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'n_ II I", 1 4' ‘ . I ~2 . A v, . ' ' " . ' .I \ I... 7.. . .- ~ . o , ~ 3 . " ..L '1- . l . -. o.’ 5055 ~’ ~ _" ‘ 913,...» 3. 'r- J ‘fy —£ ". ‘41: .Vr'i' ~7fi_ J. . , A I I . . .__ . , - try; 1 - ..< '5'. . :‘V—“" 1235;: . . ‘ ‘;:‘ ._ lFK/(‘p'l 1;“ “I” \- h "r‘ 1.. . ."fi'. , I,.::_fil “,fq H) . \ ‘v..."'... “t .I _. . ‘ z. ‘ " . -‘ I 5‘ J”: M“ ; [-5. )3 .3: 1" “I ’2'} n1. .1‘«; , '1 ‘ , ’i g‘ . v"b‘|t} a? :‘H‘x A 3 A. q\ ‘fl' Q‘. b::"‘3“ ‘ R I}? . 1‘“ .539? I 1. 5+... 2“ 2“. A STUDY OF THE EFFECT OF BACTERIOPHAGE ON PHAGOCYTOSIS A STUDY OF THE EFFECT OF BACTERIOPHAGE ON PHAGOCYTOSIS THESIS Submitted to the Faculty of the Michigan State College in partial fulfillment of the requirements for the Degree of Master of Science by John Richard Eudsen June 1931 JHESIS AGKNOWLEDGMENT The writer desires to express his grateful acknowledgment of the able assistance of Dr. N. W. Larkmm, Lecturer in Bacteriology, under wham.the work was carried out, and of the Faculty of the Bacteriology Division in this institution. 1033093 I. II. III. IV. V. VII. VIII. TABLE OF CONTENTS Introduction Review of the Literature Experimental Protocols Plate I Discussion Conclusion Bibliography Page 10 16 £3 8 A STUDY OF THE EFFECT OF BACTERIOPHAGE ON PHAGOCYTOSIS Introduction The discovery of bacteriophage in 1915 has opened up hitherto un- known and unsuspected avenues of approach to the problems of infection and immunity. The importance of these problems cannot be over estimated fbr with their solution we may look for’material advances in our efferts to control infectious diseases. Nething is at present known regarding the identity of the principle and very little as to its mode of operation. Nevertheless, there is an enormous amount of experimental work from which will eventually come a solution of the problem of the nature of bacterio- phage. Msanwhile it has found a few practical uses among which clinical application is outstanding. So striking have been the results in numerous instances that practice has greatly outstripped theory and we are now faced with the problem.of providing an explanation for'some of the occurrences reported and of building a thundation upon which advance- ments may rest. Among other factors, stimulation of phagocytosis is of considerable interest in connection with bacteriophage therapy, and although a minor amount of work has been reported, there are many phases of the problem in need of confirmation and further study. Since phagocytosis is highly considered as a factor in immunity, it seemed that any contribution showing the relationship between bacteriophage and this phenomenon.would be of considerable value. Although aware that the work to be reported in this paper by no means disposes of’the problem, it is felt that the results - g - obtained constitute a real and useful contribution in this field of endeavor. The work of which this paper is a report was undertaken with the hope of adding something to our present knowledge of one phase of the question, namely, the effect of bacteriophage upon the phenomenon of phagocytosis. In order properly to evaluate the results and to interpret their relationship to what has already been accomplished, it is necessary to summarize what the literature now contains on this question. Likewise in a subject as yet none too familiar to many biologists, it is advisable to present a brief description of the fundamental phenomenon involved. Review of Literature From time to time bacteriologists have observed that broth.cultures of bacteria, after having undergone an apparent initial period of growth, suddenly for some reason became sterile. The explanation of this occurr- ence was furnished by Twort (1) who discovered a principle capablenof producing this reaction at will. He was stopped, howezer, from.continuing his researches by the war. It remained for d'Herelle}to demonstrate the serial passage of the principle responsible for the clearing of these cultures and to designate a source from which it could easily be obtained. this principle desrelle named Bacteriophage, more specifically, Bacterio- phagus intestinale. While it is possible to conceive of a chemical substance capable of having Just such an effect, the potency of any champ ical decreases with dilution and upon serial passage the quantity necessary to produce the reaction would be diluted beyond its effective quantity. - 3 - Bacteriophage on the other hand is characterised by the fact that it runains effective far beyond the point of dilution at which chemicals would have any action. Also, the bacteriophage, transferred serially for an indefinite period, instead of decreasing in potency, actually increases both with respect to potency and to quantity. 'Hence the conclusion that this principle is living matter. In addition to the lytic action of bacteriophage, numerous other evidences of its activity can be demonstrated, among these the formation of plaques. These transfermations in colony appearance are characterised by clear, glassy and smooth areas containing the principle and became manifest when cultures of bacteria containing bacteriophage are cultured on solid media. The phenomenon of bacteriophagy has suggested farbreaching possibili- ties in the study of biological problems and in the treatment of disease. Countless investigators have studied the subject from every possible angle but at present the exact identity of the active substance and its mode of 'operation have eluded search and although numerous physicians have made extensive use of bacteriophage in the treatment of disease, its value has not yetbeen generally accepted. the literature bearing on the