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Vol. 34, No. 6 www.cmnewsletter.com March 15, 2012

Chromogenic Agar Media in the Clinical, Food, and Environmental

Testing Arenas, Part I
Elliot L. Rank, Ph.D., D(ABMM), St. Luke’s and Roosevelt Hospital Medical Centers, New York, New York
Abstract
Chromogenic agar media have become useful tools in the laboratory for the rapid recovery and identification of specific
microbial species or the isolation of bacteria that can be identified at the genus level. A wide variety of chromogenic media are
currently available and target the pathogenic microorganisms that are important in both clinical and environmental testing labo-
ratories. The greatest utility of chromogenic agar in the clinical microbiology laboratory revolves about the recovery of methi-
cillin-resistant Staphylcoccus aureus and gram-negative urinary pathogens. The greatest utility of the chromogenic agar in the
environmental laboratory revolves about the recovery of gram-negative pathogens, such as Escherichia coli O157 and Salmonella
spp. from water, foods, and the environment. In Part I of this two-part article, the development of chromogenic agar medium and
its practical utility for the detection and identification of Candida spp. and the Enterobacteriaceae are discussed.

Introduction medium upon which the colony was and the residual chromogen accumu-
The classical approach to differenti- growing. lates in the bacterial cells, leading to a
ating and identifying bacterial colonies Colony color expression in the pigmented colony by which the selected
growing on an agar medium involved a absence of selective pressure can be microorganism can be named at the
phenotypic array of individual observ- misleading, as different genera of micro- genus or species level within a statisti-
able characteristics and simple biochem- organisms can produce very similar cally reliable confidence interval.
ical tests. Colonies were described by color hues. For example the pale yellow Objective
these characteristics and then subjected hues of Micrococcus luteus and Staphy-
The aim of this review article is to
to tests that were relatively rapid, taking lococcus aureus, the purple hues of Ser-
describe succinctly the various uses and
less than 30 minutes to perform or ratia spp., the various shades of green
applications of CAM in clinical, food,
requiring overnight incubation in indi- and red for the Pseudomonas spp., and and environmental testing laboratories
vidual biochemical broth tubes to assess the natural sulfide-black color of Sal- as useful, efficient, and accurate agar
utilization. The “pigmentation” of the monella enterica serovar Typhimurium medium tools for the isolation of spe-
growing colony (1) was one of the first have long been used as a guide for pre- cific microorganisms. Secondly, among
important observable clues to use in the liminary identification. Virtually all the results and conclusions expressed
process of identifying a bacterial colony, bacterial colonies display of varying
in addition to colony size, shape, edge shades of white, off-white, or ivory, the
properties, presence and amount of sur- distinctions of which can be confound-
face polysaccharide, and the selective ing even to an experienced clinical
microbiologist.
The commercial introduction of
Editor‘s Note: Part II of this article will chromogenic agar media (CAM)
appear in the April 1, 2012 issue of CMN occurred in the 1990s. The basic scien-
(Vol. 34, No. 7). tific principles underlying this product
Corresponding author: Elliot L. Rank, relied on the use of selective media,
Ph.D., D(ABMM), Director, Molecular a proprietary carbohydrate substrate-
Clinical Infectious Diseases, St. Luke’s chromogen complex, and targeted
and Roosevelt Hospital Medical Centers,
bacterial uptake. Essentially, the carbo-
1111 Amsterdam Ave., New York, NY 10025.
Tel.: 212-523-5796. Fax: 212-523-4346. hydrate substrate complex is enzymati-
Email:[email protected] cally hydrolyzed by the microorganism,

Clinical Microbiology Newsletter 34:6,2012 © 2012 Elsevier 0196-4399/00 (see frontmatter) 43

