Technical Data Sheet

Chromocult® Coliform Agar acc. ISO 9308-1

Ordering number: 1.10426.0500

For the simultaneous detection of coliform bacteria and E. coli in drinking water, waters with
low bacterial background flora and processed food samples.

Drinking water and waters with low bacterial background: This culture medium
complies with the specifications given by EN ISO 9308-1.

Chromocult® Coliform Agar membrane filter method has been approved and accepted
by the U.S. Environmental Protection Agency (EPA) for monitoring total coliform bacteria
and E. coli under the Total Coliform Rule (40 CFR 141.21).

Processed food: Chromocult® Coliform Agar acc. ISO 9308-1 (CCA) 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.

Mode of Action

The interaction of selected peptones, pyruvate, sorbitol and phosphate buffer promotes
rapid colony growth, even for sub-lethally injured coliform bacteria. The growth of Gram-
positive bacteria as well as certain Gram-negative bacteria is inhibited by the presence of
Tergitol®7 which has no negative effect on the growth of coliform bacteria.
Further, the combination of two chromogenic substrates permits the simultaneous
detection of coliform bacteria and E. coli.
Coliform bacteria detection: The substrate Salmon-GAL is used for the detection of β-
D-galactosidase activity, which is characteristic for coliform bacteria. This interaction
results in a pink to red color of the coliform colonies.
E. oli detection: The substrate X-glucuronide is used for the detection of β-D-
glucuronidase activity, 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 pink to red color.
Some E. coli (3-4%) are β-D-glucuronidase-negative and appear as pink to red colonies,
e.g. most E. coli O157 strains. For the detection of E. coli O157 specific culture media
should be used.
Accompanying flora appears as colorless colonies, except for some organisms, which
possess β-D-glucuronidase activity. These colonies appear light blue to turquoise in color.

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 Typical Composition

Specified by ISO 9308-1 Chromocult® Coliform Agar

Enzymatic Digest of Casein 1 g/l Enzymatic Digest of Casein 1 g/l
Yeast Extract 2 g/l Yeast Extract 2 g/l
NaCl 5 g/l NaCl 5 g/l
NaH2PO4 x 2 H2O 2.2 g/l NaH2PO4 x 2 H2O 2.2 g/l
Na2HPO4 2.7 g/l Na2HPO4 2.7 g/l
Sodium Pyruvate 1 g/l Sodium Pyruvate 1 g/l
Sorbitol 1 g/l Sorbitol 1 g/l
Tryptophane 1 g/l Tryptophane 1 g/l
Secondary alcohol ethyloxylate
surfactant (CAS No. 68131-40-8) 0.15 g/l Tergitol® 7 0.15 g/l
(e.g. Tergitol® 15-S-7 surfactant)
6-chloro-3-indoxyl-beta-D-
6-Chloro-3-indoxyl-beta-
galactopyranoside (Salmon-beta-D- 0.2 g/l 0.2 g/l
D-galactopyranoside
galactosid), (CAS No. 138182-21-5)
5-Bromo-4-chloro-3-indoxyl-β-D-
glucuronic acid, cyclohexylammonium salt 5-Bromo-4-chloro-3-
monohydrate (X-beta-D-glucuronide CHX 0.1 g/l indoxyl-D-glucuronic 0.1 g/l
salt), (CAS No. 114162-64-0) acid
Isopropyl-β-D-thiogalactopyranoside Isopropyl-beta-D-
0.1 g/l 0.1 g/l
(IPTG), (CAS No. 367-93-1) thiogalactopyranoside
Bacteriological agar 9-18 g/l Agar-agar* 10 g/l
Water 1000 ml Water n.a.

* Agar-agar is equivalent to other different terms of agar.

Preparation

Dissolve 26.5 g in 1 liter of purified water. Heat in boiling water and agitate frequently
until completely dissolved. Do not autoclave, do not overheat!

Cool the medium to 45 - 50 °C and pour plates. According to EN ISO 9308-1 dispense in
Petri dishes to a depth of at least 4 mm (approximately 18 ml in a 90 mm plate).

For using by poured plate method cool the medium to 44 - 47 °C in a water bath before
use (see EN ISO 11133).