subject of bacteriophage has become so voluminous as to preclude the possibility of'making an attempt at a summary in the present work. .1 recent article states that over two thousand references on this subject exist at the present time and several reviews, among which are those by diHerelle (5) and Hadley (4), include excellent bibliographies to which the reader is referred in regard to questions not - 4 - related directly to the problun here presented. Since we are concerned in this paper with bacteriophage and its effect upon phagocytosis, it may be well to review briefly our present knowledge of this subject. no role of the leucocyte as a scavenger for the body and in immunity has long been a subject of vital interest and inquiry and methods of stimulating phagocytic activity have been investigated for over forty years. The power of the phagocyte to ingest foreign material including bacteria was known long before the time of Metchnikoff; but it was he who demonstrated that subsequent to ingestion, the process of digestion took place. For*fletchinkoff the process of phagocytosis comprised the sole protection of man against the invasion of pathogenic organisms, a point of view which gave rise to the classic battle between the cellular and humeral theories of imunity. The keen observations of this man, however, on the phagocytic activity of the polymorphonuclear leucocyte cast considerable light on this subject and laid the feundation upon which the later significance of phagocytosis was based. Subsequent discovery of other phagocytic cells of the body, partic- ularly those of the reticulo-endothelial system, have served to impart considerable impetus to the idea that the ingestion of invading bacteria by various cells of the body is of“major importance and should be taken into consideration when dealing with any infectious disease. The adequate provision made through the discovery by wright and Douglass (5) of'a method for determining accurately the phagocytic activity of cells has enabled investigators to evaluate more carefully the role - 5 - of the phagocyte in immunity and has assured the acceptance of the phenomenon as a satisfactory explanation for certain types of immunity. The work of these and subsequent investigators is so well-known that it is unnecessary to give the details of their thousands of experiments. Briefly, these experiments indicate a definite relationship between cpsonins and bacteriotropins on the one hand and the phenomenon of phagocytosis on the other. These former two substances are classed as antibodies and are, supposedly, increased by artificial immunization. Their effect upon the bacterial cell is to render it more susceptible to the phagocytic cell. Among numerous incidental observations accompanying the study of bacteriophage was a report by diflcrelle and Eliava of having observed a marked increase in the number of bacteria taken up by the phagocytic cell in the presence of bacteriophage. The details of their work, however, were not given. These workers obtained opsonic indices as high as forty and came to the conclusion that the increase was due to an effect of the bacteriophage upon the bacterial cell. This work was subsequently confirmed by leiss and Arnold (6) who also failed to give details of their work. They obtained opsonic indices as high as 9.3 and likewise concluded that the increase was dependent upon the action of bacteriophage on the bacteria. Slith (7) and Nelson (8) are the only recent contributors to the subject. Smith made an extensive study of this problem and reports having obtained indices with a maximum.value of 52.48. Smith states also that the increased index is due to an action of the bacteriophage upon the - 5 - bacterium. He also includes in the same report results of opsonic work in which bacteriophage susceptible and bacteriophage resistant strains of the same race of staphylococcus were first subjected to the action of the bacteriophage for varying periods of time and then brought into contact with phagocytes and the difference in phagocytosis noted. He observed that phagocytosis of the bacteriophage susceptible organisms increased with the time of their exposure to the bacteriophage but that there was no such increase with the bacteriophage resistant strain. He also found that while dilution greatly lowered the index, there was still the same correlation as in undiluted bacteriophage. Smith believes the susceptible organism.is capable of adsorbing bacteriophage whereas the resistant strain does not; therefore, the latter are not ingested to the extent that those susceptible are. Nelson's results also indicate a marked increase in opsonic index in the presence of bacteriophage, obtaining a.mathmm value of 13.8 with susceptible cocci. He also noted that a.much smaller’number of the resistant strain were ingested; but it is his contention that the bacterio- phage exerts its influence upon the phagocytic rather than upon the bacterial cell. Gohs and Jacobson (9) and (lO)in their investigation of the phage- cytic activity of the leucocyte did not employ bacteriophage but endeavored to study the spontaneous ingestion of bacteria by the leucocyte. They used bacteriophage susceptible and bacteriophage resistant strains of the Shiga dysentery bacillus in their work, and observed a much smaller number of resistant organisms ingested, as compared to the susceptible - 7 - strain of the same race. Similar results have been obtained by other workers. .1 study of the literature leads to some interesting comparisons and reveals certain significant disagreements but d'Herelle, Weiss and Arnold and Smith agree on one point at least, - that an increased index is