Transfer of Escherichia Coli to Lemons Slices and Ice during Handling

Abstract

The objective of this study was to determine the transfer and survival of bacteria during the handling and storage of lemons and transfer of bacteria during handling of ice. Ice and lemon slices are handled and stored in public eating places and used in beverages. During handling and storage the contamination and growth of bacteria may occur leading to the spread of disease. To fulfill the objective, hands were inoculated with Escherichia coli prior to handling of wet and dry whole lemons and in a separate experiment, ice cubes were handled. E. coli transferred to whole lemons or ice after handling were determined. The CFU per lemon and percentage of E. coli transferred were greater for wet lemons -6123 cfu and 4.62% compared to 469 cfu and .2% for dry lemons. The second experiment found from 2 to 67% of the bacteria on hands were transferred to ice by hands and from 30 to 83% of the bacteria on scoops were transferred to ice. In a third experiment, lemons were inoculated with E. coli, then sliced and stored at 4 or 22C and tested at 0, 4 and 24 hr. Lemons stored at room temperature (22°C) had an increase in E. coli population after 24 hour while those stored under refrigeration had a decrease even though bacteria did survive on lemons in either case.

Full text

Journal of Food Research; Vol. 6, No. 4; 2017
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
111
Transfer of Escherichia Coli to Lemons Slices and Ice during
Handling
Paul Dawson1, Inyee Han1, Ahmet Buyukyavuz1, Wesam Aljeddawi1, Rose Martinez-Dawson2, Rachel Downs1,
Delaney Riggs1, Carrrie Mattox1, Alejandro Kurtz1, Mary MacInnis1, Jacob Freeland1, Seth Garrison1, Taylor
May1, James McClary1, Frank Monitto1, Trinh Nguyen1, Kelly Polte1, Matthew Suffern1, Zachary Tanner1,
Alana Thurmond1 & Virginia Ellis1
1Department of Food, Nutrition and Packaging Sciences, 2Department of Mathematical Sciences, Clemson
University, Clemson, SC 29634, USA
Correspondence: Paul Dawson, Depertment of Food, Nutrition and Packaging Sciences, Clemson University,
Clemson, SC 29634, USA. Tel: 1-864-656-1138. E-mail: pdawson@clemson.edu
Received: May 1, 2017 Accepted: June 9, 2017 Online Published: June 28, 2017
doi:10.5539/jfr.v6n4p111 URL: https://doi.org/10.5539/jfr.v6n4p111
Abstract
The objective of this study was to determine the transfer and survival of bacteria during the handling and storage
of lemons and transfer of bacteria during handling of ice. Ice and lemon slices are handled and stored in public
eating places and used in beverages. During handling and storage the contamination and growth of bacteria may
occur leading to the spread of disease. To fulfill the objective, hands were inoculated with Escherichia coli prior
to handling of wet and dry whole lemons and in a separate experiment, ice cubes were handled. E. coli
transferred to whole lemons or ice after handling were determined. The CFU per lemon and percentage of E. coli
transferred were greater for wet lemons -6123 cfu and 4.62% compared to 469 cfu and .2% for dry lemons. The
second experiment found from 2 to 67% of the bacteria on hands were transferred to ice by hands and from 30 to
83% of the bacteria on scoops were transferred to ice. In a third experiment, lemons were inoculated with E. coli,
then sliced and stored at 4 or 22C and tested at 0, 4 and 24 hr. Lemons stored at room temperature (22°C) had an
increase in E. coli population after 24 hour while those stored under refrigeration had a decrease even though
bacteria did survive on lemons in either case.
Keywords: lemons, lemon slices, ice, E. coli, handling, storage
1. Introduction
The non-alcoholic beverage market is an $841 billion industry (Bailey, 2014) with the annual alcoholic beverage
market at $494 billion (beer), $319 billion (wine) and $637 billion (spirits) (Marketrealist, 2014). Beverages are
often prepared with the addition of ice and cut fruit slices such as lemons, limes and oranges. These drink items
are also often handled by a server or the individual consuming the drink which offers an opportunity for
contamination which is a possible source of contamination leading to foodborne illness.
1.1 Foodborne Illness
Thirty-one major pathogens cause 9.4 million cases of foodborne illness and about 2,612 deaths from tainted
food annually (CDC, 2011). The Economic Research Service reports that foodborne illness costs $6.9 billion in
medical expenses, lost productivity and deaths (USDA, 2014). Hand cleanliness or lack thereof, plays a major
role in transmission of infectious disease in various public sectors including the food industry (Jumma, 2005).
