Ultrafiltration Studies on the Bacteriophage.

Abstract

Studies on the magnitude of the bacteriophage corpuscle have been carried out by a number of investigators with widely different result. Thus Bechhold and Villa. 1 Prausnitz, 2 and Stassano and de Beaufort 3 assigned a diameter of about 20μμ to the lytic particle; Eliava and Suarez 4 conclude that the diameter is considerably less than 20μμ, while Zinsser and Tang 5 place it within the range from 20μμ to 100μμ.

The mechanism of ultrafiltration, a procedure usually employed in making such determinations, has recently been critically studied by Hitchcock, 6 , 7 , 8 Grollman 9 and others, who emphasize the necessity of conducting experiments of this kind under as simple and well controlled conditions as possible.

In ultrafiltration studies on the bacteriophage more ideal experimental conditions are undoubtedly realized by employing protein-free suspensions, a procedure made possible by the work of Arnold. 10 Arnold devised a method based upon the diffusion of the lytic principle into agar whereby one may prepare highly active bacteriophage suspensions which neither give any chemical reaction for proteins, nor, when injected into animals, produce antibodies directed against the proteins normally associated with the bacteriophage.

The question of the magnitude of protein-free bacteriophage is of particular interest because of Bronfenbrenner's 11 conclusion that the substance responsible for the lytic action of bacteriophage is adsorbed on colloidal particles of various sizes. We decided, therefore, to undertake ultrafiltration studies for the purpose of eliciting any difference in particle size between purified (protein-free) and non-purified bacteriophage.

A highly virulent race of anti-coli bacteriophage (Phage B) was used in our experiments and was propagated exclusively on one strain of B. coli (k-13). The non-purified lysate in Martin's broth was adjusted to the same lytic titre as that of the purified phage extracted from agar according to the method of Arnold. These latter extracts were consistently protein-free when tested with the ordinary chemical tests for proteins.