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MOV 5 V^^ 
 
 A Bacteriologic Study of the Diphtheroid Organisms with 
 Special Reference to Hodgkin's Disease 
 
 DISSERTATION 
 
 Submitted in Partial Fulfilment of the Requirements for the Degree 
 
 of Doctor of Philosophy in the Faculty of Pure Science 
 
 of Columbia University in the City of New York 
 
 BY 
 
 FREDERICK EBERSON, B.Sc, M.A., M.Sc. 
 
 NEW YORK CITY 
 1918 
 
 T\ 
 
o 
 
 

 m 
 
 PREFACE 
 
 The study reported in the following pages was undertaken with the 
 purpose of sifting out the voluminous data which have accumulated 
 on the subject of diphtheroid organisms. 
 
 The greatest impetus given to such an investigation has resulted, in 
 recent years, from the attempts of numerous workers to associate 
 Hodgkin's disease with a certain organism belonging to the diphtheroid 
 group. In a short time the literature on the subject assumed great 
 proportions. 
 
 A survey of the field revealed immediately the fact that the group 
 as a whole has been indiscriminately used as a cover for any organ- 
 ism which bore some sort of morphological resemblance, real or 
 apparent, to the diphtheria type. The material presented in this study 
 is by no means exhaustive since a great many methods will have to be 
 tried before a clear-cut answer is to be given to the question as tc 
 what a true diphtheroid is and where it belongs in the comprehensive 
 group of which these bacteria are members. 
 
 Of great moment is the idea of mutation introduced by Rosenow 
 and his school. This problem is fraught with such difficulties not 
 amenable to satisfactory control, that a reply to the findings of these 
 workers will have to be deferred. At present the outlook for a deci- 
 sive answer to this question seems to be found in at least one method 
 whereby we can control the constitution of our nutrient media by 
 means of H-ion concentration, and also in a phase of serological 
 technic which has not been studied heretofore. In the following 
 pages an effort will be made to attack the problem from these angles. 
 
 I wish to thank Professor Hans Zinsser for much valuable advice 
 and suggestions and Dr. R. E. Buchanan of the Iowa State College 
 for his criticisms on nomenclature. F. E. 
 
 Bacteriological Laboratory. Columbia University. 
 New York City, January, 1918. 
 
 93754 
 
CONTENTS 
 
 Preface 3 
 
 1. NOMENCLATURE AND CLASSIFICATION OF THE DIPH- 
 
 THEROIDS 
 
 Introduction 7 
 
 HISTORICAL REVIEW OF DIPHTHEROIDS AND EMENDATIONS 8 
 
 DISTRIBUTION OF DIPHTHEROIDS 18 
 
 Diphtheroids in Hodgkin's Disease 17, 18 
 
 Diphtheroids in Glands and Tissues 18 
 
 Cultural Study and Grouping of Organisms 18, 19 
 
 Diphtheroids from the Eye 21 
 
 Nasal Diphtheroids 21 
 
 Diphtheroids from Ascitic Fluids 21 
 
 Diphtheroids in Blood Cultures 22 
 
 Skin Diphtheroids 22 
 
 Diphtheroids from the Brain 23 
 
 Diphtheroids from the Appendix 23 
 
 Source and Fermentative Reactions 24 
 
 CLASSIFICATION OF THE DIPHTHEROIDS 25 
 
 2. SEROLOGIC STUDY 
 
 EXPERIMENT WITH HODGKIN's STRAINS 30 
 
 Agglutination Tests 31 
 
 Complement Fixation 31 
 
 APPLICATION OF AGGLUTININ ABSORPTION TESTS TO DIFFERENTIATION 
 
 OF TYPE DIPHTHEROIDS 32 
 
 RELATION OF TYPE DIPHTHEROIDS 33, 34 
 
 3. PLEOMORPHISM OF DIPHTHEROIDS, MUTABILITY OF TYPES 
 
 AND A METHOD FOR DETERMINING MUTANTS 
 
 VARIABILITY OF BACTERIA 34 
 
 CULTURAL STUDY OF MUTA'TION .' 36 
 
 Experiments with Museum Strain of C. enzymicus (Mellon).... 36 
 
 Effect of Various Mediums on Selection of Type Zl 
 
 Experiments with Mellon Strain cultivated on Blood Medium.. 39 
 
 STUDY WITH PURE LINE OF C. ENZYMICUS 39 
 
 Bacillary Type 39 
 
 "Transition" of Bacillus to Coccus 39 
 
 Coccus Type 40 
 
 Behavior of Coccus and Bacillary Types in Carbohydrates 41 
 
 SEROLOGIC EXPERIMENTS 42 
 
 Absorption of Agglutinins 42 
 
 Absorption Experiments with Coccus Serum 42 
 
 Absorption Experiments with Bacillary Serum 43 
 
 SUMMARY AND CONCLUSIONS 44 
 
 Bibliography 45, 46, 47 
 
 Plates 1-3. Explanation of plates 48, 49, 51, 53 
 
 Vita 55 
 
A BACTERIOLOGIC STUDY OF THE DIPHTHEROID 
 
 ORGANISMS WITH SPECIAL REFERENCE TO 
 
 HODGKIN'S DISEASE 
 
 Plates 1-3 
 
 Frederick Eberson 
 
 From the Department of Bacteriology, Columbia University, College of Physicians and Sur- 
 geons, New York City 
 
 I. NOMENCLATURE AND CLASSIFICATION OF THE 
 DIPHTHEROIDS 
 
 Introduction 
 
 Within the past few years the literature dealing with organisms of 
 the diphtheria group have received many new contributions. It seems 
 that most human and animal diseases of hitherto unknown etiology 
 have been ascribed to some sort of diphtheroid organism. Whether or 
 not these results will be confirmed has little bearing on the problem 
 which confronts the systematic bacteriologist who finds himself hard 
 put to it in attempting to classify the great number of organisms which 
 for no reason other than a slight resemblance to the diphtheria bacteria 
 have been thrown haphazardly into a group of "unknowns" — the 
 diphtheroids. 
 
 Since Loeftler's -discovery of the diphtheria organism, so many 
 so-called "pseudodiphtheria" bacteria have been mentioned in the litera- 
 ture that it seems necessary at this time to survey the field and come to 
 an understanding as to what constitutes a diphtheroid. 
 
 In a recent paper on the "Nomenclature of the Coccaceae," 
 Buchanan has pointed out the chaotic state of bacterial nomenclature. 
 That the naming of species, genera and higher groups needs careful 
 study and revision is a foregone conclusion. A great need is felt for 
 such an analysis in the groups of bacteria which possess common char- 
 acteristics. Such a group is the diphtheroid group. 
 
 First, it is necessary to define a "diphtheroid." The term "pseudo- 
 diphtheria" has always seemed unsatisfactory because the organisms 
 so-called dififer in so many respects from the true Klebs-Loeffler organ- 
 ism as to warrant the dropping of the term from the literature except 
 in conditions to be suggested herewith. A review of the extensive 
 
 Received for publication Feb. 6, 1918. 
 
8 Frederick Eberson 
 
 work done in this group reveals the fact that there are bacteria which 
 resemble the true diphtheria organism morphologically and culturally, 
 but which differ in one important respect, namely, that of virulence as 
 tested on guinea-pigs. The true Klebs-Loeffler has been shown to fer- 
 ment dextrose, maltose and dextrin with acid production and to possess 
 definite toxic properties on injection, resulting in the death of the 
 animal in 3-4 days with a characteristic inflammation and congestion 
 of the suprarenal glands. To those bacteria which lack this virulence 
 but which otherwise conform to the character of true diphtheria bac- 
 teria, should be assigned the species designation ''pseudodiphtheria." 
 The generic name given to the group by Lehmann and ^eumann is 
 Corynebacterium and should be retained for the organisms of this 
 group. Certain characters ought to be accepted as properly belonging 
 to the species before we can adequately define what we mean by a diph- 
 theroid. These may be enumerated as follows : Morphological resem- 
 blance to diphtheria organism, gram-positive, nonmotile, absence of 
 spores, presence or absence of metachromatic granules, no gas-produc- 
 tion in carbohydrate mediums. The fermentation of sugars may or 
 may not occur. This property gives us a basis for classification which 
 will be taken up later. A careful study of the different species which 
 have been described shows that the characters enumerated ought rightly 
 to be accepted as a basis for differentiating the diphtheroids from other 
 bacteria which have been improperly classed with them. With this end 
 in view the different species are taken up individually and examined 
 from the standpoint of validity and priority. The suitability of the 
 name is considered from the viewpoint of sufficient description, and in 
 the light of more comprehensive studies which have given us sugges- 
 tions for proper nomenclature. When the species name implies etiology 
 which is unknown, doubtful or unconfirmed, that part of it is modified 
 to suit the case. 
 
 Historical Review of Diphtheroids and Emendations 
 B. pseudodiphtheriticum (Loeffler) Migula 
 The description is given as follows : Gram-positive, no polar bodies, non- 
 motile. Grows luxuriantly on blood serum with a glistening layer. In broth 
 clouding is produced. LoeiBer described a pseudodiphtheria organism in his 
 first paper on the etiology of diphtheria. He said the organism resembled 
 diphtheria but was avirulent for guinea-pigs ; that it was smaller than the true 
 diphtheria and showed a marked absence of club-shaped forms. In 1888, 
 V. Hofmann published an account of avirulent diphtheria-like organisms iso- 
 lated from the oral cavity in 26 of 45 normal persons. He called these organ- 
 isms pseudodiphtheria bacilli and described them as follows : Clouding pro- 
 duced in broth, no acid production, grow at room temperature, no spores. 
 
Diphtheroid Organisms 9 
 
 The rightful name for this species is Corynebacterium pseudodiphtheriticum 
 (Hofmann-Wellenhof). It is questionable, however, if we can retain this name 
 for all diphtheroids which are morphologically and culturally like the one 
 described by v. Hofmann. Recent studies show that there are diflferent species 
 of saprophytic diphtheroids which conform to the type characters, yet are not 
 found in the nose or throat. At the close of this communication we shall 
 describe a few such strains which have been isolated from widely varying 
 sources. 
 
 B. PSEUDODIPHTHERITICUS ALCALIFACIENS 
 B. PSEUDODIPHTHERITICUS ACIDUM FACIENS 
 
 These names were applied by 'Kurth to diphtheroid organisms which he found 
 to be avirulent, and were capable of producing, respectively, a weakly alkaline 
 and an acid reaction in sugar broth. The alcalifaciens species conforms to the 
 following description : No granules, short forms on serum with white growth, 
 abundant growth in bouillon with clouding, no acid produced in sugar broth. 
 
 This description, although meager, suggests that Kurth was undoubtedly 
 dealing with the type Hofmanni. B. alcalifaciens should lapse into synonymy 
 with this species. As for the B. pseud, acidum faciens a new species is hardly 
 justified in view of the fact that avirulent diphtheroids differ in their fermenta- 
 tive properties in various sugars. Moreover the description is incomplete. 
 Apart from the interest which attaches to the first attempt at classifrcation 
 from the fermentation standpoint, these two species have no particular value. 
 
 B. XEROSIS (neISSER AND KUSCHBERT) MIGULA 
 
 Club-shaped, plump, and longer septate forms. On blood serum terminal 
 swellings never noted. Gram-positive, polar bodies sometimes visible, no 
 growth below 25 C, no clouding in bouillon, but granular sediment is formed. 
 Distinguished from pseudodiphtheria by its ability to grow only above 25 C. 
 
 One point needs slight modification in this description of B. xerosis, namely, 
 that of temperature requirement. On certain mediums, such as ascitic dextrose 
 agar we have noted growth of 12 strains of diphtheroids which were isolated 
 fiom the eye. These organisms complied with the description given for 
 B. xerosis as regards morphology and fermentative properties in different car- 
 bohydrate broths. Some of the strains developed very slowly at temperatures 
 ranging from 18-25 C, showing delicate growth on slant mediums after 4 
 days. Eisenberg states that B. xerosis grows only at body temperature. 
 
 B. septatum gelpke 
 
 Nonmotile, gram-positive, club-shaped forms on serum. Young cultures 
 show polar staining, no spores. Grows on serum with a delicate yellowish 
 dry appearance. In bouillon no clouding occurs but a granular flaky deposit 
 is visible. Does not grow at temperatures below 28 C. Is to be distinguished 
 from other organisms of the group by this property. Associated with conjunc- 
 tival inflammation — "schwelltjngskatarrh." 
 
 A comparison of the 2 organisms just described makes it appear questionable 
 that we are dealing with a distinct species in B. septatum. This organism 
 which appears to be identical with B. xerosis is, like the latter, not necessarily 
 associated with a conjunctivitis for B. xerosis has been isolated frequently 
 from normal eyes. Zinsser is of the opinion that the B. xerosis is a harmless 
 parasite which may occur more often in the slightly inflamed than in the nor- 
 mal conjunctiva. The term septatum is not especially characteristic for diph- 
 
10 Frederick Eberson 
 
 theroids associated with normal and diseased eyes. Large striped or septate 
 bacteria have been found in the nose and in the blood during the course of per- 
 sonal study on this group of organisms. 
 
 B. NODOSUS PARVUS (lUSTGARTEN-MANNABERG) 
 
 Club-shaped organism, disposed at angles or parallel nonmotile, spores 
 absent, slow growth at Zl C. on agar with white appearance, nonpathogenic, 
 facultative. Found in normal human urethra. Migula has changed the name 
 of this bacterium somewhat and calls it B. nodosum. Similar organisms have 
 been isolated from the urine. In one case we have found this organism asso- 
 ciated with a gonorrheal urethritis and in another instance in normal urine. 
 Marked differences have been noted in the acid-forming properties of these 2 
 strains. One gives rise to abundant acid in several sugars and the other fails 
 to attack any of them. They are gram-positive. Hine mentions 11 strains of 
 diphtheroids which he isolated from the urogenital tract. They appeared as 
 large clubbed forms, markedly segmented and exhibited granules, often of large 
 size. They fermented dextrose, saccharose, maltose and dextrin. 
 
 It is suggested that the name Corynebact. nodosum (Migula) be given to these 
 organisms and that B. nodosum parvum lapse into synonymy. 
 
 B. ENDOCARDITIS GRISEUS ( WEICHSELBAUM) 
 
 Flugge classifies this species under the head of pseudodiphtheria. It is a 
 short rod, morphologically resembling B. xerosis. Grows well at room temper- 
 ature, facultative, gram-positive, irregular staining property, js actively motile, 
 glistening growth on mediums, with grayish-white appearance. In streak cul- 
 tures a light brown or. reddish-gray growth is observed. Pathogenic for 
 guinea-pigs and white mice. Rabbits, when injected, show endocarditis. Found 
 on the heart valves in a fatal case of recurrent ulcerative endocarditis. Accord- 
 ing to the suggested definition for diphtheroid this bacillus should be ruled out. 
 The property of motility has not been observed among members of the diph- 
 theria group. 
 
 B. erythematis 
 
 Demme, in 1887, described an organism which caused erythema with death 
 in guinea-pigs. It is a delicate rod, disposed in smaller or larger groups, non- 
 motile, forms spores, grows best at Vl C. but is slow in development, facultative, 
 gram-positive. Flugge has put this bacterium in the diphtheroid group. Apart 
 from the diphtheroid picture which might be seen in a microscopic field because 
 of the configuration in groups and by virtue of the bulging forms due to spores, 
 there is no apparent reason for classifying this species as a diphtheroid. Spore- 
 formation rules the organism out of the group. 
 
 B. RENALIS BOVIS (eNDERLEN) MIGULA 
 
 Rods with thickened ends, nonmotile, gram-positive. In broth, a granular 
 precipitate is formed, the mediums remaining clear. Obligate aerobe. Does 
 not grow at room temperature. A more adequate description is given by Ernst. 
 He isolated the strain from cases of pyelonephritis in cattle. The organism 
 grows poorly as compared with the diphtheria bacterium. Babes-Ernst gran- 
 ules appear much later. It is club-shaped, lancetlike or cylindrical. No growth 
 takes place at room temperature. Nq acid produced in dextrose and glycerin, 
 no spores, facultative aerobe, nonmotile, gram-positive. Pathogenicity appears 
 doubtful. Ernst was unable to demonstrate virulence for guinea-pigs or rab- 
 
Diphtheroid Organisms 11 
 
 bits by means of intraperitoneal or intrapulmonary inoculations. Concludes 
 that the organism is not etiologic for pyelonephritis, since he could not recover 
 the organism from the lesions or reproduce the disease. 
 
 Grips named an organism which he isolated from swine Bacillus pyogenes 
 ' suis. Kiinnemann described a similar organism from cattle under the name 
 B. pyogenes hovis. Glage combined the two under the single specific name 
 B. pyogenes. Glage and Prieve, in their studies have shown that the organisms 
 are related to the influenza bacillus and are members of the group of hemo- 
 globinophilic organisms. The name C. pj'Ogenes (Glage) is perhaps most suit- 
 able for this organism. 
 
