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Efficacy of sanitizers in reducing Escherichia

coli O157:H7, Salmonella spp. and Listeria
monocytogenes populations on fresh-cut...

Article in Food Control · November 2007

DOI: 10.1016/j.foodcont.2006.09.008

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 EYcacy of sanitizers in reducing Escherichia coli O157:H7, Salmonella
spp. and Listeria monocytogenes populations on fresh-cut carrots
Saúl Ruiz-Cruz a, Evelia Acedo-Félix b, Martha Díaz-Cinco b,
Maria A. Islas-Osuna a, Gustavo A. González-Aguilar a,¤
a
Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y, Desarrollo,
A.C. Apdo. Postal 1735, Hermosillo, Sonora 83000, Mexico
b
Coordinación de Ciencia de los Alimentos, Centro de Investigación en Alimentación y, Desarrollo,
A.C. Apdo. Postal 1735, Hermosillo, Sonora 83000, Mexico

Abstract

Shredded carrots were inoculated with Escherichia coli O157:H7, Salmonella or Listeria monocytogenes and washed for 1 or 2 min with
chlorine (Cl; 200 ppm), peroxyacetic acid (PA; 40 ppm) or acidiWed sodium chlorite (ASC; 100, 200, 500 ppm) under simulated commer-
cial processing conditions. After washed, the carrots were spin dried, packaged and stored at 5 °C for up to 10 days. Bacterial enumeration
was signiWcantly (P 6 0.05) reduced by 1, 1.5 and 2.5 log CFU/g after washing with ASC 100, 250 and 500 ppm, respectively. All sanitizers
reduced pathogen load below that of tap water wash and unwashed controls. During storage at 5 °C the bacterial load of all treatments
increased gradually, but to diVerent extent in diVerent treatments. ASC inhibited bacterial growth more eVectively than the other sanitiz-
ers and also maintained the lowest pathogen counts (<1 log CFU/g) during storage. Organic matter in the process water signiWcantly
(P 6 0.05) reduced the antibacterial eYcacy of Cl, but not that of PA or ASC. Therefore, ASC shows the potential to be used as a com-
mercial sanitizer for washing shredded carrots.

Keywords: E. coli O157:H7; Salmonella; L. monocytogenes; Shredded carrots; Chlorine; Peroxyacetic acid; AcidiWed sodium chlorite

1. Introduction processing steps (peeling and cutting) enhance the suscep-

Consumption of fresh-cut produce has increased in
recent years; because of greater availability of health
information and typically busier lifestyles, consumers are
placing greater value on these fresh, nutritious and conve-
nient products (IFPA, 2000). Unfortunately, there has
been an increase in the frequency of outbreaks of illnesses
associated with the consumption of fresh produce, espe-
cially fresh-cut produce (Brackett, 1999; Hodge, 1999;
Thunberg, Tran, Bennett, Matthews, & Belay, 2002), since
* of organic matter is present in the wash water (Nguyen-the
tibility of these products to microbial growth (Tournas,
2005).
Washing is one of the most important steps during pro-
cessing of produce, since it removes soil and microorgan-
isms from the surface. However, not all washing methods
and washing solutions are equally eVective. Given that
fresh-cut products are ready to eat, and are not subjected to
further microbial killing steps, the use of eVective sanitizing
agents during produce washing is necessary to ensure prod-
uct safety. Chemical sanitizers have been widely used in
food processing to reduce pathogens. However, the eYcacy
of common sanitizers on the surface of fruits and vegeta-
bles may be limited or unpredictable when a large amount
& Carlin, 1994).
 Chlorine is the most widely used sanitizing agent for
reducing pathogens on whole and fresh-cut vegetables. The
recommended concentrations of Cl range from 50 to
200 ppm with a contact time of 1–3 min (Beuchat, 1998;
Cherry, 1999). However, under typical commercial produce
wash conditions, the eYcacy of Cl on pathogen reduction is
limited (1–2 log CFU/g). This reduction in eYcacy may be
caused by organic matter in recycled wash water, insuY-
cient cooling or exposure to light or air or a combination of
these factors. Moreover, Cl can react with organic matter to
form carcinogenic products (Beuchat, 2000). Therefore, the
fresh-cut produce industry would beneWt greatly by Wnding
alternatives to chlorine for sanitizing fresh produce.
