Microbial Detection Using Chromogenic and Fluorogenic Culture Media

As the rapid detection and identification of microorganisms becomes increasingly vital in microbiology, fluorogenic and chromogenic substrates have emerged as powerful tools, leveraging the specific enzymatic activities of certain microorganisms. By incorporating synthetic fluorogenic or chromogenic substrates into primary selective media, enumeration and detection can be conducted directly on the isolation plate.

Chromogenic and fluorogenic media are specialized microbiological growth media that contain enzyme substrates linked to a chromogen (color reaction), fluorogen (light reaction) or a combination of both. These media offer significant advantages for the enumeration, detection, and identification of microorganisms. They facilitate the screening of all major food pathogens, including Salmonella, Campylobacter, Listeria, Listeria monocytogenes, Staphylococcus/S. aureus, Coliforms, E. coli as well as specific target microorganisms such as, E. coli O157.

Section Overview

• Principle of the Fluorogenic and Chromogenic Technique
• Advantages of Chromogenic Media
• ChromoCult^® Coliform Agar
  • ChromoCult^® Coliform Agar ES
• ReadyCult^® Fluorogenic Media
  • ReadyCult^® Coliforms 100
  • ReadyCult^® Enterococci 100
• MilliChrome™ Chromogenic Culture Media
• Related Products

Principle of the Fluorogenic and Chromogenic Technique

The comparison between traditional and enhanced media formulations containing chromogenic substrates is a significant topic in microbiology today. Fluorogenic enzyme substrates typically consist of a specific substrate tailored for a particular enzyme (e.g., sugar or amino acid) and a fluorogen, such as 4-methylumbelliferone, which emits a blue fluorescence in a positive result. The substrate MUG (4-methylumbelliferyl-β-D-glucuronide) is commonly added to traditional media for the detection of Escherichia coli or E. coli O157.

Chromogenic substrates such as ONPG, X-Gal, or X-Glu, combined with a specific medium selectivity, facilitate the easy differentiation of microorganisms based on the coloration of grown bacteria. The enzyme characteristic of the target organism cleaves the chromogenic substrate into a sugar component and a chromogen, resulting in a distinct color change in liquid broth or on agar plates in the presence of oxygen. This enables direct confirmation of the target organism without the need for further testing.

Furthermore, it is now possible to detect and differentiate multiple organisms on a single plate. By integrating several chromogenic substrates with appropriate selectivity, different species or groups of microorganisms can be differentiated on one plate. Most chromogenic media are both selective and differential, inhibiting non-target organisms while allowing target pathogens to develop colored colonies due to their metabolism of one or more chromogenic enzyme substrates.

Chromogenic substrates and selective agents

In Table 1, known substrates and selective agents are listed and give some idea about additional possibilities.

