Biomaterial Database

Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes. 1994

S J Macnaughton, and A G O'Donnell, and T M Embley

Department of Agricultural and Environmental Science, University, Newcastle upon Tyne, UK.

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 coli 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 30 and 50 min hydrolysis, whereas B. subtilis, E. coli 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.

UI	MeSH Term	Description	Entries
D008828	Microbiological Techniques	Techniques used in microbiology.	Microbiologic Technic,Microbiologic Technics,Microbiologic Technique,Microbiological Technics,Technic, Microbiologic,Technics, Microbiological,Technique, Microbiologic,Techniques, Microbiologic,Microbiologic Techniques,Microbiological Technic,Microbiological Technique,Technic, Microbiological,Technics, Microbiologic,Technique, Microbiological,Techniques, Microbiological
D009171	Mycolic Acids	Long fatty acids found in the CELL WALLS of some ACTINOBACTERIA, including MYCOBACTERIUM TUBERCULOSIS.	Mycolic Acid,Acid, Mycolic,Acids, Mycolic
D002463	Cell Membrane Permeability	A quality of cell membranes which permits the passage of solvents and solutes into and out of cells.	Permeability, Cell Membrane
D005456	Fluorescent Dyes	Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.	Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D000190	Actinomyces	A genus of gram-positive, rod-shaped bacteria whose organisms are nonmotile. Filaments that may be present in certain species are either straight or wavy and may have swollen or clubbed heads.
D012240	Rhodococcus	A bacterial genus of the order ACTINOMYCETALES.
D012336	RNA, Ribosomal, 16S	Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis.	16S Ribosomal RNA,16S rRNA,RNA, 16S Ribosomal,Ribosomal RNA, 16S,rRNA, 16S
D015345	Oligonucleotide Probes	Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin.	Oligodeoxyribonucleotide Probes,Oligonucleotide Probe,Oligoribonucleotide Probes,Probe, Oligonucleotide,Probes, Oligodeoxyribonucleotide,Probes, Oligonucleotide,Probes, Oligoribonucleotide
D017403	In Situ Hybridization	A technique that localizes specific nucleic acid sequences within intact chromosomes, eukaryotic cells, or bacterial cells through the use of specific nucleic acid-labeled probes.	Hybridization in Situ,Hybridization, In Situ,Hybridizations, In Situ,In Situ Hybridizations