CHROMAgar – History, Mechanism of action, & Types

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Chromogenic media (a.k.a. CHROMAgar) are specialized selective and differential culture media that utilize synthetic chromogenic enzyme substrates in order to specifically target pathogenic species (or groups of species) based on their enzyme activity. Because the enzyme activity in these target pathogens are never completely species specific, the use of other complementary enzyme substrates (e.g., ONPG, X-Gal, or X-Glu) and/or selective agents (e.g., antibiotics) have also been included into CHROMAgar to make them more selective and differential in nature. The majority of chromogenic media are therefore both selective and differential, accommodating the inhibition of non-target organisms (e.g., using antibiotics or other inhibitors) and enabling target pathogens to grow as colored colonies due to their metabolism (usually by hydrolysis) of one or more chromogenic enzyme substrates. Chromogenic media offer a range of benefits for the enumeration, detection, and identification of microorganisms from a wide variety of clinical and environmental samples.

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Some of the advantages of chromogenic media over traditional culture media include:

  • Faster results (compared to traditional method)
  • Reliable visual detection (often no further testing required)
  • Additional testing possible directly from the media
  • Reduces waiting time to get culture results (compared to traditional method)

Chromogenic media demonstrate a proven advantage over conventional culture media due to a superior detection rate for target pathogens or a superior differentiation of mixed cultures (Figure 1). CHROMAgar is also less time-consuming than traditional culture media. More so, CHROMAgar does not consume more resources like traditional culture media that requires more resources and time for evaluating a clinical or environmental sample. Culture media containing chromogenic substrates are invariably more expensive than conventional/traditional media but this can be offset by a reduced need for complementary reagents and less labour time associated with the processing of culture plates and suspect pathogens. Due to these factors, the use of chromogenic media in diagnostic laboratories and for other microbial investigations is increasingly widespread.

Figure 1. Illustration of differences between classical/traditional culture media (LEFT) and CHROMAgar (RIGTH). Photo courtesy: http://www.chromagar.com

Principle /mechanism of action of CHROMAgar

The focus behind the development of Chromogenic media (CHROMagar) was to produce culture media that would make the detection and identification of microorganisms from both clinical and environmental samples more rapid and more reliable. Chromogenic substrate such as ONPG, X-Gal, or X-Glu, together with a specified selectivity of the medium, is the simple principle behind chromogenic media (Figure 2). The target organisms are characterized by enzyme systems that metabolize the substrates to release the chromogen.

The chromogen can then be visually detected by direct observation of a distinct colour change in the medium. Direct confirmation of the target organism without further testing is sometimes possible. This is what makes CHROMagar unique and different from the classical/traditional culture media. An ideal chromogenic substrate should be hydrolysed to release a coloured product that remains highly localized on microbial colonies. This allows clear differentiation of microbes producing the target enzyme from those that do not. This is especially important when attempting to detect specific pathogens within polymicrobial cultures. The substrate and products of hydrolysis should be non-inhibitory to microbial growth.

Figure 2. Illustration of the mechanism of action of CHROMAgar. Photo courtesy: http://www.chromagar.com

Table 1. Summary of possible enzyme activities, chromogenic substrates and selectivity system for microorganisms

