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 http://www.archive.org/details/cu31924000232821 
 
THE ROfeKEFEtLEifllSTlTUTE 
 FOR MEiliGAE R#EAR€H 
 
 No. 9 January 21, 1919 
 
 THE USirO? BLOOD AGAIttORt^E^S^ 
 JAMES HpWARR^^ 
 
 NEW YORK 
 The Rockefeller Institute for Medical Research 
 
 1919 
 
MONOGRAPH No. 9 
 
 THE USE OF BLOOD AGAR 
 
 FOR THE STUDY OF 
 
 STREPTOCOCCI 
 
 BY 
 
 JAMES HOWARD BROWN 
 
 NEW YORK ,^ r, / f 
 
 The Rockefeller Institute for Medical Research 
 
 1919 c.^ , \,\ "/R 
 
PREFACE. 
 
 The purpose of this Work is quite as much the description of methods 
 as the, recording of results, the latter being in a very high degree 
 dependent upon the former. 
 
 The close association between methods and results is the most 
 striking feature recognized in a review of the literature. Discordant 
 results can be seen in most cases to have been due to different methods 
 employed; i.e., in those cases where the authors have described their 
 methods sufficiently to permit their being understood. The review of 
 hterature is presented in tabular form because it seems to be most 
 accessible and most useful in that form. The review of so many 
 papers in textual form including all the items tabulated would have 
 occupied much more space and would have been much less convenient 
 for reference. Some but not all of the papers tabulated are discussed 
 in the text. In the tables under such headings as Media, Inoculation, 
 Incubation, etc., are mentioned any details which the authors give re- 
 garding these; if these spaces are left blank it may be assxmied that 
 the author reviewed has published no information on these subjects. 
 Under Results and Conclusions are presented only the results and 
 conclusions of the authors reviewed. All quotations are in italics. 
 By referring to the work of an author in each of the tables^-The 
 Use of Blood Agar, The Use of Blood Bouillon, etc., Fermentation 
 Reactions, Pathogenicity and Virulence, Mutations and Variability, 
 General Conclusions, etc. — a fairly complete review of' the author's 
 work is obtained. Reviews with respect to serology, epidemiology, 
 pathological anatomy, and clinical medicine have not been included 
 except incidentally. In each table papers are arranged chronologi- 
 cally so that a paper referred to in the text or bibliography is easily 
 found in the tables by seeking first the year of publication and then 
 the author's name. A historical review of each subject is obtained 
 by reference to the reviews in the order in which they are listed. 
 
 In the Bibliography are included many papers which it has not 
 been considered necessary to review in the tables. There are also in- 
 
IV PREFACE 
 
 eluded a number of papers on other subjects than Streptococci be- 
 cause certain suggestions in regard to methods or technique have been 
 obtained from them. 
 
 I take pleasure in expressing my gratitude to Dr. Theobald Smith 
 under whose direction this work was started, and from whom during 
 five years of association I obtained much of the training and many 
 of the ideals which have resulted in this work. I am also grateful 
 for the facilities of the Department of Comparative Pathology of 
 Harvard Medical School and for assistance from Harvard University 
 in the form of the Charles Elliott Ware Memorial Fellowship. 
 
 J. H. B. 
 
 May, 1917. 
 
MONOGRAPH OP THE ROCKEFELLER INSTrrUTE FOR MEDICAL RESEARCH, 
 
 NO. 9, January 21, 1919. 
 
 THE USE OF BLOOD AGAR FOR THE STUDY OF 
 STREPTOCOCCI* 
 
 By JAMES HOWARD BROWN. 
 {From the Department of Comparative Pathology, Earvard Medical School, Boston) 
 
 Plates 1 to 34. 
 (Received for publication, April 24, 1918.) 
 
 CONTENTS. 
 
 Introduction 3 
 
 Description of the Types in Blood Agar 8 
 
 Type a 8 
 
 Type |3 14 
 
 Type a IS 
 
 Type Y . ■ 17 
 
 Various Representatives of Each T3rpe 19 
 
 Type a 19 
 
 Type a'..... 26 
 
 - Type /3 26 
 
 Influence of the Amount of Blood and of Agar Used 31 
 
 Influence of the Age and Kind of Blood Used 34 
 
 Influence of the Composition of the Agar Used 40 
 
 Reaction (Acidity) _ 40 
 
 Kind of Meat and Peptone Used , 41 
 
 Influence of Dextrose in Agar 46 
 
 Influence of Anaerobiosis 56 
 
 Effect of the Growth of One Streptococcus on that of Others 60 
 
 Theories as to the Causes of the Various Appearances 65 
 
 Type p ^ 65 
 
 Type a... ? 70 
 
 * A thesis submitted to the Division of Medical Sciences of the Faculty of 
 Arts and Sciences of Harvard University in partial fulfillment of the requirements 
 for the degree of Doctor of Philosophy, with assistance derived from the Charles 
 Elliott Ware Memorial Fellowship. 
 
 In preparation for publication a number of minor changes and additions to the 
 text have been made. The number of illustrations has been reduced and the 
 remaining ones have been rearranged. 
 
 1 
 
2 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 Permanence of Cultural Characteristics 77 
 
 Appearance in Bouillon 77 
 
 Fermentation Reactions 77 
 
 Appearance in Blood Agar 79 
 
 Occurrence of Various Types of Streptococci 83 
 
 Classification of Streptococci '. 86 
 
 Summary • • ■ 94 
 
 Appendix 
 
 A Simple System of Notes for Preserving the Genealogy and Other 
 
 Details in the Study of Bacterial Cultures 98 
 
 A Method of Making Photographic Records of Growth on Agar and 
 
 Blood Agar Plates 103 
 
 Bibliography 108 
 
 Table I. Tabular Description of the Principal Strains Referred to in this 
 
 Work. 
 Table II. A Tabulation of Streptococci with Reference to Type of Ap- 
 pearance in Blood Agar and Fermentation Reactions. 
 Tabular Review of the Literature on Streptococci and Streptococcus In- 
 fections. Tables III to VIII. 
 Table HI. The Use of Blood Agar. 
 
 Table IV. The Use of Blood Bouillon and Other Fluid Media. 
 Table V. Fermentation Reactions. 
 Table VI. Pathogenicity and Virulence. 
 Table VII. Mutations and Variability. 
 
 Table VIII. General Conclusions with Reference to the Correlation 
 of Source, Pathogenicity, Hemolysis, Fermentation Reactions, Etc. 
 
INTRODUCTION. 
 
 The usual form of reporting scientific work is to start with a re- 
 view of the literature, to indicate one or more problems which have 
 attracted the author's interest, and finally to report original work on 
 the subject. Ideal though it may be thoroughly to familiarize oneself 
 with all the work that has been reported and to employ it as a point 
 of departure for new work, it is doubtful whether it is the method 
 usually followed. It has not been in the present instance. The work 
 here reported was practically complete before the thorough review 
 and tabulation of the literature was made. In thus entering upon a 
 thorough study of the appearances produced by various streptococci 
 in blood agar it was realized that others might have made similar 
 observations. Recent literature, however, indicated that the work 
 which had been done had led to no generally accepted standardiza- 
 tion of method or of definition of such terms as "hemolytic" and 
 " non-hemolytic" as applied to the appearances produced by strepto- 
 coccus colonies in or on blood agar. The importance of such factors 
 as the amount and kind of blood used, the amount and composition 
 of agar used, the method of inoculation, and the period of incubation 
 was suspected. The work of Rosenow on "Transmutations within 
 the Streptococcus-Pneumococcus Group" was an additional incentive. 
 It seemed worth while for a single author to work at the subject of 
 The Appearances Produced by Streptococci in Blood Agar from the 
 ground up, employing a system of records which would allow noth- 
 ing to escape observation and recording; and finally to compare the 
 results with those previously reported with a view to being able to 
 interpret the results of others and to explain the many disagreements, 
 among them. This is the plan here followed. 
 
 During "A Study of Streptococci Isolated' from Certain Presumably Milk-Borne 
 Epidemics of Tonsillitis Occurring in Massachusetts in 1913 and 1914" by Dr. 
 Theobald Smith and myself there came to us for study material in the form of 
 throat swabs, milk samples, pathological exudates, and a few strains of strepto- 
 cocci isolated by others. Most of the material came from cases of .epidemic 
 septic sore l!hroat, secondary lesions, and suspected milk. Attention was fixed 
 
 3 
 
4 BLOOD AGAR FOE STUDY OF STREPTOCOCCI 
 
 on the streptococci found in this material, these being the organisms commonly 
 considered to be the causative agents in such epidemics. In throats and in 
 milk as ordinarily obtained, however, there are many streptococci normally 
 present but harmless under ordinary conditions. The differentiation of the 
 pathogenic from the non-pathogenic streptococci was therefore the question of 
 primary importance. The method followed was to plate out the suitably diluted 
 material directly in blood agar, a medium adopted because known to favor the 
 growth of a large -number of organisms and one in which they produce widely 
 different appearances. This was found to be true of different strains of strepto- 
 cocci. A number of strains found pure in the material from secondary lesions 
 furnished the clue to the type of streptococcus which was probably the etio- 
 logical factor in the epidemics. In blbod agar this type of streptococcus was 
 easily distinguished by the production of clear colorless hemolyzed zones about 
 the colonies. Such streptococci were found in varying numbers in the material 
 from the sore throats and in the carefully drawn milk of only one or two cows 
 which epidemiological data had placed under suspicion. A study of fermenta- 
 tion and agglutination reactions showed that there were two or three varieties 
 of these hemolytic streptococci. Pathogenicity for rabbits was found to be 
 correlated with hemolysis and with certain fermentation reactions. There were 
 also present in the blood agar plates inoculated with material from throats and 
 with milk sediment large numbers of colonies of other streptococci producing 
 much less hemolysis and certain other characteristic features. 
 
 There arose immediately the question of naming these two most common 
 types of streptococci. Such names as Streptococcus mitior seu viridans, Strepto- 
 coccus brevis, Streptococcus longus, Streptococcus pyogenes, and Streptococcus 
 pathogenes naturally suggested themselves. Davis had recently applied the 
 name Streptococcus epidemicus to streptococci from a similar epidemic. There 
 were reasons, however, which made us hesitate to apply any of the foregoing 
 names to our strains. Not all of the more hemolytic strains produced long 
 chains in fluid media while some of the less hemolytic ones did, hence Strepto- 
 coccus longus and Streptococcus brevis did not seem to be apt terms. The cap- 
 sules or greenish color of the colonies described by Davis for Streptococcus epi- 
 demicus were not observed by us. Streptococcus pyogenes was a term introduced 
 by Rosenbach (1884) without reference to hemolysis. After considerable de- 
 bate and as the result of inoculation experiments (Petruschky, 1896) it came to 
 be regarded as synonymous with Streptococcus erysipelatos (Fehleisen, 1883), 
 both included by Schottmuller (1903) under the name of Streptococcus longus 
 pathogenes seu erysipelatos on the basis of hemolytic characters only, later limited 
 by Andrewes and Horder (1906) to hemolytic streptococci having certain fer- 
 mentative characters, but since used by various authors (Seattle and Yates, 
 1911; Rogers and Dahlberg, 1914) with regard to the fermentation reactions and 
 without regard to hemolytic characters. We avoided the use of the name Strep- 
 tococcus mitior seu viridans for the less hemolytic streptococci because many of 
 them produced little or no green color on blood agar and all did produce more or 
 
JAMES HOWARD BROWN 5 
 
 less hemolysis. In the presence of these difficulties we were impressed with the 
 greater importance of describing the essential characteristics of the strains rather 
 than of naming them. We described, "two types of streptococci, with reference to 
 hemolysis," the less hemolytic being called the "alpha type" and the markedly 
 hemolytic being called the "beta type." Occasionally streptococci were found 
 corresponding to neither of these two types but were never present in sufficient 
 numbers or with sufficient constancy to excite suspicion of their importance re- 
 specting epidemic sore throat and were, therefore, not discussed in the report 
 of our studies as published in January, 1915. 
 
 I have now to report a more detailed study of the appearances 
 produced by the growth of streptococci in blood agar. I have in 
 cultivation between 12S and 150 strains of streptococci and pneumo- 
 cocci. More than half of these have been under observation for 
 several years; others have been isolated from various sources from time 
 to time. All of them have been under the author's care exclusively 
 and there are complete genealogical records covering every trans- 
 plant and every day of the existence of each strain since it was ac- 
 quired. From these have been selected a number of representative 
 strains for more intensive study, for photography, and report. 
 These strains and their more conspicuous characteristics are Hsted in 
 Table I. It will be observed that in this table the strains are grouped 
 according to certain "Types in Blood Agar." Although the appear- 
 ances of colonies of various streptococci on standard agar or glycerol 
 agar have been described by various authors and though even since 
 blood agar has come into general use a number of authors have pro- 
 posed the use of such media as the Conradi-Drigalski litmus-lactose- 
 crystal-violet agar, blood agar is now generally employed for the 
 isolation and primary differentiation of streptococci. As already 
 mentioned, it has the double advantage of being an excellent medium 
 for the growth of streptococci and of being of differential value be- 
 cause of the different appearances produced by different strains. 
 Below are the descriptions of the alpha and beta types as described by 
 Smith and Brown (1915). 
 
 'Type a. — As observed after forty-eight hours incubation the change produced by 
 streptococci of this type may he described as a somewhat greenish discoloration and 
 partial hemolysis of the blood corpuscles immediately surrounding the colony form- 
 ing a rather indefinitely bounded zone one to two millimeters in diameter and sur- 
 rounded by a second, narrow, clearer, not discolored, partly hemolyzed zone. The 
 
6 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 inner {discolored) zones were fairly constant in size and composition on all the 
 plates. Under the microscope many of the corpuscles were seen to be present but ob- 
 viously discolored, the discoloration differing a good deal in intensity for different 
 strains of streptococci. The corpuscles remaining in the outer {clearer) zones were 
 much fewer and, never discolored. The outer zones were much more conspicuous on 
 crowded plates than on plates containing only a few colonies. The deep colonies 
 themselves were small {greater diameter, 0.5-1 millimeter) and biconvex or complex 
 {lobulated or asteroid), the formation of biconvex or complex colonies being quite 
 constant and characteristic for different strains." 
 
 There was no reason for calling this type alpha and the other beta 
 except that the a type was the one first noted and commonly found in 
 varying numbers on all the swabs whether the /3 type was also present 
 or not. The |8 type, however, was the one found in secondary lesions 
 and was regarded as the etiological factor in the epidemics. 
 
 "Type /3. — Streptococci of this type produced hemolyzed zones on horse-hload- 
 agar plates radically different from those of the alpha type. The former produced 
 sharply defined, clear, transparent, completely hemolyzed, colorless zones two to 
 four millimeters in diameter. Under the microscope no corpuscles were seen to 
 remain within the zones. The colonies themselves were simple and biconvex, never 
 complex {greater diameter, O.S-1 millimeter)." 
 
 "There was nothing particularly distinctive about the surface colonies of either 
 type on blood agar plates. All were round, glistening, grayish, translucent, flatly 
 convex colonies surrounded by hemolyzed zones corresponding to their respective 
 types." 
 
 In describing streptococci as of the alpha, beta, or gamma type, I 
 refer to 1;heir appearances in blood agar only. 
 
 In the work about to be reported the medium. used throughout, 
 unless otherwise described, was standard veal infusion agar con- 
 taining the aqueous extractives of 500 gm. of veal, 5 gm. of commer- 
 cial salt, 15 gm. of agar-agar shreds, and 10 gm. of Witte peptone 
 per liter. The final reaction was 0.8 to 1.2 per cent N acid to phenol- 
 phthalein. This agar was tubed in 12 cc. amounts. For the pouring 
 of blood agar plates it was melted, placed in a water bath at 45°C. 
 for about IS minutes, two- thirds of a cubic centimeter of defibrinated 
 blood added and evenly mixed. The tube of medium was inocu- 
 lated at once with a loop or two of properly diluted 24 hour bouillon 
 culture and poured into a Petri dish 9 cm. in diameter. The medium 
 therefore contained 5 per cent of blood and formed a layer 2 mm. 
 
I 
 
 JAMES HOWARD BROWN 7 
 
 thick in the Petri dish. After the medium had solidified, the moisture 
 which otherwise would condense on the surface of the agar and on 
 the lid of the Petri dish was allowed to evaporate by placing the m- 
 verted dish in the position shown in Plate 1, Fig. 1. Allowed to 
 remain in this position for IS to 30 minutes in a room free from dis- 
 turbance the plates were rarely if ever contaminated. Broadhurst 
 (1913) and Hohnan (1914) have emphasized the importance of em- 
 ploying the most favorable possible medium for the determination of 
 fermentation reactions. It is not just to describe an organism as 
 unable to ferment a test substance when the failure is due to the un- 
 suitableness of the medium in which the substance is dissolved. 
 Bacillus coli will not ferment dextrose in physiological salt solution. 
 A medium should be used which will bring out the largest possible 
 quota of fermentation reactions. Holman (1914) describes a suit- 
 able medium as one "in which the organisms grow well independently 
 of the carbohydrate added". The work of Gordon (1905), Andrewes 
 and Border (1906), Winslow (1912), FuUer and Armstrong (1913), 
 and even of those using Hiss serum water is open to criticism from 
 this standpoint. For similar reasons there should be emphasized the 
 importance of using a blood agar that will reveal the maximum 
 amount of differentiation. It is for this reason that the medium and 
 proportions described above have been used. The importance of 
 this will be illustrated after the fundamental types in blood agar have, 
 been described. 
 
DESCRIPTION OF THE TYPES IN BLOOD AGAR. 
 
 Type a. — In Plate 15, Fig. 1 is the photograph of a horse blood 
 agar plate of an a type streptococcus at the end of approximately 48 
 hours incubation. This plate may be described as containing "a 
 good number of colonies well distributed." To the eye the deep 
 colonies appeared as small biconvex greenish colonies (greater di- 
 ameter, 0.8 mm.) surrounded by zones of 1.3 mm. diameter in which 
 the blood corpuscles were distinctly greenish in .color. Under the 
 low power of the microscope (7 to 8 diameters) the greenish zone was 
 less apparent, but in the outer portion of the zone there was possibly a 
 slight hemolysis which in the photograph (Fig. la) appears quite 
 distinct. It must be remembered, however, that red light is quite 
 inactive to the photographic plate while a greenish Hght is less so, 
 so that the actual amount of hemolysis was less than appears in the 
 photograph. The surface colonies on this plate were round, flatly 
 convex, glistening colonies, somewhat greenish by transmitted light, 
 grayish by reflected light, and with httle apparent alteration of the 
 surrounding medium. 
 
 The word "apparent" is used because as a matter of fact , there are invisible 
 exchanges between the colonies and the surrounding medium which exert a 
 decided influence on neighboring colonies of the same or different organisms. 
 It has often been observed that in pure cultures the colonies in a plate containing 
 only a few colonies wUl be much larger than in a plate containing many colonies 
 even though in the latter plate the colonies may be well distributed and well 
 separated from one another by several millimeters of apparently unaltered me- 
 dium. The ability of a streptococcus colony so to alter the surrounding agar as 
 to render it unsuitable for the growth of a so called surface spfeader is well shown in 
 Plate 18, Fig. 5. This blood agar plate at the end of 24 hours incubation con- 
 tained only streptococcus colonies (mostly deep) with indefinite partly hemo- 
 lyzed zones about them. When observed at the end of 48 hours incubation a 
 hemolytic surface spreader had appeared as a contamination rapidly spreading 
 over nearly half the plate (Plate 18, Fig. S), but in its progress neatly circum- 
 venting every one of the deep streptococcus colonies leaving islands of sterile 
 laked blood agar about 2 to 2.5 mm. in diameter above and around them. 
 
 The agat plate shown in Plate 15, Fig. 1 has been described as 
 containing "a good number of colonies well distributed." It con- 
 
JAMES HOWARD BROWN y 
 
 tained about 100 colonies and this is a good optimum. The colonies 
 are far enough apart to exert no apparent influence on one another 
 and are able to attain their full development unhindered. Even if 
 there were 200 colonies they might still be able to do so. If there 
 were many more than this, however, two effects would be noticeable: 
 (1) the colonies would be smaller, and (2) around each of them would 
 appear a more or less distinct zone of partial hemolysis. 
 
 These effects suggest the following explanation: (1) The metabolic products 
 formed by an organism are always detrimental to its growth. In a crowded 
 plate there is less opportunity for the diffusion of these products outward because 
 they are being formed throughout the plate and rapidly reach a concentration 
 which inhibits the growth of the colonies. (2) The hemolytic substance, what- 
 ever its nature, as it diffuses outward from the colonies must reach a certain 
 concentration before it can produce hemolysis. If the colonies are few, diffusion 
 into the surrounding medium takes place quite readily. If the plate is crowded 
 with colonies the diffusion of the hemolytic substan^ce from an individual colony 
 is soon checked by the concentration of the substance throughout the agar, 
 and a concentration in the immediate neighborhood of the colony is soon 
 attained sufficient to bring about hemolysis with the formation of zones. The fact 
 that notwithstanding the general diffusion of the hemolytic substance through- 
 out the medium the zones are fairly sharply defined is probably due to the fact 
 that the corpuscles act as an indicator wifh a sharp end-point; they lake abruptly 
 and simultaneously when a certain concentration of hemolytic substance is 
 reached forming a definite zone, though the concentration of the hemolytic sub- 
 stance within the zone may be only slightly above that outside the zone and the 
 slope from one to the other very gradual. 
 
 At the end of 48 hours incubation, then, streptococcus B-7a pro- 
 duced small deep colonies surrounded by small zones of somewhat 
 greenish discolored corpuscles with or without apparent partial hemo- 
 lysis, depending upon the number of colonies in the plate. A plate 
 similar to the one described had been in the incubator 48 hours. 
 Other work prevented its being examined then. Rather than being 
 allowed to remain in the incubator another night it was placed into 
 the refrigerator. Next morning all the colonies were hemolytic. The 
 experiment was repeated, this time making careful observations be- 
 fore and after refrigeration. The hemolysis occurred during refrig- 
 eration. DupUcate plates were made, one treated as before and the 
 other left in the incubator for 96 hours. The results were as follows: 
 
10 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 Plate 1 
 
 Plate 2 
 
 (48 hours at 37°C.) 
 
 Both plates alike; 
 no hemolysis. 
 
 (48 hours in refrigerator) 
 
 Distinct hemolyzed zones 
 in less than 24 hours. 
 
 (48 hours at 37°C.) 
 
 Colonies larger but no 
 hemolysis. 
 
 It was later observed that at room temperature (18 to 20°C.) some 
 hemolysis might occur while the plates were lying about on the 
 desk waiting to be examined. Htemolysis has been seen to appear in 
 30 to 45 minutes at room temperature. The lesson is obvious. A 
 strain may appear to be non-hemolytic at one time and hemolytic at 
 another unless care is taken to examine it under the same conditions 
 each time. Although Konr^d (1909) and Sigwart (1909) noted the 
 appearance of hemolysis about the colonies of green-producing strep- 
 tococci at room temperature and in the refrigerator, the fact has not 
 been appreciated by other authors. Authors who have considered 
 hemolysis on blood agar unreliable as a differential character because 
 variable may have encountered the above factors without recognizing 
 them (i.e., variations in appearance due to the number of colonies in 
 a plate and the time and temperature of observation). The hemo- 
 lysis occurring at lower temperatures is evidently not to be regarded 
 as an artefact for it does not occur in an uninoculated plate similarly 
 exposed after incubation, but does occur in definite relation to the 
 colonies of streptococci. It is quite as definitely characteristic of 
 certain strains of streptococci as is hemolysis under any, other con- 
 dition of temperature. I have therefore adopted as a routine pro- 
 cedure the incubation of blood agar plates for 48 hours (an observa- 
 tion being made at the end of 24 hours also) followed by refrigeration 
 for 24 hours or longer and observations again made. The 48 hour 
 period of incubation was adopted because while some strains reach 
 their full differential development in 24 hours, others do not. 
 
 After refrigeration for 48 hours the blood agar plate of strepto- 
 coccus B-7a first shown in Plate 15, Fig. 1 appeared as in Fig. 2 
 with small but definite hemolyzed zones around each deep colony 
 and a less well defined hemolysis about the surface colonies. Ex- 
 amined with a good simple lens or under the low power of the micro- 
 scope the deep colonies were seen to be surrounded by what may be 
 
, JAMES HOWARD BROWN 11 
 
 called "double zones" (Fig. 2a); i.e., composed of a distinct inner 
 collection of (in this case) greenish non-hemolyzed corpuscles next 
 to the coloiiy and an outer more or less hemolyzed ring (l.S mm. in 
 diameter in this case). By comparing Figs, la and 2a of the same 
 colony before and after refrigeration it appears that the inner col- 
 lection of greenish corpuscles has remained unchanged and unaffected 
 by the hemolysis during refrigeration. Apparently the greenish dis- 
 coloration of the corpuscles was accompanied by a fixation against 
 hemolysis for (l) it preceded hemolysis, (2) the hemolytic sub- 
 stance must have passed out from the colony through the collection 
 of corpuscles, and (3) it must have been present in greater concen- 
 tration among these corpuscles next to the colony than farther out 
 where hemolysis did occur. This subject will be developed farther in 
 a later chapter. As it had been noted that the zone of greenish cor- 
 puscles was formed during incubation and that hemolysis took place 
 during refrigeration, the blood agar plate was replaced into the incu- 
 bator for another period of 48 hours. There appeared outside the 
 hemolyzed zone another ring of greenish corpuscles not so apparent 
 macroscopically in Fig. 3 but easily seen microscopically in Fig. 3a, 
 a "triple zone." Still another exposure to the -temperature of the 
 refrigerator resulted in the formation of a second ring of partial hemo- 
 lysis, a "quadruple zone" easily seen macroscopically in Fig. 4 and 
 microscopically in Fig. 4a. Apparently the alternate throwing off of 
 rings of fixed corpuscles and of hemolysis in response to alternate 
 incubation and refrigeration is limited only by the exhaustion of the 
 medium and the accumulation of metabolic products which inhibit 
 further growth and activity. Saito (1912) observed a similar ring 
 formation but apparently did not detect its relation to temperature. 
 He says: 
 
 "ich will nicht unterlassen, darauf hinzuwei^en, dass ich hei dem Studium der 
 hamolytischen Flatten mancherlei sah, wofUr ich in der Literatur noch keine Erklar- 
 ung gefunden hdbe und was ich auch selbst nicht erklaren kann. Namentlich bei 
 Streptococcus acidi lactici konnte ich nicht selten beobachten, dass unter den gut 
 gewachsenen KuUuren die Blutkorperchen besser erhalten waren als in den hamoly- 
 tischen verwaschenen Bofen in der Umgebung. Bei meinem lanceolatus 4 und 5 
 habe ich einige Male etwas- sehr Merkwurdigesbeobachtet. Es war die Kultur von 
 einem hellen Hof umgeben, in dem eine vollstandige Zerstorung der rot'en Blutkor- 
 perchen stattgefunden hatte. Dann kam ein dunkler Ring, ein zweiter heller Ring, 
 
12 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 ein zweiter dunkler Ring, ein dritter heller Ring und dann der unveranderte Ndhr- 
 boden. Es isi sekr merkwurdig, dass ein von der Kultur in den Ndhrboden diffundi- 
 erender Stof nicht am starksien an seinem Entstehungsort, sondern in einiger Enl- 
 fernung wirkt, und dass sick Maxima und Minima der Wirkung abwechseln. Es 
 beweist dies, dass bei dem Vorgang der Edmolyse und Hdmoglobinzerstorung nock 
 Dinge miispielen, die sich nock der allgemeinen Kenntnis entziehen." 
 
 The concentric rings seen by Saito were in all probability the 
 record of his taking the plates from the incubator to room tempera- 
 ture for observation and of their subsequent reincubation. I have 
 seen as many as six concentric rings appear in some cases. 
 
 Little or nothing has been said about the surface colonies. The 
 surface colonies of streptococci on blood agar are of much less dif- 
 ferential value than the deep ones. Sigwart (1909) recognized this 
 fact and it has more recently been emphasized by Krumwiede and 
 Valentine (1915). A glance at Table III on The Use of Blood Agar 
 will show that with very few exceptions, however, authors have con- 
 fined their attention to surface growth, probably merely because it 
 was found easier to keep blood agar plates on hand ready made and 
 to smear or streak them with culture material than to inoculate them 
 in fluid condition. At any rate, there has been a great neglect of the 
 deep colony. 
 
 The surface colonies of streptococcus B-7a have already been de- 
 scribed as they appeared after 48 hours incubation (Plate 15, Fig. 1). 
 They were round, flatly convex, glistening colonies, somewhat green- 
 ish by transmitted hght, grayish by reflected Ught, with Httle or no 
 alteration of the surrounding medium. If there had been many 
 more colonies on the plate their appearance would have been much as 
 it was in this plate after refrigeration (Fig. 2) ; i.e., there would have 
 been a hazy ring of partial hemolysis about the surface colony. Taken 
 from the incubator at various times for examination there may de- 
 velop about the surface colonies zones similar to those in Fig. 4 
 and since the multiple zones of the surface colonies are much less dis- 
 tinct than those of the deep colonies the finer characters of the former 
 may pass imobserved. A close examination reveals a radical differ- 
 ence in the appearance of the zones of the two kinds of colonies. 
 The quadruple zones of the deep colonies are made up from the center 
 outward of a collection ,of corpuscles, a ring of hemolysis, a ring of 
 
JAMES HOWARD BROWN 13 
 
 * 
 
 corpuscles, and an outer ring of hemolysis. The zones of the surface 
 colonies on the same plate appear triple, beginning with a ring of 
 hemolysis next to the. colony, then a ring of corpuscles, and an outer 
 ring of hemolysis. This latter is the appearance described by Natvig 
 (1905) as " Type 11" (see Table III on The Use of Blood Agar) . He 
 says : 
 
 "77. Um die Kolonien hildete sick ein ca. 1 mm. breiter, farbloser, matter Hof, und 
 ausserhalb derselben ein ebenso breiter, griin gefdrbter Hof. Ab und zu war auch aussen 
 herum ein schmaler farbloser Saum zu sehen." 
 
 He states that only two of his strains produced this reaction and that it was 
 not always equally apparent. Evidently he did not recognize the factors influ- 
 encing it, though he offers the following explanation: 
 
 "Hier schien ein HUmolyse, tkeilweise mil vollstHndiger Zersetzung des Bltitfarbstoffes 
 {inner er Ring), tKeils mit einer Umbildung des Hdmoglobins zu grilnen Parbstoffen, vor 
 sich gegangen zu sein. Dagegen wwde eine AujlSsung der Blutkorperchen nur gelegentlich 
 und in geringem Grade bemerkt {der schmale farbhse Saum). Die Umbildung des Hdmo- 
 globins in griine Farbstoffe war offenbar nur ein Vorstadium einer voUstdndigen Decom- 
 position des Blutfarbstofes, denn wenn man die Entwicklung der Reaction verfolgte, beo- 
 bachtete man zuerst nur ein schwache graugriine Fdrbung der Kolonien selbst, spdter dann 
 einen grilnen Hof dicht an den Kolonien und nock spdter entfdrbten sich sowohl die Kolonien 
 als die dieselben ndchst umgebende Partie, wdhrend der griine Hof gleichsam nach aussen 
 verlegt war." 
 
 It would appear that Natvig's study of surface colonies only led him astray. 
 I have never witnessed the hemolysis of corpuscles after they have been dis- 
 colored green and am in doubt as to whether there may be slight encroachment 
 of hemolysis on the outer edges of the collection of greenish corpuscles where the 
 discoloration is very slight or may be due to only a few of the corpuscles being 
 discolored. What Natvig observed was probably this: He took the blood agar 
 plate from the incubator and noted the "griinen Hof dicht an den Kolonien." 
 The plate was reincubated and when again examined the colony was immedi- 
 ately surrounded by a ring of partial hemolysis and outside of this a second ring 
 of greenish corpuscles. Under these circumstances it is but natural that he 
 should have regarded the greenish discoloration as a "Vorstadium" of hemolysis, 
 but as a matter of fact the "griinen Hof first observed had not been hemolyzed; 
 it was merely concealed by the overgrowing surface colony. The hemolysis 
 occurred outside the first "griinen Hof." If he had scraped off the colony from 
 the agar he would have found the collection of green corpuscles beneath. The 
 substance of the colony scraped off woU|ld have been seen to be grayish in color; 
 it appeared grayish green at the time of the first observation because the light 
 by which it was seen passed through the green corpuscles lying beneath and 
 beyond the edges of the colony ; it appeared less green later because it had increased 
 
14 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 in size and substance while the collection of greenish corpuscles' beneath had not 
 increased in size because limited by the ring of hemolysis and consequent lack of 
 corpuscles surrounding it. I have many times observed the course of events 
 here described. Mandelbaum (1907-08) describes it also, though he describes 
 the discoloration as brownish rather than greenish, a difference due to differ- 
 ences in the media employed. He observed surface colonies and his description 
 fits my own observations perfectly if it is assumed that instead of taking the cover 
 off the Petri dish to observe the colonies from the top surface, he observed them 
 through the medium from the bottom of the dish, not an uncommon procedure. 
 He says: 
 
 "Die Kolonien des Milior dagegen zeigen niemals die Fdrbung und slruklurlose Form, 
 die man bei ihrem Wachstum auf gewohnlichem Agar zu sehen gewohnt ist. Die Parbe isl 
 tiefbraun bis braurtrot oder rostbraun, die Kolonien scheinen grab gekdrnt. Untersucht 
 man nun diese Kolonien mil dem Mikroskop, so entdeckt man, dass man von der eigentlichen 
 Kolonie gar nicht sieht, sondern nur die Form oder, besser gesagt, den Abdruck, der durch 
 die roten Blutkorperchen gebildet wird, die 'grobe Kornang' ist nichts anderes als eben die 
 dicht zusammenliegenden roten Blutkorperchen. Die Rand der Kolonie ist durch eine dunk- 
 lere Linie angedeutet. Ueber diese hinaus liegen noch auf eine kurze Strecke die unauf- 
 gelSsten roten Blutkorperchen, die dieselbe Farbe zeigen wie die unmittelbar unter der Kolonie 
 Uegenden. Dann folgt der helle, farblose Hof, in dem die roten Blutkorperchen nur noch als 
 'Schatten' zu erkennen sind. Man kann die eigentlujte graue Kolonie ruhig mil der 
 PlatinSse abtragen, ohne das mikroskopische Bild zu verSndern." 
 
 Mandelbaum's deductions from the above observations are hardly justified 
 but will be considered later (page 71). 
 
 Type /3. — This type of appearance in blood agar may be described 
 as a colony surrounded by a perfectly clear, colorless zone of hemo- 
 lysis. The blood agar plate of such a streptococcus after 48 hours 
 incubation is shown in Plate 6, Fig. 1. There is no trace of dis- 
 coloration and when viewed microscopically no corpuscles are seen in 
 the medium surrounding the colony (Fig. la). The colony itself is 
 grayish by transmitted or reflected light. The distinguishing char- 
 acteristics of this type appear much earlier than do those of the 
 alpha tj^e and are often quite well developed in 18 hours at incubator 
 temperature. In 48 hours the zones and colonies are a little larger 
 than at the end of 24 hours but no different qualitatively. The sur- 
 face colonies are surrounded by similar zones. 
 
 The characteristics of this type have been recognized by most authors since 
 Schottmiiller (1903) described them as distinguishing "Streptococcus longus 
 pathogenes seu erysipelatos," and although Streptococcus pyogenes was a name 
 originally employed by Rosenbach (1884) without reference to hemolytic char- 
 
JAMES HOWARD BROWN 15 
 
 acter, the source of his strains makes it altogether probable that most of them 
 were of this type. Neither did Rosenbach describe the fermentation reactions of 
 his strains, but since the great majority of so called hemolytic streptococci from 
 wound infections have befcn found to ferment dextrose, maltose, saccharose, 
 lactose, and salicin but not raffinose, mannite, or inulin, I believe it is justifiable 
 to restrict the name Streptococcus pyogenes to streptococci of the beta type in blood 
 agar and having the fermentative characters just mentioned. This has been 
 done by Andrewes and Horder (1906), if it may be assumed that by "hemolytic" 
 they meant only this most conspicuous type of hemolysis. Many authors, how- 
 ever, have used the name Streptococcus pyogenes with reference to either the 
 hemolytic or the fermentative character alone, both of which practices are full 
 of possibility of error, since not all streptococci of the beta type produce the 
 same fermentation reactions and many strains having the same fermentative 
 characters produce radically different appearances in blood agar. Streptococcus 
 hemolyticus and Streptococcus vulgaris are in so far as can be determined from the 
 literature synonymous with Streptococcus pyogenes, though they may be more 
 inclusive. 
 
