Location: Produce Safety and Microbiology Research
Project Number: 2030-42000-050-07-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Aug 26, 2014
End Date: Apr 30, 2019
1. Determine the effect of SO2 fumigation on the survival of common foodborne pathogens under in vitro conditions. 2. Determine the best inoculum concentration of bacterial pathogens and their survival on table grapes. 3. Examine the effect of SO2 on the survival of foodborne pathogens on freshly harvested California table grapes.
Procedure to accomplish Objective 1: Bacterial culture will be prepared by inoculating single colonies of each pathogen in LB broth supplemented with rifampicin and grow at 28ºC overnight on a shaker (150 rpm). Freshly-harvested California table grapes will be shipped directly from the harvest sites. Only firm and intact grapes will be used for inoculation. Grape berries will be placed with pedicel side down on each well of a 24-well culture plate and inoculated with either 104 cells per grape (low inoculum) or 106 cells per grape (high inoculum). The inoculated grapes will then be treated with SO2 with a dose of 1000 ppm-hr (2000 ppm for 30 min), 1500 ppm-hr (3000 ppm for 30 min), 2000 ppm-hr (4000 ppm for 30 min), 2500 ppm-hr (5000 ppm for 30 min), and 3000 ppm-hr (6000 ppm for 30 min). The control group will be set up under condition identical to the fumigated samples, but without SO2 treatment. The population size of bacterial pathogen on table grapes following each treatment will be determined by plate counting to determine the survival of each pathogen following each SO2 treatment. The presence of bacterial pathogen will be detected by plating aliquots of the enrichment culture on rifampicin-containing LB plates. Once the minimum dose required to completely inactivate each pathogen on table grapes is determined, we will refine the fumigation dose in subsequent tests in 100 ppm-hr decrements to determine the minimum effective dose for each pathogen. Procedure to accomplish Objective 2: The inoculum of E. coli O157:H7 and S. enteric Thompson will be prepared as described in Objective 1. Similarly, each bacterial pathogen will be inoculated onto the grapes. For Sub-objective 1 the inoculated grapes will be quickly tempered to 60-68'F and immediately treated with SO2 with a dose of 800 ppm, 1200 ppm, 1600 ppm, and 2000 ppm for 30 min. For Sub-objective 2 the inoculated grapes will be quickly tempered to 32ºF and immediately treated with SO2 with a dose of 800 ppm, 1200 ppm, 1600 ppm, and 2000 ppm for 30 min. For both sub-objectives, the control group will be set up under conditions identical to the fumigated samples, but without SO2 treatment. The fumigated grapes will be stored at 32ºF with 95% RH for three days. The number ofviable pathogen cells on table grapes will be determined daily for three days by plate counting. Procedure to accomplish Objective 3: The inoculum of E. coli O157:H7 and S. enterica Thompson will be prepared as described in Objective 1. Before the day of the experiments, grapes will be transferred to a cold incubator at 32ºF overnight. The inoculated grapes will be treated with SO2 immediately after inoculation followed by incubation at 32ºF with 95% RH for one week. The second fumigation will be conducted on Day 7 with a same SO2 dose as the Day 0, and the viable bacterial pathogen cells on the grapes will be determined daily by plate counting until no survivors will be detected by the enrichment experiments.