Isolation and Grouping of RNA Phages

Abstract

Introduction. Loeb and Zinder (1961) first reported on a coliphage containing RNA as its genetic material. Since then, RNA phages were isolated in several laboratories from various sources (Davis et al. 1961, Paranchych et al. 1962, Marvin et al. 1963, Nonoyama et al. 1963, Bradley 1964, Bishop et al. 1965). These phages were spherical (diam. 20-25 m ji), male specific and sensitive to RNase. Furthermore, most of them were serologically related, that is, the anti-phage rabbit serum prepared by one of these phages could neutralize most of the other phages (Nonoyama et al. 1963, Bishop et al. 1965, Scott 1965, Overby et al. 1966). The only phage which was definitely unrelated in a serological aspect was an RNA phage Q,9. This phage was isolated at our former laboratory, the Institute for Virus Research, .Kyoto University, in 1961. Similar attempts to isolate RNA phages were continued in our present laboratory and approximately 30 strains were isolated in these three years. During the course to study these RNA phages, we discovered that these RNA phages were separated in three groups serologically and also by a Millipore filtration method. The procedures of isolation and grouping of RNA phages were presented in this paper. Isolation o f RNA phages. Sources of phage isolation were human feces and sewage water. The latter was more suitable for our purpose and used in most of our experiments. To isolate RNA phages, the small amount of the sample was inoculated in the culture of E, coli K12 (2)-F+, which was grown to 5 x 107 cells per ml in PGYC medium (Miyake et al., 1966). After overnight standing at 37°C, the culture was chloroformed and centrifuged at low speed (3000 rpm, 20 min.) and the supernatant was plated on the F+ strain. The plaques on these plates were tested for male specificity and sensitivity to RNase. In most of our experiments, an F-strain, E, coli K12 W3110 (2)-was used to test male specificity. Sensitivity to RNase was tested by plating the phage with soft agar medium