Standardization and Relative Purification Technique with Plant Virus Preparations. ∗

Abstract

The usual procedure in the preparation of an infective juice for inoculation or other experimental purposes, in the case of a virus like that of typical tobacco mosaic, consists first in thoroughly crushing or grinding the fresh tissue and then separating the coarser material by filtering off the clearer juice through cotton or cloth. Pressure also may be applied to the crude pulp. The crude juice thus prepared will contain the virus in what appears to be the original concentration. Often this procedure may be improved upon by a simple freezing and pressure technique. With the latter the softer tissues (young stems and leaves) are frozen for a few hours at—20°C. or below, for a period of 4 hours or less, then pressure of 10,000 lbs. or more per square inch is applied. With this treatment a clearer juice is obtainable.

By the method of grinding commonly employed, the crude juice contains varying quantities of starch, proteins and other solid and highly viscous reserve products; likewise chlorophyll, plastid, and protoplasmic particles. If ground in a mortar there is also a large fraction of comminuted cell wall material. The presence of these coarser suspension and colloidal particles is unfortunate in such studies as involve, for example, filtration, adsorption and aggregation, cataphoresis, or various other physical or chemical treatments. Moreover, these coarse particles may vary widely in quantity, and will be related to significant changes in viscosity with plants grown under different environmental conditions. They also interfere with quantitative work involving accurate standardization.

The elimination of coarser particles has been variously accomplished, filtration and centrifugation being often employed. The ordinary laboratory centrifuge alone is inadequate. Filtration through porous filter candles or even alundum cups is slow and likely to give a product very diverse in virus concentration, depending upon kind and quantity of viscous materials present.