Species	Enzyme	Substrate	Selective Agents
Bacillus cereus	β-glucosidase, Phosphatidylinositol-specific Phospholipase C	indoxyl-β-glucopyranoside, indoxyl-myo-inositol-1-phosphate	polymyxin B
Campylobacter	na	na	deoxycholate, cefoperazone, amphotericin B
Candida	β-acetylgalactosaminidase, alkaline phosphatase	indoxyl-N-acetyl-β-D-glucosaminide, indoxyl-phosphate	Chloramphenicol, Gentamicin
Clostridium perfingens	β-glucosidase (plus sucrose fermentation)	indoxyl-β-D-glucoside	D-cycloserine, polymixin B
Coliforms/E. coli	β-glucuronidase, β-galactosidase	indoxyl-β-glucuronide, Indoxyl-β-galactoside	bile salts, tergitol 7<R>, SDS, novobiocin, cefsulodin
Cronobacter (E.sakazakii)	a-glucosidase	indoxyl-a-D-glucoside	deoxycholate, crystal violet, sodium thiosulfate
E. coli O157	β-glucosidase, a-galactosidase	indoxyl-β-D-glucuronide, indoxyl-a-galatoside	bile salts, SDS, crystal violet, potassium tellurite, novobiocin, cefixime
Enterococci	β-D-glucosidase	indoxyl-β-glucoside	sodium azide, polysorbate 80
Extended Spectrum ß-Lactamase Enterobacteria (ESBL)	β-D-glucosidase	indoxyl-β-glucoside	cefpodoxime, cefotaxime, ceftazidime
Klebsiella	β-D-ribofuranosidase, β-D-glucosidase	indoxyl-β-D-ribofuranoside, indoxyl-β-D-glucoside	bile salts, SDS, carbenicillin
Listeria spp.	β- glucosidase	indoxyl-β-glucoside	lithium chloride, ceftazidime, amphotericin B, nalidixic acid, polymyxin B
L. monocytogenes	Phosphatidylinositol-specific Phospholipase C, β- glucosidase,	indoxyl-β-glucoside, indoxyl-myo-inositol-1-phosphate	lithium chloride, ceftazidime, amphotericin B, nalidixic acid, polymyxin B
Pseudomonas	β-Alanyl arylamidase	7-Amido-1-pentyl-phenoxazin-3-one	cetrimide
Salmonella	a-galactosidase, lipase	indoxyl-a-galactoside, indoxyl-fatty acid ester	sodium deoxycholate
MRSA (Methicillin Resistant Staphylococcus aureus)	a-glucosidase	indoxyl-a-D-glucopyranoside	methicillin, high concentration of sodium chloride
Staphylococcus aureus	a-glucosidase, phosphatase, deoxyribonuclease	indoxyl-a,D-glucoside, phenolphthalein phosphate, indoxyl-phosphate	tellurite, lithium chloride
Streptococci	β-glucuronidase	indoxyl-β-glucuronide	sodium azide
UTI (Urinary Tract Infections)	β-glucosidase, β-galactosidase	indoxyl-β-glucopyranoside, indoxyl-β-galactoside	-
Vibrio	β-glucosidase, β-galactosidase	indoxyl-β-glucoside, indoxyl-β-galactoside	high concentration of sodium chloride, sodium thiosulphate, sodium citrate, sodium cholate
VRE (Vancomycin Resistant Enterococci)	a-glucosidase, β-glucosidase, β-galactosidase	indoxyl-a-glucopyranoside, indoxyl-β-glucopyranoside, indoxyl-β-galactoside	vancomycin
Yeasts and Molds	β-N-acetylgalactosaminidase, β-xylosidase	indoxyl-N-acetyl-β-D-glucosaminide, indoxyl-β-D-xyloside	oxytetracycline

Advantages of Chromogenic Media

Chromogenic media are invaluable media for the detection of target microorganisms, even in mixed culture conditions, offering the following benefits:

• Faster results compared to traditional methods: Some chromogenic media provide confirmed results within 24 hours.
• Reliable visual detection: Often, no further testing or subculture is required.
• Multiple visual detection: Distinguish multiple organisms on a single plate by their different colors.
• Additional testing: Further testing is still possible directly from or on the media.
• Combination with other biochemical tests: This can include tests such as coagulase and lecithin tests.
• Cost efficient and time saving solution.

 

ChromoCult^® Coliform Agar – A Culture Medium With 2 Different Chromogenic Substances

ChromoCult^® Coliform Agar is a selective and differential chromogenic culture medium, suitable for microbiology laboratories that analyze food and water samples. This medium enables the detection, differentiation, and enumeration of E. coli and coliforms from drinking water and processed food matrices in under 24 hours. The careful selection of inhibitors in this selective media is crucial for the growth and recovery of damaged microorganisms. Notably, ChromoCult^® Coliform Agar contains Tergitol^® 7, which inhibits Gram-positive bacteria without adversely affecting the growth of the targeted coliforms or E. coli.

Benefits of using ChromoCult^® Coliform Agar:

• Time-saving: A fast method for simultaneous detection of coliforms and E. coli in water and processed food.
• Safe analytical results: Reliable detection of injured bacteria due to carefully selected inhibitors, resulting in fewer chances of false
• User-friendly: Clear detection of coliforms and E. coli even in the presence high amounts of accompanying flora. Simply add the recommend supplement
• Cost-effective: Easy confirmation of E. coli; just add a drop of indole reagent to a colony, and the cherry color indicates whether it is E. coli.
• Regulatory approval: Compliant to ISO 9308-1, approved by US-EPA, EU and AOAC™ as an alternative method for the detection of total coliforms and E. coli in water and processed food.