 Among human beings, pus cavities and suppurating wounds have been found 
 to harbor diphtheroid organisms. Whether or not these possess pyogenic prop- 
 erties is speculative. It may be of interest to note that three strains isolated 
 by the writer have been obtained from an anal pus pocket in a patient whose 
 kidney underwent suppuration and complete necrosis. These organisms have 
 been injected subcutaneously and intraperitoneally into guinea-pigs with nega- 
 tive results. Diphtheroids found in sinuses following abscess formation are 
 not rare. It is very likely that these organisms are skin inhabitants. 
 
 B. STRIATUS FLAVUS (V. BESSER) 
 
 Eisenberg's description : Found in normal nasal mucus. Short thick rods, 
 sometimes curved, with striped or barred appearance. On agar white, thick 
 growth with yellow pigment. Grows rapidly at room temperature, no spores. 
 
 B. STRIATUS ALBUS (v. BESSER) 
 
 Same source as B. striatus flavus. Appearance the same. Growth on agar 
 is white and glistening, no spores. Fliigge mentions B. striatus albus only. 
 Chester has named the organism B. striatum. 
 
 That these striped diphtheroids occur in the normal nasal mucus has been 
 shown by several investigators. Undoubtedly v. Besser was dealing with but 
 one type of organism which is capable of producing pigment varying from white 
 to yellow. In a comprehensive study made by Morse this was found to be the 
 case. Our own studies have confirmed these findings. Morse has given this 
 species diphtheroid the name B. flavidus with the following diagnosis : Thick 
 forms with clear cut bars. Large and irregular granules visible. Heavy 
 growth on serum with pigment varying in color from white to yellow. Ferments 
 dextrose and maltose but not saccharose. B. flavidus as a species designation 
 is invalid since a prior description had already been given by v. Besser and 
 later amended by Chester to conform with binomial system of nomenclature. 
 The emendation by Morse is recognized but the species should be written, 
 Coryn. striatum (Chester) nov. comb. 
 
 B. BORDONI — UFFREDUZZI (eISENBERG) 
 
 This is known as B. epidermidis and has been described as Bizzozeros Lep- 
 tothrix epidermidis. It is an avirulent spore-former growing best at 15-20 C 
 Since this organism does not conform to the genus requirements, it should not 
 be classed with the diphtheroids. 
 
 B. CLAVATUS 
 
 Fliigge, in 1894, gave this name to a club-shaped bacillus which he isolated 
 from milk heated to 100 C. for one hour. It is a long bacillus, motile, forms 
 spores. Swollen at ends, anaerobic, liquefies gelatin. 
 
12 Frederick Eberson 
 
 This same species is met with in an earlier work on dysentery by Kruse 
 and Pasquale. They isolated from the heart blood and mesenteric fluid in 
 oases of Egyptian dysentery an organism which they call "B. clavatus." It is 
 not described in any way as to warrant its recognition as a species. The char- 
 acters enumerated by Fliigge make it invalid as a type of corynebacteria. 
 
 SPORE-FORMING DIPHTHEROID OF DE SIMONI 
 
 Isolated from nasal secretion in case of ozena. Said to form spores only 
 when grown in milk or on potato. Facultative anaerobe. Grows luxuriantly 
 in dextrose, lactose and maltose broth with slight acid formation. 
 
 The author was undoubtedly misled by the appearance of an impure culture. 
 The illustration given in his article shows a complete transformation from a 
 typical diphtheroid of slender and unevelnly staining aspect to a blunt spore- 
 bearing rod in older cultures. This is highly improbable, yet were it absolutely 
 authentic, one should not be inclined to class this species with the diphtheria 
 group for reasons outlined previously. 
 
 B. DIPHTHEROIDES 
 
 Described by Klein. Isolated from purulent secretion of udder of cow. 
 Organism fails to grow below 25 C. Growth on agar slow and sparse, aerobic. 
 On serum grows best at Zl C., producing white colonies. It is oval or spherical 
 in form with a deeply staining center. Club-shaped forms present, gram- 
 positive. Produces abscess in guinea-pigs after subcutaneous or intraperito- 
 neal inoculation. The organism dies within a week when grown on artificial 
 mediums. 
 
 This diphtheroid, according to the few cultural characters given, is in all 
 likelihood identical with C. pyogenes (Glage). The description is too meager 
 to warrant retaining it as a distinct species. The fact that the organism died 
 so readily on artificial mediums indicates that it resembled the hemoglobino- 
 philic type of which Glage's is an example. 
 
 B. VARIABILIS LYMPHAE VACCINALIS 
 
 This is the first of several descriptions of a species associated with the vac- 
 cine pustules of calves. Nakanishi, in 1900, reported on an organism which he 
 isolated from a vaccine pustule. It is club-shaped, coccoid, segmented or 
 branched. Stains poorly with Gram, facultative anaerobe, nonmotile. Central 
 granule noted when stained with warm fuschin. Growth on serum is abundant. 
 Pale yellow or orange-yellow pigment produced. Poor growth on gelatin. 
 Spore-formation is questionable. The name of this species is not a binomial 
 and therefore may be discarded. A somewhat later and more adequate descrip- 
 tion by Levy and Fickler is invalid, however, for the same reason. Their 
 organisms were found to be identical with that of Nakanishi and possessed 
 pyogenic properties for guinea-pigs, mice and rabbits. The name given to the 
 species by these authors is Corynebact. lymphae vaccinalis. It is definitely 
 gram-positive. In 1904 this species is met with as Coryneb. pyogenes, so 
 described by Lewandowsky who published a paper almost simultaneously with 
 Galli-Valerio. The last named author found his species to be identical with 
 Nakanishi's and that of Levy and Fickler. Galli-Valerio proposes the name 
 Coryneb. vaccinae (Galli-Valerio). Lewandowsky's designation is a binomial 
 and valid. Galli-Valerio gave no reasons for rejecting a prior description and 
 was apparently unaware of Lewandowsky's work which appeared at the same 
 
Diphtheroid Organisms 13 
 
 time. The species is best described perhaps as Coryneb. vaccinae (Galli- 
 \'alerio) because the name C. pyogenes had already been used by Glage (q. v.) 
 for an organism isolated from cattle. 
 
 B. coryzae segmentosus Cautley ; B. diphtheroides citreus ; B. diphtheroides 
 brevis; B. maculatus ; B. diphtheroides liquefaciens ; B. auris ; B. ceruminis. 
 
 These seven strains were described by Graham Smith. He isolated the first 
 5 from the nose and mouth, and the last 2 from the ear. B. coryzae segmentosus 
 was first cited by Cautley in a local English government report. The organism 
 is characterized as follows : Nonmotile, gram-positive, polar bodies visible, 
 white growth in gelatin. In broth, clear with sediment. Slight acid produced 
 in dextrose bouillon. Avirulent. 
 
 The species' name is objectionable from the etiologic standpoint and violates 
 the rule for binomial nomenclature. This organism has been isolated from 
 normal nasal mucus on numerous occasions and is not necessarily associated 
 with coryza. It differs from C. striatum in its slight acid-producing power in 
 dextrose, its slow growth and small polar bodies. That this species may be 
 obtained from the eye as well as from the nose has been noted by the writer. 
 
 In these instances it is to be differentiated from C. xerosis by the absence 
 of acidity when grown in maltose and saccharose broths. As a rule C. xerosis 
 ferments these last named sugars (Morse). It is suggested that the name 
 Coryneb. segmentosum nom. nov. be substituted for the invalid B. coryzae seg- 
 mentosus Cautley. B. diphtheroides citreus corresponds to Coryn. striatum 
 (Chester). It produces yellow pigment. The name given by Graham Smith 
 is not a binomial and disregards priority. It should lapse into synonymy with 
 Coryn. striatum (Chester) Eberson. 
 
 B. DIPHTHEROIDES BREVIS 
 
 From pus of oral abscess, Growth on agar, white and slimy. In gelatin, 
 white and dry, clouding in bouillon, granular sediment. Highly acid in sugar 
 broth. This species will be discussed later in connection with diphtheroids 
 isolated from the ear. This organism B. maculatus is meagerly described as an 
 acid-producer. Avirulent. It is impossible to tell wherein this organism 
 agrees with or differs from the main types which have been established in 
 recent studies and for that reason can hardly be looked on as a distinct species 
 until more complete biometrical study is made for strains from a similar 
 source. 
 
 B. DIPHTHEROIDES LIQUEFACIENS 
 
 On serum growth is light yellow. Very long bacillus, somewhat curved, 
 with polar bodies. Slightly motile; gram-positive; on agar, growth is thick 
 and moist with light yellow pigment. Gelatin is liquefied in 3 days. In bouillon, 
 slight clouding with granular sediment. In sugar broth reaction is weakly 
 alkaline or neutral. Avirulent. A search through the literature indicates that 
 gelatin liquefying properties have not been associated with diphtheroids, except 
 in this one instance. In 61 strains studied by the writer, but one was found 
 to liquefy gelatin. This species will be described under isolations from ascitic 
 fluids and Graham Smith's organism will be discussed and emended. 
 
 B. AURIS 
 
 From aural pus of scarlet fever patient. On serum is similar to that of 
 Klebs-Loeflfler organism, but slower in growth. Segmented, nonmotile, gratn- 
 positive, polar bodies visible. On agar, gray colonies ; on gelatin, small trans- 
 
14 Frederick Eberson 
 
 lucent colonies which become white in three days. Bouillon is clouded slightly 
 and sediment produced. Visible acid produced in sugar broth. 
 
 B. CERUMINIS 
 
 From normal ear. Long, thin, curved organism. Gram-positive, polar 
 bodies. Bouillon remains clear with sediment. Neutral or alkaline reaction 
 in sugar broth. Avirulent. 
 
 DIPHTHEROIDS FROM THE EAR 
 
 Diphtheroids of the ear have been made the subject of a very valuable paper 
 by Alice Hamilton. In a study of over 50 cases of otitis media and scarlet 
 fever she succeeded in differentiating 2 groups of organisms. 
 
 Group 1. — Includes diphtheroids with the following characters : Short rods, 
 gram-positive (weakly staining). Abundant growth on agar. Ferment dex- 
 trose and saccharose but not maltose or dextrin. Nonpathogenic. 
 
 Group 2. — Long, barred or granular, often clubbed. Gram-positive, scanty 
 growth on agar. Ferment dextrose and maltose, but not saccharose. Often 
 virulent for guinea-pigs. 
 
 The pathogenicity of these organisms is altogether different from that of the 
 diphtheria bacterium inasmuch as diphtheria antitoxin did not protect against 
 them. Hamilton's studies showed also that the organisms isolated from otitis 
 undoubtedly have some bearing on the disease since vaccine treatment had a 
 marked effect on opsonic determinations. If we compare this type (Group 2) 
 with the B. auris described by Graham Smith it appears that we have the 
 same organism. Unfortunately the work of the last named author does not 
 mention virulence tests and the strains were too few in number to merit com- 
 parison. We are safe, however, in recognizing that the diphtheroids found in 
 the ear are not of the same type and that in cases of otitis media and post- 
 scarlatinal infections another species, not necessarily of etiologic significance, 
 seems to predominate. The name Coryn. auris (Graham Smith) is proposed. 
 
 B. CERUMINIS 
 
 This organism has been observed also by Dr. Dwyer, of this laboratory. 
 This organism fails to ferment any of the sugars and differs from C. Hofmanni 
 in morphology as well as its behavior in broth which remains clear, with sedi- 
 ment. The name should be Coryn. ceruminis (Graham Smith) comb. nov. 
 
 There remains for consideration the diphtheroids of Hamilton's Group I. 
 These organisms differ from C. hoagii (Morse) only in the absence of a salmon- 
 pink pigment. In all likelihood these strains conform to this type. Besides 
 these ear diphtheroids, there seems to be a variety which is characterized by 
 vigorous fermentative properties in a number of sugars. Two such strains have 
 been isolated by the writer. A description follows : Gram-positive, small rods, 
 abundant, white glistening growth on agar, no spores. Form large amounts 
 of acid in dextrose, lactose, saccharose, maltose, mannite, and dextrin, but do 
 not split raffinose. Acidity titrations with N/20 NaCH after 48 hours' incuba- 
 tion were as follows : 
 
 
 Dextrose 
 
 Lactose 
 
 Saccharose Maltose 
 
 Mannite 
 
 Dextrin 
 
 Strain 1 
 
 3.2 
 
 3.5 
 
 3.9 4.0 
 
 2.8 
 
 2.3 
 
 Strain 2 
 
 5.0 
 
 3.6 
 
 4.4 4.7 
 
 2.6 
 
 2.0 
 
 These strains were obtained from cases of otitis media. This species is similar 
 to the diphtheroides brevis described by Graham Smith. It is suggested that a 
 
Diphtheroid Organisms 15 
 
 binomial be used for the species with the emendation noted above, namely, 
 Coryn. acidum sp. nov. 
 
 B. PSEUDODIPHTHERITICUS GAZOGENES 
 
 This is the only gas-forming diphtheroid met with in the literature. Jacobson 
 has described it as follows : Isolated from stools of an infant. Is a vigorous 
 gas former, club-shaped with thickened ends. "One of the cultures resembled 
 streptobacilli. This character did not persist in the transplants." Gram-positive 
 and "in cultures a few days old gram-negative organisms were observed." No 
 spores. Good growth in gelatin, after 5-6 days. Litmus milk is reddened in 
 24 hours. Nonpathogenic. From the description given by the author it does 
 not seem likely that we have to deal with one organism. Several things point 
 to the impurity of the culture. Isolation from feces, as was here the case, makes 
 such contamination all the more probable. According to the definition of a 
 diphtheroid, this organism which is here described is hardly to be reckoned as 
 one of the genus Corynebacterium. 
 
 DIPHTHEROIDS AND ACNE VULGARIS 
 
 Unna, in 1891, repeatedly found in lesions of acne vulgaris an organism which 
 he concluded was the cause of the disease. He did not isolate the organism in 
 pure culture, but gave it the name "Flaschenbacillus" and described it as fol- 
 lows : Unevenly staining rod having clubbed ends and showing ellipsoid forms. 
 In 1894, Hodara, in a bacteriologic study of acne, reported the presence of two 
 types of bacteria in acne lesions. One type is that of Unna and the other 
 Hodara considers as another variety which is similar in appearance but shows 
 varying morphology. For this species he used the name "Flaschen-Kugel- 
 Bacillus" ("Bacillus a Flacons-Ballons"). 
 
 Neither of these papers goes into any detail concerning the organisms and 
 are of little, if any, value for classification purposes. The first accurate report 
 on these diphtheroids is given by Sabouraud about 3 years later. Sabouraud 
 studied the bacterial flora of the sebaceous plugs which can be squeezed out of 
 the skin. He found an organism which he claimed to be the true cause of 
 the disease according to his definition, and which he described as follows : 
 Punctiform bacillus, almost coccoid, gram-positive, less than 1 mikron in length. 
 Stains readily with anilin dyes. Requires highly acid mediums for growth. 
 Produces intense clouding in broth. Sabouraud named the organism "bacille 
 de seborrhee grasse." 
 
 It was not until 1901, when Gilchrist made a thorough study on the bacterial 
 flora of the skin, that an adequate description is found for the bacteria which 
 were mentioned by Unna and his followers. Gilchrist's description points to 
 the identity of his organism with that of his predecessors. It is gram-positive, 
 stains unevenly and very small. It is anaerobic, although many strains were 
 found to grow aerobically after continued cultivation on artificial culture 
 mediums. Surface smears made with the material from pustules failed to show 
 growth whereas stab cultures and heavily inoculated unbroken pustules always 
 showed growth. The organism is named B. acnes by the author. We are still 
 in doubt as to the cause of acne and for this reason B. acnes as a species 
 designation may be criticized. Fleming claims to have obtained beneficial results 
 by the use of autogenous vaccines prepared from similar organisms which he 
 isolated from cases of acne. As Sabouraud pointed out these diphtheroids are 
 found in sebaceous plugs and recent observations of numerous workers as well 
 as of our own, shovv that these bacteria, anaerobic in character, can be isolated 
 
16 Frederick Eberson 
 
 very readily from "blackheads" without the use of special mediums. It is 
 believed by some that "blackheads" are to be looked on as a stage of acne. In 
 order to adhere to the rules of priority we should designate this diphtheroid 
 as follows ; Coryn. acnes (Gilchrist) comb. nov. Cultural studies with this 
 organism were made by Siidmerson and Thompson in 1910. They confirmed 
 Sabouraud's findings as to the suitability of highly acid mediums for culti- 
 vating C. acnes. One part horse serum to 3 parts of 3% nutrient agar (4.0) is 
 recommended as a culture medium for isolation. Ordinary glucose agar may 
 be used just as well, however. Fermentation studies show that the organism 
 ferments dextrose, saccharose (shght), maltose, mannite and inulin. Raffinose 
 is fermented by 1 strain and not by another. The authors suggest raflfinose as 
 a means for differentiating 2 species. Several strains studied by the writer 
 formed no acid in raffinose and in dextrin. 
 