SpeciesEnzymeSubstrateSelective Agents
Bacillus cereus    ß-glucosidase, Phosphatidylinositol-specific Phospholipase Cindoxyl-ß-glucopyranoside, indoxyl-myo-inositol-1-phosphate  polymyxin B    
Campylobacter  na  na  deoxycholate, cefoperazone, amphotericin B
Candida  ß-acetylgalactosaminidase, alkaline phosphataseindoxyl-N-acetyl-ß-D-glucosaminide, indoxyl-phosphateChloramphenicol, Gentamicin  
Clostridium perfingens  ß-glucosidase (plus sucrose fermentation)indoxyl-ß-D-glucoside  D-cycloserine, polymixin B  
Coliforms/E. coli  ß-glucuronidase, ß-galactosidaseindoxyl-ß-glucuronide, Indoxyl-ß-galactosidebile salts, tergitol 7®, SDS, novobiocin, cefsulodin
Cronobacter (E. sakazakii)  α-glucosidase  indoxyl-a-D-glucoside  deoxycholate, crystal violet, sodium thiosulfate
E. coli O157    ß-glucosidase, α-galactosidase    indoxyl-ß-D-glucuronide, indoxyl-a-galatoside  bile salts, SDS, crystal violet, potassium tellurite, novobiocin, cefixime
Enterococciß-D-glucosidaseindoxyl-ß-glucosidesodium azide, polysorbate 80
Extended Spectrum ß-Lactamase Enterobacteria (ESBL)ß-D-glucosidase  indoxyl-ß-glucoside  cefpodoxime, cefotaxime, ceftazidime
Klebsiella  ß-D-ribofuranosidase, ß-D-glucosidaseindoxyl-ß-D-ribofuranoside, indoxyl-ß-D-glucosidebile salts, SDS, carbenicillin  
Listeria spp.    ß- glucosidase    indoxyl-ß-glucoside    lithium chloride, ceftazidime, amphotericin B, nalidixic acid, polymyxin B
L. monoctogenes    Phosphatidylinositol-specific Phospholipase C, ß-glucosidase,indoxyl-ß-glucoside, indoxyl-myo-inositol-1-phosphate  lithium chloride, ceftazidime, amphotericin B, nalidixic acid, polymyxin B
Pseudomonasß-Alanyl arylamidase7-Amido-1-pentyl-phenoxazin-3-onecetrimide
Salmonella  α-galactosidase, lipase  indoxyl-a-galactoside, indoxyl-fatty acid estersodium deoxycholate  
MRSA (Methicillin Resistant Staphylococcus aureus)α-glucosidase  indoxyl-a-D-glucopyranoside  methicillin, high concentration of sodium chloride
Staphylococcus aureus    α-glucosidase, phosphatase, deoxyribonuclease  indoxyl-a,D-glucoside, phenolphthalein phosphate, indoxyl-phosphatetellurite, lithium chloride    
Streptococciß-glucuronidaseindoxyl-ß-glucuronidesodium azide
UTI (Urinary Tract Infections)  ß-glucosidase, ß-galactosidase  indoxyl-ß-glucopyranoside, indoxyl-ß-galactoside–  
Vibrio    ß-glucosidase, ß-galactosidase    indoxyl-ß-glucopside, indoxyl-ß-galactoside, indoxyl-ß-galactosidehigh concentration of sodium chloride, sodium thiosulphate, sodium citrate, sodium cholate
VRE (Vancomycin Resistant Enterococci)  α-glucosidase, ß-glucosidase, ß-galactosidase  indoxyl-a-glucopyranoside, indoxyl-ß-glucopyranoside, indoxyl-ß-galactosidevancomycin    
Yeasts and Moulds  ß-N-acetylgalactosaminidase, ß-xylosidaseindoxyl-N-acetyl-ß-D-glucosaminide, indoxyl-ß-D-xylosideoxytetracycline

Differences between traditional/classical culture media and CHROMAgar

The traditional approach to the detection of pathogenic bacteria in pathological specimens has typically involved the inoculation of one or more general purpose culture media such as nutrient agar, chocolate agar, MacConkey agar, and blood agar to mention a few. Such culture media allow the growth of a wide range of bacteria, and suspect pathogens are detected on the basis of their colonial appearance (e.g., pigmentation, morphology, haemolysis). Such colonial characteristics rarely permit more than a presumptive identification and biochemical and/or serological tests are often required for definitive identification of the suspect microbial organism on the culture media. This approach frequently necessitates the testing of commensal bacteria that may resemble pathogens.

For example, one of the commonest pathogens sought from infections of skin and wounds is Staphylococcus aureus and many commensal staphylococci of the skin may need to be tested to exclude the presence of this pathogen. Detection of Salmonella spp., a frequent cause of gastroenteritis, requires isolation of the pathogen from stool samples. Culture media containing lactose, plus a pH indicator, have been traditionally used for differentiation of Salmonella(a non-lactose fermentor) from commensals such as Escherichia coli. However, it is frequently necessary to screen many other commensals that also fail to ferment lactose (e.g., Proteus spp.) to exclude the presence of Salmonella. The screening of commensal bacteria to exclude pathogens is time consuming and can be costly in terms of serological or biochemical reagents. It is this deficit in traditional culture media that CHROMAgar tries to fix and adjust.

Most commercially available chromogenic media have exploited indoxylic substrates (Table 1). Indoxyl, and its halogenated derivatives, can be derivatized to form a range of esters. Release of indoxyl via hydrolysis by a specific bacterial enzyme results in the formation of brightly coloured indigo dye. This is due to spontaneous dimerization of indoxyl molecules in the presence of oxygen. Halogenation of the indoxyl molecule has a dramatic effect on the colour and intensity of this chromogen. For example, 5‐bromo‐4‐chloro‐indoxyl forms a bright green/blue dye whereas 5‐bromo‐6‐chloro‐indoxyl forms a magenta dye. Indoxylic glycosides including glucoside, galactoside and glucuronide derivatives are widely used because of their high sensitivity, low toxicity and availability from a number of commercial sources.

