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Application

Chromocult Coliform AgarSelective agar for the simultaneous detection of total coliforms andE. coli in drinking water and processed food samples.

Intended UseChromocult Coliform Agar is a selective and differential chromogenic culture medium intended for use inmicrobiology 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 asfrankfurters, cooked chicken and non-fat dried milk.

The manufacturing process for food and water often result in metabolic injury to the microorganisms and/orreduction of microbial members. Sub lethal microbial injury is caused by chemical or heat treatment as well asstorage 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 inhibitGram-positive bacteria. These strong inhibitors however may limit the growth of the targeted microorganisms ifthey are already sub lethally damaged.

A careful selection of inhibitors used in the selective media is required to ensure the growth and recovery of thesedamaged microorganisms.

Chromocult Coliform Agar contains Tergitol7 as an inhibitor of Gram-positive bacteria which has no negativeeffect on the growth of the targeted coliforms/ E. coli. Chromocult Coliform Agar is therefore the ideal mediumfor the detection of coliforms/ E. coli in drinking water and processed foods.

Validation StudiesWater testing

Chromocult Coliform Agar membrane filter method has been approved and accepted by the U.S. EnvironmentalProtection 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 ColiformAgar 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 totalcoliforms and E. coli.

In Spain Chromocult Coliform Agar membrane filter method has been approved and accepted as an alternativemethod according to the European Union Directive on Drinking Water. In validation studies for testing theequivalence of the quantitative detection of E. coli and coliforms, the Chromocult Coliform Agar method wascompared to the reference method of the ISO 9308-1:2000 "Detection and enumeration of Escherichia coli andcoliform bacteria, Part 1: Membrane filtration method."

The equivalence study was performed according to the recommendations of ISO 17994 "Water quality - Criteriafor the establishment of equivalence between microbiological methods." It was clearly demonstrated thatChromocult Coliform Agar is "equivalent or better" when compared to the reference method (Lactose TTC Agarwith Tergitol 7).

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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Chromocult Coliform Agar has been validated by the AOAC Research Institute under the Performance TestedMethodsSM 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) wasused for method comparison testing.

The Chromocult Coliform Agar method was found to be equivalent to the AOAC official method.

Mode of ActionThe interaction of selected peptones, pyruvate, sorbitol and phosphate buffer promotes rapid colony growth, evenfor sub lethally injured coliforms. The growth of Gram-positive bacteria as well as certain Gram-negative bacteriais 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 andE. coli.

The addition of antibiotic supplements is recommended if sample material is known to have high level ofbackground 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; sodiumpyruvate 1.0; tryptophan 1.0; agar-agar 10.0; Sorbitol 1.0; Tergitol 7 0.15; 6-chloro-3-indoxyl-beta-D-galactopyranoside 0.2; 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid 0.1; isopropyl-beta-D-thiogalactopyranoside 0.1.

PreparationPreparation 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 (0.2m 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 ofvancomycin (2.5 mg/vial) and cefsulodin (2.5 mg/vial) in lyophilized form. To prepare the supplement, suspendthe lyophilisate in the original vial by adding 2 ml of sterile purified water. Shake vigorously until the solution isclear. 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 stillliquefied, 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 PreparationPrepare test samples using standard laboratory techniques such as those described in the BacteriologicalAnalytical Manual, Standard Methods for the Examination of Water and Wastewater or the ISO standard specificfor the product concerned.

To minimize possible interference between the coloration of coliforms/ E. coli and the sample (e.g. low pH), it isadvisable to dilute the sample 1:10 in a buffered solution (e.g. add 450 ml Butterfield's phosphate buffer, bufferedpeptone water, or buffered sodium chloride peptone broth to blender jar containing 50 g of sample and blend 2min) and then to perform further dilutions as needed.

ApplicationWater testing

For water testing the agar medium base should be used with the addition of antibiotics. In the USEPA study theagar medium base was supplemented with 10 mg of cefsulodin per liter. The European study in Spain wasperformed 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 mlfor surface water) using a membrane filter. Place the filter on Chromocult Coliform Agar ensuring that no air istrapped 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 obtainedusing membrane filters of cellulose mixed-ester material, e.g. Pall Gelman GN-6 (OSSMER et al., 1999).

Food testing


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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 Petridish.

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 inoculumis 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.

ResultsCount 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-glucuronidaseactivity. 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 accompanyingbacteria) 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, shouldbe diluted prior to inoculation.

Calculate the number of E. coli and other coliforms per milliliter or per gram of sample from the number ofcharacteristic colonies in the plates.

LimitationsChromocult Coliform Agar is designed for rapid colony growth, even for sub lethally injured coliforms. Theselectivity of the agar medium base is limited. Samples with a high microbial load can lead to overgrowth of thewhole plate.

For samples with high microbial load use the agar medium base with antibiotics (approved for water testing) orthe Chromocult Coliform Agar ES (approved for food testing).

Some strains of Aeromonas spp. can grow as salmon to red colonies (similar to coliforms) on ChromocultColiform Agar when using the agar medium base without cefsoludin.

Aeromonads can be easily differentiated from coliforms by the oxidase reaction. Coliforms are oxidase-negativewhereas 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.


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Performance CharacteristicsWater Testing

USEPA study: The specificity of Chromocult Coliform Agar was assessed for recovery and identification of totalcoliforms and E. coli. Both positive and negative results were evaluated according to the EPA's ATP format forthe presence or absence of total coliforms and E. coli as compared to the reference methods. Based on theverifications for total coliforms, Chromocult Coliform Agar was found to have a false positive error of 2.0 % whilethe false negative rate (undetected target error) was 0 %. For E. coli, the false positive and negative rates were0 %.