The US Food and Drug Administration (FDA)
approved the use of PA for sanitizing certain food prod-
ucts, including fruits and vegetables, at concentrations that
do not exceed 80 ppm in wash water (Anonymous, 2000a).
Park and Beuchat (1999) reported that PA at concentra-
tions of 40–80 ppm reduced Salmonella and E. coli
O157:H7 populations on “Cantaloupe” and “Honeydew”
melon surfaces in the range of 2.6–3.8 log CFU/g, but it was
less eVective on asparagus spears.
The FDA has also approved the sanitizer, ASC, for use
as spray or dip in the range of 500–1200 ppm (Anonymous,
2000b). ASC is formed by a reaction between sodium chlo-
rite (NaClO2) and citric acid in aqueous solution. The bac-
tericidal eVect of ASC for surface washing of alfalfa seeds
(Weissinger & Beuchat, 2000), and Chinese fermented cab-
bage (Inatsu, Bari, Kawasaki, Isshiki, & Kawamoto, 2005;
Inatsu, Maeda, Bari, Kawasaki, & Kawamoto, 2005b) has
been reported. Recently, González, Luo, Ruiz-Cruz, and
McEvoy (2004) found that ASC produced a substantial
reduction in E. coli O157:H7 populations on inoculated
shredded carrots when compared with Cl, citric acid and
water.
Studies on sanitizers are generally conducted with fresh
tap water (laboratory conditions) and few researchers have
compared sanitizers under simulated process water condi-
tions. During commercial processing of fresh-cut produce,
water is often reused and recycled, resulting in the accumu-
lation of a large amount of organic matter. In order to
ensure the safety of fresh-cut produce, it is necessary to
evaluate the eYcacy of sanitizers in water containing diVer-
ent concentrations of organic matter to simulate commer-
cial practices.
The objective of this study is to evaluate the eVectiveness
of Cl, PA and ASC for reducing populations of E. coli
O157:H7, Salmonella and L. monocytogenes from inocu-
lated shredded carrots under laboratory and simulated
commercial processing wash water conditions.
2. Materials and methods
2.1. Bacterial strains and media
E. coli O157:H7 ATCC 25922 and ATCC 43890, Salmo-
nella serotype Poona (donated by Laboratorio Estatal de
Sanidad Pública, Sonora, México) and S. Typhimurium
ATCC 14028, and Listeria monocytogenes ATCC 7644
were used in this study. Cultures of E. coli O157:H7, Salmo-
nella and L. monocytogenes from freezer stocks were grown
in tryptic soy broth (Difco Laboratories, Detroit, MI).
To suppress the growth of microorganisms naturally
present on carrots, nalidixic acid-resistant strains were
obtained and used in this study (Inatsu, Bari, et al., 2005;
Inatsu, Maeda, et al., 2005b). E. coli O157:H7 and Salmo-
nella strains were adapted to grow on Luria-Bertani broth
(LB; Difco, Becton Dickinson, Sparks, MD) supplemented
with 50 g/ml nalidixic acid (LB-Nal) and L. monocytoge-
nes in tryptic soy broth containing 0.6% yeast extract, sup-
plemented with 50  g/ml nalidixic acid (TSBYE-Nal) and
incubated at 37 °C. To obtain pure cultures, a loop of
E. coli O157:H7 was streaked on Sorbitol MacConkey agar
(SMAC; Difco Becton Dickinson, Sparks, MD), Salmonella
spp. on Bismuth SulWte agar (BS; Difco Laboratories,
Detroit, Ml) and L. monocytogenes on Oxford medium agar
(Ox; Difco Laboratories, Detroit, Ml). Each agar medium
was supplemented with nalidixic acid (50 g/ml) and plates
were incubated at 37 °C. After incubation, a single colony
from each plate was selected and inoculated into 10 ml of
LBB-Nal (E. coli O157:H7 and Salmonella) or 10 ml of
TSBYE-Nal (L. monocytogenes). Individual strains were
grown in each broth at 37 °C with constant agitation at
175 rpm. Cultures were transferred to each broth by a loop
at two successive 24 h intervals and one overnight (16–18 h)
before they were used as inoculants. Cells of each strain
were harvested by centrifugation (4000 £ g, 15 min) and
washed with 2 vol of sterile 0.1% peptone water and resus-
pended at a cell density of approximately 109 CFU/ml. Vol-
umes of cell suspensions of E. coli O157:H7 or Salmonella
were combined to create a two-strain mixture. The cells
were added proportionally to tap water to obtain a dip
inoculum solution of approximately 107 CFU/ml. The inoc-
ulum level was conWrmed by replica plating onto selective
agar after serial dilution in sterile 0.1% peptone water, fol-
lowed by incubation at 37 °C for 24 h.
