Agar Layer Method for Production of High Titer Phage Stocks.

Abstract

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.

Compatibility Tests between Genetic Linkage Groups in a Strain of Staphylococcus aureus

“…This was propagated at 30 "C on the various donor strains by the soft-agar overlay method of Swanstrom & Adams (1951). Bacteria were removed by Millipore filtration.…”

The relative resistance of f2 bacteriophage to inactivation by disinfectants

“…The original f2 stock from which pools of f2 bacteriophage were grown was obtained from Dr Norton Zinder, Rockefeller University, 66th St and York Avenue, New York. f2 bacteriophage pools were produced by the agar layer method of Swanstrom & Adams (1951), which was originally designed for the production of the T series bacteriophages. Escherichia coli (E. coli) k37 +.…”

An additional set of phages to characterize epidemic methicillin–resistant Staphylococcus aureus strains from Spain (1989–92)

“…The source and origin of the lysogenic and propagating strains are shown in Table 1. They were propagated by the soft agar overlay method [12] and their lytic spectra were determined (Table 2).…”