 Having seen hemolysis take place more readily during refrigeration 
 than during incubation in the case of streptococci of the alpha type 
 one might expect the zones of the beta tj^je shown in Plate 6, Fig. 1, 
 to become much enlarged during refrigeration. As a matter of fact, 
 however, after refrigeration for 48 hours they showed no change 
 (compare Plate 6, Figs. "1 and 2), nor did they change during a 
 second period of incubation and refrigeration though colonies trans- 
 ferred to bouillon were in all cases viable. The difference in behavior 
 of the two types in respect to refrigeration is so great as to suggest 
 different hemolytic substances or processes. 
 
 Type a'.— In describing the beta type it was stated that the sur- 
 face colonies were surrounded by zones similar to those of the deep 
 colonies. However,, even in trpng to distinguish streptococci of the 
 beta type the employment of surface inoculation only can result in 
 failure to recognize the alpha prime tj^e which in so far as appear- 
 ance is concerned is intermediate between the alpha and beta typts. 
 In the paper by Smith and Brown (1915) there are included among 
 the beta strains two designated B-2 and B-6 respectively. They 
 differed from all the other beta type strains in fermenting rafl&nose 
 and were non-pathogenic when injected intravenously into rabbits. 
 These were among the first strains isolated and studied by us and 
 
16 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 our notes on them are less satisfactory than on strains isolated later. 
 Replating these strains many months later I found that they did not 
 produce the clear hemolyzed zones of the heta type but belonged to 
 what I have since called the alpha prime type. One of these strains 
 (B-6q;') is shown in Plate 10, Fig. 1, after 48 hours incubation. It 
 can scarcely be called anything but a hemolytic streptococcus for there 
 are distinct hemolyzed zones and no apparent discoloration. By 
 carefully comparing this figure with one of the heta type, however, 
 {e.g., Plate 6, Fig. 1) it is seen that the deep jcolonies of the alpha 
 prime type appear somewhat hazy or poorly defined within the 
 hemolyzed zones. Under the microscope (Plate 10, Fig. la) the 
 reason for this haziness is apparent; a few corpuscles are seen to re- 
 main throughout the zone but are most numerous next to the colony. 
 They were not noticeably discolored, possibly because not sufficiently 
 numerous to impart their color to the mass. On the other hand, the 
 zones of the surface colonies were practically like those of the surface 
 colonies of the beta type because the thin collection of corpuscles near 
 the colony was completely obscured by the overgrowing surface col- 
 ony. Different strains of the alpha prime type differ with respect to 
 the relative clearness and size of their zones, and strains of the beta 
 type differ also with respect to the size of their zones, so that the 
 surface colonies of one type may be quite indistinguishable from 
 those of the other type. The deep colonies must be studied micro- 
 scopically to differentiate them. Without doubt authors devoting 
 their attention to the appearance of surface growth only have in- 
 cluded many of these strains in the Streptococcus longus pathogenes 
 group and finding them avirulent for rabbits or coming from clini- 
 cally mild infections, as they often do, and perhaps finding that these 
 strains differ also in their fermentation reactions from other hemo- 
 lytic streptococci have concluded that hemolysis or feirmentation reac- 
 tions or both are of no significance with respect to virulence or 
 pathogenicity. (For a review of the question of the relation of viru- 
 lence to hemolysis, etc., see Table VIII on General Conclusions, etc.). 
 In all probability many of the strains described {e.g., Broadhurst, 
 1915; Rosenow, 1914; Thalmann, 1911; Sigwart, 1909; Fromme, 
 1908; Heynemann, 1908; Natvig, 1905) as producing a hazy hemolysis 
 were of the alpha prime type, but most authors have been content to 
 designate strains merely as hemolytic or non-hemolytic. 
 
JAMES HOWARD BROWN 17 
 
 Superficially there is considerable resemblance between the alpha 
 prime and beta appearances while quantitatively at least there is a 
 great difference between the alpha and alpha prime appearances. 
 Nevertheless I have indicated a relationship between the latter two 
 by calling one alpha and the other alpha prime. This relationship is 
 indicated not only by the presence' of fixed corpuscles next to the 
 colony but also by the response to refrigeration. After 48 hours 
 incubation the plate shown in Plate 10, Fig. 1, was refrigerated for 48 
 hours. The zones were noticeably enlarged as shown in Figs. 2 and 
 2a. Placed into the incubator for another period of 48 hours the 
 zones became still larger and rather diffuse but no multiple zones of 
 concentric rings were formed. The relationship to the alpha type was 
 clearly demonstrated by the appearance of the same strain in a rabbit 
 blood agar plate instead of horse blood agar. In the rabbit blood agar 
 plate treated in the same manner as the one just described the typical 
 alpha appearance was produced (Plate 10, Figs. 3, 3a, 4, and 4a). 
 After refrigeration there was seen a tj^ical double zone with a broad 
 collection of green corpuscles next to the colony. It must not be as- 
 sumed, however, that the alpha prime appearance is never produced in 
 rabbit blood agar. It has often been seen in rabbit blood agar and in 
 human' blood agar, but was not produced by this particular strain. 
 
 When after cultivation for more than a year it was discovered that 
 streptococci B-2 and B-6 no longer could be classed among those pro- 
 ducing the beta type of hemolysis, the idea was entertained that their 
 hemolytic character had changed. Searching through the notes 
 carefully, however, it was discovered that from the time of their 
 isolation these strains had produced in horse blood agar zones of 
 hemolysis described as slightly hazy or a little less clear than those 
 of other hemolytic streptococci. In all probability the hemolytic 
 character had not changed. 
 
 Type 7. — There remains at least one more type of appearance in 
 blood agar to be described. By the gamma type is meant the growth 
 of the streptococcus colonies within and on the blood agar plate 
 without the production of any perceptible hemolysis or discoloration 
 of the surrounding medium during incubation or refrigeration. A 
 streptococcus of this type is shown in Plate 2. It requires no de- 
 
18 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 scription. Streptococci causing this appearance in blood agar have 
 been described by Mandelbaum (1907-08) as Streptococcus sapro- 
 phyticus, by Zangemeister (1910) as Streptococcus anhemolyticus 
 vulgaris, and were found by Rosenow (1914) among those from the 
 joint fluid of cases of rheumatism. 
 
 Each of the above described blood agar types is fairly distinct 
 from the others so that on seeing a streptococcus colony for the first 
 time in a blood agar plate there is rarely any difiiculty in deciding to 
 which type it belongs. Incubation for 24 hours is usually sufficient 
 to determine the type though it is best to confirm the diagnosis by 
 incubating the plate for 48 hours and then refrigerating it. To in- 
 sure purity the strain should always be replated until colonies of 
 only one t3^e develop. In a mixed colony the hemolytic {beta) 
 type will completely obliterate the characters of the alpha or gamma 
 type even though cocci of the latter types may be present in the 
 colony in considerable numbers. This will be illustrated later 
 (page 64). (See also Hohnan (1914) in Table VII on Mutations and 
 Variability.) 
 
VARIOUS REPRESENTATIVES OF EACH TYPE. 
 
 Belonging to each of the above types are organisms of widely dif- 
 ferent characters in other respects and the appearances in blood agar 
 produced by different strains show quantitative differences within 
 each type except the gamma type, this latter t5^e being defined by 
 purely negative characters. Streptococci of this type, however, do 
 differ with respect to size of colonies, fermentation reactions, etc. 
 
 Type a. — In so far as the author has been able to observe, not 
 only do all green-producing and brown-producing streptococci belong 
 to this type but also the Pneumococcus and Streptococcus {Pneumo- 
 coccus) mucosus. 
 
 In hor^e blood agar streptococcus A-BLa (Plate 12) differs from 
 B-7a (Plate 15) described as the t5^e form, in producing a broader 
 central collection of greenish corpuscles surrounded after refrigera- 
 tion (compare Plates 12 and 15, Figs. 2 and 2a) by a broader ring of 
 partial hemolysis. The appearances produced by the two strains 
 are, however, essentially similar. A greater difference is apparent by 
 comparing Plate 12, Figs. 3 and 3a, with Plate 15, Figs. 4 and 4a, pho- 
 tographs taken after the second period of refrigeration. Both show 
 quadruple zones about the deep colonies, but in the case of streptococ- 
 cus A-BLa the second ring of corpuscles though distinct is quite 
 narrow and is composed of relatively few corpuscles. Streptococcus 
 D-4a (Plate 14) forms zones which after the second period of in- 
 cubation and refrigeration (Figs. 3, 3a, and 3b) are much like those 
 of A-BLa. The colonies themselves of D-ia, however,! though less 
 numerous, are noticeably smaller than those of A-BLa or B-7a. 
 The deep colony in Fig. 3a viewed from the edge and appear- 
 ing biconvex shows the quadruple zone less plainly than one (Fig. 
 3b) viewed from the broad side and appearing round. While 
 streptococcus D-4a resembles A-BLa after the second period of 
 refrigeration, streptococcus E-4a (Plate 17, Figs. 4 and 4a) resembles 
 B-7a more closely, the second corpuscle rings of the latter two being 
 
 19 
 
20 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 quite prominent. For some unknown reason, however, the periph- 
 eries of the quadruple zones of E-4a and A-BLa are surprisingly 
 sharply defined (especially that of E-ia), while those of B-7a and 
 D-4a blend more gradually with the surrounding medium. The 
 plates of these four strains just described were poured from four 
 tubes of the same lot of agar, contained the same amounts of the 
 same lot of horse blood, and were inoculated and poured one after 
 the other on the same day. They were incubated, examined, refrig-' 
 erated, and photographed just as nearly simultaneously as possible. 
 The four strains (Table I) were isolated from the throats of four 
 different individuals during four different epidemics of sore throat, 
 but in no case were they considered responsible for the patient's con- 
 dition or the epidemic. Two of the strains were isolated in 1914 and 
 had similar fermentation reactions (D-4a and E-4a;) but were least 
 ahke in other respects. The other two were isolated 10 months 
 previously. In blood agar their dissimilarities were much less con- 
 spicuous than their resemblances. All produced typical alpha type 
 2ones in horse blood agar. 
 
 The deep colonies of the four strains just described and in fact the 
 deep colonies of most bacteria in agar are circular biconvex or lenticu- 
 lar bodies. This shape is so common as to be of little differential 
 value and is probably largely determined by the natural cleavage of 
 the agar itself under pressure from the growing colony. If a deep 
 •dextrose agar shake culture of a gas-forming organism is examined 
 the gas bubbles in the agar are seen to be biconvex. Very small 
 colonies may be spherical and the degree of convexity of colonies 
 is not the same for all organisms or strains, but different deep colo- 
 nies of the same strain appear round, oval, or biconvex merely be- 
 cause of their position with respect to the surface of the plate from 
 which they are observed. An attempt to illustrate this by a pro- 
 jection drawmg is shown in Plate 1, Fig. 3. Three colonies of the 
 same size and shape are shown in the upper half of the figure as they 
 might appear viewed laterally. Projected into the lower half of 
 the figure are the same three colonies as viewed looking downward 
 from the surface of the plate. The colonies appear biconvex, oval 
 and round respectively. 
 
 In plates made from throat swabs there were occasionally found 
 
JAMES HOWARD BROWN 21 
 
 by Dr. Smith and myself a certain number of colonies which we called 
 complex to distinguish them from the usual biconvex colonies just 
 described. It was natural to suppose at first that these might be 
 accidental variations of the biconvex form, due possibly to the fusion 
 of colonies developing from two or more cocci close together in the 
 medium. It was discovered, however, that the formation of either 
 form of colony was "quite constant and characteristic for different strains." 
 Strains isolated from so called complex colonies over 2 years ago have 
 been replated and reexamined. Plain agar and blood agar plates of 
 two of these strains are shown in Plate 19. Figs. 1 and 2 are of 
 streptococci F-2a and D-la respectively, in horse blood agar after 
 48 hours incubation. In both figures the colonies are quite numerous 
 and the zones of the deep colonies are plainly double and of the alpha 
 type. Under the low power of the microscope the colonies were seen 
 to be of the same form as those of the same strains in plain agar plates 
 shown in Figs. 3 and 4. All of the deep colonies of streptococcus 
 F-2a (Figs. 1 and 3) were small irregular conglomerate clumps; none 
 was biconvex. Photomicrographs of three of these are shown in Figs. 
 3a, 3b, and 3c. The deep colonies of streptococcus D-la (Figs. 2 and 
 4) were quite different from the preceding; some were biconvex (Fig. 
 4c), some were described as "ruptured biconvex colonies" (Fig. 4a), 
 others were somewhat triangular with or without rupture (Fig. 4e), 
 and still others had the form of rather elaborate radiate structures 
 (Figs. 4b and 4d). The deep colonies of streptococcus F-2ai were all 
 asymmetrical; those of D-la always symmetrical. Searching back 
 through thd notes it is found that these characteristics, have been 
 noted from the time these strains were isolated over 2 years ago. 
 Both these strains formed in bouillon sediment difficult to disintegrate 
 by shaking. Microscopically the bouillon cultures were seen to be 
 composed of clumps of crooked chains. 
 
 The characteristics in bouillon agree with the descriptions of Streptococcus con- 
 glomeratus given by Kurth (1891) and by Thalmann (1910, 1911, and 1912). It 
 has been impossible to identify the two varieties shown in Plate 28 with the two 
 varieties {A and E) described by Thalmann. 
 
 Thalmann's description of Streptococcus longissimus led to the 
 recognition of similar strains in my collection. A study of one of 
 these (D-AD Saa) is shown in Plate 18. 
 
22 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 The chief characteristics ascribed to this organism by Thalmann (1910) are: 
 (1) the formation on "feuchtem, leicht alkalischem Fkischwasserglyzerinagar" of 
 delicate round colonies of irregular size and with irregular borders (thread-like 
 projections); (2) "Auf Blutagar-bildet der Streptococcus longissimus bei Zusatz 
 von frischem Blut nach 20-24 Stunden bei 37° flache, hell bis braunlich gefarbte, 
 meist nicht ganz regelmassig geformte Kolonien, die einen grUnlichen oder griin- 
 blauen Hof aufweisen. Die Hamolyse fehlt entweder vollkomen oder sie ist 
 gering. Bei vorhandener Hamolyse sind in der Regel einzelne Blutkorperchen im 
 hellefi Mof sichtbar, oft ist die Hamolyse ringformig, da die direkt auf den Kolonien 
 aufliegenden Blutkorperchen ihren Farbstoff nicht abgegeben haben;" (3) bouillon 
 is said not to be clouded but an abundant thready fleecy sediment is formed 
 which disintegrates fairly easily when shaken; (4) microscopically bouillon cul- 
 tures are said to present very long (crossing several microscopic fields) smooth 
 chains lying parallel in skeins, the elements being generally slightly ovoid and 
 weakly Gram-positive. The characteristics of streptococcus D-AD 830; agree with 
 this description in every respect. Thalmann apparently employed only surface 
 inoculation of his plates. 
 
 In Plate 18, Fig. 7, is shown a plain agar plate inoculated in fluid 
 condition and then streaked. Only the surface colonies interest us. 
 Some from the encircled area on the plate are shown in Figs. 7a and 
 7b under two powers of the microscope. The borders are irregular 
 and filamentous (Fig. 7b). In horse blood agar after a period of 48 
 hours incubation the most striking feature was the presence around 
 the deep colonies of colorless zones which macroscopically might 
 have been mistaken for zones of the beta type (Fig. 1). They were 
 somewhat cloudy, however, and microscopically (Fig. la) a small col- 
 lection of greenish brown corpuscles or pigment was seen close to the 
 colony. Under the microscope the outer hemolyzed portion of the 
 zone could never be mistaken for the beta type; it was in fact much less 
 distinct microscopically than macroscopically, and was quite diffuse, 
 inany unhemolyzed corpuscles remaining within it. After refrigera- 
 tion the hemolysis about the deep colonies was much more distinct 
 (Figs. 2 and 2a). If we confine our attention to surface colonies as 
 did Thalmann, we find that his description is quite applicable — "Die 
 Hamolyse fehlt entweder vollkommen oder sie ist gering." Macro- 
 scopically, even after refrigeration, only a faint partly hemolyzed 
 border was seen around the surface colonies and along the edges of 
 the streaks. Microscopically it was hardly visible. By transmitted 
 light the streaks were distinctly green, due to the greenish discolored 
 
JAMES HOWARD BROWN * 23 
 
 corpuscles which lay beneath the growth and extended nearly 1 mm. 
 beyond its edges. Macroscopically the appearance of the deep colo- 
 nies was that of the alpha prime typt in horse blood agar. However, 
 the greenish discoloration visible macroscopically under the surface 
 growth and microscopically about the deep colonies causes this strain 
 to be placed among those which produce the alpha type of zones in 
 blood agar. 
 
 The strains above described producing the alpha type of appear- 
 ance have all had certain characteristics in common (Table I) . None 
 of them produced capsules; none was soluble in bile or sodium tauro- 
 cholate; and none fermented inulin. For comparison with the above 
 there have been studied a number of strain's of Streptococcus {Pneu- 
 mococcus) mucosus and of pneumococcus. These strains were all 
 encapsulated under suitable conditions, all were soluble in sodium 
 taurocholate, and all fermented inulin. In blood agar all produced 
 the alpha type of appearance. 
 
 Park and Williams (190S) and others have given reason for regarding the 
 Streptococcus mucosus of Schottmiiller as a pneumococcus rather than a strepto- 
 coccus. It is in fact the Type III pneumococcus of Cole. The name Strepto- 
 coccus mucosus was originally applied by Howard and Perkins (1901) to an or- 
 ganism isolated by them from a case of peritonitis resulting from a tubo-ovarian 
 abscess. These authors did not employ blood agar or blood bouillon, did not 
 observe the action of bile on their culture, and did not study the reaction toward 
 inulin. From their description, however, it is altogether likely that their organ- 
 ism was either identical with the Streptococcus mucosus later described by Schott- 
 miiller (1903) or may have been a strain of Streptococcus epidemicus (Davis, 
 1912). These two organisms are remarkably similar morphologically and cul- 
 turally but differ in respect to fermentation of inulin, solubility in bile, and type 
 of appearance in blood agar, the former producing the alpha and the latter the 
 beta appearance. The one might not inappropriately be called Pneumococcus 
 mucosus and the other Streptococcus mucosus if the latter name had not already 
 become so firmly associated with the pneumococcus-Uke organism. For many 
 years (since 1903) European literature has applied the name Streptococcus mucosus 
 to the organism described by Schottmiiller and known in this country as Pneu- 
 mococcus mucosus or Pneumococcus Type III. Apparently Streptococcus epidemicus 
 has not been encountered or recognized in Europe. Under these conditions it 
 would seem a mistake to try to transfer the name Streptococcus mucosus to the 
 latter organism. It would seem much better to continue to use the names 
 Pneumococcus mucosus and Streptococcus mucosus synonymously, just as Pneumo- 
 coccus, Streptococcus pneumonia, and Diplococcus lanceolatus are used. 
 
24 ■ BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 Streptococcus mucosus Brig.-IIaa; in horse blood agar differed from 
 the streptococci above described most conspicuously in the size of 
 the colonies. The deep colonies (Plate 20)^ were biconvex discs over 
 a millimeter in diameter and because of their size had a marked 
 tendency to find the top or bottom surface of the agar, especially 
 during the second period of incubation (Fig. 3) . The surface colonies 
 were even more distinctive. Instead of being rather small round 
 greenish colonies as seen by transmitted light after the first period of 
 incubation, they were quite large (many of them several millimeters 
 in diameter), roundish, glistening, almost transparent "tearful" colo- 
 nies which when first observed were quite convex but soon dried down 
 to flat transparent films (Fig. 1). Beneath and close to the surface 
 colonies were areas of pale greenish discoloration accompanied by 
 lightening of the medium (partial hemolysis?). The typical deep 
 colony, even before refrigeration was surrounded by a distinct partly 
 hemolyzed zone within which near the colony was a good collection 
 of pale greenish corpuscles (Fig. la). This zone might be called 
 double except for a faint narrow second ring of corpuscles which is 
 explained as follows: This culture was very sensitive to the lowering 
 of temperature so that during the process of examination at room 
 temperature the outer hemolyzed zone was seen to increase in size. 
 This was after 48 hours incubation, but the plate had also been ex- 
 amined after 24 hours incubation at which time the zones consisted 
 merely of light greenish, partly hemolyzed rings which, according to 
 measurements then taken, extended just to where the faint second 
 ring of corpuscles later appeared. The removal of the plate from the 
 incubator at the end of 24 hours was therefore recorded by a slight 
 hemolysis occurring during this observation followed by a little 
 more greenish discoloration and fixation of corpuscles after replace- 
 ment in the incubator. After refrigeration (Figs. 2 and 2a) the 
 outer hemolyzed zone was still larger. Figs. 3 and 3a were made 
 after a second period of incubation and refrigeration during which 
 the colony became much larger so as to encroach upon the central 
 collection of fixed corpuscles and to extend almost to the second ring 
 
 ' The plate described and photographed was of agar made with beef infusion 
 and Digestive Ferments Company peptone, but was apparently like a plate made 
 with veal infusion agar and Witte peptone. 
 
JAMES HOWARD BROWN 25 
 
 which at this time appeared darker, whether because of any alteration 
 in it or because of some optical effect of its closer proximity to the 
 colony I am unable to say. During the last period of incubation the 
 hemolysis had continued to extend outward but a ring of fixed cor- 
 puscles did not appear. 
 
 Pneumococcus Cole I 109 (Plate 21) in horse blood agar^ differed 
 from streptococcus Brig.-IIaa in fprming smaller colonies, especially 
 the surface colonies being smaller, less mucoid, and less transparent. 
 The hemolyzed zones were less influenced by low temperatures so 
 that at no time did they reach the size of those of Brig.-IIaa. After 
 the first period of refrigeration (Figs. 2 and 2a) both surface and deep 
 colonies had distinctly double zones, the central collection of cor- 
 puscles being distinctly greenish. After reincubation for 24 hours 
 there was little visible change (Figs. 3 and 3a) but the effect of this 
 incubation was revealed by the subsequent refrigeration during which 
 there appeared quadruple zones, very distinct about some of the deep 
 colonies (Fig. 4") and less so about others (Fig. 4a). The quadruple 
 zone was also apparent about the bottom surface colony (Fig. 4™) 
 but the zones of the top surface colonies were apparently double (Fig. 
 4") if viewed microscopically. Macroscopically these latter would 
 probably be described as being greenish by transmitted light and 
 surrounded by a narrow ring of partial hemolysis. 
 
 Pneumococcus Cole II 45 in horse blood agar (Plate 24)' pro- 
 duced essentially the same appearance as pneumococcus Cole I 109. 
 The most apparent difference was a more intense' green color, the dis- 
 colored corpuscles next to the colony quite obscuring the outline of the 
 deep colony itself (Figs, la and 2a) . At the end of 24 hours incuba- 
 tion the deep colonies were surrounded by simple zones of greenish 
 discoloration, apparently partly hemolyzed, but at the end of 48 hours 
 incubation the zones were distinctly double (Figs. 1 and la). The 
 effect of refrigeration (Figs. 2 and 2a) was not to intensify the hemo- 
 lysis but rather to render it more diffuse. The surface colonies showed 
 only a faint indefinite ring of hemolysis. They were flat, roundish, 
 
 ^ Made with beef infusion and Digestive Ferments Company peptone. 
 
 ' The plate photographed contained veal infusion — Witte peptone agar but 
 was apparently like one containing beef infusion — Witte peptone agar. The 
 result of using Digestive Ferments Company peptone will be described later. 
 
26 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 and green by transmitted light, but by scraping off the substance of 
 one of them it was seen to be gray, the green color by transmitted 
 light evidently being due to the discolored corpuscles beneath the 
 colony. 
 
 There is seen to be much resemblance between the growth in horse 
 blood agar of the Streptococcus mucosus and the two pneimiococci 
 just described. Other strains that I have studied resemble them. 
 They produce larger colonies than do the non-encapsulated alpha 
 type streptococci. As distinguished from the latter their deep hemo- 
 lytic colonies are more hemolytic, producing distinct double zones in 
 48 hours at 37°C. even when there are few colonies in the plate, while 
 their surface colonies are if anything less hemolytic than those of the 
 other streptococci. The latter fact probably accounts for the prev- 
 alent idea that pneimiococci are non-hemolytic. 
 
 Type a.'. — Streptococcus B-6a has been described as producing the 
 alpha prime type of appearance in horse blood agar and streptococcus 
 D-AD 82a has been referred to as approaching this type. In Plate 11 
 is shown another strain (streptococcus F-2a.') which produced zones 
 essentially like those of B-6q;'. The hemolytic activity of the former, 
 however, was somewhat the greater and was especially noticeable 
 after the second period of incubation. At this time the zones of 
 F-2a' were 5 to 6 mm. in diameter and perfectly clear except for the 
 small central collection of corpuscles visible microscopically. The 
 colonies themselves had also increased in size during the last in- 
 cubation. 
 
 Type /3. — In Plates 6, 7, and 9 are shown three strains of strep- 
 tococci producing clear hemolyzed zones without corpuscles remain- 
 ing next to the colony, the beta type. Of these the growth of A-ST/3 
 in horse blood agar has been described. In this medium strepto- 
 coccus D-AD4;8 (Plate 7, Figs. 1 and 2) was indistinguishable from 
 A-ST/3. Both strains were from human sources, one from a throat 
 and the other from peritoneal pus, but they differed in their fermenta- 
 tion reactions (Table I).^ The deep colonies and hemolyzed zones of 
 
 * Only a few strains of streptococci similar to A-ST/3 have been described. It 
 is a non-encapsulated, ^ hemolytic streptococcus, differing from 5. pyogenes (e.g., 
 
JAMES HOWARD BROWN 27 
 
 a third streptococcus, B-2b (cow)|3 (Plate 9), were also like the pre- 
 ceding except that the surface colonies of the latter were slightly 
 larger and possibly a little more moist in appearance. In the photo- 
 graphs, however, these three strains are hardly distinguishable from 
 one another. Streptococcus B-2b (cow)|8 was isolated from the udder 
 of a gargety cow in a dairy herd which from epidemiological data was 
 suspected of being responsible for a sore throat epidemic at Canton, 
 Mass., May, 1913 (Smith and Brown, 1915). It was studied in 
 comparison with strains from the epidemics of Chicago (1912), Bal- 
 timore (1912), and Boston (1911) and found to be similar to them 
 in all respects. This is the variety of streptococcus first carefully 
 described by Davis and Rosenow (1912) and later called "Strepto- 
 coccus epidemicus" by Davis (1912 and 1913). Some of the strains 
 available for comparison came directly from Davis and he had exam- 
 ined the strain from the Boston epidemic sent to him from this 
 laboratory. With regard to hemolysis, the descriptions of Davis and 
 Rosenow correspond very well with my observations of surface 
 colonies. The occasional greenish tint described by them will be con- 
 sidered later. In the case of the strain here described capsules had 
 not until recently been noticed. It is stated by Davis (1912; see 
 Table VIII on General Conclusions, etc.) to be frequently lacking. 
 If, however, some of the moist surface growth from a blood agar 
 plate (incubated barely over night) is suspended in a suitable india 
 ink, covered with a cover-slip, and examined at once, the capsules are 
 readily seen. They have been found to be present on all the strains 
 of Streptococcus epidemicus in my possession even after several years 
 of cultivation. The capsules of some strains are extremely perishable, 
 being present in young surface colonies only, and rapidly disappearing 
 
 D-AD4/3) in fermenting mannite but not lactose. Such streptococci were de- 
 scribed by Smith and Brown (1915) as the cause of an epidemic at Norton, Mass. 
 Holman (1916) has described a similar strain and in his classification of strepto- 
 cocci has grouped these together as S. hemolyticus II to distinguish them from 
 other hemolytic streptococci. It would seem to us that since this streptococcus 
 was found to be the cause of an epidemic, since the characteristics of our strains 
 have remained constant for several years, and since these characters are such as 
 to form one of the major groups in Table II, it may be justifiable to propose a 
 name for this group — Streptococcus alactosus /3 (Smith and Brown). 
 
28 BLOOD AGAR POR STUDY OF STREPTOCOCCI 
 
 after being suspended in india ink. The other characters tabulated 
 by Davis (1913; Table VIII on General Conclusions, etc.) for the dif- 
 ferentiation of Streptococcus epidemicus from Streptococcus hemolyt- 
 icus are quantitative and hardly sufficient for diagnosis. The growth 
 on an agar or blood agar slant or in a streak on a blood agar plate, 
 taken in conjunction with a knowledge of the hemolytic' type in blood 
 agar plates, is the hiost constant and reliable means of differentiating 
 these two hemolytic streptococci. In Plate 1, Fig. 2, is a drawing of 
 three agar slants {a, b, and c). These slants were inoculated by 
 placing one small loop of 24 hour bouillon culture of streptococcus 
 into the condensation fluid of the slant and then streaking the in- 
 oculated fluid upward along the slant. The tubes were incubated 
 over night in a standing position in the moist atmosphere of the 
 incubator. At the end of this time, strains of Streptococcus epidemicus 
 and of Streptococcus mucosus present the appearance shown on slant a 
 of the figure. The growth on slant a is composed of flat, almost trans- 
 parent, confluent, amebiform patches, small or large depending upon 
 the number of organisms carried up onto the slant. The streak 
 across the surface of a freshly poured blood agar plate exhibits the 
 same characteristics even more conspicuously. Streptococcus mucosus 
 is easily differentiated from Streptococcus epidemicus because the 
 former produces the alpha and the latter the beta type of hemolysis 
 in blood agar. The growth of Streptococcus mucosus on the slant 
 may be somewhat more raised or mucoid than that of Streptococcus 
 epidemicus but that does not alter the ameboid form of the growth. 
 If the upper part of the slant is somewhat dry the growth there may 
 take the form of small discrete colonies as shown on slant b of the 
 drawing, but subinoculations from such colonies into bouillon and 
 then onto suitably moist slants again reveal the characteristic 
 amebiform patches. Similar slants or plates inoculated with other 
 streptococci (including Streptococcus pyogenes) develop streaks of 
 small, round, convex colonies, discrete unless crowded, as shown in 
 slant c of the drawing. The pneumococcus often develops larger and 
 flatter round colonies which if crowded may be confluent but quite 
 distinct from the ameboid forms shown by Streptococcus (Pneumo- 
 coccus) mucosus and Streptococcus epidemicus. Slants inoculated too 
 heavily with any of the streptococci or pneumococci develop more 
 
JAMES HOWARD BROWN 29 
 
 or less homogeneous film-like streaks without differential character. 
 Ten or eleven strains of Streptococcus epidemicus in cultivation since 
 1911, 1912, and 1913 have up to the present time shown no change 
 with regard to the above characteristic. 
 
 During tonsillitis epidemics A and B described by Smith and 
 Brown (1915), milk from the cows of suspected dairies was examined 
 first microscopically, then those samples which revealed large num- 
 bers of leukocytes were studied culturally. There was mastitis in 
 both herds. From one cow of one herd there was isolated a hemo- 
 lytic streptococcus like those isolated from patients in epidemic B. 
 This streptococcus was B-2b (cow)/3. From cows of each herd there 
 were, also isolated bovine hemolytic streptococci which differed in 
 important respects {e.g., fermentation reactions, pathogenicity for 
 rabbits, and agglutination) from those of human origin. The bovine 
 streptococci from both herds had the same fermentation reactions 
 and showed some agglutinative relationship, but exhibited somewhat 
 different appearances in blood agar which will now be described. 
 
 Streptococcus B-18 (cow)/3 is representative of those isolated from 
 herd B suspected in connection with epidemic B. Small colorless 
 hemolyzed zones about the deep colonies in horse blood agar plates 
 were visible after incubation for 24 hours, while after 48 hours these 
 zones were about 3 mm. in diameter (Plate 5, Fig. 1). There were 
 no corpuscles remaining next to the colony (Fig. la) and no discolora- 
 tion. These zones were therefore of the beta type and in fact differed 
 in appearance from those of strieptococcus A-ST/3, D-AD4|3, and 
 B-2b (cow)/3 only in being slightly smaller and less sharply defined 
 at their outer borders. These zones increased slightly in size during 
 refrigeration (Figs. 2 and 2a). 
 
 Streptococcus A-18 (cow)/3 is representative of those isolated from 
 herd A suspected in connection with epidemic A. Usually in horse 
 blood agar this streptococcus had the following appearance: After 
 incubation for 48 hours the deep colony (Plate 4, Fig. 3) is surrounded 
 by a small (2 to 2.5 mm. in diameter), clear, colorless, sharply de- 
 fined hemolyzed zone, free of corpuscles or discoloration and there- 
 fore of the beta type. Outside this zone which I will call the central 
 zone is a broad (about 1 cm. in diameter), translucent, partly hemo- 
 lyzed, well defined zone containing many apparently unaltered cor- 
 
30 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 puscles. There is no discoloration. The area within the outer zone 
 but outside the inner zone is noticeably darker than the outer por- 
 tion of the outer zone. This dark area was less apparent to the eye 
 than it is in the photograph and I think it is due to the diffusion of 
 free hemoglobin outward from the inner completely hemolyzed zone. 
 A similar shading of the medium is apparent around the zones of 
 other beta hemolytic streptococci (e.g., Plates 6, 7, and 9). The 
 outer partly hemolyzed zones of streptococcus A-18 (cow)/3 were in- 
 fluenced by inconspicuous factors which are not fully understood. 
 For instance, the plate shown in Figs. 1 and 2 was inoculated just 
 6 days before the one shown in Figs. 3 and 4; both were made of agar 
 of similar composition having titratable acidities of 1.2 and 1.1 per 
 cent N acid respectively; both contained similar amounts of the same 
 lot of horse blood; yet the outer zones in Figs. 1 and 2 are hardly 
 visible. They were more readily seen in the agar plate itself. In 
 this instance the difference between the two agar plates was prob- 
 ably due to the difference of 6 days in the age of the blood used. 
 The blood was kept without apparent alteration in the refrigerator 
 in the interval, but had been drawn only 24 hours when used for the 
 plate shown in Figs. 1 and 2. Little or no change was produced in 
 the appearance of the zones of this streptococcus by refrigeration. 
 
 The broad outer zones produced by streptococcus A-18 (cow) /3 
 can hardly be considered due to acids produced since they were 
 even more conspicuous in a medium of fermented veal infusion agar 
 and washed horse blood corpuscles (Plate 4, Fig. 6). Streptococ- 
 cus B-18 (cow) |8, on the other hand, did not produce similar zones 
 in this medium. 
 
 In the descriptions. given above quotations and citations from other authors 
 have been avoided. For these the reader is referred to Table III on The Use of 
 Blood Agar and- especially to the work of the following authors: Schottmuller 
 (1903); Rosenow (1904); Kerner (1905) ; Natvig (1905); Ruediger (1906); Boxer 
 (1906); Mandelbaum (1907-08); Konrid (1909); Sigwart (1909); Thalmann 
 (1910); Saito (1912); Davis and Rosenow (1912); Davis (1912) ; Ruediger (1912); 
 Rosenow (1914); Baerthlein (1914); Smith and Brown (1915); Krumwiede and 
 Valentine (1915); Davis (1916). 
 
INFLUENCE OF THE AMOUNT OF BLOOD AND OF AGAR USED. 
 
