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An investigation of streptobacillus moniliformis and its pleuropneumonia-like L-1 variant Payne, John Irving 1952

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AN INVESTIGATION OF STREPTOBACILLUS MONILIFORMIS AND ITS PLEUROPNEUMONIA-LIKE L - l VARIANT by JOHN IRVING PAYNE  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of BACTERIOLOGY AND IMMUNOLOGY  We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS.  Members of the Department of  THE UNIVERSITY OF BRITISH COLUMBIA April, 1952  ABSTRACT  Investigations into some of the cultural,  morphological,  and biochemical characteristics of a strain of Streptobacillus moniliformis and i t s pleuropneumonia-like L - l variant are described. Experiments on the virulence and pathogenesis of these organisms indicated that virulence of the strain had been lost and could not be regained by the methods used.  Gross-agglutination and cross-  absorption tests demonstrated satisfactorily that Streptobacillus moniliformis and the L - l variant are antigenically related.  Acknowledgments. I would like to take this opportunity to express my appreciation for assistance i n this investigation to Dr. C.E. Dolman, Miss Helen Chang, Dr. D.C.B. Duff, to the other members of the Department of Bacteriology and Immunology, and to the National Research Council of Canada. I would also like to extend a heart-felt "thanks" to my good friends B i l l Wallace and Roy Griffiths, for their time and patience spent i n helping with the photomicrography, and especially to Miss G. Barbour, without whose encouragement this work would not have been completed.  TABLE OF CONTENTS PAGE I.  THE PROBLEM AND DEFINITIONS OF TERMS USED 1.  The Problem  2. II.  III.  1 1  a)  Statement of the Problem  1  b)  Importance of the Investigation  2  Organization of the Remainder of the Thesis  2  AN HISTORICAL REVIEW OF THE LITERATURE  3  1.  A Short History of Streptobacillus moniliformis .....  3  2.  The Pleuropneumonia-like L - l Variant  6  EXPERIMENTAL STUDIES A.  7  Some Cultural and Morphological Studies of Streptobacillus moniliformis and the L - l Organism ...  7  1.  Growth of S. moniliformis i n Fluid Media  7  2.  Growth of S. moniliformis and the L - l Organism on Solid Media  B.  13  The Effect of Certain Physical and Chemical Conditions upon the Growth of Streptobacillus moniliformis and. the L - l Organism  16  1.  Temperature  16  2.  Moisture  17  3.  a)  S. moniliformis  17  b)  L - l Organism  IB  Anaerobiosis  19  a)  S. moniliformis  19  b)  L - l Organism  20  PAGE 4. Percentage of Agar  5.  20.  a)  S. moniliformis  20  b)  L - l Organism  20  Percentage of Serum  20  6. Different Sera  22  a)  S. moniliformis  22  b)  L - l Organism  23  7. pH  23  8. CO2 Atmosphere  24  a)  S. moniliformis  24  b)  L - l Organism  24  9. Fermentation of Carbohydrates  25  a)  S. moniliformis  b)  L - l Organism  25 •  26  10.  Miscellaneous Biochemical Reactions ...................  27  11.  Yeast Extract  28  12.  Glucose  13.  Growth in Modified Hornibrook's Medium  14.  a)  S. moniliformis  b)  L - l Organism  29  30 ".  Osmotic Tensions and Surface Depressant Agents a)  Sodium Chloride  b)  Tween-80 and G-1690  30  31 31 32 32  C. Studies on the Effect of Antibiotics upon the- Growth of Streptobacillus moniliformis and the L - l Organism ..  34  PAGE 1.  Penicillin  2.  D.  .  34  a)  S. moniliformis  34  b)  L - l Organism  35  Dihydrostreptomycin ....  37  a)  S. moniliformis  37  b)  L - l Organism  37  The Lipoid Structures of Streptobacillus moniliformis  v  and the L - l Organism 1.  39  Appearance of the Lipoid Structures in Growing  :>  Cultures  39  2.  Examination of the Lipoidal Material  3.  Growth of S. moniliformis i n Lipoid-Free Serum, Medium.  4.  The Effect of an Ether Extractant, Cholesterol, and  E.  .  2.  3.  42  Lecithin upon the Growth of S. moniliformis.  44  a)  Ether Extractant  44  b)  Cholesterol ....  45  c)  Lecithin  46  d)  Cholesterol plus Lecithin  46  Serological Studies 1.  4-1  The Preparation of S. moniliformis Antiserum  48 48  a)  Preparation of the Antigen  48  b)  Animal Inoculations  49  The Preparation of L - l Antiserum.  50  a)  Preparation of the Antigen  50  b)  Animal Inoculations  51  Cross-Agglutination Experiments  52  PAGE U.  Cross-Absorption Tests  53  5. Growth of S. moniliformis i n Streptobacillus Antiserum.. F.  5A  The Effect of Certain Physical and Chemical Conditions upon the Virulence and Pathogenesis of Streptobacillus Moniliformis and the L - l Organism a.  Streptobacillus moniliformis 1.  Inoculation of Mice with a Stock Culture of S. moniliformis  2.  b.  55  Increased Percentages of Serum  56  L.  Glucose  56  5. Temperature  57  6.  57  lipdid-Free Serum  7. Mucin  58  8.  58  Animal Passage  L - l Organism 1.  Inoculation of-Mice with the L - l Organism  2.  Inoculation of Mice with the•Penicillin-Grown  DISCUSSION  V. SUMMARY VI. VII.  59  60  Photomicrography Photomicrographs  IV.  56  3. Rapid Subculture  L - l Organism G.  55  61 follow page  62 63 71  BIBLIOGRAPHY  72  APPENDICES  78  PAGE APPENDIX A *.  •  78  1. Strain of Streptobacillus moniliformis used  78  2. Origin of the L - l Variant  78  APPENDIX B  79  1. Stock Cultures of Streptobacillus moniliformis .......  79  2. Stock Cultures of the L - l Organism  81  3. Cultures of S. moniliformis i n Clotted Human Blood and i n Sterile Soil  .  ,....  81  a)  Clotted Human Blood  81  b)  Sterile Soil  82  APPENDIX C  84  Experimental Media APPENDIX D  84 ,  The Preparation of Idpoid-Free Serum  i  •  86 86  LIST OF TABLES  PAGE  TABLE I. Cross-Absorption tests with Streptobacillus moniliformis and the L - l Organism  follows  i  5U  I.  THE PROBLEM AND DEFINITIONS OF TERMS USED  Streptobacillus moniliformis, one of the causative agents of rat-bite fever, gives rise to a peculiar variant which is, termed "a pleuropneumonia-like variant" because of i t s morphological and cultural similarities to the causative organism of bovine pleuropneumonia.  This  variant i s commonly indicated as the " L - l " organism to distinguish i t from other organisms of the L-series.  (The "L" terminology was  introduced by Klieneberger, and has the merit of brevity only. "L  u  merely represents the i n i t i a l letter of the Lister Institute, where she was carrying out her investigations; Klieneberger calls bacillary organisms the "A"-form as distinct from the non-bacillary L-form). Minute elements of the L - l organism are always found with the parent streptobacillary form; there i s a cyclic development of filaments and b a c i l l i from the L-elements and a reversion of streptobacillary forms into the small, plastic, and amorphous shapes of the L-form.  However, the L-form can often be obtained free from the  Streptobacillus, at least on solid media, and hence studied i n i t s relationship to the Streptobacillus.  1. The Problem a) Statement of the problem.  It was the purpose of this  investigation (1) to study some of the cultural, morphological, and biochemical characteristics of both the parent Streptobacillus and the L - l organism; and (2) to compare the pathological and immunological properties of these two organisms.  2.  b)  Importance of the Investigation. There is much confusion  in the literature with regard to some of the properties of S. moniliformis and i t s L - l variant. The elucidation of the inter-relationship of these two organisms may explain certain of the described anomalies.  2.  Organization of the remainder of the thesis  The remainder of the thesis i s arranged to cover a Review of the Literature, the Experimental Studies, the Discussion, and the Summary. Since so many separate aspects of the study were undertaken, for the sake of clarity certain groups of experiments w i l l be presented and discussed consecutively. general issues.  The f i n a l discussion w i l l be reserved for more  II.  AN HISTORICAL REVIEW OF THE LITERATURE.  1. A Short History of Streptobacillas moniliformis. At the present time the controversy as to whether there are one or two agents responsible for the disease known as rat-bite fever , i s not yet settled. Both Spirillum minus and Streptobacillus moniliformis have been implicated i n the disease and evidence has been presented on behalf of each of these organisms as causative agents. Whatever the f i n a l outcome i s with regard to these organisms as inciters of this disease, Streptobacillus moniliformis, at least, i s known to cause rat-bite fever and i t i s this organism which w i l l be considered i n the following pages. Ogata was apparently the f i r s t author to describe elements . of the Streptobacillus and the L - l organism. In 1908 he gave a description of a micro-organism, Sporozoa muris. which he thought belonged to the protbzoons of the Neosporidia. The organism had been isolated from cases of rat-bite fever and could be used to infect guinea-pigs and rabbits by inoculation and by rat bite. Later, in 1913-19L4, Ogata reclassified his micro-organism as an Aspergillus, since he was then able to observe filamentous forms of the organism which excluded i t from the Sporozoa. The micro-organism, Bacillus septico muris. isolated from a case of "rat-bite disease" by Proescher (1911) (54), was reported as a tiny, straight or curved bacillus with rounded or slightly pointed ends. It stained bipolarly i f fixed i n a mixture of mercuric chloride and  alcohol and stained with dilute Giemsa's stainj but i t was not cultivable using ordinary laboratory media. In 1914, Schottmflier published a report on two cases of "Bisskrankheit".  From blood cultures of one of the patients, a laboratory  worker who had been bitten by a r a t , a Gram-positive "streptothrix" was isolated which grew only on LBffler's serum medium and on milk agar. Schottmflller called this organism Streptothrix muris r a t t i . Blake (1916) (7) cultured Streptothrix muris r a t t i from the blood of a patient bitten by a rat; positive cultures were later recovered post mortem from the heart-valve vegetations of this f a t a l case. Negative i n vitro and i n vivo cultivation results from two cases of rat-bite fever were published by. Tileston i n 1916 (63). However, micro-organisms which resembled Streptothrix muris r a t t i , obtained from the blood of one patient, were readily demonstrable under dark-field illumination as filamentous, non-motile, straight or curved organisms, some of which looked l i k e short chains of b a c i l l i or chains of cocci of various sizes.  These forms were poorly revealed by ordinary staining  methods. The descriptions of two streptothrices (Streptothrix longus and Streptothrix brevis) obtained from.two different cases of rat-bite by Litterer (1917) (44-) are generally considered to be descriptions of Streptobacillus moniliformis. Tunnicliff (1916) (65,66), isolated pure cultures of a streptothrix "similar to Streptothrix muris r a t t i " from the lungs of rats with bronchopneumonia. From 56 of 60 white rats showing acute or chronic bronchopneumonia, she observed "a long, fine, straight or wavy filamentous organism....in smear preparations or by dark-field illumination".  5. Tunnicliff and Mayer (1918) (67) cultured a streptothrix from a fatal infection resulting from the bite of a rat.  However, these authors  claimed that this latter organism was more closely related to Streptothrix putorii, an organism isolated by Dick and Tunnicliff (1918) (13) from the blood of a patient bitten by a weasel, than to Streptothrix muris r a t t i . Ebert and Hesse (1925) reportedly isolated from a case of "japanisches Rattenbissfieber" an organism resembling the organisms of Blake and Schottmflller.  In the same year Thorp (62), announced the  isolation of a "leptothrix" from wrist swellings resulting from disease following a rat bite.  This latter report i s rather inconclusive and  incomplete. An organism, differing only i n i t s Gram staining reaction from the organism of Schottmflller, was reported from the blood of a case of "acute erytheme multiforme" by Levaditi, Nicolau, and Poincloux (1926-26) (42).  No history of rat bite was recorded. These authors were apparently  unfamiliar with Schottmflller's organism or considered their organism to be divorced from rat-bite fever.  Accordingly, they named their organism  Streptobacillus moniliformis.  This latter name, now generally accepted i n  place of Streptothrix muris r a t t i , w i l l be used throughout this paper, or the  organism w i l l simply be designated the Streptobacillus. In 1926, i n the small town of Haverhill, Massachusetts, a  febrile epidemic occurred involving 86 persons. A report of the disease was made by Place, Sutton and Willner ( 5 2 ) and Place and Sutton (53), while Parker and Hudson made a complete bacteriological investigation (50). An unusual organism, apparently transmitted through the medium of contaminated raw milk, was cultured from the joint fluids and blood of the patients. Parker and Hudson proposed the name Haverhillia multiformis for the  6. organism, these authors being unaware of the organisms of Schottmfiller and Levaditi.  This type of infection, termed by the authors Erythema  arthriticum epidemicum, has since become known as Haverhill fever. Several other cases listed as Haverhill fever were subsequently reported i n which rat-bite was involved (1, 18, 2 5 , 4 6 , 56) and i n which no bite history was recorded (24, 26). It i s now generally accepted that the organism Haverhillia multiformis i s the same as Streptobacillus moniliformis. 1  2.  The Pleuropneumonia-like L - l Variant  In 1935, Klieneberger (32) reported an unusual type of organism growing i n apparent symbiosis with Streptobacillus moniliformis.  The  organism was similar i n colony formation and morphology to the causative organism of bovine pleuropneumonia, hence i t was called a  "pleuropneumonia-  like micro-organism". Dienes (14), on the other hand, took the view that the L - l ^ pleuropneumonia-like organism, was a variant form of the Streptobacillus and not a symbiont.  Subsequent work carried out by Dienes on other genera  indicated that pleuropneumonia-like organisms were common to a number of bacterial species.  Finally, i n 1949, Klieneberger-Nobel (37) acknowledged  that "the evidence i s now so conclusive that A- (typical young bacteria; Klieneberger-Nobel s terminology) and L-forms are two distinct phases of 1  the same organism that my symbiont theory i s untenable".  6a. Dolman, et a l . (19), were the f i r s t authors to report the isolation of Streptobacillus moniliformis from cases of rat-bite fever i n Canada. Not only were pure cultures of the Streptobacillus repeatedly obtained from blood cultures of the twp patients involved, but also pure cultures of the pleuropneumonia-like, L - l organism, were grown directly from blood cultures. This i s the f i r s t recorded instance of the i n vivo demonstration" of the L - l variant. Using the same strain of S. moniliformis, Dr. Dolman and Miss Chang, working i n the Department of Bacteriology and Immunology, have demonstrated the reversion of the L - l organism to the Streptobacillus, as well as the previously reported Streptobacillus -—>  L - l variation. On several occasions, L - l  colonies were noted to develop secondary S. moniliformis colonies, from which pure Streptobacillus colonies were obtained upon transfer and from which, i n turn, L-colonies were again derived. This i s conclusive proof of the reversibility of the Streptobacillus  L - l relationship.  7 III.  A.  EXPERIMENTAL STUDIES  Some Cultural and Morphological Studies of Streptobacillus " moniliformis and the L - l Organism Much has been written about the relationships between the  streptobacillary and the pleuropneumonia-like  elements; many allusions  and speculations have been made, and as a result considerable confusion has arisen. Accordingly, i n an attempt to clarify some of the points to the satisfaction of this observer, the following elementary observations were made. Note: The origin of the strain of S. moniliformis and the L - l organism i s given i n Appendix A.  1.  Growth of S. moniliformis in Fluid Media  Ordinary smear preparations made by centrifuging tubes of different ages were not satisfactory from the point of view of observing the development of the L - l organism from the streptobacillary form, or. vice versa.  In young cultures the presence of any organisms, especially  of unusual morphology, was totally obscured by a mass of f i b r i n and other serum artefacts.  Comparison with slides made from uninoculated tubes was  of l i t t l e aid. The use of a variety of fixation methods, such as osmic acid vapour, absolute alcohol, various concentrations of formalin and mercuric chloride, glacial acetic acid vapour, and Bouin's fixative, and a number of stains, such as Gram, Giemsa, Wright, Wayson, Methylene Blue, Azur, Thionine, Toluidine Blue, and Aniline Blue, did not help to clarify the picture. The f i r s t true signs of the organism were small, scattered, free  8.  b a c i l l i and thin filaments, some of which exhibited pseudo-branching. An occasional f i e l d showed granulations along the lengths of the filaments. By means of Robinow's c e l l wall and transverse septa mathod (38), thin cellular walls and septa could be demonstrated-in the filaments.  Capsular  stains and stains for endospores and metachromatic granules failed to reveal capsules, spores or granules at any age of culture. As the culture aged the filaments became twisted and entwined into compact clumps with numbers of long or short filaments coming out from the mass of growth. At no time were swollen or moniliform appearances observed i n preparations from fluid media.  (This point i s mentioned  because i t may somehow be associated with the loss of virulence of this strain).  These clumps of organisms often showed empty spaces i n the midst  of their growth as, i f something that had been there previously were now dissolved. Aging of the culture was associated with reduced stainability and the loss of any definite c e l l organization.  Clumps now stained feebly  or not at a l l , and finally, there was nothing noticeable but a few granules, cellular debris, and artefacts. Many authors have written extensively upon the type of growth that occurs i n broth media. The type of growth obtained i n different tubes seemed to depend upon how quickly growth was initiated, and this in turn seemed to depend upon the source of the inoculum and especially upon i t s size.  If a small loopful of a f a i r l y old culture were transferred to a  new tube of brothcat 37°C i t often grew very slowly, and slow growth appeared to be associated with the production of hard ball-like growth. These "balls" frequently grew to a considerable size, and when very large, there were few of them. The "flaky" type of growth was more closely  9. associated with a large inoculum and quicker development of the culture. However, no matter which form of growth resulted, the morphology of stained cultures was always similar. In passing, i t might be mentioned that the organism i t s e l f was not yellow or creamy, as often described, but rather a grey or whitish colour.  Growth in tubes was never turbid and i f  tubes were shaken u n t i l a turbidity resulted the organisms usually settled quite rapidly. Occasionally clumps of growth could be seen adhering to the sides of a tube, especially i f the tube was slightly inclined during incubation, but more normally, growth collected at the bottom of the tube leaving a clear supernatant. The most satisfactory method for the observation of ordinary stained smears was as follows:- Thin smears were made on clean glass slides and allowed to dry i n air.  Absolute methanol was applied and allowed  to act for several minutes, after which time i t was carefully washed off with distilled water. The serum proteins precipitated on the slide by the methanol were dissolved i n the water and thus removed.  Staining was carried  out, preferably with a polychromatic stain, i n very dilute staining solutions for relatively long periods.  This method usually reduced the  number of artefacts considerably and also removed the obscuring serum background. An adjunct to ordinary staining methods was the method mentioned by Tang, Wei, and Edgar (60), i n which fixed, stained preparations were observed by means of the dark-field microscope. seemed to be the most revealing.  Of the stains used Giemsa's  Unfortunately, the yellow-green colour  given off by the organisms was tiring to the eyes, although photographs taken by this method may prove worthwhile. Examination of cultures under dark-field illumination were  similarly fragmentary.  Throughout this phase of the studies a Baiisch  and Lomb paraboloid condenser was used; the condenser and microscope were housed i n a cardboard box which contained a 60-Watt lamp to serve as a source of heat.  A thermometer inserted i n top of the warm  chamber indicated the internal temperature; this could be kept f a i r l y constantly at 37 *C by opening or closing the hand-flaps i n the side of the box through which the microscope adjustments were reached. Many hundreds of paraffin sealed dark-field preparations were made and examined, some for days or even weeks.  Conditions of  culture, age of inoculum, etc. were varied so that growth could be observed under a variety of conditions.  A certain scepticism about  the elaborately described developmental cycles of the organism arose as a result of these examinations. Before an attempt i s made to describe some of the dark-field findings, i t may be pertinent to submit certain criticisms of the technique 1.  The organism did not usually live long enough to observe i t s various methods of reproduction.  There was no diffusion away of  metabolic wastes. 2 . Observations of individual elements, especially of the smaller types, was rendered extremely d i f f i c u l t by a) Brownian movement which kept the tiny bodies i n constant movement; and yet i f a particle was not seen to move, i t was d i f f i c u l t to distinguish the organism from slide and coverslip artefacts; b) convection currents caused by the heating of the slide by the substage dark-field unit and the warm box i t s e l f ; c) convection currents caused by the thermal contractions  and expansions of minute a i r bubbles which were originally trapped during preparation, or which resulted from the gradual drying up of the preparation from the heat of the warm box and the dark-field unitj d) artefacts arising from i. ii.  the serum and proteins of the medium, the slides and coverslips which could not be cleaned perfectly.  3.  The organism was often killed directly or impaired i n i t s growth by the heat of the dark-field unit when repeated observations, even of relatively short duration, were made. Young cultures in tryptose phosphate serum broth appeared  as short, hollow-looking rods and filaments, and large amorphous, granular clumps.  Most of the b a c i l l i had refractile granules at  one or both ends, while these same refractile dots occurred periodically along filaments as i f they were transverse septa. Sometimes b a c i l l i could be observed to grow into filaments.  In so  doing, the organisms turned and twisted and looped u n t i l eventually clumps were formed. The clumps appeared granular, probably because of the dense overlapping of filaments.  Usually a large number of  small or large, round, oval, or cylindrical hollow thin-walled bodies were associated with the clumps of organisms. bodies were distinctly double walled.  Some of these  In every field a mass of  •floating bodies, either alone or i n foam-like packets with large and small bodies "adhering" one to the other, could be seen drifting here and there, colliding and changing shape, and coming to rest against filaments or clumps i n such a way that they seemed to  originate from the filaments or clumps.  Occasionally the reverse  condition was seen, the bodies then floating away from clumps and filaments as i f they were being expelled from their point of origin. At no time was motility of the conventional type noticed, although convection currents caused continual movement of various structures across the f i e l d .  Myriads of tiny refractile bodies were also seen  bouncing here and there under Brownian bombardment.  Many of these  l i t t l e particles resembled the granules visible at the ends of b a c i l l i and filaments, or the granules i n the clumps of organisms. Great d i f f i c u l t y was experienced i n attempting to separate the vast assortment of f i b r i n artefacts from elements which might be connected with the organism.  Particles trailing small filaments  as well as most of the other bizarre forms seen i n growing microcultures could be seen as clearly and as frequently i n uninoculated preparations. Making the cultural conditions less favourable for growth as mentioned by Heilman (27) and Klieneberger (33) did not produce new forms or processes that could be interpreted as a general phenomenon i n normally growing cultures.  I f the amount of serum  was reduced considerably, growth did not take place. The organism also resisted a wide range of osmotic pressures without revealing any new forms, etc. The use of semi-solid agar was similarly limited i n i t s use, and furthermore, suitable preparations were d i f f i c u l t to make. Examination of wet mounts of young broth cultures under ordinary illumination with, reduced light did not show many of the elements seen under dark-field illumination.  In fact, a l l that  could be clearly observed were scattered b a c i l l i , filaments, clumps, and in some fields, a few small particles under molecular bombardment. The granules i n the filaments and clumps showed up more darkly here and there.  Preparations i n the warm box disclosed  growth of the filaments and b a c i l l i and the accumulation of great numbers of lipoid bodies in the clumps of organisms. Wet mounts of such cultures did not stain with a saturated 70$ ethyl alcohol solution of Sudan III. The lipoid bodies were apparently dissolved, since they disappeared after the stain was applied, leaving masses of b a c i l l i .  (Agar blocks of both the Streptobacillus and the L - l  organism did not take up the Sudan dye.  Osmic acid vapours also  failed to stain the lipoid bodies).  2.  Growth of S. moniliformis and the L - l Organism on Solid Media Very l i t t l e could be pieced together by making ordinary  smear preparations of colonies incubated for different lengths of time.  Young colonies consisted of regular, short b a c i l l i and  filaments of various lengths.  The filaments were generally twisted  and convoluted and showed large swellings along their lengths either as spindle-shapes in the filament or round or ovoid bodies growing eccentrically from the filament. evident.  Pseudo-branching was usually  As the colonies aged the filaments appeared to fragment  into small granules and autolytic processes took place, especially in the large bodies.  In smears of s t i l l older colonies, only  b a c i l l i and short filaments could be seen, and now and then a few large round clumps of normal b a c i l l i closely packed together i n a ball.  Stainability decreased and colonies eventually showed  u. amorphous indefinite debris. Colonies of S. moniliformis were round, greyish, glistening, slightly convexed, and transparent; they grew to a diameter of 1-2 mm.  Among such colonies appeared the L - l colonies,  small (c. 0.1 mm), rough, granular colonies, deeply embedded i n the agar.  These colonies also developed within growing S. moniliformis  colonies as chalky points of growth, which could be clearly seen by washing off the streptobacillary growth with broth or saline, thus leaving the L-growth behind i n the agar.  The L-colonies were  finally obtained free of streptobacillary elements and were propagated for twenty-four continuous plate transfers. Occasionally some of the plates made from one L-generation to the next would show a few Streptobacillus colonies among the L-colonies, indicating reversion of the L-colonies to the Streptobacillus. Ordinary smear preparations of L-colonies did not show any recognizable forms even when fixed and stained by a number of methods. Neither the L - l nor the Streptobacillus colonies showed any degree of growth when inoculated into drops of fresh serum, broth and observed under dark-field illumination.  Direct microscopic  examination of unstained colonies was also unsatisfactory. The staining of cultures i n situ on the surface of agar by the method of Dienes (15) was not successful. However, some success was achieved using the agar block fixation method employed by Salaman, et a l . (55), a modification of that of Klieneberger and Smiles (35). By means of this latter-mentioned technique certain of the characteristics of the L-organisms could be examined. Actually, only the features at the periphery of a mounted colony were worthy  15. of study since the centre of the colony was invariably a dense mass of stain due to i t s thickness. Young colonies showed the periphery to be made up of round bodies which contained packed granules. Older colonies consisted of greatly enlarged bodies often overlapping one another; the granules i n these bodies showed up quite distinctly, as a rule.  Among the large bodies s t i l l retaining granules were to  be seen large, round, empty blebs and vacuoles produced by the autolysis of the large bodies, and undoubtedly by the method of preparation.  The c e l l walls on the large bodies were occasionally  very thin and fragile-looking and the granules contained therein often appeared like a continuous mass. This i s understandable when i t i s remembered that the bodies were not f l a t but rather growing in three planes, and from some angles individual elements would be lost i n the depth of the mass. Permanent mounts of S. moniliformis colonies made by this method showed very closely packed filaments with the characteristic swellings along their lengths.  This feature was shown most clearly  when colonies were examined which were slightly spread during the fixation process.  Here, as i n the L-colonies, the periphery of the  colony was the important part to examine.  16-, B.  The Effect of Certain Physical and Chemical Conditions upon the Growth of Streptobacillus moniliformis and the L - l Organism  1.  Temperature  Tubes of 30$ serum tryptose phosphate broth were inoculated with 0.1 ml of a 27-hour culture.  Six inoculated tubes and one  uninoculated control tube were placed at each of the following temperatures a)  Refrigeration temperature ( 4 °  b)  Room temperature ( 2 0 * - 23°C).  c)  30* C.  d)  35'C  e)  37'C.  "f)  40 C  g)  45* C  a  The tubes were examined daily and smears were made and examined when growth appeared,  incubation was continued for a period  of two weeks, at which time the tubes were discarded. Growth occurred within 2 4 hours i n tubes held at 30*C, 3 5 * 0 , and 37*C and more slowly and not so heavily at 20*G, 40*C,  and 45*C.  At these latter, higher temperatures there was considerable  precipitation of serum and medium proteins, but growth was easily proved by microscopic examination of stained smears. Growth did not occur at refrigeration temperature.  A l l control tubes were negative.  The morphology of the organisms (clumps of b a c i l l i and broken filaments), was similar at a l l temperatures except for the tubes held at 45'C, where the Streptobacillus appeared as very long,  17. slender filamentous strands with more pseudo-branching than i s usually seen i n cultures kept at lower temperatures. Young cultures of Streptobacillus moniliformis heated at 50*C for ten minutes and at 60*C for five and ten minutes could no longer be subcultured and thus were considered to be killed at these temperatures.  These findings are i n keeping with those of Steen (58)  Parker and Hudson (50), and Oeding and Pedersen (4-9). Ceding and Pedersen (49), and Heilman (28) claimed that growth did not occur at room temperature, while Steen (58) and Parker and Hudson (50) gave growth temperature ranges from room temperature to 41*C.  The strain of the organism, and the length of time that  laboratory cultivation has been carried on since primary isolation, undoubtedly influence the temperature tolerance.  2. a)  Moisture  S. moniliformis • Freshly poured typtose phosphate serum agar plates  (30$ serum) were spread with a young broth culture of the Streptobacillus. Six plates were placed i n the incubator (37*C) i n the usual inverted manner, and six were placed i n a copper gas jar with a screw-thread top i n order to conserve moisture.  Very slight growth occurred on  one or two of the plates incubated under usual conditions, while heavy growth covered the surfaces of a l l the moist-incubated plates. It must be noted that a l l of the plates which were incubated i n the usual manner contained much water of condensation, both on the serum-agar surfaces and on the tops of the Petri plates.  The  18. presence of this moisture may have allowed slight growth to develop under ordinary conditions, since i t i s generally agreed among authors that growth does not develop except under conditions which allow for the conservation of moisture. On other occasions growth of the Streptobacillus has not been obtained under ordinary incubation.  Purity of stock cultures  has often been successfully checked by inoculating several plates from the same source and placing some of the plates directly i n the incubator and others i n containers.  Contaminating organisms grew up  on both sets of plates while the pure Streptobacillus grew only on the moist-incubated plates. Dried or old agar plates spread and placed i n a closed receptacle allowed growth to develop, but development was slower than i n the case of freshly poured plates, and the colonies may have been somewhat smaller. It was found that incubation i n the above-mentioned copper gas jars or, preferably, i n Petri plate tins with plate racks, was far more convenient than the sealing of individual plates with adhesive tape, Scotch tape, or Parafilm. b)  L - l Organism The L - l organism was also found to be very susceptible to  conditions of moisture and i t would not grow unless incubated i n adequate moisture.  19 3. a)  Anaerobiosis  S. moniliformis Several freshly spread tryptose phosphate serum agar  plates were placed i n a Mclntosh-Fildes jar and anaerobically. incubated.  incubated  Control plates were placed i n a copper gas jar and  Growth was compared at three days of incubation. It was found that growth occurred under both aerobic  and anaerobic conditions, and that the amount of growth and the size of the colonies were the same under the two states.  The  morphology of the organisms was not noticeably changed by anaerobiosis.  Moisture requirements were supplied i n both cases  by the closed containers. This short experiment and other similar experiments subsequently carried out teiid to verify reports i n the literature that Streptobacillus moniliformis grows equally well under aerobic and anaerobic conditions.  Lominski, et a l . (4-5), stated "the  organism i s thus aerobic but a facultative anaerobe," while Ceding and Pedersen (49) noted that in primary culture in fluid and solid media, only scanty growth was obtained anaerobically and no growth aerobically; but that the next subculture allowed equally good growth aerobically and anaerobically.  