Reference : 01-695 Scharlau Microbiology - Technical data sheet

Product :
Microinstant ® CHROMOGENIC COLIFORMS
AGAR BASE

Also known as
CCA; ACC
Specification
Selective and differential medium for the detection and enumeration of coliforms and E. coli in water samples by MF
technique.
Formula * in g/L
Peptone...................................................... 3.00 Sorbitol....................................................... 1.00
Sodium chloride......................................... 5.00 6-Chloro-3-indoxyl-
Di-sodium hydrogen phosphate................. 2.70 ß-D-galactopyranoside............................... 0.20
Sodium dihydrogen phosphate 5-Bromo-4-chloro-3-
dihydrate.................................................... 2.20 indoxyl-ß-D-glucuronic acid........................0.20
Tryptophan................................................. 1.00 Agar..........................................................13.00
Sodium pyruvate........................................ 1.00
Tergitol®7.................................................... 0.15 Final pH 6,8 ±0,2 at 25 ºC

* Adjusted and /or supplemented as required to meet performance criteria

Directions
Suspend 29,45 g of powder in 1 L of distilled water and bring to the boil until fully dissolved. Do not autoclave nor
overheat. Cool until to 45-50ºC and add the contents of 2 vial of CV Selective Supplement for Coliforms (Art. No. 06
-140LYO1). Mix well and distribute into Petri dishes avoiding bubble formation. The finished plates remain effectives for
at least one month if stored in the dark between 2-8ºC.
Description
The combined action of peptone, pyruvate and sorbitol allow rapid colony growth in this phosphate buffered medium,
which also permits simple recovery of sublethal thermally injured coliforms. Sodium chloride provides the correct osmotic
environment necessary for growth.
The selectivity is attained, partially, by the Tergitol® 7, which inhibits the growth of Gram positive bacteria and some Gram
negative without effecting the coliform bacteria. Selectivity is enhanced by the cefsulodin and Vancomycin that which acts
against pseudomonas and Gram negative oxidase positive bacteria enterococci and other Gram positive bacteria.
The colonial differentiation is due to the chromogenic mixture, composed of two enzyme substrates: 6-chloro-3-indoxyl-ß-
D-galacto-pyranoside (Salmon®-GAL) and 5-bromo-4-chloro-3-indoxyl-ß-D-glucuronide (X-Glucuronide).
The first one is cleaved by the characteristic enzyme found in coliforms, ß-D-galactosidase and gives a salmon-red
colour to the coliform colonies. The second chromogenic substance is cleaved by the ß-D-glucuronidase enzyme
characteristic of E. coli and turns the colonies of these bacteria a blue colour.
E. coli has the two enzymes and cleaves both chromogenic substances giving dark blue to violet colonies. Total coliforms
are the sum of E. coli colonies plus salmon-red colonies.
Other Gram negative bacteria produce colourless colonies except some that possess glucuronidase activity (but not
galactosidase) and they produce light blue to turquoise colonies.
To confirm the E. coli colonies in this medium a small amount of tryptophane is included verifying indol production: coat
the blue-violet colonies with a drop of Kovacs Reagent. If the reagent turns a cherry-red colour in a few seconds this
confirms the production of indol and hence the presence of E. coli.
When the Chromogenic Agar for Coliform is used with the membrane filter method, the colour and growth of the colonies
can be modified by the characteristics of the membrane filter. It is advisable to perform validation of the membrane filter
type used.
The Spanish Health Ministry (Ministerio de Sanidad y Consumo) has officially adopted this medium as an alternative
methodology for the microbiological analysis of water for human consumption, giving a new definition for Escherichia coli
("Enterobacteriaceae that express the ß-D- galactosidase and the ß-D-glucuronidase enzymes simultaneously") and
coliform bacteria: "Enterobacteriaceae that express the ß-D-galactosidase enzyme".
Limitation of the procedure:
The production of β-galactosidase, although common to all the coliforms, varies from one strain to another being
influenced by the temperature and incubation time. At temperatures above 37 ° C its production decreases, causing a
loss of reddish color intensity, while the bluish tones in the strains of Escherichia coli are accentuated.
If the membrane filtration method is used, it must be taken into account that the nature and characteristics of the filter
membrane used also influences the size and color of the colonies grown on this culture medium.

