International Journal of Food Microbiology 60 (2000) 205218

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New developments in chromogenic and uorogenic culture media
*
M. Mana
Hygiene Institute, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
Abstract
This review describes some recent developments in chromogenic and uorogenic 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 uorogenic 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 identication
1. Introduction The uorogenic enzyme substrates generally con-
sist of a specic substrate for the specic enzyme
The ability to detect the presence of a specic and such as sugar or amino acid and a uorogen such as
exclusive enzyme using suitable substrates, in par- 4-methylumbelliferone, being able to convert UV
ticular uorogenic or chromogenic enzyme sub- light to visible light. Methylumbelliferyl-substrates
strates, has led to the development of a great number are water soluble, highly sensitive and very specic.
of methods for the identication of microorganisms Because of their pH-dependance, strong diffusion in
even in primary isolation media. The incorporation solid media and the need of UV-light, the use of
of such substrates into a selective medium can these substrates is limited. Chromogenic enzyme
eliminate the need for subculture and further bio- substrates are compounds which act as the substrate
chemical tests to establish the identity of certain for specic enzymes and change colour due to the
microorganisms (Mana et al., 1991; Mana, 1996). action of the enzyme. In general, based on their
This review describes recent developments in uoro- chemical reaction, four groups of chromogenic com-
genic and chromogenic media for specic detection pounds can be distinguished and recently described
of microorganisms. by Mana (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-
*Tel.: 143-1-40490-79450; fax: 143-1-40490-9794.
E-mail address: mohammad.mana@univie.ac.at (M. Mana). bromo-6-chloro-3-indolyl (magenta) or 6-chloro-3-
0168-1605/ 00/ $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PI I : S0168- 1605( 00) 00312- 3
206 M. Mana / International Journal of Food Microbiology 60 (2000) 205218
indolyl (salmon) showing no diffusion on the agar minimum concentration sufcient for distinct indica-
plate. 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
2. The application of chromogenic and uorescence of 4-MU is pH dependent, the pH of
uorogenic media in food and water growth media containing MUG should be slightly
microbiology alkaline; otherwise alkaline solution needs to be
added to reveal uorescence. MUG can be sterilized
2.1. Enterobacteriaceae together with other medium ingredients without loss
of activity; furthermore, no inhibitory effect on E.
2.1.1. Escherichia coli coli growth has hitherto been observed. The dis-
The new generation of media uses b-D-glucuronid- advantage of incorporating MUG into solid media is
ase (GUD) as indicator for E. coli. GUD is present in that uorescence diffuses rapidly from the colonies
9496% of E. coli strains, but also some Salmonella, into the surrounding agar. MUG methodes are cited,
Shigella and Yersinia spp. (Hartman, 1989). Some e.g. in DIN 10 110 Determination on E. coli in meat
authors found GUD-positive strains of Citrobacter and meat derivates, DIN 10 183, part 3 Determi-
freundii (Gauthier et al., 1991) or some strains of nation of E. coli in milk, milk derivates, icecream,
Klebsiella oxytoca, Serratia fonticola and Yersinia baby food based on milk or ISO 11866-2 Enumera-
intermedia (Alonso et al., 1996). GUD test is used tion of presumptive E. coli Part 2: most probable
increasingly for detection of E. coli in water and number technique using MUG.
food microbiology as E. coli is an important in- A new chromogenic selective agar for the de-
dicator of fecal contamination in samples from the tection of E. coli is TBX Agar. TBX is a modi-
food processing and water purication plants. Other cation of Tryptone bile agar to which the substrate
Escherichia spp. do not produce this enzyme as 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-
described by Rice et al. (1991). Some pathogenic GLUC or BCIG) is added. GUD cleaves the
strains of E. coli such as typical E. coli O157:H7 chromogenic substrate BCIG and the released chro-
however, do not posses GUD either (Frampton and mophore causes distinct and easy to read blue-green
Restaino, 1993). coloured colonies of E. coli. Furthermore TBX Agar
GUD activity is measured by using different complies with the ISO/ DIS Standard 16649 for the
chromogenic and uorogenic substrates such as p- enumeration of E. coli in food and animal feeding
nitrophenol-b-D-glucuronide (PNPG), or 5-bromo-4- stuffs.
chloro-3-indolyl-b-D-glucuronide (X-GLUC) and 4- On a solid medium containing 8-hydroxy-
methylumbelliferyl-b-D-glucuronide (MUG). MUG quinoline-b-D-glucuronide (HQG) (200 mg/ l) and a
is hydrolyzed by GUD yielding 4-MU, which shows ferric salt, b-glucuronidase positive strains grow as
blue uorescence when irradiated with long-wave black colonies. The pigment is located only in the
UV light (366 nm). MUG has been incorporated into colony mass. The product of hydrolysis is 8-hy-
both liquid media including lauryl sulfate broth, droxyquinoline, which forms black complexes with
m-Endo broth, EC broth, Brila-broth, DEV-lactose- ferrous and ferric ions. The commercially available
pepton-broth and LMX broth. The solid media to medium Uricult-Trio (Orion Diagnostica) contains
which MUG was added, include violet red bile agar, HQG. Of 324 E. coli strains isolated from urine
ECD-agar, MacConkey-agar and m-FC agar and are samples, 92% grew black-brown colonies on dip-
already described earlier (Mana, 1996). Fluorocult slide, thus being b-glucuronidase positive (Larinkari
media (Merck) are used for determination of E. coli, and Rautio, 1995).
e.g. in drugs (Huang et al., 1994), in foods of animal
origin (Bredie and de Boer, 1992), for the isolation 2.1.2. E. coli O157:H7
of diarrhea-causing E. coli (Muto et al., 1991), E. coli O157:H7 is an important food-borne
clinical samples (Heizmann et al., 1988; Mori et al., pathogen and can cause diarrhea, hemorrhagic coli-
1991), milk and milk products (Hahn and Wittrock, tis, and hemolytic uremic syndrome (HUS). The agar
1991) and bathing water (Havemeister, 1991). The medium most commonly used for the isolation of E.
