PERMEABILIZATION OF MYCOLIC-ACID-CONTAINING ACTINOMYCETES FOR IN-SITUHYBRIDIZATION WITH FLUORESCENTLY LABELED OLIGONUCLEOTIDE PROBES

The application of whole-cell hybridization using labelled oligonucleo tide probes in microbial systematics and ecology is limited by difficu lties in permeabilizing many Gram-positive organisms. In this investig ation paraformaldehyde treatment, acid methanolysis and acid hydrolysi s were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oli gonucleotide probe designed to bind to a conserved sequence of bacteri al 16S rRNA. Methods were evaluated on stationary-phase cultures of Go rdona bronchialis. Mycobacterium fortuitum, Nocardia asteroides, N. br asiliensis. Rhodococcus equi. R, erythropolis, R. fascians, R. rhodoch rous and Tsukamurella paurometabola, none of which could be probed fol lowing 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtili s. Lactobacillus plantarum, Escherichia coil and Pseudomonas putida we re also studied. The data showed that most of the mycolic-acid-contain ing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths o f time. In general, the mycolic-acid-containing organisms required bet ween 50 and 50 min hydrolysis, whereas B. subtilis, E. coil and P. put ida were rendered permeable in only 10 min. Interestingly, L. plantaru m could not be permeabilized using acid hydrolysis even after 60 min e xposure to 1 M HCl.