 As stated above, the use of 12 cc. of standard meat infusion agar 
 plus 5 per cent of blood was found to give the maximum amount of 
 difEerentiation. Schottmiiller (1903) used 2 cc. of blood to 5 cc. of 
 agar. There is nothing in his paper to indicate what size the Petri 
 dish he used may have been. The size most commonly used is 8.5 to 
 9.0 cm. in diameter. In such a plate 7 cc. of blood agar would make 
 a layer only 1.2 mm. thick and because of the usual uneveimess of the 
 bottoms of these plates this amount of medium does not make a satis- 
 factory plate. Furthermore, a layer so thin dries out very quickly and 
 is not a favorable medium. From the descriptions of Schottmiiller 
 it appears more likely that he used a smaller Petri dish (7 to 7.5 cm. in 
 diameter) which is also on the market. 7 cc. of blood agar in this 
 dish would make a layer 1.7 to 1.8 mm. thick. The layer of agar in 
 Schottmiiller's plates was therefore none too thick, and it is prob- 
 ably for this reason that he obtained good results in spite of the 
 use of such a large proportion of blood. Unfortunately most authors 
 have not given sufficient data to enable one to determine how thick 
 the layer of agar in their plates may have been. Many of them 
 have not even stated the proportion of blood to agar used (see Table 
 III on The Use of Blood Agar). That the thickness of the layer 
 of medium is important is obvious from the following considerations: 
 If a colony is able to produce a clear hemolyzed zone 2 mm. in diame- 
 ter, this zone will barely penetrate the layer of medium 2 mm. thick 
 provided the colony is situated in the center of the layer of agar. 
 The zone of a surface colony, however, is hemispherical and if it has 
 a radius of less than the thickness of the layer of agar it will not 
 appear clear because of the underlying non-hemolyzed corpuscles. 
 Furthermore it is only the outer portion of the zone of a surface 
 colony which is visible because the central portion is obscured by the 
 overgrowth of the colony. If the zone of a deep colony has a diame- 
 ter less than the thickness of the layer of agar, it will also appear 
 clouded because it fails to penetrate the layer of medium. This 
 source of error is largely overcome by examining the colonies and 
 
 31 
 
32 BLOOD AGAR POR STUDY OF STREPTOCOCCI 
 
 zones microscopically because by focusing down onto the deep colony 
 theffe is obtained an optical section at the level of the colony. Few 
 authors have done this. 
 
 In Plate 6, Fig. 3, is shown a 48 hour horse blood agar plate cul- 
 ture of streptococcus A-ST/3. This plate contained 5 per cent of 
 horse blood in agar forming a layer 2 mm. thick. The clear colorless 
 zones of the deep colonies are well above 2 mm. in diameter and are 
 shown in the figure in marked contrast with the surrounding medium. 
 In Fig. 6 is shown the same organism in the same agar containing only 
 2.5 per cent of the same horse blood, but in a layer of medium 4 mm. 
 thick. Note the rather clouded appearance of the zones and the 
 poorer contrast between zones and surrounding medium. In Fig. 7 
 is shown the same organism in the same agar containing 5 per cent 
 of the same horse blood in a layer of medium 4 mm. thick. Note 
 that only one of the deep colonies shown has a fairly clear zone be- 
 cause of its advantageous position in the center of the layer of blood 
 agar; all other colonies located nearer one surface or the other have 
 quite clouded zones. In Fig. 5 is shown an attempt to photograph 
 a culture of the same dilution of the same organism in the same agar 
 containing 25 per cent of the same horse blood forming a layer of 
 medium 2.6 mm. thick. (SchottmuUer used 28.5 per cent of blood.) 
 This plate contained about the same number of colonies as did the 
 plates shown in Figs. 3, 6, and 7 and each colony was surrounded 
 by a laked zone of the same size as those in the other plates, but the 
 zones were quite red with the released hemoglobin. The plate was 
 therefore quite opaque to. the photographic plate. Naturally these 
 remarks regarding the most hemolytic organisms, the beta type strepto- 
 cocci, apply- even more forcibly to those of the alpha type. Many 
 of the details which have been described for the zones of the latter 
 would have been completely concealed by the use of a thicker layer 
 of medium or by a larger proportion of blood, and would have been 
 lacking altogether in plates containing much smaller amounts of 
 blood. A comparison of Figs. 4 and 5 with 6 and 7 of Plate 12 shows 
 the appearance of a culture of streptococcus A-BLa in the same agar 
 containing 5 and 10 per cent respectively of the same horse blood. 
 Both plates were subjected to the same treatment, namely 48 hours 
 incubation (Figs. 4 and 6) followed by 48 hours refrigeration (Figs. 
 5 and 7). 
 
JAMES HOWARD BROWN 33^ 
 
 About 5 per cent of blood in a layer of agar about 2 mm. thick 
 appears to be the optimum. 
 
 A review of the literature indicates that this conclusion is justified. Schott- 
 miiller (1903) using blood agar (2 : 5) makes no mention of the finer characters of 
 the zones produced by streptococci. He states that none except Streptococcus 
 longus pathogenes produced macroscopicaUy visible hemolysis. I think he would 
 undoubtedly have included some of the alpha prime zone producers under Strep- 
 tococcus longus pathogenes. Rieke (1904) tried two extremes of blood agar mix- 
 tures, 2 : S parts and 5 drops of blood to S cc. of agar. On the former his Strep- 
 tococcus mitior strains produced no hemolysis, while on the latter they all pro- 
 duced hemolysis but only one of them produced any perceptible greenish tint. 
 Natvig (1905) found blood agar 2:5 of less differential value than 12 drops of 
 blood to 10 cc. of agar. His observations were quite accurate for surface colo- 
 nies. To continue to point out the results obtained by each author who has 
 stated how much blood and agar he used would be a useless repetition of state- 
 ments to be found in Table HI on The Use of Blood Agar. Attention is called 
 to the work of the- following authors as reviewed in that table: SchottmiiUer 
 (1903); Rosenow (1904); Rieke (1904); Natvig (1905); Boxer (1906); Silber- 
 strom (1906); Mandelbaum (1907-08); Zangemeister (1909); Hoessli (1910); 
 Thalmann (1910) ; LeBlanc (1912); Saito (1912) ; Rosenow (1914); Kligler (1915); 
 Holman (1916). 
 
INFLUENCE OF THE AGE AND THE KIND OF BLOOD USED. 
 
 Until now our attention has been confined almost wholly to the ap- 
 pearances produced by streptococci and pneumococci in blood agar 
 made with defibrinated horse blood. Although a good number of 
 workers have used horse blood for this purpose, the' majority have 
 not. In this laboratory it was available, freshly drawn once or 
 twice each week. The blood used, however, was often 2 weeks old, 
 and with the exception of the difference noted above in the case of 
 streptococcus A-18 (cow)i3 no difference in the results obtained by 
 using blood 24 hours old or after being kept in the refrigerator for 
 as long as 2 weeks has been noticed. IVfost authors who have made 
 any statement on the subject, have used rabbit or human blood. It 
 was important, therefore, to make parallel series of experiments 
 using each of these kinds of blood. In the plates are shown com- 
 parable figures of horse, rabbit, and human blood agar plates sub- 
 jected to alternate periods of incubation and refrigeration. A 
 description of each of the figures would be tedious. Remarks will 
 be confined to a general statement and a description of any marked 
 variations in appearance that may have resulted from the use of 
 other than horse blood. 
 
 In general it may be said that, excluding the alpha prime type, 
 there was never any danger of confusion of the other three types 
 {alpha, beta, and gamma) by the use of horse, rabbit, or human blood 
 . agar provided the criteria above described were searched for. T|he 
 differences in appearance due to the use of different kinds of blood 
 were only quantitative. The exception in the case of the two alpha 
 prime strains (B-6a:' and F-2a') has been mentioned above (page 17). 
 In rabbit blood agar both these strains produced the alpha appfear- 
 ance (compare Plate 10, Figs. 3 and 4 with Plate 11, Figs. 3 and 4). 
 As already mentioned, this does not mean that the alpha prime ap- 
 pearance is never produ,ced in rabbit blood agar, but it occurs less* 
 frequently than in horse blood agar. It has also been seen in cat 
 blood agar. In human blood agar streptococci B-6a' and F-2q;' 
 produced zones which after 24 hours incubation had the typical 
 
 34 
 
JAMES HOWARD BROWN 35 
 
 alpha prime appearance, and in fact after 48 hours incubation (Plate 
 10, Fig. 5 and Plate 11, Fig. S) resembled the alpha prime type rather 
 than thp typical alpha type, though under the microscope a slight, 
 almost doubtful greenish discoloration of the corpuscles next to the 
 deep colony could be seen. Tiere was hardly any change during re- 
 frigeration (Plate 10, Fig. 6 and Plate 11, Fig. 6) and the zones 
 became larger (especially those of F-2a') during reincubation. The 
 appearance of thesp two strains in human blood agar, therefore, re- 
 sembled that in horse blood agar more closely than that in rabbit 
 blood agar. The most notable difference between th§ human blood 
 agar plates and the horse blood agar plates was an indefinite milky 
 clouding of the hemolyzed zones in the human blood agar. This is 
 apparent in the photographs. The point of greatest interest in con- 
 nection with these two strains is that relying upon the usual macro- 
 scopic observation either of these strains might be called "hemo- 
 lytic" in horse or human blood agar and " non-hemolytic" or "green- 
 producing" in rabbit blood agar. This shows to how much confusion 
 the use of different kinds of blood has led in the hands of different 
 workers or of the same worker at different times, and how important 
 it is for an author to state what kind of blood he has usedi 
 
 Plate IS shows some striking quantitative differences in the ap- 
 pearance of streptococcus B-7a on plates made of the same lot of 
 agar thoughout but with horse, rabbit, and human blood. The 
 blood was 24 hours old in each case. There were about the same 
 number of colonies in each plate. All three plates were subjected to 
 the same alternate periods of incubation and refrigeration. After 48 
 hours incubation (Figs. 1, 5, and 9), the three plates were almost 
 identical in appearance, and after 48 hours refrigeration the only 
 notable difference was in the amount of hemolysis, which was great- 
 ■ est in the horse blood plate (Fig. 2) and least in the human blood 
 plate (Fig. 10), while in the latter plate the greenish discoloration 
 was most pronounced. The himian blood plate was practically un- 
 influenced by further incubation and refrigeration (Fig. 11), differing 
 in this respect from both the others. The formation of distinct 
 quadruple zones in the horse blood agar plate (Fig. 4) has been de- 
 scribed (page 11). Reincubation of the rabbit bipod agar plate re- 
 sulted in a phenomenon not described above. The discoloration be- 
 
36 BLOOD AGAR FOR STXJDY OF STREPTOCOCCI 
 
 came a brownish green and appeared to extend outward from the 
 colony ahnost obliterating the ring of partial hemolysis which had 
 appeared during refrigeration (Fig. 7). Microscopic" exainination 
 showed that the brownish discoloration visible within the hemolyzed 
 zone was due not to discoloration of the corpuscles but rather to a 
 diffuse brownish stain or pigment coloring and clouding the medium. 
 The photomicrograph (Fig. 7a) fails to reveal this because the dif- 
 ferent parts of the zone were alike opaque to the photographic plate 
 whether they were brownish or greenish or contained corpuscles or 
 not. During a second refrigeration (Figs. 8 and 8a) another ring of 
 hemolysis appeared and to the eye the zones were quadruple though 
 in the photograph they appear double. 
 
 Streptococcus A-BLa (Plate 12) behaved much like B-7a towards 
 the different kinds of blood used in that there was much less hemo- 
 lysis in the rabbit and human blood plates. Greenish discoloration 
 and fixation of corpuscles were much more evident in the rabbit blood 
 plate than in the other two plates, but the brownish pigment- did not 
 appear during the second incubation as it did in the case of B-7a. 
 After a second period of refrigeration the zones were distinctly quad- 
 ruple, and after a third period of incubation and refrigeration six rings 
 could be seen though the outer two were quite faint. 
 
 Streptococcus D-4a (Plate 14) much like A-BLa in horse blood agar 
 but differing from both A-BLa and B-7a in fermentation reactions was 
 startlingly different from these two in its appearance in rabbit and 
 human blood agar. The appearance in rabbit blood agar (Figs. 4 
 to 6) could almost be called the alpha prime type. Doubtless many 
 authors seeing the growth of this strain macroscopically in rabbit 
 blood agar (Fig. 4) after 48 hours incubation would call it "hemo- 
 lytic" and would name it Streptococcus longus pathogenes rather than 
 Streptococcus viridans. The surface colonies especially resembled 
 those of ordinary hemolytic streptococci. Even after only 24 hours 
 incubation the appearance was the same except that the zones were 
 smaller. The small collection of slightly greenish corpuscles visible 
 microscopically close to the deep colony and the increase in hemoly- 
 sis during refrigeration and reincubation would serve to indicate that 
 this strain did not belong to those producing the beta appearance, 
 however. 
 
JAMES HOWARD BROWN 37 
 
 Streptococcus E-4q! (Plate 17) was essentially like B-7a in blood 
 agar made of all three kinds of blood. Its fermentation reactions were 
 different from those of B-la which it resembled in blood agar and 
 were the same as those of D-ia which it did not resemble in rabbit 
 blood agar. 
 
 The plates described of the above four strains (A-BLo;, B-Ja, 
 D-ia, and E-4a) with each kind of blood, were made at the same 
 time, with the same samples of blood, each 24 hours old, the same 
 agar, and were subjected to the same periods of incubation and 
 refrigeration. They are strictly comparable. 
 
 At a different time the strain Streptococcus longissimus D -AD 82a 
 (Plate 18) was studied in blood agar made with horse, rabbit, and 
 hvunan blood. The growth in horse blood agar has been described 
 (page 21). In this plate are given photomicrographs of both deep 
 and surface colonies from each agar plate. The appearances differed 
 conspicuously from those of the above four strains described. The 
 large collection of greenish corpuscles about the colonies in the 
 rabbit blood plates (Figs. 3 and 4) should be especially noted as con- 
 trasted with the few in the horse blood plate (Figs. 1 and 2) ; also the 
 very clear and sharply defined ring of hemolysis about the deep 
 colonies in the rabbit blood plate after refrigeration (Figs. 4 and 4a) ; 
 the lack of hemolysis about the surface colonies as compared with 
 the deep colonies; in both the horse blood and rabbit blood plates 
 after refrigeration (Figs. 2 and 4) and in the human blood plate after 
 refrigeration (Fig. 6) the marked hemolysis about the surface colo- 
 nies as compared with that about the deep colonies in the same plate 
 and as compared with the surface colonies" on horse and rabbit blood 
 agar. 
 
 The strain of Streptococcus mucosus (Brig.-IIaa, Plate 20) in rabbit 
 blood agar produced smaller zones of hemolysis than in horse blood 
 agar, but was otherwise the same in appearance. 
 
 On the other hand, Pneumococcus Cole I 109 (Plate 22) produced 
 wider and clearer but less sharply bounded zones in rabbit blood agar 
 (Figs. 1, 2, and 3) than in horse blood agar (Plate 21, Figs. 1, 2, 3. 
 and 4). 
 
 In human blood agar the Streptococcus mucosus and Pneumococc 
 Cole 1 109 and Cole II 45 looked very much alike, all producing littl 
 
38 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 hemolysi's but broad collections of greenish corpuscles about the deep 
 colonies (Plate 23, Figs. 5 and 6). 
 
 When the comparison of the appearances produced by different 
 streptococci and the pneumococci in blood agar made with different 
 kinds of blood was undertaken, it was hoped to find either that the 
 appearances were the same or nearly the same for all kinds of blood 
 or that the differences in appearance were quite uniform in direc- 
 tion; i.e., that there was more or less hemolysis in one kind of blood 
 agar and more or less greenish discoloration in another. With or- 
 ganisms of the alpha type such was not the case, however, at least 
 with reference to horse and rabbit blood. Some strains produced 
 markedly different appearances in blood agar made with different 
 kinds of blood and others did not. The differences noticed in the 
 case of some strains were quite opposite in direction from those noted 
 in the case of other strains and the differences were not apparently 
 correlated with other characters of the strain in so far as could be 
 determined. In human blood agar, however, there was a general 
 tendency for both green-producing streptococci and pneumococci to 
 produce less hemolysis and more greenish discoloration of corpuscles 
 than in horse or rabbit blood agar. , 
 
 The variations in appearance of the beta tj^e zones of hemolysis 
 produced by streptococci A-ST/3 (Plate 6), D-AD4|3 (Plate 7), and 
 B-2b (cow)/3 (Plate 9) in horse, rabbit, and human blood agar were 
 quite insignificant. The zones in horse blood agar were most sharply 
 defined, those in rabbit blood agar had rather indefinite borders, and 
 those in human blood agar were slightly smaller and had a slightly 
 milky clouded appearance not due to incomplete hemolysis. 
 
 Of the bovine strains of beta type streptococcus B-18 (cow)/3 (Plate 
 5) produced similar appearances in horse, rabbit, and human blood 
 agar. Streptococcus A-18(cow)|8 (Plate 4) in rabbit and human 
 blood agar produced smaller "inner" zones of hemolysis than in horse 
 blood agar and very faint "outer" partly hemolyzed translucent 
 zones. 
 
 It may be said, therefore, that in blood agar made with the three 
 kinds of blood used there is much less variation in the appearance of 
 
JAMES HOWARD BROWN 39 
 
 the beta type zones produced by streptococci than of the alpha type 
 zones. There appears to be no danger of mistaking a beta type strep- 
 tococcus for an alpha type streptococcus in blood agar, but certain 
 of the alpha t3^e streptococci when groAvn in certain kinds of blood 
 agar may resemble the beta type so closely that only a careful micro- 
 scopic study of the deep colonies can be relied upon to identify 
 them. 
 
 Streptococcus D-I7 (Plate 2) grew alike in horse, rabbit, and 
 human blood agar, producing no apparent hemolysis or discoloration 
 in any of them. 
 
 Table III on The Use of Blood Agar should be consulted for the work of the 
 following authors: Natvig (1905); Silberstrom (1906); Heynemann (1908); 
 Zangemeister (1909); Saito (1912). 
 
INFLUENCE OF THE COMPOSITION OF THE AGAR USED. 
 
 Reaction {Acidity). — ^A number of strains producing the alpha and 
 beta types of appearance in horse blood agar were replated under 
 comparable conditions in standard veal infusion agar, fermented^ 
 veal infusion agar, and standard (0.3 per cent) Liebig extract agar, of 
 various reactions. Media made from Liebig extract is apparently 
 sugar-free, while those made from meat infusion always contain a 
 small amount of fermentable sugar. It was to eliminate the pos- 
 sible effect of fermentable sugar that the fermented veal infusion agar 
 was also used. Any differences in the appearances produced by 
 growth in these three different kinds of agar will be described later, 
 and attention will be here confined to differences in reaction of each 
 medium. It was found that the reaction (titratable acidity) of the 
 veal infusion agar (both fermented and unfermented) might vary 
 from 0.4 per cent to 1.5 per cent n acid to phenolphthalein without 
 affecting the appearances of either alpha or beta type in blood agar, 
 and the result was the same whether hydrochloric or lactic acid was 
 added to the agar to increase its acidity. The two extremes mentioned 
 were considered far enough apart to include any probable reaction of 
 standard meat infusion agar as made in all laboratories. When 
 various amounts of acid were added to standard Liebig extract 
 medium an unexpected difficulty was encountered in that the blood 
 underwent almost immediate laking when added to extract agar 
 having a titratable acidity of about 0.7 per cent n acid or above. In 
 order to eliminate entirely the possibility of spontaneous laking of 
 blood during incubation of the extract agar it was found necessary 
 to keep its reaction at or below about 0.4 per cent n acid. There 
 were no variations in the alpha and beta appearances in this medium 
 due to reactions between 0.15 per cent and 0.4 per cent n acid. 
 
 Since the only difference between the veal infusion agar and the 
 extract agar was in the veal infusion or beef extract used, the differ- 
 
 ° Made from fermented bouillon prepared from a veal infusion which had 
 been fermented by B. coli for 16 to 18 hours, heated, reaction adjusted, peptone 
 added, and made into bouillon. 
 
 40 
 
JAMES HOWAED BROWN 41 
 
 ence in behavior towards blood must reside in these constituents. 
 Other work had led the author to titrate the hemolytic activity of 
 graduated amounts of various acids added to veal infusion bouillon 
 and to Liebig extract bouillon. The results obtained after 2 hours 
 at 37°C. by adding 5 per cent of an undiluted suspension of washed 
 rabbit blood corpuscles (i.e., a suspension equivalent to the original 
 volume of the blood) to two series of tubes containing the different 
 bouillons are shown in the following table. 
 
 Veal Infusion Bouillon. 
 
 Titratable Acidity 
 
 (percent normal) 11.3 9.4 7.45 6.2 5.1 4.15 3.35 2.4 1.4 
 Hemolysis ++++ +++ + - - _ _ _ _ 
 
 Liebig Extract Bouillon. 
 
 Titratable Acidity 
 
 (percent normal) 10.75 8.7 6.45 4.65 4.5 3.55 2.6 1.65 0.75 
 Hemolysis ++++ ++++ ++++ ++++ ++++ +++ ++ + - 
 
 The results were the same whether hydrochloric, acetic, lactic, or butyric acid 
 was used. A veal infusion bouiUon made without the addition of peptone be- 
 haved like the veal infusion bouillon containing peptone. 
 
 Walbum (1914) has shown the importance of hydrogen ion concentration for 
 hemolysis by various hemolytic agents and in the absence of other agents the 
 hemolytic power of acids themselves is dependent upon the hydrogen ion con- 
 centration. It is therefore probable from the results recorded in the above table 
 that the veal infusion bouiUon with a titratable acidity of 7.45 per cent n had 
 about the same hydrogen ion concentration as the Liebig extract bouillon with a 
 titratable acidity of 1.65 per cent n. Clark (1915) has published titration curves 
 of infusion and extract broths showing that the "buffer" or "amphoteric" prop- 
 erty of infusion broth is much greater than that of the extract broth. The same 
 is probably true of infusion and extract agars. \ 
 
 The "reaction" of the media is expressed in terms of per cent 
 normal titratable acid because it is the method in general use among 
 bacteriologists, but it should be noted that the mere statement of 
 authors that agar or bouillon of a certain reaction was used means 
 nothing unless it is also stated whether the medium was made from 
 meat infusion or extract. 
 
 Kind of Meat and Peptone Used. — All the blood agar plates so far de- 
 scribed, unless otherwise stated, were made with veal infusion and Witte 
 
42 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 peptone agar. Until a year or so ago veal and Witte peptone were 
 used for all standard media in this laboratory. Since then it has been 
 more convenient to procure beef rather than veal. At about the same 
 time our stock of Witte peptone was nearly exhausted and the con- 
 tinuance of the European war made it very difl&cult to obtain more. 
 A similar product, however, was being manufactured by the Diges- 
 tive Ferments Company in this country. Before adopting any 
 changes in the constituents of the media, however, it was determined 
 to compare carefully the appearances of various strains of streptococci 
 in blood agar made with the following substances: 
 
 (a) Veal infusion and Witte peptone. 
 
 (6) Veal infusion and Digestive Ferments Company peptone. 
 
 (c) Beef infusion and Witte peptone. 
 
 (d) Beef infusion and Digestive Ferments Company peptone. 
 
 (e) Liebig extract and Witte peptone. 
 
 (/) Liebig extract and Digestive Ferments Company peptone. 
 
 The appearances of the various alpha zone-producing strains were 
 influenced to different degrees by the above alterations in media. The 
 most constant difference was that due to the use of meat infusion agar 
 or Liebig extract agar. For most strains the colonies in extract agar 
 were slightly smaller than in meat infusion agar. In the case of 
 streptococci ArBLa, B-7a (compare Plate 16, Figs. 1 and 3), and 
 E-4q; (compare Plate 17, Figs. 5 and 7) there was less greenish dis- 
 coloration and a smaller collection of fixed corpuscles next to the 
 colony in extract agar than in infusion agar. This may result in a 
 more conspicuous zone of hemolysis and was especially noticeable in 
 the case of streptococcus B-7q; (Plate 16, Figs. 3 and 4). . J» no case 
 was the difference in appearance in these various media great enough to 
 cause confusion between the alpha and the beta types provided deep col- 
 onies were examined microscopically. 
 
 With the pneumococci the differences were more conspicuous, and 
 were different for each of the two strains studied. In Liebig extract 
 and Witte peptone agar the colonies of pneiunococcus Cole I 109 were 
 small and brownish green, surrounded by small collections of brownish 
 green corpuscles and small indefinite partly hemolyzed zones; the 
 medium surroundmg these zones was also somewhat lighter. This 
 plate was not photographed but would have appeared just about like 
 
JAMES HOWARD BROWN 43 
 
 Plate 23, Fig. 3. By comparison with the photograph of the veal 
 infusion blood agar plate (Plate 23, Figs. 1 and 2) there is seen to be 
 much less hemolysis and an obscuring of the colonies by the brownish 
 green discoloration. With pneumococcus Cole II 45 the appearance 
 in extract agar was almost the reverse of that described for Cole I 
 109. In Liebig extract Witte peptone agar (Plate 24, Figs. 5 and 6) 
 it produced more hemolysis, appearing with practically clear colorless 
 zones easily mistaken macroscopically for zones of the beta type. 
 Microscopically, however, the edges of the deep colony were seen to 
 be obscured by a thin collection of brownish green corpuscles (com- 
 pare with Figs. 1 and 2). 
 
 There were no apparent differences in the appearances of the alpha 
 type streptococci in extract media made with the two kinds of 
 peptone. 
 
 In the case of the pneumococci the appearances in extract agar 
 were noticeably influenced by the use of Digestive Ferments Company 
 peptone instead of Witte peptone. Photographically the appear- 
 ance of the Digestive Ferments Company peptone extract agar 
 plate (Plate 23, Figs. 3 and 4) of pneumococcus Cole I 109 is the 
 same as was the Witte peptone plate, but to the eye the discoloration 
 of the former plate was quite green and that of the latter brownish 
 green. The difference in color was more conspicuous in the plates of 
 pneumococcus Cole II 45; in the Digestive Ferments Company 
 peptone plate (Plate 24, Figs. 7 and 8) not only was the discoloration 
 vividly green but the collection of corpuscles was so broad and thick 
 as to conceal the deep colony itself, making it appear much larger than 
 those in Witte peptone agar (compare Figs. 5a and 7a). 
 
 There were no differences in appearance of any of the alpha t5^e 
 zone producers (streptococci or pneumococci) due simply to the use of 
 veal or beef infusion agar respectively except in the case of pneumo- 
 coccus Cole I 109 which produced a broader collection of greenish 
 corpuscles about the deep colony and less hemolysis in beef infusion 
 agar than in veal infusion agar (compare Figs. 1 and 2 of Plate 21 
 with Figs. 1 and 2 of Plate 2^). The mediimi shown in Plate 23 
 was made with Witte peptone too but other experiments indicated 
 that it was the difference in the infusions rather than in the peptones 
 that caused the differences between the two plates. 
 
44 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 In either veal or beef infusion agar, however, there were differences 
 produced by the use of Witte or Digestive Ferments Company 
 peptone. Streptococcus Brig.-IIaa and pneumococcus Cole I 109 
 showed no variation with respect to the two kinds of peptone in 
 veal or beef infusion agar. Streptococcus B-7a showed slight varia- 
 tion in this respect. In meat infusion agar made with Digestive 
 Ferments Company peptone the greenish discoloration was very 
 slightly more vivid. Streptococcus A-BLa showed . more greenish 
 discoloration when grown in agar containing Digestive Ferments 
 Company peptone than in that containing Witte peptone. Pneimio- 
 coccus Cole II 45 (compare Plate 24, Figs. 1 and 3) produced in 
 Digestive Ferments Company peptone veal or beef agar a much 
 heavier and more vivid green collection of corpuscles next to the 
 deep colonies than in Witte peptone agar. 
 
 Of the streptococci producing the beta type of hemolysis A-ST/3 
 showed no variation with the use of veal or beef infusion or of either 
 kind of peptone. In Liebig extract agar, however, hemolysis was 
 much more extensive and occurred much more readily than in meat 
 infusion agar as a comparison of Figs. 3 and 4 of Plate 6 will show. 
 The photograph in Fig. 4 (extract agar) was taken after 24 hours 
 incubation and yet the hemolysis is more extensive than that in 
 Fig. 3 (veal infusion agar) taken after 48 hours incubation. In this 
 length of time the zones in the extract agar plate shown in Fig. 4 
 were confluent over a large portion of the plate. 
 
 For streptococci C-64;3 and D-AD4|3 (both of which are strains of 
 typical Streptococcus pyogenes in the most limited sense, i.e. with re- 
 gard to hemolysis, fermentation reactions, pathogenicity, morphology,, 
 growth on agar slants, and as distinguished from Streptococcus epi- 
 demicus) the results in the various kinds of agar were the same as for 
 streptococcus A-ST/3 just described. 
 
 From the above strains Streptococcus epidemicus (Davis) differs 
 noticeably. Briefly the results were as follows: The appearances in 
 Liebig extract agar whether made with Witte peptone or Digestive 
 Ferments Company peptone were alike. There were clear colorless, 
 zones of hemolysis with no trace of discoloration about deep or sur- 
 face colonies by transmitted or reflected light. In veal infusion agar 
 (Plate 9, Figs. 3 and 4) there was clear hemolysis without discolora- 
 
JAMES HOWARD BROWN 45 
 
 tion about the deep colonies, but the surface colonies in agar made 
 with different kinds of peptone showed conspicuous differences. On 
 Digestive Ferments Company peptone veal infusion agar (Fig. 4) the 
 top surface colonies were a deep opaque green color by transmitted 
 light and surrounded by narrow clouded rings of hemolysis. If on 
 this medium surface colonies only were observed macroscopically the 
 organism might readily be called Streptococcus, viridans. When 
 viewed microscopically (Fig. 4a), however, the greenish discoloration 
 was seen to be due to a diffuse pigment obscuring the edges of the 
 colony and extending outward into the zone of hemolysis, and not 
 due to discolored fixed corpuscles as seen in zones of the alpha type. 
 On the Witte peptone veal infusion agar the surface colonies were 
 translucent with well defined edges and without discoloration except 
 for yellowish centers which appeared less opaque to the eye than 
 they do in the photograph (Fig. 3). The yellow nucleus appears as a 
 dark patch in the center of the colony shown in Fig. 3a, but the dif- 
 ferences between Figs. 3a and 4a are very conspicuous. No changes 
 occurred during refrigeration. The appearances in beef infusion 
 agar were the same as in veal infusion agar. 
 
 The differences in appearance of the top surface colonies of the 
 Streptococcus pyogenes and Streptococcus epidemicuJs on meat infusion 
 Digestive Ferments Company peptone blood agar are shown macro-, 
 scopically in Figs. 5, 6, and 7 of Plate 8. Streptococci D-AD4|3 
 and C-64;3 are strains of Streptococcus pyogenes while streptococcus 
 X-38j8 is a strain of Streptococcus epidemicus (from the Baltimore 
 epidemic, 1912). The three plates were of the same medium, inocu- 
 lated, incubated, and photographed simultaneously.' 
 
 A bovine streptococcus of the beta type of hemolysis (A-18 (cow)|8) 
 showed no variation in appearance due to the use of veal or beef or of 
 Witte or Digestive Ferments Company peptone. In Liebig extract 
 blood agar only the small clear central hemolyzed zones appeared 
 during 24 hours incubation, but during the next 24 hours at incubation 
 temperature the broad outer zones not only appeared but were so 
 nearly completely hemolyzed as almost to obliterate the inner zones. 
 
 " Note that the large thin patches of growth are bottom surface colonies, while 
 the dark round colonies are on the top surface. 
 
46 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 Streptococcus saprophyticus (D-I7) produced neither hemolysis 
 nor discoloration in any of the agars used except that around some 
 of the colonies in Liebig extract blood agar there was a very faint, 
 hardly visible, trace of indefinite lightening of the medium which 
 will be mentioned again later. 
 
 Although our present object is 'satisfied by having shown that im- 
 portant differences in appearance may result from the use of different 
 kinds of commercial peptone, in view of the experiments about to be 
 described, it was important to determine whether or not the presence 
 of fermentable sugar was responsible for the different results obtained 
 by the use of Witte or Digestive Ferments Company peptone. Solu- 
 tions of neither peptone gave any reaction for sugar with Fehling's or 
 Benedict's reagents. No recognizable trace of acid or gas was formed 
 by the growth of Bacillus coli and various actively fermenting strep- 
 tococci in either peptone solution. 
 
 Influence of Dextrose in Agar. — The point of departure for this 
 series of experiments was the paper by Ruediger (1906) on The 
 Cause of Green Coloration of Bacterial Colonies in Blood Agar. 
 Ruediger studied a number of strains of hemolytic Streptococcus 
 pyogenes and of green-producing pneumococci in blood agar with and 
 , without the addition of glucose. He says : 
 
 . " * * a large number of typical S. pyogenes cultures produce no hemolysis in plates 
 made of glucose blood agar, although they produce very extensive hemolysis made of plain agar 
 and blood. If these glucose blood agar plates are allowed to remain in the incubator for 
 36 to 48 hours, the streptococcus colonies become distinctly green and are surrounded by a 
 green halo." 
 
 We have made comparative studies of several strains of beta hemo- 
 lytic streptococci including Streptococcus pyogenes, Streptococcus epi- 
 demicus, and Streptococcus alactosus (Smith and Brown) in both 
 veal infusion blood agar and Liebig extract blood agar with and 
 without the addition of 1 per cent dextrose. The appearance of 
 streptococci A-ST;8, B-2b (cow)/3, and r)-AD4i3, in veal infusion 
 blood agar has already been described. Streptococcus pyogenes C-64/3 
 was in every respect like D-AD4/3 and Streptococcus epidemicus X-320 
 and X-38/3 were just like B-2b (cow)/3. All these strains produced 
 the same appearance in veal infusion blood agar and in Liebig ex- 
 tract blood agar except that in the latter hemolysis occurred a little 
 
JAMES HOWAED BROWN 47 
 
 more readily and a little more extensively than in the former. Clear 
 colorless zones were produced by all these strains in less than 24 hours 
 in both kinds of agar. In the case of streptococcus A-ST/3 the effect 
 of the addition of 1 per cent of dextrose to the infusion and extract 
 blood agar was the same (Plate 26, Figs. 7 and 8). As observed after 
 24 hours incubation there were distinct colorless hemolyzed zones 
 about the deep colonies, completely hemolyzed and free from cor- 
 puscles where penetrating the agar or as viewed microscopically in 
 optical section. Instead of being 2.5 or 3.0 mm. in diameter, how- 
 ever, these zones were only 1.3 mm. in diameter. The surface colo- 
 nies where well scattered apart were without hemolysis, but where 
 crowded together were surrounded by small partly hemolyzed rings 
 1.25 mm. in diameter. By transmitted light the surface colonies 
 could be imagined to have a slightly greenish tint. After 48 hours 
 incubation the zones of the deep colonies were shghtly larger and 
 microscopically there was visible a shght collection of brownish pig- 
 ment (not corpuscles) next to the colony. Beneath the area of 
 crowded surface colonies there was generalized partial hemolysis and a 
 brownish discoloration obHterating, the individual zones noted after 
 24 hours incubation. The plates had a diffuse brownish red tone 
 more intense in the extract agar plate, and the greenish tint was less ■ 
 apparent than after 24: hours incubation. As distinguished firom 
 these plates streptococci C-64/3 and X-32/3 (also others not photo- 
 graphed) produced appearances in veal infusion dextrose blood agar 
 similar to that just described (Plate 26, Figs. 1 and 4), but in Liebig 
 extract dextrose blood agar (Figs. 2 and 5) produced no hemolysis 
 except a slight indefinite diffuse lightening of the medium in the 
 neighborhood of the colonies. In the photographs of these plates 
 most of the colonies are not visible but are present in about the same 
 numbers as in the plate to which dextrose was not added. To the 
 eye by transmitted light after 24 hours incubation there was no dis- 
 coloration in the extract dextrose blood agar plate of streptococcus 
 C-64/3 (pyogenes), but in a similar plate of streptococcus X-32^ 
 (epidemicus) the patches of bottom surface growth and the top sur- 
 face colonies'where close together had a shght greenish tint. After 48 
 hours incubation the plates of both these streptococci (and others 
 like them) were of a brownish red color and the areas of greenish 
 
48 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 discoloration were less apparent. In neither kind of dextrose blood 
 agar were there distinctly green colonies surrounded by green halos 
 as described by Ruediger. ^ 
 
 Ruediger, however, used fresh defibrinated rabbit blood rather 
 than horse blood which I used in the experiments just described. 
 He also used a sugar-free agar containing Witte peptone, salt, and 
 agar, but no. meat infusion or extract, with and without the addition 
 of 1 per cent of dextrose. He says that using this medium Strepto- 
 coccus pyogenes "forms green colonies in plates of glucose blood a,gar" 
 as did also the pneumococci. In the same agar without dextrose. 
 Streptococcus pyogenes produced hemolysis while the pneumococci 
 did not "produce more than a faint trace of green" due, he thinks, to a 
 trace of sugar in the blood. Ruediger concludes that: 
 
 "The green coloration of bacterial colonies in blood agar plates is dependent upon 
 the production of acid and the action of this acid (probably lactic acid) on the red 
 corpuscles." 
 
