application are dependent to a considerable extent upon r6le of filters in removing bacteria was the only factor

Transcription

1 FILTRATION OF BACTERIOPHAGE NEWTON W. LARKUM, AND MARGARET F. SEMMES Michigan Department of Health, Lansing Received for publication August 22, 1929 Problems concerning filtration are constantly assuming a greater significance in bacteriology and in immunology. A most encouraging aspect of this situation is that the idea of adsorption is now generally accepted whereas, until quite recently, the mechanical r6le of filters in removing bacteria was the only factor considered. This has led to a considerable advance in our knowledge of the nature of the materials filtered. In the study of socalled filterable viruses and of bacteriophage in particular the rapid advance in understanding of filters has been productive of some outstanding achievements. Among these has been the demonstration by Kramer (1927) that by use of materials of positive charge, such as plaster of Paris, substances which passed the usual negatively charged filters could be removed from suspension. In the study of bacteriophage it may readily be seen that much use might be made of such findings. Problems relating to the nature of the bacteriophage as well as to its therapeutic application are dependent to a considerable extent upon filtration. Such questions as the relationship between the bacteriophage and the "dissolved" bacterial proteins, the size of the bacteriophage particle, and the charge which this particle carries, may well utilize filtration as a possible means of solution. Qualitative and quantitative changes in bacteriophage as a result of filtration have engaged the attention of many investigators, and Todd (1927), who demonstrated that in acid reaction bacteriophage is inhibited in its passage through a Berkefeld filter, has suggested a type of research which should yield much information. We have attempted to verify Todd's work and to extend it. 213

2 214 NEWTON W. LARKUM AND MARGARET F. BEMMES EFFECT OF H-ION CONCENTRATION ON FILTRATION OF BACTERIOPHAGE Mandler Filters The usual reaction for the filtration of bacteriophage is on the alkaline side of neutrality since the material is prepared in broth at a hydrogen-ion concentration of ph 7.6 to 7.8. The question naturally arises then, as to the possibility of changing the amount of bacteriophage in filtrates by changing the hydrogen-ion concentration. There are known limits to such a procedure for the principle is inactive at ph 4.0 and It is the generally accepted opinion that the principle has a negative charge. Hence, as the reaction of the medium becomes acid, it might be expected that the bacteriophage would assume a positive charge and be retained by the filter which formerly aliowed it to pass. Conversely, increase in alkalinity of the medium might result in an increase in the amount of bacteriophage passing through the filter. In brief, the problem concerns the optimum hydrogen-ion concentration for maximum yield of bacteriophage. Todd (1927) has shown that an acid reaction reduces the bacteriophage yield. At a hydrogenion concentration of ph 5.0, filtration of a particular bacteriophage race was inhibited. Whether this result holds for all bacteriophage races remains to be shown. For this reason we utilized two races, certainly different from Todd's and quite different from each other. Both were polyvirulent races, one active against gram-negative bacilli of the enteric group, the other active against staphylococci. Passage of these races through six Mandler filters of nearly identical pressure tests showed that no bacteriophage was adsorbed by any of the filters when the reaction of the liquid phase was about neutral. Determination of the amount of protein nitrogen present in the filtrates showed a maximum variation of i37 mgm. In succeeding experiments, differences in excess of this figure are considered significant. The reason for these determinations will appear later. Bacteriophage active against enteric bacilli was distributed in flasks, to each of which a quantity of hydrochloric acid or sodium

3 FILTRATION OF BACTERIOPHAGE 215 hydroxide was added sufficient to bring the reactions to ph 4.0, 5.0, 6.0, 8.0, 9.0, and 10.0 respectively. The reactions were checked by electrometric methods. About 50 cc. of the contents of each flask was filtered through one of the six Mandler filters mentioned above, and the filtrates were titrated for bacteriophage content according to the method of serial dilution. Plates were made and plaque counts were used to check the readings in the broth. The results are given in table 1. TABLE I Typhoid bacteriophage-mandler filters LYSIS IN BROTH AT DILUTION MILION NITRO- PLAQUES GEN IN FILTER ph PER CUBIC GRAMS 1-' o-9 CENTI- PER METER 100 cc. 4.0 _ 10 A _ B _ ,500 C , () , ,500 D , ,500 E , ,000 F In this exper4ment, the phage used had already been filtered once as a stock preparation. As a check upon these results and in order to determine whether development of phage in different reactions would alter the findings, flasks of broth were adjusted to the reactions indicated, inoculated with identical quantities of culture and of bacteriophage, and incubated. Control flasks were inoculated with culture but not with bacteriophage. Lysis occurred to some extent in all flasks. The contents of these flasks were then filtered through the same Mandler filters used in the previous experiment and both filtered and unfiltered lysates were titrated. The results are given in table 2.

