Chromogenic Agars
P Druggan, Genadelphia Consulting, West Kirby, UK
C Iversen, University of Dundee, Dundee, UK
Ó 2014 Elsevier Ltd. All rights reserved.
Introduction
The origins of chromogenic agars go back to the investigation
of gene control in Escherichia coli. Although the features of gene
control are taught commonly in microbiology courses, they are
not necessarily taught to food microbiologists and food
scientists. They are important to understanding how chromo-
genic agars work and how to design them.
Until the late 1940s, investigation of lactose transport and
hydrolysis mutants was based on MacConkey agar and Eosin
methylene blue agar. Improvements were made to the
techniques for detecting mutants by the introduction of the
lactose analogs o-nitropheny-b,D-galactoside (ONPG) and p-
nitrophenyl-b,D-galactoside. These are colorless substrates, but
on hydrolysis, they produce a yellow color in and around the
colony. This was a significant advance as it allowed researchers
to look for mutants in the small number of genes involved in
the utilization of lactose, rather than the large number of genes
involved in the complex system that results in its fermentation.
The nitrophenol substrates had improved specificity for
mutants in the genes of the lac operon, but they suffer from
a number of weaknesses. Nitrophenol is a weak chromophore,
with a low extinction coefficient. It is also lipid soluble, and
when hydrolyzed within the cytoplasm, it rapidly diffuses
across the cytoplasmic membrane (CM) into the surrounding
medium.
In 1966, 5-bromo-4-chloro-3indolyl-b,D-galactoside (X-
Gal) was used in genetic experiments with E. coli. This is
another colorless lactose analog that on hydrolysis, dimerizes
in the presence of oxygen to form a deep blue-green pigment
that precipitates in the colony. The use of X-Gal for aiding in
the detection of E. coli was patented in 1979, and although
ONPG was used to differentiate Salmonella from coliforms in
1964, a commercial diagnostic culture medium exploiting this
principle and the available chromogens was not commercial-
ized until 1989, when the molecular biologist Dr. Alain Ram-
bach used it as one of the components in Rambach Agar to
differentiate coliforms from Salmonella.
Fluorogens have also been used in a similar way to chro-
mogens. Instead of a chromogenic color reaction, fluorogens
produce fluorescence under long-wave ultraviolet (UV) light on
cleavage of the substrate. A key example is the MUG (glucu-
ronidase) test, where ß-D-glucuronidase (produced almost
exclusively by E. coli) cleaves 4-Methylumbelliferyl-ß-D-
glucuronide to 4-methylumbelliferone and glucuronide, with
the fluorogen 4-methylumbelliferone being detected under
a long-wavelength UV lamp.
A new class of selective agents known as InhibigensÔ have
been developed, which provide highly specific selectivity and
allow improved recovery of target organisms. In this case, an
inhibitor molecule is linked to a specific substrate in place of
a chromogen. When bound to the substrate, the inhibitor
molecule is inert, but it becomes toxic when a cell takes up and
cleaves the substrate. Instead of being aimed at differentiating
248 Encyclopedia of Food Microbiology, Volume 2
the target organism, InhibigensÔ are directed at the competing
flora.
This short piece of history was given to illustrate that
knowledge of molecular biology and organic chemistry are
important to food microbiologist. Innovation comes from
knowledge in breadth, not in depth.
Molecular Biology of Substrate Utilization
Three steps are required for a chromogenic substrate to produce
a color in a colony. These are (1) induction of genes, (2)
transport of substrate, and (3) hydrolysis of the substrate. The
genetic elements governing these steps are grouped together in
an operon. We will use the lac operon as our model for
discussion, as it is the best known, and because lactose
fermentation has been the major diagnostic test for differenti-
ating nonpathogenic coliforms from non-lactose-fermenting
Salmonella for more than 100 years.
Figure 1 shows a topographic model of the lac operon.
When an inducer binds to the control region, the genes
downstream of it are transcribed into functional enzymes. If
there is no inducer, there is no induction of the enzyme. The
control of gene expression is quite complex, and it has been
simplified for this discussion. This is a general model for gene
regulation. The structure might vary for different nutrients and
transport systems, but for di- and trisaccharides, it is sufficient
to communicate the principles around the process.
This system allows the cell to tightly control gene expression
to respond to its immediate environment at minimal cost in
resources. The system is imperfect, and this allows small
numbers of the proteins to be generated in the cell. This allows
nutrients to enter the cell and to induce the operon, if they are
present at sufficient concentration.
Induction of the operon for a sugar is fundamentally
important for the exploitation of chromogenic substrates.
Chromogenic substrates may or may not induce an operon.
This is dependent on the substrate, the chromophore, and the
species and will be discussed later.
The gene for b-galactosidase is lacZ. This enzyme hydrolyzes
b-galactosides, like lactose, into two parts. One of these is
always galactose, as this is the part of the molecule that is
recognized by the enzyme. The other part of the molecule can
be a sugar or a chromophore, and this is why chromogenic
substrates can be used to replace sugars.
Both Gram-positive and Gram-negative bacteria have
evolved systems that preferentially exclude harmful molecules
and allow for entry of nutrients in the cell. Because of the
Control lacZ lacY lacA
Figure 1 Topographic model of the lac operon. lacZ ¼ gene for the
hydrolase, b-galactosidase; lacY ¼ gene for the permease; lacA ¼ gene for
the acetylase.
http://dx.doi.org/10.1016/B978-0-12-384730-0.00416-X

