International Journal of Food Microbiology 60 (2000) 205–218

www.elsevier.nl / locate / ijfoodmicro

New developments in chromogenic and fluorogenic culture media

M. Manafi*
Hygiene Institute, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria

Abstract

This review describes some recent developments in chromogenic and fluorogenic culture media in microbiological
diagnostic. The detection of b-D-glucuronidase (GUD) activity for enumeration of Escherichia coli is well known. E. coli
O157:H7 strains are usually GUD-negative and do not ferment sorbitol. These characteristics are used in selective media for
these organisms and new chromogenic media are available. Some of the new chromogenic media make the Salmonella
diagnostic easier and faster. The use of chromogenic and fluorogenic substrates for detection of b-D-glucosidase (b-GLU)
activity to differentiate enterococci has received considerable attention and new media are described. Rapid detection of
Clostridium perfringens, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus are other application of
enzyme detection methods in food and water microbiology.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Microbiology; Fluorogenic; Chromogenic; Culture media; Rapid identification

1. Introduction The fluorogenic enzyme substrates generally con-

The ability to detect the presence of a specific and
exclusive enzyme using suitable substrates, in par-
ticular fluorogenic or chromogenic enzyme sub-
strates, has led to the development of a great number
of methods for the identification of microorganisms
even in primary isolation media. The incorporation
of such substrates into a selective medium can
eliminate the need for subculture and further bio-
chemical tests to establish the identity of certain
microorganisms (Manafi et al., 1991; Manafi, 1996).
This review describes recent developments in fluoro-
genic and chromogenic media for specific detection
of microorganisms.
*Tel.: 1 43-1-40490-79450; fax: 1 43-1-40490-9794.
E-mail address: mohammad.manafi@univie.ac.at (M. Manafi).
sist of a specific substrate for the specific enzyme
such as sugar or amino acid and a fluorogen such as
4-methylumbelliferone, being able to convert UV
light to visible light. Methylumbelliferyl-substrates
are water soluble, highly sensitive and very specific.
Because of their pH-dependance, strong diffusion in
solid media and the need of UV-light, the use of
these substrates is limited. Chromogenic enzyme
substrates are compounds which act as the substrate
for specific enzymes and change colour due to the
action of the enzyme. In general, based on their
chemical reaction, four groups of chromogenic com-
pounds can be distinguished and recently described
by Manafi (1998). Indolyl derivatives are water
soluble and heat stable and the mostly used deriva-
tives such as 5-bromo-4-chloro-3-indolyl (X), 5-
bromo-6-chloro-3-indolyl (magenta) or 6-chloro-3-

0168-1605 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0168-1605( 00 )00312-3
206 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
indolyl (salmon) showing no diffusion on the agar
plate.
2. The application of chromogenic and
fluorogenic media in food and water
microbiology
2.1. Enterobacteriaceae
2.1.1. Escherichia coli
The new generation of media uses b-D-glucuronid-
ase (GUD) as indicator for E. coli. GUD is present in
94–96% of E. coli strains, but also some Salmonella,
Shigella and Yersinia spp. (Hartman, 1989). Some
authors found GUD-positive strains of Citrobacter
freundii (Gauthier et al., 1991) or some strains of
Klebsiella oxytoca, Serratia fonticola and Yersinia
intermedia (Alonso et al., 1996). GUD test is used
increasingly for detection of E. coli in water and
food microbiology as E. coli is an important in-
dicator of fecal contamination in samples from the
food processing and water purification plants. Other
Escherichia spp. do not produce this enzyme as
described by Rice et al. (1991). Some pathogenic
strains of E. coli such as typical E. coli O157:H7
however, do not posses GUD either (Frampton and
Restaino, 1993).
GUD activity is measured by using different
chromogenic and fluorogenic substrates such as p-
nitrophenol-b-D-glucuronide (PNPG), or 5-bromo-4-
chloro-3-indolyl-b-D-glucuronide (X-GLUC) and 4-
methylumbelliferyl-b-D-glucuronide (MUG). MUG
is hydrolyzed by GUD yielding 4-MU, which shows
blue fluorescence when irradiated with long-wave
UV light (366 nm). MUG has been incorporated into
both liquid media including lauryl sulfate broth,
m-Endo broth, EC broth, Brila-broth, DEV-lactose-
pepton-broth and LMX broth. The solid media to
which MUG was added, include violet red bile agar,
ECD-agar, MacConkey-agar and m-FC agar and are
already described earlier (Manafi, 1996). Fluorocult
media (Merck) are used for determination of E. coli,
e.g. in drugs (Huang et al., 1994), in foods of animal
origin (Bredie and de Boer, 1992), for the isolation
of diarrhea-causing E. coli (Muto et al., 1991),
clinical samples (Heizmann et al., 1988; Mori et al.,
1991), milk and milk products (Hahn and Wittrock,
1991) and bathing water (Havemeister, 1991). The
minimum concentration sufficient for distinct indica-
tion was 50 mg / ml. However, the concentration
necessary for acceptable detection of E. coli depends
on the other constituents of the media. As the
fluorescence of 4-MU is pH dependent, the pH of
growth media containing MUG should be slightly
alkaline; otherwise alkaline solution needs to be
added to reveal fluorescence. MUG can be sterilized
together with other medium ingredients without loss
of activity; furthermore, no inhibitory effect on E.
coli growth has hitherto been observed. The dis-
advantage of incorporating MUG into solid media is
that fluorescence diffuses rapidly from the colonies
into the surrounding agar. MUG methodes are cited,
e.g. in DIN 10 110 ‘Determination on E. coli in meat
and meat derivates’, DIN 10 183, part 3 ‘Determi-
nation of E. coli in milk, milk derivates, icecream,
baby food based on milk or ISO 11866-2 ‘Enumera-
tion of presumptive E. coli – Part 2: most probable
number technique using MUG’.
A new chromogenic selective agar for the de-
tection of E. coli is TBX Agar. TBX is a modi-
fication of Tryptone bile agar to which the substrate
5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-
GLUC or BCIG) is added. GUD cleaves the
chromogenic substrate BCIG and the released chro-
mophore causes distinct and easy to read blue-green
coloured colonies of E. coli. Furthermore TBX Agar
complies with the ISO / DIS Standard 16649 for the
enumeration of E. coli in food and animal feeding
stuffs.
