(Czech Republic)

Vegetable samples (25g) added to 225 mL buffered peptone water and inoculated w/ hi/low numbers of E. coli cells. Innoculum levels confirmed using plate counts on NA. Samples enriched for 18 h at 37°C. All experiments were performed in triplicate. Uninoculated control samples were included in all exp'ts.

Lettuce samples were collected from the field and from commercial establishments under normal conditions of harvest or purchase. They were placed in previously sterilized plastic bags and transferred to the lab with minimum delay.

100g of the sample was weighed out, diced, and placed in 500 ml plastic containers. To which, 250 ml of sterile peptone water were added. The sample was washed for 1 h with constant agitation, after which they were left at room temperature for 2 h in order to resuscitate the organisms. Dilutions of the samples for the count of aerobic bacteria was done with peptone water.

Iceberg lettuce was stored at 4 degrees Celsius in a Ziplock plastic bag after aseptically removing outer leaves and core. Four 2- by 2-cm lettuce pieces were aseptically cut using a sterile surgical blade, weighed, and submerged in 30 ml of inoculum (which were suspended in sterile deionized water (SDW)) in a sterile 125-ml Erlenmeyer flask. Flasks were sealed with sterile serum vial stoppers. After 24 hours of incubation, lettuce pieces were rinsed for 1 min. twice with SDW and drained in sterile petri dishes. Edges of lettuce pieces (0.3 cm) were aseptically removed, and the bacterial population adhering to the edges of lettuce was determined by surface plating with the method of Takeuchi and Frank (see Reference #18 of article) except extended the stomaching to 4 minutes.

Iceberg lettuce was used. The outer three or four leaves were removed from the lettuce head and discarded. Inner leaves (50g) were inoculated by distributing 0.5ml of mixed-strain suspensions in 0.1% peptone water or bovine feces slurry on the surface of leaves.

Control and treated lettuce leaves (50g) were placed in a plastic bag and 200 ml of sterile distilled water was added. The lettuce was rinsed by vigorously shaking the lettuce and water for 20 s to simulate food-service or household practice. After decanting the rinse water, 50 ml of sterile 0.1% peptone water was added and leaves were washed by shaking for 20 s. The peptone wash was analyzed for E.coli O157:H7.

Iceberg lettuce's outer leaves and core were removed and discarded. The remaining leaves were then shredded into pices 2-5mm wide.

Inoculation: Approx. 4 kg of the lettuce was separately placed in a perforated container, submerged in sterile deionized water, gently washed for 1 min, and drained. Lettuce was then submerged in large or small population inocula or in sterile deionized water (control) for 1 min. (Note: Large population suspension was prepared by adding 10 ml of undiluted strain mixture to 10 L of sterile deionized water. A small population suspension was prepared by adding 10 ml of diluted strain mixture (10^-3 in 0.1M potassium phosphate buffer.) The vegetables were then gently mixed during the submersion period (as before).

Ennumeration: 50 g samples were combined with 200 ml of sterile 0.1% peptone in a sterile polyethylene bag and pummeled with a stomacher for 2 min. Wash fluid was serially diluted and surface plated (0.1 ml) in duplicate on modified SMA (sorbital Maconkey agar) and quadruplicate on plate count agar.

Samples in which E.coli O157:H7 was anticipated to be present at population of less than 10g were subjected to enrichment in modified Trypticase soy broth. Vegetables (25g) were combined with 225 ml of broth in 500-ml Erlenmeyer flasks and incubated at 37 degrees Celsius for 18h on a rotary shaker (150 rpm). Cultures were then streaked on modified SMA and incubated at 35 degrees before examining.

All samples aseptically taken from internal flesh or leaves of vegetable.

Isolation of E. coli: (sec 2.4 in paper--this paper has separate methods for E. coli [general] and O157) 1 g sample pummelled in Stomacher in 10 mL peptone saline diluent. Homogenate cultured onto TBX Agar, incubated at 30°C for 4 hr, followed by incubation at 44ûC for 18 hr. Plates examined for blue colonies--confirmed as E. coli by growth at 44°C on tryptone bile agar and b -glucuronidase production.

Microflora of Lettuce: fresh head lettuce stripped of unsightly leaves, torn into serving size pices. 11 g sample blended with 99 ml sterile phosphate buffer using Waring blender. Colony forming units determined aerobically with PCA. To study indvidual members of microflora of lettuce juice, 10 colonies picked by random design from each countable plates. Isolates streaked on PCA slants, incubate 32°C, 24 hr. cultured observed for gram rxn, morphology, spore formation, litmus milk action, proteolysis of skim milk agar, catalase & oxidase rxn, appearance on eosin methylene blue agar, gas production in BGLB.

Lettuce juice prep/treatment: heads of lettuce processed w/ Acme Juicerator. To obtains diff. Conc, some juice lyophilized with Virtis automatic freeze dryer. . When ready for use, lyophilized product reconstituted w/ sterile distilled H2O to give 2:1 or 4:1 conc. Relative to original juice. Did PCA and various tests as described in previous paragraph (microflora of Lettuce).

Lettuce juice also used as growth medium for known cultures. When juice is used for this, juice freed of significant contaminants by irradiation at ambient temperature (25-35°C) w/ 2 Mrad of gamma radiation from Cobalt 60 source providing approx 5 Krad/min

Vegetables were visually assessed, then a 1:4 vegetable:distilled water sample was blended for 2 minutes and the pH was recorded 2 min after blending. Each vegetable was assayed for total aerobic plate count on plate count agar, for coliforms on violet-red bile agar, and for yeasts and molds on potato dextrose agar with 100 mg/l tetracycline.