environmental niche occupied by Gram-negative bacteria, their
barrier is more complex and more effective than that of Gram-
positive organisms. Gram-negative organisms have an outer
membrane (OM) – see Figure 2. This is an asymmetric lipid
bilayer that is impermeable to the entry of toxic molecules into
the cell. The OM has a selective permeability to nutrients
because of porins that allow for the diffusion of molecules less
than 600 Da into the cell. This represents the molecular weight
cut-off for chromogenic glycoside substrates in the Gram-
negative cell, as the S-layer and the peptidoglycan layer that
sandwich the OM have size exclusion limits that are almost two
orders of magnitude greater than that of the porins.
The lacY gene encodes the permease that is located in the
CM. The CM is hydrophobic, and those water-soluble nutrients
that diffuse through the porins with molecular weight above
100 Da cannot cross this membrane. The permease accumu-
lates b-galactosides within the cell against a concentration
gradient. It is a proton symport and derives its energy from the
hydrogen ion potential across the CM. This is a common type
of transport system in Gram-negative bacteria, but it is not the
only type.
The lacA gene transcribes an acetylase transferase. This has
a similar function to chloramphenicol acetylase, which is part
of an efflux system that has evolved in bacteria to eliminate
molecules that pose a threat to the cell. The b-galactoside
transacetylase is part of a system that eliminates unmetabo-
lizable molecules out of the cell. The bacterial cell has a hier-
archy for sugars, based on the net energy yield through
catabolism. This is controlled through a series of proteins that
respond to the concentration of the various sugars in the
cytoplasm. If there was a mutation in the hydrolase gene, lacZ,
that prevents the transcription of a functional b-galactosidase,
the cell could accumulate b-galactosides in the cytoplasm. This
would prevent the bacterium from using energetically less
favorable, but metabolizable sugars that might be in the envi-
ronment surrounding the cell. The product of the lacA gene is
essentially a metabolic fail-safe system that has evolved to
minimize the effect of mutation in the hydrolytic enzyme
genes. For chromogenic substrates to be effective three steps
need to occur.
External environment
S-layer
Outer membrane Porin
Peptidoglycan layer
Periplasmic space
Cytoplamic membrane Permease
Cytoplasm
Figure 2 Idealized structure of the Gram-negative cell wall. The arrows
represent the flow of nutrients into the cell.
IDENTIFICATION METHODS j Chromogenic Agars 249
1. Induction of the operon.
2. Transportation of the substrate across the cytoplamic
membrane against a concentration gradient. This requires
a substrate-specific permease transcribed from a gene in the
operon.
3. Hydrolysis of the substrate and release of a colored mole-
cule – the chromophore.
Fermentation Media
In traditional fermentation media, like MacConkey agar, the
pH change caused by fermentation of lactose is the result of
a cascade of steps that starts with transportation of a sugar into
the cell and through an additional 16 enzyme steps (see
Figure 3) before acid is generated and the color of the medium
changes.
Including the control sequences for each gene, roughly 35
genetic elements could affect the production of acid in lactose
fermenters. Assuming a phenotypic mutation rate of 5  108
and a population density of 1  108 cfu ml1 in a pure
culture of a coliform, it can be estimated that around 1750
cells in the population with a single mutation might fail to
ferment the sugar. This conservative estimate does not take
into consideration other mutations that might affect the cells
capacity to ferment sugars in general. Mutations in the lac
operon and the common pathway are the most significant,
but expression of inactive enzymes in the two branches
would halve the total production of acid by the cell.
Depending on the buffering capacity of the medium, this may
or may not cause false positives.
The large numbers of competitive microflora in food
samples leads to a relatively large number of nonfermenting
bacterial species. During development, media for Salmonella
were designed to inhibit nonfermenting organisms as much as
possible without inhibiting the target organism. The ferment-
able sugars were included to differentiate organisms that could
not be inhibited without inhibiting Salmonella. Mutants that
interrupt the pathway shown in Figure 3 would appear as false
positives on media like Xylose lysine deoxycholate agar or
brilliant green agar. Although both of these media include
sucrose as an additional fermentable carbohydrate, it can be
seen from Figure 3 that the addition of a carbohydrate might
improve the specificity of the medium, and on hydrolysis, the
glucose and fructose would feed into the common pathway. If
there is a mutation in the genetic elements for control and
expression of the enzymes in this, it would affect the ability to
generate acid from both sucrose and lactose.
The incidence of Salmonella in cooked food is estimated to
be 1.5%. The number of positives seen in a routine testing
laboratory will depend in the material being tested and on the
type of processing involved. From this brief discussion, we
hope that we have shown why false-positives colonies are
a common feature of pathogen testing on traditional media.
The implications of this will be discussed later in this chapter.
Chromogenic Substrates
Chromogenic substrates offer a number of advantages over
traditional media based on pH indicators. A color change is
250 IDENTIFICATION METHODS j Chromogenic Agars