M. Mana / International Journal of Food Microbiology 60 (2000) 205218 207
coli O157:H7 is Sorbitol-MacConkey agar (SMAC, tellurite and 10 mg novobiocin/ l according to Stein
March and Ratnam, 1986). Strains of E. coli and Bochner, 1998) had inhibited the major back-
O157:H7, unlike the majority of E. coli strains, do ground-ora except Hafnia alvei strains isolated
not ferment D-sorbitol within 24 h and are GUD- from milk and raw meat. It was found, that RB agar
negative and do not grow at 45.58C. Sorbitol-nonfer- had a sensitivity and a specicity of 91.1% and
menting colonies, indicative of the typical E. coli 91.6%, respectively using pure cultures and only
O157:H7, are colourless on this medium. SMAC 2.1% false positive results (H. alvei) analyzing the
agar is not generally useful for Shiga-like-toxin food samples.
(Stx)-producing E. coli strains of serotypes other On BCM agar E. coli O157:H7 strains produce
than O157:H7 because no known genetic linkage convex colonies 1.52.5 mm in diameter surrounded
exists between Stx production and sorbitol fermen- by distinct blue/ black precipitates. Sorbitol positive
tation. E. coli O157:H7 also does not ferment isolates were blue to turquoise. A comparison of
rhamnose on agar plates, in contrast to the majority BCM O157:H7 and SMAC-BCIG agars using natu-
of other sorbitol-nonfermenting strains. The selec- rally contaminated beef samples was made using
tivity of SMAC agar has been improved with the presumptively positive enrichment broths identied
addition of cexime-rhamnose (CR-SMAC, Chap- by various rapid methods. The percent sensitivity
man et al., 1991), cexime-tellurite (CT-SMAC and specicity values were 90.0 and 78.5 for BCM
agar, Zadik et al., 1993), MSA-MUG (Szabo et al., O157:H7 and 7.0 and 46.4 for SMAC-BCIG. Thus,
1986) and BCIG-SMAC (Okrend et al.,1990). Re- BCM O157:H7 ( 1) medium displayed greater
cently, new selective media have been developed to sensitivity and specicity than MSA-BCIG for de-
increase the effectiveness of E. coli O157:H7 isola- tecting E. coli O157:H7 using articially and natu-
tion, including Rainbow agar O157 (RB, Biolog Inc., rally contaminated beef products (Restaino et al.,
Hayward, USA), BCM O157:H7 (BCM, Biosynth 1999b). On CHROMagar O157 (Chromagar, Paris,
AG, Staad, Switzerland), Fluorocult E. coli O157:H7 France) E. coli O157 strains produce pink colonies
(Merck, Darmstadt, Germany) and CHROMagar (Wallace and Jones, 1996; Bettelheim, 1998a). A
O157 (CHROMagar, Paris, France). collaborative evaluation of detection methods for E.
Fluorocult E. coli O157:H7 is a selective agar for coli O157:H7 from radish sprouts and ground beef
the isolation and diferentiation of E. coli O157:H7 using plating and immunological methods is de-
from food samples and from clinical material. So- scribed by Onoue et al. (1999). The plating media
dium desoxycholat inhibits the Gram positive bac- tested were CT-SMAC, BCM O157 and
teria. E. coli O157:H7 strains grow as greenish CHROMagar O157. E. coli O157:H7 was recovered
colonies and show no uoresence under UV light. well from ground beef by all of the methods except
On RB O157, E. coli strains grow, yielding direct plating with SMAC. For radish sprouts, the
colonies ranging in colour through various shades of IMS-plating methods with CT-SMAC, BCM O157
red, magenta, purple, violet, blue, and black. The and CHROMagar O157 were most efcient.
typical glucuronidase negative strains form distinc- A study was done by Taormina et al. (1998) to
tive charcoal gray or black colonies. Other recover E. coli O157:H7 cells from unheated and
glucuronidase positive strains gave red or magenta heated ground beef, and to compare the ability of
coloured colonies (Bochner, 1995). RB agar shows ve enrichment broths to recover E. coli O157:H7
good results for isolation when the background ora cells from heated ground beef. Eight selective agars
is low, or when E. coli O157 is present as a high and two non-selective agars were evaluated for their
proportion (e.g. 5%). Bettelheim (1998b) has evalu- ability to support colony formation by E. coli
ated and compared RB agar with SMAC agar. Some O157:H7 surviving heat treatment. Of the selective
other EHEC also stand out as blue-black, whereas media tested, modied eosin methylene blue agar
O113 and some other EHEC strains were mauve, red (MEMB) and RB agar O157 supported recovery of
or pink. Mana and Kremsmair (1999) have found signicantly (P ,0.05) higher numbers of heat-
that strains of E. coli O157:H7 could be readily stressed cells of E. coli O157:H7, regardless of
isolated and recognized by typical black colonies on heating time. Chromagar O157, CT-SMAC and CR-
RB agar. The mixture tellurite/ novobiocin (2.5 mg SMAC performed poorly, even in unheated samples.