On a solid medium containing 8-hydroxy-
quinoline-b-D-glucuronide (HQG) (200 mg / l) and a
ferric salt, b-glucuronidase positive strains grow as
black colonies. The pigment is located only in the
colony mass. The product of hydrolysis is 8-hy-
droxyquinoline, which forms black complexes with
ferrous and ferric ions. The commercially available
medium Uricult-Trio (Orion Diagnostica) contains
HQG. Of 324 E. coli strains isolated from urine
samples, 92% grew black-brown colonies on dip-
slide, thus being b-glucuronidase positive (Larinkari
and Rautio, 1995).
2.1.2. E. coli O157: H7
E. coli O157:H7 is an important food-borne
pathogen and can cause diarrhea, hemorrhagic coli-
tis, and hemolytic uremic syndrome (HUS). The agar
medium most commonly used for the isolation of E.
 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
coli O157:H7 is Sorbitol-MacConkey agar (SMAC,
March and Ratnam, 1986). Strains of E. coli
O157:H7, unlike the majority of E. coli strains, do
not ferment D-sorbitol within 24 h and are GUD-
negative and do not grow at 45.58C. Sorbitol-nonfer-
menting colonies, indicative of the typical E. coli
O157:H7, are colourless on this medium. SMAC
agar is not generally useful for Shiga-like-toxin
(Stx)-producing E. coli strains of serotypes other
than O157:H7 because no known genetic linkage
exists between Stx production and sorbitol fermen-
tation. E. coli O157:H7 also does not ferment
rhamnose on agar plates, in contrast to the majority
of other sorbitol-nonfermenting strains. The selec-
tivity of SMAC agar has been improved with the
addition of cefixime-rhamnose (CR-SMAC, Chap-
man et al., 1991), cefixime-tellurite (CT-SMAC
agar, Zadik et al., 1993), MSA-MUG (Szabo et al.,
1986) and BCIG-SMAC (Okrend et al.,1990). Re-
cently, new selective media have been developed to
increase the effectiveness of E. coli O157:H7 isola-
tion, including Rainbow agar O157 (RB, Biolog Inc.,
Hayward, USA), BCM O157:H7 (BCM, Biosynth
AG, Staad, Switzerland), Fluorocult E. coli O157:H7
(Merck, Darmstadt, Germany) and CHROMagar
O157 (CHROMagar, Paris, France).
Fluorocult E. coli O157:H7 is a selective agar for
the isolation and diferentiation of E. coli O157:H7
from food samples and from clinical material. So-
dium desoxycholat inhibits the Gram positive bac-
teria. E. coli O157:H7 strains grow as greenish
colonies and show no fluoresence under UV light.
On RB O157, E. coli strains grow, yielding
colonies ranging in colour through various shades of
red, magenta, purple, violet, blue, and black. The
typical glucuronidase negative strains form distinc-
tive charcoal gray or black colonies. Other
glucuronidase positive strains gave red or magenta
coloured colonies (Bochner, 1995). RB agar shows
good results for isolation when the background flora
is low, or when E. coli O157 is present as a high
proportion (e.g. 5%). Bettelheim (1998b) has evalu-
ated and compared RB agar with SMAC agar. Some
other EHEC also stand out as blue-black, whereas
O113 and some other EHEC strains were mauve, red
or pink. Manafi and Kremsmair (1999) have found
that strains of E. coli O157:H7 could be readily
isolated and recognized by typical black colonies on
RB agar. The mixture tellurite / novobiocin (2.5 mg
207
tellurite and 10 mg novobiocin / l according to Stein
and Bochner, 1998) had inhibited the major back-
ground-flora except Hafnia alvei strains isolated
from milk and raw meat. It was found, that RB agar
had a sensitivity and a specificity of 91.1% and
91.6%, respectively using pure cultures and only
2.1% false positive results (H. alvei) analyzing the
food samples.
On BCM agar E. coli O157:H7 strains produce
convex colonies 1.5–2.5 mm in diameter surrounded
by distinct blue / black precipitates. Sorbitol positive
isolates were blue to turquoise. A comparison of
BCM O157:H7 and SMAC-BCIG agars using natu-
rally contaminated beef samples was made using
presumptively positive enrichment broths identified
by various rapid methods. The percent sensitivity
and specificity values were 90.0 and 78.5 for BCM
O157:H7 and 7.0 and 46.4 for SMAC-BCIG. Thus,
BCM O157:H7 ( 1 ) medium displayed greater
sensitivity and specificity than MSA-BCIG for de-
tecting E. coli O157:H7 using artificially and natu-
rally contaminated beef products (Restaino et al.,
1999b). On CHROMagar O157 (Chromagar, Paris,
France) E. coli O157 strains produce pink colonies
(Wallace and Jones, 1996; Bettelheim, 1998a). A
collaborative evaluation of detection methods for E.
coli O157:H7 from radish sprouts and ground beef
using plating and immunological methods is de-
scribed by Onoue et al. (1999). The plating media
tested were CT-SMAC, BCM O157 and
CHROMagar O157. E. coli O157:H7 was recovered
well from ground beef by all of the methods except
direct plating with SMAC. For radish sprouts, the
IMS-plating methods with CT-SMAC, BCM O157
and CHROMagar O157 were most efficient.
A study was done by Taormina et al. (1998) to
recover E. coli O157:H7 cells from unheated and
heated ground beef, and to compare the ability of
five enrichment broths to recover E. coli O157:H7
cells from heated ground beef. Eight selective agars
and two non-selective agars were evaluated for their
ability to support colony formation by E. coli
O157:H7 surviving heat treatment. Of the selective
media tested, modified eosin methylene blue agar
(MEMB) and RB agar O157 supported recovery of
significantly (P , 0.05) higher numbers of heat-
stressed cells of E. coli O157:H7, regardless of
heating time. Chromagar O157, CT-SMAC and CR-
SMAC performed poorly, even in unheated samples.