1.2 Ice
The US Food and Drug Administration defines ice as food (FDA, 2010) and the World Health Organization has
stated that ice coming in contact with food should have the same level of safety and quality as drinking water
(WHO, 1997). Ice used for human consumption can be contaminated with pathogenic organisms and be a vector
for spreading foodborne illness (Falcao, Dias, Correa & Falcao, 2002; Gerokomou et al., 2011). In 1987, the
Centers for Disease Control reported a 4-state outbreak of Norwalk virus in Pennsylvania, Delaware and New
Jersey from contaminated ice estimated to have affected more than 5,000 people (Levine, Stephenson & Craun,
1990). Contaminated ice was also a prominent transmission vector for spreading the 1991 cholera epidemic in Peru
causing 7922 illnesses, 17 deaths and also expanding throughout Latin America (Ries et al., 1992). More recently,

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diarrheagenic E. coli has been found in commercial ice produced in Brazil (Falcao, Falcao & Gomez, 2004). In the
past, pathogens have been detected in ice from ice making machines (Stout, Yu & Muraca, 1985; Panwalker &
Fuhse, 1986; Laussucq, Baltch, Smith, Smithwick, Davis et al., 1988; Wilson, Hogg & Barr, 1997). In a survey of
over 3,500 samples of ice used to cool drinks, Nichols, Gillespie & Louvois, (2000) found that 9% contained
coliforms at greater than 100 cfu/100 ml and 11% had total aerobes at greater than 1000 cfu/ml. Ice produced at
retail outlets in Nigeria were contaminated with more than 1000 cfu/ml and isolates displayed 100, 67 and 87%
resistance to Ampicillin, Erythromycin and Tetracycline, respectively (Lateef, Oloke, Guegium-Kana & Pacheco,
2006).
1.3 Lemons
Food establishments often place lemons in open containers at room temperature throughout the day for consumer
access allowing consumers and food service workers to handle lemons for cutting and when serving slices with
beverages. Lemons naturally contain bacteria that provide a symbiotic relationship with the fruit (Gardner,
Feldman, & Zablotowicz, 1982). Similarly, human hands naturally contain bacteria such as Micrococcus luteus
and Serratia. rubidea that transfer from humans to other objects. Transfer is particularly high when fingers
contact lips (Rusin, Maxwell, & Gerba, 2002). Martinez-Gonzalez et al. (2003) found that orange surfaces
inoculated with 2.3, 3.6 and 4.4 log10CFU/cm2 Salmonella Typhimurium, E. coli and Listeria monocytogenes,
respectively resulted in 1.0, 2.3 and 2.7 log10CFU/ml for these organisms in orange juice prepared from the
inoculated oranges. Cut lemons are located at self-service drink stations for consumers to handle which increases
the number of people touching lemon slices, many who are not food service workers and not subject to hand
washing regulations. Lemons are held without refrigeration and to reduce waste, leftover lemons are sometime
placed under refrigeration overnight for use the next day. Thus lemon slices are exposed to numerous
opportunities for contamination and held unrefrigerated to allow microbial growth prior to use by consumers.
1.4 Hand Sanitation and Cross Contamination
Cross contamination in food service may play an important role in foodborne illness (Fendler, Dolan & Williams,
1998). During food preparation, bacteria on hands can be transferred to raw foods from hands and indirectly
from other surfaces (Montville, Chen & Schaffner, 2002). Hands can also be a source for contamination from
food workers that may be ill by not have overt symptoms who shed pathogens (Rocourt & Cossart, 1997; Rose
& Slifko, 1999). Numerous studies have examined the transfer of bacteria to food from food contact surfaces
including stainless steel (Kusumaningrum, Riboldi, Hazelberger & Beumer, 2003; Moore, Sheldon & Jaykus,
2003; Rodriguez & McLandsborough, 2007; Kesiken, Todd & Ryser, 2008), fabrics (Marples & Towers, 1979;
Sattar et al. , 2001; Scott & Bloomfield, 1990), gloves (Legg, Khela, Madie, Fenwick, Quynh & Hedderley, 1999;
Heal et al., 2003; Montville et al., 2001; Blom, Gozzard, Heal, Bowker, & Estela, 2002; Gill & Jones, 2002;
Mackintosh & Hoffman, 1984; Patrick, Findon, & Miller, 1997; Scott & Bloomfield, 1990; Shale, Jacoby &
Plaatjies, 2006) and hands (Scott & Bloomfield, 1990; Legg et al., 1999; Merry, Miller, Findon, Webster & Neff,
2001). A scoop, hands or other utensil is often used to deliver ice to a beverage offering the opportunity for cross
contamination. The objectives of this study were to determine 1) to what extent bacteria is transferred to lemons
when handled with contaminated hands; 2) the degree of bacterial transfer to ice when handled with
contaminated hands or scoops; and 3) if bacterial numbers will increase during the storage of contaminated
lemons.