Growth, according to Steen  (58), was not better i n an anaerobic jar than under aerobic conditions.  Good growth was obtained aerobically by Farrell, Lordi,  and Vogel (25); Thjdtta and Jonsen (61) called their organism a facultative anaerobic microbe because i t grew well both under anaerobic and aerobic conditions.  20. b)  L - l Organism The L - l organism also did not improve i n growth under  anaerobic conditions.  4. a)  Percentage of Agar  S. moniliformis Growth of the Streptobacillus was obtained on plates of  tryptose phosphate serum agar containing from 1$ to 4$ agar.  The  morphology of the colonies and .of the organisms was not affected. Routinely, 1.5$ tryptose phosphate agar was made up and filtered through number I Whatman f i l t e r paper during autoclavingj this resulted i n an extremely clear agar that served well for colony observations for both the Streptobacillus and the L-colonies and eliminated the often confusing picture given by the precipitate that forms i n tryptose phosphate broth when agar i s added.  Three  m i l l i l i t r e s of sterile serum was added to each sterile Petri plate, the final agar percentage being about 1$. b)  L - l Organism In the case of the L - l organism, growth could not be  obtained on serum agar with a concentration of agar greater than 2$.  5.  Percentage of Serum  Ox serum was added to several series of tubes of tryptose phosphate broth (pH 7.5) to give f i n a l serum percentages of from 5$ to 90$.  The tubes were inoculated with one loopful of a 22-hour  culture, incubated, and observed for one week. Control tubes without  added serum, and tiibes containing 100$ serum, inoculated and uninoculated, were also included. Growth was supported by the lower percentages of serum, 5$ and 10$, but was slower in developing than i n higher percentages. Twenty per cent, 30, 4 0 , 50, and 60$ serum produced excellent growth of the Streptobacillus. Growth appeared to diminish somewhat i n 70, 80, and 9 0 $ serum; in fact, growth was not really discernible unless the tubes were centrifuged and the sediment resuspended i n a small quantity of physiological saline. Growth also developed i n 100$ serum, as shown by smears of centrifuged growth, and by the fact that pure cultures of the Streptobacillus were isolated from the cultured heart bloods and spleens of five mice, each inoculated with 0.5 ml of a 45-hour culture grown i n 100$ sterile ox serum. The morphology of the organisms i n 80$ - 100$ serum seemed to show smaller, more poorly defined organisms than in the other serum tubes.  Control tubes were  negative. Heilman (28) reported that veal broth (pH 7.6) plus 1$ proteose peptone supported a relatively luxuriant growth of both the Streptobacillus and the L - l organism when from 10$ to 90$ of horse serum was added. Steen (58) claimed that regular rod forms were seen when the Streptobacillus was grown i n 60$ or more serum broth, and that percentages below 60$ produced peculiarities and irregularities in the morphology of the organism. He said that i n 10$ serum medium there were mainly irregular, slender filaments, while in 60 - 80$ serum medium there was a rather homogeneous picture with bacillary forms and few threads.  /  The particular strain used i n this investigation displayed  22.  a f a i r l y consistent uniformity i n morphology when grown i n media containing from 10$ serum to 80$ serum, showing short filaments, isolated b a c i l l i , and large and small clumps of organisms, from which short filaments extruded.  Perhaps the uniformity of morphology  i n high percentages of serum viewed by Steen was due to the recent isolation of his strain, since he reported moniliform organisms that no longer appeared i n the strain used above. Routinely, U ml quantities of tryptose phosphate broth at pH 8.0 were measured into tubes from a burette, plugged, and sterilized for 15 minutes at 15 lbs. pressure and stored i n the refrigerator until ready for use. Just before use, 1 ml of sterile ox or human serum was added per tube, giving a f i n a l serum concentration of 2 0 $ .  6. Different Sera a)  S. moniliformis Series of serum broth tubes containing 1 0 - 70$ serum were  made with the following sera — human, reconstituted human, ox, sheep, rabbit, guinea-pig, and cat. The tubes were inoculated with one loopful of a young ox serum culture of the Streptobacillus.  Observations were carried out  for one week. Human and ox sera supported good growth i n a l l cases, growth developed rapidly and was approximately equal i n amount. Sheep, rabbit, and guinea-pig sera supported slight growth i n the 3 0 - 50$ serum ranges.  Reconstituted human serum and cat serum did not allow  growth of the organism. It must be mentioned that ox and normal human  sera were often used interchangeably for carrying stock cultures, since reports i n the literature indicate that different strains of S. moniliformis became adapted to particular kinds of sera, and become harder to grow i n heterologous sera after a lapse of time. The morphology of the organism i n the different sera did not vary, as judged by stained preparations. Thj.Btta and Jonsen (61) reported that blood and serum from different animals such as ox, horse, rabbit and sheep gave as good media as those from man.  b)  L - l Organism It was found that the L - l organism could not be transferred  from human or ox serum to plates containing other kinds of serum, although the serum concentrations were the same.  7.  pH  Tryptose phosphate broth was made up i n 0.5 pH increments from pH 5.0 to pH 9.0.  Serum percentages were varied from 0$ to 90$  in each pH series, the serum increasing i n 10$ amounts. The nine i  pH series were inoculated with one drop of a young Streptobacillus culture per tube.  The tubes were observed for one week.  Unfortunately, the very strong buffering power of the serum overcomes the phosphate-buffering system of the broth.  The above  pH values underwent marked changes, especially as more serum was added at a given pH.  For example, the addition of up to 20$ serum  to pH 7 . 5 gave no measureable pH difference when measured on the pH metre, but 30$ serum changed the value to pH 7 . 5 2 and 90$ to pH 7.80.  Thus, although excellent growth was obtained through a range of from 10$ to 70$ serum at pH 5.0 to 4$ to 60$ at pH 8.5, these pH values cannot be regarded as accurate throughout each pH series. Brown and Nunemaker (10) claimed that a pH of 7.6  was  preferred by their strain of S. moniliformis, although adequate growth was obtained throughout a pH range of 7.0 to 8.0.  Steen (58)  presented a table showing the influence of pH on the growth of S. moniliformis i n 40$ serum broth.  According to this table the range  of growth lay between pH 8.0 and pH 6.6, optimal growth occurring at pH 7.7 and developing more slowly at the extremes.  Again, Hazard and  Goodkind (26) found that their organism was f a i r l y sensitive to variation i n hydrogen ion concentration, and placed the optimum pH between 7.4 and  8.0.  8. a)  CO2 Atmosphere  S. moniliformis Several freshly inoculated plates of tryptose phosphate  serum agar were placed in vacuum desiccators and carbon dioxide atmospheres of 5$ and 10$ were added. The desiccators were incubated together with several plates i n a copper gas jar as normal growth controls.  At the end of four days'the plates were examined, at  which time i t was observed that there was no obvious stimulation of growth in either atmospheric concentration of carbon dioxide.  The  colonial morphology was similar to that displayed on the control plates, and stained smears showed no morphological differences. No evidence of growth stimulation due to increased carbon dioxide pressures was reported by Lominski, et a l . (45), Thjfltta  25. and Jonsen (61), Steen (58), Heilman (28), Oeding and Pedersen (49)J while Topley and Wilson (64), Parker and Hudson (50) and Warren (70) claimed enhanced growth when carbon dioxide was present. b)  L - l Organism Plates similarly treated as i n the above experiment  indicated that growth of the L - l organism did not appear to be stimulated by incubation i n 5 - 10$ carbon dioxide atmospheres.  9. Fermentation of Carbohydrates a)  S. moniliformis Five tubes of 1$ arabinose, glucose, mannitol, inositol,  adonitol, sorbitol, sucrose, lactose, maltose, inulin, dextrin, and starch, (Brom-thymol Blue Indicator) inoculated with one drop of a 24-hour culture of the Streptobacillus, failed to show any signs of growth or fermentation at the end of one week of incubation i n the abscence of added serum. The addition of 20$ beef serum to tubes of the above or the use of Hiss  1  Serum Water media, showed the following carbohydrates  were fermented - glucose, sucrose, starch, and dextrin.  On the other  hand, arabinose, mannitol, inositol, adonitol, sorbitol, lactose, maltose, and inulin were not fermented at the end of one week. Uninoculated control tubes were negative. The above reactions agree generally with those of Heilman (28), although he reported no fermentation of sucrose, whereas the strain used i n the above tests showed slight fermentation of this sugar by the end of the fourth day, at which time the Streptobacillus  26. was visible i n Wayson-stained smears of the tubes.  The strain of  Parker and Hudson (50) vigorously attacked starch, dextrin, and glucose, while lactose and maltose were weakly attacked.  Oeding and Pedersen  (49) found that no acid was formed i n lactose, saccharose, maltose, mannitol, glucose, galactose, laevulose, salicin, raffinose, starch or dextrin:  on the contrary, the medium was more alkaline during growth.  Alkalinity was also indicated i n serum-carbohydrate media by Steen (58). He found that inoculated tubes of glucose, lactose, laevulose, mannitol, and soluble starch became increasingly alkaline with' incubation.  Borgen (8) stated that there was no fermentation of  carbohydrates with his strain although he did not indicate whether serum was used i n the presence of his carbohydrates. The discrepancies i n regard to carbohydrate fermentations may be explained by differences i n the strains of the Streptobacillus isolated, and by the fact that the tests for fermenting a b i l i t y have not been standardized. Each author tends to employ different basal media, different concentrations of carbohydrate, varying amounts and kinds of sera, and different methods of preparation - some carbohydrates being Seitz-filtered, others being autoclaved.  b)  L - l Organism Slight fermentation was obtained i n sucrose, maltose, and  glucose inoculated with the L - l organism.  Fermentation was very slow,  often taking a week to show up, and only a few tubes of each inoculated series revealed any activity at a l l .  27. 10.  Miscellaneous Biochemical  Reactions  Appendix G contains a partial l i s t of various media which were unsuccessfully inoculated with the Streptobacillus in an effort to eliminate the necessity of added serum, blood, or ascites f l u i d . Several other biochemical reactions are listed below.  At least five  tubes or plates were used i n each test, i n addition to control tubes. Nutrient gelatin stabs - no growth, no liquefaction of the gelatin. Voges-Proskauer Test - negative, acetylmethyl-carbinol not formed, no growth. Methyl Red Test - negative, no growth. Indole test - negative, no growth in Tryptone Broth. Urea stab cultures - no growth; ammonia not produced. Litmus milk, with and without serum - no grov/th, litmus not reduced. Sterile skim milk - no growth, no coagulation. LBffler's serum slants - excellent growth i n 24. hours; colonies appear like those on tryptose phosphate serum agar; heavy growth i n the fluid at the base of the tubes. Nutrient broth - no growth. Nutrient broth plus 20$ ox serum - f a i r l y heavy growth* Nutrient agar - no growth. Nutrient agar plus 2 0 $ ox serum - a few small, white colonies i n 3-4  days.  Glucose agar - no growth. Nitrate broth - not reduced to nitrite. Petroff's medium - f a i r l y good growth in 48 hours; colonies similar to those on LBffler's serum slants. Egg-yolk agar slants - very slight growth; a few pin-point colonies in 4 - 5 days. Sheep's blood agar plates - f a i r l y heavy growth i n 3 - 4 days; - colonies small; no hemolysis.  28. Human blood agar plates - growth as on sheep's blood agar. Rabbit's blood agar plates - growth slightly heavier than on sheep's or human blood agar; no hemolysis. Chocolate agar - f a i r l y good growth i n 3 - A days.  11.  Yeast Extract  Five per cent yeast extract solution i n d i s t i l l e d water was added to a series of tubes of tryptose phosphate broth so that the following percentages of yeast extract were obtained - 0.01$, 0.02$, 0;05$, 0.1$, 0.2$, 0.5$, and 1$.  Another series of tubes of similar  yeast extract concentrations was made i n 20$ serum tryptose phosphate broth.  A l l tubes were inoculated with 0.05 ml of a 20-hour culture  of the Streptobacillus.  Control tubes were included.  The tubes were  examined daily for one week. Within 48 hours, a l l of the tubes of serum-yeast extract showed heavy growth, but no more than control tubes without added yeast extract.  Growth did not develop i n any of the tubes containing  yeast extract without serum. The 5$ yeast extract control tube was also negative for growth. From the above experiment i t appeared that yeast extract was not a growth adjunct and did not increase growth even i n the presence of added serum. These findings agree with those of Heihnan (28),  ThjBtta and Jonsen (61), and van Rooyen (69), who found that  yeast extract did not allow growth of the Streptobacillus without serum or ascites fluid, and did not increase growth when serum or ascites fluid was also present.  29  12.  Glucose  Several series of tubes of tryptose phosphate broth were made up to contain the following percentages of glucose i n a constant volume containing 20$ ox serum:- 0 . 1 , 0 . 2 , 0 . 5 , 1 . 0 , 2 . 0 , 5 . 0 , and 10$ glucose. A control series of the same glucose concentrations, but without any serum was also included.  Each tube  was inoculated with one loopful of a 48-hour Streptobacillus culture. After 24 hours of incubation, very heavy, flocculent, flaky growth had appeared i n a l l of the glucose-serum tubes. Growth was not quite as heavy i n 10$ glucose as i n the other glucose concentrations.  Control tubes of tryptose phosphate broth plus 2 0 $  ox serum without the addition of glucose also showed good growth i n 24 hours, but i t was not quite as heavy as that i n the glucose-serum tubes.  Growth did not appear i n the glucose series without added  serum, even after one week of incubation. Subcultures made from glucose-serum tubes to tryptose phosphate serum agar plates did not grow as quickly or as heavily as corresponding plate cultures made from serum tubes without glucose, and subcultures could only be made for a few days after the serumglucose tubes had begun to show growth.  This finding i s understandable,  since the pH level i n the glucose-serum tubes dropped more rapidly due to the fermentation of the glucose. Levels i n these tubes f e l l to about pH 6 . 0 , as judged by the addition of Brom-thymol blue and Bromcresol purple indicators, while glucose-free serum tubes stayed on the alkaline side of neutrality. Smears made from the above tubes showed no difference i n  30. morphology i n the "glucose-serum series and the glucose-free series, in spite of the fact that osmotic tensions must have increased greatly due to the added glucose.  13. a)  Growth i n Modified Hornibrook's Medium  S. moniliformis Serial passages of the Streptobacillus were attempted i n the  modification of Hornibrook's medium mentioned by Heilman (28). Heavy inocula were made into this medium from young cultures i n tryptose phosphate serum broth, and 0.1 ml amounts were subsequently transferred from tube to tube at 2- or 3-day intervals. Growth was heavy and typical i n the f i r s t two transfers, probably because" of the carry-over of serum from the f i r s t subculture; thereafter, growth was d i f f i c u l t to interpret macroscopically because of the poorness of growth and because of the opalescence of the medium and the settling out of "sheets" of starch. Stained preparations were also poor because of medium artefacts and the scantiness of growth. Examination of dark-field preparations was similarly misleading. In a l l , 25 subcultures were carried through serial passage in this medium. The presence of growth was determined by adding 0.1 ml of culture to tryptose phosphate serum broth tubes: .growth resulted i n each case.  Unlike Heilman's strain, which reached maximal growth  in 24 hours, and which was subcultured 4-0 times without loss of virulence for mice, this strain never increased i n growth even up to the 25th subculture. Dumoff and Duffy (20) made substitutions for starch with commercial glycogen, dextrin, glucose, and maltose on a weight-for-weight  basis i n an attempt to determine whether starch, could be replaced by other polysaccharides.  (Heilman had been unable to replace the starch  in Hornibrook's medium with dextrin, glucose, maltose, or salicin and s t i l l maintain growth of the Streptobacillus). These workers reported that good growth did not appear on Hornibrook's starch medium even after 100 passages.  Growth for several strains of the Streptobacillus  was established in Hornibrook's medium, in which dextrin was used to replace starch, although growth was less than i n the starch-containing medium. Glycogen could also be used i n place of starch.  