by the majority of investigators in the food, water, and environmental testing. from denture wearers, patients with
articles cited below is the consistent There are many varieties of CAM offered dry-mouth syndrome, and HIV-infected
idea that the traditional microbiological by several manufacturers. A brief sum- patients; palate swabs; and dental-root
approach to growth, isolation and iden- mary of the manufacturers and their pro- caries lesions. Pfaller et al. (4) showed
tification of bacteria can be simplified ducts is shown in Table 1. This review the utility of the CC medium on stock
and achieved more rapidly with the use article will focus on a few of the research yeast isolates and the recovery of yeast
of CAM than with conventional micro- reports in which CAM have assisted in from patients’ stool specimens and
biological agar media. the scientific expansion of knowledge argued that the simultaneous identifi-
in the clinical and non-clinical arenas. cation of different species of Candida
Media The manufacturer of the CAM and the would facilitate improved appropriate
The basal composition of CAM location where the medium was manu- anti-fungal therapy for the patient.
consists of proprietary mixtures of high factured will be stated (if described in The first comparative study among
concentrations of carbohydrate and pep- the research report) in order to aid the these CAM was reported in 1996 by
tone and selective concentrations of reader in distinguishing between the Baumgartner et al. (5) in France using
agents such as NaCl or classes of anti- products, the study objectives, and CC, Albicans ID (BioMérieux, Marcy
biotics active against gram-positive or the results obtained by the various l’Etoile, France), and Sabouraud-Chlor-
gram-negative bacteria. The medium investigators. amphenicol agar plate (Diagnostics
formulations can be reviewed on-line Pasteur, Marnes la Coquette, France).
in the manufacturer’s product inserts. Use of Chromogenic Agar The authors showed that both CAM had
Media to Identify Candida spp. higher detection rates for 615 isolates
Biochemistry
in Clinical Specimens of Candida spp. from 951 clinical
Bromine and chlorine form halogen-
ated compounds with indoxyl in single-, The first report of the use of a specimens than the Sabouraud-Chlor-
double- or triple-substituted positions. CAM to identify clinically relevant amphenicol agar medium formulation.
Bromoindoxyl, chloroindoxyl, or bro- yeasts involved CHROMagar Candida The authors noted that yeast colonies
mochloroindoxyl and other derivatives (CC) (CHROMagar, Paris, France); were smaller on Albicans ID after 24 h
are the common chromogens bound to the authors, Odds and Bernaerts (2), of incubation but that the medium far
carbohydrate substrates used in CAM. obtained greater than 99% sensitivity surpassed CC in identification of C.
The carbohydrate substrate-chromogen and specificity of the new medium albicans at 24 h; however, this distinc-
complex is metabolized by the bacterial for the presumptive identification of tion was minimized after 48 h of incu-
isolate via enzymatic hydrolysis, the car- Candida albicans, Candida krusei, bation. The CC medium performed
bohydrate is utilized, and the halogen- and Candida tropicalis when evaluated better than Albicans ID medium after
ated indoxyl compounds remain to emit against growth of the same isolates on 72 h of incubation for overall number
their color. Two or more carbohydrate Sabouraud’s Dextrose Agar (SDA). of yeasts isolated.
substrate-chromogens may be incorpo- Seven hundred twenty-six isolates rep- Candida dubliniensis was identified
rated. Enzymatic hydrolysis is depen- resenting 33 strains of Candida spp. by Kirkpatrick et al. (6) in a separate
dent on a number of bacterial enzymes, were used, including stock strains and report as having a darker-green colony
including β-galactosidase, β-glucosi- clinical specimens obtained from vagi- appearance on primary CC agar medium,
dase, β-glucuronidase, α-glucosidase, nal, oral, ano-rectal, nail, and skin sites. distinguishable from the light-green
α-galactosidase, α-mannosidase, N- Distinct colors were detected for C. albi- appearance of C. albicans. C. dublin-
acetyl-β-glucosidase, N-acetyl-β-galac- cans, C. tropicalis, Candida norvegen- iensis is associated with oral candidiasis
tosidase, sulfatase, esterase, lipase, sis, C. krusei, Trichosporon spp. and in HIV-infected patients but can be con-
peptidase, and phosphatase. Geotrichum spp. The authors also fused phenotypically as an atypical-
showed that the isolates could be sub- appearing C. albicans using traditional
The Uses for Chromogenic cultured from the CC agar medium growth approaches. Fifty-one dark-
Agar Media and Types of back to the SDA medium without loss green isolates were subjected to exten-
Products Available of viability. Using CC agar medium, sive phenotypic and genotypic assays
CAM have been used in all phases of Beighton et al. (3) showed that Candida for confirmation as C. dubliniensis.
microbiological investigation, including spp. could be identified in patients’ Twenty-three were confirmed as C.
clinical isolation and identification and dental specimens, including oral rinses dubliniensis, and the other 28 were