208 M. Mana / International Journal of Food Microbiology 60 (2000) 205218
SMAC and BCM O157:H7 agar were similar to 1995) or sodium dodecyl sulfate (SDS) (Berg and
CT-SMAC and CR-SMAC in their ability to recover Fiksdal, 1988) increasing the b-D-galactosidase ac-
E. coli O157:H7 from heated beef. TBX agar tivity by improving the transfer of the substrate
performed signicantly better than these media, but and/ or enzyme across the outer membrane.
was inferior to MEMB agar and RB agar O157:H7. Commercially available media have been de-
Enrichment using tryptone soya broth with novo- veloped which permit rapid simultaneous detection
biocin or a procedure using brain heart infusion and of E. coli and coliforms in water (Table 1). These
tryptone phosphate broths recovered the highest media contain a variety of enzyme substrates for
population of heat-stressed E. coli O157:H7. detection of b-D-galactosidase (presence of
A new medium EOH-agar was developed for coliforms) and b-D-glucuronidase (presence of E.
identication of E. coli 0157:H7 by Kang and Fung coli ), and have previously been reviewed (Mana et
(1999) and was compared with SMAC agar. Indigo al., 1991; Mana, 1996).
carmine (0.03 g/ l) and phenol red (0.036 g/ l) were
found as the best combination for differentiation 2.1.3.1. Liquid commercially available media for the
between E. coli O157:H7 and other E. coli and detection of E. coli and coliforms
added to the basal agar medium (SMAC medium The Colilert system, containing ONPG/ MUG,
excluding neutral red and crystal violet). On the dark simultaneously detects the presence of both total
blue EOH medium, E. coli produced a yellow colour coliforms and E. coli. After incubation, the formula
with clear zone, whereas E. coli O157:H7 produced becomes yellow if total coliforms are present and
a red colour without clear zone. The recovery uorescent under long-wavelength (366 nm) ultra-
numbers of E. coli 0157:H7 from inoculated ground violet light if E. coli is in the same sample. Schets et
beef, pork, and turkey and of heat- and cold-injured al. (1993) compared Colilert with Dutch standard
E. coli O157:H7 also were not signicantly different enumeration methods for E. coli and coliforms in
between SMAC and EOH media (P .0.05). water and have found, that Colilert gave false-nega-
tive results in samples with low numbers of E. coli
2.1.3. Media for simultaneous detection of E. coli or total coliforms, indicating that the Colilert does
and coliforms not always support the growth of environmental E.
As both E. coli and coliforms are important coli or coliforms. They regarded the Colilert as
indicators of water pollution, there arises the necessi- unsuitable for monitoring water samples in com-
ty to create media which are able to simultaneously parison to Dutch standard method. Another study by
detect both bacteria. This would then guarantee a Landre et al. (1998) reported the false positive
better performance of microbiological quality con- coliform reaction mediated by Aeromonas spp. in the
trol. The new enzymatic denition of coliforms, Colilert system. Data obtained clearly demonstrate
which is not method related, is the possession of that A. hydrophila can elicit a positive coliform type
b-D-galactosidase gene which is responsible for the reaction at very low densities. Cell suspensions as
cleavage of lactose into glucose and galactose by the low as 1 cfu/ ml were observed to yield a positive
enzyme b-D-galactosidase. The determination of b- reaction. Similar results were obtained with other
galactosidase is accomplished by using o-nitro- members from the mesophilic group of aeromonads.
phenyl-b-D-galactopyranoside (ONPG), p-nitro- Use of Colilert for monitoring water quality will lead
phenyl-b-D-galactopyranoside (PNPG), 6-bromo-3- to overestimation of coliforms as Aeromonas spp.
indolyl-b-galactopyranoside (Salmon-Gal), 5-bromo- are known to be present in treated drinking water
4-chloro-3-indolyl-b-D-galactopyranoside (XGAL) supplies. Development of blue-green color in an
or 6-bromo-2-naphthyl-b-D-galactopyranoside initially light yellow coloured solution, indicate the

(BNGAL), 8-hydroxychinoline-b-D-galactoside, presence of coliforms using LMX broth or

cyclohexenoesculetin-b-D-galactoside and uoro- Readycult Coliforms, containing Xgal / MUG. Fluo-

genic 4-methylumbelliferyl-b-D-galactopyranoside rescence at 366 nm in the same vessel denoted the
(MUGAL). Attempts were made to enhance the presence of E. coli. An evaluation of a number of
coliform assay response by adding 1-isopropyl-b-D- presence/ absence (P/A) tests for coliforms and E.
thiogalactopyronaside (IPTG) to the media (Mana, coli, including LMX broth (Merck) and Colilert
M. Mana / International Journal of Food Microbiology 60 (2000) 205218 209
Table 1
a
Commercially available media for the detection of E. coli and coliforms (updated from Mana, 1996)
Medium Substrate/ colour Manufacturer
Coliforms E. coli
Liquid media

Fluorocult LMX broth XGAL/ blue-green MUG/ blue uorescence Merck (Germany)
Readycult coliforms XGAL/ MUG MUG/ blue uorescence Merck (Germany)
ColiLert ONPG/ Yellow MUG/ blue uorescence IDEXX (USA)
Coliquick ONPG/ Yellow MUG/ blue uorescence Hach (USA)
Colisure CPRG/ red MUG/ blue uorescence IDEXX (USA)
Solid media
R
Fluorocult agars MUG/ blue uorescence Merck (Germany)
TBX-agar BCIG/ blue Oxoid (UK), Merck (Germany)
Uricult Trio HOQ/ black Orion (Finnland)
EMX-agar XGAL/ blue MUG/ blue uorescence Biotest (Germany)
C-EC-MF-agar XGAL/ blue MUG/ blue uorescence 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 Sano (France)
E. coli / coliforms SalmonGal / red XGLUC/ purple Oxoid (UK)
ColiScan SalmonGal / red XGLUC/ purple MicrologyLab. (USA
MI-agar MUGal / blue uorescence Indoxyl / blue Brenner et al. (1993)
HiCrome ECC SalmonGal / red XGLUC/ blue Union Carbide (USA)
Other systems
ColiComplete XGAL/ blue MUG/ blue uorescence Biocontrol (USA)
ColiBag/ Water check XGAL/ blue-green MUG/ blue uorescence Oceta (Canada)
Pathogel XGAL/ blue MUG/ blue uorescence Charm Sci.(USA)
E. colite XGAL/ blue MUG/ blue uorescence 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 non-coliform bacteria such as strains of Serratia
the Environment series in the UK (Lee et al., 1995). spp., H. alvei, Vibrio metschnikovii, V. vulnicus, A.
