 27 and 30% lower than with the TTC method (this large difference is due to a high number of false-positive results on TTC especially by Klebsiella oxytoca colonies). Instead, when the confirmation was based on the MUG (where the false-positive results by K. oxytoca were eliminated), the number of confirmed E. coli colonies with the Chromocult method was between 1.7% and 12% higher than with the TTC method. A significant difference was detected in the confirmation rate between the Chromocult and TTC methods. For coliforms the portion of presumptive colonies confirmed as positive (oxidasenegative) was 1753 / 2293 (76.5%) in the case of TTC and 2192 / 2272 (96.5%) in the case of the Chromocult. For E. coli out of the 635 blue-violet colonies on Chromocult, 593 (93.4%) were indole-positive at 44C. Out of the 2969 presumptive colonies on TTC, 962 (32.4%) were indole-positive. Food Testing Chromocult Coliform Agar was evaluated for the recovery of coliforms/ E. coli in frankfurters, cooked chicken and non-fat dried milk in internal and AOAC approved external laboratories. The study compared the Chromocult Coliform Agar method to the AOAC reference method (966.24) for the enumeration of E. coli and coliform bacteria. The food samples were pre-tested to establish the titer of naturally contaminating E. coli and coliform bacteria. Three 300-gram samples of frankfurters, cooked chicken and nonfat dried milk were batch-inoculated with dry inoculum, each at a different level: "low" (targeted spike of 10 CFU/ g), "middle" (targeted spike of 10 2 CFU/g), and "high" (targeted spike of 10 3 CFU/g). One additional 300-gram sample remained uninoculated to serve as a negative control. From each 300-gram sample, five 50-gram sub samples were weighed and blended for 2 minutes with 450 mL of sterile diluent (Butterfield's phosphate buffer). Following blending, five 50 gram samples were plated individually onto Chromocult Coliform Agar. Dilutions were made using 99 mL of Butterfield's phosphate buffer as a diluent. Both the pour plate and spread plate technique was used for all foods. Plates were inverted and incubated aerobically at 35C for 24 1 hours. Enumeration of dark-blue or violet colonies yielded a presumptive E. coli count while enumeration of salmon or red-colored colonies yielded a presumptive coliform count. Confirmation of typical E. coli colonies was done according to AOAC method 966.24, followed by Gram stain, oxidase test, and analysis with MicroID test strips. Each 50-gram sub sample plated onto Chromocult Coliform Agar was also tested using a 3-tube, most probable number (MPN) series. The AOAC official method 966.24 "Most Probable Number Method for Coliform Bacteria and E. coli" was followed. Typical E. coli colonies on L-EMB were confirmed by Gram stain, oxidase test, and analysis with MicroID test strips. Merck Chromocult Coliform Agar effectively detected total coliforms and E. coli in all food types tested. Independent of the spike level, the coliform and E. coli level determined by AOAC MPN method 966.24 was consistent with results generated by the Chromocult Coliform Agar for all foods. Furthermore, presumptive E.

Merck KGaA, 64271 Darmstadt, Germany, Tel. +49(0)6151 72-2440 EMD Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA, +1-978-715-1335

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coli colonies on Chromocult Coliform Agar (violet in color) were confirmed as E. coli in all isolates. Similarly, red, presumptive coliform colonies all exhibited gassing in BGLB, indicative of confirmed coliform identification. This suggests that not only can the Chromocult Coliform Agar reliably quantify total coliforms present in a food, but also they can consistently differentiate E. coli from other coliform bacteria. Fifty three (53) pure cultures of E. coli and other coliforms were cultured on Chromocult Coliform Agar at 35C for 24 hours. All strains of E. coli and other coliforms showed characteristic growth. Overall sensitivity for Chromocult Coliform Agar was 100%. Forty four (44) pure cultures of non-coliform bacteria were cultured on Chromocult Coliform Agar at 35C for 24 hours. Most of the non-coliform bacteria showed colorless growth or they were completely inhibited. Only 3 strains showed turquoise growth. There were no false positive reactions. Overall specificity for Chromocult Coliform Agar was 100%.

Storage conditions and Shelf life

Store the dehydrated medium dry and tightly closed. Protect from light. Don't use clumped or discoloured medium. Store at 15C to 25C and use before the expiry date on the label.

Precautions
For further information please refer to the Material Safety Data Sheet (MSDS). Safety information on ingredients and culture media are summarised in ChemDAT manual. The ChemDAT manual is available on CD-ROM or can be down loaded from Internet www.chemdat.info.

Technical assistance
For technical assistance, please contact your local Merck representative or Merck KGaA 64271 Darmstadt, Germany. Tel: 49-6151-720, Fax: 49-6151-72 20 00, Email: service@merck.de.