44 0196-4399/00 (see frontmatter) © 2012 Elsevier Clinical Microbiology Newsletter 34:6,2012

Table 1. Manufacturers’ chromogenic agar mediaa
Application
BD BL BM BR Rambach Hardy Oxoid Remel
Pathogen or target organism BBLb Rainbow c chromIDd Select e CHROMagar f HardyChrom g Brillianceh Spectrai
Clinical
Acinetobacter ■
Candida ■ ■ ■ ■ ■ ■
Clostridium difficile ■
Escherichia coli O157 ■ ■
Extended spectrum ■ ■ ■
beta-lactamases (ESBL)
CTX-M ESBL ■
Carbapenemase (KPC) ■ ■
Methacillin-resistant ■ ■ ■ ■ ■ ■ ■
Staphylococcus aureus (MRSA)
Urine pathogens ■ ■ ■ ■ ■ ■
Urine pathogens (biplate) ■ ■ ■
Pseudomonas ■ ■
Salmonella ■ ■ ■ ■ ■
Salmonella-Shigella ■
Shiga-toxigenic E. coli (STEC) ■
S. aureus ■ ■ ■ ■ ■
Streptococcus B ■ ■
Vibrio ■
Vancomycin-resistant ■ ■ ■ ■ ■
enterococci (VRE)

Industrial
Bacillus cereus ▲
Cronobacter sakazakii ▲ ▲ ▲
CampyCount ▲ ▲
E. coli ▲ ▲
E. coli O157 ▲ ▲ ▲ ▲ ▲
E. coli coliform ECC ▲ ▲ ▲ ▲ ▲
Enterococci ▲
Listeria ▲ ▲ ▲ ▲ ▲
Pseudomonas ▲
Salmonella ▲ ▲ ▲ ▲ ▲ ▲
S. aureus ▲ ▲ ▲ ▲
Shigella-Aeromonas ▲
Shiga toxin E. coli ▲
Vibrio ▲
a
■ , Clinical applications; ▲, food, water and environmental testing applications.
b
BBL is the trademark of Becton, Dickinson and Company, Sparks, MD.
c
Rainbow is the trademark of Biolog, Inc., Hayward, CA, (BL).
d
chromID is the trademark of bioMérieux Corporation, Marcy l'Etoile, FR.
e
Select is the trademark of Bio-Rad Laboratories, Hercules, CA.
f
CHROMagar is a trademark of Dr. A. Rambach, Paris, FR.
g
HardyChrom is the trademark of Hardy Diagnostics, Santa Maria, CA.
h
Brilliance is the trademark of Oxoid Diagnostics, Cambridge, UK.
i
Spectra is the trademark of Remel Diagnostics, Lenexa, KS.

Clinical Microbiology Newsletter 34:6,2012 © 2012 Elsevier 0196-4399/00 (see frontmatter) 45

