IN-SITU DETECTION OF SPORES AND VEGETATIVE CELLS OF BACILLUS-MEGATERIUM IN SOIL BY WHOLE-CELL HYBRIDIZATION

Spores and vegetative cells of Bacillus megaterium were detected in li quid culture and in soil by DAPI staining and whole cell hybridization with fluorescent oligonucleotide probes after special permeabilizatio n. A SDS/DTT treatment (10 mg ml(-1) SDS, 50 mM dithiothreitol at 65 d egrees C for 30 minutes) significantly enhanced DAPI staining of spore s. An additional lysozyme treatment (0.1% at 37 degrees C for 20 minut es) made them even permeable for oligonucleotides as indicated by spec ific hybridization of a bacterial probe. Vegetative cells were permeab le for DAPI, however, they required treatments with lysozyme (0.1% at 25 degrees C for 10 minutes) to allow permeation of oligonucleotides. The differential permeabilization of vegetative cells by lysozyme trea tment alone and of spores and vegetative cells by the combination of S DS/DTT and subsequent lysozyme pretreatment facilitated quantification of spores and vegetative cells of B. megaterium growing in soil. Coun ts for spores and vegetative cells were generally higher (up to two or ders of magnitude) than those obtained by colony forming units. The de scribed approach offers a promising tool for quantitative autecologica l studies on bacilli in heterogeneous environments.