Timeline / History of CHROMAgar

Dr. Alain Rambach is the Pioneering Scientist and founder/discoverer of CHROMAgar. CHROMagar™, Rambach™, AquaCHROM™ are trademarks created by Dr. A. Rambach. The timeline of how CHROMAgar was discovered and developed by Dr. Rambach is as follows:

  • 1967: Following his studies at Ecole Polytechnique in Paris, Alain Rambach joins the Pasteur Institute where he works with Professor François Jacob, Nobel Prize winner in genetics.
  • 1973: On completing his thesis in bacterial genetics, and in face of strong opposition from the scientific community, Dr. Rambach embarks on his research in gene manipulation. Eventually this field -becomes known as genetic engineering today.
  • 1976: After two years at Stanford University as a postgraduate in biochemistry, he returns to France and joins the Pasteur Institute as one of the leading scientists in the field of genetic engineering.
  • 1979: In an unrelated field, he pioneers, invents and patents a system for detecting E. coli bacteria by ‘chromogenic differentiation’ culture media.
  • 1980: Dr. Rambach establishes the firm Genetica as a joint venture with Rhône Poulenc. Genetica has been involved in genetic engineering and has become one of the leading R&D companies in the field of pharmaceutical fermentation process. When Rhône Poulenc integrates Genetica into its structure, Dr. Rambach decides to pursue his goal and devotes his talent and energy to developing another invention.
  • 1989: He patents a ‘chromogenic’ system for detection of the Salmonella pathogen, known as Rambach Agar. This is the first commercially available chromogenic culture medium.
  • 1993: Dr. Rambach founds CHROMagar Microbiology.
  • 1993 – Present: Dr. Rambach heads Research and Development (R&D) at CHROMagar.

How does Chromogenic Culture Media technology work? 

The Chromogenic Culture Media (CHROMagar) technology is based on soluble colourless molecules (called chromogens), composed of a substrate (targeting a specific enzymatic activity in a particular microbe) and a chromophore. When the target microorganism’s enzyme cleaves the colourless chromogenic conjugate, the chromophore is released. In its unconjugated form, the chromophore exhibits its distinctive colour and, due to reduced solubility, forms a precipitate. The medium contains chromogenic substrate which is utilized by the microorganism to give colored colonies that is specific for each microorganism. Depending on the color of the result, the presence or absence of target organism is determined and also accurately differentiated from others. There are several advantages to this method

FOR THE LABORATORY TECHNICIAN: EASY TO READ AT A GLANCE!

  • The result from CHROMagar use is very specific and distinctive.
  • CHROMagar is a colour-based differentiation method.
  • Clearly distinguishable with the naked eye under normal lighting conditions. With this technique, colonies of specific microorganisms can be recognized by their colour at a glance.

ECONOMIES OF SCALE: COST SAVINGS & FAST METHOD!

  • CHROMagar allows for easy differentiation of microorganisms without the complex and costly traditional detection procedures employed in traditional agar testing techniques (No subcultures).
  • By saving time and labor, CHROMagar increases the efficiency of laboratory testing.
  • By shortening the timeframe for identifying pathogens, CHROMagar helps to prevent the spread of infections.

Some CHROMAgar Types

CHROMagar™ COL-APSE

CHROMagar™ COL-APSE is a sensitive and specific medium for the selective growth of colistin resistant bacterial pathogens. Polymyxin E (colistin) and B are increasingly used as antimicrobials in the treatment of multi-drug resistant bacterial infections. Although intrinsic in Gram-positive and some Gram-negative species (Proteus, Morganella, Serratia), polymyxin resistance is now a problem in a number of other pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Klebsiella pneumoniae).

Polymyxin resistance arises due to mutations / insertions in genes involved in LPS biosynthesis (lpx, pmrA/B, mgrB, phoP/Q) and/ or the acquisition of phosphoethanolomine transferases (PEtN). The recently described plasmid-encoded PEtN, MCR-1, is of great concern and is now found worldwide in a range of animal, human and environmental bacterial isolates. CHROMagar™ COL-APSE is particularly useful as a primary isolation medium in the surveillance and recovery of colistin resistant bacteria from complex human, veterinary and environmental samples especially those with plasmid mediated MCR-1 or novel mechanisms of polymyxin resistance.