Induction

Lac operon
Permease

-Galactosidase

Glucokinase Galactosemutarotase

Galactokinase

Leloir
Galactose-1-phosphate pathway
uridylyltransferase

UDP-galactose-4-epimerase

Phosphoglucosemutase

Glucosephosphate isomerase

6-Phosphofructokinase

Fructose-biphosphate aldolase

Triosephosphate isomerase

Glyceraldehyde-phosphate dehydrogenase

Phosphoglycerate kinase

Enolase

Pyruvate kinase

Lactate dehydrogenase

Lactic acid

Figure 3 Enzymes involved in the fermentation of lactose to lactic acid. The lac operon is shown in blue. The Leloir pathway for conversion of
galactose to glucose-6-phosphate is shown in red. The conversion of glucose to glucose-6-phosphate is shown in green. Common enzyme pathways
are shown in black.

Table 1 Probability of false-negative due to a phenotypic mutation in a cell

Lactose fermentation Chromogenic b-galactosidase

Probability of a colony with a phenotypic mutation 5  108 5  108

Number of genetic elements 35 3
Probability of no phenotypic mutation in a cell ((1(5  108))35) ((1(5  108))3)
Probability of a single phenotypic mutation in a cell 1((1(5  108))35) 1((1(5  108))3)
Number of phenotypic mutants in 1  109 cfu ml1 1750 150
Number of phenotypic mutants in 10 mm sample 17.5 1.5
 IDENTIFICATION METHODS j Chromogenic Agars 251

Figure 4 (a) 5-Bromo-4-chloro-3-indolyl-b,D-galactoside (X-Gal); (b) 5-bromo-4-chloro-3-indolyl-caprylate.

