Chromogenic culture media: how they work and when to use them

Chromogenic culture media are selective media containing synthetic substrates made of a sugar bound to a colourless chromophore. When an enzyme of the target organism hydrolyses the bond, the chromophore is released, precipitates inside the colony and gives it a stable colour. The most widely used substrate is a halogenated indolyl: 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, or X-glucuronide, is cleaved by the β-glucuronidase of Escherichia coli and produces blue-green colonies on TBX, while on CCA the same enzyme turns them dark-blue to violet. On a traditional medium the colony is recognised by morphology and then confirmed with biochemical tests; a chromogenic medium moves presumptive identification onto the primary plate, because colour is read instead of shape: fewer confirmation steps, and a reading that depends less on operator experience.

How is colony colour read?

Colour depends on which enzyme is present and which substrate the medium carries. Chromogenic Coliforms Agar (CCA) carries two substrates: β-D-galactosidase, common to all coliforms, cleaves Salmon-GAL and turns the colonies pink to red; β-D-glucuronidase, typical of E. coli, cleaves X-glucuronide and turns them dark-blue to violet. A colony expressing both enzymes shows both precipitates together. ISO 9308-1 asks for one further step: pink to red colonies count as coliforms only if oxidase negative — this is how Aeromonas spp. are excluded — together with all the dark-blue to violet ones.

Other media combine the chromogenic substrate with an opacity reaction. On Listeria Chromogenic Agar according to Ottaviani and Agosti (ALOA), β-glucosidase — present across the whole Listeria genus — turns every colony blue-green; phosphatidylinositol-specific phospholipase C (PI-PLC), produced only by L. monocytogenes and L. ivanovii, hydrolyses the phosphatidylinositol in the medium and surrounds the colony with an opaque halo. Blue-green with an opaque halo means presumptive L. monocytogenes, and presumptive only: ISO 11290-1 warns in the same clause that L. ivanovii colonies are also blue-green and surrounded by an opaque halo. Blue-green without a halo, another Listeria.

Which ISO methods prescribe a chromogenic medium?

The ISO horizontal methods for the microbiology of the food chain and for water quality name the chromogenic medium in the normative text itself. The table summarises the cases most often met in control laboratories.

What a chromogenic medium does not solve

Colour gives a presumptive identification, not a confirmation: the normative method decides whether and how the colony is confirmed. There are also precise biological limits. On CCA, β-D-glucuronidase-negative E. coli strains, such as E. coli O157, do not turn blue and end up counted among coliforms. Nor are the enzymes exclusive: some Shigella strains give light blue colonies on CCA, and 64 non-Listeria isolates, of 12 species in 7 genera, have been described growing blue-green on ALOA. This is why ISO 11290-1 prescribes two selective solid media of different principle; ALOA alone belongs to part 2, the enumeration one. Test conditions matter as well: ISO 16649-2 requires (44 ± 1) °C for 18-24 h, ISO 9308-1:2014/Amd 1:2016 requires (36 ± 2) °C for (21 to 24) h. Outside that, chromogen yield and selectivity are not the validated ones.

How is the performance of a chromogenic medium controlled?

The reference is ISO 11133:2014 + Amd 1:2018 + Amd 2:2020, which describes preparation, production, storage and performance testing of culture media for food, feed and water. Three tests are required: productivity on the target strain against a non-selective reference, selectivity towards non-target flora, specificity of the chromogenic reaction. With a ready-to-use medium the manufacturer runs the batch tests and documents them in the certificate of analysis, and the laboratory does not repeat them: provided the manufacturer works under a certified quality system and provided transport and storage conditions have been met. Supplier qualification, the incoming check, verification of the certificate and the management of storage and shelf life remain the laboratory's own. With a dehydrated medium reconstituted in house, performance testing of the prepared batch falls to whoever prepares it.

Chromogenic media in the catalogue

The references below are Condalab chromogenic media in the CABRU catalogue, in dehydrated format. The range also covers ready-to-use plates, flasks and selective supplements: the full list is in the product catalogue and in the microbiology culture media category.

For the reference suited to a given matrix and method, or for a quotation, just state the code or the name of the medium and write to CABRU.