Virulence  for mice was retained i n these media for a number of generations. These authors suggested that starch, glycogen and dextrin may neutralize an inhibitory substance (possibly fatty acids) present i n the medium and thus permit the growth of the organism.  It seemed doubtful that  these three substances would furnish the same growth factor.  b)  L - l Organism The L - l organism used i n this study could not be made to grow  i n Hornibrook's starch medium, even with the large inoculations of Lcolonies and lengthy periods of incubation.  Heilman (28) also found  that his L - l strains grew very sparsely or not at a l l on this medium.  14.  Osmotic Tensions and Surface Depressant Agents It was f e l t that the penetrating nature of the L - l organism  on solid media might somehow be related to changes i n the osmotic tension of the organism, and that changes i n salt concentration and surface tension might possibly force the dynamic reversible state of  32.  the organism i n the direction of L-fonnation in the liquid medium. Further, i t was hoped that a concentration of salt or  depressant-agent  would be found which would break up the hard, compact, ball-like growth of the organism into a more diffuse type of growth, which could be used for immunization and agglutination tests.  a)  Sodium Chloride Tryptose phosphate broth was made up to contain the following  concentrations of NaCl:  0, 0.08, 0.2, 0.8, 1.6, 2.4, 4.0, 8.0$.  Three  tubes of each of these salt concentrations were inoculated with 0.05  ml  of a 48-hour S. moniliformis culture. Three other series were inoculated with 27-day-old L - l colonies. Observations were made for 42 days. Plates and smears were made from time to time. The results were confused by the precipitation of serum and medium proteins which simulated growth.  However, the Streptobacillus  produced heavy, ball-like growth in a l l of the NaCl concentrations within 10 days of incubation. The growth in the L-tubes was very erratic, occurring i n some tubes of high and low concentrations and not in others.  Growth  was always streptobacillary i n form. Plates and smears revealed S. moniliformis in a l l of the tubes exhibiting growth. The morphology of the organism was constant throughout a l l of the salt concentrations.  b)  Tween-80 and G-1690 Concentrations of Tween-80 and G-1690 were made up so that  concentrations i n tubes ranged from 4,000 ug/ml to 0.25 ug/ml. These  concentrations of both agents were made i n series of tubes containing 20$ ox serum, and also i n tubes without serum. The tubes were inoculated with 0.05 ml of a 16-hour culture of S. moniliformis. Observations were continued for 28 days.  Smears and plates were made  from the tubes at various times. The Streptobacillus grew i n a l l of the tubes of Tween-80 plus serum within 2 4 hours and i n G-1690 plus serum by the fourth day.  Growth did not occur i n serum-Tween-80 containing 8,000 ug/ml  of the depressant, while streptobacillary growth occurred i n G-1690 plus serum (8,000 ug/ml).  Microscopic streptobacillary growth was  detected i n 31.3 - 0.25 ug/ml of Tween-80 without serum, and i n the same concentrations of G-1690 without serum. Growth did not occur in 1$ Tween-80 or G-1690 broths without serum. Plates yielded large numbers of Streptobacillus colonies i n the case of the serumdepressant broths.  The morphology of the organisms was constant  throughout. The L - l organism was not tested against surface depressant agents.  34. C.  Studies on the Effect of Antibiotics upon the Growth of Streptobacillus moniliformis and the L - l Organism  These studies were undertaken i n the hope that the presence of antibiotics i n certain concentrations i n fluid medium would tend either to force the Streptobacillus to produce the L - l form, or to encourage stabilization of the L - l form.  If such ends were achieved,  the problem of immunization and subsequent agglutination testing would be greatly simplified.  1. a)  Penicillin  S. moniliformis Serial dilutions of Crystalline Potassium Penicillin G  (Connaught Laboratories) were made so that i n 5 ml amounts the f i n a l Penicillin concentrations ranged from 40,000 units per m i l l i l i t r e to 40 units/ml.  Each tube was inoculated with 0.05 ml of a 48-hour  culture of S. moniliformis.  The tubes were observed for a period of  35 days, smears and plates being made at frequent intervals. After three days, streptobacillary growth had developed i n the tubes ranging from 625 to 4 0 units of Penicillin /ml.  Control  tubes without Penicillin showed heavy Streptobacillus growth within 24 hours.  At 8 days, growth had developed i n the tubes containing  from 10,000 units/ml to 40 units/ml.  Growth remained stable at the  8-day range, merely increasing i n quantity i n the lower Penicillin concentrations. Both smears and plates were unsatisfactory.  The Penicillin  may have affected the stainability of the organisms and prevented  35.  growth upon transfer to plates.  However, a l l evidence seemed to  indicate that the growth obtained i n each tube was streptobacillary in nature.  b)  L - l Organism A series of Penicillin dilutions was made up similar to  those used i n the experiment above with S. moniliformis.  The tubes  were each inoculated with one 12-day-old L-colony of the 16th generation of continuous L passage. stoppers to prevent evaporation.  The tubes were plugged with rubber  Daily observations were made for a  period of 35 days, smears and plates being made at frequent intervals. Growth was slow i n developing.  At the end of 6 days a very  few hard balls of growth were seen i n tubes of from 1,250 units of Penicillin /ml to 4 0 units/ml.  The control tube without any Penicillin  revealed streptobacillary growth at the f i f t h day of incubation. At 13 days, growth had appeared i n from 29th  5,000 - 40  units/ml, and by the  day two or three small balls of growth had appeared i n the tube  originally containing  10,000  units/ml.  Growth was examined by sucking some of the balls into a Pasteur pipette and expelling them on slides where they flattened and spread out as they dried.  This growth took up Wayson's stain very  rapidly and stained an intense blue colour. 1  Microscopically i t  appeared as large amorphous clumps with vacuoles or granules scattered in strata throughout i t s thickness.  No streptobacillary elements could  be demonstrated, despite repeated examinations. Some of the plates spread from the tubes showed growth of L-colonies after 7 days of incubation.  36.  On the basis of the above findings, the L - l organism was finally cultivated i n the liquid medium. Five generations of the L - l were made i n 20$ serum tryptose phosphate broth plus approximately 500 units of Penicillin per ml, and A generations i n medium containing 1,000 units/ml, before subcultures were discontinued.  Each generation  was made i n from one to two l i t r e s of medium at one time, the medium being dispensed i n large centrifuge bottles or Erlenmeyer flasks, A heavy inoculum was used from generation to generation.  There was no  obvious cultural, morphological, or serological difference between the growth obtained from medium containing 1,000  or 500 units of Penicillin  /ml. When a new generation was started, a number of L - l colonies was picked from a serum agar plate and added to the Penicillin serum medium. Growth was seen to develop slowly, often requiring a week of incubation before becoming noticeable.  First signs of growth showed  on the pieces of agar carrying the primary inoculum.  L-colonies grew  over a l l of the agar before growth became apparent anywhere else i n the medium. Later, growth appeared as large and small hard balls, some 3 - A Dim in diameter.  Examinations of the cultures were made by  fishing up balls by means of a Pasteur pipette. Growth was never very plentiful no matter how lengthy the incubation period.  Although i t  is not known how long the L - l remains viable in the presence of Penicillin, subcultures were easily made on the 15th day of incubation, and the pleuropneumonia morphology was s t i l l visible at 18 - 21 days. L - l colonies from serum agar plates could initiate growth i n the Penicillin medium when 29 days oldj but growth developed faster i f young colonies were used.  37. 2.  a)  Dihydrostreptomycin  S. moniliformis Concentrations of Dihydrostreptomycin (DHS) were made,  ranging from a real potency of 620 ug/ml to 0.15 ug/ml i n 5 ml quantities of 20$ serum tryptose phosphate broth.  Each tube was  inoculated with 0.05 ml of a 48-hour S. moniliformis culture. The tubes were observed for 33 days and then discarded.  Smears and plates  were made at frequent intervals. After 24 hours, streptobacillary growth had already appeared i n concentrations of DHS ranging from 0.5 to 0.15 ug/ml. Growth had also developed i n the normal controls.  Within 8 days of incubation,  growth had developed i n a l l of the concentrations of DHS used, from 620 - 0.15 ug/ml. Here, as i n the Penicillin experiments, smears were unsatisfactory, although growth of Streptobacillus colonies was obtained from the plates.  b)  L - l Organism A similar series of Dihydrostreptomycin dilutions was made  and inoculated with one 19-day-old L-colony per tube. were continued for 33 days.  Observations  Plates and smears were made periodically.  Growth occurred i n DHS concentrations of 0.3 ug/ml and 0.15 ug/ml within 8 days of incubation. No other growth appeared, even i n the normal controls. showed S. moniliformis.  Plates and smears of these two tubes  Some preliminary work was also carried out using Aureomycin.  However, the L - l organism was grown satisfactorily i n  Penicillin, and since this was the purpose of the investigations with antibiotics, the Aureomycin experiments were not completed.  39. D.  The Lipoid Structures of Streptobacillus moniliformis and the L - l Organism  In 1933, Strangeways (59) wrote the following description of a serum broth culture of S. moniliformis Under dark-ground.illumination the typical long forms of the streptobacillus...can be seen, and amongst these, a number of spherical globules or masses of varying sizes and luminosity. Occasionally comparatively large globules can be seen which appear to contain definite particles moving about within them. The nature of these globules, which sometimes form a foamy mass, i s unknown, but their appearance seems to be characteristic of serum broth cultures. Klieneberger (33) (1936) mentioned two different kinds of globules associated with the L - l organism, one of which appeared transparent i n the viable growth and left holes i n stained preparations. The second type of globule, she claimed, stained well throughout.  In  1941, Partridge and Klieneberger (51) and Williams (73) published papers on the lipoid structures, the f i r s t authors on those from the L - l organism, the second author on those from S. moniliformis cultures isolated from naturally infected wild mice (72). The following experiments were carried out i n an effort to duplicate some of the findings reported by the above workers on the lipoidal bodies observed i n dark-field and wet film preparations.  1.  Appearance of the Lipoid Structures i n Growing Cultures Dark-field preparations of young or old broth cultures and  emulsified colonies from serum agar revealed the same type of lipoid bodies when the centrifuged growth.was examined and also when the supernatant fluid from centrifuged cultures was observed.  These bodies  appeared as small or large foamy masses, some with a refractile double-  contoured edge, either enmeshed and entangled i n the clumps of organisms or wandering freely i n the fluid.  Other bodies were  round, cylindrical, ovoid, or any combination of these shapesj they occurred i n great numbers, and moved quite rapidly to and fro across the microscopic f i e l d as the preparation became heated from the darkfield light source.  Freely floating bodies were often seen bumping  into masses of organisms and adhering, while other bodies were seen to leave the organisms and float away. When bodies collided with each other a change of shape often resulted.  As preparations were  allowed to evaporate under the heat of the dark-field light, the bodies seemed to burst and completely disintegrate.  Staining of such  a dried preparation did not reveal any of the bodies.  However, i f  droplets of water were added to the edge of the coverslip of a dried preparation the bodies quickly reappeared.  If acetone, ether, or  chloroform were added to a preparation, the bodies dissolved, but after evaporation of the solvent, the addition of water would not produce the bodies again. Besides the above forms, most preparations showed the presence of a large number of long, thin lines or filaments running here and there, often across many microscopic fields.  In most cases,  these filaments were very straight, but now and then filaments were seen which contained one or more obtuse angles along i t s length. These filaments usually had no real beginning or ending, but occasionally masses of the above lipoid bodies would be found at one or both ends.  In places, the uniformity of the' filaments would be  broken by a number of well-defined spheres or ovoid shapes stretched out i n a straight line, or the width of the filament would suddenly  a. increase to considerable thickness or decrease to a very fine thread. Filaments similar to those observed above could be produced by pressing the edge of the coverslip with a pencil, whereupon the lipoid bodies were drawn out into long filaments.  When the pressure was relaxed some  or most of the filaments retracted into spherical shapes again, depending upon how hard the coverslip had been pressed.  These same  results could be obtained by cutting out colonies from plates, placing l  them on slides, and then pressing coverslips down on top of the colonies.  This phenomenon was especially evident when transmitted  light was used for observation with the high power lens of the microscope. 2.  Examination of the Lipoidal Material  S. moniliformis was grown i n 500 ml of tryptose phosphate broth containing 30$ ox serum.  After 72 hours of incubation the  culture was centrifuged, washed three times with physiological saline, and placed in a desiccator u n t i l dry.  (Dark-field examination of  saline-washed growth s t i l l showed abundant balloon and foamlike masses). Repeated extractions of the growth were made, f i r s t with acetone, then with absolute ethyl alcohol. The acetone-soluble fractions yielded cholesterol-like crystals after slow evaporation and crystallization. (Dark-field examination of acetone-washed growth showed a great reduction i n the number of visible lipoid bodies).  These crystals  were thin, transparent, mica-like sheets which could not be distinguished from a sample of pure cholesterol crystals.  The alcohol-soluble  fractions yielded the soft,yellowish, fatty substance which Williams claimed possesses the physical and chemical properties of lecithin.  42.  This substance darkened in colour as i t was exposed to the a i r .  (Dark-  field examination of acetone-alcohol-washed growth revealed no lipoid bodies).  When this latter fraction was observed under the light  microscope i n reduced light, i t appeared as an amorphous, greasy mass. However, a drop of water added to such a preparation caused the formation of spherical and writhing cylindrical "myelin bodies", as Williams  (73)  called them, which, upon pressure via the coverslip, appeared to simulate the globule shapes and filaments characteristic of S. moniliformis. These observations were seen more clearly and more dramatically under dark-field illumination. Myelin bodies were observed to arise when water was added to 90$ animal lecithin examined under the microscope.  Portions of the  acetone-soluble fraction and the alcohol-soluble fraction from the organisms mixed together and placed under the microscope showed the presence of lipoid bodies, thus proving that the total extracted lipoidal fraction could exist i n the form of myelin bodies. \ 3.  Growth of S. moniliformis in Lipoid-Free Serum-Medium These observations posed questions as to the source and  mechanism of production of the lipoid bodies.  Accordingly, following  the work of Williams (73), the technique of Hewitt (30) was employed, with slight modifications, to prepare a sample of lipoid-free serum (see Appendix D). Ten serial transfers of S. moniliformis were made i n 30$ lipoid-free serum tryptose phosphate broth, the transfers being made with large inocula when growth was maximal i n each generation.  The  general characteristics of growth i n lipoid-free serum medium may be  43. summarized as follows: a)  Growth was d i f f i c u l t to initiate and usually required a large inoculum; furthermore, large inocula were required to maintain the organism from one generation to another.  b)  The quantity of growth was only a fraction of that obtained i n normal serum controls.  c)  Increasing the amount of lipoid-free serum to 80$ did not improve growth.  d) ' Observed under dark-field illumination, growth was nearly completely free of lipoid bodies and crystals; normal filamentous and bacillary forms of the organism were the only visible elements. e)  Growth did not result when several attempts were made to plate the organism from lipoid-free liquid medium to lipoid-free solid medium.  f)  Excellent growth resulted when growth was transferred from l i p o i d free liquid medium to normal serum medium; the lipoid bodies also reappeared. In order to determine the amount of extractable lipoid substance  present, S. moniliformis was grown in large flasks of tryptose phosphate broth plus 30$ ox serum, centrifuged after suitable growth had developed, washed twice with saline, and dried in weighed beakers over calcium chloride.  