Comparison of Precipitating Action of Basic Dyes on Bacteriophage and Bacterial Proteolytic Enzymes

Safranin is able to remove a number of biological substances from solution. Robertson 1 observed that safranin when added to a solution of trypsin would cause a precipitate. Holzberd 2 showed that this precipitate was proteolytic and later Marston 3 proved that not only safranin but all the azin dyes removed trypsin and pepsin from solution. The azin dyes also quantitatively precipitate bacterial proteolytic enzymes. 4 Vinson 5 reported that the virus of the mosaic disease of tobacco was completely removed from the infected plant juice by safranin. Krueger and Baldwin, 6 in their studies on the inactivation of bacteriophage, used safranin as one of the inactivating agents, and observed a precipitate which partly but not completely removed the phage from solution.

We examined the action of the azin and other basic dyes on bacteriophage and compared the results with those obtained by the use of the same dyes with bacterial proteolytic enzymes.

The following chemical types of basic dyes were used: azin, thiazin, oxazin, xanthane and phenylmethane. The bacteriophage employed was a coli-phage made in synthetic medium and free from all protein except bacterial. The titer of the phage was 1x10^-9. One cc. of the phage and 7 cc. of physiologic salt-solution were placed in a 15 cc. tube and 2 cc. of M/70 solution of the dye in water added. The mixture was allowed to stand for 18 hours at room temperature, and then centrifuged. The supernatant fluid was poured off and filtered through a Berkefeld N filter. This was found to be necessary as the unfiltered supernatant fluid of both the phage and the enzyme showed the presence of small amounts of lytic and proteolytic substance. The precipitate was taken up in 10 cc. of a pH 6.8 phosphate buffer solution.

Both the precipitate and filtrate were diluted in plain broth and tested for phage in the usual manner. For the bacterial proteolytic enzymes, a Berkefeld filtrate of a 4-day culture of B. pyocyaneus in synthetic medium was used in the same manner as the bacteriophage. The test for the presence of the proteolytic enzyme was by inoculating the filtrate and suspended precipitate into carbol-gelatin, incubating at 37°C. for 48 hours, placing in the icebox for 1 hour, and then observing for liquefaction.