TEST OF A THEORY ON THE ORIGIN OF BACTERIOPHAGE l

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1 TEST OF A THEORY ON THE ORIGIN OF BACTERIOPHAGE l BY R. W. GLASER (Received for publication November 15th, 1937) Two theories concerning the nature of bacteriophage have been widely championed: first, that 'phage is a living virus which invades the bacterial cell and propagates within it; and second, that 'phage is a non-living substance derived from bacterial metabolism and having the capacity to increase in amount when brought in contact with susceptible microorganisms. According to Krueger (1936), "hypotheses embodying either of these concepts together with varying subsidiary corollaries have been advanced in considerable numbers and the arguments centering around them constitute a formidable fraction of phage literature." Although the known facts, as Krueger says, seem to favor a bacterial origin for 'phage, another theory has not been sufficiently tested experimentally. It may be that 'phage originates within the animal harboring certain bacteria and is produced as a reaction or as an autocatalytic antibody against the microorganisms. To test this notion one would have to rear an animal under perfectly sterile conditions and find it consistently free of 'phage. Then it would have to be infected with a non-lysogenic but lytically susceptible bacterium for varying periods of time, after which the animal would again be tested for 'phage. If 'phage then appeared, it would mean that the tissues of the animal had responded by forming 'phage or, at least, had participated in the reaction. The housefly (Musca domestica L.) was the animal chosen for this work because it seemed likely that this species could be reared under sterile conditions. 2 Furthermore, Shope (1927) found a bacteriophage active against four species of bacteria, in salt solution extracts of wild, 1 From the Department of Animal and Plant Pathology of the Rockefeller Institute for Medical Research, Princeton, New Jersey. The writer wishes to thank Mr. N. A. Coria for valuable technical assistance. 2 The description of a method for rearing houseflies free from microorganisms and bacteriophage has been accepted for publication by the Journal of Parasitology. 311 Downloaded from at Pennsylvania State University on February 18, 216

2 312 E. W. GLASER contaminated housefiies. This fact showed that the housefly organism and 'phage are not incompatible. Fortunately for the work, we possessed cultures of a non-lysogenic although lytically susceptible bacterium. This microorganism is a staphylococcus, S. muscae, originally isolated and described (Glaser, 1924 and 1926) from cases of a specific disease of adult houseflies. Since that time the cultures have lost their pathogenicity for flies and instead of invading the body cavity, as was originally the case, now remain exclusively within the alimentary tract. The microorganism grows readily on a variety of artificial media. It is exceedingly susceptible to the bacteriophage isolated from wild houseflies, giving complete lysis up to a dilution of 1~ 12. Nevertheless, even when originally isolated from the hemolymph of diseased flies, the staphylococcus was 'phage-free and has remained so up to the present time. For these reasons a number of investigators in this laboratory have found S. muscae a convenient indicator in their work with bacteriophages. EXPERIMENTAL Only houseflies free from bacteria and bacteriophage were used, unless, as in certain experiments, purposeful contaminations were practised. Table 1 gives the data for an initial experiment with 1 sterilized housefly eggs. A test for bacteriophage against the staphylococcus with the larval medium and the adult food yielded negative results. TABLE 1 Aseptically reared flies and their environment tested for bacterial and 'phage sterility Initial experiment 1 sterilized eggs 1st generation 3 <f 3 9 2nd generation 3 cf 3 9 3rd generation 3 cf 3 9 'Phage test against staphylococcus With medium and food With emerged flies Bacterial sterility test No. of emerged flies* * According to Hewitt and others, individual females may deposit 1 to 15 eggs. Since we used three females in our transfers more flies should have emerged than the figures in the table indicate. Crowding and other factors possibly produced this result. However, this does not matter, because the experiments were solely aimed to provide a sufficient number of adults for the tests. Downloaded from at Pennsylvania State University on February 18, 216