When dry, the beakers were reweighed and the deposit was  repeatedly extracted with acetone and alcohol.  The weight of the residues  after extraction indicated that the amount of extracted material accounted roughly for 30 - 4-0$ of the dry weight of the cultures. The strains of organism used by Williams grew well, both i n liquid and on solid media containing lipoid-free serum. It should be noted that he used horse serum, while ox serum was used in the above  studies.  From his experiments he reconciled the reduction of growth  in lipoid-free serum medium with the fact that the globules and balloons, which normally make up a large percentage of the organism's bulk, are not present to give the same amount of growth. He concluded that the lipoid material was directly associated with the presence of lipoids i n the medium (as serum) and that since growth was obtained in fat-free serum the production of lipoid structures was a non-essential part of the metabolism of the organism. Partridge and Klieneberger (51) also isolated and chemically identified cholesterol from S. moniliformis, and considered that i t was derived from the medium, while a fatty substance, also isolated from cultures, was probably liberated as a result of enzyme action occurring within the living c e l l . Williams (73) found in more extensive experiments that approximately half the dry weight of culture deposits were composed of lipoid materials.  This weight, and also that of the bacterial residue,  were dependent upon the amount of serum present.  Partridge and  Klieneberger  (51) observed that 35 - A0% of the dry weight of S. moniliformis was extracted with organic solvents.  4.  The Effect of an Ether Extractant, Cholesterol, and Lecithin upon the Growth of S. moniliformis  a)  Ether Extractant Approximately one or two m i l l i l i t r e s of dark-brown substance  with some lighter-coloured fat globules floating i n i t were obtained after concentration of the ether extractant from the preparation of 100 ml of lipoid-free serum (above).  This material consisted of a great  45. mass of globules and thin, plate-like crystals, when seen under brightand dark-field illuminations. Half of this substance was added to 100 ml of tryptose phosphate broth (pH 8.0).  The solution was very turbid, and after being autoclaved  for 10 minutes at 15 pounds pressure, dark fatty droplets were found floating i n a scum on the surface, and fine cholesterol-like particles were visible when the flask was shaken. Five m i l l i l i t r e quantities were pipetted into sterile tubes. A second generation 36-hour culture of S. moniliformis i n lipoid-free serum broth was washed with, and resuspended i n , sterile physiological saline, so that the scanty growth was concentrated.  Six  tubes of the above medium were inoculated with one drop of this suspension. A control tube of untreated serum broth was also inoculated to ensure viability of the organisms.  The tubes were incubated and observed daily  for two weeks. The tubes were plated onto serum agar at the end of one week. Growth did not develop i n any.of the tubes (except the normal growth control) or on any of the plates by the end of two weeks, at which time they were discarded.  Dark-field examinations revealed only foam-like  masses, etc.  b)  Cholesterol The following accurately weighed amounts of pure cholesterol  (recrystallized 5 times from alcohol) were each added to 100 ml of tryptose phosphate broth:- 1, 5, 10, 15, 20, and 25 mg.  Cholesterol  crystals were s t i l l present i n large amounts after autoclaving for 10 minutes at 15 pounds pressure.  (Cholesterol i s only slightly soluble  in water).  Five m i l l i l i t r e quantities of each of the above solutions  were pipetted into sterile tubes.  The tubes, were inoculated with one  drop of the culture used i n the ether-extraetant experiment.  The tubes  were examined daily for two weeks and spread on serum agar at the end of one week. Growth did not develop either i n the tubes or on the plates. Examinations of the tubes were negative by dark-field illumination. Somewhat similar results were obtained by Partridge and Klieneberger (51) who could not obtain growth when stable colloidal suspensions of cholesterol and cholesteryl palmitate were used to replace serum, although control tubes of these substances plus serum yielded adequate growth.  c)  Lecithin Approximately one gram of 90% animal lecithin was added to  100 ml of tryptose phosphate broth. The solution was very dark after autoclaving.  Five m i l l i l i t r e amounts were pipetted into sterile tubes.  The tubes were inoculated with one drop of the suspension of organisms used i n the ether-extractant experiment and incubated for 2 weeks. At the end of one week, serum agar plates were spread from the tubes. No growth developed either i n the tubes or on the plates by the end of two weeks. Dark-field examination of the tubes showed large numbers of myelin bodies.  d) Cholesterol plus Lecithin Tofeur m i l l i l i t r e samples of the broth containing 10 mg of cholesterol per 100 ml were added 0.01, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0,  47. and 5.0 ml of 1% lecithin broth. Each of these tubes was inoculated with the above-mentioned  suspension of S. moniliformis and incubated for  2 weeks. Serum agar plates were spread from the tubes at one week. Growth did not develop either i n the tubes or on the plates. Examination by dark-field demonstrated myriads of balloon forms and foam-masses. In considering these experiments on cholesterol, etc., i t must be borne i n mind that a great many factors may have accounted for the negative results obtained, for example a)  There may have been too much or too l i t t l e of the substances added to the basal medium;  b)  other growth substances may be necessary as well as those added;  c)  neither of the substances alone or combined may have been growth factors;  d)  the pH of the medium may not have been correct for the proper functioning of the substances as growth factors;  e)  the effectiveness of the growth substances may have been destroyed by the methods of preparation.  48. E.  1.  a)  Serological Studies  The Preparation of S. moniliformis Antiserum  Preparation of the Antigen  ^  f  S. moniliformis was grown i n flasks of 30% serum tryfose phosphate broth for 48 hours and then centrifuged.  The growth was  washed twice i n sterile physiological saline and transferred as a thick paste to a sterile mortar where i t was ground for one-half hour. Smears made at 5-minute intervals indicated that the clumps of Streptobacillus were being broken up slowly.  The heavy paste was then made up to a No.  4 McFarland turbidity i n sterile saline and dispensed equally into five small serum bottles. a)  These aliquots were treated as follows -  formalin was added to one bottle to give a f i n a l concentration of 0.5$ formalin.  b)  phenol was added to 0.5$  concentration.  c)  one bottle was heated to 60*C for one hour.  d)  1:10,000 dilution of Merthiolate was made i n one bottle.  e)  one bottle was autoclaved for 15 minutes at 15 pounts pressure.  These antigens were tested for s t e r i l i t y and proved to be sterile even after several months. In passing i t should be mentioned that a number of other methods of trituration were tried, especially, grinding with sterile quartz sand, agitation i n the Waring blendor, and grinding i n two closely f i t t i n g test tubes.  None of these methods held any real advantage over the above  method and in fact some had disadvantages, for example, the quartz sand became ground up into paste and added to the turbidity of the bacterial suspension.  b)  Animal Inoculations Ten rabbits were employed, two receiving each of one of the  five antigens.  Six intravenous inoculations (posterior auricular  vein) were made i n each animal.  A total of 13 ml of antigen were given  per rabbit, the f i r s t 4 inoculations being given at 4-day intervals, and the f i f t h and sixth injections (3 and 6 mis, respectively) at 2week intervals.  Periodic test bleeds demonstrated that there was no  substantial increase i n antibody t i t r e after the third inoculation. One of the rabbits receiving Merthiolated antigen died several days after the fourth inoculation.  An organism was isolated  from pieces of the lung, spleen, and kidney, which gave the typical morphology and pigment production of Serratia marcescens. although i t was not conclusively identified.  Serum obtained from this animal  agglutinated homologous antigen to a strong +2 t i t r e at Is 2560 dilution, the highest t i t r e obtained with any of the antigens.  The Merthiolated  antigen was again carefully checked and s t e r i l i t y reaffirmed. Each of the two animals receiving the same antigen showed similar antibody levels throughout.  The only significant differences  noticed i n the titres of the various antisera obtained were i)  the phenolized antigen gave the lowest titres of the antigens used (about +2 at 1:640 dilution), and  ii)  contrary to expectation, the autoclaved antigen incited the highest titres of the normal series (+1.5 titre at 1:2560). The antisera were pooled and Seitz-filtered.  Tested against  formolized S. moniliformis, this pooled antiserum produced a noticeable +2 agglutination at 1:1280 dilution.  50. 2.  a)  The Preparation of L - l Antiserum  Preparation of the Antigen Since the L - l organism invariably reverted to the  streptobacillary form when added to broth, i t was necessary in the beginning, before growth was obtained in Penicillin-medium, to make use of L-coloni>es grown on serum agar.  This necessitated the preparation  of many hundreds of tryptose phosphate serum agar plates and the picking of myriads of colonies, since the penetrating nature of the L - l growth did not allow the simple washing off and emulsification of colonial growth.  Considerable serum agar was unavoidably picked with each  colony.  These colonies were collected i n 0.5$ formol-saline to preserve  them until sufficient could be accumulated for experimental purposes,, Simple grinding i n a sterile mortar for lengthy periods produced only a "gelatinous" paste which settled out rapidly when resuspended i n physiological saline.  Centrifugation at several speeds  did not allow separation of the organisms from the agar, i n fact, the presence of any organisms was completely obscured.  Grinding with  quartz sand and crystalline alumina resulted i n milky suspensions due to the abrasives, while the particulate agar and colonies quickly sedimented.  Agitation in the Waring blendor l e f t a jelly-like mass with  a perfectly clear supernatant.  Short exposures to boiling, and hydrolysis  with acids and alkalis, only caused the precipitation of serum and medium proteins; and even i f these methods had been successful i n removing the agar, treatment by such drastic means undoubtedly would have destroyed or greatly altered the antigenic make-up of the organisms. Although inoculations were begun with picked, formolized  51. colonies which had been forced through small gauge syringe needles to break-up the clumps, the inoculations were concluded with L-antigen grown i n Penicillin-medium. This latter antigen was broken up by shaking with glass beads i n small serum bottles.  b)  Animal Inoculations Four rabbits were employed for the production of L-antiserum.  In a l l , 14 intravenous inoculations were given each animal, mostly at 4-day intervals.  The f i r s t 7 injections, a total of 6 ml, were  composed of a thick, paste-3ike mass of formolized colonies picked from agar.  (Formalin was used because i t had proved to be satisfactory i n  the preparation of the Streptobacillus antiserum).  Assessment of  antibody levels with this same antigen were most confusing, since the large flocculent, fluffy appearance of the agar, and the hard particulate nature of the colonies, simulated the appearance of agglutination i n a l l tubes, including the controls. Growth of L-organisms was finally achieved i n liquid medium in the presence of Penicillin.  This facilitated both the rabbit  inoculations and the determination of antibody levels.  Also, any  cross-reaction between ox serum antibody produced by the inoculation of ox serum agar and the challenging serum-containing antigen, were eliminated now by repeated washings of the liquid-grown antigen with subsequent removal of a l l , or. most, of the serum-medium proteins.  The  next 7 injections, a total of 15 ml of Penicillin-grown antigen, were made at about 4-day intervals; the last injection comprised 4 ml per animal. Repeated test bleeds indicated that the antibody titres against  52.  homologous L-antigen remained more or less stable after the third injection of Penicillin-grown organisms and that these levels were not greatly increased by further inoculations.  Further, there was no  noticeable difference i n the titres of any of the animals. The antisera were pooled and Seitz-filtered.  Tested against  formolized L-organisms, this pooled antiserum gave a * 2 agglutination of 1:1280 dilution, similar to the level reached i n the pooled Streptobacillus antiserum.  3.  Gross-Agglutination Experiments  Pooled Streptobacillus- and L-antisera were tested for crossagglutination by challenging serial dilutions of each serum with the heterologous and the homologous antigens.  A representative example of  the results i s contained i n Table I. Examination of this table shows that the Streptobacillus antiserum agglutinated S. moniliformis to a t i t r e of at least  1:1024,  antigen  and the L - l organism to a t i t r e of 1:512,  while the L-antiserum agglutinated L - l organisms to a t i t r e of and the Streptobacillus antigen to a level of  1:1024  1:1024.  also.  It should be mentioned that various temperatures of incubation and different incubation periods were tried.  The combination that gave  the best results was incubation i n a water-bath at 50 of not less than 15 hours.  56°C  for a period  Agglutination seemed to be quite similar i n  the case of each of the antisera, appearing as a very heavy, flocculent precipitate. The best method for antigen preparation was to make up the desired Streptobacillus and L - l turbidities i n very large test tubes of  53. saline a few hours before the suspensions were to be added to the agglutination tubes. settle out.  This allowed time for the heavy particles to  The L - l organism was often remarkably stable at room  temperature i n such large test tube suspensions and remained suspended for as long as two weeks, s t i l l yielding a turbidity suitable for testing purposes.  On the other hand, the Streptobacillus suspensions  were not as stable, and auto-agglutinated within several days.  4.  Gross-Absorption Tests  Table I.illustrates a sample series of cross-absorption experiments.  The absorbed sera (2 ml of heavy organism suspension to  2 ml of antiserum) were absorbed i n a water bath at 54*C for 20 hours, at which time absorption was complete.  (Another series of tests showed  that absorption was not complete after one hour at 54"C).  Serial  dilutions of the various absorbed sera were made and challenged with both the homologous and heterologous antigens. Comparison was made with the unabsorbed antisera, also tested against their homologous and heterologous antigens. The results indicated that absorption of the Streptobacillus antiserum with S. moniliformis subsequently prevented agglutination of either S. moniliformis or the L - l organism.  However, Streptobacillus  antiserum absorbed with the L - l organism and tested against S. moniliformis showed an agglutination of *2 i n a dilution of Is 128, while the same Labsorbed antiserum failed to show any agglutination when tried against the L - l organism.  Agglutination was not evident when L-antiserum  absorbed with S. moniliformis, and L-antiserum absorbed with L - l organisms,  were each tested against S. moniliformis and against the L - l organism. The unabsorbed Streptobacillus- and L-sera both gave relatively high titres with homologous and heterologous antigens (see section of cross-agglutination tests, above).  5.  Growth of S. moniliformis i n Streptobacillus Antiserum Excellent growth was obtained i n tryptose phosphate broth  enriched with 20$ Streptobacillus rabbit antiserum.  Growth was usually  very rapid and was as heavy as the heaviest growth obtained i n broth containing normal rabbit serum. not very definite. crystals were seen.  Preparations stained poorly and were  Under the dark-field, very large cholesterol-like In addition, there were large masses of granular  growth (either degenerate streptobacillary or L growth) with lipoid bodies, both fixed to the growth and floating freely, as well as tiny elements under the Brownian influence. these cultures were plated out.  Growth did not result when  TABLE I. Cross-Absorption tests with Streptobacillus moniliformis and the L - l Organism  Antiserum  Absorbed or Unabsorbed  Streptobacillus  Unabsorbed  Streptobacillus  Unabsorbed  L-l  Streptobacillus  Absorbed with Streptobacillus  Streptobacillus  1 256  1 512  1024  1 2048  + 4 v4 +4  +4  +4  +3  -» 1  +3 + 3  +3  +2  -  mm  -  -  -  -  -  -  16  1 32  +4  +4  ^4  +3  Streptobacillus  -  -  -  -  Absorbed with Streptobacillus  L-l  -  -  -  -  Streptobacillus  Absorbed with L - l  Streptobacillus  +3  Strept oba c i l l u s  Absorbed with L - l  L-l  .-  L-l  Unabsorbed  L-l  Unabsorbed  L-l  L-l  Absorbed with Streptobacillus  Streptobacillus  1  Tested Against  1. 