Purification of Phage by Adsorption and Elution

Abstract

A precise study of the nature of viruses is rendered difficult by various extraneous substances. Although these substances do not appear to affect the action of the virus on its specific host, they modify the behavior of the virus in tests designed to ascertain its nature. Recently two methods have been reported for purifying viruses. One is based on ultrafiltration, 1 the other on adsorption and elution. 2

The latter procedure is more easily carried out in the laboratory. However, it is difficult to standardize the technique and hence some authors have reported positive, others negative results. This paper deals with the standardization of the technique and the further purification of B. coli bacteriophage.

Media and Reaction: The character of the medium influences the adsorption, elution and amount of phage produced. In a synthetic medium the concentration of phage at the outset is low and elution is unsatisfactory. Adsorption is best in an acid and elution in an alkaline medium. The following protocol is typical:

Parallel tubes of broth and synthetic media were seeded at the same time with the same amounts of phage and culture, incubated 24 hours at 37°C, and filtered through a Seitz filter.

The phage titre in the broth was 10⁹, in the synthetic media 10⁵.

Method of Eluting. Adsorption is more complete at pH 5.5 than at pH 7.6 and the former adsorbate yields the best eluate. In the protein containing broth the phage is not so completely removed as in the synthetic medium. On the other hand it is apparently less firmly bound to the adsorbent and more readily removed by the eluting fluid.

The kaoline is also a variable factor. Each lot must be tested for optimal concentration.