 Virulence tests were made on guinea-pigs and mice. Only the latter suc- 
 cumbed to an injection of 4 loopfuls of a 5 day culture. On necropsy the ani- 
 mals showed a greatly enlarged spleen, enlarged and congested mesenteric glands, 
 enlarged, soft and anemic kidney, congested suprarenals and an enlargement 
 and congestion of the axillary and inguinal glands. 
 
 B. PSEUDOTUBERCULOSIS OVIS PREISZ (nOCARd) 
 
 This organism was originally isolated by Preisz and Guinard from necrotic 
 areas in the kidney of sheep. Later, Preisz described the organism more fully. 
 It is gram-positive, nonmotile and club-shaped. On serum it produces a heavy 
 moist growth with golden-yellow pigment. Toxin production is attributed to 
 the organism and is fatal for guinea-pigs, rabbits, goats and sheep. In guinea- 
 pigs, intraperitoneal inoculation gives rise to an orchitis. In larger animals 
 there is produced a pseudotuberculosis which can be differentiated from true 
 tuberculosis by histologic study of the pseudotubercles which do not show the 
 characteristic giant cells. In a recent paper by Hall and Stone this organism 
 was found to produce suppurative processes throughout the lymphatics in guinea- 
 pigs. The toxin which is elaborated by the bacterium resembles but is not iden- 
 tical with that of diphtheria, yet is partially neutralized by diphtheria antitoxin. 
 The authors found that the organism was hemolytic in blood agar which did 
 not contain an excess of fermentable carbohydrate. Dextrose and maltose were 
 fermented, but glycerol was not attacked, thus differing from C. striatum which 
 ferments glycerin and like the B. pseudotuberculosis ovis Preisz produces a 
 yellow pigment. Three strains of this organism (horse, sheep, calf) which were 
 sent to the Museum of Natural History by the writer were tested for acid pro- 
 duction along with the other diphtheroids. The strain from the calf produced 
 more acid in dextrose and dextrin than either of the other two. The detailed 
 results of the tests were as follows : 
 
 Dextrose 
 
 /- IN/, 
 
 Lactose 
 
 6u aciu ^ 
 Saccharose 
 
 Dextrin 
 
 (Horse) 1.8 
 (Sheep) 0.9 
 (Calf) 2.4 
 
 0.6 
 
 0.3 
 0.4 
 
 0.3 
 0.3 
 
 0.7 
 1.7 
 
 Coryn pseudotuberculosis (Prei 
 
 sz) comb. 
 
 nov. is perhaps 
 
 the correct desig- 
 
 nation. 
 
 B. HOAGII 
 
 
 
 Morse described this type in 1912 as representing one of the groups which 
 she established as the result of a biometrical study of 295 strains. The char- 
 acters may be summarized as follows : Medium-sized bacterium showing solid 
 
Diphtheroid Organisms 17 
 
 barred and wedged forms with abundant but small and imperfect granules. On 
 serum it produces a very heav}', confluent, glistening growth with a character- 
 istic salmon-pink color. Ferments dextrose and saccharose, but not maltose. 
 The organism was described by Hoag, but not named by him. The species should 
 be written Corynebacterium hoagi (Morse) comb. nov. 
 
 CORYNEBACTERIUM HODGKINIl 
 
 This name is applied to an organism said to be the cause of a glandular 
 disease about which pathologists and bacteriologists are still at odds. In recent 
 years Bunting and Yates, Rosenow, and Billings have been strong adherents to 
 the belief that Hodgkin's disease is caused by a diphtheroid organism which 
 they have cultivated from glandular material obtained from such patients. 
 Organisms similar to those of Bunting and Yates have been isolated from 
 Hodgkin's cases by Rhea and Falconer recently, and according to the earlier 
 work of Fraenkel and Much, and de Negri and Mieremet have been found in 
 similar cases. Fraenkel and Much thought that the organism was related to the 
 tubercle bacillus since it was found by them to be resistant to antiformin. This 
 character has been shown to be insignificant by de Negri and Mieremet who 
 observed the contrary. This test was made on 60 strains of diphtheroids by 
 the writer and in no case was an antiformin-resistant type observed. The 
 first description of this diphtheroid is given by Fraenkel and Much as follows: 
 Granular, antiformin-resistant, gram-positive, nonacid fast. Avirulent for guinea- 
 pigs, de Negri and Mieremet add several cultural characters : Plump short rod, 
 granular, polar staining. Grows best at Z2 C. and prefers. aerobic conditions 
 and alkaline reaction of mediums. Abundant growth on Loeffler's serum, with 
 slimy consistency. Bordet medium of blood-glycerin, potato-extract and agar 
 gives abundant growth in 24 hours. According to Bunting and Yates, gelatin 
 is not liquefied and broth is not clouded, but sediment is formed. In this paper 
 it is not proposed to discuss the etiology of Hodgkin's disease, but in review- 
 ing the work of others and from personal studies, it seems that we are not 
 yet ready to ascribe to these diphtheroids the great importance which has been 
 given to them. That organisms similar to these can be isolated from normal 
 individuals as well as from a variety of diseased conditions has been well shown 
 by Bloomfield who made a study of the bacterial flora of lymphatic glands. He 
 found avirulent organisms which were correlated with saprophytes on the body 
 surface in cases of Hodgkin's disease, lymphosarcoma, carcinoma. Another 
 group of organisms seemed to possess a relation to oxygen supply. These 
 were of frequent occurrence. They are short, pleomorphic rods, gram-positive, 
 nonacid fast, no polar bodies and exhibit clubbed forms. Bloomfield found 
 these in cases of lymphosarcoma, arthritis, carcinoma and Hodgkin's disease. 
 Virulence tests on rabbits, guinea-pigs and mice were negative. He concludes 
 that definitely diseased glands yield a greater number of successful cultures than 
 do normal glands. Saprophytic organisms are filtered out by the glands and 
 become a permanent flora of the same. None of the isolated strains appears 
 to be the etiologic factor in specific diseases. Another study by Fox has thrown 
 further light on the question of secondary invasion. Fox found that no one 
 bacterial variety with definite morphologic and cultural characters has been 
 isolated from cases of Hodgkin's disease. That these diphtheroids may he found 
 in enlarged glands in other conditions is mentioned by Fox and has been 
 observed by the writer who has isolated organisms morphologically and cul- 
 turally identical with the so-called C. Hodgkinii, from an hypertrophied tonsil 
 and from lymph nodes and other sources. For the sake of comparison the fol- 
 
18 
 
 Frederick Eberson 
 
 lowing table is given so as to show the lack of uniformity in Hodgkin's strains 
 which our study has so far illustrated. 
 
 A glance at Table 1 shows that the strains isolated from Hodgkin's cases 
 not only differ among themselves, but are not to be distinguished from organ- 
 isms obtained from widely varying sources. The study of Hodgkin's disease 
 is to be the subject of another report. Until the etiology of Hodigkin's disease 
 is more definitely ascertained, it is suggested that C. Hodgkinii should not be 
 retained as a valid species inasmuch as the two C. Hodgkinii (Rosenow) strains 
 are widely different in cultural characters. In view of the fact that other 
 organisms which are in no way related to this disease or any adenopathy 
 resembling it, are culturally and morphologically identical with this organism, 
 the name Hodgkinii serves no purpose and leads to confusion. 
 
 TABLE 1 
 Showing the Lack of Uniformity in Acid Production in Hodgkin's Stkains 
 
 
 N/20 Acid Produced in 
 
 Cul- 
 ture Source 
 
 Saccha- 
 Dextrose Lactose rose i Maltose 
 
 i i 
 
 Mannite Dextrin 
 
 1 Hodgkin's (Bunting)....... 
 
 2 Hodgkin's (Bunting) 
 
 3 Lympiiocy tic leukemia 
 
 4 Acute pseudoleukemia 
 
 5 Hodgkin's (Bunting) 
 
 6 Blood (Bunting) 
 
 0.2 
 
 0.7 — 
 0.3 — 
 0.2 2.5 
 0.2 — 
 0.2 — 
 
 1 1 1 1 1 1 
 
 0.3 
 1.1 
 0.5 
 0.5 
 0.3 
 0.3 
 2.7 
 0.2 
 0.4 
 0.3 
 0.3 
 0.2 
 1.7 
 2.9 
 2.2 
 0.3 
 0.4 
 1.7 
 
 - 
 
 0.2 
 0.3 
 
 S C. Hodgkinii (Rosenow).... 
 10 Blood (Eberson) 
 
 2.8 - 
 
 1.9 
 
 0.3 
 
 11 Tonsil (Eberson) 
 
 0.2 — 
 
 1.7 
 
 1.7 
 
 
 
 12 Anal pus pocket (Eberson). 
 
 13 C. Hodgkinii (Rosenow).... 
 15 Bone marrow (Eberson).... 
 
 28 Hodgkin's (Rosenow) 
 
 34 Hodgkin's (Rosenow) 
 
 38 Hodgkin's (Rosenow) 
 
 44 Lymph node (Eberson) 
 
 49 Hodgkin's (Eberson) 
 
 51 1 Hodgkin's (Eberson) 
 
 0.1 
 0.2 
 0.9 
 2.0 
 1.9 
 
 0.1 
 1.5 
 
 0.1 
 
 0.3 
 0.3 
 
 0.3 
 
 DISTRIBUTION OF DIPHTHEROIDS 
 
 At this point it seems convenient to tabulate the diphtheroids which 
 have been found in glands and tissues and arrange the several species 
 which have not been classified heretofore. 
 
 Diphtheroids in Glands and Tissues 
 
 The following signs indicate acid production : ±, 0.1 to 0.5 N/20 NaOH 
 required for neutralization; +, 0.6 to 1.0 N/20 NaOH required for neutral- 
 ization ; ++, 1.1 to 2.5 N/20 NaOH required for neutralization ; + + +, 2.6 
 to 4.Q N/20 NaOH required for neutralization ; — , no acid ; alk, weakly alkaline. 
 
 Sugar broths were prepared as follows: 1% by weight of each of the carbo- 
 hydrates were added to meat infusion broth prepared according to standard 
 methods. The carbohydrates used were Merck's C. P. lactose, saccharose, 
 maltose, mannite and raffinose and Kahlbaum's dextrose and dextrin. Difco 
 peptone was used in preparation of broth. To the mediums (sterilized in 
 Arnold for 20 minutes on 3 successive days and immediately cooled) was 
 
Diphtheroid Organisms 
 
 19 
 
 added sterile ascitic fluid in the proportions of 1 in 10. The mediums were 
 then incubated at 37 C. for 24 hours and 1 day at room temperature to test 
 for sterility. For inoculation, 48-hour cultures of the diphtheroids grown on 
 ascitic dextrose agar (0.5% dextrose) were used. Titrations were made in the 
 cold after 48 hours' incubation, using N/20 NaOH. Five c c of the broth were 
 used for titration. Instead of adding 45 c c of water and a few drops of phenol- 
 phthalein to each 5 c c of material, it was found more convenient to add phenol- 
 phthalein to a large volume of water and then to make this exactly neutral 
 by addition of a small amount of N/1 NaOH, as was necessary for the water 
 used in the tests. In this way, errors due to variations in reaction of dis 
 tilled water may be avoided. This method and the procedure followed in the 
 preparation of mediums were adhered to in all titrations. 
 
 TABLE 2 
 The Acid-Producing Properties of Diphtheroids Found in Glands and Tissues 
 
 Cul- 
 ture 
 
 Source 
 
 Dex- 
 trose 
 
 Lac- 
 tose 
 
 B.* 1 j Hodgkin's disease ± 
 
 B. 2 Hodgkin's disease -t- 
 
 B. 3 Axillary gland, lymphocytic leu- 
 kemia ± 
 
 B. i Cutaneous tumor, acute pseudo- 
 leukemia ± 
 
 B. 5 Hodgkin's disease ± 
 
 B. 6 Hodgkin's disease ± 
 
 B. 8 C. Hodgkinii + + + 
 
 8t Hypertrophied tonsil — 
 
 10 Blood ± 
 
 11 I Hypertrophied tonsil ± 
 
 B. 13 C. Hodgkinii ± 
 
 13t Hypertrophied tonsil — 
 
 14tl Tonsil — 
 
 15 Bone marrow, leukemia ± 
 
 27t Hypertrophied tonsil — 
 
 B. 28 Hodgkin's disease + 
 
 29 Hypertrophied tonsil — 
 
 R. 34 Hodgkin's disease +-1- 
 
 35 Hypertrophied tonsil ± 
 
 B. 38 Hodgkin's disease + + 
 
 44 Lymph node — 
 
 47 : Brain, meningitis -f-l--l- 
 
 49 Hodgkin's disease ± 
 
 51 Hodgkin's disease I ++ 
 
 + + + 
 
 Baffl- 
 nose 
 
 -f 
 
 ± 
 
 + + + 
 
 alk 
 
 alk. 
 
 +-t- 
 
 + -f- 
 
 + + + 
 
 + + 
 
 + + 
 ± 
 + + 
 
 — alk. 
 
 alk. 
 alk. 
 
 * B. =: Bunting strains; B. = Bosenow strains. 
 
 Diphtheroids isolated from glands and certain tis.sues fall into sev- 
 eral groups : ( 1 ) Those which ferment dextrose and maltose slightly ; 
 (2) those which ferment dextrose, saccharose, maltose vigorously and 
 dextrin slightly; (3) those which ferment dextrose and maltose vigor- 
 ously; (4) those which ferment dextrose and maltose vigorously and 
 dextrin slightly; (5) those which ferment lactose, maltose and rafifinose 
 vigorously, and dextrin and dextrose slightly; (6) those which ferment 
 lactose and maltose slightly; (7) those which ferment none of the 
 sugars ; and (8) those which ferment dextrose, lactose and maltose 
 vigorously. That the range of fermentative powers varies widely is 
 
20 Frederick Eberson 
 
 seen at a glance. Before any systematic classification is attempted 
 many more strains must be isolated from lymph glands and tissues. A 
 few new strains will be described here, however. 
 
 The diphtheroids which were isolated from tonsils, of which 224 were 
 studied, varied in morphology from very small rods to large club-shaped forms. 
 They were all gram-positive, nonmotile, nonacid fast, nonspore forming, aerobic, 
 and failed to produce gas in carbohydrate mediums. Four were hemoglobino- 
 philic. A few of those strains possessed hemolytic properties. One produced 
 an orange pigment when grown on ascitic dextrose agar slants. This property 
 was observed on ordinary agar as well. Diphtheroids with orange pigment 
 had not been described in the literature at this time, and it is suggested that 
 this organism be called C. aurantiacum. A similar species was obtained from 
 a Hodgkin's gland. The pigment production was studied from the standpoint 
 of oxygen and temperature requirements. At room temperature (18-25 C.) 
 under aerobic and anaerobic conditions, pigment was produced abundantly in 
 from 24-48 hours. At Zl C. under aerobic conditions, the orange color was 
 abundant in the same interval of time, whereas under anaerobic conditions, 
 a very slight coloration was observed after 10-14 days. The organism is 
 gram-positive, nonmotile, does not form spores, is not acid fast and is faculta- 
 tive. On ascitic dextrose agar, it grows abundantly with an orange tinge after 
 24 hours. It is a small bacterium, coccoid in appearance. No polar bodies or 
 granules seen when stained with the Neisser stain! It grows well in dextrose, 
 lactose, saccharose, maltose, mannite, dextrin and raffinose broth and produces 
 acid in dextrose and maltose. At room temperature abundant growth takes 
 place. Gelatin is not liquefied. 
 
 Hemolysis of rabbit corpuscles by diphtheroids is not usually attributed to 
 any but the true Klebs-Loeffler bacterium from severe cases (Schwoner). The 
 organisms isolated from the tonsils were in all cases but one, culturally unlike 
 the diphtheria organism and in this one instance proved to be an avirulent 
 diphtheroid, for which the name C. pseudodiphtheriae sp. nov. is proposed. 
 This organism grows on plain agar, is morphologically like the Klebs-Loeffler 
 bacillus. It shows no growth anaerobically after 7 days and is apparently 
 strictly aerobic. Some of the strains from tonsils required blood for growth 
 and died off rapidly unless transferred frequently. These organisms resembled 
 B. influenzae and were hemoglobinophilic. 
 
 Three strains were found which fermented dextrose, saccharose, and maltose 
 vigorously and formed slight acid in dextrin. Raffinose remained neutral or 
 faintly alkaline. These diphtheroids were isolated from glands in patients 
 with Hodgkin's disease. In sugar broths, growth was abundant with even 
 clouding. At room temperature, on ascitic agar abundant growth occurs. At 
 37 C. on the same medium growth is abundant with a heavy creamy consistency 
 and brownish tinge. The organisms, small, ovoid or coccoid, stain readily with 
 anilin dyes, are gram-positive, facultative, nonmotile, do not form spores, are 
 not acid-fast. On rabbit blood-agar plates, brown pigment is formed along the 
 streak growths. On Loeffler's serum, abundant white, glistening growth takes 
 place. One strain, on first isolation, from the gland on blood plates, exhibited 
 a remarkable likeness to B. tuberculosis in character of growth. The colonies 
 were dry and heaped up and friable. This peculiar property did not persist in 
 later transplants. Antiformin treatment showed these diphtheroids to be non- 
 resistant. The name C, glandulae sp. nov. is suggested for this species. 
 