The prepared medium is slightly turbid to turbid and yellowish. The pH value at 25 °C is in
the range of 6.6-7.0

There should be no visible moisture on the plates before use. When moisture is present,
the plates should be dried for the minimum time required to remove visible moisture,
following the procedure as described by EN ISO 11133.

Preparation and addition of optional Cefsulodin solution (US-EPA approved

method): Dissolve 5 mg of Cefsulodin in 2 ml of purified water and sterilize by
membrane filtration (0.2 μm nominal pore size). Aseptically add the solution (2 ml per 500
ml of medium) to 500 ml of liquefied medium (45 - 50 °C). Pour plates as described
above.

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 Experimental Procedure and Evaluation

Prepare test samples using standard laboratory techniques such as those described in the
applicable ISO standard, Bacteriological Analytical Manual, or Standard Methods for the
Examination of Water and Wastewater specific for the product concerned.

Drinking water and waters with low bacterial background: Chromocult® Coliform
Agar acc. ISO 9308-1 is usually combined with membrane filtration for water analysis.

Note: The type and quality of membrane filter affects the size, coloration and number
of colonies significantly. Merck Millipore mixed cellulose ester filters (gridded, 0.45 µm pore
size, EZ-PakTM, Merck, article number EZHAWG474) were used in ISO validation studies
(Lange et al. 2013) and supported the color formation and the growth of colonies
efficiently.
• Filter appropriate volume of sample (e.g. 100 ml municipal drinking water, 250 ml
bottled water) using a membrane filter.
• Place filter on CCA plate ensuring that no air is trapped underneath.
• Incubate the inoculated dishes aerobically in an inverted position at 36 °C ± 2°C
for 21-24 h

Note: Do not incubate longer than 24 h to reduce risk of counting unwanted

microorganisms with similar colony colors.

• After incubation, examine the membrane filters and count all colonies giving a
positive β-D-galactosidase and β-D-glucuronidase reaction (dark-blue to violet) as
E. coli.
• Count all colonies giving a positive β-D-galactosidase reaction (pink to red) as
presumptive coliform bacteria that are not E. coli.
• Confirm pink to red colonies by a negative oxidase reaction (to avoid false-positive
results caused by oxidase-positive bacteria like Aeromonas spp.).

Note: In ISO validation studies oxidase activity of presumptive coliform colonies was
tested using Bactident® Oxidase (article number 113300) test.

Processed food

For food analysis, Chromocult® Coliform Agar acc. to ISO 9308-1 is usually inoculated by
the pour plate method.

• Using a sterile pipette, transfer 1 ml of liquid test sample or 1 ml from the

appropriate dilution to a sterile Petri dish.
• Pour into about 15 ml of the CCA at 44 - 47 °C into each Petri dish.
• Carefully mix the inoculum with the medium by rotating the Petri dishes and allow
the mixture to solidify by leaving the Petri dishes standing on a cool horizontal
surface.
• Incubate the inoculated dishes aerobically at 35 - 37 °C in an inverted position for
24 hours.
• After incubation, examine the plates for presence of typical colored colonies of E.
coli and other coliform bacteria.

For fresh food samples with a higher microbial load, Chromocult® Coliform Agar ES (article
number 100850) is recommended.

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 Validation studies - Water testing

EN ISO 9308-1:2014. The performance of Chromocult® Coliform Agar was validated

according to ENV ISO 13843 (“Water quality – Guidance on validation of microbiological
methods”) using pure cultures and naturally contaminated water samples. Samples were
processed according to ISO 9308-1 using EZ-Pak membrane filters (Merck) for filtration.
In total 220 colonies, including typical E. coli and coliform bacteria as well as atypical
colonies, were randomly selected to determine the fundamental characteristics of
CCA.

The results show that Chromocult® Coliform Agar acc. ISO 9308-1 is a reliable
method for the quantification of both E. coli and coliform bacteria.

US-EPA. Chromocult® Coliform Agar membrane filter method has been approved and
accepted by the U.S. Environmental Protection Agency (EPA) for monitoring total coliform
bacteria and E. coli under the Total Coliform Rule (40 CFR 141.21).

In validation studies for the detection of total coliform bacteria and E. coli in
drinking water the Chromocult® Coliform Agar supplemented with 2,5 mg / L Cefsulodin
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 coliform bacteria and E. coli.