8  Streptobacillus +4  Streptobacillus + 4 +4  -  1  1 1 128 64  -  -  1  +1  -  -  -  -  -  -  -  -  • 4 + 4 +4 + 4  *3  +3  +2  •2 •3  •3  * 2  *2  -  . -  -  -  -  •»2 • 2 +2 + 2 -  -  + 4 *4  -  -  -  -  -  Controls Streptobacillus L-l  TABLE I. Cross-Absorption  - Cont'd.  tests with Streptobacillus moniliformis and the L - l Organism  Antiserum  Absorbed or Unabsorbed  Tested Against  1 8  1 16  1 32  1 64  L-l  Absorbed with Streptobacillus  L-l  -  -  -  L-l  Absorbed with L - l  Streptobacillus  -  -  L-l  Absorbed with L - l  L-l  -  -  1 128  1 256  1 512  -  -  -  -  -  -  -  -  - "  -  -  -  -  +4 Complete Agglutination +3 - Marked Agglutination +2 Moderate Agglutination +1 - Slight Agglutination - - No Agglutination s  3  1 1024  -  1 2048  -  -  -  55. F.  The Effect of Certain Physical and Chemical Conditions upon the Virulence and Pathogenesis of Streptobacillus moniliformis and the L - l Organism  a.  Streptobacillus moniliformis  i 1.  Inoculation of Mice with a Stock Culture of S. moniliformis Three mice each were inoculated intraperitoneally with the  following amounts of washed growth from a 48-hour culture i n 20% ox serum: 0.05, 0.1, 0.5, and 1.0 ml.  Two of the mice receiving one  millildtre of culture died two days later; contaminating organisms were recovered, but not S. moniliformis.  The other mice remained well u n t i l  the time they were killed, 2\ months after inoculation.  Heart bloods  and spleens from these latter mice were cultured i n 20% serum tryptose phosphate broth.  Pure, positive cultures of S. moniliformis were  obtained i n most of these tubes within 4-8 hours. During the course of these and other experiments with mice, direct smears were made from the heart bloods and from the juice of pressed spleens. Although these smears were stained ^ i t h a number of different stains (Giemsa, Wayson, Wright, Methylene Blue stains), no obvious organisms were ever seen.  Artefacts abounded, but nothing  distinctly resembling S. moniliformis was observed. Also, normal uninoculated mice were killed and cultured from time to time to act as experimental controls. recovered in these cases.  No organisms were ever  56. 2.  Increased Percentages of Serum  Intraperitoneal inoculations were made into groups of 5 mice each with 0.5 ml of whole, unwashed, 4-5-hour Streptobacillus cultures i n 20, 40,. 60, 80, and 100$ ox serum.  No signs of disease were evident  when the animals were killed 28 - 32 days after injection.  Pure cultures  of S. moniliformis were obtained i n each case from cultured heart bloods and spleens.  3.  Rapid Subculture  Five subcultures were begun from a stock culture of S. moniliformis.  Subcultures of these tubes were made at 18 — 24--hour  intervals for 15 days.  Four cultures were selected at the end of the  fifteenth day and 0.5 ml of each culture was inoculated into each of 3 mice. The mice appeared healthy when killed 38 - 40 days after inoculation.  Pure Streptobacillus cultures were obtained from cultured  heart bloods and spleens.  4. Glucose For nearly one year the organism had been transferred on glucose-free tryptose phosphate serum media.  Since the primary isolation  of this S. moniliformis strain was made i n ordinary laboratory serum tryptose phosphate broth, which probably contained glucose, i t was decided to test the effect of added glucose upon the virulence of the organism, since organisms grown i n glucose-free medium were not k i l l i n g mice.  57.. Five daily transfers were made i n 1% glucdse-tryptose phosphate serum broth.  These cultures grew consistently faster than  control glucose-free cultures. The f i f t h subculture was inoculated into tubes of the following f i n a l percentages of glucose (constant 20% ox serum) - Oil, 0 . 2 , 0 . 5 , 1 . 0 , 2 . 0 , 5 . 0 , and 10%. A second series of these concentrations was inoculated at 24 hours.  The 0.2% and 2 . 0 %  glucose growths of this second series were inoculated ( 0 . 5 ml) into each of 5 mice. Positive cultures of S. moniliformis were recovered from the heart bloods and spleens three weeks after inoculation.  The mice were  healthy when k i l l e d .  5.  Temperature  A 72-hour culture of S. moniliformis grown i n 30 ml of medium at 45*C was centrifuged and resuspended i n saline.  5  ml of sterile physiological  Five mice were inoculated intraperitoneally with 0 . 5 ml of  concentrated  suspension.  The normal-appearing mice were killed 3 1 days after injection, and pure cultures of S. moniliformis were obtained from a l l the heart bloods and spleens.  6.  Lipoid-Free Serum  A second generation culture i n lipoid-free serum broth (20% serum) was centrifuged at 4 0 hours and resuspended i n 2 ml of sterile physiological saline.  Three mice were inoculated intraperitoneally with  0 . 5 ml of this suspension.  58. The Streptobacillus was recovered i n pure culture from most of the heart bloods and spleens, 31 days after inoculation.  7. Mucin Five pert-cent gastric mucin was made up i n phosphate buffer to pH 7.4 and autoclaved for 5 minutes at 15 pounds pressure. When cool, the solution was adjusted slightly to neutrality.  Three m i l l i l i t r e s  of mucin were added to a 4-0-hour S. moniliformis culture and mixed well. H a l f - m i l l i l i t r e amounts of this mucin-organism mixture were inoculated intraperitoneally into 5 mice.  Mucin solution alone was inoculated into  another 5 mice to serve as mucin toxicity controls. A l l the mice were killed at 18 days; they appeared to be healthy and no lesions were found. By the end of 5 days of incubation a l l heart blood and spleen cultures from the inoculated mice were positive for S. moniliformis.  Cultures from the control mice were negative at  the end of one week's incubation.  8. Animal Passage A culture of S. moniliformis recovered from the heart blood of a mouse previously inoculated with the Streptobacillus was successively injected into, and recovered from, eight different series of animals. At least 3 - 5 mice were used per passage.  Pure cultures were grown  from the heart bloods and spleens from each series of mice.  At no time  did the animals show any signs or symptoms of disease, and no lesions of any kind were observed i n the viscera upon careful examination. Intervals between passage were as long as a month and as short as 5 days, yet pure cultures were always easily obtained.  59. Blind animal passage was completed i n U series of mice using the organism from the eighth passage mentioned above, that i s , the organism was passaged 12 times altogether.  The heart bloods from the  3 - 5 mice were withdrawn by means of Pasteur pipettes soon after death: a drop of the blood from each mouse was added to a tube of serum tryptose phosphate broth, and the rest was pooled i n sterile 2.5% sodium citrate i n physiological saline.  (Splenic cultures were also  made). The next series of mice was inoculated as quickly as possible, so that the organisms would not be killed by exposure to the sodium citrate (2.5% sodium oxalate proved to be immediately fatal to mice and could not be used for transfers).  In each instance the heart blood and  spleen cultures showed pure growth of S. moniliformis, thus confirming the continued transfer of the organism.  No signs of disease were ever  noticed and the morphology of the organism never noticeably changed.  b.  L - l Organism  1. Inoculation of Mice with the L - l Organism Ten mice were each inoculated with 0.5 ml of 8-day-old colonies of L - l organisms that had been kept i n the L-form on tryptose phosphate serum agar plates for 18 successivis generations.  The colonies from four  heavily grown plates were picked into 6 ml of sterile physiological saline and forced through a No. 26 gauge needle three or four times to break up the particles before injection. Two other series of 10 mice were each inoculated with 0.5 ml of 19-day-old L - l colonies and 0.5 ml of 30-day-old colonies from the  60. generation used above. The animals of each group were killed at 4-1, 37, and days, respectively.  34  S. moniliformis was recovered from nearly every  mouse of the f i r s t group, either from heart blood culture, splenic culture, or both, while only 5 mice of the second set and 4- from the last series yielded positive cultures. One of the mice accidentally received part of i t s inoculation of 8-day-old colonies subcutaneously.  This mouse was carefully noted  and at the time of death i t was discovered that a subcutaneous lump had developed at the site of injection.  This lump was about  inch  long and egg-shaped. When opened, i t yielded a thick, caseous yellow substance which when smeared revealed typical straight streptobacilli and clumps of filaments:  no moniliform shapes were seen.  Pure cultures  of S. moniliformis were obtained upon subculture.  2.  Inoculation of Mice with the Penicillin-Grown L - l Organism Five mice were each inoculated with 0.5 ml of a heavy  suspension of 14-day-old growth i n medium containing 1,000  units of  Penicillin per mlj a second series of mice received 0.5 ml of growth from medium containing 500 units of Penicillin per ml. The mice were killed at 32 days.  None of the cultures made  from the heart bloods and spleens showed any growth at the end of two weeks of incubation when the tubes were discarded.  61.  G.  Photomicrography  A large number of photomicrographs were taken on a binocular research Watson microscope replaced with a monocular attachment.  A  35-mm Leica camera (without lens) was screwed into place on a MicroIbso mounting with a cable shutter release.  The 10-power eyepiece  was screwed into the Micro-Ibso mounting and inserted into the monocular attachment.  A periscopic viewer perpendicular to the microscope allowed  viewing and centering of the slides; several squares lined i n the viewer aided the centering.  The light source was a ribbon filament lamp with a  built-in i r i s diaphragm; this diaphragm was used i n conjunction with the i r i s diaphragm on the microscope.  Proper lighting and exposure time  were judged on the basis of previous experience with photomicrography. Time was indicated with a stop-watch. Kodak Panatomic-X film was used i n i t i a l l y because i t had earlier proved to be satisfactory for this type of work.  Unfortunately,  most of these attempts with this film proved unsatisfactory; the negatives seemed to indicate proper exposure times but when enlarged, (3i  u  by A-g-") they revealed heavy graininess, despite the fact that  fresh developers (Kodak D-76 and Kodak Microdol) were used.  Contrast  papers were employed without any improvement i n the enlargements. Intensification of the film with mercuric intensifiers did not improve the  quality of the prints. Better photographic success was achieved by the use of a  View-Camera with a long extensible bellows mounted on an enlarger frame.  A monocular Bausch and Lomb microscope was used.  The light  source was provided by a picture projector, the light intensity being  62. regulated by the i r i s diaphragm of the microscope.  Kodak Super Panchro  Press Type B Sheet Film was used; exposure times were determined by the trial-and-error method. Work was conducted i n a dark room since this facilitated observation of the view plate.  Contact prints were made  on Velox papers. Results thus obtained showed a minimum of graininess and great c l a r i t y of detail.  The only objection to this method arose  from the fact that thinly smeared slides were d i f f i c u l t to observe through the view plate.  Photomicrographs produced by this method are  included. Kodak Micro-File Film (35 mm) was used i n the Leica photomicro camera set-up mentioned above.  This film was used because of i t s  extremely fine grain, which i t was hoped would allow greater clarity of enlargements than did Panatomic-X film.  Since this type of film i s  extremely "slow", t r i a l exposure times of the same f i e l d were run at 5, 10, 15, and 20 seconds; a section of the film was developed and the 20 second exposure was chosen and was used throughout the remainder of the  roll.  Certain frames were satisfactory and some of them are included. It was f i n a l l y discovered after the expenditure of much film  and time that most of the films were being obscured by the presence of dust particles i n the microscopic set-up; the negatives looked excellent in a l l respects, but enlargements proved to be greatly disappointing. Although the ocular and the objectives on the above microscope were changed and the new lenses carefully cleaned, the dust particles remained, out  of focus, but always i n the same places on the negatives.  By  elimination i t would seem that the dust must have originated i n the Micro-Ibso attachment whence i t could not be eliminated.  PHOTOMICROGRAPHS.  Figure I f Agar block L - l colony (3 days old) f i x e d through the agar with Bouin's f i x a t i v e . D i l u t e L8ffler's Methylene Blue s t a i n . Magnification 190--X. This colony shows the t y p i c a l dense c e n t r a l area and the thinner periphery. Large swollen bodies may be noted even at t h i s r e l a t i v e l y low magnification.  Figure 2 :  .  Bouin-fixed L-colonies (3 days), D i l u t e Wayson s t a i n . M a g n i f i c a t i o n 4-0 X.  Figure 3; . Enlargement of the c e n t r a l colony of Figure 2, showing the darker c e n t r a l area and large bodies around the periphery. Magnification 400 X.  Figure 4 and 5: Bouin-fixed L - l colonies (2 days o l d ) . D i l u t e Wayson s t a i n . M a g n i f i c a t i o n 1843 X. These two f i g u r e s show the young, developing, large bodies a t the peripheries of colonies. The presence of i n t e r n a l granules i s obscured i n these photographs.  Figure 6: L-colony (3 days). D i l u t e Wayson. Magnification 1000 X. This colony shows large bodies as they are beginning to develop; d i f f e r e n t i n t e n s i t i e s of s t a i n i n g are noticeable.  Figure 7: L-colony (3 days). D i l u t e Wayson s t a i n . M a g n i f i c a t i o n 1000 X. Vacuoles show up quite c l e a r l y on t h i s preparation.  Figure 8: stain.  L-colony (3 days). Dilute Wayson Magnification 1000 X,  Figure 9: L-colony (3 days). Lttffler's Methylene Blue stain. Magnification 1000 X.  Figure 10: stain.  L-colony (6 days). Dilute Wayson Magnification 1000 X.  Figure 11: Bouin-fixed L - l colony (6 days o l d ) . D i l u t e Wayson s t a i n . M a g n i f i c a t i o n 1843 X. Granules may be seen w i t h i n the amorphous, swollen, large bodies.  Figure 12: Bouin-fixed L-colony (6 days). D i l u t e Giemsa s t a i n . M a g n i f i c a t i o n 1000 X.  Fij.iZ.  Figure 13: L-growth (12 days) i n tryptose phosphate serum broth plus 500 u n i t s of P e n i c i l l i n per ml, 2nd generation. Fixed w i t h absolute methanol. D i l u t e Wayson s t a i n . M a g n i f i c a t i o n 1000 X.  Figure L4: L-growth (12 days) i n tryptose phosphate serum broth plus 1000 u n i t s of P e n i c i l l i n per ml, 3rd generation. Fixed with absolute methanol. D i l u t e Wayson s t a i n . M a g n i f i c a t i o n 1000 X. The amorphous L-growth i s f i l l e d throughout with c r y s t a l s or vacuoles.  I  F.o 14.  Figure 15: Streptobacillus grown i n tryptose phosphate broth (2 days). Fixation with absolute methanol. Dilute Wayson stain. Magnification 1650 X. What appear to be swollen forms on the filaments are actually solid areas of filaments, together with precipitated stain.  Figure 16: Streptobacillus moniliformis i n tryptose phosphate serum broth (3 day culture). Smear fixed with absolute methanol and stained with dilute Wayson. Magnification 1843 X. Only straight filaments are present, although the thickness of the preparation simulates swollen bodies.  Fiq. 15.  Figure 17; Streptobacillus grown i n tryptose phosphate serum broth (14- days). Methanol fixation. Dilute Wayson stain. Magnification 1000 X.  Figure 18: Edges of Bouin-fixed Streptobacillus colonies (6 days). (These preparations were slightly smeared during the fixation process). Dilute Wayson stain. Magnification 1000 X. Interlacing filaments with swollen bodies are clearly seen.  Figure 19; See Figure 18.  Figure 20; Edge of Bouin-fixed S. moniliformis •colony (18 days). Dilute Giemsa stain. Magnification 1000 X.  F«j-19.  63. IV.  DISCUSSION.  A l l of the forms reported here have been seen and reported previously,and nothing original has been observed that might clarify the f i n a l relationship of the L - l variant to the Streptobacillus. The dark-field method of examination has been criticized by several authors (17, 3 4 , 69) and declared to have limited usage for the observation of these developmental phenomena. Some success, however, has been achieved by means of phase microscopy ( 4 0 ) . Although a number of authors (27, 5 0 , 5 2 , 59) have described branching i n S. moniliformis i t i s now agreed that what was seen was only pseudo- or false-branching, not the true type of branching, which as a method of reproduction, i s confined to the Streptomyces ( 6 ) . Some controversy s t i l l seems to exist in the literature as to the Gram staining reaction of S. moniliformis. During the course of these studies, a large number of Gram stains were made on cultures of various ages from l  broth and from agar. When cultivated i n broth, the organism consistently stained Gram-negatively, while agar preparations often gave what might be called a localized Gram-positive  staining reaction. This was seen in  young colonies which contained filaments with large numbers of swollen bodies. The filaments, themselves stained Gram-negatively, while the  64. large bodies, spindles, and enlargements stained positively.  