208 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
SMAC and BCM O157:H7 agar were similar to
CT-SMAC and CR-SMAC in their ability to recover
E. coli O157:H7 from heated beef. TBX agar
performed significantly better than these media, but
was inferior to MEMB agar and RB agar O157:H7.
Enrichment using tryptone soya broth with novo-
biocin or a procedure using brain heart infusion and
tryptone phosphate broths recovered the highest
population of heat-stressed E. coli O157:H7.
A new medium ‘EOH-agar’ was developed for
identification of E. coli 0157:H7 by Kang and Fung
(1999) and was compared with SMAC agar. Indigo
carmine (0.03 g / l) and phenol red (0.036 g / l) were
found as the best combination for differentiation
between E. coli O157:H7 and other E. coli and
added to the basal agar medium (SMAC medium
excluding neutral red and crystal violet). On the dark
blue EOH medium, E. coli produced a yellow colour
with clear zone, whereas E. coli O157:H7 produced
a red colour without clear zone. The recovery
numbers of E. coli 0157:H7 from inoculated ground
beef, pork, and turkey and of heat- and cold-injured
E. coli O157:H7 also were not significantly different
between SMAC and EOH media (P . 0.05).
2.1.3. Media for simultaneous detection of E. coli
and coliforms
As both E. coli and coliforms are important
indicators of water pollution, there arises the necessi-
ty to create media which are able to simultaneously
detect both bacteria. This would then guarantee a
better performance of microbiological quality con-
trol. The new enzymatic definition of coliforms,
which is not method related, is the possession of
b-D-galactosidase gene which is responsible for the
cleavage of lactose into glucose and galactose by the
enzyme b-D-galactosidase. The determination of b-
galactosidase is accomplished by using o-nitro-
phenyl-b-D-galactopyranoside (ONPG), p-nitro-
phenyl-b-D-galactopyranoside (PNPG), 6-bromo-3-
indolyl-b-galactopyranoside (Salmon-Gal), 5-bromo-
4-chloro-3-indolyl-b-D-galactopyranoside (XGAL)
or 6-bromo-2-naphthyl-b-D-galactopyranoside
(BNGAL), 8-hydroxychinoline-b-D-galactoside,
cyclohexenoesculetin-b-D-galactoside and fluoro-
genic 4-methylumbelliferyl-b-D-galactopyranoside
(MUGAL). Attempts were made to enhance the
coliform assay response by adding 1-isopropyl-b-D-
thiogalactopyronaside (IPTG) to the media (Manafi,
1995) or sodium dodecyl sulfate (SDS) (Berg and
Fiksdal, 1988) increasing the b-D-galactosidase ac-
tivity by improving the transfer of the substrate
and / or enzyme across the outer membrane.
Commercially available media have been de-
veloped which permit rapid simultaneous detection
of E. coli and coliforms in water (Table 1). These
media contain a variety of enzyme substrates for
detection of b-D-galactosidase (presence of
coliforms) and b-D-glucuronidase (presence of E.
coli), and have previously been reviewed (Manafi et
al., 1991; Manafi, 1996).
2.1.3.1. Liquid commercially available media for the
detection of E. coli and coliforms
The Colilert system, containing ONPG / MUG,
simultaneously detects the presence of both total
coliforms and E. coli. After incubation, the formula
becomes yellow if total coliforms are present and
fluorescent under long-wavelength (366 nm) ultra-
violet light if E. coli is in the same sample. Schets et
al. (1993) compared Colilert with Dutch standard
enumeration methods for E. coli and coliforms in
water and have found, that Colilert gave false-nega-
tive results in samples with low numbers of E. coli
or total coliforms, indicating that the Colilert does
not always support the growth of environmental E.
coli or coliforms. They regarded the Colilert as
unsuitable for monitoring water samples in com-
parison to Dutch standard method. Another study by
Landre et al. (1998) reported the false positive
coliform reaction mediated by Aeromonas spp. in the
Colilert system. Data obtained clearly demonstrate
that A. hydrophila can elicit a positive coliform type
reaction at very low densities. Cell suspensions as
low as 1 cfu / ml were observed to yield a positive
reaction. Similar results were obtained with other
members from the mesophilic group of aeromonads.
Use of Colilert for monitoring water quality will lead
to overestimation of coliforms as Aeromonas spp.
are known to be present in treated drinking water
supplies. Development of blue-green color in an
initially light yellow coloured solution, indicate the
presence of coliforms using LMX  broth or
Readycult  Coliforms, containing Xgal / MUG. Fluo-
rescence at 366 nm in the same vessel denoted the
presence of E. coli. An evaluation of a number of
presence / absence (P/A) tests for coliforms and E.
