Abstract
It has recently been shown 1 that sedimentation boundaries could be obtained and approximate size determinations made with dilute protein suspensions in the angle centrifuge if a synthetic density gradient of sucrose or some other nonsedimentable material was added to counteract disturbances of convection. Under these conditions tubes could be removed from a centrifuge and sampled without greatly altering a boundary. These principles have been applied to the sedimentation of influenza virus in allantoic fluid, and the findings are herewith reported.
The PR8 strain of influenza A virus or the Lee strain of influenza B virus was inoculated into the allantoic sacs of 11-day-old White Leghorn embryos. After 48 hours' incubation at 37°C the eggs were chilled overnight at 4°C; the blood-free allantoic fluids were removed and cleared by centrifugation at 1800 r.p.m. for 5 minutes. Without further treatment the virus fluids were spun in celluloid tubes in a vacuum air-driven centrifuge in the presence of a sucrose density gradient with the concentration of sugar increasing from a negligible amount at the top of the fluid column to nearly 12% at the bottom of the tube. After centrifugation at a given rate 1 cc samples were removed at different levels in the fluid column by means of a sampling apparatus already described. 2 Control tubes which had been prepared in identical manner but not centrifuged were allowed to stand for a comparable time and also sampled. All the samples were immediately tested for infectivity in mice and for capacity to agglutinate chicken red blood cells. 3
The results of a typical experiment are shown in Fig. 1. Repeated tests with allantoic fluids containing either A or B virus strains after centrifugation at 12,800 r.p.m. for 28 minutes showed the following characteristics:
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