Diphtheroid Organisms 21 
 
 While this study was under waj', a paper by Torrey appeared dealing with 
 bacteria associated with lymph nodes. He tabulated 4 distinct groups, which 
 when analyzed showed 8 different groups according to fermentation reactions. 
 One of the groups included chromogenic types, chiefly orange-red. This species 
 I have named C. aurantiacum. An anaerobic diphtheroid occurring in lymph 
 nodes was described by Torrey and named B. lymphophilus. It is slender^ 
 pleomorphic, gram-positive, from 0.5 X 1.2-3.2 mikrons. It is not acid-fast, 
 occurs singly or in pairs, is nonmotile ; stab cultures in 0.5% agar show rods 
 of irregular morphology and uneven staining. On Loeffler's medium, raised 
 white growth occurs. In dextrose, high acid is produced, even to the amount 
 of 10 +. It is markedly aciduric and acidophilic. Glycerol is also fermented, 
 but lactose, mannite, inulin and dextrin are not attacked. No gas is produced. 
 In my studies, several strains were isolated which exhibited anaerobic prefer- 
 ence for a few generations only, but were not like Torrey's strain either with 
 respect to anaerobic requirements or unusual acid-production in any one carbo- 
 hydrate. Torrey's results are of special interest in showing that there are 
 several types of diphtheroids commonly present in the lymph nodes of Hodgkin's 
 disease, none of which has definite pathogenic properties or can be accepted as 
 the cause of the disease. In this respect he confirms the findings of Bloomfield 
 and myself. 
 
 EYE DIPHTHEROIDS 
 
 Diphtheroids isolated from the eye were found to vary in cultural behavior. 
 Three strains were obligate aerobes and produced only a very slight amount 
 of acid in dextrose with 0.5 c c N/20 acid maltose. Seven other strains, facul- 
 tative, produced slight acid in the same sugars. One strain grew slightly, if 
 at all, on agar and favored Loeffler's serum. All of these grev/ rather deli- 
 cately on ascitic agar and serum. At room temperature growth was slow. The 
 organisms were large, clubbed and striated. The type C. xerosis prevailed. 
 
 NASAL DIPHTHEROIDS 
 
 Ten strains were isolated from nasal discharges, mostly in persons with 
 cold in the head. Of these, 5 strains were characterized by slight acid forma- 
 tion in dextrose with marked acid in maltose. The remaining 5 were of 2 
 types, one which produced slight acid, the other abundant acid in dextrose and 
 maltose. On Loeffler's serum, growth was abundant, white and glistening. 
 Stained with anilin dyes, the organisms appeared short and thick with larger 
 striped forms predominating. They are gram-positive, facultative, nonmotile, 
 do not form spores, are not acid fast and grow well in sugar broths. At 
 room temperature moderate to abundant growth takes place. The type C. 
 striatum prevailed. 
 
 DIPHTHEROIDS FROM ASCITIC FLUIDS 
 
 Thirty specimens from cases of cirrhosis and tuberculosis were studied 
 anaerobically and aerobically for diphtheroid organisms. No strict anaerobes 
 were found. For anaerobic cultures, Buchner tubes (Zinsser's modification*) 
 were used. Three strains were isolated. All are gram-positive, nonmotile, do 
 not form spores, are not acid-fast, are facultative. Two strains grow very 
 rapidly at room temperature, a third does not. One liquefies gelatin and gives 
 rise to an offensive odor after several days. In broths, the following results 
 were noted (litmus used as indicator). 
 
 • A piece of absorbent cotton is pressed down over the pyroKallol to prevent rapid 
 mixinR with the NaOH. 
 
22 Frederick Eberson 
 
 Strain Dextrose Lactose Saccharose Maltose Mannite Dextrin Raffinose 
 9 + — + + + + _ 
 
 42 — — — — — __ 
 
 Strain 43 liquefies gelatin and produces a putrid odor. This strain, prac- 
 tically conforms to the description given by Graham Smith to a liquefying 
 diphtheroid which he named B. diphtheroides liquefaciens. Motility is lacking, 
 however. As was mentioned before, it does not seem as if this property is 
 peculiar to the group with which we are dealing. The organism isolated from 
 ascitic fluid is slender, curved and appears granular. It does not form spores, 
 is facultative, grows abundantly with confluent mass on agar at room tempera- 
 ture and at 37 C. and gives off a decidedly putrid odor. On Loeffler's serum 
 it produces a heavy white, moist and glistening layer. It is suggested that the 
 name C. putidum sp. nov. be given to this species. Strain 9 is characterized 
 by its wide range of fermenting power. It is a very small rod, not much larger 
 than B. influenzae. On first isolation, club-shaped forms predominated. It 
 is gram-positive, nonmotile, does not form spores, is facultative, forms acid 
 in dextrose, saccharose, maltose, mannite and dextrin. It grows abundantly 
 at room temperature and at 37 C. On Loeffler's serum, abundant growth occurs. 
 It does not liquefy gelatin. C. ascites sp. nov. is proposed as a name for this 
 diphtheroid. Strain 42 is a small gram-positive organism, nonmotile, forms no 
 spores, grows moderately on ascitic agar at 37 C. In the early generations this 
 organism showed a decided preference for anaerobic conditions. No growth 
 takes place at room temperature after 10 days. It does not liquefy gelatin 
 and does not ferment sugar broths. Grows best in raffinose broth. This seems 
 to be the most selective medium for obtaining abundant growth. All of the 
 diphtheroids studied grew moderately well and for the most part abundantly 
 in the presence of this trisaccharid when other sugars used for fermentation 
 studies showed little or no growth other than a sediment. C. delicatum sp. nov. 
 is suggested as the name for this species (see blood diphtheroids). 
 
 In the early generations this organism showed a decided preference for 
 anaerobic conditions. 
 
 A peculiarity noted in the isolations from ascitic fluids was the fact that 
 the first generation always had larger clubbed and knobbed forms. In suc- 
 ceeding subcultures only very small diphtheroids were seen. 
 
 DIPHTHEROIDS IN BLOOD CULTURES 
 
 Two Strains were isolated from blood cultures in cases of pneumonia. In 
 the first generation these showed larger clubbed forms and small rods. Growth 
 is delicate. The organisms are small gram-positive, nonmotile, facultative and 
 produce very slight acid in dextrose or maltose but as a rule leave all sugars 
 unfermented. Gelatin is not liquefied and growth at room temperature is 
 negative after 10 days. On Loeffler's serum moderate growth occurs. These 
 diphtheroids have been identified with C. delicatum. 
 
 SKIN DIPHTHEROIDS 
 
 ■ Hine working with 34 strains of diphtheroids found a group which was asso- 
 ciated with skin wounds or diseased cavities opening on to or near the sur- 
 face. Strains of this group were found to acidify dextrose, saccharose usually 
 and less often maltose. The author did not describe these organisms. That 
 diphtheroids can be found on the skin very readily is well established. Judging 
 by the amazing number of sources from which the organisms of this group may 
 
Diphtheroid Organisms 23 
 
 be isolated, it seems highly probable that we have to deal with saprophytes 
 which abound everywhere like numerous species of the Coccaceae group and 
 which gain entrance into the body through the various openings, both normal 
 as well as pathologic. 
 
 In the course of this study 4 strains were isolated from an anal pus pocket 
 and from the skin on the neck. Two of these strains which will be described 
 below are in accord with those mentioned by Hine ; the other 2 differ sufficiently 
 from these in fermentative and other cultural characters to warrant their 
 acceptance as other species. 
 
 (1) Skin Diphtheroid: On Loeffler's serum, abundant dull white growth is 
 produced. On agar delicate growth takes place. Gelatin is not liquefied. At 
 room temperature little growth is observed after 1 week. The organism is 
 gram-positive, nonspore forming, nonmotile, facultatively aerobic, small, ovoid 
 and stains readily. Slight acid is produced in dextrose and maltose, the latter 
 being more strongly acidified. It is avirulent. The name C. epidermidis sp, nov. 
 is suggested for this species. 
 
 (2) Anal Pus Pocket: On Loeffler's serum abundant, moist and glistening 
 growth occurs. On agar, heavy glistening growth takes place in broth, uniform 
 clouding. Ferments dextrose and maltose vigorously and usually saccharose. 
 It is gram-positive, facultative, nonmotile, does not liquefy gelatin, does not 
 form spores, and grows abundantly at room temperature. It is avirulent. To 
 this species the name C. suppuratum sp. nov. is given. Similar strains have 
 been observed by Trumpp in empyema and by Hine in a suppurating neck gland. 
 The organism in no way appears to be associated with suppuration except as a 
 secondary invader. 
 
 DIPHTHEROIDS FROM BRAIN 
 
 One strain was isolated from the brain in a case of meningitis. The organ- 
 ism is gram-positive, ovoid, or coccoid in appearance, nonmotile, nonspore 
 forming. It grows very rapidly at room temperature and at 37 C. on ordinary 
 agar. On Loeffler's serum, growth is white and moist. It is avirulent for 
 guinea-pigs and for rabbits injected intraperitoneally and subdurally. Vigorous 
 acid production takes place in dextrose, lactose and maltose. Growth in broth 
 is abundant. Gelatin is not liquefied. Ortmann has reported a diphtheroid 
 isolated from the brain in a case of meningitis but has given no description of 
 the organism. It is purely a secondary invader. The name C. cerebralis sp. nov. 
 is suggested for the species. 
 
 DIPHTHEROIDS FROM THE APPENDIX 
 
 One strain was obtained from this source. There was slight suppuration of 
 the appendix. The organism grows moderately on Loeffler's serum. On agar 
 moderate growth occurs. Few clubbed and barred types were observed in 
 the first generation and in the next few transplants. Neisser's granules are 
 present. The organism is fairly large, nonmotile, gram-positive, does not pro- 
 duce spores and does not liquefy gelatin. At room temperature slight growth 
 occurs. In dextrose, lactose, saccharose, maltose and dextrin slight acid is 
 formed. Heavy flaking with sediment, but no clouding, occurs in the broths 
 mentioned. 
 
 Hine has obtained a diphtheroid from an appendix but has not described the 
 organism. It is a saprophyte and a secondary invader. The name C. floccuiens 
 sp. nov. is suggested for this species. 
 
24 
 
 Frederick Eberson 
 
 In all, diphtheroids were obtained from 21 different sources and 
 these fell into 12 distinct fermentation groups, as shown in Table 3. 
 The greatest number of organisms comprised the group of nonfer- 
 menters and here is to be noted the fact that of 13 strains from Hodg- 
 kin's disease, 8 came under this head. However, little significance need 
 be attached to this point, inasmuch as strains from Hodgkin's glands 
 fell into four other groups besides this one. In the same way, it is to 
 be noted that strains from the eye and nose fall into 4 and 3 different 
 groups, respectively. 
 
 table 3 
 Source and Fermentative Reactions of Diphtheroids 
 
 Source 
 
 i Dextrose, Dextrose, 
 
 Dextrose Saccha- Maltose 
 
 rose 
 
 ; Acne (pustule) 
 
 Appendix 
 
 ' Asciticfluid ' 
 
 Bile I 
 
 '• Blood 
 
 , Brain 
 
 Ear 
 
 Eye 
 
 > Lymph nodes, Hodg- 
 kin's 
 
 , Lymph nodes, leuke- 
 mia \ 
 
 '■ Lymph nodes, pseu- 
 doleukemia 
 
 ; Marrow 
 
 , Nose 
 
 ^ Pus (anal pocket) 
 
 ' Tonsil 
 
 ' Urine 
 
 I Horse, ealf, sheep 
 
 . (PreiszNocard B.) 
 
 1 Unknown 
 
 Total. 
 
 Dextrose, Dextrose, Dextrose, 
 Lactose, Saccha- Saccha- 
 Saccha- rose, rose. 
 
 rose. Maltose, Maltose, 
 Maltose Dextrin Mannite, 
 Dextrin 
 
 1 1 ! 1 
 
 Dextrose, 
 Saccha- Dextrose, 
 rose. Maltose, 
 Maltose Dextrin 
 
 Dextrose, 
 
 Dextrose, Lactose, 
 
 Dextrose, Maltose, Saccha- Nonfer- To- 
 
 Lactose, Lactose, rose, raenters tal; 
 
 Maltose Dextrin Maltose, 
 
 Mannite, 
 
 Dextrin 
 
 2 (Leu- 
 
 
 
 kemia 1) 
 
 1 
 
 1 
 
 1 
 
 5 
 
 10 
 
 
 3 
 
 2 
 
 3 
 
 1 
 
 2 
 
 
 3 
 
 28 
 
 It becomes evident that neither the source nor the cultural charac- 
 ters serve to distinguish the supposed cause of Hodgkin's disease from 
 numerous organisms which may be regarded as harmless saprophytes ; 
 also that diphtheroid organisms comprise a greater number of groups 
 than hitherto supposed. 
 
 There appears to be a definite correlation between fermentative 
 properties and source with regard to those organisms which were iso- 
 lated from the nose and throat. These are characterized by relative 
 inability to ferment carbohydrates. Acid formation in dextrose 
 occurred but 6 times of 19; in dextrose and saccharose, 3 of 19; in 
 
Diphtheroid Organisms 25 
 
 dextrose and maltose, but 1 in 19, and of the entire number of strains 
 more than half failed to ferment any sugars. Acid formation, there- 
 fore, is limited in its range, never exceeding 2 carbohydrates and show- 
 ing weak splitting action in general. 
 
 classification 
 
 Classification from the cultural standpoint is difficult because of the 
 manifold variations which are observed. The resolution of all diph- 
 theroids into a few distinct "types" such as suggested by Morse is 
 possible only for a limited number of organisms from a limited number 
 of sources. Lehmann and Neumann classify "pseudodiphtheria" from 
 eye and nose as Coryneb. pseudodiphtheriticum and Coryneb. xerosis, 
 including diphtheroids from both sources under each heading. 
 
 More recent work such as that of Plotz on typhus fever (C. typhi- 
 exanthematici) indicates that the diphtheroid group may possess patho- 
 genic species which are far removed from the type C. diphtheriae. The 
 organism described by Plotz and his co-workers is an obligate anaerobe 
 with characteristic serum reactions. Some diphtheroids might be 
 classified from the viewpoint of human and animal diseases, and par- 
 ticularly striking cultural characters such as oxygen requirement and 
 sugar fermentations. C. acnes is anaerobic and might very well go 
 into this group of anaerobes. Here also belong those "oxygen tension" 
 diphtheroids which are not anaerobic in the usual sense of the word» 
 but which grow beneath the surface in solid mediums. The property 
 of gelatin liquefaction, not hitherto recognized as a diphtheroid charac- 
 ter, suggests another subgroup. 
 
 With these points in mind the following classification is offered.. 
 An effort has been made to group the species logically but it is evident; 
 that relationships between different types do not always permit the 
 most desirable grouping. Certain characters were used as a basis for 
 arranging the distinct species under a chosen type. These will become 
 evident as the tabulation is studied. In the key given here, all of the 
 known diphtheroids have been reviewed and certain new species added. 
 
 classification of diphtheroids 
 
 Group 1. — Diphtheria. Type C. diphtheriae (Klebs-Loeffler). — 
 Nonmotile, nonsporeforming, gram-positive, does not liquefy gelatin, 
 shows polar bodies in young cultures. Aerobic and grows between 19 
 and 42 C. Acid produced in dextrose, maltose and dextrin. Produces 
 
26 Frederick Eberson 
 
 toxin and gives rise to clinical symptoms known as diphtheria, for 
 which a specific antitoxin exists. 
 
 C. pseudodiphtheriae sp. nov. (Fig. 10). — Avirulent organism, 
 morphologically and culturally like C. diphtheriae but produces no 
 toxin and exerts no action when injected into animals. Isolated from 
 hypertrophied tonsils. 
 
 Group 2. — Pseudodiphtheriticum. Type C. pseudodiphtheriticum 
 (Hofmann-Wellenhoff.) — Nonmotile, nonsporeforming, gram-positive, 
 does not liquefy gelatin. It is shorter and thicker than the C. diph- 
 theriae, but variations such as knobbed, clubbed and barred forms 
 occur. It is avirulent and produces no toxin. In carbohydrates, no 
 acid is produced. 
 
 C. ceruminis (Graham Smith) Eberson. — Differs somewhat from 
 C pseudodiphtheriticum in morphology, as well as in its behavior in 
 broth which remains clear, with sediment. In carbohydrates, no acid 
 is produced. It is avirulent. Isolated from normal ear. 
 
 C. delicatum sp. nov. (Fig. 6). — Avirulent strains from ascitic 
 fluids and blood culture. The organism is small and delicate with a 
 decided preference for anaerobic conditions in the early generations. 
 Some strains form traces of acid in dextrose or maltose, but usually 
 ferment none of the sugars. 
 