coli, including LMX broth (Merck) and Colilert
 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218 209

Table 1
Commercially available media for the detection of E. coli and coliforms (updated from Manafi, 1996)a
Medium Substrate / colour Manufacturer
Coliforms E. coli
Liquid media
Fluorocult  LMX broth XGAL / blue-green MUG / blue fluorescence Merck (Germany)
Readycult coliforms XGAL / MUG MUG / blue fluorescence Merck (Germany)
ColiLert ONPG / Yellow MUG / blue fluorescence IDEXX (USA)
Coliquick ONPG / Yellow MUG / blue fluorescence Hach (USA)
Colisure CPRG / red MUG / blue fluorescence IDEXX (USA)

Solid media
Fluorocult R agars – MUG / blue fluorescence Merck (Germany)
TBX-agar – BCIG / blue Oxoid (UK), Merck (Germany)
Uricult Trio – HOQ / black Orion (Finnland)
EMX-agar XGAL / blue MUG / blue fluorescence Biotest (Germany)
C-EC-MF-agar XGAL / blue MUG / blue fluorescence Biolife (Italy)
Chromocult SalmonGal / red XGLUC / blue-violet Merck (Germany)
Coli ID XGAL / blue SalmonGlu / Rose-violet bioMerieux (France)
CHROMagar ECC SalmonGal / red XGLUC / purple Chromagar (France)
Rapid’ E. coli 2 XGAL / blue SalmonGlu / purple Sanofi (France)
E. coli / coliforms SalmonGal / red XGLUC / purple Oxoid (UK)
ColiScan SalmonGal / red XGLUC / purple MicrologyLab. (USA
MI-agar MUGal / blue fluorescence Indoxyl / blue Brenner et al. (1993)
HiCrome ECC SalmonGal / red XGLUC / blue Union Carbide (USA)

Other systems
ColiComplete XGAL / blue MUG / blue fluorescence Biocontrol (USA)
ColiBag / Water check XGAL / blue-green MUG / blue fluorescence Oceta (Canada)
Pathogel XGAL / blue MUG / blue fluorescence Charm Sci.(USA)
E. colite XGAL / blue MUG / blue fluorescence Charm Sci.(USA)
m-Coliblue TTC / red XGLUC / blue Hach (USA
a
Abbreviations: ONPG, o-nitrophenyl-b-D-galactopyranoside; Salmon-GAL, 6-bromo-3-indolyl-b-D-galactopyranoside; XGAL, 5-bromo-
4-chloro-3-indolyl-b-D-galactopyranoside; CPRG, chlorophenol red b-galactopyranoside; XGLUC, 5-bromo-4-chloro-3-indolyl-b-D-glucuro-
nide; MUG, 4-methylumbelliferyl-b-D-glucuronide; TTC, triphenyl tetrazolium chloride; HOQ, hydroxyquinoline-b-D-glucuronide.

(Idexx) has been published under the Department of
the Environment series in the UK (Lee et al., 1995).
They compared four P/A tests with UK Standard
methods and found that more coliforms were de-
tected than with membrane filtration technique; in
addition, it was shown that results in LMX broth
were the easiest to interpret. The study concludes
that there is no P/A test that is best at all locations
for both coliforms and E. coli, and as there can be
marked ecological differences between sources it is
important that particular P/A tets are validated in
each geographical area before use. The efficiacy and
rapidity of detectable reactions make LMX  broth or
Readycult  coliforms a very useful tool in routine
water and food microbiology (Hahn and Wittrock,
1991; Betts et al., 1994; Lee et al., 1995; Manafi,
1995; Manafi and Rosmann, 1998). Only a few
non-coliform bacteria such as strains of Serratia
spp., H. alvei, Vibrio metschnikovii, V. vulnificus, A.
hydrophila and A. sobria gave a false positive
reaction with Xgal (Manafi, 1995; Fricker and
Fricker, 1996). This is in accordance with Ley et al.
(1993), who found that Xgal medium, in addition to
providing a rapid test for coliforms, also detected
b-galactosidase-positive aeromonads and nonsheen-
forming members of the Enterobacteriaceae on m-
Endo agar. They reported the low sensitivity of
m-Endo for detecting Aeromonas spp. which are
considered ubiquitous waterborne organisms and
should not be present in drinking water (Moyer,
1987). It is suggested adding cefsulodin at 5–10
mg / ml to culture media inhibiting the growth of
Aeromonas and Flavobacterium species (Brenner et
al., 1993; Alonso et al., 1996; Geissler et al., 2000).
210 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
Results of these studies suggest that cefsulodin may
be a useful selective agent against Aeromonas spp.
which should be included in coliform chromogenic
media when high levels of accompanying flora are
expected.
2.1.3.2. Solid commercially available media for the
detection of E. coli and coliforms
Chromocult coliforms agar (CCA) contains
chromogenic Salmon-GAL and X-GLUC, the growth
of coliforms even sublethal damaged cells is granted
due to use of peptone, pyruvate, sorbit and a buffer
of phosphate. Gram positive and some Gram nega-
tive bacteria are inhibited by Tergitol 7. On the
CCA, non-E. coli fecal coliforms (Klebsiella, En-
terobacter and Citrobacter) (KEC) were identified
by the production of a salmon to red colour from
b-galactosidase cleavage of the substrate Salmon-
GAL, while E. coli colonies were detected by the
blue / violet colour, produced by the cleavage of X-
glucuronide by b-D-glucuronidase. CCA was com-
pared with the Standard Methods membrane filtration
fecal coliform (mFC) medium for fecal coliform
detection and enumeration (Alonso et al., 1998).
Statistically, there were no significant differences
between fecal coliform counts obtained with the two
media (CCA and mFC agar) and two incubation
procedures (2 h at 378C plus 22 h at 44.58, and
44.58C) as determined by variance analysis. In this
study E. coli represented, on average 70.5–92.5% of
the fecal coliform population. A high incidence of
false negative KEC (19.5%) and E. coli (29.6%)
colonies was detected at 44.58C. The physiological
condition of the fecal coliform isolates could be
responsible for the nonexpression of b-galactosidase
and b-glucuronidase activities at 44.58C.
Byamukama et al. (2000) described the quantifica-
tion of E. coli contamination with CC agar from
different polluted sites in a tropical environment. It
proved to be efficient and feasible for determining
fecal pollutions in the investigated area within 24 h.
Geissler et al. (2000) compared the performance
of LMX  broth, Chromocult Coliform  -agar (CC)
and Chromocult Coliform  -agar plus cefsulodin (10
mg / ml) (CC-CFS), with Standard Methods multiple
tube fermentation (MTF), for the enumeration of
total coliforms (TC) and E. coli from marine recrea-
tional waters. Data from the analysis of variance
showed significant differences (P # 0.05) between
TC counts on CC-CFS and LMX. Disagreement
between the CC-CFS agar and the two commercial
enzyme media, was primarily due to the false
positive results. Background interference was re-
duced on CC-CFS and the TC counts that were
obtained reflected more accurately the number of
TCs. The results of this study support the validity of
the LMX and CC media for the enumeration of E.
coli in marine waters. Background interference was
reduced on CC-CFS and the TC counts that were
obtained reflected more accurately the number of
TCs. Some authors evaluated agar media incorporat-
ing Xgal (Ley et al., 1993; Jermini et al., 1994).