They compared four P/A tests with UK Standard hydrophila and A. sobria gave a false positive
methods and found that more coliforms were de- reaction with Xgal (Mana, 1995; Fricker and
tected than with membrane ltration technique; in Fricker, 1996). This is in accordance with Ley et al.
addition, it was shown that results in LMX broth (1993), who found that Xgal medium, in addition to
were the easiest to interpret. The study concludes providing a rapid test for coliforms, also detected
that there is no P/A test that is best at all locations b-galactosidase-positive aeromonads and nonsheen-
for both coliforms and E. coli, and as there can be forming members of the Enterobacteriaceae on m-
marked ecological differences between sources it is Endo agar. They reported the low sensitivity of
important that particular P/A tets are validated in m-Endo for detecting Aeromonas spp. which are
each geographical area before use. The efciacy and considered ubiquitous waterborne organisms and

rapidity of detectable reactions make LMX broth or should not be present in drinking water (Moyer,

Readycult coliforms a very useful tool in routine 1987). It is suggested adding cefsulodin at 510
water and food microbiology (Hahn and Wittrock, mg/ ml to culture media inhibiting the growth of
1991; Betts et al., 1994; Lee et al., 1995; Mana, Aeromonas and Flavobacterium species (Brenner et
1995; Mana and Rosmann, 1998). Only a few al., 1993; Alonso et al., 1996; Geissler et al., 2000).
210 M. Mana / International Journal of Food Microbiology 60 (2000) 205218
Results of these studies suggest that cefsulodin may TC counts on CC-CFS and LMX. Disagreement
be a useful selective agent against Aeromonas spp. between the CC-CFS agar and the two commercial
which should be included in coliform chromogenic enzyme media, was primarily due to the false
media when high levels of accompanying ora are positive results. Background interference was re-
expected. duced on CC-CFS and the TC counts that were
obtained reected more accurately the number of
2.1.3.2. Solid commercially available media for the TCs. The results of this study support the validity of
detection of E. coli and coliforms the LMX and CC media for the enumeration of E.
Chromocult coliforms agar (CCA) contains coli in marine waters. Background interference was
chromogenic Salmon-GAL and X-GLUC, the growth reduced on CC-CFS and the TC counts that were
of coliforms even sublethal damaged cells is granted obtained reected more accurately the number of
due to use of peptone, pyruvate, sorbit and a buffer TCs. Some authors evaluated agar media incorporat-
of phosphate. Gram positive and some Gram nega- ing Xgal (Ley et al., 1993; Jermini et al., 1994).
tive bacteria are inhibited by Tergitol 7. On the They have found that coliform strains produced
CCA, non-E. coli fecal coliforms (Klebsiella, En- sharp blue colonies on the agar plate because of
terobacter and Citrobacter) (KEC) were identied insolubility of the indigo dye, which does not alter
by the production of a salmon to red colour from the viability of the colonies. The MI agar method
b-galactosidase cleavage of the substrate Salmon- (Brenner et al., 1996) containing indoxyl-b-D-gluc-
GAL, while E. coli colonies were detected by the uronide (E. coli) and 4-methylumbelliferyl-b-D-gal-
blue/ violet colour, produced by the cleavage of X- actopyranoside (coliforms), was compared with the
glucuronide by b-D-glucuronidase. CCA was com- approved method by the use of wastewater-spiked
pared with the Standard Methods membrane ltration tap water samples. Overall, weighted analysis of
fecal coliform (mFC) medium for fecal coliform variance (signicance level, 0.05) showed that the
detection and enumeration (Alonso et al., 1998). new medium recoveries of total coliforms and E. coli
Statistically, there were no signicant differences were signicantly higher than those of mEndo agar
between fecal coliform counts obtained with the two and nutrient agar plus MUG, respectively, and the
media (CCA and mFC agar) and two incubation background counts were signicantly lower than
procedures (2 h at 378C plus 22 h at 44.58, and those of mEndo agar ( ,5%). Brenner et al. (1996)
44.58C) as determined by variance analysis. In this made a comparison of the recoveries of E. coli and
study E. coli represented, on average 70.592.5% of total coliforms from drinking water by the MI agar
the fecal coliform population. A high incidence of method and the USEPA-approved membrane lter
false negative KEC (19.5%) and E. coli (29.6%) method. The current USEPA-approved membrane
colonies was detected at 44.58C. The physiological lter method for E. coli requires two media, an MF
condition of the fecal coliform isolates could be transfer, and a total incubation time of 28 h. Since
responsible for the nonexpression of b-galactosidase December 1999, MI agar method is approved for
and b-glucuronidase activities at 44.58C. detecting total coliforms and E. coli under the total
Byamukama et al. (2000) described the quantica- coliforms Rule and for enumerating total coliforms
tion of E. coli contamination with CC agar from under the Surface water Treatment Rule in USA.
different polluted sites in a tropical environment. It On COLI ID agar (bioMerieux) coliforms are blue
proved to be efcient and feasible for determining colour colonies and E. coli are rose colour with a
fecal pollutions in the investigated area within 24 h. rose zone around the colonies. Other Gram negative
Geissler et al. (2000) compared the performance bacteria are on COLI ID bright rose, small and have

of LMX broth, Chromocult Coliform -agar (CC) no surounding zone, Gram positives and yeasts are

and Chromocult Coliform -agar plus cefsulodin (10 inhibited. Similar to the Chromocult Coliform agar,
mg/ ml) (CC-CFS), with Standard Methods multiple on Coliscan and on CHROMagar ECC, E. coli
tube fermentation (MTF), for the enumeration of colonies are blue-violet, other coliforms are red
total coliforms (TC) and E. coli from marine recrea- colonies. There are two approaches to the Coliscan
tional waters. Data from the analysis of variance method, Coliscan Easygel and Coliscan-membrane
showed signicant differences (P #0.05) between lters. The sample can be added directly into the
M. Mana / International Journal of Food Microbiology 60 (2000) 205218 211
bottle of Coliscan Easygel, swirled, poured into a sensitivity, false positive error, undetected target
pretreated petri dish and incubated. error, and overall agreement indicated E. coli re-
Alonso et al. (1999) compared the performance of covery on m-ColiBlue24 was superior to recovery on
CHROMagarECC (CECC), and CECC sup- mTEC for all ve parameters. Recovery of total
plemented with sodium pyruvate (CECCP) with the coliforms on this medium was comparable to re-
membrane ltration lauryl sulfate-based medium covery on m-Endo.