seen in a colony after transportation and hydrolysis, and this

reduces the potential number of false positives due to mutation
by more than 10-fold, as fewer genes are involved in generating
a signal. How this affects the number of false positives on
a plating media is shown in Table 1.
Chromogenic substrates used in culture media for food
microbiology are either synthetic analogs of di- and trisaccha-
rides that have an ether bond (Figure 4(a)) or are esters
(Figure 4(b)).
Hydrolysis of the ether or ester bond releases the colorless
indolol. In the presence of oxygen, this dimerizes to form the
pigment indigo (Figure 5). The indolyl substrates are the most
common synthetic chromogenic substrates used in culture
media. A number of newer substrates have been synthesized in
the past decade that form a color on hydrolysis by chelating
cations, although these are not used extensively as yet.
A variety of colors are available in chromogenic substrates
due to the substitution of halogens onto the indolol molecule.
Halogens can either make the color stronger or can change the
color, depending on the electron withdrawing capacity of the
halogen and its location on the indole ring. The names, struc-
tures, and colors are shown in Table 2.
Although the color of a chromogenic substrate is the easiest
feature to appreciate, it is not the most important. For di- and
trisaccharide analogs, the most import feature is the sugar
attached as this is recognized by the permease, and it may have
a significant role in the induction of the appropriate operon.
A significant problem when reviewing the literature on chro- Figure 5 Dimerization of colorless 5-bromo-4-chloro-3-indolyl to blue-
mogenic substrates is that abbreviations regarding sugars are green 5,50 dibromo-4,40 -dichloro-indigotin (indigo).
252 IDENTIFICATION METHODS j Chromogenic Agars
Table 2 Structure, name, and color of indolyl chromophores
Structure
R, represents an organic monomer, such as monosaccharide or a fatty acid.
applied arbitrarily. In this text, we have used the system of
abbreviation for monosaccharides recommended by Interna-
tional Union of Pure and Applied Chemistry (IUPAC) (Table 3).
A major obstacle to the adoption of chromogenic substrates
is the lack of understanding how they relate to individual
sugars. A list of X-series chromogenic substrates is shown in
Table 4. What is probably most striking is that a single substrate
potentially can be used for a range of sugars, but this does not
necessarily mean that they induce the operon for a sugar – that
is a different issue (Table 5).
Description Color of dimer
3-Indolyl-R
5-Bromo-3-indolyl-R
5-Bromo-4-chloro-3-indolyl-R
5-Bromo-6-chloro-3-indolyl-R
6-Chloro-3-indolyl-R
6-Fluoro-3-indolyl-R
Glycosides are the main diagnostic tools used to differentiate
microorganisms in culture media. In traditional fermentation
media, a combination of glycosides, such as lactose and sucrose,
simply lower the pH for those organisms that ferment them, as
after initial hydrolysis they feed into the glycolytic pathway
(Figure 3) and reach the same end product. The indolyl
substrates come in a variety of colors (see Table 2). On hydro-
lysis, they dimerize and remain in the colony. This means that
two or more substrates can be combined in a medium to improve
its specificity. This is illustrated in the Venn diagram in Figure 6.
 IDENTIFICATION METHODS j Chromogenic Agars 253

Table 3 IUPAC abbreviations for monosaccharides

Name Abbreviation Name Abbreviation

Abequose Abe N-acetylglucosamine GlcNAc

Allose All Glucuronic acid GlcA
Altrose Alt Gulose Gul
Apiose Api Iduronic acid IdoA
Arabinose Ara Lyxose Lyx
Arabinitol Ara-ol Mannose Man
Fructose Fru Rhamnose Rha
Fucose Fuc Psicose Psi
Fucitol Fuc-ol Quinovose Qui
Galactose Gal Ribose Rib
Galactosamine GalN Ribose-5-phosphate Rib5P
N-acetylgalactosamine GalNAc Sorbose Sor
Glucose Glc Tagatose Tag
Glucosamine GlcN Talose Tal
Glucitol (sorbitol) Glc-ol Xylose Xyl

Table 4 5-Bromo-4-chloro-3-indolyl- analogs for naturally occurring disaccharides

