Distribution of viable and non-viable bacteria in the gastrointestinal tract of gnotobiotic and conventional rats

Abstract

To determine the proportion of viable and non-viable bacteria in the various sections of the gastrointestinal tract of rats, the contents of jejunum, cecum, proximal and distal colon from conventional rats and rats monoassociated with Bacteroides thetaiotaomicron were analyzed with four different methods. The number of viable and non-viable bacteria were determined microscopically with the fluorescence-based Bac Light staining technique (L/D) that distinguishes organisms with the nucleic acid-binding dyes Syto 9 and propidium iodide. The viable bacteria were also enumerated by fluorescent in situ hybridization (FISH) with a mixture of oligonucleotide probes targeting the 16S rRNA and by cultivation on agar plates. The number of total bacteria was determined microscopically after cell staining with 4',6-diamidino-2-phenylindol (DAPI). When applied to the contents of various sections of the rats' gastrointestinal tract the staining with L/D and FISH yielded similar cell counts. The highest percentage of viable bacteria, determined as the ratio of L/D-stained to total counts, was detected in the cecum of both rat models (77-87%). Only 50% of the DAPI-stained cells in the colonic contents of conventional rats were also detected by FISH or the viability stain. In gnotobiotic rats 70% of the cecal or colonic cells detected with DAPI were recovered by plating. In conventional rats this proportion decreased to 25%. In conclusion, (I) the culture-based method provides an incomplete picture of the viable population in conventional rats and (II) in situ methods such as L/D and FISH are an useful alternative to cultivation on agar plates for measuring bacterial viability in the rats' gut. Keywords: intestinal microflora, conventional rat, gnotobiotic rat, in situ hybridization, viability staining.