Inoculum preparation methods can impact growth or survival of organisms inoculated into foods, thus complicating direct comparison of results among studies. The objective of this study was to evaluate preinoculation culture preparation for impact on Escherichia coli O157:H7 inoculated onto leaves of romaine lettuce plants and cut leaf surfaces. E. coli O157:H7 was grown quiescently or shaken at 15, 25, or 37 degrees C to different growth phases in tryptic soy or M9 minimal salts broth or agar. Cells were harvested, washed, and suspended in 0.1% peptone, Milli Q water, or well water and refrigerated for 0 or 18 h. Prepared inoculum was spotted onto cut romaine lettuce (10 microl; 3 x 10(4) CFU/10 g) or onto romaine lettuce plants (20 microl; 3 x 10(6) CFU per leaf). Cut lettuce was sealed in 100-cm2 bags (made from a commercial polymer film) and incubated at 5 or 20 degrees C. Lettuce plants were held at 23 degrees C for 24 h. For all tested conditions, levels of E. coli O157:H7 increased at 20 degrees C on cut lettuce and decreased on cut lettuce stored at 5 degrees C or on leaves of lettuce plants. At 20 degrees C, preinoculation culture conditions had little impact on growth of E. coli O157:H7 on cut lettuce. However, survival at 5 degrees C was significantly better (P < 0.05) for cultures grown at 15 or 37 degrees C in minimal medium and to late stationary phase. Impact of preinoculation handling on survival on lettuce plants was less clear due to relatively high standard deviations observed among samples.