(mLSA) for enumeration of E. coli and non-E. coli ColiComplete is a paper disc contain MUG and
thermotolerant coliforms such as Klebsiella, En- Xgal and can be used with lauryl tryptose broth in
terobacter and Citrobacter. To establish that they the MPN test. One paper disc is added to each tube
could recover the maximum coliforms and E. coli and after incubation at 358C for 24 h, the tubes are
population, they compared two incubation tempera- observed under long-wave UV for the conrmation
ture regimens, 41 and 44.58C. Statistical analysis by of E. coli (blue uorescence). Blue colouration in the
the Fisher test of data did not demonstrate any broth indicates the presence of total coliforms and/ or
statistically signicant differences (P 50.05) in the E. coli (Feldsine et al., 1994).
enumeration of E. coli for the different media ColiBag (a pre-sterilized plastic bag), E.colite,
(CECC and CECCP) and incubation temperatures. ColiGel and Pathogel are media containing enzyme
Variance analysis of data performed on Klebsiella, substrates Xgal and MUG. ColiGel and Pathogel
Enterobacter and Citrobacter counts showed signi- contain a gelling agent, which solidies the sample;
cant differences (P 50.01) between Klebsiella, En- coliforms gave blue colonies and E. coli gave blue
terobacter and Citrobacter counts at 41 and 44.58C and uorescent colonies under UV light. ColiPAD
on both CECC and CECCP. Analysis of variance offers a multiple tube fermentation procedure for the
demonstrated statistically signicant differences (P 5 determination of E. coli and coliforms in water and
0.05) in the enumeration of total thermotolerant wastewater.
coliforms (TTCs) on CECC and CECCP compared Japanese and US Food and Drug Administration
with mLSA. Target colonies were conrmed to be E. standard methods (USDA), as well as two agar plate
coli at a rate of 91.5% and Klebsiella, Enterobacter methods, were compared with the three commercial
and Citrobacter of likely fecal origin at a rate of kits using enzyme substrates (Venkateswaran et al.,
77.4% when using CECCP incubated at 418C. The 1996). The levels of detection of coliforms were high
results of this study showed that CECCP agar with the commercial kits (7898%) compared with
incubated at 418C is efcient for the simultaneous the levels of detection with the standard methods
enumeration of E. coli and Klebsiella, Enterobacter (8083%) and the agar plate methods (5683%).
and Citrobacter from river and marine waters. Among the kits tested, the Colilert kit had highest
level of recovery of coliforms (98%), and the level
2.1.4. Other systems for detection of E. coli and of recovery of E. coli as determined by b-
coliforms glucuronidase activity with the Colilert kit (83%)
Grant (1997) described a membrane ltration was comparable to the level of recovery obtained by
medium (m-Coliblue) which detects total coliforms the USDA method (87%). Levines eosine methylene
(red colonies) and E. coli (blue colonies) simul- blue agar was compared with MUG-supplemented
taneously. Recovery of total coliforms and E. coli on agar for isolation of E. coli. Only 47% of the E. coli
this membrane ltration (MF) medium was evalu- was detected when eosine methylene blue agar was
ated with 25 water samples and the testing of the used; however, when violet red bile (VRB)-MUG
m-ColiBlue24 broth, was conducted according to a agar was used, the E. coli detection rate was twice as
US Environmental Protection Agency (USEPA) high.
protocol. For comparison, this same protocol was
used to measure recovery of total coliforms and E. 2.1.5. Salmonella
coli with two standard MF media, m-Endo broth and The conventional media for the detection of
mTEC broth. E. coli recovery on the new medium Salmonella have a very poor specicity creating an
was also compared to recovery on nutrient agar abondance of false positives (such as Citrobacter,
supplemented with MUG. Comparison of specicity, Proteus) among the rare real positive Salmonella.
212 M. Mana / International Journal of Food Microbiology 60 (2000) 205218
The workload for unnecessary examination of sus- caprylate. In the presence of C esterase the substrate
8
pect colonies is so high that the real positive is cleaved with the release of 4-methylumbelliferone
Salmonella colonies might often be missed in routine (4-MU). One drop of MUCAP has to be added to
testing. The use of new chromogenic and uorogenic each colony tested on Columbia agar and observed
media make the Salmonella diagnostic easier and under UV light (366 nm) for 15 min. Strong bluish
faster. uorescence indicates the presence of Salmonella
spp.The MUCAP test was found to be very sensitive,
2.1.5.1. SM-ID agar (bioMerieux, France) rapid and easy to perform but not very specic,
On SM-ID agar Salmonella colonies are detected giving many false-positive results. The combination
by their distinctive red colouration, while coliforms of the MUCAP test and selective media was more
appear blue, violet, or colourless. The biochemical specic. Since most of the false-positive strains are
characteristics used with SM-ID medium are acid oxidase positive, the combination of MUCAP and
formation from glucuronate combined with a neutral the oxidase test is recommended (Humbert et al.,
red indicator. Two chromogenic substrates for b- 1989).
