SUSPENSIONS. Spores of the parent strains could survive exposure to a temperature

Transcription

1 THE RECOVERY OF BACTERIOPHAGE FROM FIL- TRATES DERIVED FROM HEATED SPORE- SUSPENSIONS PHILIP B. COWLES Department of Immunology, Yale University School of Medicine, New Haven, Connecticut Received for publication, March 11, 1931 In recent papers by den Dooren de Jong (1930, 1931) dealing with bacteriophages for B. megatherium the author states that of eighteen megatherium strains which he was studying three were lysogenic, that is, their broth culture filtrates contained bacteriophages capable of causing lysis of other susceptible strains. Spores of the parent strains could survive exposure to a temperature of 100 C. for five minutes, and the filtrates of the cultures derived from these heated spores showed all of the typical bacteriophage properties. The lytic principle itself was killed or inactivated by heating at 70 C. for five minutes. On the basis of these experiments he concluded that, as the bacteriophage alone was destroyed at 70 C., the principles found in the cultures arising from the heated spores must have been developed by the living cell itself and that, therefore, d'herelle's theory of the living nature of the bacteriophage is not tenable. This conclusion seems warranted if we admit that all bacteriophage present in a culture is actually destroyed by heating at 90 or 100 C. The principle, when free, undoubtedly is so destroyed or inactivated, but if some of it is incorporated in the spores when they are formed, it is not impossible that it may be protected by the same mechanism, whatever it is, that enables the spore to withstand temperatures lethal to vegetative cells. This would permit an alternative explanation. If the latter assumption is true, the lysogenic strains mentioned above may have been contaminated with bacteriophage when 119

2 120 PHILIP B. COWLES they were originally received. Such strains are not common, for only three were found among the 18 studied, and an equal number of cultures obtained from various sources and examined in this laboratory have all been bacteriophage-free when tested against two susceptible strains, No. 1 and No. 338b. Through the courtesy of Dr. den Dooren de Jong there were secured several of his megatherium strains, including one, No. 899, which apparently produced bacteriophage, and the sensitive No. 338b, and his experimental results were readily verified. It seemed advisable, therefore, to determine whether or not lysogenic strains in which the lytic agent was carried through the spores could be produced from known bacteriophage-free strains of spore-forming organisms. For this purpose the following organisms and their homologous bacteriophages were studied. B. megatherium and B. anthracis (Cowles, 1930; 1931), a strain of B. subtilis obtained from Dr. d'herelle, and Cl. tertium. Incidentally, the bacteriophage for Cl. tertium is the first to the writer's knowledge that has been found for any of the clostridia. Its isolation is of some interest since it confirms what has always been assumed-that the phenomenon of transmissible autolysis is present among members of this group as well as among the aerobes. With B. megatheriurm and Cl. tertium, results were obtained indicating that after proper contacts between organism and bacteriophage, the latter could be carried through the heated spores, but for several reasons these two species were not so well suited to demonstration purposes as B. anthracis and B. subtilis, to which, accordingly, attention was more particularly directed. The Strasbourg strain of B. anthracis was used. This is easily lysed, but develops within twenty-four hours a secondary growth which is resistant to the lytic action. Resistance is maintained through numerous cycles of spore formation, even when the spores are heated between each cycle to kill vegetative cells and free bacteriophage, and furthermore bacteriophage can usually be demonstrated in the filtrates of such cultures. In sevteral instances spore suspensions were sealed in glass ampoules and subjected, in the water-bath, to temperatures of 80, 85, 90, 950C8. for

