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Purification, pH Stability and Sedimentation Properties of the T₇ Bacteriophage of Escherichia Coli¹

From the Department of Surgery and the Division of Dermatology and Syphilology of the Department of Medicine, Duke University School of Medicine, Durham, N.C.

Abstract

Concentration and purification of the T₇ bacteriophage of Escherichia coli, strain B, has been effected through the combined procedures of preliminary centrifugation in the Sharples supercentrifuge, filtration of the concentrate through a Mandler candle and two cycles of alternate low- and high-speed spinning in the vacuum ultracentrifuge. Preliminary chemical analysis showed contents of N 12.3; P, 3.8; and carbohydrate, 16.5 and 16.8 per cent, the latter values obtained by copper-reduction and orcinol methods, respectively. The nucleic-acid content calculated on the basis of P was about 38 per cent. The diphenylamine reaction was positive, and evidence of the presence of glucose and ribose was obtained by paper chromatography. The apparent partial specific volume was dependent on virus concentration. The value of the partial specific volume obtained with 12.1 mg virus per ml was V = 0.68. The average yield of phage was 0.61 mg per liter of lysate, giving a total of 197 mg phage from 323 liters of lysate. The lytic unit of the purified material was 10^–15.4 g.

The pH-range optimum for stability of infectivity was 6 to 8, and that for physical stability of the virus particles, studied in the ultracentrifuge, was 5 to 10. In the range of stability, a single sharp boundary was observed and the sedimentation-rate was essentially constant in this region, having a value of about 470 S. Beyond this range at both ends, degradation occurred giving rise to two components, 464 S and 19 S on the alkaline side and 381 S and 35 S on the acid side. The sedimentation-constant at pH 6.9 was dependent on virus concentration, declining from values of about 470 S at concentrations of 4.7 to 0.25 mg per ml to 325 S at 0.021 mg phage per ml.

Electron-micrographs revealed essentially spherical particles with a bilobular arrangement of internal structures; the diameter of the images in conventional micrographs was 51 mµ and that in shadowed preparations 73 mµ.

Footnotes

¹ This investigation was supported by a research grant to Duke University from Lederle Laboratories, Inc., Pearl River, N. Y.; from the National Cancer Institute, U. S. Public Health Service; and by the Dorothy Beard Research Fund.