Physical stabilization and confocal microscopy of bacteria on roots using 16S rRNA targeted, fluorescent-labeled oligonucleotide probes

Fluorescent-labeled, rRNA targeted oligonucleotide probes were used with laser scanning confocal microscopy (LSCM) for in situ detection of bacteria located on root surfaces. Inoculated Pseudomonas syringae and Rhodococcus fascians cells were directly fixed on the root surface prior to analysis by LSCM and probed using a Texas red-tagged oligonucleotide probe. Natural bacterial populations on the surface of non-sterile clover root and in physically stabilized rhizosphere soil were detected following in situ hybridization using a fluorescein-labeled, bacterial specific oligonucleotide probe. The optical sectioning capability of the confocal microscope allowed a greater depth of field to be viewed than was possible using conventional epifluorescent microscopy. As such, three-dimensional images were presented as montages of optical sections taken through the Z focal plane. This technique shows considerable potential as an alternative to selective isolation and viable counting procedures for the spatial analysis of microorganisms in situ.