Substrate Sugar IUPAC nomenclature

XbGal Allolactose 6-O-b-D-Galactopyranosyl-D-glucose

Lactose 4-O-b-D-Galactopyranosyl-D-glucose
Lactulose 2-O-b-D-Galactopyranosyl-D-fructose
XaGal Floridoside 2-O-a-D-Galactopyranosyl-D-glycerol
Galactinol 3-O-a-D-Galactopyranosyl-myo-inositol
Melibiose 6-O-a-D-Galactopyranosyl-D-glucose
XaGlc Maltose 4-O-a-D-Glucopyranosyl-D-glucose
Palatinose 6-O-a-D-Glucopyranosyl-D-fructose
Trehalose 1-O-a-D-Glucopyranosyl-D-glucose
Turnose 3-O-a-Glucopyranosyl-D-fructose
XbGlc Aesculin 6-O-b-D-Glucopyranosyl-dihydroxycoumarin
Cichoriin 7-O-b-D-Glucopyranosyl-dihydroxycoumarin
Daphnin 7-O-b-D-Glucopyranosyl-dihydroxycoumarin
Salicin 1-O-b-D-Glucopyranosyl-2-(hydroxymethyl)phenol
Arbutin 4-O-b-D-Glucopyranosyl-hydroxyphenol
Cellobiose 4-O-b-D-Glucopyranosyl-D-glucose
Gentobiose 6-O-b-D-Glucopyranosyl-D-glucose
Scillabiose 4-O-b-D-Glucopyranosyl-D-rhamnose
XaGlcNAc Chitobiose 2-Amino-2-deoxy-4-O-(2-amino-2-deoxy-b-D-glucopyranosyl)-D-glucose
XaXyl Primeverose 6-O-a-D-Xylopyranosyl-D-glucose
XaRha Rutinose 6-O-a-L-Rhamnosyl-D-glucose
XbFru or XaGlc Sucrose 1-O-a-D-Fructofuranyl-D-glucose

Substrate use is under strict control in the bacterial cell

based on the net energy yield. This means that in the case of
E. coli in Figure 5, the organism hydrolyzes the b-galactoside in
preference to the b-glucuronide; once the b-galactoside is
depleted, the E. coli cell then hydrolyzes the b-glucuronide. This
means that two separate homodimer pigments form and the
dark-blue color is due to the mixture of these in the colony.
If both substrates were hydrolyzed simultaneously, or if two
b-galactoside substrates were used, with different chromo-
phores, then because of dimerization, three pigments would
form (two homodimers and a heterodimer). This can dramat-
ically alter the color. This is summarized in Figure 7.
Chromogenic Media by Organism
Chromogenic media have been developed for use in food,
water, medical, and veterinary microbiology in which suitable
specific trait, or traits, in the target organisms have been iden-
tified. Most microbiological media companies now market
a range of proprietary chromogenic media.
The main benefit of using chromogenic media is clearer
differentiation of target colonies, which improves specificity
and reduces the number of confirmation tests needed.
Compared with traditional methods, this can prove more cost
effective in terms of both time and consumables.
 Diagnostic features of a range of chromogenic media commercially available in 2013 for analysis of food

254
Table 5

Species Examples of media Substrates Comments

IDENTIFICATION METHODS j Chromogenic Agars

Bacillus cereus Oxoid Brilliance™ Bacillus Cereus Agar X-b,D-glucopyranoside (X-Glc) B. cereus has an operon for the transportation and phosphorylation of
Biokar Compass® Bacillus cereus Agar b-glucosides and hydrolysis of the resulting b-glucoside-
6-phosphate. B. cereus hydrolyzes the substrate giving a colony
with a blue-green center and pale edge.
Chromagar® B. cereus X-b,D-glucopyranoside (X-Glc) B. cereus is b-glucosidase positive, giving colonies with a blue-green
Soy lecithin center. The organism also possesses phosphatidylinositol
phospholipase C (PIPLC), which hydrolyzes the lecithin leaving an
opaque halo around the colony.
R&F ® Bacillus cereus/Bacillus X-myo-inositol-1-phosphate B. cereus has an operon for PIPLC, which is a pathogenicity factor.
thuringiensis Chromogenic Plating Hydrolysis of X-myo-inositol-1-phosphate by PIPLC gives a blue-
Medium green colony.
Campylobacter AES Chemunex CASA® Agar Triphenyltetrazolium chloride This medium uses a tetrazolium dye that on reduction forms an
(TTC) insoluble formazan. The specificity of this type is due to the
selective agents and TTC really only acts as a visual aid.
Oxoid Brilliance™ CampyCount Agar Triphenyltetrazolium chloride Oxoid may have incorporated an L-alanyl-L-1-aminoethylphosphonic
(TTC) acid, an Inhibigen™, as this would provide high specificity against
L-Alanyl-L- Gram-negative organisms as these possess L-alanyl
1-aminoethylphosphonic aminopeptidase, Campylobacter do not.
acid
R&F® Campylobacter jejuni/C. coli Aldol acetate This is a true chromogenic Campylobacter agar. Campylobacter, like
Chromogenic Plating Medium many species, possess esterases capable of hydrolyzing acetate
esters. The short-chain indolyl esters are unstable and cannot
withstand heating. Biosynth® recently developed chromogenic
Aldol substrates that are substantially more stable than the indolyl
substrates. It looks like R&F labs have used one of these in this
medium. Specificity is due to the choice of selective agents, as
a wide range of bacteria can hydrolyze acetate esters.
Clostridium perfingens Membrane Clostridium Perfringens Indoxyl-b,D-glucopyranoside Clostridium perfringens colonies appear yellow on this medium due
(m-CP) Medium (I-Glc) sucrose fermentation. A number of taxa within the Clostridia
Phenophthalein-biphosphate ferment sucrose, so I-Glc is included in the medium to differentiate
Sucrose and phenol red these from C. perfringens, which does not have an operon for the
transport and hydrolysis of b-glucosides. C. perfringens possess an
operon for acid phosphatase. Phenolphthalein biphosphate is
included in the medium as a presumptive indicator of C. perfringens;
however, phenolphthalein has a pKa around 9.6 and is colorless at
pH below 8.6. The plate is exposed to ammonia vapor to raise the
pH of the medium to detect acid phosphatase activity.
Cronobacter spp. AES Chemunex Enterobacter Sakazakii X-a-D-glucopyranoside Many organisms within the Enterobacteriaceae are capable of
(Enterobacter sakazakii) Isolation Agar (ESIA) (XaGal) hydrolyzing a-glucosides like maltose. X-a-D-glucopyranoside
Oxoid Brilliance™ Enterobacter appears to only induce the expression of a-glucosidase in a small
Sakazakii Agar (DFI) number of taxa within this family. Cronobacter spp. appear green on
Chromogenic Cronobacter Isolation DFI due to the slight yellow coloration of the base medium; blue on
Agar (CCI) ESIA due to the background crystal violet; and blue-green on CCI
due to increased XaGlc concentration and a more defined base
medium relative to DFI agar.