2. Methods
2.1 Bacterial Inoculum
An Escherichia coli ampicillin-resistant strain with a fluorescent gene was used for the bacterial transfer and
survival studies. A non-pathogenic E. coli strain JM109 was labeled with jellyfish green fluorescent protein
according to the following protocol as described previously (Jiang et al., 2002). The competent bacterial cells
were electroporated in a Gene Pulser II (Bio-Rad) with plasmid vector pGFPuv (ClonTech, Palo Alto, CA).
Transformants were selected from isolated colonies grown on Luria-Bertani agar (LB) plates containing 100 g
ampicillin/mL. The resulting ampicillin-resistant transformants emitted bright green fluorescence under UV light.
The stability of GFP label in the E. coli strain was determined by streaking on trypticase soy agar (TSA) plates
containing 100 g ampicillin/mL for several generations. The E. coli JM 109 culture was held in a −80˚C freezer
in vials containing tryptic soy broth (TSB) (Becto™, Becton Dickinson and Company Sparks, MD, USA)
supplemented with 20% (v/v) glycerol (Sigma, St. Louis, MO, USA). The frozen vial was thawed at room
temperature prior to culturing. From this thawed vial, 0.1 mL of culture was transferred to 10 mL TSB
containing 0.5% ampicillin (Sigma, St. Louis, MO, USA) in 2 loosely screw-capped tubes and then the tubes
were incubated for 16 - 18 h at 37˚C with vigorous shaking (Thermolyne Maxi-Mix III type 65,800,

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Barnstead/Thermolyne, Dubuque, IA). The second transfer was prepared from this first transfer culture by
adding 0.1 mL from the first transfer tube to another fresh 10 mL TSB (DIFCO) with 0.5% ampicillin (Sigma),
and again incubated for 16 - 18 h at 37˚C with shaking. After incubation, the cells were harvested by
centrifugation at 3000 rpm (1200 g) (IEC HN-SII Centrifuge, International Equipment CO., Inc., Needham
Heights, MA, USA), then the pellet re-suspended in 10 mL of sterile peptone solution (0.1%) (Bacto peptone,
Becton Dickinson) to obtain a population of approximately 6-7 log CFU/mL. Initial cell populations were
verified by enumeration of the cells following surface plating in TSA containing 0.5% ampicillin (DIFCO™,
Becton Dickinson and company Sparks, MD, USA) and incubation at 37˚C for 24 h.
2.2 Experiment 1: E. Coli Transfer from Hands to Whole Lemons
2.2.1 E. Coli on Hands
Each subject washed their hands with warm water and soap, allowed their hands to air dry, and then 1 mL of the
E. coli inoculum was deposited in the center of their dominate hand. The E. coli was applied by rubbing hands
together for 30 sec, and then hands were allowed to air dry for 30 sec. To enumerate bacteria on subject’s hands,
both hands (separately) were placed into a sterile stomacher bag with 20 mL of sterile 0.1% peptone and rinsed
for 30 seconds, covering all fingers, palm, and back of the hand. Next, 1 mL of the peptone solution was
removed from the stomacher bag, placed into 9 mL of sterile 0.1% peptone and serially diluted. A 0.1 ml aliquot
from sample dilutions were pipetted and spread onto TSA plates containing 100g ampicillin/mL. Plates were
held for 5-10 minutes and were then inverted and placed in an incubator at 37oC for 24 hours. The next day the
plates were inspected under UV light and plates with 25 to 250 CFU/plate were counted and then multiplied by
the dilution number then converted to CFU/hand and log CFU/hand based on serial dilutions.