Literature
American Public Health Association. 1995. Standard methods for the examination of water and wastewater, 19th ed. American Public Health Association, Washington, D.C., USA. Byamukama, D., Mach, R.L., Kansiime, F., Manafi, M., and A.H. Farnleitner. 2005. Discrimination Efficacy of Fecal Pollution Detection in Different Aquatic Habitats of a High-Altitude Tropical Country, Using Presumptive Coliforms, Escherichia coli, and Clostridium perfringens Spores. Appl. Environ. Microbiol. 71: 65-71. Geissler, K., Manafi, M., Amoros, I., and J.L. Alonso. 2000. Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. J. Appl. Microbiol. 88: 280-285. Gonzalez, R.D., Tamagnini, L.M., Olmos, P.D., and G.B. de Sousa. 2003. Evaluation of a chromogenic medium for total coliforms and Escherichia coli determination in ready-to-eat foods. Food Microbiology 20: 601- 604. ISO International Standardisation Organisation. Microbiology of food and animal feeding stuffs - General rules for microbiological examinations. ISO 7218:1996/Amendment 1:2001. ISO International Standardisation Organisation. Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination. Part 1: General rules for the preparation of the initial suspension and decimal dilutions. ISO 6887-1:1999. ISO International Standardisation Organisation. Water quality - Sampling. Part 1: Guidance on the design of sampling programmes and sampling techniques. ISO 5667-1:2006.

Merck KGaA, 64271 Darmstadt, Germany, Tel. +49(0)6151 72-2440 EMD Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA, +1-978-715-1335

2008-08-26

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ISO International Standardisation Organisation. Water quality - General guidance on the enumeration of microorganisms by culture. ISO 8199:2005. Kilian, M. and Blow, P. 1976. Rapid diagnosis of Enterobacteriaceae. I. Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. B 84: 245-251. Ogden, I.D., Brown, G.C., Gallacher, S., Garthwaite, P.H., Gennari, M., Gonzalez, M.P., Jorgensen, L.B., Lunestad, B.T., MacRae, M., Nunes, M.C., Petersen, A.C., Rosnes, J.T., and J. Vliegenthart. 1998. An interlaboratory study to find an alternative to the MPN technique for enumerating Escherichia coli in shellfish. International Journal of Food Microbiology 40: 57- 64. Ossmer, R. 1996. Simultaneous Detection of Total Coliforms and E. coli with Chromocult Coliform Agar. HealthRelated Water Microbiology Symposium, Mallorca, Spain. Ossmer, R., Schmidt, W., Mende, U. 1999. Chromocult Coliform Agar - Influence of Membrane Filter Quality on Performance. - XVII Congresso de la Sociedad, Granada, Spain. New Zealand Dairy Industry. 1998. Microbiological Methods Manual, Section 48: Product Test Methods - Enteric Indicator Organisms. - NZTM 2; 48.5.1-48.5.10. Suwansonthichai, S., and S. Rengpipat. 2003. Enumeration of coliforms and Escherichia coli in frozen black tiger shrimp Penaeus monodon by conventional and rapid methods. International Journal of Food Microbiology 81: 113 - 121. Turner, K.M., Restaino, L., and Frampton, E.W. 2000. Efficacy of Chromocult Coliform Agar for Coliform and Escherichia coli Detection in Foods. J. of Food Prot. 63: 539-541. US-EPA approved method. Federal Register/Vol. 67, No.209, 29. October 2002, Rules and Regulations. U.S. Food and Drug Administration. 2003. Bacteriological Analytical Manual Online, Chapter 1: Food Sampling and Preparation of Sample Homogenate, AOAC International, Gaithersburg, MD.,USA.

Ordering Information
Product Chromocult Coliform Agar E. coli/Coliform SelectiveSupplement Buffered Peptone water (BPW) Sodium chloride peptone broth (buffered) Ordering No. 1.10426 .0500 1.00898.0001 1.07228.0500 1.10582.0500 Pack size 500 g 1 x 16 vials 500 g 500 g

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Quality control
Test strains Escherichia coli ATCC 11775 Escherichia coli DSMZ 502 Citrobacter freundii ATCC 8090 Enterobacter aerogenes ATCC 13408 Klebsiella pneumoniaeAT CC 13883 Salmonella enteritidis ATCC 13076 Enterococcus faecalis ATCC 19433 Bacillus cereus ATCC 11778 Inoculum (CFU/plate) 10-100 10-100 10-100 % Recovery rate 70 70 70 Colony colour dark-blue to violet blue to violet salmon to red SalmonGAL XGlucuronide

10-100

70

salmon to red

10-100

70

salmon to red

10-100

not limited

colourless

1000-2000

0.01

1000-2000

0.01

Citrobacter freundii ATCC 8090

Escherichia coli ATCC 11775

Merck KGaA, 64271 Darmstadt, Germany, Tel. +49(0)6151 72-2440 EMD Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA, +1-978-715-1335

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