Bacteriophage stocks prepared by the lysis of fluid bacterial cultures usually average 1O1O infectious particles per ml and rarely exceeded lO"/mI. A method of achieving higher virus concentrations than this is desirable. Luria( 1) suggested a modification of a method originally used by Hershey( 2 ) which under proper conditions gives a virus stock about 10 times as concentrated as those obtainable in fluid cultures. This method has been briefly described(3) and has been used successfully by Cohen(4) and by Marshak (5) for the preparation of purified phage suspensions. The purpose of the present paper is to describe the method in general terms and to point out those significant variables which should be investigated and controlled by anyone wishing to use the method with a new phage-host cell system. Materials and methods.In essence the method consists in the lysis of a dense suspension of the host bacterium by the phage in a thin agar layer which has been poured over a much thicker base layer of nutrient agar. When bacterial lysis is complete ,the upper agar layer is scraped from sthe plates and the phage is extracted with minimum amounts of broth. A sample experiment using coli phage T2r+ will illustrate the method. The basal agar contains 0.8% Difco nutrient broth, 0.5% NaCl and 1.5% agar. About *Aided by a grant from the Xational Foundation for Infantile Paralysis. This paper is the essence of a thesis submitted by M. Swanstrom in partial fulfillment of the requirements for the degree of M.S. in the Graduate School of -4rts and Sciences of S e w York University.-1. Luria, S. E., Personal communication.
It has been noticed previously that certain viruses can be rapidly inactivated by shaking or by bubbling gases through the virus suspensions. CampbellRenton (1) studied the effect of violent mechanical shaking on bacteriophages and found them to be fairly rapidly inactivated, at rates which were characteristic for each phage. Grubb, Miesse, and Puetzer (2), while studying the effect of various vapors on influenza A virus, noted that bubbling air at the rate of 1 liter a minute through the virus suspension resulted in detectable reduction in infectivity in 10 minutes. In a somewhat more extensive study McLimans (3) found that both Eastern and Western strains of equine encephalitis virus were rapidly inactivated by shaking in buffered saline suspensions. The inactivation also occurred when gases were bubbled through suspensions of the virus. The rate of inactivation was the same whether oxygen or helium was the gas used, indicating that the inactivation was probably a physical process, rather than the result of chemical interaction between virus and gas. He also noted that the rate of inactivation increased markedly as the pH was reduced from 7 to 5, though control suspensions at rest suffered no inactivation.The inactivation of certain physiologically active proteins such as enzymes (4) and toxins (5) on shaking is a familiar phenomenon. Perhaps not quite so well known is the fact that this kind of inactivation can be specifically prevented by the presence in the diluent of very small amounts of proteins. It has been demonstrated that the spreading of a protein at a gas-liquid interface results in the denaturation of the protein, since the spread protein becomes completely insoluble in water (6) Presumably the r61e of the shaking or bubbling in the inactivation of viruses and physiologically active proteins is simply that of enormously increasing the area of the gas-liquid interface, and hence increasing the chances of the susceptible protein being spread on that surface. This paper is devoted to the kinetics of the inactivation of bacteriophage by shaking and to the effect of environmental influences on the rate of inactivation. Materials and MethodsThe group of seven coli-dysentery phages studied by Demerec and Fano (7) was used.
Burnet and McKie (1) in 1930 published a report on balanced salt action as manifested in bacteriophage phenomena. They found that coli and staphylococcal bacteriophages when diluted in 0.1 N solutions of sodium, potassiam, and ammonium salts were much more susceptible to the inactivating effects of temperature (60°C.) than were the same phages diluted in broth. The addition of a small amount of calcium, magnesium, or barium salt partially or completely prevented this inactivation. They interpreted this phenomenon as another example of the physiological ion antagonism studied by Ringer, Jacques Loeb, and many other physiologists. Gratia (2) has drawn similar conclusions from experiments on the stability of a megatherium phage in salt solutions.In a study of the properties of the coli-dysentery phages in chemically defined media we have found that these phages exhibit a similar phenomenon, which, however, cannot be explained on the basis of ion antagonism. The present paper is a report on the kinetics of inactivation of phages in the presence of various ions and on the effect of the environment on the rate of inactivation. Materials and MethodsThe group of seven coU-dysentery phages studied by Demerec and Fano 0) was used. Certain properties of this group of bacterial viruses have been summarized by Delbriick (4). These phages were grown on Escherichia coli, strain B, in a chemically defined medium containing per liter 1 gin. NH4CI, 0.1 gin. MgSO~, 1.5 gm. KH~PO4, 3.5 gin. Na~HPO4, and 9 gm. lactic acid. The medium was adjusted to pH 6.5 by the addition of NaOH. Since phage T5 is not produced in the absence of calcium ion, calcium chloride to a final concentration of 0.001 ~x was added when preparing stocks of this phage. All phage stocks used contained between 10 ° and 101° plaque-forming particles per ml. All phage assays were made on strain B of E. coli using the agar layer technique of Gratia as modified by Hershey (5). The plating medium was Difco nutrient agar to which 0.5 per cent of sodium chloride was added. The broth used in certain experiments was Difco nutrient broth with 0.5 per cent of sodium chloride. The pH of these broth-containing media was 6.8.All glassware used in this study was cleaned with acid dichromate and repeatedly rinsed with hot distilled water since, as will be shown later, very small amounts of * Aided by a grant from the
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.