Three-Dimensional Observation of Soil Bacteria in Organic Debris with a Confocal Laser Scanning Microscope

抄録

Confocal laser scanning microscopy (CLSM) was applied to three-dimensional (3-D) observation of bacteria harboring in soil organic matters. Ethidium bromide (EB) was employed for staining soil bacteria, since EB did not stain strongly soil constituents other than bacteria. Staining of soil specimens in 100μg/ml of EB at pH 7.2 for 3 min followed by observation under blue excitation offered a satisfactory preparation for CLSM. A sequence of 60 confocal images of a specimen (soil organic debris 18μm thick) was obtained by optical sectioning at different levels in the specimen at intervals of 0.3μm. From the 60 sections, several types of 3-D projections were generated by image processing : a micrograph with a great depth (18μm) of focus, a pair of split stereo images which could be observed stereoscopically with the naked eye, etc. These 3-D images enabled to observe the spatial distribution of bacteria in organic debris in detail including bacterial colonization inside the organic debris. Possible use of CLSM in soil microbiology was discussed.