It i s  these latter sites which have been suggested as rich sources of chromatinic material and as centres of genetic activity on the part of the organism.  As the colonies aged and the large bodies disappeared,  the smears uniformly took up the counterstainj and f i n a l l y autolysis resulted i n the loss of true stainability. The question of motility i n the Streptobacillus has been carefully studied.  As mentioned previously, no motility of the  conventional type was ever observed under dark-field illumination, or by ordinary well-slide preparations and wet mounts. Neither was motility demonstrated i n serum tryptose phosphate semi-solid agar motility tubes of the usual type.  Only the odd isolated report (45)  claims motility for S. moniliformis and i t cannot but be wondered i f the authors of these claims have been dealing with possible motile contaminants, such as were too often encountered during the course of these investigations. A singular case of motility i n an organism of the pleuropneumonia group should be cited.  Andrewes and Welch (4)  obtained a pleuropneumonia-like organism from the lungs of mice inoculated with material believed to contain typhus rickettsia.  Growth  developed i n serum medium after 3 or 4 days' incubation and was carried in subculture both in liquid and on solid media containing serum. Dark-field illumination, especially from the f i r s t cultures, showed varying numbers of motile organisms, which seemed to crawl along on the slide like motile mycobacteria, even against or across the stream of a gentle convection current. Pseudo-colonies simulating those of pleuropneumonia-like  micro-organisms were recorded by Brown, Swift, and Watson ( 9 ) . These unusual forms were characteristic for the sera of different animal species and seemed to be largely composed of spherocrystals that stained typically with fat stains. The explanation given for their transferability was that the bits of colonies spread on new plates acted as centres specially favourable for the crystallization of ,new spherocrystals out of the medium. During the course of this investigation, a number of different crystal formations were observed on serum agar plates.  In some  respects they resembled the pseudo-colonies of Brown, et a l . , but they usually occurred on old plates and could not be transferred to new media. The selective action of antibiotics, bacteriostatic chemicals, bacteriophage, and antibodies for the isolation of L-colonies from numerous genera of bacteria need not be mentioned here.  It was not  the purpose of these studies to determine the ultimate sensitivities of the Streptobacillus and the L - l organism but rather to use these agents as a possible means for the propagation of the L - l organism in liquid media so that i t could be used to better advantage i n other investigations.  Incidentally, however, the Penicillin resistance of  this strain originally reported by Dolman, et a l . ( 1 9 ) was again illustrated, as contrasted to the marked Penicillin sensitivity of strains investigated by other workers ( 2 , 2 9 , 4 3 , 4 5 , 5 8 ) . The concentrations of Penicillin, i n which growth of either the Streptobacillus or L - l organism resulted, cannot be considered as absolute since the h a l f - l i f e of Penicillin i n solution i s normally short, and i n addition, the Penicillin dilutions were subjected to pH  66. changes and to the relatively high incubation temperature. One of the striking features noticed among some organisms exposed to sublethal concentrations of Penicillin i s the production of a number of swollen moniliform and filamentous forms that look almost identical with those of S. moniliformis (3, 31, 57).  Any  relationship between this phenomenon and the norma} production of large bodies by S« moniliformis i s unknown. In passing, i t would be pertinent to give the following recent references concerning the susceptibility of a number, of human pleuropneumonia-like organisms to the i n vitro action of antibiotics (11, 41, 47). The Streptobacillus i s resistant to considerable changes i n osmotic tensions, as evidenced by growth i n 0 - 10$ NaCl broth and i n 0.1 - 10$ glucose broth.  Also, the organism withstands the effect of  strong concentrations of surface-depressant agents. The identification of lipoid bodies as artefacts i n cultures of S. moniliformis and the L - l organism modifies considerably the elaborately described life-cycles of these and related organisms, since these bodies have been mistaken as viable elements of growth.  For  example, many of the unusual methods of reproduction or "genethodes" of the pleuropneumonia organism discussed by Turner (68) are undoubtedly artefacts of the above origin and, as suggested by Williams (73), the formation of lipoid structures may be a function common to pleuropneumonialike organisms since a l l these organisms depend on the presence of serum for growth. Many of the reproductive forms described by Turner were seen i n uninoculated preparations observed under dark-field illumination, such as "conidioids" with extruded germinal tubes and  67. unipolar,- bipolar, and multipolar germinations.  Extreme caution should  be exercised in relating such complex phenomena to the development of the organism. The relationship of cholesterol to the Streptobacillus and the L - l organism requires further investigation. Edward and Fitzgerald (22, 23) found that ethereal extracts of egg-yolk could replace serum for the growth of a number of different strains of pleuropneumonialike organisms.- Fractionation of egg-yolk extracts indicated that cholesterol was involved, and in some strains growth was obtained when pure cholesterol was added, to a basal medium. When 1% soluble starch was incorporated into the medium with the cholesterol improved results were obtained.  Bovine albumin plus cholesterol also gave satisfactory  growth, and ,it appeared that cholesterol was essential for growth since no growth of any strain could be obtained by starch or bovine albumin alone.  Two other sterols, cholestanol and stigmasterol, were equal to  cholesterol in growth promotion when used i n the basal medium with bovine albumin.  The action of the starch and albumin was attributed  to the neutralization of fatty acids, especially oleic acid, by these substances.  Inhibition of S. moniliformis by sodium oleate i n a  concentration of 9 0 gamma per ml of medium was reported by Dumoff and Duffy (21).  Here, too, adequate additions of bovine serum reversed this  inhibitory effect.  This inhibitory reversal of added starch, serum, and  albumin has already been alluded to as a possible explanation for growth of the Streptobacillus i n modified Hornibrook's medium. The results of the cross-agglutination and  cross-absorption  tests seem to confirm the work of Dawflon and Hobby (12), and of  68, Klieneberger (36), upon the antigenic similarity of S. moniliformis and the L - l organism.  The unabsorbed sera gave positive reactions  with both organisms, while either serum absorbed with i t s homologous antigen no longer reacted with either organism.  Heterologous absorption  of the Streptobacillus antiserum with the L - l organism s t i l l allowed agglutination when tested with S. moniliformis, while L-antiserum absorbed with S. moniliformis lost a l l agglutinating ability.  These  results are quite understandable when i t i s recalled that S. moniliformis contains L - l elements concomitantly with the Streptobacillary elements, and further that the removal of the L - l antiserum fraction from the Streptobacillus antiserum by L - l absorption s t i l l leaves the streptobacillary antiserum fraction to agglutinate with the streptobacillary antigen, i f challenged by S. moniliformis, while absorption of either antiserum with S. moniliformis w i l l remove a l l agglutinating power against either organism.  The other results indicate  also that the L - l antiserum contained only one kind of agglutinin while the Streptobacillus antiserum contained two. Serological similarity between L-forms and their b a c i l l i have been reported i n several other genera as further evidence for ;  the genetical identity of the L-forms and the b a c i l l i (16, 71, 74.). However, Dienes (17) reported that, i n some instances, differences between the two forms were demonstrable. Growth of S. moniliformis i n i t s antiserum was described by Nelson (48). After 48 hours of incubation excellent macroscopic growth had developed i n the antiserum and i n a normal serum control.  He  claimed that the b a c i l l i and filaments i n the normal control were replaced by large spherical bodies i n the case of growth i n the  69.. immune serum. At f i r s t i t was thought that here was an excellent method for the growth of the L - l organism, in liquid media, but unfortunately, the morphological studies did not prove conclusively that the L-l alone was growing in Streptobacillus antiserum.  Perhaps further  work on the elucidation of this point i s indicated. Enhanced virulence of S. moniliformis when inoculated simultaneously with mucin was reported by Levine and Civin  (4-3).  They inoculated each of three mice with a mixture of one m i l l i l i t r e of mucin and one m i l l i l i t r e of saline-suspended organisms. These animals died within 48 hours, while one control mouse, inoculated with mucin alone, was not affected. The disparity between their results and the results obtained in this investigation may have been due to the differences i n mucin preparations, size of inoculations, and recentness of isolation, although i t would be dangerous to make too many generalizations concerning the effectiveness of mucin in virulence enhancement on the basis of three dead mice. None of the conditions used for the enhancement of virulence were found to work. Even animal passage failed to cause death in the mice.  This latter finding is i n accord with the statement of Brown and  Nunemaker ( 1 0 ) that "the Streptobacillus....became completely avirulent for mice after 400 generations.  We were not succesful i n regaining the  virulence by animal passage, once i t had been lost".  The protective  efficacy of the Streptobacillus and the L - l antisera could not be tested because of the loss of virulence in this strain.  Unfortunately,  two lyophilized cultures of S. moniliformis made some two years ago when  70. the strain was more pathogenic could not be revived and thus could not be checked for their virulence. Just what significance the L - l organism plays i n the l i f e of S. moniliformis i s unknown. Its resistance to Penicillin seems to indicate resistance to conditions of duress.  Unknown also i s the  part that the L-organisms play in disease conditions and their relation to carrier states. The possibility of a regenerative process in bacteria brought about by L-forms must not be overlooked (39, 4-0). Again, conjecture might be raised as to whether the pleuropneumonia organisms themselves are remnant forms now no longer able to give rise to a parent or daughter bacillary stage.  In these latter cases, though,  i t must not be forgotten that the pleuropneumonia-elements are pathogenic, while i n the pleuropneumonia-like group the L-forms are apparently non-pathogenic, since pathological conditions are associated with the bacillary forms.  71. V.  SUMMARY  The L - l variant appears to be less active metabolically than the Streptobacillus.  This reduction of activity may be  associated with the survival of the organism and i s undoubtedly related to such phenomena as antibiotic resistance on the part of the L - l organism.  S t i l l , the f i n a l role of the L - l variant has not been  ascertained although certain possibilities have been put forward. The L - l organism i s apparently closely related antigenically to S. moniliformis, as judged by the results obtained during crossagglutination and cross-absorption  experiments with these two  organisms and their corresponding rabbit antisera.  The greatest  difficulty i n this project was encountered while trying to obtain L - l growth i n liquid medium, so that i t would be used for rabbit immunizations and also as a suitable antigen i n the assessment of antibody levels and cross-absorption  tests.  Satisfactory growth of  the L - l organism was finally obtained i n tryptose phosphate serum broth in the presence of 500 - 1,000 units of Penicillin per ml of medium. Morphological and cultural observations of the two organisms confirmed most of the salient features previously reported for the Streptobacillus and the L - l variant.  Dark-field observations made  over an extended period failed to indicate visually the interdevelopment of the two organisms.  However, the dark-field studies  included investigations into the nature of lipoid artefacts normally associated with the organisms; and >the resulting observations suggested that many of the forms of these organisms described in the literature were artefact in origin.  i  BIBLIOGRAPHY.  72.  VI. BIBLIOGRAPHY. 1. Allbritten, F.F., Sheely, R.F., and Jeffers, W.A. "Haverhlllia Multiformis Septicemia. Its Etiologic and Clinical Relationship to Haverhill and Rat-Bite Fevers." J. Am. Med. Assoc., 1940, 114., 2360-2363. 2. Altemeier, W.A., Snyder, H., and Howe, G. "Penicillin Therapy i n Rat Bite Fever." J. Am. Med. Assoc., 1945, 127, 270-273. 3. Alture-Werber, E., Lipschitz, R., Kashdan, F., and Rosenblatt, P. "The Effect of Incompletely Inhibitory Concentrations of Penicillin on Escherichia c o l i . " J. Bact., 1945, j>0, 291-295. 4. Andrewes, C.H., and Welch, F.V. "A Motile Organism of the Pleuropneumonia Group." J. Path. Bact., 1946, 4j£, 578-580. 5. Bergey's Manual of Determinative Bacteriology. Williams and Wilkins Co., 6th edition. 6. Bisset, K.A. "The Cytology and Life-History of Bacteria." 1950. Edinburgh, E. and S. Livingstone Ltd. 7. Blake, F.G. "The Etiology of Rat-Bite Fever." 1916, 23, 39-60.  J. Exp.  Med.,  8. Borgen, L.O. "Infection with Actinomyces muris r a t t i after a Rat Bite." Separatum. Acta pathologica, 1948, 2j5, 161-166. 9 . Brown, T.M., Swift, H.F., and Watson, R.F. "Pseudo-Colonies Simulating those of Pleuropneumonia-like Microflrganisms." J. Bact., 1940, 4 0 , 857-866. 10. Brown, T. McP., and Nunemaker, J.C. "Rat-Bite Fever. A Review of the American Cases with Revaluation of Etiology; Report of Cases." Bull. Johns Hopkins Hosp.y 1942, 70, 201-327. 11. Brown, T. McP., Wichelhausen, R.H., Merchant, W.R., and Robinson, L.B. "A Study of the Antigen-Antibody Mechanism in Rheumatic Diseases." Am. J. Med. Sci., 1951, 221, 618-625. 12. Dawson, M.H., and Hobby, G.L. "Pleuropneumonia-like Organisms as a Variant Phase of Streptobacillus moniliformis." Third Intern. Congr. Microbiol., Abstr. Communic, 1951, p. 21. 13. Dick, G.F., and Tunnicliff, R. "A Streptothrix isolated from the Blood of a Patient bitten by a Weasel (Streptothrix putorii.)" J. Infect. Dis., 1918, 23, 183-187.  73.  14. Dienes, L. "L Organisms of Klieneberger and Streptobacillus moniliformis." J. Infect. Dis., 1939, 6£, 2 4 - 4 2 . 15. Dienes, L. "The Significance of the Large Bodies and the Development of L Type of Colonies i n Bacterial Cultures." J. Bact., 1942, AA, 37-72. 16. Dienes, L., Weinberger, H.J., and Madoff, S. "Serological Reactions of L Type Cultures isolated from Proteus." Proc. Soc. Exp. Biol. Med., 1950, 7j>, 409-412. 17. Dienes, L., and Weinberger, H.J. Bact. Rev.,  1951,  "The L Forms of Bacteria."  15_, 245-288.  18. Dodd, K. "An Isolated Case of Erythema Arthriticum Epidemicum." Boston M. & S. J., 1926, 19^, 633. 19. Dolman, C.E., Kerr, D.E., Chang, H., and Shearer, A. R. "Two Cases of Rat-Bite Fever due to Streptobacillus moniliformis." Canad. J. Pub. Health, 1951, 4 ^ , 228-241. 2 0 . Dumoff, M., and Duffy, C.E. "The Substitution of Starch, Glycogen, and Dextrin for Natural Body Fluids i n the Cultivation of Streptobacillus moniliformis." J. Bact., 61, 535-539. 21. Dumoff, M., and Duffy, C.E. "Effect of Sodium Oleate on Growth of Streptobacillus moniliformis." Proc. Soc. Exp. Biol. Med., 1951, 77, 1-3.  '  22. Edward, D.G. FF., and Fitzgerald, W.A. "Observations on the Growth Requirements of Organisms of the Pleuropneumonia Group." J. Gen. Microbiol., 1951, i>, Communic. 2 3 . Edward, D.G. FF., and Fitzgerald, W.A. "Cholesterol i n the Growth of Organisms of the Pleuropneumonia Group." J. Gen. Microbiol., 1951, 5, 576-586. 2 4 . Engel, A. "Haverhill Fever (in Connection with a Case observed i n Sweden)." Acta Medica Scandinavica, 1949, 132, 562-571. 2 5 . Farrell, E., and Lordi, J.V. "Haverhill Fever. Report of a Case with Review of the Literature." Arch. Internal Med., 1939, £ 4 , 1-14. 26. Hazard, J.B., and Goodkind, R. "Haverhill Fever (Erythema Arthriticum Epidemicum): a Case Report and Bacteriologic Study." J. Am. Med. Assoc., 1932, 9_9_, 534-538. 27. Heilman, F.R. "A Study of Asterococcus muris (Streptobacillus moniliformis). I. Morphologic Aspects and Nomenclature." J. Infect. Dis., 1941, 69_, 32-44.  74. 28. Heilman, F.R. "A Study of Asterococcus muris (Streptobacillus moniliformis). II. Cultivation and Biochemical Activities." J. Infect. Dis., 1941, 69_, 45-51. 2 9 . Heilman, F.R., and Herrell, W.E. "Penicillin i n the Treatment of Experimental Infections with Spirillum minus and Streptobacillus moniliformis (Rat-Bite Fever)." Proc. Staff.' Mayo Clinic, 1944, 1?_, 257-264. 30. Hewitt, L.F.  "Optical Rotatory Power and Dispersion of Proteins."  Biochem. J., 1 9 2 7 , 2 1 , 216-224.  31. Hughes, W.H., Walker, I.R.H., and Fleming, A. "The Effect of Penicillin on Morphology." J. Gen. Microbiol., 1949, 3_, Abstract. 32. Klieneberger, E. "The Natural Occurrence of Pleuropneumonia-like Organisms i n Apparent Symbiosis with Streptobacillus moniliformis and Other Bacteria." J. Path. Bact., 1 9 3 5 , 4 0 , 93-105.  