 The above experiments have been repeated with R.uediger's agar 
 and rabbit blood, without dextrose, with 0.1 per cent of dextrose, 
 and with 1 per cent of dextrose. Streptococcus D-AD4/3 (pyogenes) 
 (Plate 8) produced clear colorless zones in the sugar-free agar (Fig. 
 2), like those in veal infusion agar (Fig. 1) except that those in 
 Ruediger's agar were a little smaller, due probably to poorer growth 
 since the colonies themselves were also smaller. In the 0.1 per cent 
 dextrose agar (Fig. 3) the zones were without discoloration but in- 
 completely hemolyzed; microscopically (Fig. 3a) there was seen a 
 small amount of reddish brown (not green) pigment close to the 
 deep colony. ■ In 1 per cent dextrose agar (Fig. 4) hemolysis was still 
 less apparent; after 24 hours incubation there was present merely a 
 small (1 mm. in diameter) partly hemolyzed zone about the deep 
 colony but after 48 hours incubation there was present outside of 
 this a second broad indefinite zone of less hemolysis, and between the 
 two a red ring of hemoglobin. Microscopically (Fig. 4a) there could 
 be seen close to the colony a collection of reddish brown pigment. 
 There was no greenish discoloration. 
 
 Streptococcus A-ST|3 (S. alactosus 0) produced clear colorless zones 
 in the sugar-free agar (Plate 6, Fig. 8, clouded by scratches on 
 
JAMES HOWARD BROWN 49 
 
 the Petri dish). In the 0.1 per cent dextrose agar (Fig. 9) the deep 
 colonies had small partly hemolyzed zones surrounded, after 48 hours 
 incubation, by broad indefinite slightly hemolyzed areas. Micro- 
 scopically next to the deep colony (Fig. 9a) there was seen a collec- 
 tion of brownish pigment. The surface colonies were a deep choco- 
 late brown color by either reflected or transmitted light. In 1 per 
 cent dextrose agar (Fig. 10) the entire plate was a deep chocolate 
 brown color and the colonies w6re obscured by the discoloration. 
 
 Streptococcus B-2b (cow)/3 {epidemicus) also produced clear color- 
 less zones in sugar-free agar (Plate 9, Fig. S). In 0.1 per cent dex- 
 trose agar there was produced a smaller hemolyzed zone with a small 
 amount of brownish pigment visible microscopically close to the deep 
 colony (not photographed). In 1 per cent dextrose agar (Figs. 6 
 and 6a) there was stiU less hemolysis but broad areas of indefinite 
 faint hemolysis about the smaller zones. Surface colonies were 
 opaque and brownish. 
 
 The above three varieties of hemolytic streptococci, therefore, 
 produced abvmdant brownish pigment and a diminished amount of 
 hemolysis in dextrose blood agar but little or no greenish discolora- 
 tion. The faint broad outer areas of slight hemolysis were prob- 
 ably due to acids formed as I will presently show. 
 
 The appearance of streptococcus B-6q;' (Plate 10, Fig. 7) in veal 
 infusion horse blood agar was scarcely influenced by the addition of 
 1 per cent of dextrose. The colonies were sUghtly . larger and 
 brownish green discoloration about the colonies slightly increased, 
 but hemolysis was not reduced. 
 
 Having stated that in plain blood agar the pneumococci produce 
 green coloration, Ruediger offers the following explanation for the 
 failure of Streptococcus pyogenes to do so: 
 
 "{!) * * these organisms probably do not readily ferment the muscle sugar 
 which is jound in plain agar, and (2) they produce such rapid hemolysis that 
 the green would not he detected, because one can see no green coloration after the red 
 corpuscles have been completely hemolyzed." 
 
 If this were true an actively fermenting organism which does not 
 produce hemolysis should produce greenish discoloration in dextrose 
 agar. Such an organism is streptococcus D-I7 (saprophyticus) which 
 
so BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 has been described as producing neither hemolysis nor discoloration 
 in meat infusion blood agar. The addition of 1 per cent of dextrose 
 to veal infusion blood agar resulted after 48 hours incubation in a 
 general brownish discoloration of the entire plate. The colonies were 
 brownish by transmitted light and when viewed microscopically the 
 edges of the deep colonies were seen to be bordered by a brownish 
 pigment. There was no hemolysis nor greenish discoloration (Plate 
 3, Figs. 1 and la). I have already mentioned the fact that in Liebig 
 extract blood agar this streptococcus produced a very shght indefinite 
 lightening about some of the colonies. In extract blood agar con- 
 taining 1, per cent of dextrose (Fig. 2) the light areas about the colo- 
 nies were distinctly visible and were in fact indefinitely bounded 
 zones of partial hemolysis more apparent macroscopically than 
 microscopically (Fig. 2a). The edges of the deep colony were ob- 
 scured by a small collection of brownish pigment. There was no 
 trace of greenish discoloration. With this streptococcus the results 
 in Ruediger's rabbit blood agar with and without dextrose were 
 qualitatively the same as in Liebig extract agar. In the sugar-free 
 agar there was seen (Fig. 3) a very slight indefinite hemolysis; in the 
 same agar plus 0.1 per cent dextrose (Fig. 4) there was more hemolysis; 
 while if 1 per cent of dextrose was added (Fig. 5) there was more 
 brownish discoloration about the colony. In Fig. 5a the appear- 
 ance is not unlike that of alpha zone-producing streptococci, but to 
 the eye the dark ring about the colony was seen to be composed not of 
 green corpuscles but of a deep red or brownish red pigment in the 
 agar and not confined to the corpuscles. There was no greenish 
 discoloration. 
 
 From the above experiments it is evident that the green colora- 
 tion of bacterial colonies in blood agar plates is not dependent wholly 
 upon the production of acid and the action of this acid on the red 
 corpuscles. It may be that the presence of a certain amount of fer- 
 mentable sugar favors the production of green coloration by those 
 organisms which are capable of producing it, but it does not foUow 
 that the acids formed produce the green coloration. I tried to test 
 this directly by adding various amounts of various acids (lactic, 
 acetic, butyric, hydrochloric) to various kinds of bouillon and agar 
 containing various kinds of blood. Lakihg or brownish discoloration 
 
JAMES HOWARD BROWN 51 
 
 was invariably produced but never any greenish discoloration. 
 Anthony (1909) adopts Ruediger's explanation and says: "In our 
 laboratory all the hemolyzing strains and also the green ones, when 
 tested and retested in glucose agar blood plates, gave green colonies." 
 I can not account for the results of Ruediger and of Anthony unless 
 their hemolyzing strains were not of the true beta type. 
 
 Inasmuch as hemolysis is reduced or inhibited by the presence of 
 fermentable, sugar in the blood agar, it is evident that the beta tj^e 
 of hemolysis is not produced by acids formed, a fact recognized by 
 Sach^ (1909) and by Braun (1912). 'It is altogether probable that the 
 partial hemolysis produced by streptococcus D-I7 in Liebig extract 
 agar and in Ruediger's agar (especially in the presence of dextrose) 
 was due to the acids formed by the colonies. It has been shown 
 above (page 41) that extract bouillon is a very poor buffer solution 
 as compared to meat infusion bouillon. Similar experimeiits have 
 shown that a peptone and salt solution (such as is the basis of 
 Ruediger's agar) is even a poorer buffer solution than extract bouil- 
 lon. In extract agar or Ruediger's agar, t^herefore, a sUght amount 
 of acid formation would raise the hydrogen ion concentration in the 
 neighborhood of the colonies sufficiently to cause some hemolysis. 
 In meat infusion agar, on the other hand, the hydrogen ion concentra- 
 tion is kept down by the buffer effect of the medium and so acid 
 hemolysis does not readily occur. Probably the outer, indefinite, 
 broad, slightly hemolyzed zones formed by the other streptococci 
 (e.g., A-ST|8, D-AD4i3, B-2b (cow)/3, etc.) in extract agar and Rue- 
 diger's agar containing dextrose are to be similarly explained. The 
 diminution in the size; of the central zones produced by the beta strep- 
 tococci in meat infusion dextrose blood agar appears to be explained 
 by the partial inhibition of hemolysin production in sugar-containing 
 media. In Ruediger's blood agar plus 0.1 per cent of dextrose can be 
 seen both the diminished inner clearer zone produced by hemolysin 
 and the broad outer indefinite clouded zone of acid hemolysis. In 
 Ruediger's blood agar or extract blood agar plus 1 per cent of dextrose 
 only acid hemolysis is present. The reason for the partial or total 
 inhibition of hemolysis in media containing sufl&cient fermentable 
 sugar is yet to be discussed. 
 
52 BLOOD AGAR FOR STTJDY OF STREPTOCOCCI 
 
 Ruediger (1906) simply noted it. Sachs (1909) states that dextrose bouillon 
 diminished hemolysis only by checking the growth of the culture earlier because 
 of a development of acid. Sachs' explanation can hardly be true of growth in 
 blood agar since in dextrose blood agar the colonies become larger than in sugar- 
 free blood agar, and yet in the former there is little or no hemolysis while in the 
 latter large clear zones are produced. Kuhn (1912) concludes that the hemoly- 
 sis in dextrose blood agar or in dextrose blood bouillon is not reduced by the 
 acids formed but that in the presence of fermentable sugar the organisms adopt 
 a saprophytic or "herbivorous metabolism," while in the absence of sugar they adopt 
 a parasitic or "carnivorous metabolism" and hence attack the blood cells. Lyall 
 (1914) found hemolysis in fluid media to be inhibited by the presence of dextrose, 
 even in the presence of calcium carbonate, and by titration of amounts of acids 
 produced in media without calcium carbonate found that the inhibition did not 
 appear to be definitely associated with the amount of acid produced. There are 
 various facts which indicate that streptolysin is a product of the protein metab- 
 olism rather than the carbohydrate metaboUsm of streptococci, as for example 
 the facts that it passes through only the coarsest filters and with difficulty 
 (M'Leod, 1911), it is inactivated by moderate degrees of heat (Besredka, 1901; 
 Ruediger, 1903, 1906, and 1907; M'Leod; 1911; Braun, 1912; von HeUens, 1913), 
 it is non-dialyzable (Besredka, 1901; Ruediger, 1903, 1906, and 1907), and is 
 destroyed by peptic digestion (Ruediger, 1903, 1906, and 1907). 
 
 Lyall (1914) also found the inhibition of hemolysis by streptococci 
 in fluid media apparent within 30 minutes after dextrose was added, 
 and I have found washed streptococci suspended in fermented veal 
 infusion bouillon much more hemolytic (during 2 hours incubation) 
 than when suspended in unfermented veal bouillon or in fermented 
 bouillon plus 1 per cent of dextrose. The fermented bouillon was 
 certainly the least favorable for the growth of the streptococci during 
 the period of incubation and the time was hardly sufl&cient for the 
 development of a high degree of acidity. Apparently, therefore, in the 
 presence of fermentable sugar the protein metabolism of the strepto- 
 cocci is reduced to a minimum and for this reason the formation of 
 streptolysin is reduced or inhibited altogether. The other possibili- 
 ties which suggest themselves are that (1) the acids formed may pre- 
 vent the formation of streptolysin by acting upon the growing strep- 
 tococci in some way; (2) the acids may inactivate the streptolysin 
 as it is formed; and (3) the acids may render the blood corpuscles in- 
 susceptible to hemolysis by the streptolysin. Inasmuch as the pur- 
 pose of this paper is descriptive rather than explanatory, it appears 
 imnecessary to consider this subject further at this time. 
 
JAMES HOWARD BROWN 53 
 
 Ruediger's view, that the green coloration of bacterial colonies 
 in blood agar is due to acids produced, was based not only on his 
 observation that hemolytic streptococci produced green colonies in 
 dextrose blood agar but also upon the fact that: 
 
 * * pneumococci which produce distinctly green colonies in ordinary plain 
 blood agar, or glucose blood agar plates do not produce more than a faint trace 
 of green in plates composed of sugar-free agar and 0.3 to 0.4 cc. of fresh defibrinated 
 rabbit blood." 
 
 He continues: 
 
 * * but if 1 per cent of glucose, lactose, or inulin is added to the sugar- 
 free agar, the cultures again produce deep green colonies in blood agar plates 
 made with it." 
 
 I have studied both pneumococci and green-producing streptococci 
 in Ruediger's sugar-free agar with and without dextrose. In Plate 
 25, Figs. 7 and 8, are shown photographs of pneiunococcus Cole II 45 
 after 48 hours incubation in these two media. In sugar-free agar 
 (Fig. 7) the deep colonies were surrounded by broad indefinitely 
 bounded partly hemolyzed zones. The surface colonies were sur- 
 rounded by scarcely any hemolysis. By transmitted light both sur- 
 face and deep colonies were distinctly greenish brown. Microscopi- 
 cally there was seen next to the deep colony (Fig. 7a) a narrow but 
 heavy collection of dark green corpuscles. In the same blood agar 
 plus dextrose (Fig. 8) there was less hemolysis but more greenish 
 brown discoloration. The result is, therefore, the same as Ruediger's 
 except that there is more than a "faint trace"oi green in rabbit blood 
 sugar-free agar plates. 
 
 A green-producing streptococcus {B-7a) was observed in (1) 
 Ruediger's rabbit blood sugar-free agar, (2) the same medium plus 
 0.1 per cent dextrose, and (3) plus 1.0 per cent dextrose. These 
 plates are shown in Figs. 5, 6, and 7 of Plate 16 after 48 hours incu- 
 bation. Brownish green discoloration was most intense in the 1.0 
 per cent dextrose agar plate and least intense in the sugar-free agar 
 plate, but even in the latter the discoloration wak quite distinct. 
 Hemolysis was most conspicuous in the 0.1 per cent dextrose agar 
 plate. (The dark ring at some distance from the colony in Fig. 5a 
 is not green but is of red hemoglobin.) 
 
54 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 Since it was suspected that the greenish discoloration in the sugar- 
 free agar might be due to the presence of a small amount of sugar in 
 the rabbit blood, washed corpuscles were used instead of defibrinated 
 blood. Horse corpuscles instead of rabbit corpuscles were also tried, 
 as it had been discovered that there was apt to be less greenish dis- 
 coloration of horse blood than of rabbit or human blood. Ruediger's 
 sugar-free agar plus washed corpuscles is such a poor medium that the 
 streptococcus colonies in it are very small and therefore not com- 
 parable with those in media containing sugar or serum. Liebig 
 extract agar or agar made with fermented veal infusion (Bacillus coli 
 16 to 18 hours) supports growth much better and is also sugar-free. 
 Several strains of green-producing streptococci were observed growing 
 in extract agar and in fermented infusion agar plus washed horse 
 corpuscles with and without the addition of dextrose. In these 
 media without dextrose all the strains produced small partly hemo- 
 lyzed zones without apparent discoloration though small collections 
 of corpuscles could still be found next to the deep colonies. On the 
 other hand, if dextrose was added to the medium there were found 
 distinct collections of brownish green corpuscles about the deep colo- 
 nies. The fermented infusion agar proved to be the better medium 
 because it supported growth better in the absence of dextrose or 
 serum and at the same time favored the production of greenish dis- 
 coloration no more than did Liebig extract agar. The three media 
 used were: 
 
 (1) Fermented veal infusion agar -\- washed horse corpuscles. 
 
 (2) Fermented veal infusion agar -|- washed horse corpuscles -{■ 0.05 per cent 
 dextrose. 
 
 (3) Fermented veal infusion agar + defibrinated horse blood. 
 
 There was no great amount of discoloration in any of the plates and 
 macroscopically they appeared very much alike. In all of them after 
 48 hours incubation the deep colonies were surrounded by small 
 partly hemolyzed zones which did not penetrate the agar and so were 
 somewhat obscured by the overlying and underlying medium. Re- 
 frigeration resulted in an increase in the size of the hemolyzed zones 
 revealing more clearly the colonies and the corpuscles about them. 
 Microscopically the following differences were revealed: In the fer- 
 
JAMES HOWARD BROWN 55 
 
 mented veal infusion agar plus washed corpuscles the deep colony was 
 surrounded by very few corpuscles the color of which was doubtful 
 because of their scarcity. In the same medium plus 0.05 per cent 
 dextrose there was around the deep colony a moderate collection of 
 slightly greenish corpuscles. In fermented infusion agar plus defibri- 
 nated blood there was around the deep colony a small collection of 
 very slightly greenish corpuscles. In the medium containing dex- 
 trose the colonies were considerably larger than those in the other 
 two media and so it is doubtful whether the greenish discoloration 
 and larger collection of corpuscles in the former were due merely to 
 better growth or to the more direct action of the dextrose. A very 
 small percentage of dextrose was used because there could hardly 
 have been more in standard unfermented infusion agar plus defibri- 
 nated blood and because a larger amount of dextrose resulted in a 
 brownish rather than a greenish discoloration of the colonies and 
 medium. 
 
 The experiments of Ruediger indicating that the formation of acid 
 is the cause of the greenish discoloration by bacterial colonies in blood 
 agar are quite inconclusive. The formation of acid alone does not 
 result in greenish discoloration in blood agar. The failure of green- 
 producing organisms to produce greenish discoloration in sugar-free 
 blood agar may be due to poor growth rather than to failure to pro- 
 duce acid. The question of the cause of the green coloration is evi- 
 dently much more complex than was supposed by Ruediger. 
 
INFLUENCE OF ANAEROBIOSIS. 
 
 Parallel series of blood agar plates of several strains were studied 
 under aerobic and anaerobic conditions. The strains studied were 
 a streptococcus producing the beta type of hemolysis (B-2b (cow);8), a 
 streptococcus producing the alpha type of appearance (A-BLa), the 
 strain of Streptococcus mucosus (Brig.-IIao;), and the two strains of 
 pneumococci (Cole I 109 and Cole II 45). The agar used was beef 
 infusion agar made with Digestive Ferments Company peptone. 
 Both horse and rabbit blood were used. Anaerobic conditions were 
 secured by placing the plates in desiccators from which the oxygen 
 was absorbed by a good amount of pyrogalic acid and sodium hy- 
 drate. The tightness of the desiccators was indicated by the exist- 
 ence of a partial vacuum when they were opened after 48 hours in- 
 cubation. The lack of oxygen within the desiccators was indicated 
 by the peculiar burgundy red color of the reduced blood agar, turning 
 rapidly to the bright red color of oxyhemoglobin soon after the 
 desiccators were opened. 
 
 Streptococcus B-2b (cow)^ (epidemicus) grew equally well anaerobi- 
 cally and aerobicaUy, and produced clear colorless hemolyzed zones of 
 the same size and appearance under both conditions. It will be re- 
 membered, however, that aerobicaUy in blood agar made with Di- 
 gestive Ferments Company peptone the surface colonies of this strain 
 and others of the same variety were slightly greenish by transmitted 
 light. This was especially noticeable in rabbit blood agar. When 
 grown anaerobicaUy there was no trace of this greenish tint, the sur- 
 face colonies being whitish and transparent by transmitted light. 
 
 Streptococcus A-BLa (viridans) grew rather poorly anaerobicaUy 
 (Plate 13, Figs. 1 to 6). The colonies in both horse and rabbit 
 blood agar were very small after 48 hours incubation and showed no 
 discoloration and very faint traces of hemolysis (even after refrigera- 
 tion) slightly more apparent in the rabbit blood agar (Figs. 1 and 4). 
 After reincubation aerobicaUy the very small hemolyzed zones were 
 sl'ghtly more conspicuous (Figs. 2 and 5), but when again refriger- 
 ated there appeared conspicuous almost clear hemolyzed zones about 
 
 56 
 
JAMES HOWARD BROWN 57 
 
 the small, partly hemolyzed zones in the horse blood plate (Fig. 3) 
 but not in the rabbit blood plate (Fig. 6). In the photomicrograph 
 (Fig. 3a) the zone of the deep colony from the horse blood plate 
 appears double, but there was no apparent discoloration of the col- 
 lection of corpuscles next to the colony. 
 
 Streptococcus Brig.-IIao! (mucosus) (Plate 20) grew better an- 
 aerobically (Figs. 4, S, and 6) than did the one just described but 
 still did not grow so well as it did aerobically. After 48 hours in- 
 cubation anaerobicaUy the colonies in the horse blood agar plate 
 (Fig. 4) were surrounded by small definite colorless zones which in op- 
 tical section appeared clear (Fig. 4a), but when viewed macroscopi- 
 cally appeared clouded because they were not large enough to pene- 
 trate the layer of agar. There was no trace of discoloration or col- 
 lection of corpuscles next to the colonies. The corresponding rabbit 
 blood agar plate (not photographed) was exactly like the horse blood 
 plate in appearance. During refrigeration the zones in the rabbit 
 blood simply became larger but those in the horse blood plate (Fig. 5) 
 not only became larger but in doing so left a faint thin ring of cor- 
 puscles at the place formerly marking the edges of the first zone. 
 We have in this case what may be called a "reversed alpha zone" 
 (Fig. Sa) ; i.e., instead of there being a collection of corpuscles next to 
 the colony there is a ring of corpuscles separated from the colony by a 
 small zone of hemolysis. In this case the corpuscles were not per- 
 ceptibly discolored, but this may have been due to their being so few 
 in number. After another period of incubation aerobically and 
 refrigeration another faint ring of corpuscles and another ring of 
 hemolysis appeared (Figs. 6 and 6a) making a quintuple zone. There 
 was a doubtful greenish discoloration of the second ring of corpuscles. 
 The rabbit blood plate similarly incubated and refrigerated showed 
 but one ring of corpuscles of which discoloration was doubtful. 
 
 Pneumococcus Cole I 109 (Plates 21 and 22) formed smaller 
 colonies anaerobicaUy than aerobically. Unlike the streptococci 
 described it did not produce similar appearances in horse and rabbit 
 blood agar during 48 hours incubation anaerobicaUy (Plate 21, Fig. 
 5 and Plate 22, Fig. 4). In the horse blood agar there was only a faint 
 trace of hemolysis about a few of the colonies (Plate 21, Fig. 5'), 
 while in rabbit blood agar there were broad almost completely hemo- 
 
58 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 lyzed zones (Plate 22, Figs. 4 and 4a). There was no discoloration and 
 no collection of corpuscles next to the colony in either plate. As 
 usual, by refrigeration hemolysis was increased (Plate 21, Fig. 6 and 
 Plate 22, Fig. 5). Reincubation aerobically of the rabbit blood plate 
 resulted in the formation of a dark intensely greenish pigment (not 
 corpuscles) about the deep colonies (Plate 22, Figs. 6 and 6a) which 
 remained unchanged during a second period of refrigeration (not 
 photographed). After similar periods of aerobic reincubation and 
 re-refrigeration the zones in the horse blood plate resembled those of 
 streptococcus Brig.-IIao; just described except that there was less 
 uniformity among the zones of the pneumococcus (Plate 21, Figs. 7 
 and 8). 
 
 Pneumococcus Cole II 45 (Plate 25) grew as well anaerobically 
 as aerobically and produced essentially similar appearances in horse 
 and rabbit blood agar; During 48 hours incubation anaerobically 
 t}^ical clear colorless hemolyzed zones of the beta type (Figs. 1 and 4) 
 were formed. It will be remembered that in the same medium under 
 aerobic conditions typical alpha type zones were produced exactly 
 like those shown in Fig. 3 of Plate 24. Refrigeration caused no per- 
 ceptible change in the plate which had been incubated anaerobically 
 except a very slight increase in size of the zones in rabbit blood agar 
 (Plate 25, Figs. 2 and 5). Reincubation aerobically for 24 hours 
 caused the zones to become still larger (Figs. 3 and 6), and in the 
 rabbit blood agar there appeared a collection of corpuscle-free greenish 
 pigment about the deep colonies (Fig. 6''~0- ^ horse blood agar 
 there was a very conspicuous difference between pneumococcus Cole 
 I 109 and pneumococcus Cole II 45 after 48 hours incubation an- 
 aerobically. The former produced hardly any perceptible hemolysis, 
 while the latter produced clear beta type zones (compare Plate 21, 
 Fig. 5 with Plate 25, Fig. 1). This experiment was repeated many 
 times with exactly the same medium for both strains, incubating 
 them simultaneously in the same desiccator. The result was always 
 the same. Thinking that possibly this difference might be diagnostic 
 for all pneimaococci of types I and II (Cole), I obtained two other 
 strains from Dr. Cole. The strains were labeled I 125 and II 37. 
 Aerobically these grew like the ones studied but anaerobically pro- 
 duced no visible growth. A strange phenomenon agreeing quite well 
 
JAMES HOWARD BROWN 59 
 
 with the above distinction did appear, however. Horse blood agar 
 plates streaked with a loop of 24 hour bouillon culture of pneumococ- 
 cus Cole II 37 showed no apparent growth anaerobically even under 
 the low power of the microscope, but in the line of the streak there 
 was a quite conspicuous Une of partial hemolysis without discoloration 
 {Plate 25, Fig. 9) . This was also repeated several times with the same 
 result. Pneumococcus Cole I 125 similarly treated produced no 
 perceptible alteration of the medium. An attempt will be made 
 later to interpret this result, but it is sufficient now to point out 
 that anaerobically both strains of pneumococcus Cole II produced 
 hemolysis in horse blood agar while neither strain of Cole I did so. 
 The general results of the anaerobic incubation of blood agar plates 
 of the organisms described may be siunmarized as follows: (1) the 
 production of greenish discoloration and the fixation of corpuscles, or, 
 in other words, the formation of alpha type zones was inhibited in all 
 cases; (2) in many cases the cultures grew less well anaerobically than 
 aerobically, and in such cases there was correspondingly less hemo- 
 lysis, but (3) in general hemolysis was not decreased by anaerobic 
 cultivation except in the case of pneumococci type I (Cole) which 
 were practically non-hemolytic in anaerobic horse blood agar plates. 
 
EFFECT OF THE GROWTH OF ONE STREPTOCOCCUS ON THAT 
 
 OF OTHERS. 
 
 Marmorek (1902) announced that the filtrates of bouillon cultures of strepto- 
 cocci of human origin would no longer serve as media for the growth of other 
 streptococci of human origin. This has become known as Marmorek's test and 
 has been used by various authors as a means of differentiating pneumococci from 
 streptococci. Marmorek regarded this reaction as highly specific and as indi- 
 cating "I'unitS des streptocoques pathoghne pour l'homme.'\ He regarded as a 
 "variant" a strain of streptococci isolated from a scarlet fever patient, which 
 grew slightly in streptococcus filtrates. A "Streptococcus der Druse" {Strepto- 
 coccus equi) grew well in the filtrates from strains of human origin and was there- 
 fore regarded as belonging to a distinct species. In reviewing the literature it is 
 surprising'to find that Marmorek's test has not met with more skepticism. 
 
 In applying the test to various strains of streptococci there have 
 been encountered some strains which behaved as Marmorek described 
 and others which did not. It was found also that more accurate and 
 more graphic results could be obtained by modifying Marmorek's 
 technique as follows: Instead of bouillon filtrates, standard agar or 5 
 per cent serum agar plates are used, preferably the latter. 12 cc. of 
 the melted agar at 45°C. are inoculated with several loops of a 24 hour 
 culture of a given strain of streptococcus; the plate is poured, and 
 incubated for 48 hours. At the end of this time there are no macro- 
 scopically visible colonies but the agar appears slightly clouded, due 
 to the presence of myriads of minute colonies which can be seen 
 microscopically throughout the mediiun. Growth appears to be com- 
 plete in this length of time since no change is noticed if the plate is 
 incubated longer. Inasmuch as the arrest of growth of an organ- 
 ism under such conditions may be due either to the accumulation of 
 metabolic products or to the exhaustion of certain essential nutritive 
 substances from the medium, it is well not to speak of the medium as 
 being saturated or exhausted but to speak of it as being metabolized 
 by the organism with which it is inoculated. If a metabolized 
 standard agar, serum agar, or blood agar plate is. streaked with the 
 same strain as that by which it has been metabolized no growth will 
 occur in the line of the streak. Parallel streaks of other strains may 
 
 60 
 
JAMES HOWARD BROWN 61 
 
 also be made on the same plate and the amount of growth observed 
 after 24 or 48 hours incubation. In Plate 28 is shown photographically 
 the result of such an experiment with four strains of streptococci and 
 one pneumococcus. The streaks of these different strains are num- 
 bered as follows on each serum agar plate: 
 
 (1) Pneumococcus Cole II 45 
 
 (2) Streptococcus A-BLa 
 
 (3) Streptococcus B-2b (cow) /3 
 
 (4) Streptococcus D-AD4/3 
 
 (5) Streptococcus A-18 (cow) j8 
 
 The plates shown were of standard agar plus 5 per cent horse serum. 
 The plates shown in Figs. 1, 2, 3, 4, and 5 were metabolized as de- 
 scribed above for 48 hours by strains 1, 2, 3, 4, and 5 respectively, 
 the metaboUzing strain being indicated by a circle around the number 
 in each figure {e.g. (1) in Fig. 1). In Fig. 6 is shown a control plate 
 of the same medium which was incubated sterile (not metabolized) 
 for 48 hours. After 48 hours incubation each agar plate was streaked 
 with 24 hour bouillon cultures of each of the five strains mentioned. 
 After another period of 48 hours incubation the results were as shown 
 in the photographs. 'Hie relative amount of growth in each streak 
 is indicated by the following signs placed under the numbers on each 
 plate: 
 
 — = no visible growth, macroscopically or microscopically. 
 
 + = slight growth, visible microscopically but hardly visible macro- 
 
 scopically. 
 + + = fair growth, visible macroscopically. 
 -f- + + = good growth. 
 
 Naturally only macroscopically visible growth is shown in the 
 photographs. On the control plate (Fig. 6) all strains grew about 
 equally well. In no case was there any growth of the strain by which 
 a plate had been metaboHzed. If Marmorek's reasoning were ap- 
 plied to the result shown in Fig. 1, it would be concluded that strain 
 2 was a pneumococcus since it failed to grow on a medium metabolized 
 by a pneumococcus. In Fig. 2, however, it is seen that the pneumo- 
 coccus (strain 1) did grow on the medium metabolized by strain 2 
 which therefore would be regarded as a streptococcus. However, if 
 
62 BLOOD AGAR TOR STUDY OF STREPTOCOCCI 
 
 it is a streptococcus, a medium metabolized by strain 2 should not 
 support the growth of other human streptococci, but as a matter of 
 fact strains 3 and 4 (both streptococci of human type) did grow fairly 
 well on this medium. On the other hand, it is seen in Fig. 3 that 
 strain 2 did not grow on a medium metabolized by strain 3 (a human 
 strain isolated from a cow's udder by Smith and Brown, 1915), but 
 did grow slightly on a medium metabolized by strain 4 (Fig. 4) which 
 is a typical Streptococcus pyogenes hominis, while on this latter me- 
 dium the pneumococcus (strain 1) also grew as well as did strepto- 
 coccus strain 2. Finally, although strain 5 is a bovine streptococcus 
 and grew on media metabohzed by any of the four strains of human 
 origin, none of the latter sjiowed a trace of growth on medium me- 
 tabolized by the bovine strain (Fig. 5). Marmorek's strain of Strep- 
 tococcus equi grew well in the filtrates of streptococci of human origin 
 and is therefore regarded by him as of a different species. I suspect 
 that none of the streptococci of human origin would have grown in a 
 filtrate from his strain of Streptococcus equi, but Marmorek does not 
 mention having tried this. The above and other experiments indi- 
 cate that Marmorek's test is not specific. A pneumococcus may grow 
 readily in standard bouillon, but if the bouillon is metabolized by 
 Bacillus coli, the pneumococcus may fail to grow in it. It will often 
 fail to grow in such a bouillon even after the reaction has been re- 
 stored and a fermentable sugar added to it. Yet no one would 
 think of regarding this as a specific reaction indicating even a remote 
 relationship between the pneumococcus and Bacillus coli. Appar- 
 ently the ability of a streptococcus or any other organism to grow 
 in a medium metabolized by another organism is dependent wholly 
 upon the ability of the former to tolerate the metabolic products of 
 the latter or to utilize certain nutritive substances not utilized by the 
 latter. This is well illustrated by the general characteristics of 
 streptococcus A-18 (cow);S (streak 5 in Figs. 1 to 6 of Plate 28). 
 This strain was more vegetative than the other four strains ; it produced 
 more acid in sugar media, indicating a higher acid tolerance; it grew 
 more abundantly in all standard media; it remained viable for a 
 longer time in stock culture tubes; thus it probably metabolized the 
 medium more thoroughly than did the other strains, rendering it 
 unfit for their growth, and on the other hand was abldtto utilize media 
 
JAMES HOWARD BROWN 63 
 
 which had been metabolized by the others, carrying the- metabolism 
 still farther. It must not be assumed, however, that the ability of 
 strains to utilize metabolized media is wholly a quantitative relation- 
 ship. It is quite conceivable that a relatively poorly growing organ- 
 ism may produce certain substances which even in small quantities 
 may inhibit the growth of organisms of greater but different metabolic 
 activity. 
 
 The above described plate method of metabolizing media was the 
 most satisfactory. Metabolizing the agar in tubes was also tried. 
 After 48 hours incubation the tubes of agar full of minute colonies 
 were melted (thereby sterilized at the same time), poured into plates, 
 and streaked with various strains. The results were quite irregular 
 and unsatisfactory. Such results may have been due to the fact that 
 in the tubes metabolization took place largely under anaerobic con- 
 ditions, while after pouring the plates the streaks grew under aerobic 
 conditions, or the irregularity of the results may have been due to 
 the effect of heat required to melt the agar. The following quota- 
 tion from Rahn (1917) illustrates this possibility and also the possi- 
 bility of error in the use of bouillon culture filtrates as used by 
 Marmorek. 
 
 " Eijkmann, as the first, found that B. coli reached its maximum growth in gelatin 
 at 37° in a few days, and that this gelatin, after hardening at 20°, would not support 
 growth after streaking with a young culture of the same organism; but after this 
 gelatin had been heated at 6(y for half an hour, B. coli grew on it as well as on fresh 
 gelatin. Broth in which B. coli had grown became fit again for growth of the same 
 bacillus after filtration through porcelain. The inhibition of growth is, in this case, 
 due to a compound which resembles a toxin in many respects.''' 
 
 In Plate 29, Figs. 2 and 3, are shown blood agar plates metabol- 
 ized by streptococcus B-7q; and pneumococcus Cole II 45 respec- 
 tively, and then streaked with streptococci A-18 (cow)|8, B-2b (cow)|i3, 
 D-AD4/3, and A-ST;3. Growth occurred in all streaks but least in 
 the streak of B-2b (cow)/3 which is a strain of Streptococcus epidemicus. 
 The control plate is shown in Fig. 1. 
 
 The appearance of mixed growths on blood agar has received little 
 attention. Holman (1914) found that "hemolytic streptococci, growing 
 
 ' Rahn in Microbiology, Marshall, 1917. 
 
64 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 on blood agan in mixed culture with a green-producing form, show an 
 apparent preponderance, * * ." I have been able to confirm this 
 observation and to extend it to mixed cultures of hemolytic strepto- 
 cocci and pneimiococci or non-hemolytic {gamma type) streptococci. 
 In mixed growths on blood agar the appearance of the hemolytic 
 streptococci will completely overshadow that of the green-producing 
 or non-hemolytic strains so that the presence of the latter would not 
 be suspected. In Plate 29, Fig. 4, is shown a blood agar plate streaked 
 as follows: 
 
 Streak II = streptococcus D-1t, pure. 
 
 Streak III = streptococci D-I7 and D-AD4/3, mixed. 
 
 Streak IV = streptococcus D-AD4;8, pure. 
 
 Streak III resembles streak IV in appearance, so that the presence 
 of the non-hemolytic streptococcus in the streak is completely ob- 
 scured. In Fig. 5 of Plate 29 is shown a blood agar plate streaked as 
 follows: 
 
 Streak 1 = streptococcus A-BLa, pure. 
 
