Washington Tree Fruit Research Commission

Research Reports

Assessment of Apple Packing for Listeria Risk (2016)

WTFRC Project #
YEAR 0/0
Organization Project #
Title:Assessment of Apple Packing for Listeria Risk
PI:Karen Killinger, Ph.D.
Organization:WSU/ School of Food Science (509) 335-2970 karen_killinger@wsu.edu Box 646376 Pullman, WA 99164-6376
 PDF version of report


Ines Hanrahan


(509) 669-0267   


2403 S. 18th St., Suite 100   

Union Gap, WA  98903-1637


Trevor Suslow  


(530) 754-8313


103 Mann Laboratory

Davis, CA, 95616


Yen-te Liao   


(509) 335-3842 


PO 646376       

Pullman,  WA  99164



Multiple industry partners. As of August 2015, Ines Hanrahan assumed PI status, since Dr. Killinger left WSU to take a position with FDA. Dr. Killinger remains as a co-PI on the project. The assistance of Tonia Green, Lauren Walter, Kyu Ho Jeong, and Andy Liao are acknowledged and greatly appreciated. Special thanks to Dr. Meijun Zhu (WSU) for help with oversight of WSU based budgets upon Dr. Killingers departure.


1)  Examine current industry standard practices for control of Listeria in packing environments and compare sampling and detection methods for Listeria spp.

2)  Examine the prevalence of generic Listeria spp. and Listeria monocytogenes associated with Fuji apples stored under refrigerated storage and controlled atmosphere storage with and without ozone application

Significant findings


Objective 1:  Environmental Listeria spp. risks in tree fruit packing operations         

Partnering organizations and packing houses were identified for preliminary meetings to assess potential high risk areas for Listeria spp. contamination.  Consideration was given to equipment design, materials used as food contact surfaces as well as cleaning and sanitation regimes.  Data collected at each packinghouse were blinded for confidentiality.  Each packinghouse was interviewed to understand current practices related to cleaning and sanitation practices as well as environmental monitoring to prioritize areas for environmental sampling.

Three facilities were interviewed for participation; based on responses, two were selected for sampling.  Selection of target organisms, Listeria spp., total coliforms and generic E. coli, was discussed.  The scope of sampling was explored, examination of food contact surfaces (Zone 1)  as well as areas immediately adjacent to food contact surfaces (Zone 2), production areas (Zone 3) and areas outside of production (Zone 4).  The approach taken for sampling and study design were different based on input from the facility and their specific needs.  Aggressive environmental sampling was conducted in each facility on multiple days. In one facility, an evaluation of Listeria prevalence was performed in the packing operation and cold storage rooms, including Zone 1-4 testing prior to cleaning and sanitation. 

In one facility, a comparison of sanitation practices was performed.   Two cold storage rooms were designated for the study.  Cold storage rooms were sampled before and after typical sanitation versus before and after a more, aggressive sanitation protocol.  Samples were collected from Zone 3 and some Zone 4 locations.  In some cases, samples were composited to reduce the number of samples processed and increase the number of areas examined in the cold storage rooms.  For example, all high wall samples were composited and all the low wall samples were compostied into one sample to create only two samples.  Samples prior to cleaning and sanitation were collected from both rooms on one day.  In another facility, samples were collected prior to sanitation from Zones 1-4 to evaluate areas that may require aggressive sanitation and potential routes of microbial contamination.

Samples were collected using PUR-Blue ™ swabs (World Bioproducts), Quick swabs ™ (3M™), Swab Samplers™ (3M™) or EZ Reach ™ (World Bioproducts) sponge samplers that were appropriate for each type of sampling site.  Previous results from our laboratory indicated that some surfaces, such as wood bins, would benefit from aseptic sampling with small pieces of 3M Scotch-Brite™ pads, so in some cases, samples were collected with commercially available swabs and using treated 3M Scotch-Brite™ pads. Efforts were made for most commerical sampling supplies to use Dey-Engley (D/E) neutralizing broth, but in some cases, supplies using letheen broth were used.  Environmental samples were collected and held on ice for transport to the laboratory.  Liquid from the sampling device was collected and brought up to a 10ml volume of D/E neutralizing broth to align sampling volumes among swabbing devices.

Several methods of analysis were used for isolation of presumptive positive generic Listeria spp.  The FDA-BAM method (FDA-BAM, 2011) with some modifications was used.  The same pre-enrichment steps were used for all methods; samples were pre-enriched in Buffered Listeria Enrichment Broth (BLEB) for 24 hours at 30°C; after 4 hours of incubation, acriflavin HCL (10mg/L), nalidixic acid (40mg/L) and natamycin (25mg/L) were added to reduce background flora levels.  Serial dilutions of the pre-enriched sample were inoculated  onto Environmental Listeria Petrifilm™ (3M™) for determination of generic Listeria spp..   At 24 and 48 hours incubation,  the pre-enriched sample was plated onto Modified Oxford Listeria selective agar.  At 24 hours incubation, the pre-enriched sample was plated onto HardyChrom Listeria agar for differentiation of generic Listeria spp. from Listeria monocytogenes and ivanovii.  Results from these methods should be considered presumptive positive, not confirmed, for generic Listeria or Listeria monocytogenes.  Some isolates remain to be tested for further confirmation using methods described below.

Selected samples were processed as duplicate swabs for comparison and examination of other methods, including the use of HardyChrom Listeria agar, a proprietary test for Listeria spp., qPCR and immunomagnetic separation followed by qPCR.

Objective 2:  Examine the prevalence of generic Listeria and Listeria monocytogenes on Fuji apples stored under differing conditions

There is a need to understand the prevalence of Listeria spp. on fruit upon arrival from the orchard and after storage.  Apples were harvested from WSU orchards and were not intended for the commercial market, so there were no negative commercial implications associated with the results.  Microbial tests performed at harvest in October of 2014 did not include Listeria spp., but did examine fruit that was directly contacted by water and fruit that was not directly contacted by irrigation water from an open surface water source.  Apples were examined for the presence of total coliforms, generic E. coli using 3M™ E. coli/Coliform Petrifilms™.  The need to include Listeria spp. was identified and approved for funding, which allowed for examination of apples held under refrigerated storage and controlled atmosphere storage with and without ozone treatment.  With input from post-harvest experts, a chemical supplier and a partnering facility, apples were stored refrigerated storage for 2 months and 3 months and in controlled atmosphere storage with and without ozone application for 6 months and 8-9 months.  The presence of presumptive Listeria spp. and Listeria monocytogenes were examined as described above.  Apples were also examined for pathogenic E. coli O157:H7 and Salmonella.  The isolation of E. coli O157 was performed using immunomagnetic separation (IMS), standard plating techniques, and latex agglutination (LeJeune et al., 2001; Wright et al., 1994).  Salmonella spp. were isolated by standard plating techniques and latex agglutination (FDA-BAM  2011).  Confirmation of presumptive positive samples for E. coli O157, Salmonella spp., generic Listeria and Listeria monocytogenes will be performed by third-party laboratory serotyping. 

Results and discussion

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NT= not tested

  Table 2.  Percentage of positive samples within sampling categories by testing media or method for presumptive (;
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