2.2.2 E. Coli Transferred to Lemons
Four treatments were employed to determine bacterial transfer from hands: 1. Un-inoculated hands handling dry
lemons, 2. inoculated hands handling dry lemons, 3. Un-inoculated hands handling wetted lemons, and 4.
Inoculated hands handling wetted lemons. Each subject washed their hands then handled a lemon for 30 seconds
by rolling the lemon between hands.
For the inoculated treatments (2 and 4) the procedure was repeated as described for un-inoculated hands only
instead, hands were inoculated as described for section 2.2.1, then lemons were handled for 30 seconds. Lemons
handled by inoculated or un-inoculated hands were placed into separate filter stomacher bags, each with 20 mL
of sterile 0.1% peptone solution. The lemon and peptone were mixed for 30 sec in the bag. Then 1 mL samples
of the liquid from the bags were taken in duplicate, serially diluted as previously described then plated on TSA.
Samples were incubated and counted as described for the hand sample.
Serial dilutions were then prepared, plated, and spread in duplicate and plates were incubated and counted as
previously described. Bacteria were counted 24 hours after plating by identifying colonies under a UV light then
converted to CFU/lemon and log CFU/lemon as described for hands (2.3.1).
The % transfer of E. coli from hands to lemons was calculated using (1):
% transfer = (1)
2.3 Experiment 2: Transfer of E. Coli to Ice from Hands and Metal Scoops
The bacterial inoculum was prepared for the ice transfer experiments in the same manner as described under 2.1
for lemons.
2.3.1 Inoculation of E. Coli on Hands and Scoops
Each subject will wash their hands with warm water and soap, allow their hands to air dry, and then 1 mL of the
E. coli inoculum will be deposited in the center of their dominate hand. The E. coli will be applied by rubbing
hands together for 30 sec, and then hands will be allowed to air dry for 30 sec. For scoops, a sanitized scoop was
inoculated by placing 1 mL of E. coli inoculum in the center of the scoop then spread across the scoop surface
using a sterile glass rod then allowed to dry for 30 seconds.
2.3.2 E. Coli Transferred To Ice from Scoops and Hands
Four treatments were employed to determine bacterial transfer from hands to ice: 1. Un-inoculated hands
handling ice, 2. inoculated hands handling ice, 3. Un-inoculated scoop handling ice, and 4. Inoculated scoops
CFU recovered from lemons
CFU recovered from hands +CFU recovered from lem
X 100

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handling ice. Each subject washed their hands then picked up a handful of ice, the immediately placed the ice in
a filter stomacher bag containing 20 mL of sterile 0.1% peptone solution. This procedure was repeated with
inoculated hands and both inoculated and un-inoculated scoops. The ice and peptone water were mixed for 30
sec in the bag. To enumerate bacteria on subject’s hand the dominant hand used to pick up ice were placed into a
sterile stomacher bag with 20 mL of sterile 0.1% peptone and rinsed for 30 seconds, covering all fingers, palm,
and back of the hand. For inoculated scoops, 5 pieces of ice were placed into the scoop and gently moved back
and forth for 5 seconds then the ice was immediately placed into a filter stomacher bag containing 20 ml of
sterile 0.1% peptone solution.
Next, 1 mL of the peptone solution was removed from each stomacher bag, placed into 9 mL of sterile
0.1% peptone and serially diluted. Nine ml test tubes of sterile peptone solution (0.1%) were used for serial
dilution of samples. 0.1 ml from sample dilutions were pipetted and spread onto TSA plates containing 100g
ampicillin/mL. Plates were held for 5-10 minutes and will be then inverted and placed in an incubator at 37oC for
24 hours. The next day the plates were inspected under UV light and appropriate petri dishes will be chosen for
counting. Plates with a number of colonies ranging from 25 to 250 CFU/plate were counted and converted to
CFU/ml based on the dilution. Plates were examined under the UV light and only the fluorescent bacteria
counted. Bacterial counts were converted to CFU/hand or scoop and log CFU/hand or scoop based on the
amount of rinse solution used. Percentage of E. coli transferred from hands to ice will be calculated using (1):
% transfer = (1)
Another experiment (2-1) was conducted to determine the transfer of E. coli from metal scoops to ice at 4
different times after inoculation (0, 1hr, 1.5hr and 2hr) and for three sequential times using the same scoop.
