PHYSICAL STABILIZATION AND CONFOCAL MICROSCOPY OF BACTERIA ON ROOTS USING 16S RIBOSOMAL-RNA-TARGETED, FLUORESCENT-LABELED OLIGONUCLEOTIDE PROBES

Fluorescent-labeled, rRNA targeted oligonucleotide probes were used wi th 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 pr ior to analysis by LSCM and probed using a Texas red-tagged oligonucle otide probe. Natural bacterial populations on the surface of non-steri le clover root and in physically stabilized rhizosphere soil were dete cted following in situ hybridization using a fluorescein-labeled, bact erial specific oligonucleotide probe. The optical sectioning capabilit y of the confocal microscope allowed a greater depth of field to be vi ewed than was possible using conventional epifluorescent microscopy. A s such, three-dimensional images were presented as montages of optical sections taken through the Z focal plane. This technique shows consid erable potential as an alternative to selective isolation and viable c ounting procedures for the spatial analysis of microorganisms in situ.