EPA

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.

Intended User
Microbiological examinations are to be performed by trained
staff only.

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 EPAapproved 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
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 -Dgalactosidase, 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; 6chloro-3-indoxyl-beta-D-galactopyranoside 0.2; 5-bromo-4chloro-3-indoxyl-beta-D-glucuronic acid 0.1; isopropyl-beta-Dthiogalactopyranoside 0.1.

Preparation
Preparation of agar medium
Suspend 13.25 g in 500 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.

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Chromocult Coliform Agar

Preparation of optional antibiotic supplements

(for water testing)
Cefsulodin solution:
Dissolve 5 mg of cefsulodin in 2 ml of purified water and sterilize
by membranefiltration (0.2 m nominal pore size). Aseptically
add the solution (2 ml per 500 ml of medium) to 500 ml of liquefied medium (45-50C).
E. coli/Coliform Selective Supplement (Merck
Cat.No.1.00898.0010, EMD Chemicals Cat.No. 1.00898.0017):
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 1 vial (2 ml per 500 ml
of medium) to 500 ml of liquefied medium (45-50C).
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 the liquefied medium (45-50C) in Petri dishes to a
depth of at least 4 mm (approximately 18 ml in a 90 mm plate).
Allow the medium to solidify on a cool horizontal surface.
The medium is slightly opalescent and yellowish.
pH: 6.8 0.2 at 25 C.
Prepared plates can be stored in plastic pouches or bags for up
to 6 months (or up to 1 month if antibiotic supplements have
been added) at 2 - 8C and protected from light.
The agar plates must be dry before use.

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 litre. 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
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 millilitre
or per gram of sample from the number of characteristic colonies
in the plates.

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Chromocult Coliform Agar

Limitations
Chromocult Coliform Agar is designed for rapid colony growth,
even for sublethally 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 have to be dry before use. In case the agar surface is wet,
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 technique as the preferred
method.

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 falsepositive 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
(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 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 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 102 CFU/g), and "high" (targeted spike of 103 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 redcolored 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. 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.

Merck Microbiology Manual 12th Edition

Chromocult Coliform Agar

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
Both, contaminated and not used culture media must be disposed
in a way which is safe and meets state or national regulations.
The Material Safety Data Sheet (MSDS) provides detailed information on disposal of each medium. Safety information on
ingredients and culture media can be down loaded from Internet
www.merck-chemicals.com.

Technical assistance
USA - EMD Chemicals 480 S. Democrat Road, Gibbstown, NJ
08027-1297
Phone: 800-222-0342 or 856-423-6300
Fax: 856-423-4389
Email: emdinfo@emdchemicals.com
Website: www.emdchemicals.com
Europe/Rest of World - Merck KGaA, 64271 Darmstadt, Germany.
Tel.: +49-6151-72 20
Fax: +49-6151-723380
Email: microbiology@merck.de
Website: www.merck-chemicals.com

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.

ISO International Standardisation Organisation. 2006. Water quality Sampling. Part 1: Guidance on the design of sampling programmes and sampling techniques. ISO 5667-1:2006.
ISO International Standardisation Organisation. 2005. 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. 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. Health-Related 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.
U.S. Environmental Protection Agency. 2002. 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, http://www.cfsan.fda.gov/~ebam/bam-1.html, accessed June 24, 2008

Ordering Information
Product

Ordering No.

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.

Chromocult Coliform
Agar

1.10426.0500

500 g

E. coli/Coliform SelectiveSupplement

1.00898.0010

1 x 10 vials

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.

Buffered Peptone water

(BPW)

1.07228.0500

500 g

Sodium chloride peptone

broth (buffered)

1.10582.0500

500 g

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.

Pack size

ISO International Standardisation Organisation. 2001. Microbiology of

food and animal feeding stuffs - General rules for microbiological examinations. ISO 7218:1996/Amendment 1:2001.
ISO International Standardisation Organisation. 1999. 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.

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Chromocult Coliform Agar

Quality control
Test strains

Inoculum
(CFU/plate)

%
Recovery rate

Colony
colour

SalmonGAL

XGlucuronide

Escherichia coli ATCC 11775

10-100

70

dark-blue to
violet

Escherichia coli DSMZ 502

10-100

70

blue to violet

Citrobacter freundii ATCC 8090

10-100

70

salmon to red

Enterobacter aerogenes ATCC 13408

10-100

70

salmon to red

Klebsiella pneumoniaeATCC 13883

10-100

70

salmon to red

Salmonella enteritidis ATCC 13076

10-100

not limited

colourless

Enterococcus faecalis ATCC 19433

1000-2000

0.01

Bacillus cereus ATCC 11778

1000-2000

0.01

Citrobacter freundii ATCC 8090

Escherichia coli ATCC 11775

Merck Microbiology Manual 12th Edition