2.2. Vegetable preparation
Fresh carrot (Daucus carota L. cv. sativus) roots were
purchased from a local wholesale market in Hermosillo,
Sonora, México, transported to the laboratory and used
within 24 h following storage at 5 °C. Carrot root tips and
leaf ends were removed and roots with visible damage were
discarded. Carrots were washed with tap water to remove
soil, shredded with a food processor (Quick’N Easy Plus,
Towson, Maryland) and divided into individual 200 g por-
tions contained in nylon mesh bags (Linens N’ Things, Clif-
ton, NJ).
2.3. Inoculation procedure
Since the immersion process is a possible point of con-
tamination in the food industry, dip inoculation is the most
suitable method that can be used to simulate such a process
(Beuchat et al., 2001). Shredded carrots were immersed in
the inoculum solution (sample: inoculum ratio D 1:5 w/v)
and kept under constant agitation for 30 min. After dip-
ping, the samples were drained for 30 s and spin-dried for
30 s in a manually activated salad spinner (Metro Essore-
use, Metro marketing, Gardena, CA). The samples were
then placed into plastic containers and maintained for 1 h
at room temperature until treatment with the sanitizer solu-
tions.
2.4. Treatment procedure
The following sanitizer treatments were evaluated for
their eYcacy in killing or reducing E. coli O157:H7, Salmo-
nella and L. monocytogenes on shredded carrots. The treat-
ments were 200 ppm sodium hypochlorite of a commercial
bleach preparation, with pH adjusted to 6.5 with HCl
(Cloralex®, NL, México, 6% NaOCl), 40 ppm PA; pH 3.72
(Ecolab, St. Paul, MN) and 100, 250 and 500 ppm ASC; pH
2.71, 2.55 and 2.47, respectively (Sanova®, Alcide Corp.,
Redmond, WA). Both tap water washed and untreated car-
rot samples were used as controls.
The chemical oxygen demand, COD, was chosen to esti-
mate the organic matter content of the process water, and a
level close to that of commercial fresh-cut reused process
water (3,500 mg/liter, as determined by our previous survey)
was obtained by repeatedly dipping freshly shredded car-
rots of known mass in a Wxed volume of tap water (Gon-
zález et al., 2004). COD levels were determined by the
reactor digestion method (Chemical Oxygen Demand,
Method 8000, HACH, Loveland, Colo.) (HACH, 2002;
Jirka & Carter, 1975) approved by the EPA (EPA method
410.4) (Environmental Protection Agency, 1993). All treat-
ments were then tested under regular tap water and simu-
lated process water conditions.
Each mesh bag of inoculated shredded carrots was
dipped into one sanitizer solution (sample to wash water
ratio of 1:10 w/v) with a contact time of 1 min for ASC
treatment and 2 min for the rest of the treatments with con-
stant stirring. Shredded carrots were drained for 30 s and
spin-dried for 30 s using a salad spinner to remove excess
water. Samples of carrot shreds weighing 25 g were pack-
aged in polypropylene bags and stored at 5 °C for 10 days.
2.5. Procedures for microbial enumeration
On day 0, an enrichment step was carried out by adding
225 ml of sterile tryptic soy broth, lactose broth or tryptic
soy broth containing 0.6% yeast extract, supplemented with
50 g/ml nalidixic acid to E. coli O157:H7, Salmonella and
L. monocytogenes, respectively. Enrichment samples (25 g)
were transferred aseptically into sterile stomacher bags,
225 ml of Dey-Engley (DE) neutralizing broth was added
and carrots were macerated. The homogenized carrots were
serially diluted by a factor of ten in phosphate buVer saline.