observed in the presence of bacteriophage. Significant as these observations may be, the problem is by no means solved and many questions remain which have a very important bearing on the ultimate use of bacteriophage in clinical medicine. In the first place it is well known that individual races of bacterio- phage have marked differences and it is still unknown whether the results so far reported are due to peculiarities, characteristic of certain bacteriophage races, or whether they represent a general phenomenon common to all bacteriophages. This problem.can only be solved by extend- ing the number of bacteriophage races used in experiments of this kind. All the work so far reported involved the use of impure bacteriophage, that is, bacteriophage in combination with numerous other substances such as those composing the medium for growth, metabolic byproducts, endotoxins, exotoxins and proteins derived from.the lysed bacterial cells. Smith observed this fact and stated that he was unable to determine whether his results were due to bacteriophage as such or to some other of the various constituents of the filtrates he employed. Although.numerouszmethods fior preparing pure bacteriophage have been suggested, it was not until the publication of Krueger's and Tamada's method that purified bacteriophage suitable for such work could be prepared. Although there is some unanimity regarding the role of the bacterio- - a .- phage in this phenomenon, it has by no means been determined that the increased ingestion of bacteria by leucocytes is dependent upon a pre- liminary action by the bacteriophage on the bacterimn and furthermore, a matter of considerable significance from the standpoint of therapy, it has not been shown that the phenomenon repeats itself in vivo. ' The knowledge of these unsolved problems and the recognition of the importance of attempting to clarify somewhat the present status of bacterio- phage and phagocytosis, led to the undertaking of experiments which are to follow. They by no means cover the problem nor can it be said that the work is in any sense completed. Nevertheless, they do constitute a significant and valuable addition to our knowledge of this subject and represent an advance over what was previously known. Experimental For convenience in the presentation of this part of the investigation, each experiment has been separately listed and its specific purpose clearly stated, although, of course, they are all more or less related. The procedure in each operation is also stated, except in cases of identical repetition. Discussion of the results and their interpretation will be found in a subsequent section of the paper. Before taking up the experimental work, it may be advisable to define such toms as “cpsonins“, “opsonic index“ and the method used in the determination of the index. The term 'opsonin' literally means 'to make ready for food" and “phagocytosis", "to eat", - so that these antibodies are substances effective in increasing the natural tendency of the leucocytes - 9 - for eating or ingesting bacteria by producing a change in the bacterial cell, rendering it more desirable as food for the leucocyte. The method used for making the opsonic tests is that outlined by Fright and Douglass and consists, with minor changes, briefly in obtaining leucocytes from rabbits by bleeding from.the marginal ear vein.and collecting in 1% sodium citrate solution. The blood is centrifuged slowly to allow the red cells D to settle, then at increased speed the leucocytes form a white layer covering the red cells. The clear supernatant fluid is discarded and the leucocytes removed with a pipette and washed by resuspending in saline and centrifuging. The supernatant fluid is removed as before, and the white cells again resuspended in saline. They are then ready for use. {i twenty-four hour agar slant culture of the organism is washed off with saline and a suspension of approximately 1000 millions per cubic centimeter prepared. Equal portions of the leucocytic suspension, bacterial suspen- sion and serum or bacteriophage are taken up with a pipette, well mixed and incubated for 15 minutes at 37° C. it the end of the incubation period and following a second thorough mixing, smears are prepared and opsonic counts 'made. The smears are stained by the methylene blue method. The bacterio-' phage used throughout the present work is different in race from that used in the literature cited. The method of Wright and Douglass was also followed in making the opsonic counts, and consists briefly in finding the average number of’bacteria ingested by the leucocytes, a definite number of the latter being counted. In the present work an attempt was made to find the number of organisms in two hundred polymorphonuclear leucocytes and the average number obtained for one leucocyte is known as - 10 - the'phagocytic index". The opsonic index (the opsonic power of the fluid being tested) is obtained by dividing the figure representing the phagocytic index of the material being tested, the opsonic power of which is unknown, by the phagocytic index of normal phagocytes, that is, spontaneous in- gestion.of the same organism by the phagocyte, or phagocytosis in the absence of any stimulating substances such as cpsonins. I Although dffierelle obtained his high indices by incubation of leuco- cytes with bacteria and bacteriophage, several investigators have been unable to confirm his results and as the belief is. held by many that the increased index is due to an action of the bacteriophage upon the bacterial cell rather than upon the phagocytic cell or upon the process of phagocyb tosis itself, it seemed advisable to repeat part of