CHROMagar™ Campylobacter

Campylobacter bacteria are a major cause of foodborne diarrhoeal illness in humans and are the most common bacteria that cause gastroenteritis worldwide. In developed and developing countries, they cause more cases of diarrhoea than foodborne Salmonella. The high incidence of Campylobacter diarrhoea, as well as its duration and possible consequences makes it very important from a socio-economic perspective. In developing countries, Campylobacter infections in children under the age of two years are especially frequent, sometimes resulting in death.

The most common gastrointestinal infections in humans are caused by the Campylobacter jejuni and C. coli species. Currently, screening for Campylobacter is a culture based method using a charcoal-based selective media or a blood containing media. These culture plates are incubated for up to 72 hours and their lack of selectivity allows for the growth of large amounts of other bacterial flora. Therefore ISO (10272-1 :2006) requires the use of a second medium to work around these deficiencies.

CHROMagar™ Campylobacter is a chromogenic medium that is both selective and differential for the isolation of this enteric pathogen. This medium also requires incubation in a microaerophilic atmosphere at 42°C, however distinct red colonies of Campylobacter will grow after 24 hours and a negative report can be issued after 48 hours incubation rather than 72 hours.

CHROMagar™ Campylobacter medium can decrease the turnaround time from sample to result and therefore significantly improve patient diagnosis and workflow of food testing laboratories. With CHROMagar™ Campylobacter, no compromises! This medium combines highly specific and easy to read chromogenic technology with an unrivalled growth agar base.

Key Features and Benefits of CHROMagar™ Campylobacter medium

  • EASY TO READ – 24-48 h incubation at 42°C. The intense red colored colonies on a translucent agar facilitates the reading compared to charcoal based agar.
  • HIGH RECOVERY RATE – ~100 % enumerating Campylobacter has never been so simple and so reliable.
  • HIGHLY SELECTIVE – a greater selectivity results in a very clean plate, even for samples with high background flora.

CHROMagar™ C. perfringens

Clostridium perfringens is involved in both food poisoning and animal infections. CHROMagar™ C. perfringens allows for the detection and enumeration of C. perfringens  in food and water samples. Specific and selective, colonies of C. perfringens appear orange and also fluorescent under UV light at 365nm, any other microorganisms being blue, metallic blue or are inhibited from growing. One drawback of TSC medium that is often used for C. perfingens is that bacteria have to be placed between two layers of agar in order to grow as black colonies.

Also, this medium will make any sulfate-reducing bacteria, including the non pathogens appear as black colonies. Moreover, there is tendency for the black colonies to spread, making colony counting difficult. However, CHROMagar™ C. perfringens can be used with either pouring or surface methods (by direct streaking, spreading or with the filtration technique), the medium is specific for C. perfringens and the orange coloration makes the visualization very easy.

CHROMagar TM Mastitis

CHROMagar TM Mastitis detects most common pathogens in mastitis infections. Clinical mastitis remains an important animal health issue, affecting from 20% to 30% of dairy cows at least once during lactation. Lack of Mastitis infection control in cattle causes heavy economic losses to milk producers and to the dairy industry as it causes a reduction in the quality of milk and output, and increases veterinary expenses due to excessive use of medications, and risk of residues in the milk/ meat. Consequently, this becomes a public health issue.

Estimated costs per infection of clinical mastitis range from $178 to $489 depending on stage of lactation, milk prices and production level in affected cows. The most common causes of antibiotic use in dairy cows are the treatment and prevention of mastitis. In order to avoid its indiscriminate antibiotic use and to reduce the economic burden of clinical mastitis, it is critical to implement an on-farm rapid identification of the pathogens.

CHROMagarTM Mastitis is a new commercially available tool for the rapid and simple differentiation of the key bacteria involved in Mastitis infections. This new product is supplied as a kit with two separate media, one for Gram-positive bacteria (GP), and the other for Gram-negative bacteria (GN).