For each dilution, 1 ml was plated on each of sorbitol Mac
Conkey agar, bismuth sulWte agar, Oxford medium agar,
and plate count agar for E. coli O157:H7, Salmonella spp.,
L. monocytogenes and total aerobic bacteria, respectively,
and incubated at 37 °C for 24 or 45 h. Agars for pathogenic
bacterial growth were supplemented with 50 g/ml nalidixic
acid.
2.6. Statistical analyses
All experiments were repeated three times, and values
represent the means of duplicate determinations for each
sample. Data were subjected to the Number Cruncher Sta-
tistical Systems’97 (NCSS97) Statistical Software, ver. 6.0
(NCSS, Kaysville, Utah, USA) for analysis of variance.
Duncan’s multiple range test was used for comparison of
means to determine diVerences in microbial counts for
treatments.
3. Results and discussion
3.1. EYcacy of sanitizers on pathogens reduction
Analysis of the shredded carrots that had not been inoc-
ulated revealed the absence of E. coli O157:H7, Salmonella
and L. monocytogenes. Preliminary studies were conducted
to determine the level of inoculum that could be retained on
the surface of shredded carrots. The level of inoculum used
for the untreated shredded carrots was >7 log CFU/ml of
E. coli O157:H7, Salmonella and L. monocytogenes; how-
ever, the amount of pathogens attached to the surface was
5.81, 5.84 and 3.54 log CFU/g, respectively (Fig. 1).
The eYcacies of the three sanitizers used in this study
were statistically diVerent (P 6 0.05), when compared with
untreated and water washed. Water treatment by itself did
not caused a signiWcant change in pathogens count. The
reduction of less than 0.5 log CFU/g of E. coli O157:H7,
Salmonella, and L. monocytogenes observed on shredded
carrots was similar to that reported previously (González
et al., 2004; Ibarra-Sánchez, Alvarado-Casillas, Rodríguez-
García, Martínez-González, & Castillo, 2004; Park & Beu-
chat, 1999). These studies have demonstrated that the use of
water is insuYcient to eliminate these pathogens on fresh-
cut fruits and vegetables. Therefore, the use of eVective san-
itizers is necessary to inactivate pathogens.
Although Cl is an eVective sanitizer for fruits and vegeta-
bles, its antimicrobial activity is diminished by organic mat-
ter in the wash water (Beuchat, 1998). Since water wash is
often recycled, high organic matter content reduces the
activity of Cl (Brackett, 1992) and increases the likelihood
for contamination of fresh-cut produce. For example, Garg,
Churey, and Splittstoesser (1990) found that maintaining
the desired level of free available Cl in wash solution in the
processing plant is diYcult due to the accumulation of
organic material. In this study, the eYcacy of Cl was tested
in tap water (no recycling) and in process water (recycled
tap water). The eVectiveness of Cl treatment was signiW-
cantly higher than PA, only when it was used in tap water
Fig. 1. EYcacy of sanitizers on the reduction of E. coli O157:H7 (a, b), Salmonella (c, d) and L. monocytogenes (e, f) populations from artiWcially inoculated
shredded carrots. Bars represent the standard errors of the mean resulting from triplicate experiments. The limit of detection was 1.0 log CFU/g of shred-
ded carrots.
with reductions of 2–3 log CFU/g, but lower than ASC.
These results are in agreement with those reported previ-
ously in diVerent produce at a laboratory scale (González
et al., 2004; Park & Beuchat, 1999; Taormina & Beuchat,
1999a, 1999b). However, the eYcacy of the Cl was signiW-
cantly reduced when process water was used (Fig. 1(b, d
and f)).
In general the PA treatment was the least eVective in
reducing the pathogens followed by Cl and ASC treat-
ments. PA was more eVective than chlorine under process
water conditions with its most signiWcant eVect observed on
the reduction of Salmonella (2.1 log CFU/g); however, PA
only reduced E. coli O157:H7 and L. monocytogenes by
1.24 and 0.83 log CFU/g, respectively, under tap and pro-
cess water conditions. Our results agree with those of Park
and Beuchat (1999), who showed that treatment with 40 or
80 ppm PA reduced the population of Salmonella by
2.6 log CFU/g. However, the reduction of E. coli O157:H7
observed in our study was less than those reported by oth-
ers (González et al., 2004; Wright, Summer, Hackney, Pier-
son, & Zoecklein, 2000).