 Streak 2 = streptococci A-BLa and D-AD4j8, mixed. 
 
 Streak 3 = streptococcus D-AD4/S, pure. 
 
 Streak 4 = streptococcus D-AD4|8 and pneumococcus Cole II 45, mixed. 
 
 Streak 5 = pneumococcus Cole II 45, pure. 
 
 Streaks 2 and 4 are exactly like streak 3 in appearance, the presence 
 of the green-producing streptococcus and the pneumococcus being 
 completely concealed. Subcultures in blood agar of growth scraped 
 from streaks 2 and 4 showed that the growth in streak 2 consisted of 
 about equal numbers of the green-producing and hemolytic strepto- 
 cocci, while in streak 4 there were actually about ten times as many 
 viable pneumococci as hemolytic streptococci. 
 
THEORIES AS TO THE CAUSES OF THE VARIOUS APPEARANCES 
 IN BLOOD AGAR. 
 
 Type |8. — In looking at the clear, colorless, sharply defined, hemo- 
 lyzed zones about the colonies of beta type hemolytic streptococci 
 in blood agar (for example, Plate 6, Fig. 1) one's first impression is 
 that the blood in these zones has been totally destroyed or consumed. 
 This appearance gave rise to such descriptive expressions as: 
 
 " * * eUgante aurSole d'hSmoglobine dissoute" {MaxTDortk., 1902). 
 
 " * * vollige Resorption des Hamoglobins" (Schottmtlller, 1903; Kerner, 
 1903). 
 
 " * * vollstdndige Decomposition des Blutfarhstofes" (Natvig, 1905). 
 
 "Diese Eigenschaft, die roten Blutkorperchen zur Auflosung zu bringen und deren 
 Blutfarbstof zu zerstoren" (Mandelbaum, 1907-08). 
 
 ," * * deutliche Resorptionshofe" (Laabs, 1910). 
 
 " Zerstorung von Blutfarhstof" (Loening, 1910). 
 
 "Resorptionshofe des Pyogenes" (Thalmann, 1912). 
 
 " Hdmopepsie" and " Hdmoglobinopepsie" (Baerthlein, 1914). 
 
 It is to be noted, however, that in blood bouillon hemolytic strepto- 
 cocci never produce more than a laking of the blood. In the lower 
 part of the undisturbed tube there is a change in the color of the 
 hemoglobin from a bright cherry red to a "burgundy red" color. 
 These phenomena attracted the attention of Zangemeister (1909) 
 who found by spectroscopic analysis that the burgundy red dis- 
 coloration was due to the reduction of hemoglobin. Zangemeister cites 
 the following experiments: 
 
 (1) On laked blood agar plates hemolytic streptococci did not produce color- 
 less zones, provided "wirklich alle roten Blutkorper aufgelost waren." 
 
 (2) Pulverized saponin scattered on the surface of sterile blood agar plates 
 produced clear colorless zones. (Zangemeister cites Neufeld and Handel to the 
 effect that saponin produces only release of hemoglobin from blood corpuscles.) 
 
 From these experiments Zangemeister concludes that "Die Aufhel- 
 lung entsteht: dadurch, dass Blurfarbsioff aus dem betroffenen Bezirk 
 austritt und sich durch Diffusion im umgebenden Agar verteilt" The 
 diffusion of the hemoglobin was shown by pouring over the inocu- 
 
 65 
 
66 BLOOD AGAR FOE STUDY OF STREPTOCOCCI 
 
 lated plate of blood agar a layer of plain agar which became red 
 above the colonies of growing streptococci. Before the reading of 
 these experiments similar conclusions had been reached as a result 
 of the following experiments (Plate 30). 
 
 (1) In Fig. 2 is shown a horse blood agar plate which was heavily- 
 inoculated with a typical hemolytic (beta) Streptococcus pyogenes. As 
 shown in Fig. 2 after 24 hours incubation the blood of the plate 
 was completely laked and the mediima filled with myriads of minute 
 colonies visible microscopically. There was apparently no less hemo- 
 globin, however, than in the sterile laked horse blood agar control 
 plate- shown in Fig. 3. In Fig. 1 is shown a sterile plain agar plate 
 merely for photographic comparison with the other two plates. AU 
 three plates were handled in a parallel manner and photographed 
 simultaneously as shown. If the plate shown in Fig. 2 had been 
 incubated longer than 24 hours, it would have become brownish, 
 but it must be remembered that streptococci of this type produce 
 clear, colorless, hemolyzed zones in 18 hours. When crowded, as in 
 Fig. 2, however, only laking of blood is produced and there is no 
 apparent disappearance of hemoglobin. 
 
 (2) In Figs. 4, 5, and 7 are shown three beta type hemolytic strep- 
 tococci (B-2b (cow)|3, D-AD4;8, and A-ST/S respectively) growing in 
 laked blood agar. There is no trace of zones or of discoloration about 
 the colonies. The apparent slight indefinite lightening of the medium 
 about the surface colonies in the photographs is an artefact due to 
 refraction or reflection of light from the convex surface colonies. 
 The method of preparing these plates was to add to defibrinated horse 
 blood two volimies of sterile distilled wat^r, to centrifuge the laked 
 blood at a speed of 3,000 revolutions per minute for 30 minutes and 
 then to add to each tube of melted agar 2 cc. of the laked blood from 
 the top layer in the centrifuge tube. It will be appreciated that 2 
 cc. of the laked blood contained the hemoglobin from f cc. of blood. 
 The lowering of the salt content of the medium by the addition of the 
 hypotonic laked blood was compensated for by using tubes of agar 
 which originally had contained 12 cc. of agar but which had con- 
 tracted to the extent of about 1.5 cc. by being allowed to "dry" in 
 the room for some days. In the course of these experiments an 
 unexpected dif&culty was encountered which probably has led to 
 
JAMES HOWARD BROWN 67 
 
 wrong conclusions on the part of others who have worked with laked 
 blood agar. Loening (1910) and Hohnan (1916) assert that hemo- 
 lytic streptococci produce hemolyzed zones on laked blood agar. 
 If one or two volumes of distilled water are added to defibrinated 
 blood (horse, sheep, rabbit, or human) there appears to be instantly 
 a complete laking, the fluid becoming practically transparent. If, 
 however, this laked blood is added to clear agar, it can be seen that 
 the resulting mixture is quite clouded, much more so than was either 
 the laked blood or the medium before mixing the two. The clouding 
 of agar by laked blood led to the following observations: 
 
 A few drops of laked blood added to standard bouillon or physiological salt 
 . solution shows the same clouding. The addition of a small crystal of chemically 
 pure sodium chloride to 1 cc. or less of laked blood in a test-tube causes marked 
 cbuding. 
 
 If the laked blood is examined microscopically under a cover-slip no corpuscles 
 can be seen, but after the clouding by any of the above methods the corpuscles 
 are seen to have reappeared, not as shadows but as well formed corpuscles having 
 a deeper color than the surrounding fluid almost like that of the corpuscles of 
 unlaked blood. 
 
 If a' drop of blood is placed under a cover-slip and a drop of distilled water 
 at the edge of the cover-slip, hemoglobin is seen diffusing outward from the cor- 
 puscles exposed to the distilled water and the corpuscles themselves appear to 
 become slightly smaller, due to the fact that they have become spherical through 
 the taking in of water. During this process the outlines of the corpuscles become 
 faint and finally disappear from view. If, next, a small crystal of salt is placed 
 at the edge of the cover-slip the corpuscles will be seen to reappear and to assume 
 a disc-like form. 
 
 If laked blood is centrifuged at 3,000 revolutions per minute for 30 minutes, 
 there is thrown down a light voluminous poorly refractive fluffy sediment of 
 so called corpuscle shadows which in this case are really intact spherical cor- 
 puscle membranes filled with water or rather fluid of the same osmotic tension, 
 presumably, as the surrounding fluid. 
 
 If a crystal of salt is added to laked blood and it is then centrifuged there is 
 thrown down a compact, dark colored sediment occupying about one-third the 
 volume of the sediment mentioned in the previous paragraph. The sediment 
 from the salted blood is composed of disc-like corpuscles. 
 
 The problem throughout appears to be one of osmosis. When the fluid out- 
 side the corpuscles is made hypotonic there is an outward diffusion of hemo- 
 globin and an inward diffusion of water resulting in a spherical corpuscle which 
 occupies more volume. At the same time the exchange of solutes for solvent and 
 the stretching of the corpuscle membrane lowers the refractive index of the cor- 
 
68 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 puscle until it approaches that of the surrounding fluid, the corpuscle becoming 
 invisible and the fluid transparent. When salt is added to the laked blood the 
 reverse process takes place; water passes out .from the corpuscle allowing it to 
 contract to its normal discoid form. The contraction of the cell membrane and 
 the concentration of its contents result in its having more color and a higher 
 refractive index so that it is again visible. 
 
 If a blood agar plate is made from laked blood which has not been 
 thoroughly centrifuged to remove all the corpuscle membranes, these 
 undergo the restoration described above so that the agar is somewhat 
 clouded. Colonies of hemolytic streptococci growing in such a me- 
 dium produce hemolyzed zones (Plate 30, Fig. 6). Inasmuch as 
 apparently completely laked blood had been used for this plate, it 
 would be but natural to suppose that the zones formed were due to 
 destruction of hemoglobin. I think the evidence indicates, however, 
 that the zones were due not to a destruction of hemoglobin but to a 
 dissolution of the remaining corpuscle membranes. 
 
 Baerthlein (1914) tabulates 18 strains of streptococci among those organisms 
 which in blood agar produced " Hamopepsie," defined as a complete destruction 
 of the entire blood, hemoglobin and stromata, causing the medium to become 
 hemoglobin-free and transparent. This conclusion was arrived at by staining 
 sections of the colonies and zones in blood agar and finding that in the central 
 portions of the zones no corpuscle remains could be found. Baerthlein's asser- 
 tion that the hemoglobin is also destroyed is less conclusive, for the virtual 
 absence of hemoglobin in the zones may be accounted for by diffusion outward. 
 
 It must be noted that as long as the hemoglobin remains within 
 the corpuscles embedded in the agar the latter remains hemoglobin- 
 free, and that if in the neighborhood of the colony the hemoglobin is 
 released from the corpuscles it will tend to diffuse outward into the 
 surrounding hemoglobin-free agar; i.e., among the corpuscles out- 
 side the zone. That this diffusion alone can account for 1;he appar- 
 ently colorless zones is shown by the following experiment in which 
 no use is made of saponin or other hemolyzing reagent. 
 
 (3) A sterile standard blood agar plate containing 5 per cent of 
 defibrinated horse blood was poured. After it had solidified, a num- 
 ber of small holes (about 5 mm. in diameter) were made in the agar 
 by means of suction by the mouth applied to a piece of sterile glass 
 tubing. These holes in the agar were then filled with laked blood 
 
JAMES HOWARD BROWN 69 
 
 ugar containing a little more than 5 per cent of laked blood so that 
 when first photographed the holes filled with laked blood agar ap- 
 peared darker than the surrounding blood agar (Plate 30, Fig. 8). 
 After incubation over night the hemoglobin from the laked blood agar 
 plugs had diffused outward and could to some extent be seen as a 
 diffuse darkening in the surrounding agar. The plugs of laked blood 
 agar appeared as clear colorless artificial zones (Fig. 9). From this 
 experiment it is evident that laking of the blood about a bacterial 
 •colony and diffusion of the hemoglobin outward are sufficient to pro- 
 duce clear colorless hemolyzed zones. That these are the factors re- 
 sulting in the formation of the beta type zones by streptococci is -indi- 
 cated by Experiments 1 and 2 described above and by the following 
 observations: 
 
 (a) The zones produced by a beta type hemolytic streptococcus in 5 per cent 
 blood agar can be seen to be surrounded by more or less definite dark rings of 
 outwardly diffusing hemoglobin.* 
 
 (b) If a blood agar plate contains too many colonies of beta hemolytic strepto- 
 cocci so that there is little room for diffusion outside the zones, these appear 
 reddish though transparent. 
 
 Although destruction of the corpuscle membranes and outward dif- 
 fusion of the released hemoglobin are sufficient to account for the pro- 
 duction of beta type zones and are essentially the only factors in- 
 volved in the production of these zones during 24 or even 48 hours 
 incubation, yet it does not follow that beta type hemolytic streptococci 
 are incapable of causing a visible alteration of hemoglobin. In fact, 
 if a thickly seeded blood agar plate is incubated for longer than 48 
 hours there sooner or later occurs a gradual brownish discoloration 
 and fading of the laked blood agar until after a time (several days) 
 it appears only a little darker than a blood-free agar plate. Similar 
 changes are apparent in blood bouillon. During 24 or 48 hours in- 
 
 * If diffusion alone is responsible for the disappearance of the hemoglobin 
 from the zone, there must remain within the zone just as great a concentration of 
 hemoglobin as in the dark ring surrounding the zone, but whereas within the clear 
 zone the remaining hemoglobin is relatively inconspicuous because so much 
 lighter than the surrounding blood agar, the same amount of hemoglobin in the 
 surrounding blood agar causes it to be much darker than the unaltered medium 
 farther out. These dark rings can be seen in practically all the photographs of 
 beta hemolytic streptococci. 
 
70 BLOOD AGAR FOR STUDY OF STfeEPTOCOCCI 
 
 cubation there is apparent only a laking of the blood accompanied 
 by a reduction of the hemoglobin to the burgimdy red color of re- 
 duced hemoglobin. In time, however, the medium becomes slightly 
 brownish yellow and gradually fades out until almost as light as 
 blood-free bouillon. 
 
 Most authors who have noted the different appearances produced by hemolytic 
 organisms in blood agar and in blood bouillon have regarded the hemolytic pro- 
 cesses in the two media as qualitatively different in some respect. It has been 
 suggested that the temperature at which it is necessary to add the blood to the 
 agar has an injurious effect on the blood corpuscles (Kraus, 1903) ; that the cor- 
 puscles are injured by the solid media "(Bitter, 1886) ; that "In der Bouillonkultur 
 hat der Vibrio freike Hand in der Auswahl seiner Nahrmittel; in der AgarkuUur ist 
 er auf seine ndchste Umgebung angewiesen. In der Bouillonkultur wird er leicht 
 losliche Eiweissstofe zundchst angreifen, und diese ig>erden daher Idngere Zeit i>or 
 der Zerstorung schiitzen. In der AgarkuUur muss er alles erreichbare Ndhrma- 
 terial aufzuzehren suchen, da er auch mit seinen loslichen Fermenten nur auf mehr 
 oder weniger kurze Entfernungen wirken kann" (Meinicke, 1904); or that "durch 
 den kolloidalen Charakter der Blutagarplatte eine Beschleunigung der Hdmolyse 
 herbeigefuhrt werden" (Pribram as quoted by Mandelbaum). 
 
 Nothing in the experiments and observations described above indi- 
 cates a difference in the hemolytic process as it occurs in blood agar or 
 in blood bullion. The differences in appearance are due solely to the 
 soUd or liquid character of the medium, with the possibility of colony 
 formation and localized hemolysis (zone formation) in the former. 
 
 It has been definitely shown by Besredka (1901), Ruediger (1903), Kerner 
 (1905), M'Leod (1911), Braun (1912), and von Hellens (1913) that markedly 
 hemolytic streptococci in suitable culture media produce a filterable hemolytic 
 substance known as "streptolysin." It is natural to assume that this is the sub- 
 stance produced by similar streptococci causing hemolysis in blood agar. Since 
 the further investigation of streptolysin involves the use of fluid media rather 
 than solid media it is not thought necessary to discuss it at length here. (See 
 Table IV on The Use of Blood Bouillon and Other Fluid Media.) 
 
 Type a. — -Natvig (1905), employing surface inoculation of blood agar plates, 
 noted that some streptococci produced colonies which were surrounded by zones 
 of hemolysis bordered on the outside by rings of greenish corpuscles. Natvig 
 has already been quoted (page 13) and an interpretation of the appearance which 
 he describes has been given. His view that the greenish discoloration of corpuscles 
 is a " Vorstadium einer vollstdndigen Decomposition des Blutjarbstoffes" is not 
 supported by the appearance of the zones of deep colonies which are made up of 
 
JAMES HOWARD BROWN 71 
 
 greenish corpuscles next to the colony and partial hemolysis farther out. In the 
 formation of these "double" alpha type zones the hemolytic substance must 
 pass outward from the colony through the greenish corpuscles, and if these cor- 
 puscles were subject to hemolysis one would expect them to be hemolyzed first. 
 Mandelbaum (1907-08) recognized the significance of this appea;rance and formu- 
 lated rather an elaborate theory to account for it. He says: 
 
 "Rote BluthSrperchen, die unmittelbar mil den Kohhenleihern des Mitior in BerUhrung 
 kommen, kSnnen von dessen Hdmolysinen nicht mehr angriffen werden." 
 
 "Der Streptococcus mitior lieferl also zundchst ein Toxin, dass an den Kokkenleib ge- 
 bunden zu sein scheint und dass nicht in das umgebende NShrmedium zu difundieren ver- 
 mag. Dasselbe wirkt schadigend auf den Blutfarbstoff der roten Blutkorpercken ein. Der- 
 selbe wird in eine Verbindung von braungelber bis rostbrauner Farbe iibergefiihrt (Jlitmo- 
 globinloxin) . Dieses Toxin wird erst bei der BerUhrung mit roten BluthSrperchen von def 
 lebenden Bakterienzelle gebildet. * * * Rote BlutkSrperchen, deren Farbstoff durch 
 das HSmoglobintoxin der Mitior geschddigt wurde, konnen durch das HSmotoxin [Hiimo- 
 lysin) des Mitior nicht mehr aufgelost werden. Denn sonst miissten doch die roten Blut- 
 kSrperchen, die unmittelbar unter der Kolonie des Mitior liegen, zuerst aufgelost werden, 
 da die Hamolysine erst durch diese wie durch ein Filter in die Umgebung diffundieren. In 
 der Tat aber bleiben sie ungelSst, wShrend die anderen roten BlutkSrperchen, die nicht durch 
 das Hdmoglobintoxin gewissermassen ' immunisiert' wurden, durch die Hamolysine zur 
 AuflSsung gebracht werden, was auf der Agarplatte makroskopisch in der ' Hofbildung' 
 erkannt wird." 
 
 He accounts for the lack of hemolysis by Streptococcus mitior in blood bouillon 
 by the same theory. 
 
 "In der Blutbouitton nun kom/tfien wohl alle BlutkSrperchen mit dem Bakterienleib in 
 BerUhrung, der Mitior bildet nun,- genau wie auf der Blutagarplatte, ein Hdmoglobintoxin, 
 durch das die roten BlutkSrperchen unempfi-ndlich gegen das Hamolysin dieses Bacteriums 
 
 Aside from the objectionable use of the terms "Toxin" a.nd " immunisiert" 
 there is also open to criticism Mandelbaum's statement that this so called toxin 
 is bound to the bodies of the cocci and does not diffuse into the surroTinding me- 
 . dium. He studied the colonies and their zones under the microscope and it 
 seems strange that he did not appreciate the fact that many of the discolored 
 " immunisiert" corpuscles are not in contact with the bacterial bodies but are at a 
 considerable distance from the colony. Had he studied deep colonies this could 
 not have escaped his notice. The formation of alternate rings of discolored cor- 
 puscles and of hemolysis as observed by Saito (1912) and described above (pages 
 8 to 14) as being caused by variations in temperature, is directly opposed to Man- 
 delbaum's hypothesis of "ein Toxin, dass an den Kokkenleib gebunden zu sein 
 scheint." 
 
 With the failure of this hypothesis there naturally disappears 
 Mandelbaum's explanation of the behavior of Streptococcus mitior 
 
72 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 in blood bouillon. The following hypotheses would appear to meet 
 the known facts more accurately. 
 
 (1) It may be assumed that there are elaborated by the Strepto- 
 coccus mitior colony two substances one of which (a) produces hemoly- 
 sis and the other of which (b) produces changes in the corpuscles vis- 
 ible as a greenish or brownish green discoloration of the intact cor- 
 puscles rendering them resistant to hemolysis by the first substance. 
 It must be assumed that in the development of the colony the second 
 substance (b) is elaborated first or at any rate reaches a concentration 
 sufficient to produce fixation of the corpuscles next to the colony 
 •before the hemolytic substance (a) is present in sufficient concentra- 
 tion to produce hemolysis. The hemolytic substance acts well at 
 refrigerator temperature, but at temperatures much below that of the 
 incubator the fixing substance (&) either is not produced or is in- 
 active. Alternate exposure to the temperatures of the incubator 
 and of the refrigerator results in the production of alternate rings 
 of fixed corpuscles and of hemolysis. 
 
 The above hypothesis would explain the appearances m blood agar 
 very well and is partially supported by the fact that streptococci have 
 been found to produce an extracellular streptolysin, but this substance 
 has been demonstrated only in cultures of markedly hemolytic strep- 
 tococci (probably only those of the beta hemolytic type). On the 
 other hand, it is doubtful whether there has been demonstrated any 
 substance corresponding to the hypothetical fixing or green-producing 
 substance. Ruediger (1906) considered green production due to 
 acid formation but there have already been given (page 46 and fol- 
 lowing) reasons for rejecting this view. They are briefly as follows: 
 
 (1) There are actively fermenting streptococci (gamma type) which even in 
 •dextrose blood agar produce no greenish discoloration. 
 
 (2) In the standard blood agar which I have used there is very little ferment- 
 able sugar and yet there is abundant greenish discoloration by alpha type strep- 
 tococci. 
 
 (3) Although in dextrose blood agar, streptococci of the beta type may pro- 
 duce a small amount of greenish color in the substance of surface colonies (5. 
 epidemicus) or of brownish pigment about the deep colonies (5. pyogenes and 
 others), they never produce the collection of fixed- and discolored corpuscles next 
 to the deep colonies characteristic of the alpha type zones. 
 
JAMES HOWARD BROWN 73 
 
 Before describing a modification of the above hypothesis there will 
 be described some experiments which show that blood corpuscles 
 which have been fixed under the influence of the growth of alpha 
 type streptococci are no longer subject to hemolysis by streptococci 
 of the beta type. 
 
 A 12 cc. tube of horse blood agar in fluid condition at 4S°C. was 
 heavily inoculated with several loops of streptococcus B-Ta. The agar 
 was poured into a Petri dish and incubated 48 hours. At the end of 
 this time the plate contained no macroscopically visible colonies and 
 showed no hemolysis but was of a brownish green tint and contained 
 many small colonies visible microscopically. At this time the plate 
 was streaked with four strains of streptococci of the beta hemolytic 
 type and the plate reincubated for 48 hours. The plate was photo- 
 graphed and is- shown in Fig. 2 of Plate 29. There was no trace of 
 hemolysis associated with any of the streaks and consequently it was 
 necessary to photograph the plate by reflected light to show the 
 growth in the streaks. A plate was similarly heavily inoculated with 
 pneumococcus Cole II 45 and later streaked with the same four hemo- 
 lytic streptococci. The results were exactly the same as with the 
 plate just described (Plate 29, Fig. 3). 
 
 A standard blood agar plate was streaked across with a loop of 24 
 hour bouillon culture of streptococcus B-7q: and immediately after- 
 ward this streak was crossed by simUar streaks of hemolytic strepto- 
 cocci A-ST|3 and D-AD4;8. ^ After 24 hours incubation the plate ap- 
 peared as in Plate 27, Fig. 1. Streak I is of streptococcus B-7a and 
 is greenish by transmitted light but without hemolysis. Streaks Ila 
 and Ilia are of streptococci A-ST/3 and D-AD4/3 respectively and 
 show wide zones of hemolysis without discoloration. The hemolysis 
 is not interrupted at the crossing of streak I by streaks Ila and Ilia. 
 At the crossing, however, edges of the hemolyzed streaks are indented 
 where the corpuscles under streak I had become fixed before the 
 hemolysis had advanced that far. At this time streak I was again 
 crossed by streaks of the two hemolytic streptococci and the plate 
 again incubated. After reincubation for 24 hours the result was as 
 shown in Fig. 2. It is seen that in crossing streak I the hemolysis of 
 streaks lib and Illb is interrupted, the corpuscles underlying the 
 growth of streptococcus B-7q: having been fixed by it and rendered 
 
74 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 resistant to hemolysis by streptococci A-ST/3 and D-AD4;8. Exactly 
 similar 'results were obtained by making streak I of pneumococcus 
 Cole II 45 and streaks II and III as before (Figs. 3 and 4). 
 
 The above experiments tend to discredit another conclusion of 
 Mandelbaum's, arrived at as follows: Mandelbaum (1907-08) found 
 that the brownish red corpuscles sedimented from a 48 hour bouillon 
 culture of Streptococcus mitior and suspended in fresh bouillon were 
 not hemolyzed by Streptococcus mitior but were hemolyzed by Strep- 
 tococcus pathogenes. He concluded that the hemolysin formed by 
 Streptococcus mitior was different from that formed by Streptococcus 
 pathogenes. Mandelbavun's observation was correct; the brownish 
 discolored corpuscles from blood bouillon cultures of Streptococcus 
 mitior are readily hemolyzed by Streptococcus pyogenes in bouillon. 
 If, however, these same brownish discolored corpuscles are mixed 
 with agar, poured into a Petri dish, the plate streaked with Strepto- 
 coccus pyogenes, and incubated, no hemolysis is produced. There are 
 several possible explanations. There is no reason to believe that 
 hemolysis by Streptococcus pyogenes takes place any less readily in 
 blood agar than in blood bouillon. Unfortunately it is impossible to 
 remove the brownish corpuscles from the blood bouillon culture 
 without including also many streptococci, so that when the cor- 
 .puscles are mixed with agar the medium is at the same time very 
 heavily inoculated with Streptococcus mitior which continues to grow 
 and to influence the blood corpuscles during incubation of the plate, 
 so that by the time the streak of Streptococcus pyogenes has also grown 
 sufficiently to show any hemolytic action the brown corpuscles can 
 no longer be assumed to be in the same condition as when added to 
 the agar. No way to overcome this experimental difficulty has 
 been found. It is suspected that blood corpuscles in bouillon are 
 less profoundly altered by Streptococcus mitior than they are in agar, 
 possibly because of lower oxygen tension in the Douillon. 
 
 There are, however, other reasons than that urged by Mandelbaum for sus- 
 pecting that the hemolytic activity of streptococci of the alpha type is different 
 qualitatively from that of streptococci of the beta type. Apparently all authors 
 who have given attention to the subject agree that the hemolyzed zones of Strep- 
 tococcus pyogenes on a blood agar plate are practically fully developed within 24 
 hours, while hemolysis by Streptococcus mitior or by pneumococci develops after 
 a much longer period of incubation and in many instances is not apparent within 
 
JAMES HOWARD BROWN 75 
 
 48 hours. Furthermore, it has been noted by Sigwart (1909) and is apparent 
 from the work described above that if plates containing colonies of alpha type 
 streptococci or pneumococei are placed in the refrigerator, hemolysis appears or 
 becomes much more extensive, the zones becoming larger, while the zones of 
 colonies of Streptococcus pyogenes and other heta type streptococci change little 
 or not at all. We must assume that if the same hemolysin is produced by both 
 types of streptococci it must be present in larger amoimt about the colonies of 
 the more hemolytic beta type, and if, therefore, it is suspected that refrigeration 
 exerts its influence by lowering the resistance of the blood corpuscles to hemo- 
 lysis, those about the more strongly hernolytic colonies should show the influ- 
 ence of refrigeration most. Such, however, is not the case. 
 
 Further discussion of this problem must be introduced by reference to a recent 
 theory as to the cause of the discoloration of blood corpuscles by pneumococei 
 and streptococci. By spectroscopic analysis it has been found that the brownish 
 discoloration of blood corpuscles by certain streptococci in blood bouillon con- 
 sists of a change within' the corpuscles of oxyhemoglobin to methemoglobin 
 (Rieke, 1904; Boxer, 1906). It is stated by Cole (1914) that the brownish or 
 brownish green discoloration of corpuscles in blood agar is also the result of methe- 
 moglobinization. As a result of studies with pneumococei in fluid media Cole 
 concludes that: 
 
 " * * the formation of methemoglobin by pneumococei occurs as a result of re- 
 duction and oxidative processes occurring in the neighborhood of the bacteria * * , and 
 ■without producing substances capable of isolation." 
 
 Blake (1916) repeated Cole's experiments using Streptococcus viridans instead 
 of pnevunococci and arrived at the same conclusions as did Cole. The results 
 of these authors are also in agreement with those of Rieke (1904) who found 
 that methemoglobinization was enhanced by free access of oxygen and that 
 killed bouiEon cultures (60°C. for 1 hour) or sterile filtrates did not produce 
 methemoglobinization. He concludes that: 
 
 "Diese Umwandlung des OxyhSmoglobins in neutrales MethSmoghbin ist eine Folge 
 der physiologischen Funktionen der Streptokokken, sie ist gekniipft an den Lebensprozess 
 derselben." 
 
 I In agreement with these conclusions are our observation that in the 
 /refrigerator, where the life processes may be assumed to be reduced 
 to a minimum, the fixation and discoloration of corpuscles about the 
 colonies of streptococci and pneunaococci in blood agar are brought 
 to a standstill while hemolysis is not inhibited, and that on being again 
 incubated the fixation and discoloration of corpuscles is resumed. 
 Fixation and discoloration of corpuscles did not occur under anaerobic 
 conditions. Possibly the methemoglobinization of corpuscles about 
 the colonies of alpha type streptococci and pneumococei is due to 
 
76 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 processes of reduction and oxidation in the neighborhood of the 
 colonies rather than to some methemoglobinizing substance elabor- 
 ated by the colonies. If such is the case, however, it is surprising- 
 to note at what distances from the colonies methemoglobinization 
 occurs, as is shown by the formation of rings in response to refrigera- 
 tion and reincubation. 
 
 (2) Let us now return to a discussion of hemolysis by alpha type 
 streptococci and pneumococci. Reasons are mentioned above (page 
 74) for suspecting that the hemolysis produced by such organisms 
 is due to a different factor than that which is instrumental in pro- 
 ducing the zones of markedly hemolytic streptococci. 
 
 Cole (1914) has described a "hemolytic endotoxin" which is released from pneu- 
 mococci by their autolysis or disintegration by other causes. I have been able 
 to repeat Cole's experiments to the extent of obtaining a hemolytic autolysate. 
 It seems possible tRat the marked hemolysis occurring in the refrigerator about 
 colonies of pneumococci after 48 hours previous incubation may be due to a 
 hemolytic substance released by autolysis of the pneumococci. The resemblance 
 of the zones produced by pneumococci to those produced by alpha type strepto- 
 cocci strongly suggests that they are due to similar processes. However, these 
 streptococci are much less easily disintegrated or autolyzed than pneumococci, ■ 
 and I have been unable to obtain from streptococci anything resembling a 
 "hemolytic endotoxin." Holman (1916) regards methemoglobin production as 
 due to reduction. He says: 
 
 " The bacteria use the oxygen in the medium in which the red cells are embedded and 
 thereby change the osmotic tension, this is followed by the abstraction of oxygen from the 
 oxyhemoglobin, until we have the various stages of the reduction indicated. * * The 
 red blood cells containing this reduced hemoglobin are, I believe, more liable to spontaneous 
 hemolysis," 
 
 He would, therefore, regard the hemolysis produced by green-producing strep- 
 tococci as "spontaneous," due to reduction, and not due to the action of a 
 hemolysin. 
 
 More work must be done before any conclusions as to the validity 
 of one or the other of the above hypotheses can be reached. 
 
 The work of the following authors as reviewed in Table III on The Use of 
 Blood Agar and Table IV on The Use of Blood Bouillon is of special interest: 
 Rieke (1904); Natvig (1905); Ruediger (1906); Boxer (1906); Mandelbaum 
 (1907-08); Zangemeister (1909); Sachs (1909); Anthony (1909); Zangemeister 
 (1910); Loening (1910); Thalmann (1912); Cole (1914); Blake (1916); Holman 
 
 (1916); 
 
PERMANENCE OF CULTURAL CHARACTERISTICS. 
 
 The literature of this subject is reviewed ia Table VII on Muta- 
 tions and Variability. In this table have been included not only all 
 references which have been encountered with reference to mutability 
 or variabihty of streptococci but also the references to constancy of 
 cultural characteristics. Even if mere loss of virulence be excluded, a 
 majority of the authors who have expressed themselves in any way 
 with reference to variability or constancy have reported instances of 
 variation or have expressed themselyes as regarding such variation as 
 a fact. Except for mere loss of virulence, the variations reported 
 consist principally of inconstancy of appearances in bouillon, in blood 
 agar, and of fermentative power. 
 
 Some of our strains have been in cultivation for about 6 years, 
 notably those from the Boston epidemic (1911). Most of the strains 
 here reported were isolated in the early summers of 1913 and 1914. 
 
 Appearance in Bouillon. — The appearance in bouillon, usually 
 associated with length of chain must be regarded as of minor im- 
 portance. It is true that most of the more virulent beta hemolytic 
 streptococci grow in fairly long chains, cloud the bouiUon slightly or 
 only moderately, and produce a correspondingly abundant sediment, 
 but length of chain or failure to cloud bouillon is not necessarily asso- 
 ciated with virulence. Avirulent strains may produce longer chains 
 with abundant sediment and no clouding of bouillon or may produce 
 short chains and no sediment, clouding the bouillon well. Some 
 strains which when first isolated do not cloud the bouillon well, do so 
 after prolonged cultivation, but others produce no clouding after 3 
 years of cultivation. Among the strains which do not cloud bouillon 
 are those which produce both the alpha and beta t)T)es of hemolysis. 
 
 Fermentation. — Broadhurst (1913) and Holman (1914) have shown 
 that supposed variations in fermentation reactions may be due to the 
 use of inferior media. The latter has rightly advised the use of a 
 medium in which the organisms will grow well in the absence of fer- 
 mentable substance. Fermented bouillon (plus 1 per cent of test 
 substance) was used by Smith and Brown (1915). In view of Hol- 
 
 77 
 
78 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 man's (1914) recommendation of the use of serum bouillon many 
 of the "negative" tests of the strains reported by us have been re- 
 peated, fermented bouillon plus unheated sterile horse serum being 
 used. After an interval of many months the fermentation reac- 
 tions of the strains described in this paper have also been retested, 
 plain fermented bouillon being used in most cases. In neither set of 
 experiments has there been any variation from the results of earlier 
 tests. The validity of our results in fermented bouillon (without 
 serum) may be attributed to the fact that a result was never consid- 
 ered negative unless there was macroscopically a distinct growth in 
 the medium. Fermented bouillon as prepared in this laboratory has 
 been found to be a much better medium than Liebig extract bouillon. 
 There are various ways of preparing fermented bouillon, however. 
 The fermentation is usually carried out with Bacillus coli because it is 
 readily available in all laboratories and is an active fermenter of 
 many sugars. 
 
 Gordon (1905) used "ordinary beef broth freed from sugar by cultivating B. coli 
 therein for 3 days at 37°C. and then sterilized, filtered, rendered slightly alkaline and 
 tinted with litmus." Andrewes and Horder (1906) probably used the same me- 
 dium or beef extract bouillon. Walker (1911) used Gordon's medium for at least 
 part of his tests {"The test media were prepared as directed by Gordon, at first with 
 ordinary bouillon freed from sugar, but later, and for the bulk of the results * * 
 from 'lemco' "). Although Broadhurst (1913) made a comparative study of re- 
 sults obtained with fermented and meat extract bouillon and found the former 
 to be superior, she used meat infusion bouiEon that had been fermented for 7 
 days by Bacillus coli. A number of authors state that they used sugar-free or 
 fermented bouUIon without stating the manner of its preparation. The fer- 
 mented bouillon used in this laboratory was prepared according to the method of 
 Smith and Walker (1897) by fermenting the meat infusion with Bacillus coli 
 for 16 or 18 hours at 37°C., boUing, neutralizing, adding 1 per cent of peptone 
 and 0.5 per cent of salt, and finishing as standard bouillon. Each lot of fer- 
 mented bouillon was tested for the presence of fermentable sugar before being 
 used {Bacillus coli in fermentation tube). Heinemann (1915) made fermented 
 bouillon from meat infusion fermented 24 hours by Bacillus coli. The fermented 
 bouiUon used by Gordon and that used by Broadhurst are subject to criticism 
 in two respects: (1) fermentation by Bacillus coli for more than 18 hours is not 
 only unnecessary to remove all the fermentable sugar but is positively injurious 
 to the medium, since after the exhaustion of the sugar Bacillus coli attacks the 
 protein constituents forming ammonia and other products of protein decomposi- 
 tion which inhibit the growth of other organisms; (2) since Witte peptone (also 
 
JAMES HOWARD BROWN 79 
 
 Digestive Ferments Company peptone) contains no fermentable sugar, nothing is 
 to be gained by its presence in the medium at the time of fermentation, and in fact 
 a better medium is produced if the peptone is not acted upon by Bacillus coli 
 which grows perfectly well in the meat infusion only. 
 