 Group 3. — Xerosis. Type C. xerosis (Neisser and Kuschbert). — 
 Nonmotile, nonsporeforming, gram-positive, does not liquefy gelatin. 
 Resembles C. diphtheriae closely morphologically. Acid is produced 
 in dextrose, saccharose and maltose. The organism can be isolated 
 from normal as well as diseased eyes. Growth solid medium is rather 
 delicate. 
 
 C. epidermidis sp. nov. (Fig. 7). — Gram-positive, facultatively 
 aerobic, non-sporeforming, does not liquefy gelatin. It is small ovoid 
 in form. Little growth takes place after one week at temperature 
 below 25 C. On Loeffler's serum abundant, dull white, growth. Slight 
 acid is produced in dextrose and maltose. It is avirulent. Isolated 
 from skin and pus pockets. 
 
 C. suppuratum sp. nov. (Figs. 3, 4). — Gram-positive, nonspore- 
 forming, does not liquefy gelatin. Heavy glistening growth on agar 
 and on Loeffler's serum. Ferments dextrose and maltose vigorously 
 and usually saccharose. Isolated from pus pockets and abscesses. 
 Avirulent. 
 
Diphtheroid Organisms 27 
 
 C. auris (Graham Smith) Eberson. — Gram-positive, nonsporeform- 
 ing, does not liquefy gelatin. Long barred or granular, often clubbed. 
 Grows scantily on agar. Acid in dextrose and maltose, but not saccha- 
 rose. It is often virulent for guinea-pigs. Isolated from ear. 
 
 C. cerebralis sp. nov. (Fig. 11). — Gram-positive, ovoid or coccoid, 
 honmotile, nonsporeforming, does not liquefy gelatin. Grows rapidly 
 at room temperature and at 37 C. on agar. On Loeflfler's serum, growth 
 is white and moist. Vigorous acid production in dextrose, lactose and 
 maltose. Avirulent for guinea-pigs and rabbits. Isolated from brain. 
 
 Group 4. — Nodosum. Type C. nodosum (Migula) Eberson. — 
 Large, clubbed forms markedly segmented. Does not form spores, is 
 gram-positive, nonmotile, does not liquefy gelatin, white growth on 
 agar. Acid in dextrose, saccharose, maltose, and dextrin. Found fre- 
 quently in urine and the urogenital tract. Avirulent. 
 
 C. acidum sp. nov. (Figs. 1, 2). — Gram-positive, nonmotile, small 
 rods, with abundant white, glistening growth on agar. No spores, is 
 aerobic and does not liquefy gelatin. Produces large amounts of acid 
 in dextrose, lactose, saccharose, maltose, mannite and dextrin, but does 
 not attack raffinose. Isolated from urine. Avirulent. 
 
 C. ascites sp, nov. (Fig. 8). — Gram-positive, small rod with numer- 
 ous club-shaped forms on first isolation. Nonmotile, nonsporeforming, 
 does not liquefy gelatin, grows abundantly on agar and Loefifler's 
 serum. Forms acid in dextrose, saccharose, maltose, mannite and 
 dextrin. Avirulent. Isolated from ascitic fluid. 
 
 C. flocculens sp, nov. (Fig. 13). — Gram-positive, nonmotile, clubbed 
 or barred forms, does not form spores or liquefy gelatin. The organ- 
 ism grows moderately on agar and Loeffler's serum at 37 C. At room 
 temperature slight growth. Neisser's granules present. Produces acid 
 in dextrose, lactose, saccharose, maltose and dextrin. Heavy flaking 
 with sediment without clouding in broths. Isolated from a suppurat- 
 ing appendix. 
 
 Group 5. — Pigment- forming Diphtheroids. Type C. hoagii 
 (Morse) Eberson. — Medium sized bacterium with solid, barred and 
 wedge-shaped forms with abundant but imperfect granules. On serum 
 a heavy, confluent, glistening growth with a characteristic, salmon-pink 
 color. Gram-positive, forms no spores, does not liquefy gelatin, non- 
 motile. Produces acid in dextrose and saccharose, but not in maltose. 
 
 C. aurantiacum sp. nov. (Fig. 5), — Gram-positive, small and coc- 
 coid, nonmotile, nonsporeforming, does not liquefy gelatin, not acid- 
 
28 Frederick Eberson 
 
 fast. Grows abundantly on agar at room temperature and at 37 C. 
 and produces a bright orange pigment. No polar bodies or granules. 
 Grows well in sugar broths and produces acid in dextrose and maltose. 
 Pigment production in 24 hours under aerobic and anaerobic condi- 
 tions at 18-25 C., but at 37 C. pigment in this length of time only under 
 aerobic conditions. Under anaerobic conditions, at 37 C. a trace of 
 pigment was evident after 10-14 daysi. 
 
 C. glandulae sp. nov. (Fig. 9). — Small ovoid or coccoid bacterium. 
 Grows abundantly at room temperature or at 37 C. with a heavy creamy 
 consistency and brownish tinge. Gram-positive, nonmotile, nonspore 
 forming, does not liquefy gelatin. Grows abundantly in sugar broths 
 with uniform clouding. On LoefHer's serum abundant white glistening 
 growth occurs, changing to a brownish color after a few days. Pro- 
 duces acid in dextrose, saccharose and maltose vigorously and slight 
 acid in dextrin. RafSnose neutral. One strain on first isolation from 
 the gland on blood plates showed a remarkable likeness to B. tubercu- 
 losis in character of growth ; the colonies were dry and heaped up and 
 friable. This peculiarity did not persist in later transplants. Anti- 
 formin treatment showed these diphtheroids to be nonresistant. They 
 were not acid-fast. Isolated from glands of Hodgkin's disease. 
 
 C. striatum (Chester) Eberson. — Thick form, with clear cut bars. 
 Large and irregular granules visible. Heavy growth on serum with 
 pigment from white to yellow. Ferments dextrose and maltose, but 
 not saccharose. Gram-positive, does not form spores, nonmotile, does 
 not liquefy gelatin. Found in normal nasal mucus. 
 
 C. segmentosum nom. nov. — Nonmotile, gram-positive, nonspore- 
 forming, does not liquefy gelatin. Polar bodies demonstrable. In 
 broth, growth is clear with sediment. Slight acid is produced in dex- 
 trose. Avirulent, from nasal mucus. 
 
 Group 6. — Gelatin-liquefiers. Type C. putidum sp. nov. (Fig. 12). 
 — A slender, curved granular bacterium. It produces no spores, is 
 facultative, grows abundantly with confluent stringy mass on agar at 
 room temperature and at 37 C., giving ofif a decidedly offensive odor. 
 On Loeffler's serum, a heavy, white, moist and glistening mass. It 
 forms no acid in dextrose, lactose, saccharose, maltose, mannite, dex- 
 trin and raf¥inose. Isolated from ascitic fluid. 
 
 Group 7. — Anaerobic Diphtheroids Associated with Specific Clin- 
 ical Manifestations. Type C. typhi-exanthematici (Plotz). — Small, 
 pleomorphic, gram-positive organism, not motile, not acid-fast. Most 
 
Diphtheroid Organisms 29 
 
 of the bacteria are straight, some slightly curved, coccoid forms also 
 occur. The ends are slightly rounded or pointed. Nonspore forming. 
 It is an obligate anaerobe and can be isolated on special glucose medi- 
 ums containing ascitic fluid of a definite specific gravity. Produces acid 
 in dextrose, galactose, maltose and inulin. Found in typhus fever with 
 regularity ; specific serum reactions ; pathogenic. 
 
 C. acnes (Gilchrist) Eberson. — Gram-positive, small organism, 
 resembling C. typhi-exanthematici. Stains unevenly ; anaerobic, but 
 many strains grow aerobically after continued cultivation. Surface 
 smears fail to show growth with material from pustules, whereas deep 
 stab cultures and heavily inoculated unbroken pustules always give 
 growth. Mediums with large amounts of acid especially adapted for 
 isolating this organism which produces acid in dextrose, saccharose 
 (slight), maltose, mannite and inulin. Raffinose is fermented by some 
 strains. Pathogenic for mice and gives rise to cliaracteristic lesions. 
 
 Group 8. — Pyogenic Diphtheroids in Animals. Type C. pseudotu- 
 berculosis (Preisz) Eberson. — Gram-positive, nonmotile, club-shaped. 
 On serum heavy moist growth with golden-yellow pigment. Toxin 
 production is attributed to the organism and it is fatal for guinea-pigs, 
 rabbits, goats and sheep. Blood agar with a minimum amount of fer- 
 mentable carbohydrate shows hemolysis. Acid in dextrose and maltose, 
 but not glycerin. Produces suppurative processes in the lymphatics. 
 
 C. pyogenes (Glage). — Gram-positive, nonmotile, club-shaped or 
 cylindrical. Does not grow at room temperature. Is facultatively 
 aerobic, produces no spores and is hemoglobinophilic. Pathogenicity 
 doubtful. No acid produced in dextrose or glycerol. 
 
 C. vaccinae (Galli-Valerio). — Gram-positive, nonmotile, club- 
 shaped, coccoid, segmented or branched. Facultatively anaerobic, non- 
 spore forming. On serum, growth abundant; pale yellow or orange- 
 yellow pigment produced ; gelatin not liquefied. Pathogenic for guinea- 
 pigs, mice and rabbits. Associated with vaccine pustules in calves. 
 
 Group 9. — Anaerobic Diphtheroids Associated with Diseased 
 Lymph Glands. Type C. lymphophilus (Torrey). — Slender, pleomor- 
 phic, gram-positive, nonmotile ; markedly aciduric and acidophilic ; fer- 
 ments dextrose and glycerol; avirulent. From lymph nod€S in 
 Hodgkin's disease. 
 
 DISCUSSION 
 
 From this study, which is far from complete, it is evident that the 
 sources of diphtheroid organisms have not yet been exhausted. That 
 many strains are yet to be described is a foregone conclusion. The 
 
30 Frederick Eberson . 
 
 bacteriologc study of body fluids and glands and of secondary invaders 
 in common diseases will undoubtedly give us material for ultimate 
 classification. Apart from Morse's biometric study of the diphtheria 
 group, no other classification has been presented. In the light of the 
 material given here it seems as if Morse's classification on the basis of 
 chromogenesis, vigor of growth and fermentative properties is not 
 entirely adapted to the great number of strains which have already been 
 described and which are forthcoming. Serologic studies as a means of 
 classification in this group of bacteria have not been worked out suffi- 
 ciently to merit much discussion; the little work done has not been of a 
 very searching nature. It seems that here are the most prom.ising 
 possibilities. Agglutination has been tried by Nicholas, Nicolle, Land- 
 steiner, Bruno, Lubowski, Lesieur, and Fraenkel with varying results, 
 which were not specific. The consensus of opinion seems to be that 
 the organisms do not produce agglutinating substances in the serum. 
 Complement fixation has not been sufficiently studied. The results of 
 Morse show that cross-reactions m.ay be obtained for the diphtheroid 
 antigens and that C. diphtheriae does not fix with any of the diph- 
 theroid antigens. The results, however, are not striking enough to be 
 conclusive. The method of preparing antigen undoubtedly will modify 
 the results in such tests. Ground bacteria were used and specific serum 
 obtained by the injection of formalized suspensions of whole organ- 
 isms. Perhaps Olitzky's method for complement fixation in typhus 
 fever, would be preferable. According to his technic, nonspecific fixa- 
 tion is prevented by the use of Berkefeld filtrates of autolyzed bacteria. 
 It is hoped that by means of group reactions the diphtheroids may be 
 classified definitely. Precipitation tests suggest good possibilities. The 
 reaction is exceedingly delicate and the organisms lend themselves to 
 ready disintegration and extraction. 
 
 II. SEROLOGICAL 
 
 experiments with hodgkin^s strains 
 
 In order to determine the identity of cultures isolated from cases of 
 Hodgkin's disease, two methods were utilized — agglutination and com- 
 plement fixation. The first method enables us to identify the specifi- 
 city of the organisms by means of serums obtained from patients suf- 
 fering with the disease as well as by serums prepared with the dififerent 
 strains of bacteria isolated. Obviously, if the C. Hodgkinii is the cause 
 
Diphtheroid Organisms 31 
 
 of Hodgkin's disease we ought to be able to show a definite relationship 
 between serums from patients and organisms isolated from these and 
 other cases. By the delicate method of complement fixation we may 
 detect similar antigenic substances and establish the identity, if such 
 there be, of the several strains isolated. 
 
 Agglutination 
 
 Four serums, obtained from 4 authentic cases of Hodgin's disease were 
 studied. Eleven strains of bacteria were tested, of which 8 were isolated from 
 glands in Hodgin's disease and the remaining 3 from a case of leukemia, 
 ascitic fluid in cirrhosis and blood. 
 
 Forty-eight-hour agar cultures were suspended in salt solution and emul- 
 sions made up to approximately the same density. These were shaken with 
 glass beads for thirty minutes and then centrifugated for 5 minutes at low 
 speed to throw down coarser particles. Equal volumes of the different serums 
 (diluted with NaCl solution) were mixed with the bacterial suspensions in a 
 hanging drop. The preparations were well sealed with vaselin and incubated at 
 room temperature for 1 hour, then at 38 C. for another hour, when the results 
 were read. 
 
 The results were entirely negative and confirm the results of Fox. 
 
 Complement Fixation 
 
 In these experiments the aim was to show by tests for cross fixation what 
 relationship, if any, existed between the different types of bacteria isolated 
 from cases of Hodgkin's disease. The cultures, as in the preceding, were 
 obtained from Drs. Bunting, Yates and Rosenow and from cases studied by 
 me. These strains have been described fully in Part I. 
 
 Four strains were used in the production of immune serums from rabbits. 
 These cultures are designated 2, 8, 13 and 57. No. 2 was isolated by Dr. C. H. 
 Bunting from a patient who had a very acute form of Hodgkin's disease ; it 
 fermented dextrose and saccharose with acid. No. 8 was sent to me by Dr. 
 E. C. Rosenow, and labeled B. hodgkinii; it produced acid in dextrose, maltose, 
 saccharose, and dextrin. No. 13 was obtained from the same source; it fer- 
 mented dextrose and maltose. No. 57 was isolated by me from a case of acute 
 Hodgkin's disease. This organism fermented none of the carbohydrates and 
 was identical in this respect with a strain (No. 1) obtained from Dr. Bunting. 
 
 Healthy rabbits, weighing about 1,600 gm., were systematically treated with 
 each of these cultures heated to 58 C. for 30 minutes. Injections were made 
 with increasing doses every week for a period of 6 weeks and the serums drawn 
 10 days after the last injection. 
 
 Antigens were prepared by grinding to a fine powder the flocculent precipi- 
 tate obtained from a mixture of bacteria with an excess of absolute alcohol. 
 The powder was dried in vacuo and then ground in a mortar with solid NaCl. 
 Subsequently sterile distilled water was added to isotonicity and the anti- 
 complementary unit determined. 
 
 Complement was obtained from normal guinea-pigs and titrated in the 
 usual way. Sensitizer was prepared with thoroughly washed sheep blood cells 
 injected into rabbits and the unit determined, after which a 5% suspension 
 of sheep cells was sensitized with 2 units of the amboceptor. Readings were 
 taken after the tubes were incubated for 15 minutes in a i7 C. water bath, and 
 8 hours in the ice-chest. 
 
32 
 
 Frederick Eberson 
 
 The results showed no correspondence among the separate strains and hence 
 prove them to be nonidentical, a fact which confirms the cultural differences 
 observed in the same organisms. Fox failed to obtain fixation with the serum 
 of a patient and a strain isolated from the glands. 
 
 TABLE 1 
 Relationship of Type Diphtheroids 
 
 Serum Antl-hoagil 
 
 
 
 
 
 Absorbed with 
 
 
 
 Before Absorption 
 
 
 
 
 
 
 
 
 Pseudodiphtlieriae 
 
 Plavidus (Mellon) 
 
 Dilution 
 
 
 
 
 
 
 
 
 Pseudo- 
 
 flavidus 
 
 
 Pseudo- 
 
 
 Flavidus 
 
 
 Hoagii diph- 
 
 (MeUon 
 
 Hoagii 
 
 diph- 
 
 Hoagii 
 
 (Mellon) 
 
 
 theriae 
 
 Strain) 
 
 
 theriae 
 
 
 
 1-20 
 
 1 ++ : + 
 
 + + 
 
 + + 
 
 _ 
 
 ++ 
 
 — 
 
 1-40 
 
 I ++ i + 
 
 + + 
 
 ++ 
 
 — 
 
 ++ 
 
 — 
 
 1-80 
 
 ++ + 
 
 + 
 
 + + 
 
 — 
 
 + + 
 
 — 
 
 1-160 
 
 ++ ± 
 
 + 
 
 + + 
 
 — 
 
 ++ 
 
 — 
 
 1-320 
 
 ++ 
 
 + 
 
 + + 
 
 — 
 
 + + 
 
 — 
 
 1-500 
 
 ++ 
 
 ■+■ 
 
 ++ 
 
 
 ++ 
 
 
 1-1,000 
 
 + 
 
 — 
 
 + 
 
 
 ■+■ 
 
 
 1-1,500 
 
 + 1 
 
 
 ± 
 
 
 — 
 
 
 1-2,000 
 
 ± ! 
 