4 216 NEWTON W. LARKUM AND MARGARET F. BEMMES Our stock staphylococcus bacteriophage is in many respects strikingly different from the principle active against Gramnegative organisms. Its action is less rapid; it does not permit the development of secondary cultures; and it is in many other particulars a very peculiar and well defined race. Nevertheless, when studied in the same manner as the bacteriophage used in FILTEr A B C D E F A B C D ph TABLE 2 Typhoid bacteriophage-mandler filters 10 LYSIS IN BROTH AT DILUTION 10-' I ' I LMILION PLAQUES PER CUBIC CENTI- METER , ,500 1,200 2,500 1,500 3,000 2,000 2,000 1,000 2,500 1,200 2, NITROGEN IN GRAMS PER 100 cc the preceding experiment, it yields identical results as is shown in the protocol in table 3. filters The above experiments were repeated many times with Mandler and with filters. The latter were used not only to confirm the results obtained with Mandler filters, but to investigate the alleged inferiority of this type of filter for bacteriophage work. Some statements have appeared in the literature relative to the 1+7 +

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6 218 NEWTON W. LARKUM AND MARGARET F. BEMMES FBILTE ph TABLE 4 Staphylococcus bacteriophage- filters ' LYSIS IN BROTH AT DILUTION ' " t4tt-t-tt 44- tt t t a. I _ TABLE 5 Typhoid bacteriophage- filters MILLION PLAQUIB PER CUBIC CENTI- METER PLAQUES LYSIS AT DILUTION IN NITRO- MILLONS GUN IN FILTER ph - -PER GRAMS CUBIC PER ' 10' ' 10-' CENTI- 100 cc _ , , _ NIVTRO- GEN IN GRAMS PER 100 cc o-

7 FILTRATION OF BACTERIOPHAGE adsorption of bacteriophage by filters which did not check with daily experience in our laboratory. Gildmeister and Herzberger (1924) specifically claim that filters allow less lysin to pass than do Berkefeld filters; that they change bacteriophage quantitatively; and that with this type of filter adsorption plays no r6le. The latter statement can definitely be refuted by a simple experiment: Kramer (1927) has shown that negatively charged filters permit the passage of negatively charged dyes and retain those that are positively charged. Thus, a Mandler filter will decolorize Victoria blue and permit the passage of Congo red. filters behave in exactly the same manner, except that more dye is required to saturate the filter than is required with Mandler ifiters of equivalent size. Obviously then, specific adsorption is as much a property of as of Mandler or of Berkefeld filters. The effect of filters on bacteriophage is shown in the protocols in tables 4 and 5. These protocols are but two of many similar experiments, all giving equivalent results. The differences between filtered, and unfiltered material within the range ph 6.0 to 8.0 are not sufficient to indicate that there is an appreciable loss of lytic principle on filtration. There is much to indicate a qualitative change. Thus if two samples give lysis at the same dilution but the degree of lysis varies as is shown below there is no doubt but that a qualitative and not a quantitative difference exists: Unfiltered Filtered Such a comparison between filtered and unfiltered phage is practically an invariable result regardless of the type of filter employed. Hence, although Gildmeister and Herzberger (1924) are correct in their statement that filters change phage qualitatively, they cannot be substantiated in their inference that Berkefeld filters do not cause such a change. To be sure we are assuming that in this respect Berkefeld and Mandler filters are alike. If they are not, the criticisms which are directed against filters must likewise be applied to the Mandler type.

