TY - JOUR
T1 - PERMEABILIZATION OF MYCOLIC-ACID-CONTAINING ACTINOMYCETES FOR IN-SITU HYBRIDIZATION WITH FLUORESCENTLY LABELED OLIGONUCLEOTIDE PROBES
AU - Macnaughton, SJ
AU - O'Donnell, A.G.
AU - Embley, TM
PY - 1994
Y1 - 1994
N2 - The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis. Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis. Rhodococcus equi. R, erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis. Lactobacillus plantarum, Escherichia coil and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 50 and 50 min hydrolysis, whereas B. subtilis, E. coil and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.
AB - The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis. Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis. Rhodococcus equi. R, erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis. Lactobacillus plantarum, Escherichia coil and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 50 and 50 min hydrolysis, whereas B. subtilis, E. coil and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.
U2 - 10.1099/00221287-140-10-2859
DO - 10.1099/00221287-140-10-2859
M3 - Article
C2 - 8000549
VL - 140
SP - 2859
EP - 2865
JO - MICROBIOLOGY-UK
JF - MICROBIOLOGY-UK
ER -