Direct microscopic observation and viability determination of Campylobacter jejuni on chicken skin

A method was developed to determine the survival of Campylobacter jejuni at specific sites on chicken skin, and this method was used to observe the survival of C. jejuni at various locations on the skin during storage. This method uses confocal scanning laser microscopy to visualize C. jejuni transformed with P(c)gfp plasmid (GFP-Campylobacter) and stained with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). The green fluorescence of dead C. jejuni cells and the red fluorescent CTC-formazan in viable Campylobacter cells were clearly visible on chicken skin. The GFP-Campylobacter remaining on the chicken skin surface after rinsing was mostly located in crevices, entrapped inside feather follicles with water, and entrapped in the surface water layer. Most viable cells were entrapped with water in the skin crevices and feather follicles. These sites provide a suitable microenvironment for GFP-Campylobacter to survive. The population of C. jejuni on chicken skin decreased by 1 log unit during storage at 25 degrees C for 24 h. C. jejuni located in sites 20 to 30 microm beneath the chicken skin surface maintained viability during incubation at 25 degrees C. C. jejuni on chicken skin stored at 4 degrees C maintained constant numbers during a 72-h incubation with no significant changes in population feather follicles or crevices. Live and dead cells were initially retained with water on the skin and penetrated into the skin follicles and channels during storage. Microscopic observations of GFP-producing cells allowed the identification of survival niches for C. jejuni present on chicken skin.