(Continued)
Species Examples of media
Escherichia coli TBX Agar (ISO 16649-2:2001)
E. coli/Coliforms Merck Chromocult® Coliform Agar
Bio-Rad Rapid’ E. coli 2™ Agar
Oxoid Brilliance™ E. coli/coliform
Selective Agar
E. coli O157 Sorbitol MacConkey Agar with BCIG
CHROMagar™ O157
Listeria spp. and ALOA Agar (ISO 11290:2004)
L. monocytogenes Oxoid Brilliance™ Listeria Agar
Bio-Rad Rapid’ L.mono™ Medium
CHROMagar™ Identification Listeria
Pseudomonas aeruginosa
Substrates
X-b,D-glucuronide (X-GlcA)
X-b,D-glucuronide, (X-GlcA)
Salmon-b,D-galactopyranoside
(Salmon-Gal)
X-b,D-glucuronide (X-GlcA)
Sorbitol and neutral red
Magenta-a-D-
galactopyranoside (MaGal)
X-b,D-glucuronide (X-GlcA)
X-b,D-glucopyranoside (X-Glc)
X-b,D-glucopyranoside (X-Glc)
and soy lecithin
X-myo-inositol-1-phosphate,
xylose and phenol red
Salmon-a,D-mannoside
(Salmon-aMan)
Soy lecithin
b-Alanyl-7-amido-1-pentyl-
3H-phenoxazin-3-one (b-
ala-APP)
Comments
E. coli have an operon for the uptake and hydrolysis of E. coli b-
glucuronides, and it is found in more than 97% of strains.
Hydrolysis of the substrate produces blue-green colonies.
E. coli colonies are blue as they hydrolyze both substrates, whereas
coliforms are red.
E. coli O157:H7 has a frame shift mutation in the uid gene resulting in
expression of a nonfunctional b-glucuronidase. The combination of
sorbitol fermentation with BCIG (X-GlcA) hydrolysis improves the
specificity of this medium. E. coli O157:H7 appear colorless.
There are many taxa within the Enterobacteriaceae that have an operon
for the transportation and hydrolysis of a-galactosides, such
a melibiose. E. coli O157:H7 hydrolyzes MaGal giving a pale mauve
colony. Other strains of E. coli also hydrolyze X-GlcA and the
combined chromophores give a blue colony. Much of the specificity
of the medium comes from the use of antibiotics, but X-Glc is
included in the medium to reduce false positives from any members
of the Enterobacteriaeae that might be resistant to the selective
agents. The hydrolysis of X-Glc and MaGal gives a blue colony.
Listeria spp. hydrolyze the X-b,D-glucopyranoside producing green-
blue colonies. L. ivanovii and L. monocytogenes possess
phosphatidylcholine phospholipase C and PIPLC, these enzymes
hydrolyze lecithin forming an opaque halo around the colony.
L. monocytogenes and L. ivanovii possess phospholipase C (opaque
halo) and appear as blue colonies due to phenol red in the medium.
Xylose is included in the medium to differentiate xylose fermenting
IDENTIFICATION METHODS j Chromogenic Agars
L. ivanovii from L. monocytogenes. All other colonies on the medium
are presumptive Listeria spp.
L. monocytogenes is unique within the genus Listeria as it has
a functional operon for uptake and hydrolysis of a-mannosides that
is induced by Salmon-aMan. L. monocytogenes colonies appear
pink on this medium. L. monocytogenes and L. ivanovii possess
phospholipase C (opaque halo). All other white colonies on the
medium are considered other Listeria spp.
Pseudomonas aeruginosa possesses the enzyme b-alanyl arylamidase.
b-ala-APP is yellow, on hydrolysis, this forms a red nondiffusible
pigment in the colony. This is one of the few peptidase substrates
that have been used in chromogenic culture media and is
a significant contribution to the future of this type of technology.
There are some challenges to the use of amino peptides in culture
media, as media based on peptones contain a great number of
peptides that compete with peptide permeases. Many of these
media are based on chemically defined media, similar in many ways
to cell-culture media.
(Continued)
255