dVHerelle's work, using his method. EXperimental Work ' W 1. Object: This experiment was designed to confirm.and to extend deerelle's observations on opsonic indices approximating forty obtained by incubation of leucocytes, bacteriophage and susceptible bacteria. Procedure: The test organisms used were B. typhosus and Staphylococcus eureus. Eighteen hour agar slants were prepared and the growth washed off with normal saline to give .a suspension of approximately 1000 million bacteria per cubic centhmeter as determined by nephelometer. Bacteriophage active against these bacteria was employed. It should - 11 - be noted that d'Berelle made use of a Shiga bacteriophage and B. dysen- teriae Shiga. Thus while this experiment is in part intended to repeat dinerelle's work, it is also intended to extend his observations through the use of different races of bacteriophage and different bacterial cultures. Leucocytes were obtained by the method described on page nine. Formal serum.was procured from.human bloods sent to the laboratory for diagnostic purposes. These sera when pooled contain a very small amount of opsonin. Immune serum against B. typhosus was a portion of a stock supply prepared at the State Biological Plant and used in the State Laboratory for'routine diagnostic work. No antistaphylococcus serum was available. The various combinations of leucocytes, bacteria and opsonising substances are shown in Table I. The method of making the determinations is identical throughout all the experiments. Capillary tubes of‘about .5 mmt outside diameter and 20 em. in length were used. A.merk was placed on the capillary about 2 cm. above the tip and the leucocyte suspension drawn up into the tube by gentle suction as far as the mark. .A column of air about .5 cm» was then admitted to the tube and next the opsonising substance, (bacteriophage or serum), was drawn up to the same mark. Last the organisms were similarly measured and the ingredients mixed by blow- ing the contents of the tube onto a glass slide. By drawing the mixture several times into the tube and forcing it out again, a thorough mix- ing was accomplished. The fluid was then drawn into the tube several .. 12 .. centimeters above the tip which was then sealed in a flame. Incubation in a water bath at 37 degrees for fifteen minutes followed immediately after sealing the tube. The seal was then broken, the contents of the tube ejected onto a glass slide and a film prepared by smearing the slide. These films were then allowed to dry at room tanperature. The method of staining resulted from a mnnber of experiments de- signed to determine which was most satisfactory. In view of the fact that in other bands other methods might prove better, it does not some advisable to draw any conclusion as a result of these trials or to recommend the method selected as superior to some others. under the conditions prevailing, the use of the wright triple stain proved best adapted to these particular experiments. This gave a blue color to all organisms used as well as to the nuclei of the cells. The cytoplasm stained a light pink which gave good contrast. A Leitz research microscope with a mechanical stage, oil imersion objective and a fifteen x occular giving a calculated magnification of 1800 diameters was used for all counts. As a rule two hundred leuco- cytes were examined including mononuclear cells but not lymphocytes and the umber of organisms within the cytoplasm of each cell was recorded. From this a phagocytic index was obtained by dividing the number of organisms by the number of leucocytes counted. The results of these experiments are found in Table I. No attmnpt to analyse or interpret these results will be made in this section. All results will be presented in Part IV. -13- TABLE I. A. Cells Organisms Phagocytic Opsonic Counted Ingested Index \ grid“ 1.B.typhosus e I.B.C.(control) 200 108 - .54 1 ~ .00 z. " e W.B.C.+ normal corms ' 200 199 .90 1.66 3. " e " e imune serum 200 21'! 1.06 1.96 4. " a. " e bacteriophage 200 80'! 1.53 8.85 5. " O ' 4' ' dil. 1.1.000,000 300 173 e86 1.60 6e . 0 ' O I hosted 200 156 e78 lo“ B. Cells Organisms Phagocytic cpsonic Counted Ingested Index Index l.Staph.aureus O'cBeCe‘CODtl‘Ol) 200 120 e50 LOO 2. " eureus +I.B.C.+ normal serum 200 178 .89 1.48 so I ' *WeBeCeObQCtOI'lOPm. 200 155 e77 1.28 4. ' fl . . . . all. 1-1.000'000 800 m 09‘ 1.56 5. " " 4- " e " heated , 200 180 .60 1.00 EXPERIMENT II. EXPERIMENT II. Object: The following experiment was undertaken in an attempt to determine the effect of time or of preliminary incubation of bacteria with bacteriophage upon phagocytosis. This semned particularly advisable in view of the fact that it was by such a method that Smith and Weiss and Arnold obtained their exceptionally high indices. Procedure: Leucocytes were obtained as described on page nine. The bacterial suspension was prepared from an eighteen hour agar slant culture of B. typhosus as in Experiment I. Equal quantities of the bacteriophage or serum and the bacterial suspension were well mixed, placed in small Wessemen tubes and incubated at 37° C. At the end of fifteen minutes a portion of each mixture was removed and the bacteria washed twice with saline to remove all bacteriophage and finally resus- pmrded in saline. The tubes were returned to the incubator for additional - 14 - incubation. To one part of the suspension of washed bacteria was added an equal part of the leucocytic suspension, as described on page eleven, and the:mixture incubated for fifteen.minutes at 37° C. Following this in,- cubation smears were prepared and stained and opsonic counts made. In the same way a test was made after the one hour incubation of bacteria and bacteriophage. The results of this test follow. TABLE II. .A. Fifteen.minutee preliminary incubation Cells Organisms Phagocytic Opsonic Counted Iggested Index Index l.B.typhosus ¥W.B.C. (control) 200 127 .63 1.00 2. ' e l.B.C.