Scoops were inoculated with the E. coli ampicillin-resistant strain with a fluorescent gene as described in section
2.3.1. prior to exposure to ice. Bacteria were enumerated using the method that was previously described in
section 2.2.2. at each of the storage times and for each of the sequential exposure to ice.
2.4 Experiment 3: Survival of E. Coli on Sliced Lemons
Survival of E. coli on lemon slices was tested at three different time intervals (0, 4, and 24hr) and at refrigerated
(4±2°C) and room (21±2°C) temperatures. Lemons were inoculated with the E. coli ampicillin-resistant strain
with a fluorescent gene as described in section 2.1 by placing each lemon in sterile bag containing 20 ml of a ~6
log CFU/mL of E. coli solution which was shaken for 30 sec then the lemon removed and allowed to dry for 5
min. Lemons were then sliced into quarters. One set of slices were enumerated for E. coli after 10 min while
other lemons were stored for 4 and 24hr at room or refrigerated temperature. Bacteria were enumerated using the
method that was previously described in section 2.2.1. at each of the storage times.
2.5 Statistical Analysis
All three experiments were conducted as completely randomized designs and simple mean, standard deviation,
minimum and maximum values were determined for treatment using the Statistical Analysis System (SAS, 2014).
Experiment 1 (bacterial transfer from hands to lemons) had 11-13 subjected per each of 3 replications with each
observation duplicated for a total of 70 observations per treatment (wet or dry lemons). Experiment 2 (bacterial
transfer from hands or scoops to ice) had 11 subjects per each of 3 replications with each observations duplicated
for a total of 66 observations per treatment (hand or scoop). In a separate experiment 2.1 (bacterial transfer from
3 sequential scoops held for up to 2 hours) was conducted using three replications having 2 variables of (1) 1-3
scoops in sequence and (2) holding time (0, 1, 1.5 and 3 hours). Two scoops were utilized for each of 3
replications and each scoop was analyzed in duplicate yielding 12 observations per treatment. Experiment 3
(bacterial survival on stored lemons) had 2 variables of (1) storage temperature (room or refrigerated) and (2)
storage time (0, 4 and 24 hours) with duplicates for each of 5 replications for a total of 10 observations for each
combined storage temperature and storage time treatment combination. Treatments were subjected to an analysis
of variance, and since the treatments had a significant effect (p > 0.05), were separated using the pdiff command
of SAS (2014).
3. Results and Discussion
3.1 Experiment 1: E. Coli Transfer from Hands to Whole Lemons
No fluorescent E. coli were recovered from lemons handled with un-inoculated hands. One interesting finding
was that all (100%) of the lemons that were wet prior to handling with inoculated hands showed bacterial
CFU recovered from ice
CFU recovered from hands (scoops) + CFU recovered fro
X

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transfer while only 30.3% of lemons that were dry prior to handling with inoculated hands had bacteria detected
after handling. The average CFU per lemon when wet was 6123 with an average transfer of 4.82% (Table 1).
Conversely, the dry lemons had an average CFU of 469 (Table 1) and a transfer of 0.2%.
Table 1. Mean, median, range of the population and % transfer of bacteria on lemons from hands inoculated with
E. coli
CFU/lemon
LogCFU/lemon
%CFU transferred
Dry lemons
Wet lemons
Dry lemons
Wet lemons
Dry lemons
Wet lemons
Mean
469b (1502)
6912a
2.67b (3.2)
3.84a
0.20b (0.65)
4.86a
Stand Error
223
1644
0.16
0.09
0.07
0.8
Median
0
2180
0
3.34
0
2.48
Maximum
11720
62400
4.07
4.8
2.57
29.52
Minimum
0
40
0
1.60
0
0.02
a,bmeans with different superscripts are significantly different (p≤0.05). n=70
(values in parenthesis are calculated from only the 30% having transfer)
Patrick, Findon & Miller (1997) also found the wetness of hands (degree of drying) was directly related to the
percentage of bacteria transferred to food. In the current study, the transfer of bacteria was greater when lemons
were wet. Perez-Rodriquez et al. (2008) summarized the modelling of bacterial transfer between recipient and
donor surfaces including intrinsic factors of bacterial hydrophilicity/hydrophobicity and biofilm development,
and environmental factors including contact time, pressure, surface roughness and surface moisture. Based
previously reviewed research, Perez-Rodriquez et al. (2008) concluded that increased moisture increased
bacterial transfer from surfaces to food. Kusumaningrum et al. (2003) found a greater transfer of bacteria to
cucumber slices than roasted chicken from inoculated stainless steel indirectly supporting the finding that
moisture facilitates bacterial transfer. Shale, Jacoby & Plaatjies (2006) reported that transfer of Staphylococcus
spp. was greater between meat and surfaces than between airborne bacteria in meat abattoirs. Furthermore, there
was no difference in the degree which bacteria adhered to hands or gloves according to Legg et al. (1999).
