APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2008, p. 1240–1242
Vol. 74, No. 4
Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes?
Iris Valentin-Bon, Andrew Jacobson, Steven R. Monday, and Peter C. H. Feng*
Division of Microbiology, United States Food and Drug Administration, College Park, Maryland 20740
Received 4 October 2007/Accepted 13 December 2007
Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log10CFU/g
and a broad range of <4 to 8.3 log10CFU/g. Most probable numbers (MPN) of >11,000 /g coliforms were found
in 55 samples, and generic Escherichia coli bacteria were detected in 16 samples, but no E. coli count exceeded
Increases in the worldwide consumption of ready-to-eat
(RTE) produce have resulted in increases in food-borne illness
associated with these products (11). In the United States in
2006, a multistate outbreak of Escherichia coli O157:H7 was
traced to bagged spinach (1) and, a few months later, another
outbreak implicated bagged lettuce in fast-food restaurants,
thus raising concerns about the microbiological quality of RTE
The quality of RTE leafy greens has recently been surveyed
in the United Kingdom (8, 10), Spain (12), and Brazil (3), but
there are no recent data on the microbial quality of bagged
leafy greens in the United States. In this study, we tested 100
bags of RTE leafy greens purchased in 2007 from stores in the
Washington, DC, metropolitan area for total, coliform, and
generic E. coli bacterial counts to assess their microbiological
quality. Although recent outbreaks had implicated specific
brands and products, we opted to do a broad survey rather
than to focus on specific produce types or brands. Hence, the
100 samples, many labeled “triple-washed” or “ready to eat,”
consisted of 45 spinach and 55 lettuce mixes (12 different
varieties), including organic products. The samples included
five brands, of which 20% were local store brands that may
only have regional distribution, but the rest were national
brands that are widely available. The samples were tested using
the U.S. FDA Bacteriological Analytical Manual (2; http://www
.cfsan.fda.gov/?ebam/bam-4.html) methods. Briefly, 50 g of
the product was blended with 450 ml of buffered peptone
water, from which 1:10 serial dilutions were made. Total bac-
terial counts were done by standard plate count, where 0.1 ml
of each dilution was plated in duplicate on Trypticase soy agar
(BD Diagnostics, Sparks, MD). The coliform and E. coli bac-
terial counts were done with the most probable number (MPN)
method with ColiComplete discs (Biocontrol, Bellevue, WA)
(AOAC official method 992.30). The discs contain X-Gal (5-
bromo-4-chloro-3-indolyl-?-D-galactopyranoside), which in co-
liforms are cleaved by ?-galactosidase to yield a blue product.
The discs also contain 4-methylumbelliferone-?-D-glucu-
ronide, which in E. coli bacteria is cleaved by ?-glucuronidase
to yield blue fluorescence (365-nm UV). After 48 h at 37°C, a
combination of coliform-positive (blue) and E. coli-positive
(fluorescence) tubes was used to estimate the levels of each
indicator from the MPN table.
The mean total count of the samples examined was around
7.0 log10CFU/g, with a broad range of ?4 to 8.3 log10CFU/g
(Table 1). Our data are consistent with the results of a 1998
U.S. study of 52 bagged salads which found a mean total
bacterial count of 7.0 log10CFU/g (4). Similarly, a study from
Spain showed that the total bacterial counts for 140 RTE
lettuce samples at 16 university restaurants ranged from 3.01 to
7.81 log10CFU/g (12), while an analysis of 133 RTE leafy
salads in Brazil found that 51% had counts that were ?6.0
log10CFU/g (3). Interestingly, we saw a large variation in
counts not only among the samples, but also within same-brand
products that had identical “use by” dates and were tested on
the same day. For example, five such seemingly “identical”
romaine and spinach samples had bacterial counts that ranged
from 5.3 to 7.0 log10CFU/g and ?4.0 to 7.4 log10CFU/g,
respectively. Also, except for 23 samples that came in plastic
tubs, condensation moisture was observed at the bottom of
many bags. Since water is essential for microbial growth, con-
densation moisture in the bags would be expected to promote
microbial growth; hence, 80% of the bags were opened and
sampled from the bottom. The top-sampled bags had a mean
total count of 6.96 log10CFU/g, with a range of 4.8 to 7.86 log10
CFU/g. In contrast, the bottom-sampled bags had a mean total
count of 7.65 log10CFU/g and a range of ?4 to 8.3 log10
CFU/g. Although bottom sampling yielded higher counts,
these findings are preliminary, and additional studies, in which
the same bags are sampled from both the top and bottom, are
needed to fully establish the effects of condensation moisture
on bacterial counts.