33. Klieneberger, E. "Further Studies on Streptobacillus moniliformis and i t s Symbiont." J. Path. Bact., 1 9 3 6 , £2,  587-598.  34. Klieneberger, E. "Pleuropneumonia-like Organisms of Diverse Provenance: Some Results of an Enquiry into Methods of Differentiation." J. Hyg., 1938, 38, 458-476. 35. Klieneberger, E., and Smiles, J. "Some Observations on the Developmental Cycle of the Organisms of Bovine Pleuropneumonia and Related Organisms." J. Hyg., 1 9 4 2 , 42,  110-123,  36. Klieneberger, E. "Some New Observations bearing on the Nature of the Pleuropneumonia-like Organism known as LI Associated with Streptobacillus moniliformis." J. Hyg., 1942, A2,  485-497.  37. Klieneberger-Nobel, E. "Origin, Development and Significance of L-Forms i n Bacterial Cultures." J. Gen. Microbiol.,  1949, 3, 434-443.  38. Klieneberger-Nobel, E. "Methods for the Study of the Cytology of Bacteria and Pleuropneumonia-like Organisms." Quart. J.  Mic.  Sci.,  1950,  9JL,  340-347.  39. Klieneberger-Nobel, E. "Filterable Forms of Bacteria." Bact. Rev., 1951, 15_, 7 7 - 1 0 3 . 4 0 . Klieneberger-Nobel, E. "The L-Cycle: a Process of Regeneration in Bacteria." J. Gen. Microbiol., 1951, 5_, 5 2 5 - 5 3 0 .  75.  41. Leberman, P.R., Smith, P.F., and Morton, H.E. "The Susceptibility of Pleuropneumonia-like Organisms to the i n vitro Action of Antibiotics: Aureomycin, Chloramphenicol, Dihydrostreptomycin, Streptomycin, and Sodium Penicillin G." J. Urol., 1950, 6£, 167-173. 4-2. Levaditi, C , Nicolau, S., and Poincloux, P. "Sur le role etiologique de streptobacillus moniliformis (nov. spec.) dans l'erytheme polymorphe aigu septicemique. Compt. rend. Acad. d. sc., 1925, 180, 1188-1190. M  43. Levine, B., and Civin, W.H. "Streptobacillus moniliformis Bacteremia with Minor C l i n i c a l Manifestations." Arch. Intern. Med., 1947, 80, 53-60. 44. Litterer, W. "Study of the Streptothrix isolated i n two Cases of Rat-Bite Fever." Tr. Sect. Path. & Physiol., 1917, 275-287. 45. Lominski,. I.R.W., Henderson, A.S., and McNee, J.W. " Rat-Bite Fever due to Streptobacillus moniliformis." Brit. Med. J., 1948, 2, 510-515. 46. Lubsen, N., van der Plaats, A.B.J., and Wolthius, F.H. "Een geval van rattebeetziekte, veroorzaakt door Streptobacillus moniliformis." Nederland Tijdschr. Geneesk., 1950, 9_4_, 102-106. Found i n Biol. Abstr., 25_, April 1951, entry 11250. 47. Melen, B. "The Susceptibility of Pleuropneumonia-like Organisms to the i n vitro Action of some Antibiotics." Acta Patha. et Microbiol. Scandinav., 1952, J O , 98-103. 4 8 . Nelson, J.B.  "The Reaction of Antisera for B. actinoides.  J. Bact., 1933,  26,  11  321-327.  49. Oeding, P., and Pedersen, H. "Streptothrix muris r a t t i (Streptobacillus moniliformis) isolated from a Brain Abscess.'•' Acta Patha. et Microbiol. Scandinav., 1950, 27,  436-442.  50. Parker, F. Jr., and Hudson, N.P. "The Etiology of Haverhill Fever (Erythema Arthriticum Epidemicum)." Am. J. Path., 1926, 2, 357-379. 51. Partridge, S.M., and Klieneberger, E. "Isolation of Cholesterol from the Oily Droplets found i n Association with the H Organism separated from Streptobacillus moniliformis." J. Path. Bact., 1941, 12, 219-223.  76. 52. Place, E.H., Sutton, L.E., and Willner, 0. "Erythema Arthriticum Epidemicum; Preliminary Report." Boston M. & S. J., 1926, 194. 285-287. 53. Place, E.H., and Sutton, L.E. Arch. Intern. Med., 1934,  "Erythema Arthriticum Epidemicum." 659-684.  54. Proescher, F. "Rat-Bite Disease, with Report of a New Case." Internat. Clinics (21st Series), 1911, L., 77-107. 55. Salaman, M.H., King, A.J., Bell, H.J., Wilkinson, A.E., Gallagher, E., Kirk, C , Howorth, I.E., and Keppich, P.H. "The Isolation of Organisms of the Pleuropneumonia Group from the Genital Tract of Men and Women." J. Path. Bact., 1946, 58, 31-35. 56. Scharles, F.H., and Seastone, C.F., Jr. "Haverhill Fever following Rat-Bite." New Eng. J. Med., 1934, 211, 711-714. 57. Shanahan, A.J., and Tanner, F.W. "Further Studies on the Morphology of Escherichia coli exposed to Penicillin." J. Bact., 1948, 55, 537-544. 58. Steen, E. "Rat Bite Fever. Report of a Case with Examination of Haverhillia moniliformis." Acta Patha. et Microbiol. Scandinav., 1951, 28, 17-26. 59. Strangeways, W.I. "Rats as Carriers of Streptobacillus moniliformis." J. Path. Bact., 1933, 37, 45-51. 60. Tang, F.F., Wei, H., and Edgar, J. "Further Investigations of the Causal Agent of Bovine Pleuropneumonia;" J. Path. Bact., 1936, 4 2 , 45-51. 61. Thjfitta, Th., and Jonsen, J. "Streptothrix (Actinomyces) muris r a t t i (Streptobacillus moniliformis) isolated from a Human Infection and studied as to i t s Relation to Emmy Klieneberger s L - l ) . Separatum. Acta. Patha., 1947, 24., 334-351. 1  u  62. Thorp, E. "Rat-Bite Fever i n an Infant." 1925, 2, 255.  Brit. Med. J.,  63. Tileston, W. "The Etiology and Treatment of Rat-Bite Fever." J. Am. Med. Assoc., 1916, 16, 995-998. 64. Topley and Wilson's "Principles of Bacteriology and Immunity." 1947. Third edition. Edward Arnold and Co., London. 65. Tunnicliff, R. "Streptothrix i n Bronchopneumonia of Rats similar to that i n Rat-Bite Fever. A Preliminary Report." J. Am. Med. Assoc., 1916, 66, 1606.  77.  6 6 . Tunnicliff, R. "Streptothrix i n Bronchopneumonia of Rats similar to that i n Rat-Bite Fever." J. Infect. Dis., 1 9 1 6 , 19_, 767-771.  6 7 . Tunnicliff, R., and Mayer, K.M. J. Infect. Dis., 1918, 2JS,  "A Case of Rat-Bite Fever." 555-558.  6 8 . Turner, A.W. "A Study of the Morphology and Life Cycles of the Organism of Pleuropneumonia Contagiosa Bourn (Borrelomyces Peripneumoniae Nov. Gen.) by Observation i n the Living State under Dark-Ground Illumination.'! J. Path. Bact., 1935, £ 1 , 1 - 3 2 . 6 9 . van Rooyen, C.E. "The Biology, Pathogenesis and Classification of Streptobacillus moniliformis.'? J. Path. Bact., 1936, 34., 455-472.  70. Warren, J. "Observations on some Biological Characteristics of Organisms of the Pleuropneumonia Group." J. Bact., 1942,  4 J , 211.  71. Weinberger, H.J., Madoff, S., and Dienes, L. "The Properties of L Forms isolated from Salmonella and the Isolation of L. Forms from Shigella." J. Bact., 1 9 5 0 , 52,  765-775.  72. Williams, S. "An Outbreak of Infection due to Streptobacillus moniliformis among Wild Mice." Med. J. Austral., 1941, 1, 3 5 7 - 3 5 9 . 7 3 . Williams, S. "The Occurrence of Lipoid Structures i n Cultures of Streptobacillus moniliformis." Austral. J. Exp. Biol. Med. Sci., 1 9 4 1 , 19_, 255-259. 7 4 . Wittier, R.G. "The L-Form of Haemophilus pertussis." J. Gen. Microbiol. Supplement, . 1 9 5 1 , 5_, 1024-1031.  APPENDICES.  78 711. APPENDIX A.  1. Strain of Streptobacillus moniliformis used. The strain of Streptobacillus moniliformis used throughout this investigation was isolated by Dolman, et a l . (19) from two Indian children i n Vancouver, British Columbia (1949). The organism has been cultivated upon laboratory media from the time of isolation and through the period of this study, that i s , for approximately three years.  2. Origin of the L - l Variant used. The L - l , pleuropneumonia-like variant, was isolated from the above strain of Streptobacillus moniliformis through the kind assistance of Miss Helen Chang of the Department of Bacteriology and Immunology. This organism has been kept i n the pleuropneumonialike form on tryptose phosphate agar for at least twenty-four successive  generations.  79 APPENDIX B. 1. Stock Cultures of Streptobacillus moniliformis. Many authors have written that the Streptobacillus dies out very rapidly in liquid media. For example, Brown and Nunemaker (2) made the following observations As a general rule, subcultures must be made within 24 hours of the time of inoculation of a tube... (in dextrose starch medium) we have found that the streptobacillus may not be transferable i f one goes beyond the 24. hour limit by even 1 or 2 hours. With the tryptose medium i n which the pH drop i s not so great, the safe period before subculturing may be as long as 36 hours. Only occasionally have subcultures been made at 48 hour intervals with success. Heilman (28) stated that the Streptobacillus died out rapidly after 24 hours in serum veal infusion broth... ''all the elements i n the culture frequently were dead in 48 hours." Lominski, Henderson, and McNee ( 4 5 ) claimed that their strain of S. moniliformis in carbohydrate-containing  fluid media  had to be subcultured every 48 hours, while i n carbohydrate-free media the organism lived for five days but was dead i n seven days. Again, Hazard and Goodkind ( 2 6 ) , Bergey's Manual ( 5 ) , Parker and Hudson ( 5 0 ) , and Steen ( 5 8 ) , speak of the rapid death of the organism in liquid media, On the other hand, the organism was generally considered to survive longer on solid media, although here, too, the period of longevity was short in comparison with more common micro-organisms.  80.  It was found during the course of this investigation that the Streptobacillus could easily be isolated by plate culture from 20% serum tryptose phosphate broth cultures (5 ml quantities) without carbohydrate, which had been incubated for 24--4.S hours and l e f t at room temperature for periods up to seventeen weeks - and this i n spite of the fact that the small liquid cultures had almost completel y dried up. A larger number of L-colonies seemed to result from such older cultures than from fresher cultures; this i s not surprising i f the L-forms are admitted to be the more resistant elements of the Streptobacillus. Sometimes the plates had to be incubated for some 1 0 - 1 2 days before Streptobacillus colonies appeared, and i t may well be that this relatively long incubation period has caused at least some workers to discard cultures before growth had time to develop. Serum tryptose phosphate agar plates with f i l t e r papers in the tops of the Petri plates serve as good stock culture reservoirs for up to several months i f stored i n closed containers. Lfiffler's serum slants stoppered with rubber plugs are also very convenient for carrying stock cultures, especially i f there i s a large quantity of fluid present at the bottom of the tube. Cultures may be recovered from Lfiffler's slants several months after inoculation.  2. Stock Cultures of the L - l Organism. The L - l organism used i n this study could only be maintained for any time by continuous passage on serum tryptose phosphate agar. Plates yielded pure L-colonies after a month of incubation i n closed containers, but as time went on fewer and fewer plates seemed to show growth upon subculture. The agar-block cut out method did not appear to possess any real advantage over ordinary spreading techniques as long as the colonies were cut into with the spreading loop. No fewer than 5 plates per generation were ever used i n maintaining the stock L - l organism, and during most of this investigation when plates were being used as sources of antigen, as many as 20 to 60 plates were spread at a time, thus allowing a large reserve of suitable colonies.  3. Cultures of S. moniliformis i n Clotted Human Blood and i n Sterile Soil. The following experiments were conducted i n an attempt to find a suitable stock culture medium. a. Clotted Human Blood. Clotted blood has been recommended as a stock medium for the Streptobacillus. Twenty-five m i l l i l i t r e s of freshly drawn human blood were dispensed into five sterile tubes of 5 ml each. To each tube was added 0.5 ml of a 22-hour culture of the Streptobacillus. The tubes were covered with Scotch tape and single tubes were placed  82. under the following conditions 1. 2. 3. 4.  Held indefinitely at room temperature. Held at 3TQ indefinitely. Held in the refrigerator indefinitely. Incubated at 37*C for 2 4 hours, then refrigerated indefinitely.  5. Held at room temperature as a blood control. One and one-half months later the tubes were subcultured onto tryptose phosphate serum agar. Many streptobacillary colonies were obtained from the tube of blood that was held at 37 C indefinitely; 9  the other tubes did not reveal the presence of any organisms. The colonies showed typical Streptobacillus organisms when smeared and stained. Two years later, 30% serum tryptose phosphate broth was added to the above tubes; the tubes were incubated at  37°C  for  7  days without revealing any signs of growth. At the end of this time, large inocula from these tubes were subcultured into more broth tubes. Growth did not develop i n any of the tubes.  b. Sterile Soil. Soil was used because a number of fastidious organisms, such as Brucella, have been found to survive i n i t for lengthy periods. Tubes of s o i l were autoclaved for six hours. S t e r i l i t y tests showed that no organisms had withstood the autoclaving. One m i l l i l i t r e of a 22-hour Streptobacillus culture was added to each tube, single tubes then being subjected to the following conditions:  83. 1. 2. 3. 4.  Held at room temperature indefinitely. Incubated at 37*C for 24 hours then held at room temperature indefinitely. Refrigerated indefinitely. Incubated at 37*C for 24 hours then refrigerated indefinitely.  5.  Incubated at 37'C indefinitely. One uninoculated control tube was included with each of the  above experimental tubes. No growth appeared on serum tryptose phosphate agar plates when culture was attempted one and one-half months after inoculation of the tubes.  Growth did not develop 2 years later when tryptose  phosphate serum broth was added to the tubes.  84, APPENDIX C. Experimental Media The following media were inoculated with the Streptobacillus in an attempt to find out i f growth would occur i n the absence of complex protein like serum, blood, or ascites fluid.  Plates were  incubated both under ordinary conditions and i n moist chambers. Tubed media were incubated under ordinary conditions.  Both tubes and  plates were examined daily for one week. A l l the media listed were either negative for growth, or i f slight growth did occur i n i t i a l l y , i t could not be propagated further upon the same medium. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.  Trypsin digest agar plates Peptone sucrose agar plates Prune agar plates Oatmeal agar plates Peptone starch agar plates Potato dextrose agar plates Starch agar plates Bismuth sulphite agar plates S.S. agar plates Endo's agar plates Peptone glucose acid agar plates Purple lactose agar plates Crystal violet agar plates Tellurite agar plates Tomato juice agar plates Sodium,Albuminate agar plates Streptomyces assay agar plates Sodium thiosulphate agar plates Yeast lactate agar plates Wright's agar plates Beef infusion agar (pH 8.3) slants Nutrient agar (ph 8.3) Proteose peptone water (pH 8.3) Kligler's agar slants M-79 agar slants Greave's agar slants Ashby's agar slants Greenberg's semi-solid deeps Glucose peptone beef infusion deeps Simmon's citrate agar slants  85, 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.  Reed's deeps Crone's deeps Bean extract plus caffein agar slants Tetrathionate broth Brilliant green bile broth Sagen's fluid medium Starch suspension agar slants Hoffer's fluid medium Czapek's fluid medium Dubo's fluid medium Tyrosin agar slants Yeast tryptone broth Yeast lactate broth Yeast litmus milk Czapek's agar slants Dextrin nitrate agar slants Yeast glucose agar shakes Sodium hippurate Aesculin Arginine broth  86 APPENDIX D. The Preparation of Lipoid-free Serum (After the Method of Hewitt) One hundred m i l l i l i t r e s of sterile ox serum at 0*C was added slowly, with constant stirring, to a mixture of 4-20 ml of absolute ethyl alcohol plus 180 ml of anhydrous ethyl ether.  The mixture had  been cooled previously to -15C by placing i t in a l i t r e beaker i n a bucket of chopped ice and sodium chloride. A large Petri dish was placed over the top of the beaker after the addition of the serum. The salt and ice mixture was piled over the beaker and the bucket with i t s contents was placed i n the refrigerator to prevent rapid thawing.  Repeated thermometer readings over a period of 2 hours showed  that the temperature of the serum-precipitating mixture never rose above -15 C. 9  At the end of 2 hours the bucket was removed from the refrigerator to room temperature.  The precipitated proteins were  filtered off on a Btlchner funnel, utilizing strong water suction. The f i l t r a t i o n was extremely slow, requiring nearly 3 hours for completion.  No new alcohol-ether mixture was added to the funnel.  About 150 ml of ether at -15*C were added to the precipitate in the funnel. The proteins were quickly transferred to Soxhlet extraction thimbles.  Extraction was carried out with anhydrous ether and metallic  sodium for 2 hours.  The thimbles were placed in the refrigerator  overnight. Extractions with ether plus sodium were made until a total  87, of 22 hours was completed.  The Soxhlet thimbles were then  transferred to a desiccator over calcium chloride. of sulphuric acid was also placed i n the desiccator.  A small beaker The desiccator  was attached to a vacuum pump and evacuated for 2 hours at 20 inches of mercury (4-0.8 cm Hg).  The stop-cock was turned so as to seal the  vacuum and the desiccator was l e f t at room temperature for 2 days. The l i d of the desiccator was loosened after the second, day to allow the proteins to become moistened with the a i r of the room. The proteins were broken into small pieces and powder and stirred into 100 ml of d i s t i l l e d water.  The proteins took some 5  hours, with constant stirring, to go into solution. The resuspended proteins were then Seitz-filtered. procedure took about 3 hours to complete).  (This  

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