Centrifugation in Field-aligning Capsules: Preparative Microcentrifugation1

Journal/Book: Reprinted from SCIENCE February.27 1953 Vol. 117 No. 3035 pages 221-223.. 1953;

Abstract: Virus Laboratory University of California Berkeley 1This research has been supported in large part by a grant from the American Cancer Society upon recommendation of the Committee an Growth of the National Research Council. Preparative high-speed centrifugation is commonly employed for fractionating and concentrating many biological entities of small size. Some of these materials notably viruses may be obtained as highly active agents even in extremely small quantities. This circumstance makes it appear that microcentrifugation methods can be advantageously applied to those cases where only fractional milliliter samples of source material are available-quantities too small to be handled with the usual preparative centrifugation methods. A microtechnique for improving the specific activity of minute amounts of sedimentable materials serves also as a useful adjunct to microchemical and microphysical assay methods. Severe difficulties arise in standard centrifugation procedures when attempts are made to secure successive depositions of a small amount of material in round-bottom plastic tubes of the size commonly employed in angle rotors since excessive losses of recoverable material occur owing to "shelving" and back-diffusion from the thinly spread deposits. Capillary centrifugation in swinging-cup rotors has previously been employed (1-3) bat the largest values of centrifugal acceleration obtainable with such rotors are too small to pellet most viruses and macromolecules. A method that might be described as "field-aligning capsule centrifugation" has been employed in this laboratory for the subfractionation of very small pellets obtained from 10-ml volumes of clarified supernatant fluid of virus-infected tissue cultures and controls. Pellets obtained initially by use of conventional equipment are resuspended in 0.01-0.1 ml of diluent fluid wild centrifugation of this resuspension in small glass capsules compacts the particulate components into tips tapering to microscopic dimensions. The capsules are so prepared for centrifugation as to include an air babble which serves initially to float the capsules vertically in standard plastic centrifuge tubes filled with a liquid adjusted to a suitable density. During the centrifugation cycle the long axis of the capsule is constantly aligned with the vector resultant of the three forces acting upon it: gravitation and the radial and tangential forces resulting from the rotational motion. Effectively the alignment of the capsule is as perfect as though it were a cup swinging on a truly universal bearing. Two stages of preparation are required prior to centrifugation (a) encapsulation of the material to be centrifuged and (b) flotation of the filled capsule. ... ___MH

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