confirmed as C. albicans. In 2001, C. glabrata (n = 9), C. krusei (n = 5), (13), over 1,400 gram-negative bacilli
Jabra-Rizk et al. (7) published a com- Candida lusitaniae (n = 3), Candida and 74 enterococci were tested. Previ-
parison study assessing the difference parapsilosis (n = 3), Candida guillier- ously well-characterized and identified
between the original formulation of CC mondii (n = 2), Candida kefyr (n = 2), microbial isolates were tested for their
medium and a re-formulation by BBL, Candida firmetaria (n = 1),and Candida expected color development on CO, and
also called CC (Becton Dickinson BBL, rugosa (n = 1). Each strain was used as positive percentages of agreement were
Cockeysville, MD). Approximately its own control by growth on SDA and calculated. The authors concluded that
90 stock yeast isolates and 522 clinical subculture to CC. Plates were compared the medium was suitable for use in the
specimens were examined, from which for visual color disparities, but there clinical microbiology laboratory for
173 yeast species were recovered. The were none. Murray et al. (11) showed primary isolation and differentiation of
CAM were equivalent in performance that the CC medium (Becton Dickinson, the most commonly encountered gram-
as defined by examination of colony Sparks, MD) could be used for the dir- negative bacilli but that less frequently
growth rates and size, but there appeared ect isolation of yeast from throat, urine, isolated gram-negative bacilli and
to be little difference in color develop- and genital specimens. In addition, they Enterococcus spp. required further
ment to distinguish yeast species by obtained 69 “signal-positive” clinical investigation.
colonial growth at 24 h. Plates were blood culture specimens that were shown In an article by Hengstler et al. (14),
re-incubated for an additional 24 h, at to be “smear positive” for the presence 658 urine specimens were evaluated for
which point the authors reported that of yeast and were subcultured onto CC colony color development on CO () and
colonies growing on the BBL re-formu- and SDA. All yeast isolates recovered the results were compared to a traditional
lation were larger and deeper in color from blood grew on both medium types. rapid indole spot test or conventional
intensity. Willinger et al. (8) reported Tan and Peterson (12) similarly showed commercial biochemical identification
upon the results of their study compar- that CC (BD Diagnostics, Sparks, MD) panels. Growth of gram-negative bacilli
ing a re-formulation of Candida ID could be used for direct susceptibility and Enterococcus spp. on CO was
(BioMérieux) to CC. They used colony testing of yeasts isolated from blood shown to outperform similar growth on
morphology and pigmentation as com- cultures. Ninety-five stock and clinical Columbia agar with 5% sheep blood,
parative criteria. Four hundred twenty- specimens were seeded into blood cul- MacConkey agar, and CPS ID2 medium.
four yeast isolates were recovered from ture bottles monitored on an automated It was noted that recovery of the Proteus-
596 clinical specimens. At 24 h of incu- blood culture instrument. An aliquot of Morganella-Providentia group of micro-
bation, the sensitivity of Candida ID signal-positive blood culture medium organisms was not as robust on CO as
was 96.8% compared to a sensitivity was used to produce a confluent lawn on traditional agar media. All cultures
of 49.6% with CC. After 48 h, the sta- of bacteria on CC agar medium plates, were compared at ≥105 and ≤105 CFU/
tistical sensitivity gap closed to 99.7 and a 25-μg disc of fluconazole was ml concentration breakpoints.
and 98.9%. The authors indicated that affixed. Zones of inhibition were read BBL CO medium (Becton Dickinson)
the color intensity was more easily and compared to both standardized disc was compared with a standard two-plate
distinguishable at 24 h with Candida diffusion and minimal inhibitory con- setup for urine cultures by D’Souza et
ID than with CC. centration (MIC) methods specifically al. (15). Over 1,000 consecutive urine
Hospenthal et al. (9) addressed a for the determination of fluconazole specimens were sampled with a 1-μl
practical issue for mycology laborato- resistance. The results for direct sus- calibrated loop, followed by inoculation
ries with part-time staffing schedules. ceptibility obtained with CC compared onto tryptic soy agar with 5% sheep
They examined color development favorably to standardized disc diffusion blood, MacConkey agar, and CO
in the reading of mycology cultures and MIC methods. The authors con- medium. There were 250 positive cul-
growing on CC (DRG International, cluded that direct susceptibility testing tures, 199 of which were an exact match
Mountainside, NJ.) daily for 7 days of with CAM had the potential to provide on all media. However, there were an
incubation. They determined that color rapid and clinically useful information additional 51 discrepant culture results.
intensity did not change for the majority but stopped short of recommending Forty of the 51 discrepant results con-
of Candida spp. The authors stated that routine use due to difficulties in the sisted of pure isolate culture results; 28
C. albicans and C. krusei were easily comparison of interpretation zones of these results were correctly identified
distinguishable after 24 h of incubation and MICs observed with C. glabrata by the CO agar medium, and 12 of the
but that incubation, examination, and and C. paropsilosis. results were correctly identified by con-
interpretation of colonies growing on ventional media. The remaining 11
the medium could be extended out to Use of CAM to Identify mixed-isolate culture results yielded
7 days with reliability. Enterobacteriaceae spp. in discrepant findings, but the majority of
Direct detection of Candida spp. Clinical Specimens the correct isolate identifications were
was reported by Horvath et al. (10) in CHROMagar Orientation (CO) obtained by use of the CO compared to
an analytical study showing that CC (Becton Dickinson, Heidelberg, the conventional media. Additionally,
could be used for direct isolation of Germany), was designed to differenti- automated susceptibility test results
yeast from blood cultures. Fifty strains ate the aerobic gram-negative bacilli were compared between the micro-
of Candida spp. were inoculated into and the two major Enterococcus spp. organisms picked from the traditional
blood cultures including: C. albicans from clinical specimens. In an article media and the CO medium. There were
(n = 12), Candida tropicalis (n = 12), published in 1996 by Merlino et al. 0.4% major errors and 2% minor errors

