Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Evaluation of ALOA plating medium for its suitability

to recover high pressure-injured Listeria monocytogenes
from ground chicken meat
M.M. Jantzen1,2, J. Navas1, M. de Paz1, B. Rodrı́guez1, W.P. da Silva2, M. Nuñez1
and J.V. Martı́nez-Suárez1
1 Departamento de Tecnologı́a de Alimentos, Instituto Nacional de Investigación y Tecnologı́a Agraria y Alimentaria (INIA), Madrid, Spain
2 Departamento de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas, Pelotas, RS,
Brazil

Keywords
agar Listeria according to Ottaviani and
Agosti, chicken meat, high-pressure, injured
cells, Listeria.
Correspondence
Joaquı́n V. Martı́nez-Suárez, Departamento de
Tecnologı́a de Alimentos, Instituto Nacional
de Investigación y Tecnologı́a Agraria y
Alimentaria (INIA) (Edificio Principal), Carretera
de La Coruña km 7’5, 28010 Madrid, Spain.
E-mail: joaquin@inia.es
2005/1185: received 6 October 2005, revised
21 February 2006 and accepted 27 March
2006
doi:10.1111/j.1472-765X.2006.01950.x
Abstract
Aims: To evaluate a chromogenic plating medium for the isolation of suble-
thally injured cells of Listeria monocytogenes from processed foods.
Methods and Results: The inactivation of L. monocytogenes at pressures up to
400 MPa and 12C in ground chicken meat was employed to examine the
recovery of high-pressure injured cells. Before and after different repair incuba-
tion periods at 30C in a nonselective broth, samples were plated onto a select-
ive and differential agar [Agar Listeria according to Ottaviani and Agosti
(ALOA)] and in the same medium supplemented with 4% sodium chloride
(ALOA-S), and incubated at 37C. Sublethally injured cells were able to grow
when directly plated onto the ALOA medium, without a previous repair incu-
bation period. However, only uninjured cells grew on the ALOA-S medium.
Conclusions: Sublethally injured cells of L. monocytogenes can be quantified by
subtracting counts on ALOA-S medium from counts on ALOA medium.
Significance and Impact of the Study: Possible applications include direct enu-
meration on ALOA of stressed cells of L. monocytogenes in foods with more
than 100 colony forming units per gram.

colonies formed on agar overlays and those formed on

Introduction
Detection of viable cells of bacterial pathogens that may
be injured following food processing is essential to food
safety (Ryser and Marth 1999). Most of the media recom-
mended for isolation of sublethally injured Listeria mono-
cytogenes do not possess antimicrobial agents because they
can inhibit the recovery of injured cells (Donnelly 2002).
Few attempts have been made in the past to develop
selective and differential media for isolation of sublethally
injured Listeria cells from contaminated foods (Kang and
Fung 1999; Wu et al. 2001). Agar overlays or underlays of
nonselective media for detecting sublethally injured food-
borne pathogens in foods (Kang and Siragusa 1999) have
limitations that include the difficulty of picking well-iso-
lated colonies from agar underlays, as well as the disparity
between the colony morphologies and colour reactions of
the agar surface. Simpler, more effective methods for
recovery of injured food-borne pathogens from contamin-
ated foods are necessary. They can be based on appropri-
ate choice of selective agents, as it is known that the
degree to which selective agents inhibit the recovery of
sublethally injured Listeria cells is variable (Smith and
Archer 1988). Furthermore, they should be differential for
L. monocytogenes or pathogenic Listeria spp.
Recently, the Agar Listeria according to Ottaviani and
Agosti (ALOA) medium, developed primarily as a select-
ive and differential medium for L. monocytogenes (Ottavi-
ani et al. 1997), has been shown to efficiently recover
injured cells of listeriae (Vlaemynck et al. 2000). On
ALOA plates all Listeria spp. produce turquoise colonies,
and the pathogenic species L. monocytogenes and Listeria
ivanovii appear surrounded with a distinct opaque halo-

