ARS | Publication request: Use of florescent dyes for visualization of bacterial attachment to lettuce leaves

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 10, 2008
Publication Date: August 3, 2008
Citation: Keskinen,L.,Cooke,P.,Annous,B. 2008. Use of florescent dyes for visualization of bacterial attachment to lettuce leaves [abstract].International Association for Food Protection.Columbus, OH. p.1.

Technical Abstract: Produce safety research often involves the use of gfp-labeled bacterial cells for ease of visualizing the attachment of the targeted cells to produce tissues. This genetic manipulation, frequently tied to plasmids coding for antibiotic resistance, results in changes in cell physiology which could have unintended effects on bacterial interaction with produce tissues. The purpose of this study was to develop a non-genetically labeled fluorescent microorganism. An overnight culture of Escherichia coli O157:H7 RM6044 (tryptic soy broth, 37C) was centrifuged and washed in sterile deionized water (dH2O) once, then resuspended in dH2O. SYTO 9 or 85 dye was added to the culture and incubated at 4C for 3 h. The culture was centrifuged at 9000 rpm for 2 min, washed twice with dH2O, resuspended in dH2O, and was incubated at room temperature (RT; with or without light) and 4C (without light) for up to 7 d. Also, the cut base of romaine lettuce leaves were immersed in SYTO 9-stained E. coli O157:H7 (8 log CFU/ml) for up to 5 d at 4C. All samples were observed via fluorescence, confocal and scanning electron microscopy. All stained cultures remained viable and fluorescent following 15 d of storage at RT and 4C. Unlike SYTO 9, SYTO 85-stained cells exhibited rapid photo bleaching during imaging. Colonization of romaine lettuce leaves by the stained E. coli O157:H7 cells was easily visualized via fluorescence microscopy. Cells forming a biofilm on the leaf, were further examined via confocal and SEM imaging. No internalized E. coli O157:H7 were observed in romaine lettuce leaves. This easy method of staining, which does not require specialized equipment or training, allows for the observation of static bacterial population attachment to produce surfaces.