They have found that coliform strains produced
sharp blue colonies on the agar plate because of
insolubility of the indigo dye, which does not alter
the viability of the colonies. The MI agar method
(Brenner et al., 1996) containing indoxyl-b-D-gluc-
uronide (E. coli) and 4-methylumbelliferyl-b-D-gal-
actopyranoside (coliforms), was compared with the
approved method by the use of wastewater-spiked
tap water samples. Overall, weighted analysis of
variance (significance level, 0.05) showed that the
new medium recoveries of total coliforms and E. coli
were significantly higher than those of mEndo agar
and nutrient agar plus MUG, respectively, and the
background counts were significantly lower than
those of mEndo agar ( , 5%). Brenner et al. (1996)
made a comparison of the recoveries of E. coli and
total coliforms from drinking water by the MI agar
method and the USEPA-approved membrane filter
method. The current USEPA-approved membrane
filter method for E. coli requires two media, an MF
transfer, and a total incubation time of 28 h. Since
December 1999, MI agar method is approved for
detecting total coliforms and E. coli under the total
coliforms Rule and for enumerating total coliforms
under the Surface water Treatment Rule in USA.
´
On COLI ID agar (bioMerieux) coliforms are blue
colour colonies and E. coli are rose colour with a
rose zone around the colonies. Other Gram negative
bacteria are on COLI ID bright rose, small and have
no surounding zone, Gram positives and yeasts are
inhibited. Similar to the Chromocult Coliform agar,
on Coliscan and on CHROMagar ECC, E. coli
colonies are blue-violet, other coliforms are red
colonies. There are two approaches to the Coliscan
method, Coliscan Easygel and Coliscan-membrane
filters. The sample can be added directly into the
 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
bottle of Coliscan Easygel, swirled, poured into a
pretreated petri dish and incubated.
Alonso et al. (1999) compared the performance of
CHROMagarECC (CECC), and CECC sup-
plemented with sodium pyruvate (CECCP) with the
membrane filtration lauryl sulfate-based medium
(mLSA) for enumeration of E. coli and non-E. coli
thermotolerant coliforms such as Klebsiella, En-
terobacter and Citrobacter. To establish that they
could recover the maximum coliforms and E. coli
population, they compared two incubation tempera-
ture regimens, 41 and 44.58C. Statistical analysis by
the Fisher test of data did not demonstrate any
statistically significant differences (P 5 0.05) in the
enumeration of E. coli for the different media
(CECC and CECCP) and incubation temperatures.
Variance analysis of data performed on Klebsiella,
Enterobacter and Citrobacter counts showed signifi-
cant differences (P 5 0.01) between Klebsiella, En-
terobacter and Citrobacter counts at 41 and 44.58C
on both CECC and CECCP. Analysis of variance
demonstrated statistically significant differences (P 5
0.05) in the enumeration of total thermotolerant
coliforms (TTCs) on CECC and CECCP compared
with mLSA. Target colonies were confirmed to be E.
coli at a rate of 91.5% and Klebsiella, Enterobacter
and Citrobacter of likely fecal origin at a rate of
77.4% when using CECCP incubated at 418C. The
results of this study showed that CECCP agar
incubated at 418C is efficient for the simultaneous
enumeration of E. coli and Klebsiella, Enterobacter
and Citrobacter from river and marine waters.
2.1.4. Other systems for detection of E. coli and
coliforms
Grant (1997) described a membrane filtration
medium (m-Coliblue) which detects total coliforms
(red colonies) and E. coli (blue colonies) simul-
taneously. Recovery of total coliforms and E. coli on
this membrane filtration (MF) medium was evalu-
ated with 25 water samples and the testing of the
m-ColiBlue24 broth, was conducted according to a
US Environmental Protection Agency (USEPA)
protocol. For comparison, this same protocol was
used to measure recovery of total coliforms and E.
coli with two standard MF media, m-Endo broth and
mTEC broth. E. coli recovery on the new medium
was also compared to recovery on nutrient agar
supplemented with MUG. Comparison of specificity,
211
sensitivity, false positive error, undetected target
error, and overall agreement indicated E. coli re-
covery on m-ColiBlue24 was superior to recovery on
mTEC for all five parameters. Recovery of total
coliforms on this medium was comparable to re-
covery on m-Endo.
ColiComplete is a paper disc contain MUG and
Xgal and can be used with lauryl tryptose broth in
the MPN test. One paper disc is added to each tube
and after incubation at 358C for 24 h, the tubes are
observed under long-wave UV for the confirmation
of E. coli (blue fluorescence). Blue colouration in the
broth indicates the presence of total coliforms and / or
E. coli (Feldsine et al., 1994).
ColiBag (a pre-sterilized plastic bag), E.colite,
ColiGel and Pathogel are media containing enzyme
substrates Xgal and MUG. ColiGel and Pathogel
contain a gelling agent, which solidifies the sample;
coliforms gave blue colonies and E. coli gave blue
and fluorescent colonies under UV light. ColiPAD
offers a multiple tube fermentation procedure for the
determination of E. coli and coliforms in water and
wastewater.
Japanese and US Food and Drug Administration
standard methods (USDA), as well as two agar plate
methods, were compared with the three commercial
kits using enzyme substrates (Venkateswaran et al.,
1996). The levels of detection of coliforms were high
with the commercial kits (78–98%) compared with
the levels of detection with the standard methods
(80–83%) and the agar plate methods (56–83%).
Among the kits tested, the Colilert kit had highest
level of recovery of coliforms (98%), and the level
of recovery of E. coli as determined by b-
glucuronidase activity with the Colilert kit (83%)
was comparable to the level of recovery obtained by
the USDA method (87%). Levine’s eosine methylene
blue agar was compared with MUG-supplemented
agar for isolation of E. coli. Only 47% of the E. coli
was detected when eosine methylene blue agar was
used; however, when violet red bile (VRB)-MUG
agar was used, the E. coli detection rate was twice as
high.
2.1.5. Salmonella
The conventional media for the detection of
Salmonella have a very poor specificity creating an
abondance of false positives (such as Citrobacter,
Proteus) among the rare real positive Salmonella.
212 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
The workload for unnecessary examination of sus-
pect colonies is so high that the real positive
Salmonella colonies might often be missed in routine
testing. The use of new chromogenic and fluorogenic
media make the Salmonella diagnostic easier and
faster.