+ normal serum 100 115 1.50 2.38 3. ' + 7 e immune serum. 10 8 .80 1.86 4. ” 4 " e bacteriophage 67 80 1.20 1.90 B. One hour prelhminary incubation l.B.Typhosus +W.B.C.¢normal serum 150 215 1.43 2. 26 2. ' o W.B.C. + immune serum 100 230 2.30 3.65 3. ' e ' e bacteriophage 100 176 1.76 , 2.79 EXPERIMENT III. EXPERIMENT III. Object: The following set of experiments was performed in order to study the effect of purified bacteriophage upon phagocytosis and to extent the work involving prolhminary incubation of bacteria and bacteria» phage upon phagocytosis. Procedure: The method for the purification of bacteriophage as out- lined by Krueger and Tamada (11) is fundamentally one of cataphoresis. Bacteriophage being supposedly negatively charged should migrate towards the anode and protein impurities, on the other hand, at the hydrogen ion concentrations employed are neutral in charge and do not migrate. A - 15 - medium.free of protein and from which bacteriophage can be easily re- moved has been discovered in pure agar. The apparatus devised by Krueger and Tamada and used in this experi- ment is drawn in Plate I, page sixteen. Purified agar used in the process is prepared according to the method of Dominickiwice (12), which follows: From.thirty to forty grams of agar are dissolved in 100 cc. to 200 cc. of water, enough water being added so that the solution will flow in a thin stream when poured. While still hot, it is poured slowly into 800 cc. to 1000 cc. of 95% alcohol strongly acidified with acetic acid. The alcoholic solution is stirred vigorously while the agar is added. The result is a white precipitate which upon standing settles to the bottom. The clear supernatant fluid is than decanted off and the precipitate freed of all liquid by means of auction. The precipitate is now washed repeatedly with pure 95% alcohol until all traces of the acetic acid have been removed. This step is extremely hmportant fer the presence of acid will inhibit solidification of the pure agar, especially when a low percentage concen- tration is desired. Then precipitate is dried at 37° C. for several days or until the odor of alcohol is removed. it this time the coarsest particles are rendered.more usable by grinding in a.mortar. After the apparatus has been sterilized, the bridges C and D are filled with sterile purified agar 3% containing .8% NaCl. This is done by inverting the bridge and resting the lower end on a flat surface until the agar has solidified. Then the glass tube I is filled half full with sterile purified .5% agar containing .8% NaCl. When this has become solidified, the remainder of the tube is filled nearly to the top with 16 APPARATUS FOR PREPARATION OF amnvm PURE BLCTERI OPHLGE H (-l §3V98§§V55QQ>SS§§SV§ MI. ‘1. Bacteriophage suspension. B. Gel of .5‘ purified agar C.& D. B. I. G. H. I. “a e“ N‘Cle Glass bridges coup taining 31 purified agar and em NICle Porcelain dish with anal; crystals in distilled water. Porcelain cup with real crystals in distilled water. Positive electrode (silver foil) dips into crystals or I‘Cle Negative electrode (copper) dips into crystals of Ouclz. Glass tube. -17- the bacteriophage, leaving only room for the stopper. The negative electrode H is composed of copper which dips into crystals of CuClz, distilled water being added to the desired level. The positive electrode G is composed of silver foil which dips into a solution containing crystals of NaCl. .1 current of from 100 volts to 125 volts and from.5 milliamperes to 12 milliamperes is passed through the apparatus for 18 hours.to 20 hours. This does not necessarily have to be a continuous current. The bacterio- phage above the agar is changed at least once during this time. .at the end of this period, the supernatant fluid is removed with a pipette and discarded. The lower bridge and stopper are then removed and the agar allowed to slide out the upper end of the tube. ‘1 layer of the agar, one- half inch thick, which had been in contact with the bacteriophage is sliced off with a sharp sterile instrument, and the remainder sliced into a sterile grinding machine and a few cubic centhmeters of sterile saline added. After being macerated for two to three hours, it is taken out and the coarse particles removed by filtering through a coarse filter. Since no information was available concerning the effects of filtration on purified agar, two different types of filters were used, Seitz and mandler. At the time of Smith's investigations no method of obtaining bacterio- phage in a pure state was known, that is, bacteriophage from.which a large part of the proteins contained in the medimm such as products of bacterial metabolic activity and of disintegration, as well as toxins, etc., have been removed, and the later discovery of such a method by Krueger and Ihmmda offered an opportunity to study the properties of purified bacterio- phage as regards opsonic work. It is a well known fact that the specific -18- effect of the various proteins cannot be predicted in every case and it is well within the limits of probability that these proteins might in- terfere with the action of bacteriophage. A purified bacteriophage might, therefore, reveal certain specific characteristics and powers which would not appear in opsonic testing carried out or performed with simple stock bacteriophage. During the process of purifying the several bacteriophages used in the present work, a very interesting and perhaps valuable observation was made, namely, that thile all other bacteriophages studied collected at the anode, thus carrying a negative charge, the staphylococcus bacterio- phage collected at the cathode and is therefore the only positively charged bacteriophage at present known. This experiment is identical in every respect with Experiment II with the exception of the addition of purified bacteriophage in the tests, the test organisms being E. coli, B. typhosus, and staphylococcus eureus. Following are the results of this experiment in tabulated form. TABLEIIII. A. Fifteen.minutes(prelnminary incubation (1) Cells Organisms Phagocytic Opsonic Counted Igggsted Index Index l.B.typhosus 'F WeBeCe (Contml) 122 232 1.90 1.00 2. " + WtB.C.