 Fermented bouillon as prepared in this laboratory has been found 
 satisfactory for bringing out the fuU complement of fermentation 
 reactions of all the streptococci studied except Streptococcus mucosus 
 which failed to ferment lactose, rafl&nose, and saHcin in fermented 
 bouillon alone but did ferment them in fermented bouillon plus horse 
 serum. On the other hand, several strains of pneumococci, in- 
 cluding those described in this paper, require the addition of serum 
 to fermented bouillon to bring out their full complement of fermenta- 
 tion reactions. It is suspected that many of the strains of pneumo- 
 cocci reported by various authors not to ferment inuliti would have 
 done so in more suitable media. I have not encountered any pneu- 
 mococci which did not ferment inulin in serum bouillon. The 
 amount of serum added need not be large; 10 per cent is ample and 
 usually 1 drop of serum to 1 cc. of fermented bouillon is sufl&cient. 
 
 Appearance in Blood Agar. — Variations in appearance on blood 
 agar have been reported by many authors since Schottmiiller's (1903) 
 introduction of this medimn for the differentiation of streptococci. 
 It was about 2 years ago that the first photographs of the blood agar 
 plates here shown were made. In the report published by Smith 
 and Brown (1915) it is stated that in repeated tests extending over a 
 period of 14 months we had observed some loss of hemolytic activity 
 for certain strains. This loss of hemolytic activity never took the 
 form of the production of less clear zones of hemolysis but always the 
 formation of slightly smaller zones. Never was there observed the 
 greenish discoloration of corpuscles by strains which had not pro- 
 duced it from the first; in other words, there was never a transition 
 from beta to alpha types. "Strain B-15 from a cow forms an appar- 
 ent exception, but this strain has in the course of our studies split up 
 into a series of forms differing in their taking capacity, some being 
 non-hemolytic at present'^ (Smith and Brown, 1915). The strain B-15 
 just referred to was one of the first isolated by us and in view of what 
 is now known about the influence of commonly ignored factors on the 
 gross appearance of streptococcus colonies in blood agar it seems 
 
80 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 likely that this strain may either have been impure to start with, or 
 its variable appearance may have been due to unrecognized influences. 
 TWhen the strains of streptococci described in this paper were first 
 photographed most of them had been in cultivation for 1 or 2 years, 
 but in so far as could be determined from careful notes their appear- 
 ances in blood agar had not changed since they were isolated. Since 
 then they have been replated many times, but no noteworthy changes 
 have occurred. J In the plates there can be compared photographs 
 taken more than a year apart. ' 
 
 Streptococcus. 
 D-1 7 Compare Fig. 1, Plate 2 with Fig. 3, Plate 2. 
 
 A-18 (cow)/3 Compare Fig. 3, Plate 4 with Fig. 5, Plate 4. 
 B-18 (cow)/3 Compare Fig. 1, Plate 5 with Fig. 3, Plate 5. 
 A-ST,8 Compare Fig. 1, Plate 6 with Fig 3, Plate 6. 
 
 D-AD4,8 Compare Fig. 1, Plate 7 with Fig. 1, Plate 8. 
 B-2b (cow) /3 Compare Fig. 1, Plate 9 with Fig. 3, Plate 9. 
 A-BL a Compare Figs. 1 and 2, Plate 12 with Figs. 4 and 5, Plate 12. 
 B-7 a Compare Figs. 1 and 2, Plate IS with Figs. 1 and 2, Plate 16. 
 
 E-4 a Compare Figs. 1 and 2, Plate 17 with Figs. 5 and 6, Plate 17. 
 
 In the case of the last four strains listed above slight differences 
 appear between the photographs taken on the two dates. In the 
 photograph of streptococcus B-2b (cow)/8 taken in March, 1916 
 (Plate 9, Fig. 3), the surface colonies appear darker than in the 
 photograph taken a yeai' earlier (Plate 9, Fig. 1), though the deep 
 colonies appear the same. The same difference to a lesser degree is 
 noticeable in the case of streptococcus A-ST|8 (Plate 6, Figs. 1 
 and 3). The notes do not indicate that this difference was apparent 
 to the eye and it appears to be largely an artefact due to the use of 
 different kinds of photographic plates each year. 
 
 In the case of streptococci A-BLa, B-7a, and E-4q! it will be seen 
 that in 1916 the characteristic double zones had appeared during 48 
 hours incubation, while in 1915 they were fully developed only after 
 refrigeration. ■ It has already been shown that the early development 
 of double zones may be brought about by the presence of large num- 
 bers of colonies in a plate, but this does not appear to be the cause in 
 the instances under consideration. This difference can apparently 
 be due to very slight and unknown differences in the medium. It does 
 
JAMES HOWARD BROWN ^ 81 
 
 not indicate a change in the properties of the streptococci for it has 
 been seen to come and go during intervals of only a few days in dif- 
 ferent plates inoculated from the same culture tube. It presents an- 
 other reason for regarding the appearances in blood agar as incomr 
 plete until the plates have been refrigerated. A comparison of the 
 1916 plates with the 1915 plates after refrigeration shows remark- 
 able uniformity in appearance. A comparison of rabbit and human 
 blood agar plates made a year apart showed the same permanence of 
 characteristic appearances. Jn none of these strains was there the 
 slightest tendency to change from an alpha to a beia type or vice versa. 
 The permanence of apparently minor characteristics of all the strains 
 studied has been surprising. It is difficult to believe that there are 
 to be found in the human throat a dozen or more fixed varieties of 
 streptococci, and yet there have been isolated from a single swab 
 eight or nine strains which have retained individual characteristics- 
 for many months. This will seem to be contrary to the results of 
 Rosenow (1912) and others who have reported variations in the cul- 
 tural characteristics of streptococci, but it is not necessarily so. 
 Many of the variations reported may easily have been due to unrecog- 
 nized differences in media or technique employed over various inter- 
 vals of time, but it should be pointed out that the present effort for 
 several years has been to keep strains of streptococci undtet as uniform 
 and as favorable conditions as possible so as to determine whether or 
 not under these conditions cultural characteristics could be rehed 
 upon for the diffjerentiation of varieties of streptococci. These 
 strains have not been subjected to conditions likely to cause varia- 
 tions. The nearest approach to such conditions has been (1) passage 
 of some strains through rabbits as described by Smith and Brown 
 (1915), (2) keepiag of some strains on plain agar slants rather than 
 blood agar slants for several months, and (3) the drying of droplets 
 of bouillon cultures of several representative strains on sterile cover- 
 glasses for 3 months. No changes were brought about by any of these 
 procedures. Rosenow, on the other hand, subjecting strains to re- 
 peated animal passage and to various adverse conditions has re- 
 ported many fundamental changes in the characters of strains so 
 treated. I have made no attempt to repeat his work. It is be- 
 lieved, however, that a clear definition of various types of appear- 
 
82 BLOOD AGAR FOR STXJDY OF STREPTOCOCCI 
 
 ance in blood agar under carefully defined conditions wiU do much to 
 discourage the mistaking of differences in appearance due to vari- 
 able conditions for tnje variations or mutations of the organisms 
 themselves. 
 
 In this connection it may be worth while to mention the manner 
 in which our stock cultures are kept. To the surface of standard meat 
 infusion agar slants two or three drops of defibrinated horse blood are 
 added and the tubes incubated 48 hours to test their sterility. The 
 condensation fluid of these slants is inoculated and streaked upward 
 along the slant. Longevity of the culture is much greater if the 
 inoculated tubes are incubated only long enough for a fair amount of 
 visible growth to appear, usually just over night. They are then placed 
 in the refrigerator at 40 to 4S°F. and kept there until discarded. The 
 cotton plugs of these tubes have been dipped quickly into sterile 
 paraffin and at the time the tubes are inoculated are set afire and 
 pushed down into the tubes. The paraffin in the plugs is not suffi- 
 cient to make them air-tight but probably does help to prevent rapid 
 drying of the medium. No caps or seals of wax or paraffin are 
 necessary. Molds never cause trouble. On this medium all strains 
 except Streptococcus mucosus and the pneumococci are ordinarily 
 renewed once in 4 or 5 months. Of fifty strains of various types 
 which were recently allowed to go 7 months without renewal all 
 but two remained viable. The pneumococci and Streptococcus 
 mucosus are usually renewed each month but are viable after 2 
 months. All inoculations of animals or test media are made from 
 fresh bouilldn cultures which are always inoculated directly from the 
 stock tubes. Experiments are never conducted with cultures inocu- 
 lated from one test medium to another in an indefinite sort of way, 
 but always from^ fresh bouillon cultures taken directly from stock 
 cultures. The stock cultures are kept in the refrigerator and are 
 used for no other purpose than this. The keeping of stock cultures 
 has not been entrusted to assistants. 
 
 For a review of the hterature the reader is referred to Table VII 
 on Mutations and Variability. 
 
OCCURRENCE OF VARIOUS TYPES OF STREPTOCOCCI. 
 
 The markedly hemolytic {beta type) streptococci are commonly 
 present in various suppurations (otitis media, osteomyelitis, adeni^ 
 tis), erysipelas, and in sore throats, rarely in clinically normal throats. 
 Green-producing {alpha tj^e) and indifferent {gamma t)^e) strepto- 
 cocci are commonly found in normal throats, but though often pres- 
 ent in mixed culture they have not been found by me under con- 
 ditions that would indicate them as the cause of acute suppurations. 
 The blood agar plates inoculated with material from acute sore throats 
 or from normal throats are, therefore, quite homogeneous and char- 
 acteristic in appearance. On the other hand, plates inoculated with 
 material from chronic sore throats resemble those from convalescent 
 throats; they are very heterogeneous in appearance often containing 
 colonies of the alpha, alpha prime, beta, and gamma types with a 
 great variety of quantitative differences among themselves, so that 
 as stated above (page 81) as many as eight or nine distinguishable 
 varieties of streptococci may be obtained from a single swab. Three 
 such plates are shown in Plate 32. The finding of the alpha prime 
 type is characteristic of convalescent or chronic sorfe throats. In 
 several cases of acute sore throat material was obtained at intervals 
 of a day or two. The beta t)^e colonies were present in large num- 
 bers at first, gradually yielding to the appearance of the alpha prime 
 type and the great variety of alpha type colonies characteristic of 
 convalescent throats, until finally only the typical alpha axiA gamma 
 types remained in the normal thtqat. The succession of types fol- 
 lowed in this way strongly suggests the change of one type into an- 
 other. Yet when these seemingly changing strains have been iso- 
 lated they have remained for months or years in cultivation with 
 their characters fixed just as they were when isolated. It can not be 
 said whether or not they were actually changing in the throats from 
 which they were taken. It seems strange that there should be this 
 succession of tj^es from markedly hemolytic to moderately hemolytic, 
 less hemolytic, and green-producing. Still it must be remembered 
 that the latter are always present in greater or less numbers. The 
 
 83 
 
84 BLOOD AGAR TOR STUDY OF STREPTOCOCCI 
 
 fact that the great variety of tjrpes to be found in plates from con- 
 valescent throats can be arranged in an almost perfect series with 
 reference to their appearance does not mean that they are derived 
 one from another. The various strains of streptococci illustrated in 
 the plates can be arranged in an almost perfect series as is shown in 
 Plate 31, but each of these strains came from a different individual 
 and from five different epidemics in five different localities; there 
 can scarcely be supposed any genetic relationship among them. 
 But if they all appeared on the same plate (and just as good a series 
 has been seen on a single plate) the temptation to regard them as 
 derived one from another would be strong. One is reminded, how- 
 ever, of what occurs in a bed of cultivated plants which is later aban- 
 doned. During the abnormal condition of artificial cultivation there 
 is an almost pure culture of the plant cultivated. Abandoned the 
 next season the loose upturned soil supports the growth of a great 
 variety of weeds. Finally, however, after another season or two the 
 native grass crowds in from the edges of the bed choking out most 
 of the weeds. In the case of the streptococci a close series of forms 
 can be noted by fixing the attention on a single character; i.e., the 
 amount of hemolysis. If in the case of the flower bed the atten- 
 tion is similarly fixed on a single character, e.g. the height of the 
 plants or the color of the flowers, there can also be arranged a close 
 series among the weeds. To indicate an actual genetic series, how- 
 ever, either the various forms must be found to differ with respect 
 to the single character only or the same series must be confirmed by 
 other variable characters. With these streptococci such a series is 
 not apparent. The fermentation reactions and the morphology of 
 the various strains do not suggest the same series as do the hemo- 
 lytic types. 
 
 Reviewing the literature one is impressed with the similarity of blood agar 
 plates inoculated with vaginal or uterine secretions to those inoculated with ma- 
 terial from throats. Most authors working with vaginal streptococci agree that 
 the streptococci found in cases of severe puerperal infection are of the markedly 
 hemolytic type. They also agree that ante partum in normal cases there are 
 usually to be found only non-hemolytic or green-producing streptococci. Post 
 partum in clinically normal cases there are usually found only the non-hemolytic 
 or green-producing types, though in a minority of cases hemolytic streptococci 
 may also be found. Heynemann (1908) and Zangemeister (1910) regarded the 
 
JAMES HOWARD BROWN 85 
 
 finding of hemolytic streptococci in clinically normal cases as of grave signifi- 
 cance or at least as potentially grave. Fromme (1908) and Heynemann (1908) 
 mention having found intermediary (nioderately hemolytic) types in conval- 
 escent cases of puerperal infection. 
 
 The general subject of the relation of hemolysis to the source of strains is 
 reviewed in Table Vm on General Conclusions, etc. The work of the following 
 authors as reviewed in Table VIII on General Conclusions, etc., and in Table 
 III on The Use of Blood Agar should be especially noted: Heynemann (1908); 
 Fromme (1908); Ludke and Polano (1909); Konrad (1909); Sigwart (1909); 
 Zangemeister (1910); Rosenow (1910); Fabre and Bourret (1910); Gmindei' 
 <1912); Puppel (1912); Ruediger (1912); Sitzenfrey and Vatnick (1913); Davis 
 <1914) ; GUmer and Moody (1914) ; Hartzell and Henrici (191S) ; Rosenow (1915) ; 
 Broadhurst (1915); Rosenow and Dunlap (1916); Rosenow and Oftedal (1916); 
 Davis (1916); Smillie (1917). 
 
CLASSIFICATION OF STREPTOCOCCI. 
 
 The difficulty encountered in tiying to identify the strains of strep- 
 tococci isolated by Dr. Smith and myself has already been men- 
 tioned. The reason for such difl&culty is that various authors in 
 attempting to name or classify streptococci have fixed their attention- 
 upon different characters as criteria. Such names as 5. pyogenes,. 
 S. erysipelatos, S. epidemicus, S. lacticus, S. equinus, S. equi, S. an- 
 ginosus, S. fecalis, S. salivarius, S. rheumaticus, S. saprophyticus, and 
 S. masHtidis were suggested by consideration of the source of the 
 organisms. Such names as 5. longus, S. longissimus, S. brevis, S. 
 conglomeratus, and 5. lanceolatus were suggested by morphological 
 peculiarities. Such names as 5. pathogenes, S. mitior, and S. mitis 
 have reference to pathogenicity or virulence. Such names as S. 
 Hemolyticus, S. viridans, S. anhemolyticus, S. mucosus, and 5. acidi 
 lactici refer to certain cultural characteristics. 
 
 Comparatively few authors have considered all the above criteria, 
 in grouping or classifying the strains studied by them. Generally 
 speaking, the German authors have given most of their attention to 
 the morphology of streptococci and their appearance on blood agar, 
 with little attention to fermentation reactions; the English authors 
 have given more attention to ftermentation reactions, but very little 
 to appearance on blood agar; American authors have been divided in 
 giving their attention to one or the other set of characters and a. 
 number have tried to correlate the two. Sourcfe and pathogenicity 
 for laboratory animals have engaged the attention of authors of all 
 nationahties. 
 
 In view of the emphasis being placed on one or another character 
 it is to be expected that the various names and classifications of strep- 
 tococci should overlap. Such questions as the following arise. I& 
 S. pyogenes a 5. hemolyticus and what are its fermentation reactions? 
 Is 5. mitior (Schottmiiller) s3aion5anous with 5. mitis (Andrewes and 
 Horder)? Is S. longus seu erysipelatos (Schottmiiller) synonymous 
 with 5. longus (von Lingelsheim) or with S. erysipelatos (Fehleisen)? 
 Do S. longissimus and 5. conglomeratus belong to the hemolytic^ 
 
 ae 
 
JAMES HOWASD BROWN 87 
 
 non-hemolytic, or viridans groups? Do authors regard hemolytic 
 green-producing streptococci as S. hemolyticus or as S. viridans? A 
 review of the literature reveals the need for answers to these and 
 many similar questions. The present nomenclature of streptococci 
 is almost chaotic. 
 
 The designation of streptococci as S. longus and 5. brevis as proposed by von 
 Lingelsheim (1891) appears to be of little value not only because it is a matter 
 of common laboratory experience that length of chain is readily influenced by 
 conditions of cultivation, but also because 5. longus, defined by von Lingelsheim 
 (1899) as including those streptococci which have a tendency to grow in chains 
 of six or more elements, includes practically all streptococci. In fact, under 
 S. longus {pyogenes) von Lingelsheim (1912) includes in addition to the ordinary 
 5 pyogenes (1) Obligate anaerobic streptococci; (2) 5. conglomeratus; (3) S. lon- 
 gissimus; (4) 5. mitis sen viridans. He excludes S. lacticus, S. mucosus, and the 
 pneumococci. His inclusion of "S. mitis seu viridans" (presumably S. mitior 
 seu viridans) under 5. longus (pyogenes) is in direct disagreement with the classi- 
 ^cation of Schottmiiller (1903) who distinguished (1) 5. longus pathogenes seu 
 erysipelatos, (2) 5. mitior seu viridans, and (3) S. mucosus. Von Lingelsheim 
 fixed his attention upon morphology, Schottmiiller his upon source and appear- 
 ance on blood agar. Adopting the test substances proposed by Gordon (1905), 
 Andrewes and Horder (1906) distinguished (1) S. eguinus, (2) 5. mitis, (3) 5. 
 pyogenes, (4) S. salivarius, (5) S. anginosus, (6) 5. fecalis, and (7) pneumococci. 
 This classification was based largely upon the correlation of fermentative char- 
 acters and source. 5. pyogenes and S. anginosus are merely mentioned as being 
 "hemolytic" while S. fecalis is indicated as "non-hemolytic" on blood agar. 
 Mandelbaum (1907-08.) described 5. saprophyticus as producing neither hemo- 
 lysis nor discoloration on blood agar. He also recognized S. mitior as described 
 by Schottmiiller. 5. longissimus and 5. conglomeratus were described by Thal- 
 mann (1910) who says that they produced on blood agar either no hemolysis or 
 greenish partly hemolyzed zones. He describes S. brevis as being non-hemolytic 
 and usually producing more or less green color. Thalmann (1911) described 
 strains from four cases of angina which produced on blood agar neither hemolysis 
 nor discoloration and which he regarded as varieties of 5. pyogenes though he had 
 previously (1910) described the latter as producing clear hemolyzedzones. Zange- 
 meister (1910) recognized "hdmolytische" streptococci and 5. viridans as de- 
 scribed by Schottmiiller, and designated those streptococci which produced 
 neither hemolysis nor discoloration on blood agar as 5. anhemolyticus vulgaris 
 which is therefore apparently synonymous with 5. saprophyticus (Mandelbaum). 
 Roily (1912) distinguished (1) 5. hemolyticus (apparently synonymous with S. 
 longus pathogenes seu erysipelatos (Schottmiiller), (2) 5. anhemolyticus, (3) 5. 
 viridans, and (4f 5. mucosus. Davis (1913) characterized (1) S. hemolyticus, 
 (2) 5. epidemicus, (3) 5. mucosus, (4) S. pneumoniae, and (5) 5. viridans with ref- 
 
88 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 erence to appearance on blood agar, presence or absence of capsules, solubUity in 
 bile, fermentation of inulin, and pathogenicity for rabbits. His use of the 
 terms S. hemolyticus and 5. viridans is evidently much more restricted than is 
 their use by RoUy and SchottmiiUer respectively. 
 
 Lyall (1914) proposed to differentiate streptococci as (1) Herdolytic, (2) Methemo- 
 glohin Producers, and (3) Indifferent, according to their action on a suspension of 
 sheep corpuscles. By taking into consideration also the fermentation of inulin, 
 salicin, and rafBnose, he distinguished (1) Pneumococci and Pneumococcus mucosus, 
 (2) S. pyogenes, (3) 5. viridans and Variants, and (4) 5. fecalis and other strepto- 
 cocci. L/all's scheme may be criticized from two standpoints: (a) the diflferen- 
 tiation of Methemoglohin Producers from Indifferent strains by his method is an 
 artificial one since some strains may be Methemoglohin Producers with respect to 
 the blood of one species of animal but Indifferent with respect to the blood of 
 another species; (J) too many strains fall into groups 3 and 4; i.e., S. viridans and 
 Variants and S. fecalis and other streptococci; furthermore strains of 5. fecalis 
 are green producers and therefore belong to the S. viridans group as originally 
 defined by SchottmuUer. The grouping proposed by Kligler (J915) is incomplete 
 and not sufficiently definite. 
 
 The classification of Holman (1916) has much to commend it. It is prac- 
 tical, definite, and recognizes the names which have already been applied to 
 various streptococci. Holman classifies streptococci with reference to their 
 being hemolytic or non-hemolytic on blood agar and with respect to their be- 
 havior towards. lactose, salicin, and mannite, though he also recognizes inulin- 
 fermenting varieties of most of the non-hemolytic streptococci. 
 
 The scheme proposed by Hohnan should be extended and rear- 
 ranged, howevej. The alpha, beta, and gamma appearances in blood 
 agar should be recognized instead of dividing all streptococci into 
 hemolytic and non-hemolytic groups. Although lactose, man- 
 nite, and salicin are of primary differential value, rafl&nose and sac- 
 charose should not be ignored. Notwithstanding the statements of 
 Broadhurst and Holman that the fermentation of raffinose and of 
 inulin run almost parallel to each other, I do not find it so, nor 
 does it appear so in Broadhurst's tables. Of ,666 strains tabulated 
 (Table II), 102 act differently towards rafl&nose and inulin, and of 
 these strains 59 are reported by Broadhurst (1915). With three 
 substances there are eight fermentative complexes. By duplicating 
 these eight complexes under hemolytic and non-hemolytic groups 
 Holman was able to differentiate all the type forms of Andrewes and 
 Border and some others. With four substances there are sixteen 
 complexes, with five there are thirty-two, and with six there are 
 
JAMES HOWARD BROWN 89 
 
 sixty-four. If streptococci are divided into a hemolytic and a non- 
 hemolytic group the number of complexes is doubled, and if three 
 types of appearance in blood agar are recognized the number of 
 complexes is tripled. If, as seems advisable, the alpha, beta, and 
 gamma types are recognized in blood 'agar' and if six test substances 
 for fermentation, are employed, there would be 192 possible com- 
 plexes, many of which have not been encountered among the strep- 
 tococci. This would seem to be an impossible number of complexes 
 to keep in mind or to work with. Even Andrewes and Horder tabu- 
 lated only 94 complexes. However, as a working basis in the: absence 
 of a natural classification I have employed a tabulation of 192 com- 
 plexes which obviates the reduplication and 'ambiguity of some of 
 Andrewes and Herder's so called "variants" anA which preserves the 
 simplicity of Hohnan's scheme without ignoring the differential v^lue 
 of saccharose, raflBnose, and inulin as test substances. 
 
 In Table II streptococci are placed according to "types" in blood 
 agar; "groups" with fespect to fermentation of mannite, lactose, and 
 salicin; and "subgroups" with rtespect to fermentation of saccharose, 
 inulin, and rafl&nose. The types are indicated by a, |8, and y. The 
 groups are numbeired 1 to 8, and the subgroups 0.1 to 0.8 inclusive. 
 By this means the hemolytic and fermentative properties of a strep- • 
 tococcus can be referred to by a letter, a number, and a decimal. 
 Examplies: q;1.2 indicates a gre'en-producing streptococcus which fer- 
 ments lactose, salicin, saccharose, and raffinose but does not fer- 
 ment mannite or inulin. /31.2 indicates a streptococcus having the 
 same fermentative powers as the, above but a different type of ap- 
 pearance in blood agar, al.l indicates a streptococcus differing from 
 q;1.2 only with respect to raf&nose. It is surprising how soon and 
 how easily the different munbers are carried in mind. This is made 
 possible by the orderly alternation of positive and negative signs so 
 that, for instance, it is soon learned that groups 1 to 4 are mannite 
 non-fermenters while groups 5 to 8 are mannite fermenters; the 
 groups indicated by odd numbers are salicin fermenters while the 
 non-fermenters are represented by even numbers, etc. The signs in 
 the subgroups are just the reverse of those in the groups. It may 
 be asked what has determined the particular order in which the test 
 substances are tabulated. The order is evidently mannite, lactose, 
 
90 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 salicin, saccharose, inulin, and raffinose.' This order was adopted 
 not because it represents any natural relationship of the test sub- 
 stances or any metabolic gradient, but because it was found to bring 
 the well known groups to the top of the table and the typical members 
 or subgroups to the left where they are easily remembered. It ap- 
 pears rather remarkable that so many of the well known strains 
 which have been given specific names by various authors should by 
 this means fall into subgroup 0.1 of groups 1 to 5. What may be 
 called the "typical member" of each group is therefore indicated by 
 the numbers 1.1, 2.1, 3.1, or 4.1, etc. This means simply that the 
 "typical members" ferment saccharose but do not ferment inulin or 
 rafl&nose, while the "atypical members" act differently towards one or 
 more of these three substances. In describing Streptococcus sali- 
 varius (a2.1) as not fermenting ra£&nose it is realized that Andrewes 
 and Horder describe the " type form" as =<= with regard to raffinose. 
 However, the more restricted use of the name is justified by the 
 fact that only 24 per cent of the Streptococcus salivarius strains tabu- 
 lated by these authors did ferment rafl&nose while 76 per cent did 
 not ferment it. The non-rafSnose-fermenting strains should be re- 
 garded as being "typical members" of this group. 
 
 In Table II is indicated the number of strains belonging to each 
 subgroup which have been reported by Smith and Brown (1915), 
 Hartzell and Henrici (1915), Kligler (1915), Broadhurst (1915), 
 Smillie (1917), and Davis (1916). The strains of these authors have 
 been selected because they have employed all the six test substances 
 listed in this table and have indicated whether their strains produced 
 greenish discoloration, hemolysis, or neither on blood agar, corre- 
 sponding roughly to the alpha, beta, and gamma types." It should 
 be noted that of the 666 strains listed in this table 381 or nearly 60 
 per cent of them are " typical members" of the various groups. Among 
 the beta type streptococci there are very few "atypical members," 94 
 per cent being "typical." Streptococci of the gamma type are the 
 
 ' Dextrose and maltose are assumed to be fermented in all cases unless speci- 
 fically stated otherwise. I have never encountered a streptococcus which failed 
 to ferment these two sugars in a favorable medium. 
 
 '" From Broadhurst's paper I have not included strains of doubtful types such 
 as are labeled "haze," " haze-hemolysis ," "hemolysis?," and "no color-ham." 
 
JAMES HOWARD BROWN 91 
 
 least homogeneous with regard to the fennentation reactions, the least 
 numerous, and the least studied. The name of the "typical mem- 
 ber" of each group should not be employed for the "atypical mem- 
 bers" of the group since many of these names as Streptococcus sali- 
 varius, Streptococcus equinus, etc., indicate the common habitat or 
 source of these strains, and there is at present no reason to believe 
 that such strains as q;5.4 or aS.S come from the same sources as aS.l 
 or that they are any more nearly related to aS.l than to a\A or al.S. 
 The use of the term "variant" as used by Andrewes and Horder is 
 also objectionable since there is no evidence that their so called vari- 
 ants are variants of their "type forms." There is no apparent objec- 
 tion, however, to calling group al the Streptococcus mitis group or 
 «5 the Streptococcus fecalis group, etc., so long as it is understood that 
 the inclusion of the various subgroups or atypical members' within 
 the groups is purely an arrangement for convenience based upon a 
 certa'n similarity of fermentative characters which may br may not 
 indicate a natural relationship. The tabulation employed has been 
 very useful. It is perfectly definite. It provides space for all the 
 streptococci so far described with sufficient detail to be classified 
 according to hemolytic and fermentative characters and for many not 
 yet described. The group may be determined and indicated as 
 Holman has done, without indicating the subgroup, if desired. Recog- 
 nition is given to the names of streptococci already described by 
 various authors. 
 
 There should also be mentioned certain names which have been applied by 
 various authors to streptococci the description of which with reference to fer- 
 mentation or appearance in blood agar is not sufficiently complete to enable 
 them to be placed in the table just described. Streptococcus rheumaticus (Poynton 
 and Paine, 1902) represents probably a number of different varieties found in cases 
 of rheumatism, belonging probably to the alpha, alpha prime, and gamma types 
 (Rosenow, 1914). Streptococcus bovinus (Winslow, 1908) belongs to group 1.2, 
 and from the fact that it was found in large numbers in bovine feces, I suspect 
 that it belonged to the alpha type, but its appearance on blood agar was not 
 described by the author. Most of Broadhurst's (1915) strains were of the alpha 
 type. Streptococcus gracilis (Escherich) can not be grouped because of insuffi- 
 cient data. Streptococcus versatili; (Broadhurst, 1915) apparently belongs to 
 group 5.2 and to the alpha or the gamma type, mostly to the former. Streptococcus 
 mucosas (Howard and Perkins, 1901) and the pneumococci belong to the alA 
 or the aSA type and groups, and are further distinguished by the presence of 
 
92 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 capsules, solubility in bile, etc. Streptococcus longissimus (Thalmann, 1910) in 
 all probability belongs to the alpha type but its fermentation reactions are not 
 given. Strain D-ADSja (described above, page 21) which is undoubtedly a 
 Streptococcus longissimus is also a Streptococcus salivarius (a2.1). Strains D-la 
 and F-2a of Streptococcus conglomeratus (Kurth, 1891) belong to groups al.l 
 (Streptococcus mitis) and a2.2 respectively. Thalmann (1912) came to the con- 
 clusion that Streptococcus conglomeratus was not a distinct species or variety but 
 merely a degeneration form of various other streptococci brought about by 
 growth under unfavorable conditions. This view may be supported by the fact 
 that I have found strains of Streptococcus conglomeratus belonging to the beta 
 type as well as those of the alpha type. Heinemann (1906) came to the conclu- 
 sion that Streptococcus lacticus (Kruse) is not a distinct species but merely a 
 name for various non-pathogenic, vigorous lactic-acid-producing streptococci 
 commonly found in mUk as contaminations. This view would seem to be sup- 
 ported by the work of Saito (1912) whose descriptions would indicate that his 
 strains of Streptococcus acidi lactici belonged to the alpha type. They all coagu- 
 lated milk and fermented lactose and dextrose, but differed with respect to man- 
 nite. These facts are in harmony with these strains belonging to groups a2 
 (Streptococcus salivarius) and aS (Streptococcus fecalis) . In the last edition of 
 Kolle and Wassermann's Handbuch der pathogenen Mikroorganismen (1912) 
 von Lingelsheim gives a tabular summary of characteristics of Streptococcus 
 longus, Streptococcus lacticus, the pneumococcus, and Streptococcus mucosus. 
 There is nothing in this table to differentiate various non-hemolytic or green- 
 producing streptococci from Streptococcus lacticus. 
 
 The descriptions of the following streptococci are not sufficiently complete to 
 classify them with respect to appearance in blood agar and fermentation of 
 sugars. Streptococcus pyogenes bovis is stated by a number of authors (Glage, 
 in Kolle and Wassermann, 1912) to be indistinguishable from Streptococcus pyog- 
 enes hominis but distinct from Streptococcus agalactice contagiosa bovis. The 
 streptococci causing mastitis in cows have been called Streptococcus mastitidis. 
 Streptococcus agalactia, Streptococcus mammitis bovis, etc. From the literature 
 it cannot be determined whether they refer to the same or different organisms. 
 Mastitis in sheep and goats is produced by Diplococcus lanceolatus avium 
 (Gaertner) and Streptocotcus ovis (Wiemann) . The former is described as being 
 encapsulated but non-hemolytic^ The latter has no capsule but is hemolytic. 
 
 The streptococci mentioned in the preceding two paragraphs need 
 to be again studied by modern methods. 
 
 Addendum. — Siace this article was written there has appeared a paper by 
 Francis G. Blake on The Classification of Streptococci.^^ He proposes a scheme of 
 classification "for clinical purposes." On the basis of appearance in blood agar 
 
 " Blake, F. G., /. Med. Research, 1917, xxxvi, 99-133. 
 
JAMES HOWARD BROWN 93 
 
 plates the streptococci are divided into two divisions (1) Streptococcus hemolysans 
 and (2) Streptococcus viridans. Blake says: 
 
 "Further subdivision of Streptococcus hemolysans by means of fermentation reactions is 
 of no practical value. Streptococcus viridans, however, is divided into three subgroups by 
 its fermentation reactions in lactose and mannite, a procedure which is of value in that it 
 gives some indication of the source of the strain in question. These subgroups are (a) Strepto- 
 coccus buccalis characterized by the fermentation of lactose and failure to ferment mannite, 
 (6) Streptococcus fecalis characterized by the fermentation of both lactose and mannite, and 
 (c) Streptococcus equinus characterized by the failure to ferment either lactose or mannite. 
 Limitation of the carbohydrate-fermentation reactions to the use of lactose and mannite 
 reduces the number of unclassified or variant strains to a negligible minimum." 
 
 While this procedure may serve most clioical purposes the introduction of 
 new specific names is a question of biological nomenclature and is not justified 
 entirely by chnical purposes. If the names proposed by Blake are to indicate 
 so few differential characters, would it not serve clinical purposes just as well to 
 speak of a lactose + m&nmte— Streptococcus viridans (or more briefly aL+M— ) 
 as to introduce new names of less specific significance than those already in use? 
 Progress would seem to lie in the direction of better differentiation of the 
 species or varieties already recogm'zed. Only in this way can it be discovered 
 whether or not such varieties are of clinical or other practical or theoretical 
 importance. 
 
SUMMARY. 
 
 In the foregoing pages there have been illustrated and, described in 
 greater detail than heretofore the alpha and beta t)^es of appearance 
 produced by streptococci in blood agar. There have also been de- 
 scribed the alpha prime and gamma tj^es not heretofore described as 
 such. 
 
 There has been emphasized the importance of studying the deep 
 colony microscopically, the finer differential characters of the sur- 
 face colonies being obscured by the overgrowth of the colonies. 
 
 Between 5 and 10 per cent of blood in suf&cient agar to make a 
 layer of medium 2 mm. thick reveals the maximum amount of dif- 
 ferentiation by colonies of various streptococci. 
 
 The beta and gamma types streptococci produce essentially similar 
 appearances in blood agar made with defibrinated horse, rabbit, or 
 human blood". Some strains of alpha type streptococci and pneumo- 
 cocci produce macroscopically widely different appearances in blood 
 agar made with different kinds of blood, differences so great that a 
 strain may appear hemolytic in one medium and non-hemolytic in 
 another; but microscopically the differences are seen to be quanti- 
 tative rather than qualitative so that there need be no confusion 
 among the various types. 
 
 Streptococci of the alpha type when numerous in a blood agar 
 plate produce hemolysis more readily than when there are fewer in 
 the plate. 
 
 Defibrinated blood is suitable for use in blood agar so long as there 
 is no marked laking, the corpuscles are readily shaken up, and the 
 blood is in apparently good condition. 
 