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Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 313–317 313
Pressure-injured Listeria and ALOA
like precipitation zone (Reissbrodt 2004). This and other
chromogenic plating media allow direct counts of patho-
genic Listeria spp. and are now recommended in most
protocols and standards (Reissbrodt 2004).
The aim of the present study was to investigate a new
method to quantify sublethally pressure-injured L. mono-
cytogenes in chicken meat. Repair of injured cells during
incubation in nonselective broth was analysed by the dif-
ferential plating technique (Beuchat et al. 1986; Smith
and Archer 1988) with ALOA as the ‘nonselective’ agar
and ALOA supplemented with 4% NaCl (ALOA-S) as the
selective medium.
Materials and methods
Bacterial strain, culture media, and growth conditions
Listeria monocytogenes EGD-e, a serotype 1/2a strain (Gla-
ser et al. 2001), was kindly provided by Dr Pérez-Dı́az
(Hospital Ramón y Cajal, Madrid), and it was maintained
in 15% glycerol at )20C. A frozen stock culture was
transferred twice in 3 ml of tryptic soy yeast extract broth
followed by incubation at 30C for 18 h prior to each
experiment. Repair was carried out with UVM base med-
ium (modified University of Vermont broth without anti-
microbial supplement). For the determination of cell
numbers, the cultures or enrichments were serially diluted
(1 : 10) in 0Æ1% (wt/vol) peptone in water and duplicate
0Æ1 ml portions of each dilution were surface plated onto
ALOA and ALOA-S. All media were obtained from Biolife
Laboratories (Milan, Italy) except ALOA medium (Cro-
mogen Listeria), which was obtained as dehydrated med-
ium (with separate enrichment and selective supplements)
from Biomedics (Madrid, Spain). ALOA medium was
prepared according to instructions of the manufacturer.
Plates were examined after 24, 48, and 72 h of aerobic
incubation at 37C for CFU counts, colony size, and col-
our and halo formation. Extended incubation times were
needed due to the cell injury (48 h) or the effect of NaCl
(72 h).
Preparation of ground chicken meat
Ground chicken meat was used as a model of highly
contaminated food matrix. Commercial ground meat
was compared with pure ground meat prepared at the
laboratory with raw breast chicken purchased from a
local supermarket. Portions of c. 800 g were minced
with a domestic grinding machine for 10 s, and indi-
vidual 250 g portions were aseptically weighed into ster-
ile bags and frozen at )20C for a maximum of
2 months. Subsamples of 25 g tested negative for
Listeria by the USDA-FSIS procedure (http://www.
314 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 313–317
M.M. Jantzen et al.
fsis.usda.gov/Ophs/Microlab/Mlg_8_04.pdf, accessed 10
March 2005).
Inoculation and vacuum-packaging of chicken meat
samples
Frozen chicken meat samples (250 g) were held at 4C for
24 h before being inoculated. The L. monocytogenes cells
were harvested by centrifugation (10 000 g, 10 min, 4C),
washed twice with peptone water, and serially diluted in
the same diluent to a concentration (107 CFU ml)1) that
would give c. 105 CFU g)1 of chicken meat product. Each
package of chicken meat was inoculated with 2Æ5 ml of the
L. monocytogenes culture dilution. Each inoculated pack-
age was massaged manually for about 1 min to spread the
inoculum into the meat. Subsamples of 25 g of inoculated
ground meat were aseptically removed from the original
bulk package and repackaged into vacuum bags. All
inoculated packages were vacuum-sealed and kept at 4C.
High pressure treatment
A portion of the inoculated packages of chicken meat
(4C) was treated at the Instituto Nacional de Investiga-
ción y Tecnologı́a Agraria y Alimentaria (INIA) Food
Technology Department Facility, which has a hydrostatic
food processor (ACB, Nantes, France). To examine the
influence of high pressure on L. monocytogenes injury in
chicken meat, the inactivation kinetics under different
conditions and in different kind of samples were exam-
ined. Samples were treated from 200 to 400 MPa at a
constant temperature of 12C for 2–15 min and the high-
est pressure was established to be used. Samples were kept
at 4C for <1 h until they were analysed.
Microbiological analysis
Each package of pressure-treated samples and untreated
controls was aseptically opened and samples were trans-
ferred to stomacher bags. UVM base medium was added
to each meat sample followed by pummelling for 2 min
in a laboratory stomacher (Lab-Blender 400, Tekmar,
Cincinnati, OH, USA). Before and after different repair
incubation periods (3, 6, 9 and 24 h) at 30C, L. monocy-
togenes were enumerated in duplicate on ALOA and
ALOA-S plates. Typical L. monocytogenes colonies on
ALOA plates were counted after 48 h (ALOA) or 72 h
(ALOA-S) of incubation at 37C.
Data analysis
To compare recoveries of L. monocytogenes on ALOA or
ALOA-S at 0 and 6 h, the mean cell populations of three
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M.M. Jantzen et al. Pressure-injured Listeria and ALOA

