The property of influenza virus suspensions to agglutinate red blood ceils appears to be closely associated with the infective activity of the virus (1). The complement-fixing antigen, on the other hand, has been shown to consist of two distinct fractions: one is intimately associated with the hemagglutinin and infective virus particle, while the other has a smaller size and is readily separable from the virus particle by centrifugation or adsorption with red blood ceils (2). A preliminary report (3) showed that the infectivity and hemagglutinin of the PR8 and Lee strains of influenza virus were associated with particles which were at least 60 m/z in diameter. Subsequent papers by other workers (4, 5) have confirmed this latter finding and have set the size of the PR8 virus particle at 80 m# and that of the Lee virus particle at 100 m#.The present paper presents a further study of the various properties of influenza virus and their interrelationships, by means of experiments in the angle centrifuge and the optical ultracentrifuge, and by filtration with graded collodion membranes. It will be shown that the application of a synthetic density gradient, already described (6), to influenza virus suspensions minimizes convective disturbances in the angle centrifuge and permits the demonstration of sedimenting virus boundaries which can be identified by infectivity, agglutination, and complement fixation tests. MethodsPreparation of Virus Suspension.--The PR8 strain of influenza A virus (7) and the Lee strain of influenza B virus (8) were used in these experiments. Mlantoic fluid suspensions of each virus strain were prepared by a technique already described (9). Briefly, it consisted of inoculating the allantoic sac of 11 day old white Leghorn embryos with 0.2 cc. of a 10 -s dilution of allantoic fluid known to contain virus in high titer. After incubation at 37°C. for 48 hours, the eggs were chilled overnight at about 4°C. The blood-free allantoic fluids were then removed, pooled, and cleared by centrifugation at about 1800 R.1,.~r. for 5 minutes. Without further treatment the fresh allantoie fluids were subjected to the experimental procedures to be described, and all tests were made on the same day.Infectivity Tests.--The virus suspensions were diluted in steps of 10 -°'5, and 0.05 cc. samples were instilled intranasally into albino Swiss mice under light ether anesthesia. 301

A method of estimating the purity of preparations of elementary bodies of vaccinia is described. It depends on the comparison of the number of infective units of virus in a given material with the number of elementary bodies. The latter figure is estimated from the dry weight of the preparation by means of a calculated value for the weight of a single dehydrated elementary body. Values for the ratio of infective units of vaccine virus to elementary bodies varied between 1 : 2.4 and 1 : 9.2 in seven consecutive experiments; the average was 1:4.2. These ratios indicate a high degree of purity of the preparation. Moreover, they indicate that a relatively high percentage of the elementary bodies in the preparations was infective.

Ultracentrifugal studies on the CL dermal strain of vaccine virus indicate the following characteristics of the elementary bodies:— 1. A stable suspension of Paschen bodies in a dilute buffer solution of pH 6.2 to 8 sediments with the formation of a characteristic primary boundary which consistently shows a spread of approximately 14 per cent. 2. The principal sedimentation boundary is accompanied frequently by one or several more rapidly moving boundaries which probably are produced by groups of agglutinated elementary bodies consisting of two or more particles. 3. Occasionally the principal boundary may exhibit an irregular or peculiar behavior, a fact which necessitates a careful selection of material and the performance of many experiments for accurate interpretation of results. 4. The sedimentation constant of the slowest moving particles forming the principal boundary is computed to be 49.1 x 10–11 cm./sec./dyne. On the basis of this sedimentation rate, the average diameter of the smallest virus particles in appreciable amounts is estimated at 236 mµ. If the boundary spread is due principally to slight differences in particle size, then the largest single elementary bodies are approximately 252 mµ in diameter.

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1. A description is given of the construction details and operation characteristics of an improved type of air-driven ultracentrifuge operating in vacuum and suitable for the determination of sedimentation constants of protein molecules. 2. The rotor of the centrifuge is made of a forged aluminum alloy; it is oval in shape, measures 185 mm. at its greatest diameter, and weighs 3,430 gm. It carries a transparent cell located at a distance of 65 mm. from the axis of rotation and designed to accommodate a fluid column 15 mm. high. 3. The rotor has been run repeatedly over long periods at a speed of 60,000 R.P.M., which corresponds to a centrifugal force of 260,000 times gravity in the center of the cell. At this speed no deformation of the rotor nor leakage of the cell has been observed. 4. The sharp definition of sedimentation photographs taken at high speed serves to indicate the absence of detectable vibrations in the centrifuge. 5. When a vacuum of less than 1 micron of mercury is maintained in the centrifuge chamber, the rise in the rotor temperature amounts to only 1 or 2°C. after several hours' run at high speed. 6. There has been no evidence of convection currents interfering with normal sedimentation of protein molecules in the centrifugal field. 7. A driving air pressure of about 18 pounds per square inch is sufficient to maintain the centrifuge at a steady speed of 60,000 R.P.M. With a driving pressure of 80 pounds per square inch, it can be accelerated to this speed in less than 20 minutes, and also brought to rest in about the same length of time by the application of the braking system. 8. The adaptation of Svedberg's optical systems to this centrifuge for photographically recording the movement of sedimentation boundaries is described.