Technical data sheet - page 1 of 4 Revision date : 17/03/2021

 Reference : 01-695 Scharlau Microbiology - Technical data sheet
Product :
Microinstant ® CHROMOGENIC COLIFORMS
AGAR BASE

Technique
The water sample is filtered trough a membrane filter of 0.45 µm pore diameter validated according to the ISO Standard
7704:1985. The membrane is then placed on the surface of the CCA medium avoiding entrapment of air bubbles
between the membrane and agar surface.
The petri dish with the membrane is incubated for 18-24 hours at 36 ± 2ºC. If in 18 h there is growth of red or colourless
colonies, extend the incubation until 24 h to include late reactions of ß-galactosidase or ß-glucuronidase. Count ß-
galactosidase positive colonies and ß-glucuronidase negative colonies (all colonies coloured from salmon-rose to red) as
Coliform bacteria not-E. coli.
Count ß-galactosidase positive colonies and ß-glucuronidase positive colonies (all colonies coloured from deep blue to
violet) as E. coli.
Total Coliform count is obtained by the addition of the salmon-rose to red colonies plus the deep blue to violet colonies.
Calculate the concentration of Coliform bacteria and E. coli in 100 mL from the initial volume of water filtered and the
number of characteristic colonies counted on the membrane. The results are expressed as Colony Forming Units per
millilitre (CFU/mL).

Supplement:

Coliform CV Selective Supplement

Vial Contents:
Necessary amount for 500 mL of complete medium.
Cefsulodin.................................................................... 2.50 mg
Vancomycin.................................................................. 2.50 mg

Distilled water (Solvent)

Quality control
Incubation temperature: 36ºC ±2.0 Incubation time: 18-24 h
Inoculum: Practical range 100 ± 20 CFU. Min. 50 CFU (Productivity)/ 104-106 CFU (Selectivity) / ≥ 10³ CFU (specificity)
according to ISO 11133:2014.
Microorganism Growth Remarks
Pseudomonas aeruginosa ATCC® 10145 Inhibited ( w. inh. supplement) w/o supplem. Inh. : Colorless colonies. Indol (-)
Escherichia coli ATCC® 25922 Productivity > 0.70 Blue-violet colonies. Indol (+)
Escherichia coli ATCC® 8739 Productivity > 0.70 Blue-violet colonies. Indol (+)
Enterobacter aerogenes ATCC® 13048 Productivity > 0.70 Salmon to red colonies. Indol (-)
Salmonella typhimurium ATCC® 14028 Good Colorless colonies. Indol (-)
Citrobacter freundii ATCC® 43864 Productivity > 0.70 Salmon to red colonies. Indol (-)
Enterococcus faecalis ATCC® 19433 Inhibited (w. Inh. supplm.) -

Salmonella typhimurium ATCC 14028 Salmonella typhimurium ATCC 14028 Escherichia coli ATCC 25922
Escherichia coli ATCC® 8739

Technical data sheet - page 2 of 4 Revision date : 17/03/2021

 Reference : 01-695 Scharlau Microbiology - Technical data sheet
Product :
Microinstant ® CHROMOGENIC COLIFORMS
AGAR BASE

References
· ADAMS, M.., R.GRUBB, S.M. HAMER & A. CLIFFORD (1990) Colorimetric enumeration of Escherichia coli based on
ß-glucuronidase activity. Appl. Environ. Microbiol. 56:2021.
· ISO 7704 Standard (1985) Water Quality - Evaluation of membrane filters used for micropbiological analyses.
. ISO 11133:2014/ Adm 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and
performance testing of culture media.
· KILIAN, M. & P. BÜLOW (1976) Rapid Diagnostic of Enterobacteriaceae. I. Detection of bacterial glycosidases. Acta
Pathol. Microbiol. Scand. Sect. B 84:245-251.
· MANAFI, M & W. KNEIFEL (1989) A combined chromogenic-fluorogenic medium for the simultaneous detection of total
coliform and E. coli in water. Zentralbl. Hyg. 189:225-234.
· MINISTERIO DE SANIDAD Y CONSUMO (2009) Orden SCO/778/2009 de 17 de marzo sobre métodos alternativos
para el análisis microbiológico del agua de consumo humano. BOE. n.º 78 de 31-04-2009. Sección I, Págs. 30417
-30420. Madrid.
.TURNER, K.M., L. RESTAINO & E.W. FRAMPTON (2000) Efficacy of Chromocult Coliform Agar for coliform and
Escherichia coli detection in Foods. J.Food Protect. 63(4):539-541
Storage
For laboratory use only. Keep tightly closed, away from bright light, in a cool dry place (+4 ºC to 30 ºC).
Packaging

Technical data sheet - page 3 of 4 Revision date : 17/03/2021