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Overview of Chromogenic Media

Jvo Siegrist

AnalytiX Issue 3

Chromogenic media offer a range of benefits for the enumeration, detection, and identification of microorganisms

The use of traditional versus improved media formulation containing chromogenic substrate is currently an important topic in the field of microbiology. The focus behind such developments was to produce media that would make the detection and identification of microorganisms more rapid and more reliable. Chromogenic substrates such as ONPG, X-Gal, or X-Glu, together with a specified selectivity of the medium, is the simple principle behind chromogenic media. 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.

HiCrome™ Rapid Coliform Broth

Figure 1.HiCrome™ Rapid Coliform Broth

SpeciesEnzymeSubstrateSelective Agents
Bacillus cereus
 
ß-glucosidase,Phosphatidylinositol-specificPhospholipase Cindoxyl-ß-glucopyranoside,indoxyl-myo-inositol-1-phosphatepolymyxin B
 
Campylobacternanadeoxycholate, cefoperazone,amphotericin B
Candida ß-acetylgalactosaminidase,alkaline phosphataseindoxyl-N-acetyl-ß-D-glucosaminide,indoxyl-phosphateChloramphenicol, Gentamicin
Clostridium perfingensß-glucosidase (plus sucrosefermentation)indoxyl-ß-D-glucosideD-cycloserine, polymixin B 
Coliforms/E. coliß-glucuronidase,ß-galactosidaseindoxyl-ß-glucuronide,Indoxyl-ß-galactosidebile salts, tergitol 7®, SDS,novobiocin, cefsulodin
Cronobacter (E. sakazakii)α-glucosidaseindoxyl-a-D-glucoside deoxycholate, crystal violet,sodium thiosulfate
E. coli O157
 
ß-glucosidase, α-galactosidase
 
indoxyl-ß-D-glucuronide,indoxyl-a-galatosidebile salts, SDS, crystalviolet, potassium tellurite,novobiocin, cefixime
Enterococciß-D-glucosidaseindoxyl-ß-glucosidesodium azide, polysorbate 80
Extended Spectrum ß-LactamaseEnterobacteria (ESBL)ß-D-glucosidaseindoxyl-ß-glucosidecefpodoxime, 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-specificPhospholipase C,ß-glucosidase,indoxyl-ß-glucoside,indoxyl-myo-inositol-1-phosphatelithium chloride, ceftazidime,amphotericin B, nalidixic acid,polymyxin B
Pseudomonasß-Alanyl arylamidase7-Amido-1-pentyl-phenoxazin-3-onecetrimide
Salmonellaα-galactosidase, lipaseindoxyl-a-galactoside,indoxyl-fatty acid estersodium deoxycholate 
MRSA (Methicillin ResistantStaphylococcus aureus)α-glucosidaseindoxyl-a-D-glucopyranosidemethicillin, high concentrationof sodium chloride
Staphylococcus aureus
 
α-glucosidase, phosphatase,deoxyribonucleaseindoxyl-a,D-glucoside,phenolphthalein phosphate,indoxyl-phosphatetellurite, lithium chloride
 
Streptococciß-glucuronidaseindoxyl-ß-glucuronidesodium azide
UTI (Urinary Tract Infections)ß-glucosidase, ß-galactosidaseindoxyl-ß-glucopyranoside,indoxyl-ß-galactoside-
Vibrio
 
ß-glucosidase, ß-galactosidase
 
indoxyl-ß-glucopside,indoxyl-ß-galactoside,indoxyl-ß-galactosidehigh concentration of sodiumchloride, sodium thiosulphate,sodium citrate, sodium cholate
VRE (Vancomycin ResistantEnterococci)α-glucosidase, ß-glucosidase,ß-galactosidaseindoxyl-a-glucopyranoside,indoxyl-ß-glucopyranoside,indoxyl-ß-galactosidevancomycin
 
Yeasts and Mouldsß-N-acetylgalactosaminidase,ß-xylosidaseindoxyl-N-acetyl-ß-D-glucosaminide,indoxyl-ß-D-xylosideoxytetracycline
Table 1Summary of possible enzyme activities, chromogenic substrates and selectivity system for microorganisms

Advantage of Chromogenic Media:

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

Within recent years, great strides have been taken in the sector of chromogenic media. Initial research concentrated on the use of synthetic substrates for the detection of enzymatic microbial activities. Nitrophenol and nitroanilline compounds were used at this time, producing a yellow coloration. The color of nitrophenol, however, is influenced by a pH-change, making it difficult to use reliably in microbiology. Later developments included the use of naphtol or naphtylamine. Today, while diverse modern chromogenic substrates are available, most of the modern substrates are based on the indoxylsubstrate. The use of different chromophore and metabolite derivatives then makes it possible to detect diverse enzyme activities all in one assay. The color of the indoxyl-substrates can be as follows: blue (5-bromo-4-chloro-3-indoxyl- = X, 3-indoxyl- =Y), magenta (5-bromo-6-chloro-3-indoxyl-), salmon (6-chloro-3-indoxyl-), purple (5-iodo-3-indoxyl-) and green (N-methylindoxyl-). One of the major advantages of the indoxyl-substrate and these chromophores is that they remain in the cell, making the characterization of a single cell possible (no diffusion into the media).