Table 5 Diagnostic features of a range of chromogenic media commercially available in 2013 for analysis of fooddcont'd
Species Examples of media Substrates
Salmonella Rambach™ Agar X-b,D-galactopyranoside
(X-Gal)
Propylene glycol
Oxoid Salmonella Chromogenic Agar Magenta-octanoate
(OSCM) (M-octanoate)
Conda Salmonella Chromogenic X-b,D-galactopyranoside
Medium (X-Gal)
Oxoid Brilliance™ Salmonella Agar Magenta-octanoate
(M-octanoate)
X-b,D-glucoopyranoside
(X-Glc)
L-Alanyl-L-
1-aminoethylphosphonic
acid
COMPASS® Salmonella Agar Magenta-octanoate
CHROMagar™ Salmonella Plus (M-octanoate)
X-b,D-glucoopyranoside (X-Glc
LabM Harlequin™ Salmonella ABC X-a-D-galactopyranoside
Medium (XaGal)
CHE-b,D-galactopyranoside
(CHE-Gal) and ammonium
iron (III) citrate
Staphylococcus aureus bioMérieux chromID®: S. aureus X-a,D-glucopyranoside
(XaGlc)
256
IDENTIFICATION METHODS j Chromogenic Agars
Comments
Fermentation of propylene glycol is used as a marker for Salmonella
spp. and these colonies are a pink color typical of neutral red at low
pH. X-Gal is included in the medium to differentiate other
organisms that potentially may ferment propylene glycol.
Salmonella hydolyzes M-octanoate giving mauve colonies. There are
a few other taxa within the Enterobacteriaceae that also hydrolyze
this substrate, so X-Gal is included to improve the specificity of
the medium. Those organisms that are able to hydrolyze both
substrates have dark blue colonies.
This medium is similar to OSCM, but X-Gal has been replaced by X-Glc
to improve the sensitivity of the medium for Salmonella Group IV, as
these taxa have the operon for b-galactosidase. Although the use of
X-Glc would decrease the specificity of the medium relative to
X-Gal, this has been tackled by the addition of Inhibigen™
technology. Salmonella spp. are significantly less sensitive to this
molecule than many other genera within the Enterobacteriaceae,
and this leads to significantly fewer competitors on these plates
relative to other media.
The chromogen system in this medium is similar to Oxoid Brilliance™.
This formulation is an improvement on those that use X-Gal for
differentiating the coliforms as some Salmonella spp., have an
operon for the transport and hydrolysis of b-galactosides. None
of the taxa within Salmonella have an operon for transport and
hydrolysis of b-glucosides.
Salmonella, like many taxa within the Enterobacteriaceae are capable
of fermenting melibiose, an a-galactoside. Salmonella spp.
hydrolyze XaGal giving a green colony. CHE-Gal is included in
the medium to differentiate coliforms from Salmonella, as most
coliforms also hydrolyze XaGal.
This medium uses XaGlc to detect a-glucosidase in S. aureus. The
operon for transport and hydrolysis of a-glucosides is induced
by XaGlc in few other Staphylococci species.
 IDENTIFICATION METHODS j Chromogenic Agars 257