+ normal serum. 200 380 1.90 1.00 3. ' e ” e immune serum 200 490 2.45 1.28 4. ' e ' e bacteriophage 200 530 2.60 1.36 5e I 'F . If ' 381132 purified 200 555 2.70 1.42 6. . ¢ I! O . Mandler . 800 490 2.45 l.% (2) One hour preliminary incubation zeBetnhomI§WeBQCe+nom1 80m 200 85? 1am e67 3e ' § W.B.C.¢ MO .01.“ 200 540 2e70 1e48 4. ' e ' e bacteriophage 200 542 2.71 1.42 3. ' + ' e ' Seitz purified 200 550 2.75 1.44 6. ' e 7 e ' Handler ' 200 500 2.55 1.34 -19.. TABLE III. (cont'd.) .B. Fifteen.minutes preliminary incubation (1) Cells Organisms Phagocytic Opsonic " Counted Ingested Index Index l.B.typhosus e l.B.C. (control) 200 192 .96 1.00 2. " e l.B.C. 4 normal serum 200 281 1.40 1.45 3. " e " e immune serum 200 152 .76 .79 4. " e * e bacteriophage 200 382 1.96 2.04 5., ' e ' e * purified 200 260 1.30 1.35 (2) ‘Qne hour preliminary_incubation 4.B.typhosus+w.B.C.+bacteriophage 200 321 1.60 1.66 5e ' 4 to § '0 purified 200 342 1e7l 1e78 c. (l) Fifteen minutss_preliminary incubation_ Organisms Phagocytic Opsonic Cells Counted Ingested Index Index l.B.typhosus e W.B.C. (control) 200 334 1.67 1.00 2e . O 'eBeCe 9 110131131 serum 200 470 2e35 1e40 3. " + ” e immune serum 200 374 1.87 1.11 4. ” e ' e bacteriophage 200 666 3.33 1.99 5. " o ' ' ' purified 200 426 2.13 1.26 (2) One hour preliminary incubation 4.B.typhosus+W.B.C.+bacteriophage 200 848 4.24 2.54 _ 5. " e ' e ' purified 200 740 3.70 2.21 D. Fifteen minutes preliminary incubation (1) Cells Organisms Phagocytic Opsonic Counted ‘Iggpsted Index Index leBeCOli 4' WeBeCe (control) 200 254 leg? 1.00 8e I 4' 'eBeCe" normal 80m 200 410 2.05 1e61 3. ' e ' e bacteriophage 200 454 2.27 1.78 4. ' e ' + ' purified 200 774 3.87 3.04 (2) One hour preliminary incubation 3.B.coli+W.B.C.+bacteriophago 200 100‘ 5002 3e95 4. ' e ' o ' purified 200 950 4.75 3.74 E. Fifteen minutes preliminary incubation (1) . Cells Organisms Phagocytic Opsonic Counted Ingested Index Index l.Staph.aureus + W.B.C.(control) 200 252 1.26 1.00 2. ' eureus +WtB.C.+normal serum. 200 285 1.42 1.12 3. ' ' + ' +bacteriophage 200 210 1.05 .83 4. ” ” e ' + ' purified 200 585 2.97 2.35 (2) One hour prelhminary incubation 3.Staph.aureus+w.B.c.+bacteriophage 200 294 1.47 1.16 4. ‘ " e 7 e ' purified 200 427 2.13 1.69 EXPERIMENT IV. EKPERIMENT IV. Object: Smith's report includes work in which susceptible and resis- tant strains of the same race of staphylococcus were used to determine the relative effect of bacteriophage upon the phagocytosis of these two strains. The following experiment was designed to verify Smith's results. Procedure: This consisted in obtaining leucocytes in the usual manner and preparing suspensions of the two strains of staphylococcus to be used in the test. With these suspensions two series of bacteria-bacteriophage mixtures were made, each mixture consisting of 1 cc. bacterial suspension, .5 cc. bacteriophage and 3.5 cc. of slightly alkaline broth. The various mixtures of each series were made at different times so as to incubate for periods varying from one-half hour to four hours. Following incubation, the organisms were washed twice with saline by centrifuging, resuspending in saline and re-centrifuging. The suspensions of washed bacteria were then mixed with leucocytes and incubated for fifteen minutes at 37°C. Smears, staining and opsonic counts were made as in other tests. Results of this experiment follow: TABLE IV. Staphylococcus 8-1122 Staphylococcus 1631 Susceptible Strain Resistant Strain Period of Contact Phagocytic Opsonic Phagocytic Opsonic _(houre) Index Index Index Index 1,2 Staph.+W.mod-bacteriophage 2e17 1e14 1.88 leO” 1 ' + 7 + " 2.95 1.55 2.13 1.21 2 I! 9 . 4 . 2e86 1050 1.63 e93 4 ' e * e 7 4.86 2.55 1.12 .64 Control (not incubated) 1.90 1.00 1.75 1.00 - 21 - TABLE IV. (cont'd.) .1 second test identical with the preceding one except for a control follows: Staphylococcus B-1122 Staphylococcus 1631 Susceptible Strain Resistant Strain Period of Contact (hours) Phagacytic Index Phagocytic Index :72 Staph. ‘0 We Be Cubacteriophage 2.37 Jul-6 1 . ‘0' R 9 fl 3e10 1460 2 " + " + R 2.70 1.33 4 “ e " 4» " 4.21 1.32 No control EXPERIMENT V. W V. Oblect: Since results in vitro can never be taken to indicate what results will be in vivo, experiments similar to those above were performed on living rabbits. Procedure: To this and rabbits of approximately the same weight were selected and given an'injection of a suspension of either the bacteriophage susceptible cocci or the resistant strain of cocci of the same race immediately followed by an injection of bacteriophage. The amount of bacteriophage and bacterial suspension injected was determined on the basis of 1 cc. of the material for each kilogram body weight. Control rabbits received no bacteriophage. Administration of these materials given by way of the marginal ear vein. at stated intervals bleedings were made from the marginal ear vein collecting several drops of blood in either 1% sodium citrate or saline solution. Leucocytes