 
 — 
 
 
 ~ 
 
 
 Serum Anti-pseudodiphtheriae 
 
 
 Before Absorption 
 
 
 Absorbed with Flavidus (Mellon) 
 
 Dilution 
 
 Pseudo- 
 
 Flavidus 
 
 Hoagii 
 
 Pseudo- 
 
 Flavidus 
 
 
 diphtheriae 
 
 (Mellon) 
 
 
 diphtheriae 
 
 (Mellon) 
 
 1-20 
 
 + + 
 
 + 
 
 + 
 
 ++ 
 
 
 
 1-40 
 
 + + 
 
 +, 
 
 + 
 
 ++ 
 
 — 
 
 1-80 
 
 + + 
 
 + 
 
 -h 
 
 + + 
 
 — 
 
 1-160 
 
 + + 
 
 -+- 
 
 — 
 
 + + 
 
 — 
 
 1-320 
 
 + + 
 
 — 
 
 
 + 
 
 
 1-500 
 
 + + 
 
 — 
 
 
 ■+■ 
 
 
 1-1,000 
 
 + ■ 
 
 
 
 — 
 
 
 1-1,200 
 
 
 
 
 
 
 Serum Antiflavidus (MeUon) 
 
 Before Absorption 
 
 Absorbed with Flavidus (Morse) 
 
 Dilution 
 
 Flavidus 
 
 Pseudo- 
 
 Flavidus 
 
 Flavidus 
 
 
 (Mellon) 
 
 diphtheriae 
 
 (Mellon) 
 
 (Morse) 
 
 1-20 
 
 + 
 
 ± ': 
 
 + 
 
 _ 
 
 1-40 
 
 + 
 
 — i 
 
 4- 
 
 — 
 
 1-80 
 
 + 
 
 — 1 
 
 + 
 
 — 
 
 1-160 
 
 + 
 
 — 
 
 + 
 
 — 
 
 1-320 
 
 + 
 
 — ; 
 
 + 
 
 — 
 
 1-500 
 
 + 
 
 ~" 
 
 + 
 
 " 
 
 AGGLUTININ ABSORPTION TO DIFFERENTIATE TYPE DIPHTHEROIDS 
 
 The literature on diphtheroids, to my knowledge, does not contain 
 any reference to agglutinin absorption as a method for showing rela- 
 tionships which might exist among overlapping, dosely-related types. 
 In view of the fact that the differences which characterize the accepted 
 
Diphtheroid Organisms 33 
 
 forms are so slight, bacteriologically speaking, such a method suggested 
 possibilities for more definite classification. 
 
 The strains studied were obtained from the Museum of Natural History and 
 were marked as follows : C. hoagii, C. flavidus (Mellon), C. pseudodiphtheriae, 
 C. xerosis, C. enzymicus (Mellon), and C. flavidus (Morse). 
 
 Agglutinating serums were prepared in rabbits by giving intravenous injec- 
 tions of fresh agar cultures heated at 56 C. for 30 minutes. Increasing doses 
 were given every week until a whole culture was reached. The animals gained 
 weight throughout treatment and were bled 10 days after the last injection. 
 Preliminary titrations were made with each of the serums against homologous 
 and heterologous strains, and double absorption of each serum was made with 
 organisms which evidenced some affinity for the agglutinins present. 
 
 The results of the absorption experiments indicate that C. hoagii is 
 more inclusive in its antigenic elements, since it possesses group agglu- 
 tinins for all the other strains, excepting C. xerosis. C. flavidus (Mel- 
 
 TABLE 2 
 C0.WPARATIVE Results 
 
 Flavidus (Morse) Flavidus (Mellon) 
 
 Dextrose 3.3 cc 2.0 c c 
 
 Maltose 2.0 1.7 
 
 Saccharose 2.5 i 1.4 
 
 Glycerol 3.3 2.6 
 
 The figures represent the amount of N/20 NaOH required to neutralize 5 c e ol broth 
 culture incubated 1 day at 37 C. Growth was heavy in both sets. The Mellon strain gave 
 a flocculate growth and the Morse culture was uniformly clouded. 
 
 Ion) has no group agglutinins for any of the others and appears to be 
 highly specific. The results vary somewhat as compared with Morse's 
 complement fixation results. Mellon's flavidus shows no group agglu- 
 tinins at all, although Morse obtained partial cross fixation with her 
 strain. It appeared likely that the former culture sent to the Museum 
 of Natural History as a **B. flavidus," isolated by Mellon, was not a 
 flavidus type. When its homologous serum was absorbed with C. 
 "flavidus" (Morse), the specific agglutinins remained unchanged. That 
 complete absorption by the heterologous strain took place is evident 
 from the table. Macroscopically, the Morse culture differs markedly 
 on plain agar from the Mellon strain. The latter develops a dull, dry 
 growth, with a tendency to flocculate readily in NaCl solution, whereas 
 the former is moist and glistening in its growth. In carbohydrates the 
 comparative results were as shown in Table 2. 
 
 The relative rate of acid-production by both strains is obviously 
 different. Prolonged cultivation may give somewhat different results, 
 but it has been shown bv several workers that the maximum acid- 
 
34 Frederick Eberson 
 
 production in the usual carbohydrate concentrations is at a maximum 
 after 24 hours. In any case the antigenic properties of both strains are 
 not in agreement, and the cultural tests seem to bear out this difiference. 
 Mellon's flavidus may be a subspecies, but it differs from Morse's tyoe 
 too markedly to go by the same species name. 
 
 Closer agreement exists in the case of C. hoagii, for which we find 
 on the one hand neither group agglutinins nor cross-fixation for C. 
 hofmanni and C. xerosis, while, on the other hand, there are both 
 cross-fixation and group-agglutination with the flavidus antigen. C. 
 hofmanni shows group agglutinins for C. hoagii and C. flavidus (Mel- 
 lon), but not for C. xerosis. 
 
 
 Agglutination Tests 
 
 TABLE 3 
 
 AND Complement 
 
 Fixation Study 
 
 
 Antiserum 
 
 Antigen 
 
 C. flavidus 
 
 C. hoagii , ! C. xerosis 
 
 C. hofmanni 
 
 C. flavidus 
 
 C. hoagii 
 
 0.025 (500) 
 
 0.05 (600) 
 
 (160) 
 
 1 0.1 ( - ) 
 
 0.025 (2000) 
 
 (80) 
 
 0.1 ( - ) 
 ... (-) 
 
 0.4 ( 20 ) 
 ... ( 160) 
 
 C. hofmanni 
 
 (1000) 
 
 
 The figures and signs enclosed in parentheses represent the highest dilution in which 
 partial agglutination occurred, and the decimals indicate the amount of antigen in mg. which 
 was necessary for fixation with the difTerent serums. Absorption experiments with a strain 
 of Morse flavidus omitted. 
 
 There is no apparent correlation between the reactions in carbo- 
 hydrates and the antigenic properties. Nonspecific agglutinins appear 
 to be readily absorbed, whereas the specific agglutinins are left intact. 
 
 Morse's complement fixation study and the group agglutination tests 
 are shown in Table 3. 
 
 III. PLEOMORPHISM OF DIPHTHEROIDS, MUTABILITY 
 
 OF TYPES, AND A METHOD FOR DETERMINING 
 
 MUTANTS 
 
 VARIABILITY OF BACTERIA 
 
 Bacteria are unicellular organisms which are produced from other 
 like organisms by the process of fission — an act in which the entire 
 parent substance may be said to divide itself equally into two fractions, 
 both of which constitute the resulting pair of bacteria. Under such 
 conditions the offspring cannot possess what was not present in the 
 parent. This, in essence, is the pure line concept of bacteria and 
 implies the descendants from any single bacterial cell. The hereditary 
 charaqteristics of such a biotype do not change as a result of changed 
 
Diphtheroid Organisms 35 
 
 environmental conditions. In order to explain variations in bacterial 
 types it is necessary to consider both Darwin's theory and that of 
 deVries. The former conception is based on natural selection in which 
 such variations as are better adapted to the struggle for existence will 
 be perpetuated. The theory of deVries holds that the tendency toward 
 variation in the germ plasm may give rise to permanent variations as 
 opposed to fluctuating variations. 
 
 When bacteriology was in its infancy it was a popular notion that 
 types could be transmuted readily from one into the other and back 
 again. Strange as it may seem the idea has been reborn and given 
 great impetus within recent years. Pleomorphism is highly developed 
 among the diphtheroids and it is in this group that much confusion has 
 arisen not only as a natural result of the well known overlapping of 
 closely related types, but chiefly because of the ease with which appar- 
 ently pure cultures of the organisms undergo changes in their mor- 
 phology. In none of the work presented has the technic been of such a 
 nature as to preclude error. Billings and Rosenow state that single 
 colonies of diphtheroids from Hodgkin's disease "in dextrose agar 
 which showed bacilli only in smears, yielded in subcultures a pure 
 culture of staphylococci aerobically and forms of the bacillus either 
 pure or in mixture anaerobically on the same medium. These facts 
 suggest strongly that the associated staphylococcus is derived from the 
 bacillus." Certainly, no evidence is given by the authors that the 
 single colonies were pure. There is only one method whereby the 
 fallacy may be precluded and that method was not used in this par- 
 ticular instance. It may or may not be true that the staphylococcus 
 was associated with the bacillus, but the word "derived" is misleading. 
 We know from common practice that a single colony even when 
 obtained from highly diluted plate cultures, does not necessarily repre- 
 sent the descendants of a single bacterial cell. Such a mistake was 
 made by Goodman when he claimed to have separated the diphtheria 
 organism into an acid-producing and an alkali-producing type by the 
 plate method, on the assumption that he had a pure line. 
 
 I shall deal exclusively with C. enzymicus described and named by 
 Mellon. This organism exhibits the pleomorphism of most diph- 
 theroids but is of especial interest because of the remarkable morpho- 
 logical changes the author claims to have produced. He was able to 
 show a seeming relationship between the diphtheroids and the strepto- 
 cocci and described a method whereby he transformed the bacillary type 
 of diphtheroid into a coccus and back again. The old question of sta- 
 
36 Feederick Eberson 
 
 bility of bacterial type comes up and it is essential that the cardinal 
 points be investigated. Two methods of attack were followed in my 
 studies with C. enzymicus : cultural and serological. 
 
 Cultural Study of Mutation 
 
 Two strains of C. enzymicus were studied. One was obtained from the 
 Museum of Natural History and had been cultivated on plain agar for several 
 months. Smears stained with Loeflfler's methylene blue showed typical diph- 
 theroid bacillary forms with the usual coccoidal or short rod and extremely 
 small forms (Fig. 14). The second culture was sent by Dr. Mellon. This strain 
 had been cultivated on blood agar for about 1 year and in smear preparations 
 appeared to be practically free from coccoidal forms, showing large unevenly 
 staining bacillary types, varying in size and morphology. 
 
 Museum Strain of C. Enzymicus. — A subculture was made on plain agar 
 slants and the 24-hour growth examined. Stains were made from dififerent 
 areas and examined with a ^2 immersion, using a magnification of approxi- 
 mately 1,400. In all instances diplococci and coccilike organisms were seen 
 grouped like staphylococci and at times singly. Careful examination showed 
 clusters of bacillary forms in small numbers (Fig. 15). 
 
 The next step was to obtain a pure culture of the coccus type. Agar plates 
 (meat infusion agar 1.0 to phenolphthalein) were streaked with a platinum 
 loop touched to opiginal culture and several discrete colonies fished. In all 
 cases a single colony was used to prepare a second plate culture and from this 
 generation of single colonies, other plates streaked successively until the 10th 
 generation. Each generation was studied for morphology before seeding. 
 Pure coccus forms were obtained in every case (Fig. 16). No difficulty what- 
 soever was experienced in procuring a coccus form from the original culture. 
 The real hardship lay in trying to obtain the bacillary type in relatively pure 
 culture, free from cocci. 
 
 It is a well known fact that blood and serum mediums exert a favorable 
 influence on the diphtheroid bacteria, causing the development of large clubbed 
 and barred forms. Taking advantage of this fact, and the fortunate "trans- 
 formation" eflfected on plain agar, it becomes possible to demonstrate the 
 mechanism of variability in diphtheroids. 
 
 Again using the original culture of C. enzymicus I transplanted it to sheep 
 blood agar and examined the culture after 24 hours' incubation. Smears 
 showed cocci and numerous bacillary forms. When subcultured on the same 
 medium the bacillary forms had almost completely outgrown the cocci and a 
 typical diphtheroid picture was seen. As generation after generation on this 
 medium was studied, it was found that cocci disappeared, although not entirely. 
 At times it required careful examination of the microscopic field to discern 
 them among the larger barred and granular forms. A most interesting point 
 was next observed. When subculturing on sheep blood, transfers were made to 
 plain agar slants from the first generation on sheep blood medium. After 
 24 hours' incubation the agar tubes were found to contain cocci only. As 
 progressive generations on blood medium were transferred to plain agar it 
 became more and more difficult to suppress the bacillary types and to demon- 
 strate the coccus. By the use of various mediums and numerous cross sub- 
 inoculations it appeared quite clear that the selection of biotypes was dependent 
 on the environment. These details are shown in Chart 1. Certain cardinal 
 points may be observed by studying the diagram in which evidence is adduced 
 

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38 Frederick Eberson 
 
 that we are dealing with distinct biotypes side by side in a cuhure of bacteria. 
 When conditions are optimum for a particular biotype, variations do not occur 
 readily. A new type which may have been brought out in company with the 
 predominating type by a change to a less favorable environment can be sup- 
 pressed as conditions are restored to normal. It is to be noted, however, that 
 prolonged cultivation under what were at the outset unfavorable conditions, 
 often, if not as a rule, leads to adaptation, and it becomes impossible to obtain 
 a single biotype. 
 
 This hypothesis is supported strongly by the following observation : A sub- 
 culture on plain agar made from the 4th generation on sheep blood medium 
 (bacillary forms predominating) developed a peculiar surface growth of very 
 heavy opaque masses lying in a more or less translucent delicate growth which 
 composed the greater part of the transplant. Microscopic examination of the 
 heavy growth showed purely bacillary forms with isolated cocci, whereas the 
 translucent area gave cocci only. Touching a platinum needle lightly to the 
 heavy mass a second tube of plain agar Avas inoculated and exactly the same 
 picture was obtained (Figs. 17, 18, and 19, etc.). This was observed on blood 
 medium also (Chart 1). As to the microscopic appearance of the culture it 
 was found that no hard and fast rule can be laid down regarding the character 
 of the bacillary or coccus growth. On any medium the former developed moist 
 and opaque while the latter grew more delicately and translucent or trans- 
 parent. These characteristics were observed with the agar strain of C. enzy- 
 micus and are diametrically opposed to Mellon's description. The strain 
 cultured on blood medium conformed somewhat to his statement, yet transfers 
 to plain agar gave heavy, moist growth as well as the more delicate and trans- 
 lucent. On fresh Loeffler's medium or blood agar the coccus type could not be 
 distinguished macroscopically from the bacillary form. 
 
 To settle the question whether the coccus form is a mutant it is necessary 
 to isolate in pure culture the two biotypes which cultural experiments seem to 
 reveal. A true mutation must appear in a pure line which has been observed 
 over a definite period and shown to consist of a single biotype. As soon as 
 reversion occurs when environmental conditions are restored to normal, we are 
 no longer dealing with a mutation. Different types, to be genuine mutants, 
 must have developed when conditions remained constant. According to Mellon 
 the diplococcus form of C. enzymicus when obtained from the barred, long 
 bacillary type, remains quite constant, and although it is possible, as he states, 
 to cause it to assume a bacillary form again, this is accomplished with great 
 difficulty. It is obvious that these characteristics are not those of a true mutant, 
 since the coccus, according to the author, is not obtained until conditions are 
 changed and since it may be transformed to the bacillary type although this is 
 attended with difficulty. Mellon's view is that the organism is extremely pleo- 
 morphic and he disregards the likelihood of the coccus being a mutant or a 
 distinct biotype in his original pure culture. It is difficult, admittedly, to prove 
 that the coccus is an entity in the original culture, yet it can be done, especially 
 since there are so many factors in favor of this conception. Mellon goes on 
 to describe the source of his culture and says in part "the rabbit had received 
 intravenous injections of the strain in bacillary form and was recovered from 
 the gallbladder in long chains of streptococci." Is pleomorphism the only pos- 
 sible explanation for this strange transformation? It is just as plausible and 
 simpler to account for this by selective action on the part of the organs for 
 the coccus biotype. 
 
Diphtheroid Organisms 39 
 
 Experiments with Mellon Strain Cultivated on Blood Medium. — This culture, 
 obtained directly from Dr. Mellon, was morphologically like a typical diph- 
 theroid containing barred, granular, split and clubbed forms, and was relatively 
 free from very minute ovoid bacteria, so far as could be revealed by a routine 
 smear examination. The strain had been cultivated on blood agar for about 
 1 year and showed well-developed adaptability to this medium. 
 
