Use of a simplified cell blot technique and 16S rRNA-directed probes for identification of common environmental isolates

Abstract

A simple technique in which rRNA-targeted oligodeoxynucleotide probes are used to identify bacteria immobilized on membranes is described. By using colony lifts, bacteria are directly transferred from plates to untreated nitrocellulose membranes. Alternatively, cells resuspended from colonies can be applied to membranes by using a vacuum manifold under high-salt conditions. Blotted cells are baked and hybridized under stringent conditions by using standard protocols. Treatment of blotted cells with sodium dodecyl sulfate, urea, formaldehyde, or protease had no apparent effect on hybridization signals. Hybridization to rRNA from cells that had been stored refrigerated for 6 days was readily detected; however, fivefold more cells (approximately 10(7) cells) were required to obtain hybridization signals comparable to those generated by cells not subjected to storage. The sequences of oligonucleotide probes specific for Pseudomonas cepacia, Comamonas testosteroni, and Acinetobacter calcoaceticus and a group probe identifying Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas fluorescens, Comamonas acidovorans, and "Flavobacterium" lutescens are presented. In conjunction with the colony lift hybridization procedure, bacteria isolated from river water were identified by using these probes.