´
2.1.5.1. SM-ID agar ( bioMerieux , France)
On SM-ID agar Salmonella colonies are detected
by their distinctive red colouration, while coliforms
appear blue, violet, or colourless. The biochemical
characteristics used with SM-ID medium are acid
formation from glucuronate combined with a neutral
red indicator. Two chromogenic substrates for b-
galactosidase (Xgal) and b-glucosidase (Xglu) allow
differentiation of Salmonella (negative) from other
enterobacteria acidifying glucuronate (positive:blue
to purple). The selective agents are bile salts and
brilliant green (Dusch and Altwegg, 1995).
2.1.5.2. Rambach agar ( Merck, Germany)
Rambach agar is composed of propylene glycol,
peptone, yeast extract, sodium deoxycholate, neutral
red, and Xgal. After incubation of plates at 378C for
24 h, the formation of acid from propylene glycol
causes precipitation of the neutral red in Salmonella
colonies yielding a red colour (Rambach, 1990).
Salmonella strains show a bright red colour,
coliforms blue (b-D-galactosidase activity) or violet
(the formation of acid from propylene glycol and
b-D-galactosidase activity) and Proteus spp. remain
colourless. Sodium deoxycholate inhibits the accom-
panying Gram-positive flora. The main disadvantage
of Rambach agar is that it does not detect S. typhi, S.
paratyphi nor some rare strains such as S. moscow
and S. wassenaar. Furthermore, Salmonella strains
which are able to produce b-D-galactosidase such as
S. arizona or, in particular, subspecies IIIa, IIIb, and
VI, show blue-violet colonies on both media (Kuhn ¨ Salmonella species. Even some newer Salmonella
et al., 1994). Pignato et al. (1995) have observed that
the total analysis time for salmonellae in foods can
be reduced to 48 h by using the combination of
Salmosyst broth (Merck) as a liquid medium and
Rambach agar as isolation plate.
2.1.5.3. MUCAP-test ( Biolife, Italy)
MUCAP is a confirmation test for Salmonella
species based on the rapid detection of caprylate
esterase, using fluorogenic 4-methylumbelliferyl-
caprylate. In the presence of C 8 esterase the substrate
is cleaved with the release of 4-methylumbelliferone
(4-MU). One drop of MUCAP has to be added to
each colony tested on Columbia agar and observed
under UV light (366 nm) for 1–5 min. Strong bluish
fluorescence indicates the presence of Salmonella
spp.The MUCAP test was found to be very sensitive,
rapid and easy to perform but not very specific,
giving many false-positive results. The combination
of the MUCAP test and selective media was more
specific. Since most of the false-positive strains are
oxidase positive, the combination of MUCAP and
the oxidase test is recommended (Humbert et al.,
1989).
2.1.5.4. CHROMagar Salmonella ( CHROMagar,
France)
A comparison of CHROMagar Salmonella
medium (CAS), based on the esterase acitivity, and
Hektoen enteric agar (HEA) for isolation of Sal-
monellae have been done by Gaillot et al. (1999)
with Salmonella isolates and stool samples. All stock
cultures, including cultures of H 2 S-negative isolates,
yielded typical mauve colonies on CAS while 99%
isolates produced typical lactose-negative, black-cen-
tered colonies on HEA. Sensitivities for primary
plating and after enrichment were 95 and 100%,
respectively, for CAS and 80 and 100%, respective-
ly, for HEA. The specificity of CAS (88.9%) was
significantly higher than that of HEA (78.5%; P ,
0.0001).
2.1.5.5. Rainbow Salmonella agar ( Biolog, USA)
The growth of distinctive black colonies of Sal-
monella on the clear Rainbow Agar makes it easier
to detect Salmonella in mixed cultures. The advan-
tages of using Rainbow Agar Salmonella versus
traditional media is the sensitivity and specificity for
media such as XLT4 utilize additives such as
Tergitol 4 that are inhibitory to S. typhi and S.
choleraesuis.
2.1.5.6. Chromogenic Salmonella esterase agar
( PPR Diagnostics Ltd, UK)
Cooke et al. (1999) described a novel agar
medium, chromogenic Salmonella esterase (CSE)
agar. The agar contains peptones and nutrient ex-
tracts together with the following 4-[2-(4-
 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-
quinolinium-1-(propan-3-yl-carboxylic-acid)-bro-
mide (SLPA-octanoate bromide form). SLPA-oc-
tanoate is a newly synthesized ester formed from a
C 8 fatty acid and a phenolic chromophore. In CSE
agar, the ester is hydrolyzed by Salmonella spp. to
yield a brightly coloured phenol which remains
tightly bound within colonies. The typical Salmonel-
la strains were burgundy coloured on a transparent
yellow background, whereas non-Salmonella spp.
were white, cream, yellow or transparent. The sen-
sitivity (93.1%) of CSE agar for non-typhi Salmonel-
lae compared favorably with those of Rambach
(82.8%), xylose-lysine-deoxycholate (XLD; 91.4%),
Hektoen-enteric (89.7%), and SM ID (91.4%) agars.
The specificity (93.9%) was also comparable to
those of other Salmonellamedia (SM ID agar,
95.9%; Rambach agar, 91.8%; XLD agar, 91.8%;
Hektoen-enteric agar, 87.8%). Strains of Citrobacter
freundiiand Proteus spp. giving false-positive re-
actions with other media gave a negative colour
reaction on CSE agar. CSE agar enabled the de-
tection of . 30 Salmonella serotypes, including
agona, anatum, enteritidis, hadar, heidelberg, infan-
tis, montevideo, thompson, typhimurium, and vir-
chow.
2.1.5.7. Compass Salmonella agar ( Biokar diagnos-
tics, France)
Perry and Quiring (1997) described an agar
medium based on the detection of C8-esterase activi-
ty using 5-bromo-6-chloro-3-indolyl-caprylate.
Magenta coloured colonies indicating the presence of
Salmonella spp.