ChromoCult^® Coliform Agar ES (Enhanced Selectivity)

Fresh food typically contains a high microbial load and generally does not include stressed or injured bacteria. The presence of accompanying flora requires a high selective culture medium to inhibit unwanted bacteria while promoting the growth of target organisms. To address this challenge, the well-established ChromoCult^® Coliform Agar was modified. Tergitol^® 7 was replaced with a combination of bile salts and propionate, which effectively inhibits accompanying bacteria.

ChromoCult^® Coliform Agar ES is a selective medium that allows for the simultaneous detection of coliforms and E. coli in samples with high bacterial bioburden. This makes it an ideal medium for detecting and counting total coliforms and E. coli in fresh foods as well as in water samples with higher bioburden. The combination of carefully selected peptones and the buffering capacity of MOPS creates optimal conditions for the rapid growth of coliforms and enhances the transformation of chromogenic substrates. The simultaneous detection of total coliforms and E. coli is achieved through the use of two specific chromogenic substrates.

 

ReadyCult^® - A Culture Medium with A Combination of Fluorogenic and Chromogenic Substances

ReadyCult^® media enable detection of enterococci, coliforms, and E. coli. These media are supplied in a ready-to-use snap-pack format for fast and easy preparation. Results are obtained within 18 to 24 hours of incubation. Any color change of the broth to blue-green, even if only in the upper section, is a positive result.

ReadyCult^® Coliforms 50/100

ReadyCult^® Coliforms 50 (for 50 mL water test sample) and ReadyCult^® Coliforms 100 (for 100 mL water test sample) provide a rapid presence / absence test for the simultaneous detection of total coliforms and E. coli in drinking water. ReadyCult^® is supplied as a premeasured, single use, sterilized packet of media.

When coliforms are present in the water sample, their β-galactosidase enzymes cleave the colorless X-GAL, causing the culture medium to change color from yellow to blue green. If E. coli is present among the coliforms, their β-glucuronidase enzymes cleave the substrate MUG (4-methylumbelliferyl-β-D-glucuronide), producing blue fluorescence that is visible under UV light (366 nm). However, it is important to note that the MUG reaction is not entirely specific to E. coli – a few other bacterial species can also cleave MUG (mug positive), and certain strains of E. coli may be MUG-negative. In case of any doubts, ReadyCult^® Coliforms enables in contrast to other similar product available on the market, to a very simple and effective confirmation of the presence of E. coli:  of a biochemical test can be performed directly in the sample by adding KOVAC's indole reagent. This reagent reacts with indole to produce a red ring in the presence of E. coli, indicating its ability to generate indole from the amino acid tryptophan. A positive result provides 99% certainty of the presence of E. coli and eliminates the need for additional 24-48 hour confirmation steps.

ReadyCult^® Enterococci 100

ReadyCult^® Enterococci 100 is a selective enrichment broth for the detection of Enterococcus and group D Streptococcus species in bacteriological water examinations. The peptone mixture ensures rapid growth of enterococci, while sodium azide effectively inhibits accompanying flora, particularly Gram-negative bacteria. In a reaction that is stimulated by selected peptones, the chromogenic substrate X-GLU is cleaved by the enzyme β-D-Glucosidase, which is characteristic of enterococci. This results in a color change of the broth to an intensive blue-green.

MilliChrome™ Chromogenic Culture Media – Our Newest Chromogenic Media

The new MilliChrome™ chromogenic culture media offer a convenient and rapid method for detecting common microbial contaminants and pathogens. They are well-suited for testing target organisms in quality control or research laboratories, as well as in non-critical areas of manufacturing facilities. These culture media contain substrates that specific bacterial species, genus, or serotypes can convert into a chromogen, a substance that imparts a specific color. This reaction is performed by an enzyme unique to the target organism. Consequently, colonies of the target organism on the agar exhibit the color of the chromogen, allowing for easy identification by the naked eye, often without the need for further testing. The cells remain culturable in or on any suitable medium.

MilliChrome™ media is the latest addition to our portfolio of chromogenic culture media. They include a dehydrated base medium, agar, and the chromogenic substrate for the identification of the target organism. Necessary supplements, as well as optional supplements that further enhances specificity, should be added after autoclaving, once the medium has cooled to between 45 and 50 °C. The MilliChrome™ media are suitable for a wide range of applications, including research and hygiene monitoring. Although, MilliChrome™ media do not have specific approvals, some of them are also useful for quality control of food samples in accordance with the relevant ISO guidelines.

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