Colony Appearance on CHROMagar Mastitis GP

  • Streptococcus agalactiae → blue-green
  • Streptococcus uberis → metallic blue
  • Staphylococcus aureus → mauve with mauve halo
  • Gram negative bacteria → inhibited

Colony Appearance on CHROMagar Mastitis GN

  • E. coli → red
  • Klebsiella, Enterobacter, Citrobacter → metallic blue
  • Proteus → brown halo
  • Pseudomonas → cream, translucent
  • C. albicans → white, opaque, small
  • Gram positive bacteria → inhibited

New CHROMagar™ Campylobacter for Food and Clinical Samples

Campylobacter bacteria are a major cause of foodborne diarrhoeal illness in humans and are the most common bacteria that cause gastroenteritis worldwide. In developed and developing countries, they cause more cases of diarrhoea than foodborne Salmonella. The high incidence of Campylobacter diarrhoea, as well as its duration and possible sequelae, makes it highly important from a socio-economic perspective. In developing countries, Campylobacter infections in children under the age of two years are especially frequent, sometimes resulting in death.

The most common gastrointestinal infections in humans are caused by the Campylobacter jejuni and C. coli species. Current screening for Campylobacter is a culture based method using a charcoal-based selective media or a blood containing media. These culture plates are incubated for up to 72 hours and their lack of selectivity allows for the growth of large amounts of bacterial flora. Therefore ISO (10272-1 :2006) requires the use of a second medium to palliate these deficiencies. CHROMagar™ Campylobacter is a new chromogenic medium that is both selective and differential for the isolation of this enteric pathogen.

This medium also requires incubation in a microaerophilic atmosphere at 42°C, however distinct red colonies of Campylobacter will grow after 24 hours and a negative report can be issued after 48 hours incubation rather than 72 hours. This new CHROMagar™ Campylobacter medium can decrease the result turnaround time and therefore significantly improve patient diagnosis and workflow of both clinical and food testing laboratories. With CHROMagar™ Campylobacter, no compromises! This medium allies the highly specific and easy to read chromogenic technology with an unrivalled growth agar base.

Key Features and Benefits of the New CHROMagar™ Campylobacter for Food and Clinical Samples:

  • EASY READING – 24-48h incubation at 42°C. The intense red colored colonies on a translucent agar facilitates the reading compared to charcoal based agar.
  • HIGH RECOVERY RATE – ~100% Enumerating Campylobacter has never been so simple and so reliable.
  • HIGHLY SELECTIVE – a reinforced selectivity allows for a very clean plate, even with heavily flora loaded samples

Other CHROMAgar types include

  • CHROMagarTM E. coli
  • CHROMagarTM ECC
  • CHROMagarTM Salmonella
  • CHROMagarTM Orientation
  • CHROMagarTM Candida
  • CHROMagarTM O157
  • CHROMagarTM MRSA
  • CHROMagarTM Salmonella Plus
  • CHROMagarTM Pseudomonas
  • CHROMagarTM Vibrio
  • CHROMagarTM Listeria
  • CHROMagarTM Identification Listeria
  • CHROMagarTM VRE
  • CHROMagarTM StrepB
  • CHROMagarTM ESBL
  • CHROMagarTM KPC

Further reading

Aamlid, K.H., Lee, G., Smith, B.V., Richardson, A.C. and Price, R.G. (1990) New colorimetric substrates for the assay of glycosidases. Carbohydr Res 205, c5– c9.

Ainscough, S. and Kibbler, C.C. (1998) An evaluation of the cost‐effectiveness of using CHROMagar for yeast identification in a routine microbiology laboratory. J Med Microbiol 47, 623– 628.

Carricajo, A., Boiste, S., Thore, J., Gille, Y., Aubert, G. and Freydière, A.M. (1999) Comparative evaluation of five chromogenic media for detection, enumeration and direct identification of urinary tract pathogens. Eur J Clin Microbiol Infect Dis 18, 796– 803.

D’Souza, H.A., Campbell, M. and Baron, E.J. (2004) Practical bench comparison of BBL CHROMagar orientation and standard two‐plate media for urine cultures. J Clin Microbiol 42, 60– 64.

Dusch, H. and Altwegg, M. (1993) Comparison of Rambach agar, SM‐ID medium, and Hektoen enteric agar for primary isolation of nontyphi Salmonellae from stool samples. J Clin Microbiol 31, 410– 412.

Perry J.D and Freydiere A.M (2007). The application of chromogenic media in clinical microbiology. Journalof Applied Microbiology, 103(6):2046-2055. https://doi.org/10.1111/j.1365-2672.2007.03442.x

https://www.rapidmicrobiology.com/news/selective-growth-of-colistin-resistant-bacteria-on-chromagar-col-apse
http://www.chromagar.com/p-chromogenic_agar_technology.html#.Xw2wUxRlBdg
https://www.sigmaaldrich.com/technical-documents/articles/analytix/overview-chromogenic-media.html

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