526 THE BACTERIOPHAGE 



Since active filtrates consisting of water with a very small percentage of solids 

 (comprising, in addition to active principle, salts, tissue extractives from broth, met- 

 abolic products of bacteria, and bacterial debris) can still be diluted lo'" times be- 

 fore reaching the limit of their activity, it may be supposed that the units of the ac- 

 tive agent are free molecules of some inanimate material. 



It is possible that the lytic principle is present in the medium in a state of true 

 solution, or as a highly dispersed colloid, and that it is merely adsorbed on bacterial 

 debris and other particles, which thus determine its distribution in the medium. It is 

 possible also that the particles in the filtrate represent the "corpuscles" of autonomous, 

 organized parasites,' and can be identified with the granules observed by D'Herelle 

 within the infected bacteria under an ultra-microscope.^ 



Later investigations have indicated, however, that these particles do not repre- 

 sent autonomous units of the active agent, but merely serve as a vehicle on which 

 the active principle is adsorbed, and from which, under proper experimental condi- 

 tions, it can be detached.^ For instance, if these particles, carrying the phage, are 

 deposited on an ultra-filter through which they cannot pass, and washed repeatedly 

 with water or buffer solution, they do not give up any of the active agent. But if 

 they are washed with broth, the phage reappears in the filtrate.^ Similarly, when 

 phage is adsorbed on bacteria, it cannot be set free by washing with water, but can 

 be freed by broth. ^ 



There exists further evidence that the particles observed by D'Herelle^ and 

 measured by Bechhold,^ Prausnitz,^ and others are not likely to represent the auton- 

 omous "corpuscles" of the parasite. The lytic principle spreads on agar from a focus 

 radially. This spreading is independent of the progress of "invasion" of bacteria, 

 since it continues at 4° C* (at which temperature the invasive activity of the phage 



' d'Herelle, F.: Compl. rend. Acad, dc Sc, 165, 373. 1917; Compt. rend. Soc. bioL, 83, 247. 1920. 



^ d'Herelle, F.: Immunity in Natural Infectious Disease, p. 255. 1924; The Bacteriophage and 

 Its Behavior, p. 273. 1926. According to some investigators (Bechhold, H., and Villa, L.: Ztschr.f. 

 Hyg. n. Infektionsl^rankh., 105, 601. 1926; Stassano, H., and Beaufort, A. C: Compt. rend. Soc. dc 

 biol.,g3, 1378. 1925; Prausnitz, C: Klin. Wchnschr., i, 1639. 1922; von Angerer, K.: Arch. f. Hyg., 

 92, 312. 1924), these particles are fairly uniform in size, approaching 20 /x^ in diameter and a cal- 

 culated mass of about I TT io-'8 gm. Prausnitz, C: CentralU. f. Bakteriol., Abt. I, Orig., Suppl., 93. 

 148. 1924. 



3 Bronfenbrenner, J.: /. Exper. Med., 45, 873. 1927. 



4 Eguchi, C: quoted from Centralbl. f. Bakteriol, Ref., 78, 40. 1924-25. 



5 d'Herelle, F. : Immunity in Natural Infectious Disease, p. 255. 1924. 



^Bechhold, H., and Villa, L.: loc. cit. 'Prausnitz, C: Klin. Wchnschr., i, 1639. 1922. 



* Unpublished experiments of the writer in collaboration with Korb in 1924. Agar containinsi 

 phage and bacteria was poured into plates and incubated at 37° C. As soon as formation of plaques 

 became visible the plates were placed overnight at 4° C. The next day they were returned to the 

 thermostat. It will be seen that phage dilTused during the night beyond the periphery' of original 

 plaques. When, on the next day, the growth of bacteria was resumed (at 37° C), a halo of lysis 

 around original plaques resulted (Fig.i). 