3 THEORY ON ORIGIN OF BACTEKIOPHAGE 313 A similar test against the emerged adults was also negative. In each test with adults, 25 newly emerged flies were ground fine, under aseptic conditions, in 1 cc. of bouillon. This material was incubated for 48 hours, filtered through a Berkefeld "N" candle and the filtrate, in dilutions up to 1~ 12, tested against the indicator microorganism. In the initial tubes.5 cc. of a 48-hour bouillon culture of the staphylococcus was added to.5 cc. of the filtrate and the tubes were incubated for 48 hours. Following this procedure six passages from each tube to fresh bouillon were always made, with 48-hour incubation periods, to be certain of the results. Sterile maggots and eggs were also, at times, tested for bacteriophage with negative results. In such cases an extract prepared from 25 maggots or from 1, eggs was used. In column four the results of bacterial sterility tests are given and, in column five, the number of adults obtained. This experiment covers a period of three generations and the results were entirely negative for the presence of both bacteria and bacteriophage. Table 2 shows the data for three separate experiments obtained through eight generations when the sterile breeding medium of sterile TABLE 2 Attempts to produce 'phage in sterile flies by feeding a non-lysogenic Experiment no. Age in days of adult flies Results of 'phage tests on 8 generations of flies Control (with naturally contaminated flies) 4 I 4-13 I + Results of 'phage tests on breeding medium for 6 transfers staphylococcus Recovery of staphvlococcus "for 8 generations flies was experimentally contaminated with a 48-hour bouillon culture of S. muscae. Although the staphylococcus was recovered at each generation, no bacteriophage was present. A control experiment (no. 4) was performed in an identical manner with the exception that naturally contaminated flies, reared in horse manure, were employed. In this case, the insects and the breeding medium showed the presence of bacteriophage up to the eighth generation, at which time the experiments were terminated. In the above outline the sterile, adult flies were tested on emergence. It was thought that perhaps not enough time had intervened to produce a reaction in the adult forms. As the experiments show, the Downloaded from at Pennsylvania State University on February 18, 216

4 314 K. W. GLASER staphylococcus and its products were in contact with sterile fly tissues through eight generations extending over a period of many weeks. However, it was possible that the adult stage alone, if given more time, might react and produce 'phage. The mature forms do not live very long and under the conditions of the experiments, rarely survived over 13 days. Nevertheless, experiments were performed by contaminating the larval breeding medium with the staphylococcus and testing the adults 4, 8 and 13 days after emergence. The microorganism was recovered in the pure state from all these flies, but tests for 'phage carried through six transfers in bouillon were invariably negative. An extract of wild contaminated adults gave complete lysis of the staphylococcus through the sixth passage in bouillon up to a titer of 1~ 12. Flies caught in nature and bred in natural horse manure for eight generations yielded 'phage at each generation of adults. Flies reared under sterile conditions were negative for 'phage at each generation during eight generations. The same was true of sterile flies contaminated with S. muscae. This shows that the staphylococcus has not the ability to stimulate the fly organism to form 'phage. Further experiments showed that 'phage did not survive long in sterile flies unless a living, susceptible bacterium was present. In one set, the maggot medium was artificially contaminated separately with 'phage and with a culture of the staphylococcus killed by subjecting it to a temperature of 65 C for one hour. 'Phage was recovered from the first crop of adults, but failed to appear again from the second to the conclusion of the experiment at the eighth generation. In a second set, the medium was purposely contaminated with 'phage alone. 'Phage was again recovered from the first crop of adults, but failed to appear in any of the later generations. In a third set, the medium was experimentally contaminated with 'phage and with a living culture of the microorganism with the result that 'phage was recovered at each generation up to the concluding, eighth crop of adults. SUMMARY AND CONCLUSIONS Houseflies caught in nature or bred in the contaminated state invariably harbored bacteriophage. By establishing, in the alimentary tract of sterile houseflies, a non-lysogenic staphylococcus known to be susceptible to lysis, no 'phage was formed. In the absence of this microorganism, 'phage, when given to sterile flies, survived for one generation; whereas, in the presence of the bacterium it persisted for eight generations and probably would have survived indefinitely. Downloaded from at Pennsylvania State University on February 18, 216

5 THEORY ON ORIGIN OP BACTERIOPHAGE 315 These results do not support the theory that 'phage is the result of the interaction of host and bacteria. BIBLIOGRAPHY GLASER, R. W A bacterial disease of adult house flies. Amer. Jour. Hyg., 4 : Further experiments on a bacterial disease of adult flies with revision of the etiological agent. Ann. Ent. Soc. Amer., 19: HEWITT, C. G The House-fly, Musca domestica Linn.; its Structure, Habits, Development, Relation to Disease and Control. Cambridge, University Press, 382 pp. KRUEGER, A. P The nature of bacteriophage and its mode of action. Physiol. Rev., 16: SHOPE, R. E Bacteriophage isolated from the common house fly (Musca domestica). Jour. Exp. Med., 45: Downloaded from at Pennsylvania State University on February 18, 216