Moore et al. (2003) found varying results for transfer from inoculated stainless steel to wet or dry lettuce. For
example, transfer of Salmonella Typhimurium from stainless steel to dry lettuce ranged from 6 to 66%
(depending on how long the bacteria were on the surface before lettuce contact) and from 23 to 31% for wet
lettuce. Also for Campylobacter jejuni transfer for was from 16 to 38% for dry lettuce and from 15 to 27% for
wet lettuce. Gill & Jones (2002) reported greater transfer of E. coli from meat to gloves and from gloves to meat
when gloves were wet with between 2 to 4 log cfu/piece of meat transferred from hands and gloves contaminated
by handling inoculated meat.
3.2 Experiment 2. Transfer of E. Coli to Ice from Hands and Metal Scoops
Ice is a known transmission vector of pathogenic microorganisms in human foodborne illness (Levine,
Stephenson & Craun, 1990; Reis et al., 1992; Falcao et al. 2004). In these studies the pathogen was carried in water
used to create the ice however, cross contamination due to handling food and ice is also a cause of foodborne
illness (Fendler, Dolan & Williams, 1998; Montville, Chen & Schaffner, 2002). Bacteria can reside on hands
(Rocourt & Cossart, 1997; Rose & Slifko, 1999) and stainless steel food contact surfaces (Kusumaningrum,
Riboldi, Hazelberger & Beumer, 2003; Moore, Sheldon & Jaykus, 2003; Rodriguez & McLandsborough, 2007;
Kesiken, Todd & Ryser, 2008) and transfer bacteria to food. In the current study an average of 19.5 % of the
bacteria on hands were transferred to ice and 66.2% of bacteria on scoops were transferred to ice (Table 2). The
higher level of transfer from scoops compared to hands is expected due to the lack of attachment on stainless
steel compared to skin.
Table 2. Mean, median, range of the population and % transfer of bacteria on ice from hands and scoops
inoculated with E. coli
CFU/hand or scoop
LogCFU/hand or scoop
%CFU transferred
Hand Ice
Scoop Ice
Hand Ice
Scoop Ice
Hand Ice
Scoop Ice
Mean
23676b
506800a
4.1b
5.6 a
19.5b
66.2 a
Stand Error
3284
60773
0.07
0.04
1.9
1.2
Median
16698
369380
4.2
5.6
13.9
67.8
Maximum
1102465
1900030
5.0
6.3
67.0
82.5
Minimum
607
93610
2.8
0.3
1.9
30.0
a,bmeans with different superscripts are significantly different (p≤0.05). n=66.

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Transfer of bacteria from contaminated ice holding bins or scoops to ice may be an issue since Hampikyan,
Bigol, Cetin & Colak (2017) reported finding E. coli in 6.7 %of ice samples, 22 % of ice chest samples but no
positive E. coli from water samples used to make the ice.
In a separate experiment, the time after inoculation and sequential scoops were evaluated as factors affecting E.
coli transfer to ice. A significant effect (p≤0.05) of holding scoops after inoculation and for taking multiple ice
samples in sequence using the same scoop was found on the population of E. coli transferred to ice (Figure 1).
Less bacteria was transferred in the second scoopful of ice at 0 and 1 hour after inoculation but relatively high
levels of bacteria were still transferred from the scoop to the ice in the third scoopful. The percentage of bacteria
transferred at 0 hours for scoop sample 1, 2, and 3 was 71, 53 and 49% respectively. This trend held for all of the
holding times with a decreasing number of bacteria as holding time increased. The overall difference in %
transfer between the 3 scoopfuls taken in sequence was 11% between scoopful 1 and 2 and 16% between
scoopful 1 and 3. Overall the percentage of bacteria transferred to ice was significantly different between
scoopfuls 1 and 2 and between 1 and 3 but not 2 and 3 (p≤0.05). . This repeated transfer of E. coli from scoops to
ice is supported by previous research that reported bacteria residing on surfaces could shed during repeated
contact with other surfaces (Moore et al., 2003).