3 RECOVERY OF BACTERIOPHAGE FROM FILTRATES 121 ten minutes and to 100 C. for five minutes. Following the heating, small amounts from each tube were plated and the remainder inoculated into broth. In every case abundant growth resulted from the samples heated at 80, 85, and 90 C., but only a few spores, if any, survived 950, and none 100'C. Filtrates from broth cultures of the first three consistently showed lytic power. Those from cultures of the 950 specimens did not. Forty colonies were picked from the various plates and inoculated into broth, but in no case could bacteriophage be demonstrated in the filtrates, although all 40 sub-strains were resistant to lytic action. Mass inoculations from the plates, however, did show the presence of the lytic agent. Apparently the percentage of spores of this strain, so modified that they carried or produced bacteriophage, was relatively small at this stage in its history. With the strain of B. 7negatherium obtained from Dr. de Dooren de Jong (No. 899) this was not the case. Spores of this strain, subjected to the same treatment showed ready survival at 80, 85, and90 C.,but not at 95 or 100 C. All broth culture filtrates of the first three, and all colonies picked from plates seeded with the heated spores showed the presence of bacteriophage. This particular strain seems to have been well stabilized in its relationship to the lytic agent. The subtilis strain differed somewhat from the above in its characteristics. The race of bacteriophage used was of great potency and caused complete and permanent lysis. A resistant form of growth, however, was obtained by heavily seeding an agar plate with organisms, streaking it with bacteriophage, and then culturing on agar the growth which resulted along the bare lysed area. Several transfers of this resistant variant gave a strain, the spores of which seemed to carry bacteriophage just as had those of B. anthracis and B. megatherium. The subtilis spores could withstand a temperature of 90 C. for ten minutes or 100 C. for five minutes, and the growth resulting from these spores contained bacteriophage. Some, but not all, of the colonies picked from plates seeded with such heated spores gave cultures yielding lytic filtrates.

4 122 PHILIP B. COWLES Some evidence was obtained, particularly with the anthrax strain, that repeated sporulation, heating and growth, weakened or eliminated the bacteriophage (or the ability to produce it), without affecting the resistance of the cells to the lytic agent. Tests on the thermal death points of the bacteriophages used in these experiments showed that 75 C. for ten minutes was sufficient to inactivate them, indeed, the lytic powter of the anthrax bacteriophage was destroyed at 60 C. in that time. The observations reported above indicated that den Dooren de Jong's results in his experiments nmay bear another interpretation. Since heating spores to temperatures of 80, 90, or 100 C is not necessarily sufficient to render them bacteriophage-free a fact which has been tacitly assumed oir explicitly stated in most papers dealing with this subject, it follows that lysogenic strains may very well have been developed by contact with bacteriophage at some stage in their history. Experiments designed to show that bacteriophage can arise spontaneously in a culture have always been criticized rather severely, although some of the evidence is very suggestive and has been summarized by Hadley. MIore recently Smith and Jordan have reported observations on a culture of C. diphtlheriae in which, at irregular intervals, bacteriophage could be demonstrated, although in the interims none could be found. The difficulties of carrying on such experiments in laboratories where much bacteriophage wa7ork is being done are obvious, since the possibilities of contamination, due to the ease with which bacteriophage corpuscles are dissemninated, are ever present. In the experinents reported in this paper it may be, of course, that the bacteriophage as such does not survive the heating process, but that it has so stimulated the bacterial cell that upon subsequent growth the lytic agent is again produced. However, until more is known of the mechanism by which spores are rendered thermotolerant, it does not seem possible to reject without consideration the idea that the bacteriophage, if incorporated in the cell Nwhen sporulation occurs, may be protected from the effects of heat by that samr-e mechanism.

5 RECOVERY OF BACTERIOPHAGE FROM FILTRATES 123 SUMMARY That bacteriophage may be demonstrated in growth resulting from spores which have been heated to a much higher temperature than that tolerated by free bacteriophage was shown by den Dooren de Jong, who on this basis concluded that the lytic principle was generated in the cell. In this paper evidence is presented to show that known bacteriophage-free strains of several spore-forming organisms may be so changed by the action of the bacteriophage that the principle can be demonstrated in filtrates of cultures developed from heated spores. This does not necessarily mean that the lytic agent as such survives the heating process, but it does mean that the recovery of a lytic principle from a pasteurized culture is not conclusive proof of the spontaneous generation of bacteriophage. REFERENCES COWLES, P. B. (1930) The bacteriophagy of a spore-forming organism. Jour. Bacterial 20, COWLES, P. B. (1931) A bacteriophage for B. anthracis. Jour. Bacteriol, 21, 161. DEN DOOREN DE JONG, L. E. (1930) The bacteriophage of B. megatherium. Proe. Kon. Akad. Wetensch te Amsterdam, 33, 1-5. DEN DOOREN DE JONG, L. E. (1931) Studien iber Bakteriophagie I,II. Cent. f. Bakt. I, Orig., 120, HADLEY, PHILIP (1928) The Twort-d'Herelle phenomenon. Jour. Infect. Dis., 42, 263. HADLEY, P., AND JIMENEZ, B. (1931) Production of bacteriophage by enforced dissociation. Jour. Infect. Dis., 48, 176. SMITH, GEO. H., AND JORDAN, E. F. (1931) Distribution of diphtheria bacteriophage. Yale Jour. Biol. and Med., 3, 423.