QUANTITATIVE FLUORESCENCE IN-SITU HYBRIDIZATION OF BIFIDOBACTERIUM SPP WITH GENUS-SPECIFIC 16S RIBOSOMAL-RNA-TARGETED PROBES AND ITS APPLICATION IN FECAL SAMPLES

Three 16S rRNA hybridization probes were developed and tested for genu s-specific detection of Bifidobacterium species in the human fecal flo ra. Variable regions V2, V4, and VS of the 16S rRNA contained sequence s unique to this genus and proved applicable as target sites for oligo deoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole cetl hybridization with fluore scein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicro scopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridizatio n experiments. One of the probes developed was used to determine the p opulation of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. S ince both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the t otal culturable counts were only a fraction of the total microscopic c ounts, the contribution of bifidobacteria to the total intestinal micr oflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.