replicates were recorded and converted into logarithm 7

values (log10) to determine the significance of differences 6
(P < 0Æ05) by Student’s t-test.
5

log10 CFU ml–1

4
Results
3

High pressure-induced inactivation of Listeria 2

monocytogenes
The inactivation of L. monocytogenes at <400 MPa was at
a level that led to a low effect. In order to reach a >99%
injury on the population of L. monocytogenes, a logarith-
mic inactivation of more than 2 log10 cycles was needed.
Because of the differences in sample composition, inacti-
vation times were very different with commercial (78%
chicken meat and 22% added fat) or pure chicken ground
meat samples. At 400 MPa and 12C there was a logarith-
mic inactivation of 1 log10 cycle every 3Æ29 min (D-value)
with the commercial chicken meat and every 1Æ41 min
with the pure chicken meat (data not shown). Survivor
plots showed first-order death behaviour characteristic of
short treatments, that allowed calculation of D-values.
After 3Æ5 min these conditions were usually enough to
reach a logarithmic inactivation of >2 log10 cycles in the
pure chicken meat samples (Fig. 1). For a better repro-
ducibility, subsequent experiments were carried out with
pure chicken samples.
Growth of Listeria monocytogenes on ALOA plates
Cell numbers in untreated chicken meat enrichments
increased more than 1 log10 unit after 6 h. No increase in
numbers of survivors in treated samples occurred before
6 h, i.e. in the case of treated samples ALOA recovered
the same number of cells at 0 h and after 6 h of nonselec-
tive enrichment (Table 1 and Fig. 1). If injured cells did
not grow on ALOA, as in most selective media, the num-
ber of colonies on ALOA would increase during enrich-
ment due to the repair of injury. We concluded that
1
0
0 3·5 3 6 9 24
Injury (min) Repair (h)
Figure 1 Recovery of Listeria monocytogenes with Agar Listeria
according to Ottaviani and Agosti (ALOA) (h) and ALOA supplemen-
ted with 4% NaCl ( ) after treatment for 3Æ5 min and enrichment for
24 h.
ALOA medium allowed the recovery of most injured bac-
terial cells in spite of its selective supplement.
Growth of Listeria monocytogenes on ALOA-S compared
with ALOA
In untreated samples the recovery of L. monocytogenes on
ALOA and ALOA-S was not significantly different
(P > 0Æ05), but in treated samples the recovery of
L. monocytogenes with ALOA was significantly higher than
with ALOA-S (P < 0Æ05) (Table 1). As in the typical
repair curves (Doyle et al. 2001; Ngutter and Donnelly
2003), during treatment, the decrease in CFU on ALOA
represents the lethality and the difference between the val-
ues obtained on ALOA and ALOA-S represents the injury
(Fig. 1). The recovery of uninjured cells with ALOA-S
varied according to the incubation time at 30C in the
nonselective broth, i.e. according to the repair time.
Before broth incubation (0-h samples), more than 99% of
the cells were injured and could not grow on ALOA-S;
during repair (3- and 6-h samples) the CFU obtained on

