Lysed areas suggestive of phage activity were observed on a plate in the course of isolation of a strain of Chondrococcus columnaris from trout at the Fisheries Center of the University of Washington. C. columnaris is an aquatic fruiting myxobacterium responsible for epizootics in salmonid and other fishes (Ordal and Rucker, 1944) . Subsequently a bacteriophage was isolated which infected and lysed this particular strain of C. columnaris. Up to this time bacteriophages have not been reported

more »

... members of either the Myxobacteriales or the Spirochaetales, the orders of flexible bacteria characterized by the absence of rigid cell walls. In view of the basic difference in structure of the eubacteria and the myxobacteria, a study was undertaken to compare the properties of the myxophage with those of the well studied eubacterial phages. For this purpose a system for a quantitative assay of myxophages had to be developed. MATERIALS AND METHODS Stock cultures of C. columnaris were grown at 20 to 25 C in agar deeps consisting of tryptone, 0.05 per cent; yeast extract, 0.05 per cent; sodium acetate, 0.02 per cent; beef extract, 0.02 per cent; and agar at pH 7.2, 0.4 per cent. Liquid cultures were grown in tryptone broth containing tryptone, 0.4 per cent; yeast infusion, 3 per cent; and "Tween 80" at pH 7.2, 0.1 per cent. All broth cultures were shaken in a reciprocal shaker at approximately 27 C. The method employed for plating phase suspensions was similar to that described by Adams (1950) , except that the compositions of the media were adapted for the myxophage-host I This investigation was supported in part by the State of Washington fund for medical and biological research. 2 Portion of a dissertation presented by the senior author as partial fulfillment of the requirement for the degree Master of Science at the University of Washington. system. The overlay agar consisted of 0.4 per cent tryptone, 2.5 ml; NaCl, 0.06 M; and agar, 0.7 per cent. The base agar contained 0.4 per cent tryptone, 30 ml; and agar, 1.5 per cent. The base agar was poured into plates and dried overnight at 30 C. One-tenth ml of a broth culture containing approximately 4 X 108 cells/ml and 1 ml of the phage dilution were added to the overlay agar. Plaques were counted after incubation for approximately 40 hr at 28 C. A preparation for electron microscopy was made from a plaque by the pseudoreplica technique of Hillier and Baker (1946) . One-step growth experiments were performed according to the method of Delbruick and Luria (1942). RESULTS Phage morphology. Since preliminary attempts to purify the phage by centrifugation were unsuccessful, an electron micrograph was made of a plaque replicate. Figure 1 shows that the myxophage is about 80 mu in diameter and polyhedral in shape. Short tails appear to protrude from several of the particles, but the presence or absence of a tail cannot be definitely established until the particles are purified and prepared for electron microscopy by treatment less drastic than air drying. Plaque morphology. Twenty plaques from areas selected at random from each of two plates were measured and found to vary in diameter from 0.1 to 1.5 mm with an average of 0.8 mm. Myxophage plaques produced in overlay agar are round with clear centers and relatively sharp margins. When the plaques are viewed from a low angle, the surfaces of the plaques are seen to be elevated slightly above the surrounding bacterial growth. Development of plating technique. In preliminary experiments it was found that the plating procedure described under Materials and Methods would give reproducible plaque counts. 738 on May 8, 2020 by guest