U = Gram-negative organism growing on bile-containing medium

-Galactosidase

Citrobacter spp.
Enterobacter spp. E. coli
O157:H7 Klebsiella spp.
E. coli

Salmonella spp. Shigella

spp.
Proteus spp.
Pseudomonas spp.
Salmonella spp. Shigella
-Glucuronidase
spp.

Figure 6 Venn diagram of a dual chromogen medium containing bile salts as a selective agent for Gram-negative organisms.

Figure 7 The formation of 6,60 -difluoro-indigotin (25%), 5,50 -dibromo-6,60 -dichloro-indigotin (25%), and 5-bromo-6-chloro-60 -fluoro-indigotin (50%)
from the dimerization of a 6-fluoro-indolol and 5-bromo-6-chloro-indolol mixture.
258 IDENTIFICATION METHODS j Chromogenic Agars
See also: Bacillus: Introduction; Campylobacter; Clostridium;
Enterobacteriaceae, Coliform, and Escherichia coli: Classical
and Modern Methods for Detection and Enumeration;
Enterococcus; Escherichia coli: Escherichia coli; Escherichia
coli O157: E. coli O157:H7; Listeria: Introduction;
Pseudomonas: Introduction; Salmonella: Introduction;
Staphylococcus: Introduction; Vibrio Introduction, Including
Vibrio parahaemolyticus, Vibrio vulnificus, and Other Vibrio
Species; Yersinia: Introduction; Cronobacter (Enterobacter)
sakazakii; Escherichia coli: Pathogenic E. coli (Introduction).
Further Reading
Beckera, B., Schulera, S., Lohneisb, M., Sabrowskib, A., Curtisc, G.D.W.,
Holzapfela, W.H., 2006. Comparison of two chromogenic media for the detection of
Listeria monocytogenes with the plating media recommended by EN/DIN 11290-1.
International Journal of Food Microbiology 109, 127–131.
Demchick, P., Koch, A., 1996. The permeability of the wall fabric of Escherichia coli
and Bacillus subtilis. Journal of Bacteriology 178, 768–773.
Deutscher, J., Francke, C., Postma, P.W., 2006. How phosphotransferase system-
related protein phosphorylation regulates carbohydrate metabolism in bacteria.
Microbiology and Molecular Biology Reviews 70, 939–1031.
Druggan, P., Iversen, C., 2009. Culture media for the isolation of Cronobacter spp.
International Journal of Food Microbiology 136, 169–178.
Frey, P., 1996. The Leloir pathway: a mechanistic imperative for three enzymes to
change the stereochemical configuration of a single carbon in galactose. Feder-
ation of American Societies for Experimental Biology Journal 10, 461–470.
Manafi, M., 2000. New developments in chromogenic and fluorogenic culture media.
International Journal of Food Microbiology 60, 205–218.
Manafi, M., Kneifel, W., Bascomb, S., 1991. Fluorogenic and chromogenic substrates
used in bacterial diagnostics. Microbiological Reviews 55, 335–348.
Murray, I., Shaw, W., 1997. O-Acetyltransferases for chloramphenicol and other
natural products. Antimicrobial Agents and Chemotherapy 41, 1–6.
Nikaido, H., Vaara, M., 1985. Molecular basis of bacterial outer membrane perme-
ability. Microbiology and Molecular Biology Reviews 49, 1–32.
Tavakoli, H., Bayat, M., Kousha, A., Panahi, P., 2008. The application of chromogenic
culture media for rapid detection of food and water borne pathogen. American-
Eurasian Journal of Agriculture and Environmental Science 4, 693–698.
Webster, K.A., 2003. Evolution of the coordinate regulation of glycolytic enzyme genes
by hypoxia. Journal of Experimental Biology 206, 2911–2922.