The coliform counts of the samples we tested ranged from
* Corresponding author. Mailing address: HFS-711, FDA, 5100
Paint Branch Parkway, College Park, MD 20740. Phone: (301) 436-
1650. Fax: (301) 436-2644. E-mail: firstname.lastname@example.org.
?Published ahead of print on 21 December 2007.
TABLE 1. Total microbiological counts of 100 bagged spinach and
Product and type
?0.47 to ?4.0 log10MPN/g (Table 2), similar to the range of
?0.47 to 3.38 log10MPN/g reported for RTE lettuce in Spain
(12). We were unable to obtain a mean coliform count, as
55/100 samples exceeded our counting limit of ?11,000
MPN/g. Generic E. coli bacteria were detected in 10 lettuce
and 6 spinach samples (Table 2). The highest E. coli level
found was 9.2 MPN/g, but 12/16 samples had counts of 3.6
MPN/g or less. Most of the E. coli-positive samples (11/16) had
total counts of ?6.0 log10CFU/g, and 14/16 samples had co-
liform counts of ?11,000 MPN/g. There are no E. coli limits for
bagged produce in the United States, but guidelines and limits
exist in other countries. The Brazilian standard for salads that
are minimally processed before consumption has a fecal coli-
form limit of 100 CFU/g, and analysis of 133 salad samples
showed that 73% exceed this limit (3). The guidelines of the
United Kingdom Public Health Laboratory Service for RTE
foods, including bagged produce, have set E. coli count limits
of ?20 CFU/g as satisfactory, 20 to ?100 CFU/g as acceptable,
and ?100 CFU/g as unsatisfactory (9). In two large surveys in
the United Kingdom, 3,200 organic and 3,852 conventional
RTE salads were tested and it was found that 0.5% exceeded
the 100-CFU/g E. coli limit and were unsatisfactory (8, 9).
Although we found 16% of our samples to have E. coli bacte-
ria, none exceeded 10 MPN/g, but whether low E. coli counts
are prevalent in other bagged leafy greens in the United States
remains to be determined.
These surveys show that the microbial flora and content of
RTE produce are highly variable and complex. One study (6)
looked at the microbiological quality of fresh, uncut produce
from production through packing and showed that indicator
levels in mustard greens and spinach remained fairly constant
but that for cilantro, parsley, and, especially, cantaloupe, the
indicator levels actually increased during packing, hence show-
ing that the microbial load can vary by processing but also
depends on the produce type. The processing for whole pro-
duce is probably distinct from that for cut, bagged produce, so
it is uncertain if the high counts or the variations in counts we
observed in the bagged leafy greens are due to produce type
variations or to microbial growth, if any, during processing.
Our finding that there are wide ranges, as well as large varia-
tions, in counts among samples and even among seemingly
“identical” samples, coupled with the rare observation of a
spinach leaf with visible filth (Fig. 1), suggests that differences
or inconsistencies in processing parameters may also have an
effect on microbial load. Lastly, the finding that bagged leafy
greens (3, 4; this study) can have ranges of total (4 to 7 log10
CFU/g) and coliform (1 to 4 log10MPN/g) bacterial counts
similar to those for produce sampled in the field (5, 7) also
suggests that, microbiologically, these products may be very
difficult to clean and process.
We thank Deanne Deer and John Callahan for their assistance in
procuring the produce samples used in this study.
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FIG. 1. Photograph of a spinach leaf obtained from a bagged
TABLE 2. Coliform and E. coli bacterial contents of 100 bagged spinach and lettuce mixes
Product and type
(no. of samples)
Coliform bacteria (MPN/g)E. coli bacteria
No. of positive samplesRange (MPN/g)
VOL. 74, 2008MICROBIOLOGICAL QUALITY OF BAGGED LEAFY GREENS 1241
of open ready-to-eat salad vegetables: effectiveness of food hygiene training Download full-text
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1242VALENTIN-BON ET AL.APPL. ENVIRON. MICROBIOL.