ASC was the most eVective treatment in reducing patho-
gens at all concentrations evaluated. This sanitizer reduced
the three pathogen populations to undetectable levels (with
10 CFU/g detection limit), achieving reductions of 4.81, 4.84
and 2.5 log CFU/g of E. coli, Salmonella and L. monocytog-
enes, respectively, under both water conditions. The results
are similar to those reported by other researchers (Gon-
zález et al., 2004; Park & Beuchat, 1999). In addition, an
enrichment step was performed to recover bacteria suble-
thally injured by the treatments. This enrichment was
applied to determine whether the sanitizers were able to
eliminate the pathogens or merely to suppress their growth
(Fig. 2). As shown in Fig. 2, cells viable of E. coli O157:H7
and Listeria monocytogenes were recovered from the sam-
ples washed with ASC, which provided a bacteriostatic
eVect on these pathogens. However, no viable cells of Sal-
monella were recovered at concentrations of 250 and
500 ppm, producing a bactericidal eVect. González et al.
(2004) observed that ASC treatment at 1000 ppm reduced
the E. coli O157:H7 population in a similar manner to the
reduction of Salmonella achieved with 250 and 500 ppm
Fig. 2. Recovery of E. coli O157:H7 (a, b), Salmonella (c, d) and L. monocytogenes (e, f) from artiWcially inoculated shredded carrots after treatment with
sanitizers and 24 h of enrichment. Bars represent the standard errors of the mean from triplicate experiments. The limit of detection was 1.0 log CFU/g of
shredded carrots.
ASC, in this study. However, we found that 1000 ppm ASC
aVected the overall quality of shredded carrots (Ruiz-Cruz,
Luo, Gonzalez, Tao, & González-Aguilar, 2006). These
results conWrmed that the eVectiveness of ASC to maintain
quality and reduce pathogen counts is inXuenced by the
concentration of the sanitizer and the contact time.
The antibacterial capacity of ASC could be attributed to
the degradation of chlorous acid, which forms a calculable
fraction of the chlorite ion in solution. The level of
degraded chlorous acid depends on the hydrogen ion con-
centration (i.e. pH) of the mixed solution of chlorite and
acid (FSANZ, 2003). Moreover, the low pH environment
created by organic acids (i.e. citric acid) and the highly oxi-
dative intermediates of ASC which are broad spectrum ger-
micidal agents, aVected antibacterial capacity (Paris et al.,
2003). Recent attention has been focused on the ability of
bacterial pathogens such as E. coli O157:H7, Salmonella,
and L. monocytogenes to develop acid tolerance in response
to acidic conditions (Davis, Coote, & O’Byme, 1996; Jor-
dan, Oxford, & O’Byrne, 1999; Marques, Worcman-Barn-
inka, Lannes, & Landgraf, 2001; Rowbury, 1995);
therefore, the ability of the pathogens to adapt to adverse
environments present in food is an interesting area that
requires more investigation.
The contact time of shredded carrots with ASC appears
to inXuence the bacterial count reduction. Therefore, lower
concentrations for longer contact times are recommended
in order to completely eliminate pathogens without aVect-
ing the quality of produce. However, the wash system, the
type of produce and its susceptibility to tissue damage and
to microbial growth must be considered before practical
recommendations are proposed for sanitizer concentrations
to control microbial loads in the fresh-cut industry.
The microbial counts of the pathogens analyzed in this
study were reduced during the storage period, following a
similar pattern in all treatments used, for both tap and pro-
cessed water (Fig. 1). ASC treatment maintained the lowest
values of pathogens during the whole storage period. At the
end of the storage, untreated shredded carrots and those
treated with Cl and PA had similar microbial counts
(ranged 1.5–2.3 log CFU/g) of E. coli O157: H7 and Salmo-
nella (5 and 7 days, respectively). Besides bacterial death as
a result of drying over the storage period and possible over-
growth by the microXora of carrots, cell injury also may
have prevented recovery of pathogenic survivors, especially
on selective medium. Several studies have demonstrated an
antagonism of L. monocytogenes by microorganisms native
to plant surfaces. Ukuku, Fett, and Sapers (2004) described
this eVect of antagonism of native microXora towards L.
monocytogenes on fresh-cut and whole melons.
