Overview of Chromogenic Media

By: Jvo Siegrist Product Manager, 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.

Figure 1. HiCrome™ Rapid Coliform Broth

Figure 1. HiCrome™ Rapid Coliform Broth

 

Species Enzyme Substrate Selective Agents
Bacillus cereus

 
ß-glucosidase,
Phosphatidylinositol-specific
Phospholipase C
indoxyl-ß-glucopyranoside,
indoxyl-myo-inositol-1-phosphate
 
polymyxin B

 
Campylobacter
 
na
 
na
 
deoxycholate, cefoperazone,
amphotericin B
Candida
 
ß-acetylgalactosaminidase,
alkaline phosphatase
indoxyl-N-acetyl-ß-D-glucosaminide,
indoxyl-phosphate
Chloramphenicol, Gentamicin
 
Clostridium perfingens
 
ß-glucosidase (plus sucrose
fermentation)
indoxyl-ß-D-glucoside
 
D-cycloserine, polymixin B
 
Coliforms/E. coli
 
ß-glucuronidase,
ß-galactosidase
indoxyl-ß-glucuronide,
Indoxyl-ß-galactoside
bile 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
Enterococci
ß-D-glucosidase
indoxyl-ß-glucoside
sodium azide, polysorbate 80
Extended Spectrum ß-Lactamase
Enterobacteria (ESBL)
ß-D-glucosidase
 
indoxyl-ß-glucoside
 
cefpodoxime, cefotaxime,
ceftazidime
Klebsiella
 
ß-D-ribofuranosidase,
ß-D-glucosidase
indoxyl-ß-D-ribofuranoside,
indoxyl-ß-D-glucoside
bile 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
Pseudomonas
ß-Alanyl arylamidase
7-Amido-1-pentyl-phenoxazin-3-one
cetrimide
Salmonella
 
α-galactosidase, lipase
 
indoxyl-a-galactoside,
indoxyl-fatty acid ester
sodium 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-phosphate
tellurite, lithium chloride

 
Streptococci
ß-glucuronidase
indoxyl-ß-glucuronide
sodium 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-galactoside
high 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-galactoside
vancomycin

 
Yeasts and Moulds
 
ß-N-acetylgalactosaminidase,
ß-xylosidase
indoxyl-N-acetyl-ß-D-glucosaminide,
indoxyl-ß-D-xyloside
oxytetracycline
 

Table 1. Summary 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 colouration. The colour 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 (See Tables 1 and 2).

 

Organisms Media               
Bacillus cereus
Sigma-Aldrich
92325
HiCrome™ Bacillus Agar
Candida albicans
Sigma-Aldrich
94382
Candida Ident Agar, modified
Cl. perfringens
 
Sigma-Aldrich
Sigma-Aldrich
12398
75605
CP ChromoSelect Agar
m-CP Agar
Cl. perfringens

 
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
70722
09142
92435
HiCrome™ E. coli Agar B
HiCrome™ ECD Agar with MUG
TBX Agar
E. coli & Coliforms



 
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
81938
73009
85927
51489
39734
HiCrome™ Coliform Agar
HiCrome™ ECC Agar
HiCrome™ ECC Selective Agar
HiCrome™ Rapid Coliform Broth
Membrane Lactose Glucuronide Agar
E. coli 0157:H7


 
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
39894
72557
80330
83339
HiCrome™ EC O157 Agar
HiCrome™ EC O157:H7 Selective Agar, Base
HiCrome™ Enrichment Broth Base for EC O157:H7
HiCrome™ Mac Conkey Sorbitol Agar
Thermotolerant E. coli
Sigma-Aldrich
90924
HiCrome™ m-TEC Agar
Enterobacter sakazakii (Cronobacter spp.)
 
Sigma-Aldrich
Sigma-Aldrich
92324
14703
HiCrome™ Cronobacter spp. Agar
HiCrome™ Cronobacter spp. Agar, modified
Enterococci
 
Sigma-Aldrich
Sigma-Aldrich
52441
51759
HiCrome™ Enterococci Broth
HiCrome™ Rapid Enterococci Agar
Enterococcus faecium
Sigma-Aldrich
90919
HiCrome™ Enterococcus faecium Agar Base
Klebsiella
Sigma-Aldrich
90925
HiCrome™ Klebsiella Selective Agar Base
Listeria
 
Sigma-Aldrich
Sigma-Aldrich
53707
77408
HiCrome™ Listeria Agar Base, modified
Listeria mono Differential Agar (Base)
Proteus, enteropathogenic gram-positive organisms
Sigma-Aldrich
16636
HiCrome™ UTI Agar, modified
Salmonella



 
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
00563
90918
78419
05538
84369
HiCrome™ MM Agar
HiCrome™ RajHans Medium, Modified
HiCrome™ Salmonella Agar
HiCrome™ Salmonella Agar, Improved
Salmonella Chromogen Agar
Staphylococcus aureus
 
Sigma-Aldrich
Sigma-Aldrich
05662
68879
HiCrome™ Aureus Agar Base
Phenolphthalein Phosphate Agar
Methicillin-resistant Staph. aureus
Sigma-Aldrich
90923
HiCrome™ MeReSa Agar Base
Vibrio
Sigma-Aldrich
92323
HiCrome™ Vibrio Agar
Yeasts and fungi
Sigma-Aldrich
66481
HiCrome™ OGYE Agar Base

Table 2. Sigma-Aldrich’s product line of chromogenic media according to organisms detected (Fluorogenic media are not listed. Complete product listings are available at sigma-aldrich.com/microbiology)

 

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. sordelli, C. bifermentans or C. tetani. Only 3 strains (0.6%) gave false positive results and were identifi ed as C. fallax, C. 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.

 

Figure 2. HiCrome™ ECC Agar

Figure 2. HiCrome™ ECC Agar (73009)

 

Figure 3.HiCrome™ Coliform Agar

Figure 3. HiCrome™ Coliform Agar (81938)

 

Figure 4.CP ChromoSelect Agar Cl. Perfringens appears as green colonies

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

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Materials