Figure 1. The population of E. coli recovered from ice exposed to scoops for 5 seconds then held for different
times after inoculation and then exposed three sequential times using the same scoop
a-emeans with different superscripts are significantly different (p≤0.05). n=12. Standard error for the 12 treatments ranged from 0.16 to 0.35
logcfu/ice sample.
3.3 Experiment 3. Bacterial Survival during Holding of Lemons
During the storage of lemons, bacterial population was highest for refrigerated lemons at time t=0. Lemons held
at room temperature lemons had the highest E. coli populations at 0 and 24 hours (Figure 2). Refrigeration
reduced E. coli populations from about 5 logs cfu/lemon to about 2 logs after 4 hours which did not further
diminish after 24 hours of refrigerated storage. Beumer & Kusamaningrum (2003) found that leftover foods
stored at 10°C increased by 2-3 log cycles in 3 days. Overall, to prevent growth and transfer, lemons should be
handled dry and kept refrigerated. Enteropathogenic bacteria (Shigella and Salmonella spp.) increased in
population several log cycles in 6 hours on cut papaya and watermelon when stored at 25-27°C and the
application of lemon juice to the fruit surface reduced the population of S. typhii but the bacteria began to
increase in population after 2 hours (Escartin, Ayala & Lozano, 1989). Cross contamination in food service
environments is a major factor in many foodborne illness outbreaks (Bloomfield & Scott, 1997; Guzewich &
Ross, 1999).

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Figure 2. Population of E. coli on lemons after different storage times held at room temperature or under
refrigeration
a,bmeans with different superscripts are significantly different (p≤0.05). n=10. Standard deviation for the 6 treatments ranged from 0.26 to
1.9.
Chen, Jackson, Chai & Schaffner (2001) modeled the transfer of a surrogate bacterium Enterobacter aerogenes,
starting from raw chicken to hands, then after washing of hands, the transfer of bacteria remaining of hands to
lettuce. These researchers found transfer rates as high as 100% with over 3 logs recovered on lettuce from an
initial inoculation of 8 logs on chicken despite the two transfer steps and hand washing before touching lettuce.
Chen et al (2001) also demonstrated touching other surfaces such as spigots to turn on water would create
surfaces that could, in turn be sources of contamination.
3.5 Bacteria in Beverages
Harmful bacteria can be added to beverages by handling of ice and other garnishes. A common fallacy is that
acidic and alcoholic beverages will protect the consumer from harmful microorganisms. In fact, Dickens,
DuPont & Johnson (1985) found that 4 pathogens frozen in ice and allowed to melt for 30 minutes in cola, soda,
Scotch (80 proof), a mixture of Scotch/soda and Tequila (86 proof) were not eliminated. The following
percentages were recovered in each of the following; 100% in club soda, 55-74% in cola, 64-94% in scotch and
soda, 11-16% in pure Scotch and 5-10% in pure Tequila. Two other studies found a slight inhibitory effect of
drink acidity but only when the bacteria were exposed to acidic drinks for a day or more. In a study to determine
the best beverage to consume to avoid “traveler’s diarrhea” or “Montezuma’s revenge.” Sheth, Wisniewski &
Franson (1988) reported that wine, diet cola and sour mix eliminated Salmonella and E. coli after 48 hours of
exposure while beer and regular cola had a strong inhibitory effect but did not eliminate these bacteria. A second
study examined orange drinks with pH levels of 3.0, 4.9 and 6.8 finding that only the 3.0 pH drink reduced E.
coli and Salmonella spp. populations but only to low levels after 30 hours of exposure (Massa, Facciolongo,
Rabasco & Caruso, 1998). In these studies, extremely long exposure times were used, a scenario not likely to
occur with drinks served with ice or fresh cut lemons. Thus the contamination of ice or cut fruit such as lemons
to drinks could be a potential vehicle to transmit bacteria. Food service workers are the primary of contamination
of food with norovirus, hepatitis A and Shigella spp. and can transfer other pathogens such as E. coli and
Salmonella spp. to food (Lynch, Tauxe & Hedberg, 2009) thus sanitation of surfaces contacting ice and lemons
served in beverages should be considered in minimizing food contamination.
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Technical Contribution No. 6670 of the Clemson University Experiment Station.