46 0196-4399/00 (see frontmatter) © 2012 Elsevier Clinical Microbiology Newsletter 34:6,2012

detected. The authors calculated cost CHROMagar Candida for rapid screen- 10. Horvath, L.L. et al. 2003. Direct isolation
savings and labor efficiencies with CO ing of clinical specimens for Candida of Candida spp. from blood cultures on
medium compared to the conventional albicans, Candia tropicalis, Candida the chromogenic medium CHROMagar
media and concluded that the use of krusei, and Candida (Torulopsis) Candida. J. Clin. Microbiol. 41:2629-
glabrata. J. Clin. Microbiol. 34:58-61. 2632.
CO in the clinical microbiology labo-
ratory was faster than the conventional 5. Baumgartner, C., A. Freydiere, and Y. 11. Murray, M.P., R. Zinchuk, and D.H.
methods, decreased overall labor output Gille. 1996. Direct identification and Larone. 2005. CHROMagar Candida as
recognition of yeast species from clini- the sole primary medium for isolation
related to interpretation, and resulted
cal material by using albicans ID and of yeasts and as a source medium for
in an overall cost saving by reducing CHROMagar Candida plates. J. Clin. the rapid-assimilation-of- trehalose
individual reagent costs. Microbiol. 34: 454-456. test. J. Clin. Microbiol. 43:1210-1212.
Editor’s note: Part II of this article 6. Kirkpatrick W.R. et al. 1998. Detection 12. Tan, G.L. and E.M. Peterson. 2005.
will appear in the April 1, 2012 issue of Candida dubliniensis in oropharyn- CHROMagar Candida medium for
of CMN (vol. 34, No. 7). geal samples from human immunodefi- direct susceptibility testing of yeast
ciency virus-infected patients in North from blood cultures. J. Clin. Microbiol.
References America by primary CHROMagar 43:1727-1731.
1. MacFaddin, J. 1980. Identification Candida screening and susceptibility 13. Merlino, J. et al. 1996. Evaluation of
schemas, p. 346, 372, 442. In Biochem- testing of isolates. J. Clin. Microbiol. CHROMagar Orientation for differenti-
ical tests for identification of medical 36:3007-3012. ation and presumptive identification of
bacteria. Williams and Wilkins, 7. Jabra-Rizk, M.A. et al. 2001. Evaluation gram-negative bacilli and Enterococcus
Baltimore, MD. of a reformulated CHROMagar Candida. species. J. Clin. Microbiol. 34:1788-
2. Odds, F.C. and R. Bernaerts. 1994. J. Clin. Microbiol. 39:2015-2016. 1793.
CHROMagar Candida, a new differen- 8. Willinger, B. et al. 2001. Performance 14. Hengstler, K.A., R. Hammann, and
tial isolation medium for presumptive of Candida ID, a new chromogenic A. Fahr. 1997. Evaluation of BBL
identification of clinically important medium for presumptive identification CHROMagar orientation medium for
Candida species. J. Clin. Microbiol. of Candida species, in comparison detection and presumptive identification
32:1923-1929. to CHROMagar Candida. J. Clin. of urinary tract pathogens. J. Clin.
3. Beighton, D. et al. 1995. Use of Microbiol. 39:3793-3795. Microbiol. 35:2773-2777.
CHROMagar Candida medium for iso- 9. Hospenthal, D.R. et al. 2002. Persis- 15. D’Souza, H.A., M. Campbell, and E.J.
lation of yeasts from dental samples. tence of pigment production by yeast Baron. 2004. Practical bench compari-
J. Clin. Microbiol. 33:3025-3027. isolates grown on CHROMagar Can- son of BBL CHROMagar Orientation
4. Pfaller, M.A., A. Houston, and S. dida medium. J. Clin. Microbiol. and standard two-plate media for urine
Coffmann. 1996. Application of 40:4768-4770. cultures. J. Clin. Microbiol. 42:60-64.

Clinical Microbiology Newsletter 34:6,2012 © 2012 Elsevier 0196-4399/00 (see frontmatter) 47