 Study with Pure Lixes of C. Enzymicus Bacillary Type 
 
 Sheep blood dextrose agar plates were smeared with a platinum loop lightly 
 inoculated with the original culture. After 24 hours' incubation at 37 C. single 
 colonies were taken for seeding a second series of plates. This process was 
 repeated for 10 generations, using in each instance but 1 colony for the 
 subculture. A single colony, when examined in a smear, always showed the 
 typical bacillary appearance, yet was not "pure" insofar as type was con- 
 cerned. From 40 to 50 separate colonies selected from each generation were 
 studied microscopically and invariably ovoid and very small coccoid forms 
 could be found in goodly numbers. By carefully examining numerous fields 
 the interesting fact is revealed that a small diplococcus or coccus type is 
 present. Painstaking study of very many microscopic fields is necessary before 
 passing judgment on the presence or absence of these types, but they can be 
 found none the less. Whether or not these diplococci are appearances caused 
 by unusually short ovoid or coccoid forms, I cannot say, since the line of 
 demarkation between these types is not marked, but, as seen, they were dis- 
 tinctly spherical and appeared in pairs. Although the contrast was sufficiently 
 striking with a magnification of about 1,500 diameters, a high power Jens with 
 a magnification of 2,500 dispelled any doubt. Where two such organisms were 
 united, one could distinguish the point of union from that which is observed 
 in coccoid and small rod-forms. With these observations to go by, Mellon's 
 contention regarding his bacillary culture becomes untenable, and it is perhaps 
 inaccurate to say that "a culture of the C. enzymicus was prepared from a 
 single colony whose antecedents had shown no diplococci." The9retically as 
 well as practically the culture is absolutely pure but we are not justified, under 
 any circumstances, in assuming that a single biotype can be so obtained. It is 
 not that it is difficult to obtain the bacillary form from the coccus, but 
 that it is difficult, if not impossible, to get the bacillus without the coccus. 
 Agai' slants, inoculated with single colonies from various generations plated out 
 according to the technic described, always contained cocci. 
 
 "Transition" of Bacillus to Coccus. — The technic of Mellon was repeated 
 carefully in all details. Meat infusion veal glucose broth 1.6 to phenolphthalein 
 was enriched with sterile rabbit serum to the amount of about 5 c.c. per liter. 
 Flasks and tubes were prepared containing 30 c.c. and 10 c.c. of the medium, 
 respectively, and tested for sterility before use. A single colony from the 
 8th generation of plates made with the bacillary types, previously described, 
 was planted in a tube of this medium, incubated at 37 C. for 24 hours and 
 then at 28 C. for 3 days. From this tube the sediment was transferred to a 
 flask of the same broth and incubated at 37 C. After different intervals 
 smears were made from supernatant fluid and sediment and examined. At 
 no time was any change to a coccus observed, and the picture did not differ 
 in any degree from that seen under ordinary conditions on agar slants. 
 Before we can affirm that the morphologic appearance represents the actual 
 mechanism of transformation, it is essential that a culture of cocci be obtained 
 and propagated for several generations from material showing this "transi- 
 
40 Frederick Eberson 
 
 tional" picture. Repeating Mellon's technic I have not succeeded in demon- 
 strating this and I find that his paper contains no evidence that such a 
 postulate has been fulfilled. Subcultures were made on agar, after 12, 24, 30, 
 48, 72, and 96 hours, from individual flasks which had been seeded with the 
 sediment obtained from tubes incubated at 25-28 C. for 3 days. In none of 
 these transplants, incubated at Zl C. for 24 hours, was a culture of cocci 
 obtained. There was, however, an abundance of extremely short forms — 
 coccoids and some diplococcoids, hut nothing to indicate that the bacillary type 
 had undergone any definite change. When cocci are present, the picture is 
 unmistakable and there can be no confusion of this type with the very short 
 forms which look like diplococci (Fig. 26). ' 
 
 It is questionable whether the refined technic of Mellon is needed to demon- 
 strate what is erroneously called a transformation. That all diphtheroid cul- 
 tures show two distinct forms of organisms is beyond question and that these 
 types may be seen under usual laboratory conditions, is also a matter of 
 common experience. Great caution is necessary in interpreting a morphologic 
 appearance as the actual process responsible for the occurrence of different 
 forms in bacterial culture. Repeated subcultures gave negative results and at 
 no time was it possible to show that complete disintegration had taken place. 
 The "mucus-like debris" and "concentration of chromatin" described by Mellon 
 was seen after 24 hours, not only in broth cultures made according to the 
 special technic described, but also in agar slants grown under conditions to 
 be given. Transfers from broth containing "disintegrated bacillary forms" 
 naturally failed to give mucus-like shreds which are found in smears, but 
 the explanation for this is very simple. Detritus can hardly be expected to 
 grow in Tiransplants. As for the "chromatin masses approaching each other in 
 transplants until the figure cannot be told from a diplococcus," the fact remains 
 that these forms only resemble diplococci. In order to study the effect of lower 
 temperatures on the possible suppression of bacillary forms which might occur 
 during the interval- when the broth cultures are incubated at 25-30 C, a few 
 experiments were performed with agar cultures. 
 
 Kinyoun's method for the staining of diphtheria was used. This stain gives 
 most marked granular appearance and is best adapted to differentiating cocci 
 from bacillary bodies with chromatin substance so disposed as to mislead one 
 into taking these for true cocci. Agar slants were prepared from a bacillary 
 culture of C. enzymicus and incubated at Zl C. for 24 hours. As usual, some 
 coccus forms could be discerned in smears. The picture, however, was bacillary. 
 Such cultures when incubated further at room temperatures, 21-23 C. for 12 
 hours, showed in smears numerous diplococci and a marked overgrowth of 
 very short forms. Subcultures maintained at these temperatures always yielded 
 the same results. When transfers were made to agar and incubated at 37 C, 
 however, the diplococci became rare and finally could not be found at all. 
 At 28 C. the effect on morphology was evident also. After 48 hours at this 
 temperature, diplococci became numerous, and after 5 days they were present 
 in practically pure culture. Transfers to agar incubated at 28 and 2il C. gave, 
 respectively, numerous diplococci and short forms and few diplococci. Broth 
 cultures, kept at 28 C. for 1 week, were found to contain numerous diplococci 
 and extremely short forms which could not be distinguished readily from these. 
 
 Coccus Type. — A pure culture of cocci was obtained from the original 
 diphtheroid strain, as described previously, and attempts were made to alter 
 its morphology. The results of the experiments showed conclusively that 
 when working with such a pure strain of cocci it is impossible to transform 
 
Diphtheroid Organisms 41 
 
 it into a bacillus. In the serologic experiments described subsequently it is 
 shown that the coccus is antigenically distinct from the bacillary type and that 
 the extremely short types which are identical in appearance with cocci are 
 merely altered bacillary forms. These "cocci," to be designated here as 
 Strain 28, are antigenically the same as the long, granular rods. 
 
 Diphtheroid cocci were grown on Loeffler's serum for 27 generations, and no 
 change was noted in morphology. The organisms remained cocci. On glucose 
 serum medium the forms developed considerably larger and thicker, but were 
 typical diplococci in outline. On agar of various H-ion concentrations ranging 
 from 6.1-5.2, no change in form was observed. The use of potassium bichro- 
 mate in broth containing various amounts of acid failed to alter the coccus. 
 
 A bacillary strain of C. enzymicus was cultivated on plain agar at 28 C. for 
 several days and subcultures made from day to day. After one week at this tem- 
 perature, stained specimens from different slants showed forms which could 
 hardly be distinguished from diplococci. These proved on close examination 
 to be minute rods unevenly stained and bipolars. Such cultures, if transferred 
 to serum medium, assumed the typical bacillary form. Figure 18 illustrates 
 clearly how readily one may err in calling these forms cocci. By comparing 
 them with the true cocci shown in Figure 16 the difference can be seen. 
 
 Behavior of Coccus and Bacillary Types in Carbohydrates. — Sugar broths 
 (1.0+ to phenolphthalein) were made in the usual manner, and duplicate sets 
 
 TABLE 1 
 Acid Production 
 
 C. Enzymfcus— Coccus Control 0. Enzymicus— Bacillary 
 
 CC GO CO 
 
 Dextrose 7.4 1.6 7.0 
 
 Lactose 6.1 1.4 7.1 
 
 Saccharose 7.2 1.7 6.9 
 
 Maltose 8.0 1.8 ' 7.8 
 
 Inulin 2.1 1.8 2.0 
 
 Dextrin 3.8 1.4 4.4 
 
 Glycerol 2.4 1.8 7.4 
 
 of tubes were inoculated from a 24-hour old agar culture of C. enzymicus 
 (coccus) and C. enzymicus (bacillary). After 8 days' incubation at 37 C. the 
 titrable acid formed in each of the carbohydrates was determined by addition 
 of N/20 NaOH, using phenolphthalein as an indicator. Titrations were done 
 in the cold. Five c.c. of broth were used in a total volume of 50 c.c. with 
 distilled HaO. 
 
 Examination of the tubes after inoculation with both strains showed the 
 marked difference in rate of growth of the organisms. After 8 hours the coccus 
 type had clouded all of the sugar broths heavily. The bacillary type developed 
 more slowly, and the intensity of clouding after 24 hours approximated that 
 produced by the coccus in a third of the time. Both series showed fine granu- 
 lation and sedimentation, but this was more pronounced in the case of the 
 coccus. 
 
 The acid-production is given in Table 1. The figures represent the amount 
 of N/20 NaOH required to neutralize acid. 
 
 The tabulation shows at a glance that, with the exception of glycerol and 
 dextrin perhaps, both types behaved similarly in the carbohydrates used. This 
 might be an argument for similarity or relationship of both organisms. The 
 difference observed in glycerol, however, was too marked to escape notice and 
 
42 Frederick Eberson 
 
 on the basis of this alone would indicate a distinct property not common to 
 both organisms. In order to control the fermentation test, stained preparations 
 were made from both sets of tubes just before titrating, and the apparent 
 similarity of reaction was explained by the selective action of certain mediums 
 on the type of organism. Glycerol was found to inhibit the development of 
 cocci completely; dextrin had a similar action to a lesser degree. In all of 
 the remaining carbohydrates, the bacillary forms had been suppressed to a 
 remarkable extent. 
 
 Serologic Experiments 
 
 Absorption of Agglutinins. — The object of these experiments was to estab- 
 lish, if possible, by means of highly agglutinating serums, the relationship 
 of the bacillary diphtheroid to the diplococcus. Mellon, in his paper, concludes 
 that the streptococci are related to the diphtheroids and can be derived from 
 these. To establish such a claim it would be necessary to obtain pure strains 
 of each of these two types and show that they are mutually interchangeable. 
 This has not been shown by Mellon, and in the work reported here the results 
 
 TABLE 2 
 Absorption Experiments with Coccus Serum 
 
 Serum C. Enzymicus— Coccus 
 
 1-20 1-40 1-80 1-160 1-320 1-500 1-1000 1-1500 1-2000 1-3000 1-4000 
 
 C. enzymicus (coccus) ± + ++ ++ ++ ++ +-|- 4. -+- 
 C. enzymicus (bacillary) — — — — — — 
 
 After absorption with C. enzymicus (bacillary) 
 
 C. enzymicus (coccus) +.f4--t- ++ ++ ++ + -j- 
 C. enzymicus (bacillary) — — — — — -^ 
 
 After absorption with C. enzymicus— Strain 28 
 
 0. enzymicus (coccus) -^-^^--f ++ ++ ++ + ± 
 
 C. enzymicus (28 strain) ______ _ 
 
 do not substantiate his claim. It is evident that two organisms, derived one 
 from the other, should have similar, if not identical, antigenic properties. To 
 test the validity of such an assumption the most delicate method available is 
 that of agglutinin absorption, when the organisms in question are capable of 
 producing agglutinating serums. If the coccus type, obtained from a diph- 
 theroid culture, is truly derived from the bacillus, then a serum prepared with 
 one strain should agglutinate the other, and the absorption of agglutinins 
 should be demonstrable. With this idea in view, two serums were prepared 
 with the coccus strain and the bacillary strain, respectively. 
 
 Healthy male rabbits were injected intravenously at 4-6 day intervals 
 with increasing doses of culture heated to 56 C. for 20 minutes. The dosage 
 progressed from l^o of an agar slant to a whole culture. Ten days after the 
 last injection the animals were exsanguinated and the serum, after heating to 
 53 C. for 15 minutes, was stored at 4 C. until ready for use. The tests were 
 made with serum less than 6 days old. 
 
 The technic of absorption was as follows : 24-hour old agar cultures of 
 the organisms were emulsified in a small volume of NaCl solution, heated 
 to 56 C. for 20 minutes, and centrifugated at high speed for a short time. 
 To the sediment so obtained the heterologous serum was added after deter- 
 mining by a preliminary agglutination test the potency of the homologous 
 serum. In order to ensure complete absorption the serums were diluted. The 
 
Diphtheroid Organisms 43 
 
 mixture of bacteria and serum was incubated at TH C. for IVa hours, kept at 
 4 C. over night, and then centrifugated for 20 minutes at high speed. The 
 supernatant serum, pipetted off, was added to another heavy sediment of 
 organisms, obtained as in the preceding, and the absorption repeated, after 
 which agglutination tests were made. A series of dilutions were prepared 
 with each original serum and tubes set up at the same time as the absorbed 
 serum. 
 
 The results of these absorption experiments are decisive. A serum 
 prepared with the coccus possesses no agglutinins for the bacillary 
 diphtheroid and when absorbed completely with this bacillary strain 
 evidences no reduction in its agglutination titer against the homologous 
 coccus. The coccus serum when absorbed with a bacillary strain altered 
 morphologically by cultivation at 28 C. for 10 days so that the organism 
 
 TABLE 3 
 Absorption Experiments with Bacillary Serum 
 
 Serum C. Enzymicus— Bacillary 
 
 1-10 1-20 1-50 1-100 1-20O 1-400 1-800 1-1600 1-20CO 1-30OO 
 0. enzymicus (bacillary) ++ ++ ++ + ± — 
 
 C. enzymicus (coccus) + — — — — — 
 
 After absorption with C. enzymicus (coccus) 
 C. enzymicus (bacillary) ++++ ++ ++ +, ± 
 
 After absorption with C. enzymicus— Strain 28 
 C. enzymicus (bacillary) ± ++ + — — 
 
 could not be distinguished from cocci, lost none of its agglutinating 
 power for the coccus. Again evidence is adduced that the change on 
 the part of the bacillus was merely an appearance, and that the organ- 
 isms were not true cocci, but extremely short bacilli, altered perhaps by 
 changes in surface tension to assume a form remarkably like cocci. 
 Antigenically these organisms are therefore distinct from the coccus 
 type. In Table 3 they are shown to be identical with the original bacil- 
 lary form. The conclusion to be drawn from this experiment is that 
 the coccus has no antigenic elements in common with the bacillus. 
 
 The next experiment, made with a serum produced with the bacil- 
 lary strain of C. enzymicus, gave additional evidence in another direc- 
 tion and confirmed the difference of the two organisms. The serum, 
 although agglutinating its homologous culture as high as 1-2000, gave 
 agglutination in 1-10 for the coccus. This in itself would be an argu- 
 ment against antigenic relationship. The fact that a bacillary strain is 
 never free from cocci, however, would readily explain slight agglutina- 
 tion which might occur. This interesting point is shown quite clearly 
 
44 Frederick Eberson 
 
 in the absorption test. The serum, after complete absorption with the 
 coccus, showed a reduction in agglutinating titer, stopping at 1-1600, 
 whereas the nonabsorbed serum was positive at 1-2000. This removai 
 of agglutinins might suggest antigenic similarity on the part of the 
 coccus for the bacillus. The important point to be noted here, how- 
 ever, is the cumulative action of multiple doses of bacillary diphtheroids 
 containing cocci in mixture. Repeated injections of mass cultures 
 have resulted in the formation of agglutinins specific for each type of 
 organism, and the slight reduction in titer is thereby accounted for. As 
 a further corollary, Strain 28 absorbs most of the agglutinins out of 
 the bacillary serum, showing that the organisms are antigenically the 
 same. That the absorption is not more complete may be due to the 
 fact that but one exposure was made with the organism in question, 
 whereas in the case of the coccus strain the serum was doubly absorbed. 
 From these findings the conclusion may be drawn that the bacillus is 
 antigenically distinct from the coccus. 
 
 Two possibilties are suggested in explanation of the presence side 
 by side of two biotypes ; one is that of mutation and the second a 
 symbiotic relationship of a contaminating coccus and a typical bacillary 
 diphtheroid. The possibility of mutation is not far-fetched, although 
 highly improbable in this instance. As to a symbiosis, the likelihood 
 is less remote. In either case the question can be answered categorically 
 by applying the single bacterial-cell method to the conditions which 
 prevailed in the experiments given here. These experiments will be 
 described in a future paper. 
 