L. monocytogenes population declined rapidly with
storage and was not detected after 2 days at 5 °C. Previous
studies reported that carrots contain phenolic compounds
known as “anti Listeria” that are responsible for the
growth inhibition of L monocytogenes (Babic, Nguyen-the,
Amiot, & Aubert, 1994; Nguyen-the & Lund, 1991, 1992).
This eVect was conWrmed in raw carrots stored at 5 or at
15 °C contaminated with other microorganisms but not
with L. monocytogenes. Beuchat and Brackett (1990) found
that L. monocytogenes do not grow well on carrots unless
they received a cooking treatment. Moreover, Beuchat and
Doyle (1995) reported that lettuce treated with a solution
containing 20–50% of carrot juice caused a signiWcant
reduction of L monocytogenes, during storage at 5 °C. Far-
ber, Wang, Cai, and Zhang (1998) reported the survival of
L. monocytogenes in vegetables as caesar salad, carrot,
coleslaw, onion and squash. The population of L monocyt-
Fig. 3. The eVect of sanitizer treatment on the reduction of total aerobic counts. Bars represent the standard errors of the mean resulting from triplicate
experiments. The limit of detection was 1.0 log CFU/g of shredded carrots.
ogenes remained constant on these fresh-cut vegetables
stored at 4 °C for 9 days, except for carrots, where the pop-
ulation decreased to levels of 2 log CFU/g over a 9 day
storage period. In other study, an inhibitory or killing
eVect of carrots tissue Xuid on the E. coli O157:H7 popula-
tion was also suggested Abdul-Raouf, Beuchat, and
Ammar (1993).
3.2. EYcacy on total aerobic count reduction
The initial population of total aerobic bacteria from
untreated shredded carrots was 4.28 log CFU/g (Fig. 3).
These results are consistent with the Wndings previously
reported (González et al., 2004; Sinigaglia, Albenzio, &
Corbo, 1999). Washing shredded carrots with sanitizers
caused a signiWcant reduction (P 6 0.05) of total aerobic
count on day 0 when compared to the untreated carrots.
However, the eYcacy of Cl treatment under process water
conditions was aVected by the presence of organic matter
and only reduced 0.18 log CFU/g. Treatment with ASC 100,
250, 500 ppm caused a 0.86, 1.46 and 2.3 log CFU/g reduc-
tion under both water conditions, respectively. The eYcacy
of ASC and PA was not aVected by the organic matter. Park
and Beuchat (1999) reported similar results on total aerobic
bacteria reduction for ASC treatment (850 and 1200 ppm)
of asparagus, cantaloupes and honeydew melons. Likewise,
González et al. (2004) reported that shredded carrots treated
with 1000 ppm of ASC caused at least a 3.27 log CFU/g
reduction of total aerobic count. However, the concentra-
tions used by these authors were not able to maintain the
quality of shredded carrots (shelf-life 4–6 days at 5 °C).
Although the aerobic bacteria grew rapidly during cold
storage, all concentrations of ASC treated samples main-
tained the lowest bacterial counts throughout the storage
period. Our earlier studies showed that the concentrations
of ASC used in this study maintain the quality of shredded
carrots for 17 days at 5 °C (Ruiz-Cruz et al., 2006).
4. Conclusion
The results obtained in the present study indicate that
ASC at concentrations of 250 and 500 ppm is an alternative
to Cl for sanitization of shredded carrots. ASC has the
advantage of being more stable and preserve its eYcacy in
the presence of organic matter. Also, it caused a higher
reduction of E. coli O157:H7, Salmonella and L. monocyt-
ogenes. However, further studies are needed to determine
whether ASC might have more lethal eVects when lower
levels of bacteria are present on produce.
Acknowledgements
The authors gratefully acknowledge the technical assis-
tance of Rosalba Pérez-Morales, Alfonso Garcia-Galaz
and Monica Villegas-Ochoa and Centro de Investigación
en Alimentación y Desarrollo (CIAD, AC), Project
CYTED XI.22 “Desarrollo de Tecnologias para la Conser-
vación de Productos Vegetales Frescos Cortados”, the
Mexican National Council of Science and Technology
(CONACYT) and USDA, for their Wnancial support. The
in-kind support of ASC from Alcide Corp. (Redmond,
WA) is also greatly appreciated.
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