Use of trypsin to increase bacteriophage yield and to facilitate the isolation of bacteriophage-resistant mutants.

Lysis of bacteriophage-infected cells is often accompanied by the release of excess phage-induced lytic enzyme into the medium. Presumably, this enzyme facilitates lysis from within at the termination of the latent period, although details of the mechanism are lacking. The release of this enzyme by the first cells to lyse can cause premature lysis of the remaining cells, thus resulting in a reduced phage yield. Moreover, the isolation of phage-resistant, lytic enzyme-sensitive mutants is rendered difficult if not impossible. To overcome these problems, the enzyme must be destroyed or inactivated without affecting the infected or resistant cells. Micrococcus lysodeikticus strain 1 was grown in 25 ml of 1.5c% peptone, 0.7%O yeast extract, 0.2%c glucose, and 0.2% NaCl (pH 7.5) in 125-ml Erlenmeyer flasks. Incubation was at 35 C on a reciprocal shaker. The Ni bacteriophage used throughout the study was described previously (2) and was quantitated by the overlay method of Adams (1). Lysates were prepared by infecting a 20%,inoculum of a 24-hr M. lysodeikticus strain 1 culture with the desired level of Ni phage and by incubating with shaking at 35 C until clearing was complete (usually 3 to 4 hr). It was previously determined that the Ni-induced lytic enzyme which appears in the lysate is sensitive to several proteolytic enzymes (2). Since the conditions existing in the growth medium closely approximated ideal conditions for trypsin activity (i.e., pH 7.5), this enzyme was chosen for further study. Addition of filter-sterilized trypsin (1:250 trypsin, Difco) to the 0.04% level in the growth medium had no detectable effect on either the growth of M. lysodeikticus strain 1 or on the adsorption of NI phage to the cells. However, the presence of trypsin in the growth medium did result in increased phage yields at all phage to cell ratios. The magnitude of the increase varied from 3 to 1,300% in various experiments, but the flask containing trypsin always yielded the largest num-