Table 1 Recovery of Listeria monocytogenes

from treated samples and untreated controls log10 CFU ml)1*
with two plating media and before and 0h 6h
after 6 h of enrichment
Samples ALOA ALOA-S ALOA ALOA-S

Untreated 4Æ93 ± 0Æ31b 4Æ80 ± 0Æ32b 6Æ59 ± 0Æ22a 6Æ24 ± 0Æ22a

Treated 3Æ62 ± 0Æ60c 0Æ00e 3Æ85 ± 0Æ62c 2Æ58 ± 0Æ39d

ALOA, agar Listeria according to Ottaviani and Agosti; ALOA-S, ALOA supplemented with 4%
NaCl.
*Data represent mean ± SD of three measurements before or after treatment (400 MPa and
12C for 2Æ5 min). Within each row, values followed by different letters are statistically different
(P < 0Æ05).

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Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 313–317 315
Pressure-injured Listeria and ALOA
ALOA-S approached the CFU on ALOA, and after 9 h
they were similar (Fig. 1).
Discussion
Distinction of viable and nonviable organisms is a basic
challenge of food safety programs. Nucleic acid-based
amplification techniques can distinguish dead from live
organisms (Navas et al. 2005; Rudi et al. 2005), but detec-
tion of injured viable cells can only be accomplished if
they are allowed to repair in appropriate media (Donnelly
2002). As much of the reported injury repair research
employs culture media or different buffer systems (Smith
and Archer 1988; Ryser and Marth 1999; Donnelly 2002),
we sought to test the efficacy of nonselective enrichments
from a highly contaminated food system to resuscitate
sublethally injured L. monocytogenes. When we evaluated
the plating efficiency of ALOA for injured cells, we found
that both injured and uninjured cells grew on this med-
ium.
To estimate the total number of viable cells and the
proportion of the cell populations which were sublethally
injured by different stressors, differential plating methods
(Beuchat et al. 1986; Smith and Archer 1988) were usu-
ally employed. Briefly, these methods are based on the
observation that both uninjured and sublethally injured
cells of L. monocytogenes can form colonies on nonselec-
tive plating media, but injured cells fail to produce colon-
ies on plating media containing 4% NaCl (Beuchat et al.
1986; Smith and Archer 1988). Farkas et al. (2003) have
previously shown that pressure-injured L. monocytogenes
showed increased salt sensitivity. They found that supple-
menting brain heart infusion agar plates with 5% NaCl
did not influence the recovery of nonpressurized cells but
residual populations of treated cells were reduced by sev-
eral log10 cycles in case of NaCl containing plates (Farkas
et al. 2003).
We have shown here that ALOA and ALOA-S can be
used as differential plating media for enumerating injured
listeriae from processed foods. Our data indicate that
ALOA is an efficient medium for recovery and subsequent
selective culture of sublethally injured micro-organisms,
and offers several advantages over other injury repair
techniques. The ability of ALOA to efficiently recover
healthy and sublethally injured L. monocytogenes cells can
be very useful for the direct enumeration of stressed cells
in certain processed foods with levels of listerial contam-
ination above the sensitivity of the direct plating tech-
nique (>100 CFU g)1). Using ALOA as the selective agar
with a highly contaminated food (untreated chicken meat
samples) retained colony morphology and selectivity to
differentiate L. monocytogenes from a mixture containing
different organisms.
316 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 313–317
M.M. Jantzen et al.
Furthermore, composition of ALOA is public (http://
www.cfsan.fda.gov/ebam/m10a.html, accessed 10 March
2005) and can be prepared in-house, although in-house
preparation of media with so many different components
is difficult to standardize (Reissbrodt 2004). However, it
can be bought as a dehydrated medium, less expensive
than the ready-to-use plates (or bottles) of most other
chromogenic media for pathogenic Listeria spp.
Acknowledgements
This research was supported by the Spanish Ministry of
Education grants CAL03-027-C2-1, PTR1995-0789-OP
and RTA2005-00202-C02-01, and Fellowships of CNPq
from Brazil (MMJ) and INIA from Spain (JNF). The
authors thank S. Ortiz and P. Lopez for their technical
assistance.
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