 SUMMARY AND CONCLUSIONS 
 
 The diphtheroids have been studied with regard to nomenclature 
 and revised according to accepted standards for the naming of valid 
 species. 
 
 A tentative classification is offered with a view toward grouping 
 prominent biologic characters. Nine distinct groups of diphtheroids 
 are outlined with a type species for each group and corresponding 
 subsf>ecies. 
 
 It is evident that the groups hitherto accepted as types for all diph- 
 theroids are insufficient and not representative of a broad classification. 
 
 Diphtheroids isolated from glands and tissues — 21 different sources 
 — fall into 12 distinct fermentative groups. The greatest number of 
 organisms are nonfermenters. Strains isolated from the eye and nose 
 
Diphtheroid Organisms 45 
 
 do not attack carbohydrates vigorously, and are usually found either in 
 the nonfermenting or in the dextrose-splitting group. 
 
 The diphtheroids associated with Hodgkin's disease have been 
 studied. It has been shown that neither the source nor the cultural 
 characters serve to distinguish the supposed cause of Hodgkin's disease 
 from numerous saprophytic diphtheroids. By means of complement 
 fixation tests the conclusion is reached that the cause of this disease is 
 not the organism described by numerous workers. 
 
 Agglutinin absorption studies indicate more clearly the relationship 
 which exists between certain members of the diphtheroid group of bac- 
 teria. The results are correlated in the main with complement fixation. 
 
 Diphtheroids conform to the fundamental laws of bacteriology and 
 are not readily transformed into cocci and back again. Two distinct 
 biotypes have been isolated from a culture of C. enzymicus. 
 
 The coccus associated with a bacillary diphtheroid (C. enzymicus) 
 is antigenically distinct from the bacillus and vice versa. Under cer- 
 tain conditions a bacillary strain may be made to assume a diplococcus 
 picture, but absorption experiments show that the organisms are not 
 cocci. 
 
 Certain mediums and cultural technic have been found to exert a 
 definite selection on diphtheroid cultures. One or the other biotype 
 will prevail, depending on the extent of suppression of the first or 
 second. 
 
 Two explanations for the existence of coccus and bacillus are sug- 
 gested, either that of mutation or a symbiotic relationship of a contam- 
 inating coccus. The question can be settled by applying the single-cell 
 method to the study. 
 
 BIBLIOGRAPHY 
 
 Abbot: Bull. Johns Hopkins Hosp., 1891, 2, p. 143. 
 
 Baumgarten's Jahrensbericht, 1891, p. 317. 
 
 V. Besser cited by Migula. 
 
 Biggs, Park and Beebe : Centralbl. f. Bakteriol., 1895, 17, p. 765. 
 
 Billings and Rosenow : Jour. Am. Med. Assn., 1913, 61, p. 2122. 
 
 Bloomfield: Arch. Int. Med., 1915, 16, p. 197. 
 
 Bruno: Berl. klin. Wchnschr., 1898, 35, p. 1127. 
 
 Buchanan: Jour. Infect. Dis., 1915, 17, p. 528. 
 
 Bunting and Yates: Arch. Int. Med., 1913, 12, p. 236. 
 
 Bunting and Yates: Jour. Am. Med. Assn., 1914, 62, p. 516. 
 
 Cautley: Rep. Med. Officer of Health, Local Govt. Board, 1894-5, p. 455. 
 
 Chester: Manual of Det. Bact., 1901, p. 171. 
 
 Demme : Fortschritte d. Medizin, 1888. 1, p. 7. 
 
 Eisenberg: Bakt. Diagnostik, 1891. 
 
 Enderlen : Ztschr. f. Tiermedezin, 1891, p. 321. 
 
46 Frederick Ederson 
 
 Ernst: Ztschr. f. Hyg. u. Infektionskr., 1889, 5, p. 428. 
 
 Ernst: Centralbl. f. Bakteriol., I. O., 1906, 40, p. 79. 
 
 Fleming: Lancet, 1909, 1, p. 1035. 
 
 Fliigge-: Ztschr. f. Hyg. u. Infektionskr., 1894, 17, p. 290. 
 
 Fliigge : Die Mikroorganismen, 1896, 2, p. 477. ' 
 
 Fox: Jour. Med. Research, 1915, 32, p. 309. 
 
 Fox: Arch. Int. Med., 1915, 16, p. 465. 
 
 Fraenkel: Berl. klin. Wchnschr., 1897, 34, p. 1087. 
 
 Fraenkel and Much: Ztschr. f. Hyg. u. Infektionskr., 1910, 67, p. 159. 
 
 Galli-Valerio : Centralbl. f . Bakteriol., I. O., 1904, 36, p. 465. 
 
 Gerber and Podack : Deutsch. Arch. f. klin. Med., 1895, 54, p. 262. 
 
 Gilchrist: Johns Hopkins Hosp. Reports, 1901, 9, p. 409. 
 
 Glage : Ztschr. f . Fleisch, u. Milch hygiene, 1903, 13, p. 166. 
 
 Goodman: Jour. Infect. Dis., 1908, 5, p. 421. 
 
 Graham-Smith: Centralbl. f. Bakteriol, Ref. 1905, 36, p. 358. 
 
 Grips : Ztschr. f. Fleisch u. Milch hygiene, 1898, 8, p. 166. 
 
 Gromakowsky: Centralbl. f. Bakteriol., 1900, 28, p. 142. 
 
 Hall and Stone: Jour. Infect. Dis., 1916, 18, p. 195. 
 
 Hamilton: Jour. Infect. Dis., 1907, 4, p. 326. 
 
 Hine: Jour. Path, and Bacteriol., 1914, 18, p. 75. 
 
 V. Hofmann: Wien. klin. Wchnschr., 1888, 1, p. 65. 
 
 Hodara : Monatschr. f . prakt. Dermat., 1894, 18, p. 573. 
 
 Jacobson: Annals de I'lnst. Pasteur, 1908, 22, p. 308. 
 
 Jacobson: Centralbl. f. Bakteriol., Ref., 1909, 43, p. 213. 
 
 Klein : Centralbl. f. Bakteriol., 1900, 28, p. 418. 
 
 Kolmer : Jour. Infect. Dis., 1912, 12, p. 56. 
 
 Kruse and Pasquale : Ztschr. f. Hyg. u. Infektionskr., 1891, 16, p. 1. 
 
 Kunneman : Arch. f. wiss. u. prakt. Tierheilk., 1903, 29, p. 128. 
 
 Kurth: Ztschr. f. Hyg. u. Infektionskr.. 1898, 28, p. 409. 
 
 Landsteiner: Wien. klin. Wchnschr,, 1897, 10, p. 439. 
 
 Lehman and Neumann : Atlas, 1912, 2, p. 570. 
 
 Lesieur : Les Bacilles dit. Pseudo-diphteriques, 1902. 
 
 Levy and Fickler : Centralbl. f. Bakteriol., 1901, 30, p. 470. 
 
 Lewandowsky : Centralbl. f. Bakteriol., I. O., 1904, 36, p. 473. 
 
 Loeffler: Centralbl. f. Bakteriol., 1887, 11, p. 105. 
 
 Lubowski: Ztschr. f. Hyg. u. Infektionskr.. 1900, 35. p. 87. 
 
 Lustgarten : Vierteljahrsschrift f. Derm. u. Svphilis, 1887, p. 914. 
 
 Mellon: Jour. Bacteriol., 1917, 2, p. 81. 
 
 Migula: System der Bakterien, 2, 1900. 
 
 Morse: Jour. Infect. Dis., 1912, 11, p. 251. 
 
 Nakanishi: Centralbl. f. Bakteriol., 1900, 27, p. 641. 
 
 de Negri and Mieremet : Centralbl. f. Bakteriol., I. O., 1913, 68, « . 292. 
 
 Neisser: Ztschr. f. Hyg. u. Infektionskr., 1888, 4, p. 165. 
 
 Neisser and Kuschbert : Bresl. artztl. Ztschr., 1883, 4. 
 
 Nicholas: C. R. de la Soc. de Biol.. 1896. 
 
 Nicolle: Annals de I'Inst. Pasteur, 1898, 12, p. 186. 
 
 Olitzki: Jour. Infect. Dis., 1915, 17, p. 1. 
 
 Ortmann: Berl. klin. Wchnschr., 1889, 16. 
 
 Plotz: Jour. Infect. Dis., 1915, 17, p. 20. 
 
 Preisz and Guinard : Kolle Wassermann Handb. d. path. Mikroorganism, 
 
 1913. 5. p. 784. 
 Prochaska: Ztschr. f. Hyg. u. Infektionskr., 1897, 24, p. 373. 
 Rhea and Falconer: Arch. Int. Med., 1915, 15, p. 438. 
 
Diphtheroid Organisms 
 
 47 
 
 Sabouraud: Annals de I'lnst. Pasteur, 1897, 11, p. 134. 
 
 Schwoner: Wien. klin. Wchnschr., 1902, 15, p. 1275. 
 
 de Simoni: Centralbl. f. Bakteriol., 1898, 24, p. 294. 
 
 de Simoni : Centralbl. f. Bakteriol., 1899, 26, p. 673, 757. 
 
 Sternberg: Manual of Bacteriology, 1892. 
 
 Siidmerson and Thompson : Jour. Path, and Bacteriol., 1910, 14, p. 224. 
 
 Torrev: Jour. Med. Research, 1916, 34, p. 65. 
 
 Trumpp: Centralbl. f. Bakteriol., 1896. 20, p. 721. 
 
 Unna: Monatschr. f. prakt. Dermat., 1891, IS. 
 
 Weichselbaum : Beitrage zur. patholog. Anatomie und zur allgem. Pathologie, 
 
 4, p. 119. 
 Zinsser and Hiss : Textbook of Bacteriology, 1915. 
 
48 Frederick Eberson 
 
 EXPLANATION OF PLATES 1-3 
 Fig- 1. — C. acidum. Large forms developing in mediums enriched with serum. X 1000. 
 Fig. 2. — Same after prolonged cultivation on ordinary mediums. X 1000. 
 Fig. 3. — C. suppuratum. On serum medium after 1st isolation. X 1000. 
 F'g- 4. — Same after prolonged cultivation on ordinary medium. X 1000. 
 Fig. 5. — C. aurantiacum. X 1000. 
 Fig. 6. — C. delicatum. X 1000. 
 Fig. 7. — C. epidermidis. X 1000. 
 Fig. 8. — C. ascites. X 1000. 
 Fig. 9. — C. glandulae. X 1000. 
 Fig. 10. — C. pseudodiphtheriae. X 1000. 
 Fig. 11. — C. cerebralis. X 1000. 
 Fig. 12.— C. putidum. X 1000. 
 Fig. 13. — C. flocculens. X 1000. 
 
 Fig. 14.— I. Plain agar slant inoculated with material taken from a culture of C. enzymicus 
 grown on sheep blood glucose agar for 4 generations. The sheep blood agar showed numerous 
 rod forms with cocci intermingled. A.^ heavy white growth superimposed on a delicate trans- 
 parent background. 
 
 II. Plain agar slant inoculated with material taken from opaque, heavy white growth 
 indicated by^'. A, transparent surface growth. B, heavy white opaque growth. Smears 
 from A^ and B always showed bacillary forms, typical diphtheroids. A yielded pure cocci 
 only. Natural size. 
 
 Fig. 15. — Smear from heavy growth indicated by /4' and B in Figure 14. 
 
 Fig. 16. — Smear from transparent delicate growth marked A in Figure 14. Stained with 
 Loeffler's methylene blue. X 1000. 
 
 Fig. 17. — C. enzymicus. Original culture. 
 
 Fig.l8.— Bacillary type developing on Loeffler's serum. Transferred from sheep blood 
 agar on which the original culture had been inoculated. When the original strain (Fig. 17) 
 was planted on plain agar, bacilli could not be demonstrated. 
 
 Fig. 19. — Suppression of bacillary type on glucose agar. After 3 generations on Loeffler's 
 serum preceded by 2 generations on sheep blood agar, cocci were rare. Note the predom- 
 inating type here. 
 
 Fig. 20. — Transplant from 3rd generation on sheep blood medium to plain agar. The 
 sheep blood culture showed rare cocci and practically a pure culture of bacillary forms. 
 
 Fig. 21. — Same culture before transplanting to plain agar. X 1000. 
 
 Fig. 22. — C. enzymicus cultivated on blood agar, then transferred to plain agar and incu- 
 bated at 28 C. "Concentration of chromatin" clearly shown. 
 
 Fig. 23. — Transplant from 48-hour veal glucose broth culture of C. enzymicus. Note 
 absence of "transformed" bacilli. The technic according to Mellon. 
 
 Fig. 24. — C. enzymicus grown at 28 C. in broth. Curious chromatin staining. 
 
 Fig. 25. — C. enzymicus 3rd generation on agar from blood medium. This picture, as well 
 as Figure 22, is identical with the "transitional" phase. 
 
 Fig. 26. — C. enzymicus grown at 28 C. on plain agar for 10 days. Note the diplococcus 
 appearance. 
 
 Fig. 27. — Previous incubation at 28 C. in broth and subsequent growth at 37 C. Stained 
 according to Kinyoun's method. X 1000. 
 
Plate 1 
 
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 4 
 
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 2 
 
 
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 ^ 
 
 
 
 ^4' • 
 
 
 
 ^A 
 
 y. 
 
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 12 
 
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 13 
 
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VITA 
 
 Frederick Eberson was born in New York City, February 10, 
 1892. He received his elementary and high school education in the 
 city of Brooklyn, N. Y., and his first college training at the College 
 of the City of New York, where he took the degree of Bachelor of 
 Science in June, 1912. In October, 1912, he was appointed Research 
 j\ssistant in the Department of Sanitary Science and Public Health 
 at the Massachusetts Institute of Technology, Boston. Thence he 
 matriculated under the Faculty of Medicine and Pure Science at 
 Columbia University, specializing in Bacteriology and Biological 
 Chemistry, and received his Master degree in June, 1914. In Septem- 
 ber, 1914, he was appointed Research Fellow and Assistant Instructor 
 in the Department of Bacteriology and Hygiene at the Iowa State 
 College, where he specialized in Bacteriology and Pathology and 
 obtained his Master of Science degree in June, 1915. In September 
 of the same year he was appointed Research Scholar under the tenure 
 of a President's University Scholarship at Columbia University and 
 continued work in the Department of Bacteriology until 1916, when 
 he was appointed Research Bacteriologist to the North Manchurian 
 Plague Prevention Service, Harbin, China, and was placed in charge 
 of the Division of Bacteriology and Pathology. Toward the end of 
 1917 he was appointed to the stafif of the Rockefeller Institute for 
 Medical Research in the Department of Bacteriology and Pathology. 
 
 Publications 
 
 1912 — The Effect of Drying on the Viability of Bacteria (with C. E. A. 
 
 Winslow) Proceedings Soc. for Exp. Biol, and Medicine, May 5. 
 1915 — A Bacteriologic Study of Secondary Invaders in Hog Cholera. Jour- 
 nal of Infectious Diseases, Vol. 17, No. 2. 
 Separation of the Antibody Fractions in Hog Cholera Serum. Jour- 
 nal of Infectious Diseases, Vol. 17, No. 2. 
 A Milk-Borne Paratyphoid Outbreak in Ames, Iowa (with M. Levine). 
 Journal of Infectious Diseases, Vol. 18, No. 2. 
 1917 — Plague Poisons and Virulence. Journal of Infectious Diseases, Vol. 
 20, No. 2; National Medical Journal of China, Vol. 3, No. 1. 
 Transmission of Pneumonic and Septicemic Plague among Marmots. 
 Journal of Infectious Diseases, Vol. 20, No. 2; National Medical 
 Journal of China, Vol. 3, No. 2; English Journal of Hygiene. 
 Vol. 16. No. 1. 
 
56 Frederick Iu^erson 
 
 Nature of Plague Proteotoxius. Journal of Infectious Diseases, 
 Vol. 21, No. 1 ; National Medical Journal of China. Vol. 3. No. l' 
 1918 — Active Immunity to Systemic Plague Infection. Journal of Infectious 
 Diseases, Vol. 22, p. 62. 
 
 Experiments with Rosenow's Antipoliomyelitic Serum (with H. L. 
 Amoss). Journal of Experimental Medicine. Vol. 27, No. 2; Proc 
 Soc. for Exp. Biol, and Medicine. April. 
 
 Physiological Study of the Choroid Plexus and Experimental I 'olio- 
 myelitis (with Simon Flexner and H. L. Amoss), Journal of 
 Experimental Medicine, Vol. 27, No. 6. 
 
 Experiments with Nuzum and Willy's Antipoliomyelitic Serum (with 
 H. L. Amoss). Journal of Experimental Medicine, Vol. 28, No. 1.; 
 
 Effect of Carbohydrate Concentration on Acid Production. Biochemical 
 Bulletin (in press). 
 
 Scientific Societies 
 Society of American Bacteriologists. 
 
 American Association for the Advancement of Science. 
 National Medical Association of China. 
 Biochemical Society, Columlna University. 
 
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