Charles Korb

On the factors influencing the appearance of placques of bacterial lysis.

If the activity of “bacteriophage” is studied upon agar cultures spots or so-called placques of lysis are seen to appear, instead of a homogeneous growth. As a rule, these clear spots stand out quite distinctly against the background of agar, but occasionally one observes among them a certain number of spots which may be considerably less distinct than others. The variations among the spots are even more marked as regards their size. The placques may vary in the same plate from the size of a pin-prick to that of a size larger than the head of the pin. A number of authors have succeeded in demonstrating that by isolating and propagating each of the variants one can obtain pure strains of “bacteriophage”—showing very little if any variation in the size or distinctness of the placques (or spots). In their opinion the co-existence in one plate of spots of different size is indicative of impurity of the lytic strain. Having in our possession a strain of “bacteriophage” which showed a tendency to give rise to spots of lysis of various sizes, we considered it impure and attempted to isolate the several component strains by the usual method of daily transfers. When we found that pure strains of “bacteriophage” thus secured would unexpectedly show marked changes in respect to the average size of spots or their distinctness, we attempted to ascertain the cause of the changes. 1. Within certain limits, the average size of the placque changes inversely with the number of bacteria susceptible to lysis present in each cc. of agar. 2. The average size of the placque depends upon the relative concentration of young and old individuals in the culture—the greater the number of old bacteria, the smaller the spots.

On variants of B. pestis caviae resistant to lysis by the bacteriophage

In preliminary experiments last year we found that bacteriophage therapy did not influence the course of experimental mouse typhoid, produced by the feeding of B. pestis caviæ to mice. We were led, therefore, to inquire into the tendency of this organism to develop resistance to lysis, and to compare the virulence of such resistant strains with that of the original culture. It was found that therapeutic administration of bacteriophage did not lead to production of resistants in vivo during the ten days period. Nevertheless, the mortality of animals treated during this period was the same as that among the untreated, infected controls. Moreover, when the resistants were produced from the culture of B. pestis cavia in vitro, it was found that such resistants are avirulent. When fed to or injected into mice these resistants did not recover their susceptibility to lysis, and when recovered from animals killed for this purpose they were found to remain resistant and avirulent. These findings indicate, therefore, that the failure of bacteriophage therapy in experimental mouse typhoid is due not to production of resistants, but to failure on the part of the bacteriophage to destroy all the susceptible bacteria. It was found that resistants isolated from the lysed cultures of B. pestis caviæ maintained their resistance to lysis during twentyfive passages on agar, at frequent intervals. When transferred to broth, however, one group of resistants (namely, those which yielded an agglutinated growth upon first transfer to broth) became susceptible to lysis after 5 to 7 daily passages. The other group of resistants (yielding a diffuse growth in broth) failed to become susceptible to lysis even after 120 daily passages in broth. Simultaneously with the recovery of susceptibility, the cultures of the first group regained the degree of virulence comparable to that of the parent culture of B. pestis caviæ.