2.1.5.8. Chromogenic ABC medium ( Lab M. Ltd.,
UK)
Perry et al. (1999) described a new chromogenic
agar medium, ABC medium (ab-chromogenic
medium), which includes two substrates, 3,4-
cyclohexenoesculetin-b-D-galactoside (CHE) and 5-
bromo-4-chloro-3-indolyl-a-D-galactopyranoside (X-
aGal), to facilitate the selective isolation of Sal-
monella spp. CHE-Galactoside is utilised by most
Enterobacteriaceae, yielding black colonies. The X-
5-Gal is hydrolysed by Salmonella spp. to produce
characteristic, easy to distinguish, green colonies.
This medium exploits the fact that Salmonella spp.
may be distinguished from other members of the
213
family Enterobacteriaceae by the presence of a-
galactosidase activity in the absence of b-galacto-
sidase activity. A total of 1022 strains of Salmonella
spp. and 300 other Gram-negative strains were
inoculated onto this medium. Of these, 99.7% strains
of Salmonella spp. produced a characteristic green
colony, whereas only one strain of non-Salmonella
produced a green colony.
2.2. Enterococci
The use of chromogenic and fluorogenic substrates
for detection of b-D-glucosidase activity to differen-
tiate enterococci has received considerable attention
(Dufour, 1980; Littel and Hartman, 1983).
2.2.1. Enterolert and Microtiter plate MUST
4-methylumbelliferyl-b-D-glucoside (MUD), when
hydrolyzed by enterococcal b-D-glucosidase, releases
4-methylumbelliferone, which exhibits fluorescence
under a UV lamp (366 nm). Enterolert (IDEXX
Laboratories Inc., Westbrook, Maine), and a Microti-
ter plate MUST (Sanofi, France), containing MUD
detecting b-D-glucosidase. Niemi and Ahtiainen
(1995) desribed the evaluation of MTP method with
Slanetz–Bartley and KF-Streptococcus agar using
pure cultures. Both media yielded high recoveries of
the target species. The MUD method tended to give
slightly lower recoveries than the agar cultivation
methods with some target species at 448C but
recoveries were better than at 418C. It has to be
mentioned, that there may be some problems with
other glucosidase positive bacteria such as Aerococ-
cus spp., in particular in natural contaminated water
samples. Budnick et al. (1996) evaluated the En-
terolert for enumeration of enterococci in recreation-
al waters as a semiautomated MPN method and
compared with the standard membrane filter method
by parallel testing of 138 marine and freshwater
recreational bathing water samples. No statistical
significant difference and a strong linear correlation
were found between the two methods. Culturing of
501 Enterolert test wells resulted in false-positive
and false-negative rates of 5.1 and 0.4%, respective-
ly.
2.2.2. mEI agar
Dufour (1980) described a medium (mEI agar) for
use in a single-step, 24-h MF procedure to enumerate
214 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
enterococci in marine water and freshwater. The
medium contained nalidixic acid, cycloheximide,
triphenyltetrazolium chloride (TTC) and indoxyl-b-
D-glucoside. Enterococci strains produce an insoluble
indigo blue complex, which diffuses into the sur-
rounding media, forming a blue halo around the
colony. Rhodes and Kator (1997) evaluated mEI
agar with respect to specificity and recovery of
enterococci from environmental waters. Extending
incubation from 24 to 48 h improved enterococci
recovery but 77% of the colonies classified as non-
target were confirmed as enterococci. Decreasing the
concentration of or eliminating indoxyl-b-D-gluco-
side from mE did not significantly affect recovery of
purified isolates. Messer and Dufour (1998) recently
modified the medium by reducing the TTC from 0.15
to 0.02 g / l and adding 0.75 g of indoxyl b-D-
glucoside per liter. The new MF medium, mEI
medium, detected levels of enterococci in 24 h
comparable to those detected by the original mE
medium in 48 h, with the same level of statistical
confidence. The comparative recovery studies, spe-
cificity determinations, and multilaboratory evalua-
tion indicated that mEI medium has analytical per-
formance characteristics equivalent to those of mE
medium. The simplicity of use and decreased incuba-
tion time with mEI medium will facilitate the
detection and quantification of enterococci in fresh
and marine recreational waters.
2.2.3. Chromocult enterococci broth ( CEB) and
Readycult enterococci
Using chromogenic substrate 5-bromo-4-chloro-3-
indolyl-b-D-glucopyranoside (XGLU), Chromocult
Enterococci broth (CEB) (Merck) and Readycult
Enterococci (Merck) utilises the b-D-glucosidase
reaction as an indicator of the presence of enterococ-
ci. XGLU is liberated and rapidly oxidized to
bromochloroindigo which produces blue colour in
Chromocult broth as well as blue coloured en-
terococci colonies in Chromocult Enterococci agar.
Other b-D-glucosidase-producing organisms being
suppressed by the sodium azide content of the broth
(Manafi and Windhager, 1997). The results obtained
with pure cultures showed that 97% of the strains,
which gave positive results, were identified as en-
terococci (E. fecalis, E. faecium, E. durans, E.
casseliflavus and E. avium). The false-positive
strains (3%) were Leucononostoc, Lactococcus lactis
lactis and Aerococcus spp. No false-negative results
were found. This medium was used to enumerate
enterococci in water using a presence / absence pro-
cedure and gave still positive results with 10 cfu / 100
ml water samples after 24 h incubation.
2.2.4. RAPID9 enterococcus agar
RAPID9 enterococcus agar (Sanofi, France) is a
new medium using chromogenic Xglu for the de-
tection of enterococci and a selective mix for the
inhibition of Gram negative and other b-glucosidase
positive bacteria other than enterococci.
2.2.5. Comparative studies
In most literature, Slanetz and Bartley (1957) agar
is treated as a suitable MF medium to detect fecal
enterococci from a water sample. Amoros ´ and
Alonso (1996) presented a study including a com-
parison of Slanetz–Bartley agar and Enterococci agar
(with XGLU) using samples of irrigation channels
and seawater. They found no statistical differences
between both media in seawater, although the speci-
fity of the Enterococci agar decreased in marine
water and there was a considerable number of false-
positives.
