Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.
A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.
Amann
R I
RI
Lehrstuhl für Mikrobiologie, Technische Universität München, Germany.
Zarda
B
B
Stahl
D A
DA
Schleifer
K H
KH
D001105:Archaea / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002463:Cell Membrane Permeability; D006735:Horseradish Peroxidase; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes; D011387:Prokaryotic Cells / Q000382:microbiology*; D015347:RNA Probes; D012335:RNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification
D015345:Oligonucleotide Probes; D015347:RNA Probes; D012335:RNA, Ribosomal; D006735:Horseradish Peroxidase