Chromogenic and Fluorogenic Culture Media

B-7450_substrate_on_agar_plate

Culture media are used for growth, isolation and detection of micro-organisms. They generally consist of nutrients, such as peptones, amino acids, meat- and yeast-extracts; minerals and vitamins, inhibitors, solidifying agents (mostly agar-agar) and detection systems.

Detection systems of conventional culture media combine various carbohydrates, amino acids, and glycosides (e.g. esculin) with a suitable indicator-system. The incorporation of chromogenic or fluorogenic enzyme substrates as a detection system has led to the development of numerous primary isolation media. Chromogenic or fluorogenic enzyme substrates consist of a so-called chromophor or fluorophor linked to an enzyme-recognizing-part, such as a carbohydrate, amino acid or phosphate. Specific enzymes produced by the target micro-organism cleave the chromogenic or fluorogenic substrate liberating the chromophor or fluorophor, which highlight the micro-organism by fluorescence in culture media or coloration of the grown colony.

Two main groups of chemical compounds used in culture media are indolyl-derivatives and 4-methylumbelliferyl-substrates. The latter are water-soluble and detectable by UV only. The use of the highly sensitive fluorogenic substrates in solid media is limited because they diffuse and UV-light is needed for detection. The indolyl-derivatives are mostly non-diffusible, and the chromophor precipitates in the colony. Moreover, recognition and handling of colored colonies is an advantage in practical microbiological work.

Most recently, the first of a kind of fluorogenic plating media that function in analogy to the well established chromogenic plating media has been launched (BCM® MRSA ELF® and BCM® S. aureus ELF® ). Just like their chromogenic counterparts these media contain an enzyme substrate which stains growing colonies that produce a more or less specific marker enzyme. Colonies growing on ELF® based BCM® plating media are stained by ELF-OH which is a molecule which is highly fluorescent in the solid state emitting green light upon absorption of UV light in 370 nm range.

Chromogenic culture media utilizing virulence factors of the target microorganisms have become an attractive alternative, because they combine both rapid detection and quantification of microorganisms, including pathogenic bacteria, at low costs. Supplementation of these chromogenic plating media with inhibitors, such as antibiotics mostly or completely inhibits contaminants occurring in the samples to be tested.


Typical Enzyme Reaction Utilizing a Chromogenic Substrate

Example-chromogenic-enzyme-reaction



Download: Application Note

Cultural Response Tests using Chromogenic and Fluorogenic Substrates

Properties of Important Chromophors and Fluorophors:

Chromophor / FluorophorAbbreviationAbsorption(nm)/Color/Fluorescence
5-Bromo-4-chloro-3-indoxyl- "X" 615 Blue-turquoise to mint-green
5-Bromo-3-indoxyl- Lapis™ 610 Blue
5-Bromo-6-chloro-3-indoxyl- Magenta™ 564 Purple to magenta
6-Chloro-3-indoxyl- Salmon™ 540 Rose to salmon
N-Methylindoxyl- Green™ 665 Green
2-Nitrophenyl- ONP 405 Yellow
4-Methylumbelliferyl- MU 460 Blue (UV-light 365 nm)
ELF® ELF® upon UV-light, Green, emission 377 nm




Selected Chromogenic and Fluorogenic Substrates and their Applications:

SubstrateTarget MicroorganismApplicationChromogenicFluorogenic
-beta-D-glucuronide E. coli Water, food, clinical samples yes yes
-beta-D-galacto-pyranoside Enterobacteriaceae, coliforms, research Water, food, clinical samples yes no
-beta-D-gluco-pyranoside Enterococci, Klebsiella spp.,Enterobacter spp. Water, food,clinical samples yes no
-N-acety-beta-D-galactosaminide Candida albicans Clinical samples yes no
-N-acetyl-beta-D-glucosaminide Candida albicans Clinical samples yes yes
-caprylate-nonanoate Salmonella spp. Food, clinical samples, environmental samples yes yes
-myo-inositol-1-phosphate Pathogenic Listeria spp., B. cereus,B. thuringiensis, B. mycoides Food, clinical samples, hygiene monitoring yes yes
-phosphate S. aureus, C. perfringens Food, clinical samples yes yes

 


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