were obtained as described on page nine, nears prepared and stained and the phagocytic index detemined. In part A rabbit number 298 weighed 1690 grams, was brown in color - 22 - and received 1.7 cc. of the susceptible strain of cocci. This rabbit served as the control in the first test. Rabbit number K 285 weighed 1525 grams, was black in color and received 1.5 cc. each of the sus- pension of susceptible cocci and of the bacteriophage. In part B, rabbit number I 300 served as the control: weighed 1760 grams, was brown in color and received 1.7 cc. of the susceptible cocci and 1.7 cc. sterile broth. Rabbit number K 295 weighed 1485 grams, was black in color and received 1.5 cc. each of bacteriophage and the bacterial suspension (susceptible cocci). Parts 1 and B are identical except for the intervals of bleeding. In part c, the resistant strains of cocci were used to determine the effect of bacteriophage on their phagocytosis in vivo. Otherwise, this test was identical with the other parts of the experinent. Rabbit number I 294 served as control, receiving no bacteriophage, weighed 2065 grams, was black in color and received 2 cc. of the suspension of resistant cocci. Rabbit number 299 weighed 1775 grams, was brown in color and received 1.8 cc. each of the bacterial suspension and of the bacteriophage. All rabbits used in this experiment died within twenty»- four hours to one week. . The results of this experiment follow: TLBIE V. a. Time of test Rabbit { 298 Rabbit # 285 after injections Received staphylococcus Received staphylococcus flours) . No bacteriopha§___ Received " bacteriophage Phagocytic Index Phagocytic Index 1 1.67 2.50 3 2.38 2.59 6 1.18 1.88 18 2.81 2.41 24 2.17 2.67 A .1 .. 1 . at .41 II U 1.1.1. 5 ‘ .J— ‘- 4 ‘ . ‘ ‘ . b . . he . ' " ' ' ‘ \ 4 ' e A | C O ' f . - - - r . .l. . I C .. . .- 23 .— TABLE Va (Cont'de) Be Time of test after injection Rabbit # K 300 Rabbit # K 295 Received staphylococcus Received staphylococcus No bacteriophage Received " bacteriophage Phagocytic Index Phagocytic Index 10 minutes .43 .95 20 " .82 .84 45 " 1.00 1.09 1 hour 1.05 1.05 2 hours e55 lelfi 3 ” .96 .75 18 " 1.26 2.25 2.4 " 1.00 1.15 Another, the third experiment was designed to show the effect in vivo of bacteriophage on the phagocytosis of resistant organisms. C. Rabbit # 294 Received resistant staphylococcus 1631. Rabbit f 299 Received resistant staphylococcus 1631 Time of test after injection (hours) No bacteriophage. Receged bacteriophage . Phagocytic Index Phagocytic Index 1 hour .55 .30 3 hours .72 .99 a " .76 2.22 18 " 3.30 .46 8‘ ” D10d e30 Discussion is pointed out in the beginning of this paper, much of the work is in the nature of an attempted confirmation of previous experiments with an extension of the work along the lines suggested by the omissions of previous investigators. Thus, for example, the obtaining of indices as high as forty by d‘Rerelle through a direct incubation of bacteriophage and bacteria in the presence of leucocytes has never obtained confirm- ation, although there have been numerous reports of high indices following a preliminary action of bacteriophage upon the bacterium. This obviously - 24 - is a very significant difference. The ultimate use of this apparent stimulation of‘phagocytosis in clinical medicine is, however, not depen- dent upon the mechanics of'the reaction and while the observation that a prelhminary contact of the bacteriophage and the bacterium.is essential to an increased index may not limit the therapeutic properties, it cer- tainly has its effect upon procedure. dVHerelle made use of only one organism.and one bacteriophage race. It is obviously impossible to generalize on the bases of such an experiment, hence, there is a distinct obligation on the part of anyone attempting to repeat dtRerelle's experi- ment to employ, if possible, a diversity of bacteriophage races and susceptible organisms. This is exactly what was attempted in Experiment 1, although the addition of two different bacteriophage races comes far short of the ideal. The results are very far from confirming dWHerelle's observations, although it is true that in the presence of undiluted bacteriophage in the case of B. typhosus the maximum opsonic index was obtained. The results of the staphylococcus experiment, on the other hand, are absolutely negative and it will be found that in general throughout these experflments that staphylococcus bacteriophage gave similar results. This is particularly significant in view of the fact that the staphylococcus bacteriophage used was the polyvirulent strain, originally isolated by Gratis, which is in general use throughout the world wherever therapeutic work is carried on. This particular bacteriophage has repeatedly been demonstrated to be entirely different from any other well studied lytic principle. In connection with the work presented here in the presence - 25 - of purified bacteriophage, one very significant difference in this particular respect was very definitely determined. While the other known bacteriophages are negatively charged and collect at the anode in cataphor- esis, the staphylococcus bacteriophage, on the other hand, was found to collect at the cathode, thus indicating a positive charge. The signifi- cance of this result is likely to become far reaching. It may be considered as one of’the most important contributions of the entire study. It should be pointed out that the staphylococcus bacteriophage used by anith and with which he obtained his high indices was not the polyvirulent race used in these experiments. The effects of preliminary incubation of bacteriophagm and bacteria are revealed in all of the experiments with the exception