galactosidase (Xgal) and b-glucosidase (Xglu) allow
differentiation of Salmonella (negative) from other 2.1.5.4. CHROMagar Salmonella (CHROMagar,
enterobacteria acidifying glucuronate (positive:blue France)
to purple). The selective agents are bile salts and A comparison of CHROMagar Salmonella
brilliant green (Dusch and Altwegg, 1995). medium (CAS), based on the esterase acitivity, and
Hektoen enteric agar (HEA) for isolation of Sal-
2.1.5.2. Rambach agar (Merck, Germany) monellae have been done by Gaillot et al. (1999)
Rambach agar is composed of propylene glycol, with Salmonella isolates and stool samples. All stock
peptone, yeast extract, sodium deoxycholate, neutral cultures, including cultures of H S-negative isolates,
2
red, and Xgal. After incubation of plates at 378C for yielded typical mauve colonies on CAS while 99%
24 h, the formation of acid from propylene glycol isolates produced typical lactose-negative, black-cen-
causes precipitation of the neutral red in Salmonella tered colonies on HEA. Sensitivities for primary
colonies yielding a red colour (Rambach, 1990). plating and after enrichment were 95 and 100%,
Salmonella strains show a bright red colour, respectively, for CAS and 80 and 100%, respective-
coliforms blue (b-D-galactosidase activity) or violet ly, for HEA. The specicity of CAS (88.9%) was
(the formation of acid from propylene glycol and signicantly higher than that of HEA (78.5%; P ,
b-D-galactosidase activity) and Proteus spp. remain 0.0001).
colourless. Sodium deoxycholate inhibits the accom-
panying Gram-positive ora. The main disadvantage 2.1.5.5. Rainbow Salmonella agar (Biolog, USA)
of Rambach agar is that it does not detect S. typhi, S. The growth of distinctive black colonies of Sal-
paratyphi nor some rare strains such as S. moscow monella on the clear Rainbow Agar makes it easier
and S. wassenaar. Furthermore, Salmonella strains to detect Salmonella in mixed cultures. The advan-
which are able to produce b-D-galactosidase such as tages of using Rainbow Agar Salmonella versus
S. arizona or, in particular, subspecies IIIa, IIIb, and traditional media is the sensitivity and specicity for
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 media such as XLT4 utilize additives such as
the total analysis time for salmonellae in foods can Tergitol 4 that are inhibitory to S. typhi and S.
be reduced to 48 h by using the combination of choleraesuis.
Salmosyst broth (Merck) as a liquid medium and
Rambach agar as isolation plate. 2.1.5.6. Chromogenic Salmonella esterase agar
(PPR Diagnostics Ltd, UK)
2.1.5.3. MUCAP-test (Biolife, Italy) Cooke et al. (1999) described a novel agar
MUCAP is a conrmation test for Salmonella medium, chromogenic Salmonella esterase (CSE)
species based on the rapid detection of caprylate agar. The agar contains peptones and nutrient ex-
esterase, using uorogenic 4-methylumbelliferyl- tracts together with the following 4-[2-(4-
M. Mana / International Journal of Food Microbiology 60 (2000) 205218 213
octanoyloxy-3,5-dimethoxyphenyl)-vinyl]- family Enterobacteriaceae by the presence of a-
quinolinium-1-(propan-3-yl-carboxylic-acid)-bro- galactosidase activity in the absence of b-galacto-
mide (SLPA-octanoate bromide form). SLPA-oc- sidase activity. A total of 1022 strains of Salmonella
tanoate is a newly synthesized ester formed from a spp. and 300 other Gram-negative strains were
C fatty acid and a phenolic chromophore. In CSE inoculated onto this medium. Of these, 99.7% strains
8
agar, the ester is hydrolyzed by Salmonella spp. to of Salmonella spp. produced a characteristic green
yield a brightly coloured phenol which remains colony, whereas only one strain of non-Salmonella
tightly bound within colonies. The typical Salmonel- produced a green colony.
la strains were burgundy coloured on a transparent
yellow background, whereas non-Salmonella spp. 2.2. Enterococci
were white, cream, yellow or transparent. The sen-
sitivity (93.1%) of CSE agar for non-typhi Salmonel- The use of chromogenic and uorogenic substrates
lae compared favorably with those of Rambach for detection of b-D-glucosidase activity to differen-
(82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), tiate enterococci has received considerable attention
Hektoen-enteric (89.7%), and SM ID (91.4%) agars. (Dufour, 1980; Littel and Hartman, 1983).
The specicity (93.9%) was also comparable to
those of other Salmonellamedia (SM ID agar, 2.2.1. Enterolert and Microtiter plate MUST
95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; 4-methylumbelliferyl-b-D-glucoside (MUD), when
Hektoen-enteric agar, 87.8%). Strains of Citrobacter hydrolyzed by enterococcal b-D-glucosidase, releases
freundiiand Proteus spp. giving false-positive re- 4-methylumbelliferone, which exhibits uorescence
actions with other media gave a negative colour under a UV lamp (366 nm). Enterolert (IDEXX
reaction on CSE agar. CSE agar enabled the de- Laboratories Inc., Westbrook, Maine), and a Microti-
tection of .30 Salmonella serotypes, including ter plate MUST (Sano, France), containing MUD
agona, anatum, enteritidis, hadar, heidelberg, infan- detecting b-D-glucosidase. Niemi and Ahtiainen
tis, montevideo, thompson, typhimurium, and vir- (1995) desribed the evaluation of MTP method with
chow. SlanetzBartley and KF-Streptococcus agar using
pure cultures. Both media yielded high recoveries of
2.1.5.7. Compass Salmonella agar (Biokar diagnos- the target species. The MUD method tended to give
tics, France) slightly lower recoveries than the agar cultivation
Perry and Quiring (1997) described an agar methods with some target species at 448C but
medium based on the detection of C8-esterase activi- recoveries were better than at 418C. It has to be
ty using 5-bromo-6-chloro-3-indolyl-caprylate. mentioned, that there may be some problems with
Magenta coloured colonies indicating the presence of other glucosidase positive bacteria such as Aerococ-
Salmonella spp. cus spp., in particular in natural contaminated water
samples. Budnick et al. (1996) evaluated the En-
2.1.5.8. Chromogenic ABC medium (Lab M. Ltd., terolert for enumeration of enterococci in recreation-
UK) al waters as a semiautomated MPN method and
Perry et al. (1999) described a new chromogenic compared with the standard membrane lter method
agar medium, ABC medium (ab-chromogenic by parallel testing of 138 marine and freshwater
medium), which includes two substrates, 3,4- recreational bathing water samples. No statistical
cyclohexenoesculetin-b-D-galactoside (CHE) and 5- signicant difference and a strong linear correlation
bromo-4-chloro-3-indolyl-a-D-galactopyranoside (X- were found between the two methods. Culturing of
aGal), to facilitate the selective isolation of Sal- 501 Enterolert test wells resulted in false-positive
monella spp. CHE-Galactoside is utilised by most and false-negative rates of 5.1 and 0.4%, respective-
Enterobacteriaceae, yielding black colonies. The X- ly.
