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 liquid culture and in soil by DAPI staining and whole cell hybridization with fluorescent oligonucleotide probes after special permeabilization. A SDS/DTT treatment (10 mg ml(-1) SDS, 50 mM dithiothreitol at 65 degrees C for 30 minutes) significantly enhanced DAPI staining of spores. An additional lysozyme treatment (0.1% at 37 degrees C for 20 minutes) made them even permeable for oligonucleotides as indicated by specific hybridization of a bacterial probe. Vegetative cells were permeable 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 treatment alone and of spores and vegetative cells by the combination of SDS/DTT and subsequent lysozyme pretreatment facilitated quantification of spores and vegetative cells of B. megaterium growing in soil. Counts for spores and vegetative cells were generally higher (up to two orders of magnitude) than those obtained by colony forming units. The described approach offers a promising tool for quantitative autecological studies on bacilli in heterogeneous environments.