8 220 NEWTON W. LARKUM AND MARGARET F. SEMMES Plaster of Paris filters A continuation of a study of this type would naturally involve an attempt to alter the charge of the filter concerned. As a matter of fact it is quite necessary to determine the extent to which the hydrogen-ion concentrations involved could change the TABLE 6 Typhoid bacteriophage-plaster of Pari8 filter PLAQUES LY ISI BROTH AT DILUTION IN NITO- MTTJLONS GEN IN FILTER ph PER GRAMS CUBIC PER ? 10- ciu:m- 100 cc. METER Plaster Plaster Plaster Staphylococcus bacteriophage-plaster of Paris filter PLAQUES LYSIS IN BROTH AT DLmUTION IN NITRO- MILLIONS GUN IN FILTR ph PER GRAMS CUBIC PER C CENTI- 100 Co Plaster ,000 Plaster Plaster charge of the filter. In order to study this, use was made of the phenomenon of electroendosmosis. By the addition of hydrochloric acid to the fluiid passing through the filter it was impossible to reverse the charge as shown by a reversal in the direction of the flow of the water, at least within the range of hydrogen-ion con-

9 FILTRATION OF BACTERIOPHAGE centration not destructive of bacteriophage. In the case of the Mandler filters this result was checked by cataphoresis. Finely ground filter particles were suspended in water and on passage of a current of electricity they migrated towards the anode. The addition of acid failed to change the direction of this migration. Thanks to the researches of Mudd (1922) and of Kramer (1927) however it was possible to study the effect of reversal of charge on the passage of bacteriophage by substituting a positively charged filter for the negatively charged and Mandler. The results are shown in the protocols in table 6. There are many practical difficulties concerned with the use of plaster of Paris filters. They are relatively less porous than Mandler or filters; they have smaller pores and consequently filter more slowly. Their action may be hastened by the use of more pressure, but their strength is not sufficient to stand more than a few pounds. They are extremely liable to contain flaws, some very minute, which permit the passage of particles which are otherwise adsorbed by the filter. The use of Congo red or some other negatively charged dye in the material to be filtered serves to indicate the presence of gross leaks. Under most favorable circumstances these filters become quickly saturated and fail to retain positively charged particles. Thus, at ph 7.0, bacteriophage may be absent from the first cubic centimeter of filtrate or even from the first several cubic centimeters depending upon the size of the filter. Sooner or later, however, it appears. Because of these difficulties we feel that our results with these filters are somewhat less reliable than those previously reported. Repeated experiments, however, have sufficiently confirmed the results shown in the two protocols to justify general conclusions. DISCUSSION A consideration of the charge of the bacteriophage particle, as well as of many problems concerning the nature of the principle, is dependent upon an understanding of the relationship between the bacteriophage and the proteins with which it is associated. The data that we have presented show that the curves for the yield of protein and of bacteriophage are parallel, regardless of the 221

10 222 'NEWTON W. LARKUM AND MARGARET F. SEMMES charge of the filter used. Thus the possibility of mechanical retention of coagulated protein and such bacteriophage as it might hold may be excluded. Whether the bacteriophage is adsorbed to the protein or whether it is affected in an identical manner by change in hydrogen-ion concentration it is impossible to state. No doubt, the use of purified phage such as is described by Weiss (1927) and by Ashehov (1928) would throw considerable light on this question. Our attempts to produce purified phage have not resulted in a product of sufficient concentration to be used in such experiments. We feel however that the results of our investigations are of sufficient interest in themselves to warrant their publication without awaiting the data that might result from the use of such material. CONCLUSIONS Bacteriophage in passing Mandler and filters is removed from suspension at hydrogen-ion concentrations of ph 4.5 to 5.0 and ph 9.0 to The behavior of these two filters in this respect is identical. Both and Mandler filters have a slight qualitative effect upon bacteriophage although neither affects quantity of phage at a neutral reaction. Plaster of Paris filters remove bacteriophage from suspensions at a hydrogen-ion concentration of ph 7.0 but permit its passage at ph 4.5 to 5.0 and ph 9.0 to They reduce the amount of protein in a similar manner. Bacteriophage, as well as negatively charged dyes, saturate plaster of Paris filters rapidly. It appears from these observations that bacteriophage is ad- Rorbed to the protein in the suspensions. REFERENCES ABHEBHOV, I. N Compt. Rend. Soc. Biol., 98, 770. GILDm1EIBTER, E. AND HERZBERGER, K Centralb. F. Bakt., 1. abt., Orig., 91. KRAMER, S. P Jour. Inf. Dis., 40, 343. MUDD, S Amer. Jour. Physiol., 63, 429. TODD, C Brit. Jour. Exp. Path., 8, 369. WEISS, E Jour. Immunol., 13, 311.