 Standard meat infusion agar should be used rather than meat 
 extract agar. 
 
 The titratable acidity of the meat infusion agar shotild be in the 
 neighborhood of 1 per cent N acid to phenolphthalein but may vary 
 as much as 0.5 per cent below or above this reaction without exerting 
 a marked influence on the appearances produced by growth of the 
 streptococci. 
 
 94 
 
JAMES HOWARD BROWN 95 
 
 Either veal or beef may be used in making the agar. Differences 
 in appearance produced by the use of Witte or Digestive Ferments 
 Company peptone are relatively slight. 
 
 The presence of sm,all amounts of dextrose in the medium (as 
 little as 0.05 per cent) produces a marked effect. Actively ferm.ent- 
 ing gamma type streptococci may produce an appreciable amount of 
 acid hemolysis, especially noticeable in meat extract agar, probably 
 because of the lower "buffer effect'.' of this medium as compared with 
 meat infusion agar. Greenish or brownish discoloration similar to 
 the alpha appearance is not produced by beta or gamma type strepto- 
 cocci in dextrose blood agar, though if sufl&cient fermentation takes 
 place there may be a general brownish discoloration of the entire 
 plate beginning in the neighborhood of the colonies. The most 
 noticeable effect of the presence of dextrose in the blood agar is the 
 partial or total inhibition of hemolysis by actively hemolytic beta 
 type streptococci. In sugar-free media the alpha type streptococci 
 still produce their characteristic double zones, though growth may 
 be so scant as to make their differentiation difficult. The discolora- 
 tion and fixation of corpUscles by alpha type streptococci is encouraged 
 by the presence of a small amount of dextrose in the medium. 
 
 Under anaerobic conditions alpha type streptococci and pneimio- 
 cocci produce beta type zones. 
 
 If a beta type streptococcus is present in a colony or streak mixed 
 with 3,13. alpha or gamma type organism the presence of either of the 
 latter may be completely masked by the former. 
 
 Corpuscles in blood agar which have become discolored greenish 
 or brownish in the neighborhood of colonies or streaks of alpha tj^e 
 streptococci or pneumococci are no longer subject to hemolysis by 
 beta type streptococci. 
 
 Colonies of beta type streptococci in laked blood agar do not pro- 
 duce zones of hemolysis or decolorization; they produce no apparent 
 alteration of the Surrounding niedium. A blood agar plate heavily 
 inoculated with beta type streptococci is laked but not decolorized 
 during 24 or 48 hours incubation. More prolonged incubation re- 
 sults in gradual brownish discoloration and decolorization. A simi- 
 lar result is obtained by the growth of beta type streptococci in blood 
 bouillon. As a result of the above and other observations it is con- 
 
96 BLOOD AGAR FOE STUDY OF STREPTOCOCCI 
 
 eluded that the clear colorless zones produced by beta type strepto- 
 cocci within 24 hours are the result of laking of the blood corpuscles 
 and diffusion outward of the released hemoglobin. 
 
 The alpha -type zones are probably best explained by the theory 
 that (1) in the neighborhood of the colony there are processes of re- 
 duction and oxidation which result in methemoglobinization of the 
 blood corpuscles, apparent as a brownish or greenish discoloration; 
 and (2) there is released a hemolytic substance which diGfusing outward 
 from the colony attacks the corpuscles outside the region of methe- 
 moglobinization. Methemoglobinization occurs only at incubator 
 temperature; hemolysis occurs readily at room or refrigerator tem- 
 perature. A combination of these factors results in the formation of 
 alternate concentric rings of methemoglobinized corpuscles and of 
 hemolysis about the alpha type colonies when blood agar plates are 
 exposed to alternate periods of incubation and refrigeration. 
 
 There is reason to suspect that the hemolytic substances produced 
 by alpha and beta types streptococci may not be the same. 
 
 When kept under suitable conditions the minute cultural charac- 
 teristics of different strains of streptococci persist during long periods 
 (several years) of cultivation on artificial media. This fact does not 
 prejudge the possible effect of adverse conditions on streptococci. 
 
 The succession of various types of streptococci noted in convales- 
 cent throats and reported by others as occurring on other mucous 
 membranes is regarded as a true succession of different strains accom- 
 plished by changing conditions rathfcr than as a variation in the 
 characters of the strains present. 
 
 In the absence of a natural classification of streptococci there is 
 presented a convenient tabulation in which the names already applied 
 have been retained and definitely defined with respect to type of ap- 
 pearance in blood agar and fermenta;tion reactions. Although the 
 specific names retained are in some instances given a more limited 
 application than that intended by the authors who have described 
 them, they are in no case given a different meaning. 
 
 In any system of classification or characterization? that may be 
 adopted it is urged that the loosely used terms "hemolytic" and "non- 
 hemolytic" be replaced by recognition of the alpha, beta, and gamma 
 types. 
 
JAMES HOWARD BROWN 97 
 
 In view of the influence of many commonly ignored factors on 
 the appearances in or on blood agar it is urged that ia describing such 
 appearances authors give full details regarding the composition and 
 amount of medium used, the period of incubation, and subsequent 
 treatment, method of inoculation, etc. 
 
APPENDIX. 
 
 A Simple System of Notes for Preserving in A ccessible Form the Genealogy 
 and Other Details in the Study of Bacterial Cultures. 
 
 In view of the fluctuating variations and mutations of streptococci 
 reported by a number of observers it was realized at the beginning 
 of the work by Dr. Smith and myself that it was important to pre- 
 serve a complete genealogical history of every strain studied by us 
 from the time it came into our possession. Such a record we had and 
 it has been continued and elaborated. It is quite comttion for bac- 
 teriologists to attach a "generation" number to each transplant of 
 their strains so that they are able at any time to determine how 
 many transplants or so called generations a particular strain has 
 passed through. However, the kinds of media on which the strain has 
 been cultivated; the age, reaction, and composition of each lot of 
 medium; the length of time spent on it; the time and temperature of 
 incubation and of storage; the kind, age, and condition of blood or 
 ascitic fluid (if any) added to the mediimi, and if necessary the con- 
 dition of the animal from which the blood or ascitic fluid was drawn, 
 are all details which may be quite as important as the number of 
 generations through which a strain has passed. To say that a strain 
 has undergone "spontaneous" mutation or variation is merely to say 
 that the causes or conditions of such changes have escaped recogni- 
 tion. It is important, therefore, to employ a system of notes which 
 will record as many details as possible. It is equally important that 
 such a system shall be simple and elastic, requiring practically no 
 time or attention beyond the recording of observations, essential with 
 any system of notes. Finally, when recorded, the notes must be 
 accessible, for the most accurate observations are of little value if 
 lost in a mass of notes. 
 
 A knowledge of the way in which records are kept is of importance 
 in the proper valuation of work involving the question of variation. 
 An apparent mutation may quite as readily be the result of an error 
 in. record as of an error in observation or of working with a mixed 
 
 98 
 
JAMES HOWARD BROWN 99 
 
 culture. No apology is therefore due for describing a system of 
 notes which is by no means entirely original but has been made 
 possible by and is a development of methods found in use in this 
 laboratory. 
 
 Media. — The laboratory assistant who makes the media keeps a 
 journal in which is described in detail the making of each lot of me- 
 dium; the materials entering into it, especially the source and condi- 
 tion of the meat, the temperature and time relations, and the reac- 
 tion as determined by titration at various stages and that of the 
 finally tubed and sterilized product. A serial number entered in the 
 journal and recorded on each tube or flask of the medium makes it 
 possible for one using the medium to refer to the journal at any time. 
 
 Animals: — ^The assistant having charge of the animals gives to 
 each animal entering the laboratory a serial number which is entered 
 on a blank with the animal's identification, history, and occasional 
 observations as to its weight and general condition. When the ani- 
 mal is assigned to a worker for use, the blank passes to his care and 
 on it he records his own notes regarding the use of the animal, clini- 
 cal notes, and finally the autopsy. The blank is finally filed with 
 those of all other animals used in the laboratory. 
 
 Cultural Notes. — Each strain of bacteria is given a certain designa- 
 tion, number, or name. Many bacteriologists designate strains by a 
 serial number or by the date of their isolation. Rather than a serial 
 numbier which soon becomes quite large and is not suggestive I em- 
 ploy one or more letters followed by a small number. The letters 
 may represent the epidemic (as is the case with many of the strains 
 reported here and by Smith and Brown in 1915) or may suggest 
 the name of the patient or of the person, locality, laboratory, or ma- 
 terial from which the strain was obtained. The number following 
 the letter indicates the number of the strain obtained from the source 
 indicated by the letter. 
 
 Examples. — B-6 represents the 6th strain isolated from epidemic B. 
 
 Ad4 represents the 4th strain isolated from a patient by the name of Adams. 
 
 D-Ad4 indicates the 4th strain isolated from Mr. Adams during epidemic D. 
 
 Any system of designation convenient to the bacteriologist may be 
 used. It is important that it shall be brief for convenience, suggestive 
 
100 BLOOD AGAR FOR STXIDY OF STREPTOCOCCI 
 
 SO that the origin and facts connected with it are constantly suggested, 
 and elastic so that it will answer all purposes. The remainder of the 
 system of notes here described is illustrated by the sample cards in 
 Plate 34. 
 
 "W4" is the designation of a swab received and the card accom- 
 panying the swab is filed at the beginning of the notes which are to 
 be recorded on cards, each card representing a separate tube or 
 plate culture. On the upper left-hand corner of each card is the 
 designation (W4) followed by a fraction. The denominator of the 
 fraction is the number of the individual card. Note that the de- 
 nominators run serially, 1, 2, 3, 4, etc. The numerators, on the 
 other hand, follow no apparent order; they are the numbers of 
 the tube or plate from which the culture in hand was inoculated. 
 For example, tube 3 (number in denominator) of bouillon (yellow 
 card) was on April 4, 1915 (date of inoculation in upper right-hand 
 comer of card), inoculated from a colony on agar plate 1 (blue card). 
 This is indicated by transferring the nimiber 1 from the denominatior 
 of the blue card to the numerator of the yellow card. A glance at 
 the fractions on yellow cards Nos. 2, 4, and 5 (denominators) shows 
 that each of these also has 1 for a numerator and therefore that they 
 also were inoculated from colonies on agar plate 1. It will be no- 
 ticed that some small letters (a, b, c, and d) have been added to the 
 designation (W4) on cards Nos. 2, 3, 4, and 5 respectively. These 
 small letters indicate the colonies on plate 1 from which each 
 tube of bouillon was inoculated and the colonies will be found de- 
 scribed in the notes on blue card No. 1. While W4 was the designa- 
 tion of the swab, W4a, W4b, W4c, and W4d are, therefore, the des- 
 ignations of strains isolated from the swab. Cards Nos. 6 and 7 
 (denominators) represent plates which, as indicated by their numer- 
 ators, were inoculated from bouillon cultures Nos. 2 and 3 respec- 
 tively; furthermore a comparison of dates in the upper right-hand 
 corners shows that these bouillon cultures were about 24 hours old 
 -when plates 6 and 7 were inoculated from them. The condition of 
 the bouillon cultures at this time is described in the notes on cards 
 Nos. 2 and 3 under date of 5-IV (April 5). Observations of cultures 
 are noted under the proper date (and hour, if significant) on the 
 corresponding cards, the numbers being found on the culture tube and 
 
JAMES HOWARD BROWN 101 
 
 on the upper left-hand corner of the card, and the cards being filed 
 in the order of the serial denominator numbers and consequently 
 according to the dates of inoculation found on the upper right-hand 
 corner of the cards. 
 
 Suppose there is taken from the refrigerator a culture tube bearing 
 the number W4big represented by the card of the same number (the 
 last card on Plate 34) and that it is desired to know the history of this 
 strain. The fraction j^ refers to the card with 17 in the denomi- 
 nator {i.e., 0) which in turn refers to card 9 (i.e., 5) and this to | and 
 and thence to 5 and j, the' "0" indicating the "original" specimen. 
 
 The above series of cards is therefore: 
 
 1 
 
 1 
 
 3 
 
 I 
 7 
 
 7 
 
 9 
 
 17 
 
 jg Taking these cards in 
 hand it is found by counting them that the strain is in its sixth gen- 
 eration or transplant. Furthermore, there is on these six. cards the 
 complete genealogy of the strain and the notes of each generation 
 with exact dates and a key to full details regarding each tube of 
 medium. ' It is found that the strain was isolated into bouillon from 
 a blood agar plate, replated on blood agar, grown in bouillon once 
 more, passed through a rabbit, and recovered from the rabbit onto 
 the agar slant which has been taken from the refrigerator. One can 
 delve as much farther into the details as is desired without being 
 confused by the mass of notes representing collateral experimental ex- 
 cursions, but if one cares to examine the records of any of the latter 
 this can readily be done. 
 
 So much may be said for the use of the completed record, but 
 what of the making.oi it? The simplicity of recording the cultures 
 and taking the notes can be appreciated only by trying it. There is 
 on the desk a small box of blank cards on which have been stamped, 
 by means of a cheap rubber stamp with movable type, various head- 
 ings which are convenient for the work in hand. The use of these 
 stamped headings is in itself a great saver of time and adds to the 
 legibility. The use of cards of different colors for different media. 
 
102 
 
 BLOOD AGAR FOE STUDY OF STREPTOCOCCI 
 
 etc., facilitates the picking out of the right card from either the box 
 of unused cards or from the card index. The colors are soon fixed in 
 memory so that the selection of the right color requires no addi- 
 tional thought or labor. Each tube of medium that comes to hand is 
 
 labeled, e.g. 
 
 Bouil. 1027 
 
 the number referring to the record of the 
 
 assistant who has prepared the medium and which may be consulted 
 if desired. It is desired to inoculate this tube of bouillon from a 
 
 culture bearing the following label 
 lated it is labeled 
 
 Bouil. 1027 
 W4b^ 
 
 Agar 800 
 W4b TT 
 
 Having been inocu- 
 
 A bouillon (yellow) card is selected, 
 
 and the number of the medium recorded under the word Bouillon with 
 any other remarks regarding the medium that may be of particular 
 interest. The date is written in the upper right-hand corner of the 
 card and the culture number, W4b^^, in the left corner. But one 
 thing is lacking, the denominator. Turning to the card index it 
 is found that the last card filed under this strain had 18 for a de- 
 nominator. The next one will therefore be 19 which is placed on 
 
 the tube label 
 
 BouU. 1027 
 W4b -H 
 
 and on its card. The tube is then incu- 
 
 bated and the card filed. Any notes subsequently made on this cul- 
 ture are written under their respective dates on the card. Both sides 
 of the card are used if necessary and occasionally a second card is 
 inserted. The cards belonging to each strain or from each swab 
 (as in the present illustration) are set apart in the card index drawer 
 by slipping a little tab over the edge of the first card. 
 
 The labor involved is no greater than that of keeping any system 
 of notes; the tube must be labeled; the number, date, and medium 
 entered; and subsequent notes recorded. The labor of making these 
 entries on cards is no greater than that of entering them on papers 
 or in a book. The cards in the index need be referred to only to 
 find the denominator of the last card, certainly no more trouble than 
 to find the right page in a book. In fact the same system of number- 
 ing may be followed in a book or on loose sheets of paper, but the 
 colored cards have the advantage of making the notes readily acces- 
 sible; the colored edges of the cards are always in view and indi- 
 
JAMES HOWARD BROWN '103 
 
 vidual cards are easily taken out, studied (as in the tracing of the 
 genealogy), and replaced. 
 
 To the system as above described may be added another refine- 
 ment which does require a little extra labor but which need be used 
 only occasionally. There is a so called "comb" (Plate 34) on the 
 rulings of which are printed certain suggestive titles or key-words to 
 facts in the notes about which one may wish to inquire. The red and 
 black rulings of the comb are continued along the top edges of the 
 cards. (The edges of 1,000 new cards may be ruled in a few min- 
 utes by ruling them all at once in the box in which they are bought.) 
 The ruUngs on the edges of the cards are hardly visible from the face 
 of the individual card but are quite conspicuous forming continuous 
 lines when the cards are viewed from their upper edges as they are 
 packed together in the index drawer. By means of a card punch, 
 using the edge of the comb placed over the edge of the card as a guide, 
 notches can be cut into the edge of the card corresponding to the 
 key-words on the comb. The comb can be moved along in the index 
 and by means of the appropriate notch the record of any particular 
 feature {e.g., a capsule stain, or a fermentation of lactose) can be 
 immediately selected from among hundreds of cards. The cards 
 may be punched at any time after takihg the notes and this feature 
 may be used as much or as little as desired, but I often find it con- 
 venient to mark in this way some special record or experiment that I 
 may wish to refer to later. Together with the colors of the cards 
 this feature renders the notes readily accessible. 
 
 1,000 cards occupy a space of 8^ inches in the index drawer. Dur- 
 ing the past year I have used about 3,000 cards costing $1.50 per 
 thousand. The files of old cultures are conveniently stored in the 
 pasteboard boxes in which the cards were bought. With very little 
 expenditure of time, effort, space, or money I have the complete and 
 readily accessible genealogical records of nearly one hundred fifty 
 strains of streptococci. 
 
 A Simple and Inexpensive Method of Making Photographic Records oj 
 Growth on Agar and Blood Agar Plates. 
 
 Although detailed genealogical records were kept for aU the strains 
 reported by Smith and Brown (1915) and for all strains isolated by 
 
104 ' BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 me since then, and although from the first careful descriptions with 
 measurements of colonies and zones of hemolysis or discoloration on 
 blood agar plates were made, it was realized that in a series of experi- 
 ments which were to extend over a period of several years even the 
 memory assisted by notes might make mistakes and that certain 
 details which escaped observation might be preserved photographi- 
 cally. About 2 years ago, therefore, while the strains then being 
 studied were fresh in memory a series of representative strains for 
 intensive study was selected; and of them together with many strains 
 obtained since photographic records consisting now of well over 500 
 photographs have been made. The advantages are: 
 
 1. The photographs are permanent records of appearances in blood 
 agar, not subject to the suspicion of faulty notes or memory. 
 
 2. Details, unnoticed or the importance of which was not realized 
 at the time the culture was observed, are preserved and discovered 
 in the photographs many months later when, except for the photo- 
 graphic record, they might be regarded as newly acquired charac- 
 teristics. It is surprising how the emphasis in one's observations 
 will shift from certain characteristics to others after intervals of 
 several months. 
 
 3. Photographs of the same plate or colony on successive days of 
 incubation or other treatment can be viewed and compared side by 
 side. In no other way can the gradual development of a culture and 
 of its minute characteristics be appreciated so well. 
 
 4. The appearance of the growth of the same or different strains 
 on the same or different media can be compared at the same or dif- 
 ferent times, even though one or more of the strains may have been 
 lost long ago. 
 
 Obvious though these and other advantages are, to be practical 
 the method of making the photographs could not be too laborious 
 or expensive. The object was to develop methods which could be 
 employed in almost any well equipped bacteriological laboratory and 
 therefore to avoid the use of expensive apparatus on the market for 
 such purposes. The apparatus used for making all the photographs 
 shown in the plates accompanying this thesis is shown in Plate 33.'^ 
 
 '^ The white back-ground was placed back of the apparatus merely for making 
 this photograph. 
 
JAMES HOWARD BROWN ' 105 
 
 Simple though it is, it is the result of numerous experiments with 
 different arrangements of lights, diaphragms, lenses, distances, and 
 photographic materials. 
 
 The apparatus for photographing the agar plates (natural size) 
 consists merely of a strong (48 candle power) "Mazda" electric light 
 enclosed in a tin can at a distance of 6 feet above the table (see upper 
 left-hand corner of the photograph). The light escapes downward 
 onto the table through an opening 2.5 cm. in diameter in the side 
 of the tin can, this opening being covered with waxed cloth or paper 
 to make it translucent. The light is turned on or off at the switch 
 board on the table below. The entire apparatus is in a dark room. 
 To make a photograph, a photographic plate is placed on the table 
 directly under the light (film side up) and the agar plate to be pho- 
 tographed is placed directly on the photographic plate, the cover of 
 the Petri dish removed, and the light turned on for about 5 seconds 
 and then off again. The agar plate is covered and removed, and the 
 photographic plate developed and fixed in the ordinary way. The 
 picture is therefore a direct print or photograph by transmitted light 
 rather than by reflected light. No lenses or camera are required. 
 This method is much more satisfactory for photographing blood agar 
 plates than is photographing with the ca:mera by reflected light in the 
 usual way, for in order to show hemolyzed zones and deep colonies 
 the light must pass through the plate. The deep colonies are photo- 
 graphed perfectly by this simple direct method but because of re- 
 fraction of light by the raised edges of the top surface colonies these 
 appear with a double contour which if borne in mind serves to dif- 
 ferentiate top surface colonies from bottom surface colonies which 
 appear flat and with a single contour (Plate 6, Fig. 1). In photo- 
 graphing many blood agar plates half the plate was covered by a 
 fine net so that the distinctness of the image of the net in the hemo- 
 lyzed zones might indicate the degree of transparency of the zones, 
 but later this device was abandoned as being unnecessary, the dis- 
 tinctness of the deep colony itself within the zone being quite as good 
 an index of transparency (see Plates 4 and 7). 
 
 The photomicrographic apparatus consists pf a wooden box about 
 lOx 12x18 inches in size. The box stands on edge as shown in 
 Plate 33, one side, the front, being open but provided with a curtain 
 
106 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
 of black light-proof cloth. A hole in the top edge of the box is pro- 
 vided with a collar of the black cloth and a draw-string by means of 
 which the collar is drawn tightly about the protuding tube of the 
 microscope within the box. Inside the box is also a "Mazda" light 
 (20 candle power) enclosed in a tin can like that described above and 
 controlled from the same switchboard. The interior of the box is 
 ' coated with a dull black paint. (For making Plate 33 a piece of white 
 paper was placed in the back of the box merely for photographing 
 the apparatus so that the interior arrangement could be seen better.) 
 For photographing the colonies shown throughout the plates there 
 was used an objective of very low power, no condenser, and no 
 ocular. The agar plate containing the colony to be photographed is 
 placed on the stage of the microscope and the image of the colony 
 focused onto a piece of ground glass supported by the ring of a ring- 
 stand and about 10 inches above the tube of the microscope. When 
 the colony is in focus the light is turned off, the ground glass replaced 
 by a photographic plate, and the light turned on again for a few sec- 
 onds' exposure, after which the photographic plate is developed and 
 fixed in the usual way. It was found necessary to sKp a sleeve of 
 black paper inside the tube of the microscope to prevent reflection 
 from the sides of the tube. 
 
 This apparatus is particularly good for taking low power photo- 
 micrographs but I have also taken with it a fairly good photograph 
 of encapsulated streptococci under the oil immersion objective. 
 With low power objectives the condenser is not used but must be used 
 with high. power objectives. 
 
 Many kinds of photographic plates were tried before one that was 
 entirely satisfactory was found. The essential qualities of the 
 plate are (1) that it shall be slow so that the exposure can be regu- 
 lated satisfactorily from the switchboard, and (2) that it shall pre- 
 sent a great deal of detail and not too much contrast. For the pho- 
 tographs taken in 1915 "Wellington Special Lantern Slide Plates" 
 were used with very good results, but in 1916 it was found that the 
 "Wellington Ortho-process Plate" showed more detail. Some slight 
 differences in the photographs of 1915 and those of 1916 are due to 
 the differences in the plates used. The plate of most convenient 
 size is 3jx4| inches. For the photomicrographs these plates are 
 cut into four parts. 
 
JAMES HOWARD BROWN 107 
 
 After the methods are thoroughly standardized so that the proper 
 exposure for a blood agar plate of standard size containing a stand- 
 ard amount of agar and blood is known, one can photograph a plate 
 and a colony in 2 minutes in the dark room, the exposed photographic 
 plates being stored in light-proof boxes until a number have a,ccumu- 
 lated before being developed. The cost is about 5 cents for the plate 
 and the first print and less than 2 cents for each duplicate print. 
 
 , <t s > f^ n /: r, , '^Z' 
 
 \'i vrr.if7,i;Yf ot 1 1 ( I I' 
 <>, / ^ '■- 
 
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110 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
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JAMES HOWARD BROWN 111 
 
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112 BLOOD AGAR POE STUDY OF STREPTOCOCCI 
 
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JAMES HOWAED BROWN 113 
 
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114 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
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 Klein, E., Ueber zwei neue pyogene Mikroben: Streptococcus radiatus und 
 Bacterium diphtherioides, Centr. Bakteriol., 1900, xxviii, 417. 
 
 KUgler, I. J., A Study of the Correlation of the Agglutination and the Fermenta- 
 tion Reactions Among the Streptococci, /. Infect. Dis., 1915, xvi, 327. 
 
 Klinger, R., Untersuchungen uber menschliche Aktinomykose, Centr. Backteriol., 
 Ite Abt., Orig., 1912, Ixii, 198. 
 
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 oberflache des Menschen und seiner Umgebung, Z. Hyg. u. Infectionskrankh., 
 1907-1908, Iviii, 287. 
 
 Koch, J., and Pokschischewsky, N., Ueber die Artverschiedenheit des Strepto- 
 coccus longus seu erysipelatos und des Streptococcus equi (Druse-Strepto- 
 coccus), Z. Hyg. u. Infectionskrankh., 1913, Ixxiv, 1. 
 
 Kbnr&d, E., Weitere Beitrage zur Vaginalstreptokokkenfrage, Beitr. Geburtsh. 
 u. Gynak., 1908-1909, xiii, 364. 
 
 Kraus, R., Zur Differenzierung des Choleravibrio von artverwandten Vibrionen, 
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 1895, xix, 409. 
 
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 Ite Abt., Orig., 1912, Ixiii, 97. 
 
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 men derselben, insbesondere des Streptococcus conglomeratus, bei Scharlach, 
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JAMES HOWAED BfeOWN 115 
 
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JAMES HOWARD BROWN 117 
 
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JAMES HOWARD BROWN 119 
 
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120 BLOOD AGAR FOR STUDY OF STREPTOCOCCI 
 
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JAMES HOWARD BROWN 121 
 
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 ismen im Korper der Warmbluter, Z. Hyg. u. Infectionskrankk., 1886, i, 3. 
 
 Zangemelster, W., Der heutige Stand der Streptokokkenfrage insbesondere fiir 
 die GeAurtshilfe, MUnch. med. Woch., 1907, liv, 1021. 
 
 Zangemeister, W., Die Hamolyse der Streptokokken, Deutsch. med. Woch., 
 
 1909, XXXV, 427, 477. 
 
 Zangemeister, W., Die bakteriologische Untersuchung im Dienste der Diagnostik 
 und Prognostik der puerperalen Infektionen, Berlin, 1910. 
 
 Zangemeister, W., Ueber der Verbreitung der Streptokokken im Hinblick auf 
 ihre Infektiositat und ihre hamolytische Eigenschaft, MUnck. med. Woch., 
 
 1910, Ivii, 1268. 
 
 Zangemeister, W., and Meissl, Th., Untersuchungen uber die Verwandtschaft 
 saprophytischer und pathogener Puerperalstreptokokken und uber die 
 , Streptokokkenimmunitat, Z. Geburtsh. u. Gyndk., 1906, Iviii, 425. 
 
 Zopprltz, B., Ueber Streptokokkenversuche, Med. Klin.,'l909, v, 1112. 
 
 Zschokke, Forschungen uber den gelben Gait, Schweiz. Arch. Tierheilk., 1897, 
 xxxix. 
 
 Zschokke, Forschimgen uber den gelben Gait, Schweiz. Arch. Tierheilk., 1904, 
 
 113. 
 
TABLE I. 
 Tabular Description of the Principal Strains Referred to in this Work. 
 
 
 Type in 
 blood agar. 
 
 
 
 Growtll in Iwuillon. 
 
 Fermentation. 
 
 p 
 
 l-S 
 
 
 Designation. 
 
 Source. 
 
 Date of 
 isolation or 
 possession. 
 
 Gross appearance. 
 
 Morphology. 
 
 + 
 
 + 
 + 
 
 + 
 
 4> 
 
 S 
 
 + 
 + 
 
 
 
 + 
 
 
 d 
 
 "(3 
 
 CO 
 
 + 
 
 • Names as they might be used by various authors. 
 
 D-1 
 
 7 
 
 Septic sore throat 6th day. 
 Same case as D-la. 
 
 Mar., 1914. 
 
 Somewhat clouded; good amount of fine floccu- 
 lent sediment, disintegrated with difficulty. 
 
 Short and medium chains of fairly large round 
 or flattened cocci. 
 
 5. saprophylicus (Mandelbaum). 
 S. anhemoivticus (Zangemeister) . 
 5. equimis (Andrewes and Horder). 
 " (Holman). 
 
 A-18 (cow) 
 
 ^ 
 
 Milk from single cow in suspected herd. 
 
 June, 1913. 
 
 Not clouded; abundant loose fleecy growth on 
 bottom, easily disintegrated by shaking. 
 
 Very long rope-like chains of fairly large round 
 or flattened cocci, often arranged in pairs. 
 
 5. anginosus (Andrewes and Horder). 
 " (Holman). 
 
 B-18 (cow) 
 
 ^ 
 
 Milk from single cow in suspected herd. 
 
 May, 1913. 
 
 Well clouded; some fine flocculent sediment. 
 
 Short and medium chains of fairly large round 
 or flattened cocci, often arranged in pairs. 
 
 5. anginosus (Andrewes and Horder). 
 " (Holman). 
 
 A-ST 
 
 /3 
 
 Peritoneal pus during epidemic of septic sore 
 throat. 
 
 May, 1913. 
 
 Clouding variable; fine flocculent sediment. 
 
 Long chains of round elements, sometimes 
 twisted into clumps. 
 
 + 
 
 + 
 + 
 
 + 
 
 + 
 + 
 + 
 
 + 
 
 + 
 
 + 
 + 
 4- 
 
 + 
 + 
 -f 
 
 + 
 + 
 
 S.longus pathogenes sen erysipelatos (Schottmiiller) . 
 5. pyogenes (Rosenbach). 
 5. hemolyticus (Roily). 
 " (Davis). 
 5. hemolyticus II (Hohnan). 
 5. alactosus (Smith and Brown). 
 
 D-AD4 
 
 /3 
 
 Throat of dairyman nearly 3 wks. after clinical 
 recovery from tonsillitis. 
 
 Mar., 1914. 
 
 Not clouded; granular sediment. 
 Later fairly well clouded with small suspended 
 particles; flocculent sediment. 
 
 Medium sized round cocci in moderately long 
 tortuous chains forming tangled clumps. 
 
 + 
 + 
 
 + 
 + 
 
 - 
 
 - 
 
 5. longus pathogenes seu erysipelatos (Schottmiiller). 
 S. pyogenes (Rosenbach). 
 
 " (Andrewes and Horder). 
 vHolman). 
 S. hemolyticus (Roily). 
 (Davis). 
 
 B-2b (cow) 
 
 /? 
 
 Milk from a supposedly sound quarter of the 
 udder of a cow known to have mastitis in another 
 quarter. 
 
 May, 1913. 
 
 Fairly well clouded with fine suspended particles; 
 good amount of fine flocculent sediment. 
 
 Fairly long chains and clumps of rather large 
 round cocci, swollen elements not uncommon. 
 
 S. epidemicus (Davis). 
 
 5. mucosiis (?) (Howard and Perkins). 
 
 B-6 
 
 a' 
 a 
 
 Throat of convalescent dairyman. 
 Same patient as Ji-7a. 
 
 May, 1913. 
 
 Moderately clouded; some fine flocculent sedi- 
 ment. 
 
 Rather small round or oval elements in chains 
 of various lengths, often crooked. 
 
 + 
 + 
 
 + 
 + 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 + 
 + 
 
 S. pyogenes or 5. mitis <*)) (Andrewes and Horder). 
 S. pyogenes or S. mitis (?) (Holman). 
 
 F-2 
 
 a' 
 a 
 
 Septic sore throat 9th day. 
 Same case as F-2a. 
 
 May, 1914. 
 
 Moderately clouded; small amount of compact 
 
 sediment. 
 
 Short straight chains of rather small oval ele- 
 ments. 
 
 S. pyogenes or 5. mitis (?) (Andrewes and Horder) . 
 S. pyogenes or S. mitis (?) (Hohnan). 
 
 A-BL 
 
 a 
 
 Throat, during epidemic of tonsillitis. 
 
 May, 1913. 
 
 Well clouded; small amount of sexliment. 
 
 Small, mostly oval or elongated elements in 
 clumps of tangled chains. 
 
 + 
 
 + 
 + 
 
 5. mitior seu viridans (Schottmiiller) . 
 S. mitis (Andrewes and Horder). 
 " (Holman). 
 
 D-4 
 
 a 
 
 Septic sore throat 8th day. 
 
 Mar., 1914. 
 
 Growth rather scant; rather faintly clouded; 
 fair amount of fine sediment. 
 
 Long straight chains of fairly large oval or elon- 
 gated elements. 
 
 5. mitior seu viridans (Schottmiiller) . 
 S. sdivarius (?) (Andrewes and Horder). 
 (Holman). 
 
 B-7 
 
 a 
 
 Throat of convalescent dairyman. 
 Same patient as B-6a'. 
 
 May, 1913. 
 
 Well clouded; little or no sediment. 
 
 Short chains of rather small round or oval ele- 
 ments. 
 
 S. mitior seu viridans (Schottmiiller) . 
 
 S. mitis or 5. salivarius (variant) (Andrewes and Horder). 
 
 5. mitis (Holman). 
 
 E-4 
 
 a 
 
 Throat of child with sore throat, adenitis, and 
 erysipelas. 
 
 Mar., 1914. 
 
 Well clouded; little or no sediment. 
 
 Short chains of small, mostly slightly elongated 
 elements. 
 
 + 
 + 
 
 S. mitior seu viridans (Schottmiiller). 
 
 S. salivarius (Andrewes and Horder). 
 
 (Holman). 
 
 D-AD82 
 
 a 
 
 Throat, convalescent, 11th day. 
 
 Apr., 1914. 
 
 Not clouded; abundant fleecy growth on bottom 
 of tube, thready when shaken. 
 
 Skeins of very long parallel chains of fair sized 
 round and ovoid elements. 
 
 S. mitior seu viridans (?) (Schottmiiller). 
 
 S . longiss imus (Thalmann). 
 
 5. salivarius or S. anginosus (?) (Andrewes and Horder). 
 
 S. salivarius or S. anginosus (?) (Holman). 
 
 D-1 
 
 a 
 
 Septic sore throat 6th day. 
 Same case as D-IT. 
 
 Mar., 1914. 
 
 Well clouded; small amount of fine flocculent 
 sediment. 
 
 Short and medium chains of rather small round 
 and elongated elements; chains often crooked or 
 clumped. 
 
 + 
 
 + 
 
 + 
 + 
 
 + 
 
 + 
 
 + 
 + 
 
 S. mitior seu viridans (Schottmiiller). 
 S. conglomeratus (Kurth). 
 
 (Thalmann). 
 5. »K% (Andrewes and Horder). 
 " (Holman). 
 
 F-2 
 
 a 
 
 Septic sore throat 9th day. 
 Same case as F-2a'. 
 
 May, 1914. 
 
 Slightly or moderately clouded with small sus- 
 pended particles; good amount of sediment break- 
 ing up into small flakes when shaken. 
 
 Short and medium chains of rather small round 
 and elongated elements. 
 
 5. mitior sen viridans (Schottmiiller). 
 S. conglomeratus (Kurth). 
 
 (Thalmann). 
 5. salivarius (Andrewes and Horder). 
 (Holman). 
 
 Brig.-IIa 
 
 a 
 
 Sputum from a supposed case of influenza. 
 Pneumonia recognized next day after this sample 
 was taken. 
 
 Dec, 1915. 
 
 Fairly well clouded; good amount of ropy sedi- 
 ment. 
 
 Short and medium chains of round or slightly 
 oval elements, the entire chain enclosed in a broad 
 capsule. 
 
 S. mucosus (?) (Howard and Perkins). 
 
 " (SchottmuUer). 
 Pn. " (Park and Williams). 
 
 Cole I 109 
 
 cc 
 
 Dr. Rufus Cole, Rockefeller Institute. 
 
 May, 1915. 
 
 Well clouded; little or no sediment. 
 
 Encapsulated lanceolate diplococci. 
 