Food testing

AOAC-RI (License Number 020902). 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.

Storage

Store the dehydrated medium dry and tightly closed. Protect from light. Do not use
clumped or dis-colored medium. Store at +15 °C to +25 °C and use before the expiry date
on the label.

According to EN ISO 9308-1, self-prepared plates can be stored at +2 °C to +8 °C in the

dark and protected against evaporation for at least one month.

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 Quality Control

Reference Method of
Function Control strains Incubation Expected results
medium control
Escherichia coli
Quantitative Recovery ≥ 70
ATCC 25922 18-24 h at Tryptic
with membrane %, dark-blue to
Escherichia coli 35-37 °C Soy Agar
filtration violet colonies
ATCC 8739
Enterobacter
Productivity
aerogenes
Quantitative
ATCC 13048 18-24 h at Tryptic Recovery ≥ 70
with
Citrobacter 35-37 °C Soy Agar %, pink to red
membrane
freundii colonies
filtration
ATCC 43864
Qualitative with
Enterococcu Partial inhibition,
18-24 h at membrane
Selectivity s faecalis colorless colonies
35-37 °C filtration
ATCC 19433
quantitative
Qualitative with
Pseudomona No recovery limit
18-24 h at membrane
Specificity s aeruginosa specified,
35-37 °C filtration
ATCC 10145 colorless colonies
quantitative

Please refer to the actual batch related Certificate of Analysis.

The performance test is in accordance with the current version of EN ISO 11133 and EN
ISO 9308-1 using Merck Millipore mixed cellulose ester filters (0.45 µm pore size).

A recovery rate of 70 % is equivalent to a productivity value of 0.7.

E. coli ATCC 11775 Citrobacter freundii ATCC 8090

1.10426.0500 Page 5 of 6
 Literature

ISO International Standardisation Organisation. Water quality – Enumeration of

Escherichia coli and coliform bacteria. Part 1: Membrane filtration method for waters with
low bacterial background flora. EN ISO 9308-1:2014.

ISO International Standardisation Organisation. Microbiology of food, animal feed

and water – Preparation, production, storage and performance testing of culture media.
EN ISO 11133:2014.

US-EPA approved method. 40 CFR Part 141 (sec 141.21) Federal Register/Vol. 67, No.
209, Tuesday October 29, 2002/Rules and Regulations.

New Zealand Dairy Industry. (1998): Microbiological Methods Manual, Section 48:
Product Test Methods - Enteric Indicator Organisms. NZTM 2: 48.5.1-48.5.10.

Byamukama, D., Kansiime, F., March R.L. and Farnleitner A.H. (2000): Determination of
Escherichia coli contamination with Chromocult Coliform Agar showed a high level of
discrimination efficiency for differening fecal pollution levels in tropical waters of
Kampala, Uganda. Appl. Environ. Microbiol. 66: 864-868.

Geissler, K., Manafi, M., Amoros, I. and Alonso J.L. (2000): Quantitative determination of
total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic
media. J. Appl. Microbiol. 88: 280-285.

Lange, B., Strathmann, M. and Oßmer, R (2013): Performance validation of chromogenic

coliform agar for the enumeration of Escherichia coli and coliform bacteria. Lett. Appl.
Microbiol. 57: 547-553.

Ordering Information

Product Cat. No. Pack size

Chromocult® Coliform Agar acc. ISO 9308-1 1.1.0426.0500 500 g
ReadyPlate™ CHROM Chromocult ®
1.46689.0020 20 x 90 mm plates
Coliform Agar acc. ISO 9308-1:2014
ReadyPlate™55 Chromocult® Coliform 1.46757.0020 20 x 55 mm plates
Agar acc. ISO 9308-1:2014
ReadyPlate™55 Chromocult® Coliform 1.46757.0200 200 x 55 mm plates
Agar acc. ISO 9308-1:2014
Chromocult® Coliform Agar ES 1.00850.0500 500 g
EZ-Pak™ cellulose mixed ester filter 4 bands x 150
EZHAWG474
(gridded, 0,45 μm pore size) membrane filters
S-Pak™ cellulose mixed ester filter 4 boxes of 600 membrane
HAWG047S6
(gridded, 0,45 μm pore size) filters, individually packed
Bactident® Oxidase 1.13300.0001 50 strips

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