of'the first and the last, but are best demonstrated in Table III, although even here no very striking increases are obtained. Two conclusions might be drawn from these results, either that the technique was inadequate or'that the indices previously reported are peculiar only to certain organisms and certain bacteriophage races. Certainly, until further study of this problem is available, it cannot be stated that increased opsonic indices as a result of preliminary incubation of bacteriophage and bacterium are to be generally expected. In view of these results, it is rather difficult to say very much about the effects of purified bacteriophage. Such increases as could be observed were in general equally good with the purified material and it would further appear, although on very weak evidence, that the Seitz filter is to be selected rather than the Mandler in preparing material ‘1 for this purpose. That there is a difference in the ability of leucocytes to ingest different organisms has long been recognized and that this difference may in part be explained by the susceptibility or resistance to bacteriophage of the bacterium.selected has been indicated by Gohs and Jacobson, by Smith and by Nelson. It is again shown in Experiment IV where a susceptible and a resistant strain of staphylococcus were employed. In the final analysis, the results of any observation of opsonic indices upon which application to therapeutic work is based should depend upon the ability to reproduce this phenomenon in vivo. The observations of the behavior of leucocytes long after they are removed from the body and after various manipulations is scarcely an indication of the true state of affairs within the body. Efforts to study the ability of circulating phagocytic cells to ingest bacteria have been largely confined to tissue cultures. There is no obvious reason why a living animal cound not serve as a test tube for the study a! the ability of such cells to ingest bacteria. But it would appear that the experiments reported under‘V are the first attempts at a study of this nature. That the results are sanewhat disappointing from the standpoint of indicating any effect of bacteriophage does not detract from.the observation that such a method although entailing many difficulties may be practically applied. L.closer analysis of the results reported in the various experiments is possible, but in view of the fact of the very slight differences to be found in most instances, it is felt that such analysis would lead to entirely erroneous conclusions. It is realized that much further work along - 27 - this line is required. The results so far obtained indicate the direction in which subsequent investigations might be directed. Opsonic work is both tedious and time consuming and it is felt that the work presented represents about all that could be accomplished in the time available and while there may be some disappointment at the inability to report definite conclusions on some of the phases of the problem, it is felt that enough has been done to justify one or two statements which should have their effect on the development of'this entire subject. Conclusion. 1. The incubation of bacteriophage with susceptible bacteria and leucocytes leads to a small but definite increase in the opsonic indices, an increase measured in part by the character of the organisms used and by the selection of the race of the bacteriophage. 2. Such increases as are obtained are apparently materially affected by the preliminary incubation of bacteriophage and bacteria. 3. Purified bacteriophage is apparently equal to the unaltered material in its ability to stimulate phagocytosis. 4. The use of living animals for the determination of phagocytic activity of living cells appears feasable, but there is no indication that the presence of bacteriophage in such animals affects the degree of phagocytic activity. 5. Staphylococcus bacteriophage as opposed to all other known bacteriophage races carries a positive electrical charge. 1. 2. 3. 4. 5. 6. '7. 8. 9. 10. 11. 12. BIBLIOGRAPHY Twort, F. '91., An Investigation on the Nature of Ultra- Microscopic Viruses. Lancet, 189,11:1241-1243, 1915. d'Rerelle, I. The Bacteriophage and Its Behavior. Williams and Wilkins Company, 1926. d'Herelle, 1". Immunization in Natural Infectious Diseases. Williams and Wilkins Company, 1924. Hadley, P., The Tivort-d'Herelle Phenomenon. Journal of Infectious Diseases, 42:263-434. 1928. Wright, 1. 3., Studies on Dnmunization. William Wood and Company, 1920. Weiss, E. and Arnold, L., A Study of Antigenic Properties of Bacteriophage. Journal of Infectious Diseases, 34:317,1924. anith, G. B., Bacteriophage and Phamcytosis. Journal of Insuunology, 15 No. 2, March 1928.- Nelson, A. R.. The Effect of Bacteriophage upon the‘ Phenomena of Leucocytes and Phagocytosis. Journal of Immunology, Vol. 15, No. 1, January 1928. Cohs and Jacobson, Zt. f. Imunitatsf. 49:412, 1926. Gohe and Jacobson, Zt. f. Immunitatsf. 49:17, 1926 Krueger, A. P.,and Tamada, H. T., The Preparation of Relatively Pure Bacteriophage. Journal of General Physiology, V01. 13’ NO. 2, Ppe 145.151, NOVflnbel' m, 19”. Dominikiewics, 11.. Zur Frags aber die Einheit der lusannnen- setsung und Herstellungsweisen von Nlhrsubstraten m- Bakterie. Zentralblatt m Bakt. l, Abt. Original, 47:666, 1908. 1 . . “V. '1 ‘fig'fit 2!“. "‘J- Jig-'6 I “if “v? ' .- ' [A 9 n{‘ ‘ I ' ‘i . , , 9“ . - n. "",d'«-’ ~'.. -.-r«r~ "‘4‘“ Eta"; 6362:? rm“ ~ 51W “.9. . . ‘ 1. _. 9E ‘ ' .,.' '41. ‘1'; :_, v "a ‘I . 1?. ’7 ,‘IjLe'r-“l‘y'ii-zfi‘!‘ “.5511.- ‘v: . . r '. um /? no.“ .:.-~..1- . ' " I. t, 1' ~{"‘“" ‘-".' . I 1' .. 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