5-Gal is hydrolysed by Salmonella spp. to produce
characteristic, easy to distinguish, green colonies. 2.2.2. mEI agar
This medium exploits the fact that Salmonella spp. Dufour (1980) described a medium (mEI agar) for
may be distinguished from other members of the use in a single-step, 24-h MF procedure to enumerate
214 M. Mana / International Journal of Food Microbiology 60 (2000) 205218
enterococci in marine water and freshwater. The lactis and Aerococcus spp. No false-negative results
medium contained nalidixic acid, cycloheximide, were found. This medium was used to enumerate
triphenyltetrazolium chloride (TTC) and indoxyl-b- enterococci in water using a presence/ absence pro-
D-glucoside. Enterococci strains produce an insoluble cedure and gave still positive results with 10 cfu/ 100
indigo blue complex, which diffuses into the sur- ml water samples after 24 h incubation.
rounding media, forming a blue halo around the
colony. Rhodes and Kator (1997) evaluated mEI 2.2.4. RAPID9 enterococcus agar
agar with respect to specicity and recovery of RAPID9 enterococcus agar (Sano, France) is a
enterococci from environmental waters. Extending new medium using chromogenic Xglu for the de-
incubation from 24 to 48 h improved enterococci tection of enterococci and a selective mix for the
recovery but 77% of the colonies classied as non- inhibition of Gram negative and other b-glucosidase
target were conrmed as enterococci. Decreasing the positive bacteria other than enterococci.
concentration of or eliminating indoxyl-b-D-gluco-
side from mE did not signicantly affect recovery of 2.2.5. Comparative studies
puried isolates. Messer and Dufour (1998) recently In most literature, Slanetz and Bartley (1957) agar
modied the medium by reducing the TTC from 0.15 is treated as a suitable MF medium to detect fecal
to 0.02 g/ l and adding 0.75 g of indoxyl b-D- enterococci from a water sample. Amoros and
glucoside per liter. The new MF medium, mEI Alonso (1996) presented a study including a com-
medium, detected levels of enterococci in 24 h parison of SlanetzBartley agar and Enterococci agar
comparable to those detected by the original mE (with XGLU) using samples of irrigation channels
medium in 48 h, with the same level of statistical and seawater. They found no statistical differences
condence. The comparative recovery studies, spe- between both media in seawater, although the speci-
cicity determinations, and multilaboratory evalua- ty of the Enterococci agar decreased in marine
tion indicated that mEI medium has analytical per- water and there was a considerable number of false-
formance characteristics equivalent to those of mE positives.
medium. The simplicity of use and decreased incuba-
tion time with mEI medium will facilitate the 2.3. Staphylococcus aureus
detection and quantication of enterococci in fresh
and marine recreational waters. On the new chromogenic medium, CHROMagarE
S. aureus medium (CHROMagar, France), colonies
2.2.3. Chromocult enterococci broth (CEB) and of S. aureus are typical pink coloured. In supple-
Readycult enterococci menting CHROMagar S. aureus with an appropriate
Using chromogenic substrate 5-bromo-4-chloro-3- antibiotic, for instance methicillin, one can obtain
indolyl-b-D-glucopyranoside (XGLU), Chromocult information related to some methicillin resistant S.
Enterococci broth (CEB) (Merck) and Readycult aureus strains. This medium has been evaluated by
Enterococci (Merck) utilises the b-D-glucosidase using pure cultures and clinical specimens. Out of
reaction as an indicator of the presence of enterococ- the 181 S. aureus and 81 coagulase negative Staphy-
ci. XGLU is liberated and rapidly oxidized to lococcus strains belonging to 18 different species, S.
bromochloroindigo which produces blue colour in aureus, S. epidermidis, S. caprae and S. schleiferi
Chromocult broth as well as blue coloured en- strains yielded pink colonies (Freydiere et al., 2000).
terococci colonies in Chromocult Enterococci agar.
Other b-D-glucosidase-producing organisms being 2.4. Clostridium perfringens
suppressed by the sodium azide content of the broth
(Mana and Windhager, 1997). The results obtained The identication of C. perfringens is possible
with pure cultures showed that 97% of the strains, using Fluorocult TSC-agar (Merck, Germany) using
which gave positive results, were identied as en- a uorogenic enzyme substrate. D-Cycloserine inhib-
terococci (E. fecalis, E. faecium, E. durans, E. its the accompanying bacterial ora and causes the
casseliavus and E. avium). The false-positive colonies to remain smaller. C. perfringens colonies
strains (3%) were Leucononostoc, Lactococcus lactis can be detected using 4-MU-phosphate. Acid phos-
M. Mana / International Journal of Food Microbiology 60 (2000) 205218 215
phatase is a highly specic indicator for C. perfrin- colonies (1.02.5 mm in diameter) from PI-PLC
gens which shows a light blue uorescence on this activity on the chromogenic substrate, 5-bromo-4-
medium (Baumgart et al., 1990). chloro-3-indoxyl-myo-inositol-1-phosphate. L.
monocytogenes was distinguished from L. ivanovii
2.5. Bidobacteria and lactic acid bacteria by either its uorescence on BCM conrmatory
plating medium or acid production from rhamnose.