2.3. Staphylococcus aureus
On the new chromogenic medium, CHROMagarE
S. aureus medium (CHROMagar, France), colonies
of S. aureus are typical pink coloured. In supple-
menting CHROMagar S. aureus with an appropriate
antibiotic, for instance methicillin, one can obtain
information related to some methicillin resistant S.
aureus strains. This medium has been evaluated by
using pure cultures and clinical specimens. Out of
the 181 S. aureus and 81 coagulase negative Staphy-
lococcus strains belonging to 18 different species, S.
aureus, S. epidermidis, S. caprae and S. schleiferi
strains yielded pink colonies (Freydiere et al., 2000).
2.4. Clostridium perfringens
The identification of C. perfringens is possible
using Fluorocult TSC-agar (Merck, Germany) using
a fluorogenic enzyme substrate. D-Cycloserine inhib-
its the accompanying bacterial flora and causes the
colonies to remain smaller. C. perfringens colonies
can be detected using 4-MU-phosphate. Acid phos-
 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
phatase is a highly specific indicator for C. perfrin-
gens which shows a light blue fluorescence on this
medium (Baumgart et al., 1990).
2.5. Bifidobacteria and lactic acid bacteria
The use of chromogenic X-a-D-galactoside for
differential enumeration of Bifidobacterium spp. and
lactic acid bacteria was described by Chevalier et al.
(1991). The X-a-D-galactoside-based medium is
useful to identify bifidobacteria among Lactobacillus
or Streptococcus strains. Bifidobacteria shows blue
coloured colonies on the agar plate.
2.6. Listeria monocytogenes
L. monocytogenes is a human and animal pathogen
that is widespread in nature. The organism is a
transient constituent of the intestinal flora excreted
by 1–10% of healthy humans. It is an extremely
hardy organism and can survive for many years in
the cold in naturally infected sources. L. monocyto-
genes has been isolated from a wide variety of foods,
including dairy products, meats, and fish. Although
most of the foodborne listeriosis outbreaks have been
linked to the consumption of dairy products, recent
sporadic cases have been associated with meats, as
well as other foods. All strains of L. monocytogenes
are pathogenic by definition although some appear to
be more virulent than others. Cultural methodology
for isolating the organism from foods has been in a
state of flux since 1985.
2.6.1. BCM L. monocytogens detection system
The new BCM L. monocytogens detection system
(BCM-LMDS, Biosynth, Switzerland) consists of a
selective pre-enrichment broth, selective enrichment
broth, selective / differential plating medium, and
identification on a confirmatory plating medium. The
BCM pre-enrichment broth, allowed the growth of
Listeria and resuscitation of heat-injured L. mono-
cytogenes, which contains the fluorogenic substrate
4-methylumbelliferyl-myo-inositol-1-phosphate and
detects phosphatidylinositol phospholipase C (PI-
PLC) activity, provided a presumptive positive test
for the presence of pathogenic L. monocytogenes and
L. ivanovii after 24 h at 358C. On BCM-plating
medium, L. monocytogenes and L. ivanovii were the
two Listeria species forming turquoise convex
215
colonies (1.0–2.5 mm in diameter) from PI-PLC
activity on the chromogenic substrate, 5-bromo-4-
chloro-3-indoxyl-myo-inositol-1-phosphate. L.
monocytogenes was distinguished from L. ivanovii
by either its fluorescence on BCM confirmatory
plating medium or acid production from rhamnose.
False-positive organisms (Bacillus cereus, S. aureus,
B. thuringiensis, and yeasts) were eliminated by at
least one of the media in the BCM LMDS. The
efficacy of the BCM-LMDS was determined using
pure cultures and naturally and artificially contami-
nated environmental sponges (Restaino et al.,
1999a). In an analysis of 162 environmental sponges
from facilities inspected by the US Department of
Agriculture (USDA), the values for identification of
L. monocytogenes by BCM LMDS and the USDA
method were 30 and 14 sites, respectively, with
sensitivity and specificity values of 85.7 and 100.0%
vs. 40.0 and 66.1%, respectively. No false-positive
organisms were isolated by BCM LMDS, whereas
26.5% of the sponges tested by the USDA method
produced false-positive results.
2.6.2. Rapid L’ MONO agar
Rapid L’MONO agar (Sanofi, France) is based on
the detection of phosphatidylinositol phospholipase
C and Xylose fermentation. In this way, the colonies
of L. ivanovii appear blue sourrounded by a yellow
halo (Xylose positive) whilst the colonies of L.
monocytogenes are blue without the halo (Xylose-
negative). The colonies of other Listeria spp. remain
white because they do not posses phosphatidylino-
sitol phospholipase C activity. Further evaluation of
these media is still needed.
2.7. Bacillus cereus
BCM B. cereus and B. thuringiensis plating
medium (Biosynth, Switzerland) is a differential and
selective medium which alows B. cereus and B.
thuringiensis to grow, but inhibits growth of many
potential false positives including other Bacillus
species. Based on the detection of phosphatidylino-
sitol-specific phospholipase C (PI-PLC) activity by
Bacillus species, B. cereus and B. thuringiensis
strains produce turquoise flat dull colonies with and
without turquoise halos to the enzymatic reaction of
PI-PLC with 5-bromo-4-chloro-3-indoxyl-myo-
inositol-1-phosphate.
216 M. Manafi / International Journal of Food Microbiology 60 (2000) 205 – 218
3. Conclusions
Rapid detection and identification of microorga-
nisms is of high importance in a diverse array of
applied and research fields. By incorporation of
synthetic enzyme substrates into primary isolation
media, enumeration and detection can be performed
directly on the isolation plate. These enzyme sub-
strates have proved to be a powerful tool, utilizing
specific enzymatic activities of certain microorga-
nisms. Many of them are offering enhanced accuracy
and performance to the microbiologist. The incorpo-
ration of such substrates into a selective medium can
eliminate the need for subculture and further bio-
chemical tests to establish the identity of certain
microorganisms. These enzymatic assays may consti-
tute an alternative method for enumerating micro-
organisms in water and foods, which is specific,
sensitive and rapid.
4. Uncited reference
Okrend et al., 1990.
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