EU Equivalence Study in Spain: The Chromocult Coliform Agar method was compared to the ISO 9308-1:2000reference method (Lactose TTC Agar with Tergitol 7). The coliform enumeration was 43% higher with theChromocult method compared to the TTC method. The E. coli enumeration was strongly dependent on theconfirmation method. When confirmation was based only on indole at 44C, the number of confirmed E. colicolonies with the Chromocult method was between 27 and 30% lower than with the TTC method (this largedifference 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 wereeliminated), 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 theChromocult and TTC methods. For coliforms the portion of presumptive colonies confirmed as positive (oxidase-negative) 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 ofthe 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 chickenand 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 theenumeration of E. coli and coliform bacteria. The food samples were pre-tested to establish the titer of naturallycontaminating E. coli and coliform bacteria. Three 300-gram samples of frankfurters, cooked chicken and non-fat 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-gramsample 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 mLof sterile diluent (Butterfield's phosphate buffer).

Following blending, five 50 gram samples were plated individually onto Chromocult Coliform Agar. Dilutions weremade using 99 mL of Butterfield's phosphate buffer as a diluent. Both the pour plate and spread plate techniquewas used for all foods. Plates were inverted and incubated aerobically at 35C for 24 1 hours. Enumeration ofdark-blue or violet colonies yielded a presumptive E. coli count while enumeration of salmon or red-coloredcolonies yielded a presumptive coliform count. Confirmation of typical E. coli colonies was done according toAOAC 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 probablenumber (MPN) series. The AOAC official method 966.24 "Most Probable Number Method for Coliform Bacteriaand E. coli" was followed. Typical E. coli colonies on L-EMB were confirmed by Gram stain, oxidase test, andanalysis 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 wasconsistent with results generated by the Chromocult Coliform Agar for all foods. Furthermore, presumptive E.


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Application

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 35Cfor 24 hours. All strains of E. coli and other coliforms showed characteristic growth. Overall sensitivity forChromocult Coliform Agar was 100%.

Forty four (44) pure cultures of non-coliform bacteria were cultured on Chromocult Coliform Agar at 35C for 24hours. Most of the non-coliform bacteria showed colorless growth or they were completely inhibited. Only 3 strainsshowed turquoise growth. There were no false positive reactions. Overall specificity for Chromocult ColiformAgar was 100%.

Storage conditions and Shelf lifeStore 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.

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

Technical assistanceFor 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: [email protected].

LiteratureAmerican 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 ofFecal Pollution Detection in Different Aquatic Habitats of a High-Altitude Tropical Country, Using PresumptiveColiforms, 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 andEscherichia 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 mediumfor 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 rulesfor microbiological examinations. ISO 7218:1996/Amendment 1:2001.

ISO International Standardisation Organisation. Microbiology of food and animal feeding stuffs - Preparationof test samples, initial suspension and decimal dilutions for microbiological examination. Part 1: General rules forthe 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 ofsampling programmes and sampling techniques. ISO 5667-1:2006.


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Application

ISO International Standardisation Organisation. Water quality - General guidance on the enumeration of micro-organisms 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. Aninterlaboratory 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. Health-Related Water Microbiology Symposium, Mallorca, Spain.

Ossmer, R., Schmidt, W., Mende, U. 1999. Chromocult Coliform Agar - Influence of Membrane Filter Qualityon Performance. - XVII Congresso de la Sociedad, Granada, Spain.

New Zealand Dairy Industry. 1998. Microbiological Methods Manual, Section 48: Product Test Methods - EntericIndicator Organisms. - NZTM 2; 48.5.1-48.5.10.

Suwansonthichai, S., and S. Rengpipat. 2003. Enumeration of coliforms and Escherichia coli in frozen blacktiger shrimp Penaeus monodon by conventional and rapid methods. International Journal of Food Microbiology81: 113 - 121.

Turner, K.M., Restaino, L., and Frampton, E.W. 2000. Efficacy of Chromocult Coliform Agar for Coliform andEscherichia 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 Samplingand Preparation of Sample Homogenate, AOAC International, Gaithersburg, MD.,USA.

Ordering InformationProduct Ordering No. Pack size

Chromocult Coliform Agar 1.10426 .0500 500 g

E. coli/Coliform Selective-Supplement

1.00898.0001 1 x 16 vials

Buffered Peptone water (BPW) 1.07228.0500 500 g

Sodium chloride peptone broth(buffered)

1.10582.0500 500 g


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Quality controlTest strains Inoculum

(CFU/plate)%

Recovery rateColony colour

Salmon- GAL

X- Glucuronide

Escherichia coliATCC 11775

10-100 70 dark-blue toviolet

Escherichia coliDSMZ 502

10-100 70 blue to violet

Citrobacterfreundii ATCC8090

10-100 70 salmon to red -

EnterobacteraerogenesATCC 13408


KlebsiellapneumoniaeATCC 13883


Salmonellaenteritidis ATCC13076

10-100 not limited colourless - -

Enterococcusfaecalis ATCC19433

1000-2000 0.01

Bacillus cereusATCC 11778

1000-2000 0.01

Citrobacter freundii ATCC 8090 Escherichia coli ATCC 11775


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Chromocult Coliform Agar

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