Additional advancements in the knowledge about enzyme and species specificity have also occurred within the past year. These recent gains in the development of selective agents and diverse chromogenic substrates have led toward an impressive range of chromogenic media available to meet our customers’ unique analytical emphases (Tables 1 and 2).

OrganismsMedia             
Bacillus cereusSigma-Aldrich92325HiCrome™ Bacillus Agar
Candida albicansSigma-Aldrich94382Candida Ident Agar, modified
Cl. perfringensSigma-AldrichSigma-Aldrich12398
75605
CP ChromoSelect Agarm-CP Agar
Cl. perfringens
 
Sigma-AldrichSigma-AldrichSigma-Aldrich70722
09142
92435
HiCrome™ E. coli Agar BHiCrome™ ECD Agar with MUGTBX Agar
E. coli & Coliforms


 
Sigma-AldrichSigma-AldrichSigma-AldrichSigma-AldrichSigma-Aldrich81938
73009
85927
51489
39734
HiCrome™ Coliform AgarHiCrome™ ECC AgarHiCrome™ ECC Selective AgarHiCrome™ Rapid Coliform BrothMembrane Lactose Glucuronide Agar
E. coli 0157:H7

 
Sigma-AldrichSigma-AldrichSigma-AldrichSigma-Aldrich39894
72557
80330
83339
HiCrome™ EC O157 AgarHiCrome™ EC O157:H7 Selective Agar, BaseHiCrome™ Enrichment Broth Base for EC O157:H7HiCrome™ Mac Conkey Sorbitol Agar
Thermotolerant E. coliSigma-Aldrich90924HiCrome™ m-TEC Agar
Enterobacter sakazakii (Cronobacter spp.)Sigma-AldrichSigma-Aldrich92324
14703
HiCrome™ Cronobacter spp. AgarHiCrome™ Cronobacter spp. Agar, modified
EnterococciSigma-AldrichSigma-Aldrich52441
51759
HiCrome™ Enterococci BrothHiCrome™ Rapid Enterococci Agar
Enterococcus faeciumSigma-Aldrich90919HiCrome™ Enterococcus faecium Agar Base
KlebsiellaSigma-Aldrich90925HiCrome™ Klebsiella Selective Agar Base
ListeriaSigma-AldrichSigma-Aldrich53707
77408
HiCrome™ Listeria Agar Base, modifiedListeria mono Differential Agar (Base)
Proteus, enteropathogenic gram-positive organismsSigma-Aldrich16636HiCrome™ UTI Agar, modified
Salmonella


 
Sigma-AldrichSigma-AldrichSigma-AldrichSigma-AldrichSigma-Aldrich00563
90918
78419
05538
84369
HiCrome™ MM AgarHiCrome™ RajHans Medium, ModifiedHiCrome™ Salmonella AgarHiCrome™ Salmonella Agar, ImprovedSalmonella Chromogen Agar
Staphylococcus aureusSigma-AldrichSigma-Aldrich05662
68879
HiCrome™ Aureus Agar BasePhenolphthalein Phosphate Agar
Methicillin-resistant Staph. aureusSigma-Aldrich90923HiCrome™ MeReSa Agar Base
VibrioSigma-Aldrich92323HiCrome™ Vibrio Agar
Yeasts and fungiSigma-Aldrich66481HiCrome™ OGYE Agar Base
Table 2Our product line of chromogenic media according to organisms detected (Fluorogenic media are not listed.)

The NEW CP ChromoSelect Agar

The European Directive on drinking water quality recommends mCP agar in the reference method for recovering C. perfringens. In the present study, three media (mCP, TSCF and CP ChromoSelect Agar) were evaluated for recovery of C. perfringens in different water samples. Out of 139 water samples tested, using a membrane filtration technique, 131 (94.2%) of the samples analyzed were found to be presumptively positive for C. perfringens on at least one of the culture media.

Green-colored colonies on CP ChromoSelect agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.9% (465 colonies, indole negative) were identified as C. perfringens, 15 colonies (3.1%) were indole positive and were identified as C. sordelliC. bifermentans or C. tetani. Only 3 strains (0.6%) gave false positive results and were identifi ed as C. fallaxC. botulinum, and C. tertium. Variance analysis of the data showed no statistically significant differences in the counts obtained between various media employed in this work.

The mCP method is very onerous for routine screening, and bacterial colonies could not be used for further biochemical testing. Conversely, the colonies on CCP and TSC were easy to count and subculture for confirmation tests. TSCF detects all sulfite-reducing clostridia, not only C. perfringens; however, in some cases, excessive blackening of the agar frustrated counting of the lower dilutions. If the contamination was too high, TSCF did not consistently produce black colonies and, as a consequence, the colonies’ white color provided false negative results.

The identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most specific medium for C. perfringens recovery in water samples.

HiCrome™ ECC Agar (73009)

Figure 2.HiCrome™ ECC Agar (73009)

HiCrome™ Coliform Agar (81938)

Figure 3.HiCrome™ Coliform Agar (81938)

CP ChromoSelect Agar Cl. Perfringens appears as green colonies

Figure 4.CP ChromoSelect Agar (Cl. Perfringens appears as green colonies, 12398)

Materials
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