ARS | Publication request: Fluorescent dye technique as an alternative to gfp-labeled plasmid for visualization of Escherichia coli O157:H7 cells on romaine lettuce leaves following sanitizer treatment

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 15, 2009
Publication Date: May 12, 2009
Citation: Keskinen,L.,Cooke,P.,Annous,B.2009.Fluorescent dye technique as an alternative to gfp-labeled plasmid for visualization of Escherichia coli O157:H7 cells on romaine lettuce leaves following sanitizer treatment [abstract].American Society for Microbiology.Philadelphia,PA.p.1.

Technical Abstract: The task of imaging Escherichia coli O157:H7 cells on artificially inoculated produce often requires genetic modification of the cells through the introduction of gfp-labeled plasmid. However, these modified cells do not behave as the parent cells and the auto fluorescence of lettuce leaves interfere with localization and imaging of these cells. The objectives of this study were: A) to develop SYTO9-staining technique that does not require genetic manipulation; B) to determine whether SYTO 9-stained E. coli cells or inoculated Romaine lettuce were removed and/or inactivated by sanitizing treatments at similar levels to unstained cells. Freshly cut Romaine lettuce leaves were dip inoculated using SYTO 9-stained E. coli O157:H7 to a final concentration of 7.56 +/- 0.04 log CFU/g. Samples were examined for fluorescent cells under fluorescence microscopy (FM), and stored at 4 deg C overnight. Samples (10 g) were then rinsed for 2 min in dH2O, 200 ppm chlorine (Cl2), or were left untreated, then were blended in neutralizing buffer, and plated on the selective media CTSMAC for enumeration of E. coli O157:H7. Samples were observed under FM before and after each treatment, followed by scanning electron microscopy (SEM). Enumeration of stained cells of E. coli O157:H7 indicated that populations were reduced by 1.09 +/- 0.22 and 0.55 +/- 0.15 log CFU/g by Cl2 and dH2O rinses, respectively, which were not significantly different from results obtained using unstained cells. Although, dyed-cells on inoculated lettuce lost fluorescence activity following Cl2 rinse only, SEM imaging indicated that these cells were still present on all lettuce leaf samples. This simple and easy method of staining does not require specialized molecular microbiology training and allows for the observation of static bacterial population attached to produce surfaces. Also, unlike gfp-labeled cells, auto fluorescence of lettuce did not interfere with observing SYTO9-dyed cells. The loss of fluorescence in chlorine-treated cells seemed to indicate that chlorine was in contact with these cells, but was not capable of inactivating them. These results merit further investigation.