The use of chromogenic X-a-D-galactoside for False-positive organisms (Bacillus cereus, S. aureus,
differential enumeration of Bidobacterium spp. and B. thuringiensis, and yeasts) were eliminated by at
lactic acid bacteria was described by Chevalier et al. least one of the media in the BCM LMDS. The
(1991). The X-a-D-galactoside-based medium is efcacy of the BCM-LMDS was determined using
useful to identify bidobacteria among Lactobacillus pure cultures and naturally and articially contami-
or Streptococcus strains. Bidobacteria shows blue nated environmental sponges (Restaino et al.,
coloured colonies on the agar plate. 1999a). In an analysis of 162 environmental sponges
from facilities inspected by the US Department of
2.6. Listeria monocytogenes Agriculture (USDA), the values for identication of
L. monocytogenes by BCM LMDS and the USDA
L. monocytogenes is a human and animal pathogen method were 30 and 14 sites, respectively, with
that is widespread in nature. The organism is a sensitivity and specicity values of 85.7 and 100.0%
transient constituent of the intestinal ora excreted vs. 40.0 and 66.1%, respectively. No false-positive
by 110% of healthy humans. It is an extremely organisms were isolated by BCM LMDS, whereas
hardy organism and can survive for many years in 26.5% of the sponges tested by the USDA method
the cold in naturally infected sources. L. monocyto- produced false-positive results.
genes has been isolated from a wide variety of foods,
including dairy products, meats, and sh. Although 2.6.2. Rapid LMONO agar
most of the foodborne listeriosis outbreaks have been Rapid LMONO agar (Sano, France) is based on
linked to the consumption of dairy products, recent the detection of phosphatidylinositol phospholipase
sporadic cases have been associated with meats, as C and Xylose fermentation. In this way, the colonies
well as other foods. All strains of L. monocytogenes of L. ivanovii appear blue sourrounded by a yellow
are pathogenic by denition although some appear to halo (Xylose positive) whilst the colonies of L.
be more virulent than others. Cultural methodology monocytogenes are blue without the halo (Xylose-
for isolating the organism from foods has been in a negative). The colonies of other Listeria spp. remain
state of ux since 1985. white because they do not posses phosphatidylino-
sitol phospholipase C activity. Further evaluation of
2.6.1. BCM L. monocytogens detection system these media is still needed.
The new BCM L. monocytogens detection system
(BCM-LMDS, Biosynth, Switzerland) consists of a 2.7. Bacillus cereus
selective pre-enrichment broth, selective enrichment
broth, selective/ differential plating medium, and BCM B. cereus and B. thuringiensis plating
identication on a conrmatory plating medium. The medium (Biosynth, Switzerland) is a differential and
BCM pre-enrichment broth, allowed the growth of selective medium which alows B. cereus and B.
Listeria and resuscitation of heat-injured L. mono- thuringiensis to grow, but inhibits growth of many
cytogenes, which contains the uorogenic substrate potential false positives including other Bacillus
4-methylumbelliferyl-myo-inositol-1-phosphate and species. Based on the detection of phosphatidylino-
detects phosphatidylinositol phospholipase C (PI- sitol-specic phospholipase C (PI-PLC) activity by
PLC) activity, provided a presumptive positive test Bacillus species, B. cereus and B. thuringiensis
for the presence of pathogenic L. monocytogenes and strains produce turquoise at dull colonies with and
L. ivanovii after 24 h at 358C. On BCM-plating without turquoise halos to the enzymatic reaction of
medium, L. monocytogenes and L. ivanovii were the PI-PLC with 5-bromo-4-chloro-3-indoxyl-myo-
two Listeria species forming turquoise convex inositol-1-phosphate.
216 M. Mana / International Journal of Food Microbiology 60 (2000) 205218
EHEC belonging to other serogroups. J. Appl. Microbiol. 85,
3. Conclusions
425428.
Bettelheim, K.A., 1998b. Studies of E. coli cultured on Rainbow
Rapid detection and identication of microorga-
agar O157 with particular reference to enterohaemorrhagic E.
nisms is of high importance in a diverse array of
coli EHEC. Microbiol. Immunol. 42, 265269.
applied and research elds. By incorporation of
Betts, R., Murray, K., MacPhee, S., 1994. Evaluation of Fluoro-
cult LMX broth for the simultaneous detection of total synthetic enzyme substrates into primary isolation
coliforms and E. coli. Technical Memorandum No. 705,
media, enumeration and detection can be performed
Campden Food and Drink Research Association, UK.
directly on the isolation plate. These enzyme sub-
Bochner, B.R., 1995. Rainbow Agar VTEC, a new chromogenic
strates have proved to be a powerful tool, utilizing
culture medium for detecting verotoxin-producing E. coli,
specic enzymatic activities of certain microorga-
Abstracts of the Australian Society for Microbiology, Poster
nisms. Many of them are offering enhanced accuracy
2.1.
Bredie, W.L., de Boer, E., 1992. Evaluation of the MPN, Ander- and performance to the microbiologist. The incorpo-
son-Baird-Parker, Petrilm E. coli and Fluorocult ECD method
ration of such substrates into a selective medium can
for enumeration of E. coli in foods of animal origin. Int. J.
eliminate the need for subculture and further bio-
Food Microbiol. 16, 197208.
chemical tests to establish the identity of certain
Brenner, K., Rankin, C.C., Roybal, Y.R., Stelma J.R., G.R.,
microorganisms. These enzymatic assays may consti-
Scarpino, P.V., Dufour, A.P., 1993. New medium for simulta-
tute an alternative method for enumerating micro- neous detection of total coliforms and E. coli in water. Appl.
Environ. Microbiol. 59, 3534-3544.
organisms in water and foods, which is specic,
Brenner, K.P., Rankin, C.C., Sivaganesan, M., Scarpino, P.V.,
sensitive and rapid.
1996. Comparison of the recoveries of E. coli and total
coliforms from drinking water by the MI agar method and the
US environmental protection agency-approved membrane lter
method. Appl. Environ. Microbiol. 62, 203208.
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Budnick, G.E., Howard, R.T., Mayo, D.R., 1996. Evaluation of
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Okrend et al., 1990.
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