Isolation of Salmonella Spp. from Laboratory Mice and from Diet Supplements.

ml of phosphate-buffered saline and once with 5 ml of maintenance medium (mixture 199 with 5% calf serum). Virus suspension in 0.5 ml volume was introduced onto cell monolayers and allowed to adsorb for 4 hr at 35 C; 4 ml of primary overlay medium, heated to 45 C, were added and cell monolayers were then incubated 4 days at 35 C. The overlay medium contained 1 part calf serum, 10 parts 2.2% Difco Special Agar (Noble), and 10 parts nutrient medium. The latter consisted of 1% LAH, 0.2% yeast extract, and 0.04% streptomycin in twofold concentrated Earle's salt solution. A 1-ml amount of second overlay medium, differing only from primary overlay medium by the addition of 0.015% neutral red, was added onto cell monolayers that were incubated an additional 1 to 3 days at 35 C. On the fifth or sixth day after cell monolayers were exposed to virus suspension, minute macroscopic plaques appeared as clear discrete areas ranging in size from 0.2 to 0.5 mm. Enumeration was facilitated by employing a dissecting microscope at a magnification of 15 X. The size of plaques remained constant on subsequent incubation; their number increased gradually and reached a maximum on the seventh day. By the tenth day, plaques tended to fade because of cell degeneration. Plaques produced by psittacosis virus were similar in size and appearance to those described for meningopneumonitis virus on strain L cell monolayers (Higashi and Tamura, Virology 12:578, 1960). The maximal diameter of plaques produced by meningopneumonitis virus was attained on the eleventh day, which suggests a difference between the rates of plaque formation by the viruses. This may be related, however, to the use of different cell lines. Plaque formation by different concentrations of virus was linear over a dilution range of 1.5 log units (Fig. 1), suggesting that each plaque was initiated by a single virus particle (Dulbecco, Proc. Natl. Acad. Sci. U.S. 38:747,1952). Significant reductions in plaque formation by varied dilutions of rooster psittacosis antiserum mixed with a standard suspension of virus confirmed the virus specificity of l)laques. In sensitivity, the plaque assay compared favorably with another technique, that of yolksac inoculation of 7-day chick embryonated eggs, based on dilution endpoint. Mean values of replicate determinations made by both techniques with a standard virus suspension were 2.7 X 105 plaque-forming units (PFU) and 2.8 X 105 egg LD50 per ml, respectively. The feasibility of employing a plaque assay for psittacosis virus is indicated by these preliminary findings.