 Pnciimococcus (Frankel) . 
 
 M. lanceolatus. 
 
 S. piietimonice (Davis, Park, and Williams, and others). 
 
 Cole II 45 
 
 a 
 
 Dr. Rufus Cole, Rockefeller Institute. 
 
 May, 1915. 
 
 Well clouded; little or no sediment. 
 
 Encapsulated lanceolate diplococci. 
 
 + 
 
 + 
 
 Pn; .imococcus (Frankel). 
 
 M. I mceolatus. 
 
 S. pneumonia, (Davis, Park, and Williams, and others). 
 
 * All the strains were Gram-positive. Only the strain of 5. mucosus and the Pneumococci were soluble in sodium taurocholate. It is not to be understood that the strains 
 the strains tabulated were selected merely as representatives of different varieties of streptococci. 
 
 described were considered responsible for the conditions described under ''Source;" 
 
TABLE V — Continued. 
 Fermentation Reactions. 
 
 TABLE V — Continued. 
 Fermentation Reactions. 
 
 Organisms. 
 
 10 strains from horses of 
 which 5 were from "Druse- 
 eiter." 
 
 2 strains from human sup- 
 purations. 
 
 2 strains from the milk of 
 cows with mastitis. 
 
 1 strain from the organs 
 of an aborted calf. 
 
 Many strains from various 
 sources. 
 
 14 strains of streptococci 
 from joint fluid of 14 cases of 
 acute rheumatism. 
 
 247 strains of streptococci 
 from various human patho- 
 logical conditions. 
 
 Media. 
 
 Composition. 
 
 3 parts of 2 per cent pep- 
 tone water -(- 1 part of fil- 
 tered horse serum. 
 
 To 9 parts of the above 
 added 1 part of sterile 10 
 per cent solution of the test 
 substance in litmus solution. 
 
 Distributed 10 cc. 
 amounts into test-tubes and 
 heated 2 hrs. at 58° on 3 
 successive days. 
 
 Hiss serum water. 
 
 Also dextrose bouillon 
 used. 
 
 (Test substances in 10 
 per cent aqueous solution, 
 "sterilized by steaming for 
 twenty minutes," and then 
 added in the proportion of 
 1-9 cc. of serum water which 
 had been previously auto- 
 claved.) 
 
 Test substances. 
 
 Arabinose, rhamnose, xy- 
 lose, dextrose, levulose, gal- 
 actose, mannose, lactose, 
 saccharose, maltose, raffi- 
 nose, soluble starch, dex- 
 trin, glycogen, inulin, arbu- 
 tin, amygdalin, salicin, glyc- 
 erol, erythrite, adonite, 
 mannite, sorbite. 
 
 See under Results and 
 Conclusions. 
 
 Dextrose, saccharose, lac- 
 tose, maltose, mannite, inu- 
 lin, rafSnose. 
 
 Dextrose, mannite, inulin. 
 
 Dextrose, saccharose, lac- 
 tose, salicin, rafEnose, man- 
 nite, inulin. 
 
 Litmus milk. 
 
 Indicator or titration. 
 
 Litmus. 
 
 Titrated, in some 
 cases at least. 
 
 Neutral red, in 
 medium for all 
 strains. 
 
 One-half of 
 strains also titrat- 
 ed, cold (phenol- 
 phthalein). 
 
 Period of incubation. 
 
 1 days. 
 
 1 wk. 
 
 Results and conclusions. 
 
 All strains produced acid from dextrose, mannose, galactose, levulose, saccharose, maltose, soluble starch, 
 dextrin, salicin, and arbutin. None attacked arabinose, xylose, erythrite, or adonite. There remained 9 
 substances which fermented as indicated in the following table (compiled) : 
 
 Organisms. 
 
 
 si 
 
 o 
 
 1 
 
 Raffinose. 
 
 o 
 
 1 
 
 h- 1 
 
 ■B 
 
 M 
 
 < 
 
 1 
 
 o 
 
 *3 
 
 (3 
 
 
 Pferdestammen (5) 
 
 Drusestdmmen (5) 
 
 Human strains (2) 
 
 Bovine " (3) 
 
 3 
 
 2 
 
 4 
 
 1 
 
 1 
 
 2 
 
 4 
 2 
 
 2 
 3 
 
 1 
 
 2 
 
 1 
 
 2 
 
 2 
 3 
 
 (3) 
 
 1 
 2 
 
 (3) 
 
 3 
 
 1 
 
 2 
 
 3 
 
 Note. — The figures indicate the number of strains fermenting the substances indicated. 
 The figures in parentheses indicate strains said by the author to have been doubtful. No 
 figure indicates that none of the strains fermented the substance. 
 Tests in fermentation tubes showed that none of the strains produced the smallest trace of gas. The lit- 
 mus was reduced in the closed arm of the tubes by some strains at times. 
 
 The author concludes: "Es hahen sich aus dem Verhalten der gepriiften 15 Streptokokkenstdmme gegeniiber 
 23 verschiedenen Kohlehydraten oder kohlehydratdhnlichen Substanzen bei der gewdhlten V ersuchsanordnung Anhalts 
 punkte filr eine Charakterisierung bestimmter Streptokokkenarten oder -gruppen nicht 
 
 S. equi fermented dextrose, mannose, galactose, fructose, maltose, cellobiose, saccharose, dextrin, glycogen, 
 soluble starch, salicin, and arbutin (feebly). 
 
 It did not ferment sorbose, xylose, arabinose, rhamnose, glycoheptose, trehalose, formose, gentiobiose, lac- 
 tose, rafiBnose, inulin, sorbite, mannite, dulcite, adonite, glycerol, erythrite, perseite, or amygdaline. 
 
 Records of fermentation reactions are not given in full for all strains. No titration figures are given. 
 
 Most of the pneumococcus forms and none of the streptococcus forms tabulated fermented inulin; only 1 
 pneumococcus is tabulated as fermenting mannite, but about half the streptococci fermented this substance. 
 All strains fermented dextrose and nearly all fermented lactose, saccharose, and maltose. 
 
 " The results obtained (with reference to variability) show clearly why the classification of streptococci based 
 on fermentative powers alone has proven to be unsatisfactory." 
 
 " Three of the strains of the first group (green producers on blood agar) and all of the strains which produced 
 muscle lesions ferment mannite but not inulin and produce a high acidity in dextrose broth (i-7 per cent)." 
 
 The authors divide 247 strains into 6 groups comprising no less than 51 combinations of action on the test 
 substances including hemolysis of blood agar. 
 
 Notwithstanding the authors' statements the groups apparently do not correspond with those of Andrewes 
 and Horder. 
 
 Especially noteworthy are the large number of non-fermenters, and the presence of some (21 strains) which 
 fermented lactose or acidified m^lk but did not ferment dextrose. 
 
 "It is possible to make, more or less arbitrarily, divisiotis of streptococci from human sources, according to their 
 fermentative properties in media containing the test substances selected by Gordon. * * The agglutination tests 
 and complement fixation tests support the evidence yielded by cultural tests." 
 
 Reference. 
 
 Maass 1913. 
 
 Zur Frage der Streptokokken-Differenzierung 
 durch kohlehydrathaltige Nahrboden. 
 
 Bemelmans 1913. 
 
 La specificite des streptocoques de la gourme. 
 
 Rosenow 1914. 
 
 Transmutations Within the Streptococcus- 
 Pneumococcus Group. 
 
 Rosenow 1914. 
 
 The Etiology of Acute Rheumatism, Articu- 
 lar and Muscular. 
 
 Floyd and Wolbaeh 1914. 
 
 On the Differentiation of Streptococci; Pre- 
 liminary Notes. 
 
TABLE Y~Continued. 
 Fermentation Reactions. 
 
 TABLE Y— Continued. 
 Fermentation Reactions. 
 
 Organisms. 
 
 Media. 
 
 Indicator or titration. 
 
 Period of incubation. 
 
 
 
 
 
 
 
 
 
 
 
 
 Reference. 
 
 
 
 Composition. 
 
 Test substances. 
 
 
 
 18 throat strains. _ 
 1 strain from cervical ade- 
 nitis. 
 9 strains from milk slime. 
 
 Hiss serum water. 
 
 Dextrose, lactose, saccha- 
 rose, salicin, rafiinose, man- 
 nite, inulin, dulcite. 
 
 Milk. 
 
 Presumably lit- 
 mus. 
 
 No titrations. 
 
 7 days. 
 
 All strains fermented dextrose, lactose, and saccharose; none fermented mannite, inulin, or dulcite. The 
 strains were divided into 4 groups by their action on salicin and raihnose; 
 Group A: Salicin -f, RafBnoSe -|- 
 " B: " -, " H- 
 " C: " -f, " - 
 
 « "rj . « tt 
 
 Strains from 2 cows with inflamed udders and 4 throat strains fell into Group D. 
 
 North, White, and Avery 
 
 A Septic Sore Throat Epidemic in 
 and Homer, N. Y. 
 
 1914. 
 Cortland 
 
 42 strains fro,m milk. 
 
 14 strains from cows' 
 feces. 
 
 51 strains from infected 
 udders. 
 
 39 strains from cows' 
 mouths. 
 
 Beef extract 0.4 per cent, 
 peptone 1 per cent, dihasic 
 potassium phosphate 0.5 per 
 cent, and test substance 2 
 per cent. 
 
 Gelatin. 
 
 Dextrose, saccharose, lac- 
 tose, raifinose, starch, inu- 
 lin, manxiite, glycerol, ado- 
 nite, dulcite. 
 
 Litmus milk. 
 
 Titrated, phe- 
 nolphthalein. 
 
 7 days at SO^C. 
 
 Any result above 1 per cent N acid was considered positive. 
 
 " * * the 'metabolic gradient' * * , in our opinion, can he correct only j or the particular group under 
 consideration." 
 
 83 per cent of strains from the mouths of cows, and only 7, 23.5, and 37.5 per cent of those from the udders, 
 milk, and feces respectively reduced litmus in milk. 
 
 Adonite and dulcite were fermented by only 1 or 2 strains and hence were of no value. 
 The strains studied were classified as follows: 
 From udders Dextrose. Lactose. Saccharose. Mannite. 
 Group 1 . . -f- -|- -|- Occasionally. 
 
 " * * agrees with the published description of S. pyog." 
 
 Group 2 . . Dextrose. Lactose. Saccharose. Mannite. Glycerol. Gelatin. 
 -1- -+- -f Usually. Usually. Often 
 
 liquefied. 
 
 Rogers and Dahlberg 
 
 The Origin of Some of the Streptoco 
 in Milk. 
 
 1914. 
 :ci Found 
 
 
 From feces.. 
 
 Dextrose. 
 
 + 
 
 Lactose. 
 
 Saccharose. 
 
 + 
 
 Raifinose. 
 
 + 
 
 Mannite. 
 
 Starch. 
 Often. 
 
 Inulin. 
 Often. 
 
 Gelatin. 
 Liquefied. 
 
 
 
 From mouth. 
 
 Differ from feces strains in more frequently fermenting rafEuose, mannite, and inulin, and in failure 
 to ferment starch. 
 
 
 
 From milk. 
 
 2 strains belonged to the feces group; "all others to one of the two udder groups." 
 
 
 105 strains from various 
 pathological processes, and 
 from milk, normal throats, 
 and human stools. 
 
 Unfermeuted veal infu- 
 sion bouillon -f 1 per cent 
 of test substance. 
 
 Lactose, saccharose, sal- 
 icin, rafSnose, mannite, inu- 
 lin. 
 
 (Starch, glycerol, dex- 
 trose, arabinose.) 
 
 Milk. 
 
 Titrated, phe- 
 nolphthalein. 
 
 24 hrs. 
 
 " We determined to disregard other tests and group them strictly according to their fermentation reactions, * * . " 
 Starch, glycerol, dextrose (fermented by all) and arabinose (fermented by none) were of no value. Using the 
 6 substances mentioned below the classification arrived at was as follows: 
 
 1 Lactose. | Saccliarose. ] Salicin. j Raffinose. | Mannite. | Inulin. 
 
 Hopkins and Lang 
 
 Classification of Pathogenic Streptc 
 Fermentation Reactions. 
 
 1914. 
 )Cocci by 
 
 
 A. Pathogenic t)q3e 
 
 Group 1 
 
 B. Saprophytic t3^es 
 (a) Salivary 
 
 Group 2 
 
 " 3 
 
 " 4 
 
 " 5 
 
 (J) Fecal.... 
 
 + 
 
 + 
 + 
 -h 
 + 
 
 + 
 
 + 
 + 
 + 
 + 
 
 + 
 + 
 
 + 
 + 
 + 
 
 - 
 
 + 
 
 
 
 Group 6 
 
 " We derived no useful in 
 test." 
 
 formation from 
 
 + + - -1- 
 our titrations that could not have been obtained by the qualitative litmus 
 
 
TABLE Y—Continiied. 
 Fermentation Reactions. 
 
 TABLE V — Continued. 
 Fermentation Reactions. 
 
 Organisms. 
 
 Many strains. 
 
 Media. 
 
 Composition. 
 
 6 strains of 5. hemolyticus. 
 
 5 strains of pneumococci. 
 
 24 strains of S. viridans. 
 (All from vajious pathologi- 
 cal conditions in man.) 
 
 7 strains from throats. 
 
 3 strains from suspected 
 cow's milk. 
 
 2 strains from Chicago 
 epidemic (1912). 
 
 263 strains from various 
 sources, mostly pathological 
 conditions. 
 
 S3 strains of streptococci 
 from sore throats and sec- 
 ondary lesions in man. 
 
 12 strains from cow's milk. 
 
 2 strains of 5. laclicus 
 from ice cream and from 
 milk. 
 
 17 strains from appendix 
 tissue of 17 of 20 cases of 
 appendicitis. 
 
 Double strength Liebig 
 extract broth + 2 per cent 
 test substance, sterilized 
 fractionally and then mixed 
 with an equal volume, of 
 filtered (unheated) 50 per 
 cent beef serum water. 
 
 Test substances. 
 
 Indicator or titration. 
 
 Andrade's indi- 
 cator (decolorized 
 acid fuchsin). 
 
 Saccharose, lactose, ara- 
 binose, raffinose, inuiin, dex- 
 trin, mannite, salicin. 
 
 "Agar containing sugar- 
 free broth from fresh meat 
 and made neutral to litmus 
 ■was used." 
 
 "The carbohydrates were sterilized separately for only 15 
 minutes in the Arnold sterilizer and added to the melted 
 agar, * * ." 
 
 Hiss serum water -f 1 per 
 cent of test substance. 
 
 Sterilized fractionally af- 
 ter addition of test sub- 
 stances. 
 
 Fermented veal infusion 
 bouillon; 1 per cent N acid 
 (infusion fermented 8 hrs. 
 by B. coli, boiled, neutral- 
 ized, and made into bouil- 
 lon). 
 
 Autoclaved 13 per cent 
 aqueous solutions of test 
 substances, then added 1-12 
 cc. of fermented bouillon. 
 
 Fermented beef infusion 
 bouillon (infusion fermented 
 24 hrs. by B. coli, strained, 
 boiled, peptone and salt 
 added, neutralized with cal- 
 cium carbonate). 
 
 Test substances added 
 dry and then sterilized (?). 
 
 Ascites broth. 
 (No details.) 
 
 Dextrose, lactose, saccha- 
 rose, maltose, raffinose, 
 mannite, inuiin. 
 
 Salicin, raffinose, mannite, 
 inuiin, (saccharose, lactose, 
 dextrose) . 
 
 Milk. 
 
 Dextrose, maltose, sacCjha- 
 rose, lactose, raffinose, man- 
 nite, inuiin, salicin. 
 
 Milk. 
 
 Dextrose, saccharose, lac- 
 tose, raffinose, inuiin, salicin, 
 mannite. 
 
 Litmus milk. 
 
 Dextrose. 
 
 Litmus. 
 
 Period of incubation. 
 
 Litmus. 
 
 Titrated (phe- 
 nolphthalein). 
 
 Titrated (phe- 
 nolphthalein) . 
 
 10 days. 
 
 1 wk. 
 
 3 days. 
 
 Results and conclusions. 
 
 This medium is not coagulated by the acids produced. 
 
 ^ It is, I believe, fundamental in the study of the fermentation reactions of the streptococci, to haiie a medium in 
 which the organisms grow well independently of the carbohydrate added. Although many of the streptococci will 
 ferment a certain carbohydrate in broth, serum -water, or serum broth, others grow so poorly in the former that they 
 fail to attack the carbohydrate, and the result would appear negative if no further study -were undertaken^" 
 
 _ By repeated plating out of supposedly pure cultures the author found different individual colonies to give 
 rise to strains with different fermentative reactions. 
 
 All strains were alike in fermenting dextrose, saccharose, lactose, and maltose, but not mannite, inuiin, or 
 raffinose, except for 1 throat strain which failed to ferment lactose and 1 bovine strain which failed to ferment 
 saccharose. 
 
 Recognized all the groups of Andrewes and Border and 1 other, fermenting salicin and raffinose. 
 
 "Dextrose, lactose, and saccharose were tested with over 100 strains. With few exceptions all strains produced 
 acid from these carbohydrates and they were consequently of no significance in diferentiation. Dulcite was used 
 with over one half of the series, giving only one positive result, a Pneumococcus mucosus. Litmus milk was em- 
 'lyed throughout, but does not appear to give results which are sufficiently definite to be of value!' 
 
 (See also Table VIII on General Conclusions, etc.) 
 
 The a strains, the hemolytic bovine strains, and non-virulent strains generally produced more acid than the 
 virulent ^ strains. 
 All the strains fell into 8 groups according to their fermentative reactions. 
 
 Group. 
 
 No. of Strains. 
 
 i 
 
 i 
 
 i 
 
 , 
 
 
 i 
 
 
 
 
 
 
 
 ■s 
 
 s, 
 
 
 d 
 
 .g 
 
 •S* 
 
 
 a 
 
 /3 
 
 a 
 
 S 
 
 ^ 
 
 s 
 
 
 % 
 
 c 
 
 00 
 
 I 
 
 4 
 
 2 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 
 
 
 + 
 
 II 
 
 6 
 
 n 
 
 + 
 
 + 
 
 + 
 
 + 
 
 — 
 
 — 
 
 — 
 
 + 
 
 HI 
 
 
 4 
 
 + 
 
 + 
 
 -t- 
 
 — 
 
 — 
 
 + 
 
 — 
 
 + 
 
 IV 
 
 
 1 
 
 + 
 
 + 
 
 + 
 
 — 
 
 ~ 
 
 
 — 
 
 + 
 
 V 
 
 1 
 
 
 + 
 
 + 
 
 — 
 
 + 
 
 
 — 
 
 — 
 
 + 
 
 VI 
 
 2 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 — 
 
 — 
 
 
 vn 
 
 3 
 
 8 
 
 -t- 
 
 + 
 
 + 
 
 + 
 
 
 — 
 
 — 
 
 
 VUI 
 
 1 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 — 
 
 — 
 
 + 
 
 + 
 
 (See also Table VIII on General Conclusions, etc.) 
 
 Both strains originally fermented dextrose, lactose, saccharose, and salicin. 
 also,. Neither strain fermented raffinose or inuiin. 
 (See also Table VII on Mutations and Variability.) 
 
 1 of them fermented mannite 
 
 " The strains of streptococci which were found to have an affinity for the appendix formed short chains, much 
 acid, and a diffuse turbidity in ascites dextrose broth." 
 
 Reference. 
 
 Holman 1914. 
 
 A Method for Making Carbohydrate Serum 
 Broth of Constant Composition for Use in the 
 Study of Streptococci. 
 
 Thro 1914. 
 
 Experiments on the Variability of the Fer- 
 mentative Reaction of Bacteria, Especially the 
 Streptococci. 
 
 Capps and Davis 1914. 
 
 An Epidemic of Streptococcus Sore Throat in 
 
 Jacksonville, 111., Which Was Traced to the 
 
 Milk of Cows Affected with Streptococcus 
 
 Mastitis. 
 
 Lyall 1914. 
 
 On the Classification of the Streptococci. 
 
 Smith and Brown 1915. 
 
 A Study of the Streptococci Isolated from 
 
 Certain Presumably Milk-Borne Epidemics of 
 
 Tonsillitis Occurring in Massachusetts in 1913 
 
 and 1914. 
 
 Helnemann 1915. 
 
 The Variability of Two Strains of Strepto- 
 coccus lacticus. 
 
 Rosenow 1915. 
 
 The Bacteriology of Appendicitis and Its 
 Production by Intravenous Injection of Strep- 
 tococci and Colon Bacilli. 
 
TABLE V — Continued. 
 Fermentation Reactions. 
 
 TABLE y— Continued. 
 Fermentation Reactions. 
 
 
 Media. 
 
 Indicator or titration. 
 
 Period of incubation. 
 
 
 Reference. 
 
 
 Composition. 
 
 Test substances. 
 
 
 
 
 {a) Plain broth -|- 1 per 
 cent of test substance. 
 
 (6) Beef serum broth 
 sterilized at 60-70°C. on 
 several days. 
 
 (c) Holman's carbohy- 
 drate broth -f filtered beef 
 serum. 
 
 Lactose, saccharose, man- 
 nite, rafiinose, salicin, inulin. 
 
 Litmus milk. 
 
 (a 1 Litmus. 
 (ii Titrated. 
 
 (c! .Andrade's in- 
 dicator. 
 
 
 (a) This medium was soon abandoned "as the organisms grew in it very slowly or not at all." 
 
 (b) "The results were so irregular and so at variance with our other experiments that we felt there was some unde- 
 tected source of error." 
 
 (c) "With this medium we obtained uniform and clear cut results, * * ." 
 
 29 green-producing strains are tabulated with reference to their action on the test substances. All but 3 
 acidified milk and most of them coagulated it; all but 4 fermented lactose; all but 3 fermented saccharose; about 
 half fermented rafiinose and salicin; only 1 fermented mannite; and none fermented inulin. 
 
 "From the fermentation reactions these belong to the classes S. mitis, S. salivarius, and S. fecalis of Andrewes 
 and Harder." 
 
 Hartzell and Henriel 1915. 
 A Study of Streptococci from Pyorrhea Alveo- 
 laris and from Apical Abscesses. 
 
 60 strains from various 
 human pathological condi- 
 tions. 
 
 "Meat infusion broth" 
 with 1 per cent or 2 per cent 
 of peptone. 
 
 Lactose, saccharose, sal- 
 icin, raffinose, mannite, inu- 
 lin. 
 
 Titrated (phe- 
 nolphthalein). 
 
 3 days. 
 
 "It was found in agreement with Hopkins that a broth containing 2 per cent peptone gave more uniform results," 
 than did one containing 1 per cent peptone. < 
 (See also Table VIII on General Conclusions, etc. and Table VII on Mutations and Variability.) 
 
 Kllgler 1915. 
 A Study of the Correlation of the Aggluti- 
 nation and the Fermentation Reactions Among 
 the Streptococci. 
 
 Strains of S. mridans from 
 various human pathological 
 conditions. 
 
 "Sugar-free broth (made 
 from fresh meat), three parts, 
 and sterilized ascitic fluid, 
 one part." 
 
 Added 1 per cent of test 
 substance, sterilized sepa- 
 rately 20 min. at 100°. 
 
 Salicin, raffinose. (Man- 
 nite, inulin.) 
 
 Milk. 
 
 Titrated (phe- 
 nolphthalein). 
 
 
 _ Plated out supposedly pure cultures and tested the fermentation reactions of strains from different colo- 
 nies. The only variations reported are with respect to salicin and rafiinose. 
 
 ! 
 
 Thro 1915. 
 Further Experiments on the Variability of 
 the Fermentative Reactions of Bacteria, Es- 
 pecially the Streptococci. 
 
 Hemolytic streptococci 
 from sore throats, 1 strain 
 from secondary peritonitis, 
 and 2 from udder of cow, 
 compared with various other 
 hemolytic and non-hemolytic 
 strains. 
 
 
 
 
 
 "Serum water is unsuited for the determination of sugar fermentation. All of the type strains failed to ferment 
 salicin using this msdium, although prompt fermentation took place using serum broth or serum water containing 
 one per cent of peptone." 
 
 The strains reported from the epidemic at Rockville Centre fermented salicin but not raffinose or mannite. 
 
 (See Table VIII on General Conclusions, etc.) 
 
 Krumwlede and Valentine 1915. 
 A Bacteriological Study of an Epidemic of 
 Septic Sore Throat. 
 
 : "sJi 
 
 Over 500 strains from vari- 
 ous sources, human and ani- 
 mal. 
 
 "Sugar-free broth" plus 1 
 per cent of the test sub- 
 stances. 
 
 (See under Broadhurst, 
 1913.) 
 
 Sterilized fractionally 
 after addition of test sub- 
 stances. 
 
 Saccharose, lactose, sal- 
 icin, raffinose, mannite, inu- 
 lin. 
 
 Litmus milk. 
 
 Titrated cold 
 (phenolphthalein). 
 
 3 days. 
 
 The fennentative reactiolns and other characters of over 500 strains are tabulated. The author's tables 
 show that "the main representatives of a species are usually from the habitat designated by Andrewes and Harder" 
 * * "plus two fermentative combinations common in my own strains." "On the other hand, strains from a 
 selected habitat are scattered through a number of species; *. * ." 
 
 " * * the combinations most frequent here are also those occurring most often in the 'family tree' * * ." 
 
 (See Table VII on Mutations and Variability.) 
 
 "A comparison of the amounts of acid formed yields little of value." 
 
 Broadhurst 1915. 
 Environmental Studies of Streptococci, with 
 Special Reference to the Fermentative Reac- 
 tions. 
 
 
 
 
 
 
 (See Table VII on Mutations and Variability.) 
 
 Irons, Brown, and Nadler 1916. 
 The Localization of Streptococci in the Eye. 
 
 
 
 
 
 
 Merely the following statement with regard to fermentation reactions: "It might be said, however, that the 
 fermentative powers of some of the strains having affinity for the appendix suggest the latter origin {i.e., udders of 
 cows), while those of strains producing parotitis suggest the former source {i.e., human souices)." 
 
 Rosenow and Dunlap 1916. 
 
 An Epidemic of Appendicitis and Parotitis 
 Probably Due to Streptococci Contained in 
 Dairy Products. 
 
 7 strains of streptococci 
 from tonsils and pyorrhea! 
 pockets of cases of herpes 
 zoster; 1 strain from spinal 
 
 fluid. 
 
 "Broth." 
 
 "Various sugars." 
 
 
 
 " The fermentative powers of 7 of these strains that were proved to have affinity for the ganglia were tested on vari- 
 ous sugars in broth. All but 1 produced acid in saccharose; all but 2 in salicin; 3 fermented raffinose, and 2 man- 
 nite; none fermented inulin. In short, these streptococci had the features of a pneumococcus , except high virulence, 
 capsule, and inulin fermentative powers." 
 
 i 
 
 Rosenow and Oftedal 1916. 
 
 The Etiology and Experimental Production of 
 Herpes Zoster. 
 
TABLE V — Concluded. 
 Fermentation Reactions. 
 
 TABLE V — Concluded. 
 Fermentation Reactions. 
 
 Organisms. 
 
 R&ume of 2463 strains 
 ,tudicd by the author and 
 
 s 
 others. 
 
 1 strain of hemolytic strep- 
 tococci (human type) from 
 udder of cow without gross 
 evidence of mastitis. 
 
 1 strain of human type 
 streptococcus isolated by 
 Davis (1912) from bovme 
 mastitis. 
 
 1 strain of 5. eptdemicus 
 isolated by Davis (1912) from 
 peritoneal pus of fatal case 
 of peritonitis. 
 
 79 of 85 strains of hemo- 
 lytic streptococci from 328 
 samples of bottle milk. 
 
 5 strains: (1) "A strongly 
 hemolytic streptococcus" from 
 a case of erysipelas; (2) "a 
 hemolytic strain from a scar- 
 latinal sore throat;" (3) "o 
 hemolytic streptococcus from 
 the tonsils in a case of rheu- 
 matic fever ;" (4) " a non-kemo- 
 lytic streptococcus" from 
 pneumonic sputum; (5) "a 
 green-producing streptococcus" 
 from the same source as 
 No. 3. 
 
 Many strains of hemolytic 
 streptococci from cases of 
 tonsillitis, otitis media, and 
 scarlet fever; also from nor- 
 mal throats and suspected 
 milk. 
 
 51 strains of "Streptococcus 
 hemolysans." 
 
 79 strains of "Streptococcus 
 buccalis." 
 
 8 strains of "Streptococcus 
 fecalis." 
 
 4 strains of "Streptococcus 
 eqidnus." 
 
 Media. 
 
 Composition. 
 
 Unheated beef serum 
 broth -1- 1 per cent test 
 substance. 
 
 (See under Holman, 1914.) 
 
 '1 per cent sugar broth." 
 
 Test substances. 
 
 Indicator or titration. 
 
 Lactose, mannite, salicin, 
 inulin. 
 
 Sugar-free veal infusion 
 bouillon. 
 
 Test substances auto- 
 claved in 10 per cent aque- 
 ous solution, and 1 cc. added 
 to 9 cc. of bouillon. 
 
 Dextrose, lactose, saccha- 
 rose, maltose, salicin. 
 
 Glucose, lactose, maltose, 
 saccharose, ralTinose, salicin, 
 mannite, inulin. 
 
 Milk. 
 
 Andrade's indi- 
 cator. 
 
 Period of incubation. 
 
 Results and conclusions. 
 
 Reference. 
 
 At least 1 wk. 
 
 Titrated (results 
 for only 14 strains 
 recorded). 
 
 Dextrose, lactose, saccha- 
 rose, maltose, mannite, and 
 raffinose. 
 
 " Two per cent peptone 
 meat-infusion broth, 0.0- 
 0.5 -{- acidity, containing 
 one per cent of the test-car- 
 bohydrate and sterilized in 
 flowing steam on three suc- 
 cessive days." 
 
 Dextrose, lactose, mal- 
 tose, saccharose, mannite, 
 salicin, inulin, and raffinose. 
 
 Lactose, mannite, salicin, 
 saccharose, raffinose, and 
 inulin. 
 
 Titration against 
 0.2n NaOH, using 
 phenolphthalein as 
 an indicator. 
 
 1 wk. 
 
 7 days. 
 
 Titration (phe- 
 nolphthalein). 
 
 Also observed 
 reaction to litmus. 
 
 5 days. 
 
 "Quantitative carbohydrate acid tests by titration are not as useful as the qualitative tests." 
 (See Table VIII on General Conclusions, etc.) 
 
 Holman 
 
 The Classification of Streptococci. 
 
 1916. 
 
 Dextrose, lactose, saccharose, maltose, and salicin were fermented by all 3 strains mentioned. 
 
 Mathers 
 
 1916. 
 
 Different Types of Streptococci and Their 
 Relation to Bovine Mastitis. 
 
 "Of7P strains of these streptococci tested on 8 sugars, all fermented dextrose, lactose and maltose IJ failed to 
 ferment saccharose, 65 failed to ferment mannite, 11 failed to ferment salicin, 3 fermented inidtn, and 3 fermented 
 
 "Tnthe fermentable carbohydrate broths the milk strains grow more profusely, acidify more quickly, with generally 
 a higher terminal acidity," than do the human strains. 
 Litmus milk was rapidly acidified and coagulated by the milk strains. 
 
 Davis 
 
 1916. 
 
 Hemolytic Streptococci Found in Milk. 
 
 The only statement in regard to fermentation is the followmg: 
 tose, and maltose; Organism 2 fermented lactose, mannite, raffinose ^^ 
 
 saccharose, lactose, maltose, and dextrose; Organism 4 fermented maltose only. 
 
 Organisms 1 and 5 fermented saccharose, lac 
 maltose, atid dextrose; Organism 3 fermented 
 
 From all sources all but a few of the strains regarded as of pathological significance fermented dextrose, mal- 
 tose, lactose, saccharose, and salicin but did not ferment mannite, inulm, or raffinose. 
 
 Becker , 1^16. 
 
 The Necessity of a Standard Blood-Agar 
 Plate for the Determination of Hemolysis by 
 Streptococci. 
 
 Smlllie 1917. 
 
 Studies of the Beta Hemolytic Streptococcus 
 (Smith and Brown). 
 
 "An increase in acUity of 1.2+ or more above the initial acidity of the media was considered as indicative of fer- 
 mentation. In a considerable series, as stated above, litmus was added to the remaining^ media m the tube, parallel 
 results being obtained throughout." * * "Litmus or Andrade's decolorized acidfuchsm should be used as an indi- 
 cator, titration of the acidity produced being of no additional value." ,- r 7 !> 
 
 "In the classification proposed only lactose and mannite were retained as being of differential value. 
 
 The Classification of Streptococci. 
 
 1917. 
 
TABLE VI. 
 Pathogenicity and Virulence. 
 
 TABLE VI. 
 
 Pathogenicity and Virulence. 
 
 Organisms. 
 
 Animals. 
 
 Inoculation. 
 
 Period of observation. 
 
 Results and conclusions. 
 
 Reference. 
 
 Method. 
 
 Amount. 
 
 Strains isolated from ^the milk of 
 cows nilii i:iironic mastitis. 
 
 2 cows, 2 goats, 
 several pups, kids, 
 guinea pigs, rabbits, 
 and kittens. 
 
 Cows and goats in- 
 oculated into the 
 milk ducts by cathe- 
 ter or directly into 
 the udder by punc- 
 ture. 
 
 Small animals fed 
 with infected milk; 
 also inoculated intra- 
 peritoneally and in- 
 travenously. 
 
 1 cc. of bouillon 
 culture. 
 
 Up to 5 mos. 
 
 Both cows and 1 goat developed mild chronic mastitis persisting in 1 cow and 1 goat for 5 mos. The 
 other goat developed no lesions. 
 The smaller animals developed no symptoms or lesions whatever. 
 
 Noeard and Mollereau 1887. 
 Sur une mammite contagieuse des vaches 
 laitifires. 
 
 
 Rabbits. 
 
 Intravenous. 
 
 
 
 "In the course of my experiments my attention became directed to the relatively early hemolysis of the blood 
 invariably present in rabbits dead from a rapidly fatal dose of the pneumococcus ." 
 
 "In a number of animals there was a slight hemolysis shortly before death, while the blood was crowded with pneu- 
 mococci." 
 
 Rosenow 1904. 
 
 Studies in Pneumonia and Pneumococcus In- 
 fections. 
 
 11 strains from various patliologi- 
 cal conditions and from normal hu- 
 man mouths. 
 
 Rabbits. (Mice.) 
 
 
 
 
 The author was unable to raise the virulence of avirulent strains by passage through rabbits and mice. 
 (See also Table VIII on General Conclusions, etc.) 
 
 Kerner 1905. 
 
 Experimenteller Beitrag zur Hamolyse und 
 zur Agglutination der Streptokokken. 
 
 "5. mitis." 
 
 Gray mice. 
 
 
 0.5 cc. of 24 hr. 
 bouillon culture. 
 
 
 Found strains of "S. mitis" which were pathogenic for mice, 0.5 cc. of 24 hr. bouillon culture killing them 
 in 3-6 days. 
 
 Frankel 1905. 
 
 Ueber menschenpathogene Streptokokken. 
 
 22 strains from vulval, vaginal, and 
 uterine secretions of 10 women dur- 
 ing and 4-9 days after labor. 
 
 1 strain from a felon. 
 
 1 strain from throat (angina). 
 
 Mice, rabbits. 
 
 Actively immunized animals with each 
 strain (4 injections) and then tested their 
 resistance to a lethal dose of 5. pyogenes 
 (strain from the felon). 
 
 
 Succeeded in producing more or less immunity to 5. pyogenes with 14 strains, but not with others. In these 
 tests the 14 and other strains used for immunization were not necessarily 5. pyogenes, but the immunity was 
 tested ineach case by injection of a lethal dose of a single strain of S. pyogenes. 
 
 The author concludes that the 14 strains were identical with or nearly related to 5. pyogenes. 
 
 Natvlg 1905. 
 
 Bakteriologische Verhaltnisse in weiblichen 
 Genitalsekreten. 
 
 
 Mice. 
 
 
 
 
 Percentage of strains isolated from pathological sources: S. equimis, none; 5. mitis, rarely; 5. pyogenes, 90 per 
 cent; S. salivarius, 33 per cent; S. anginosus, 54 per cent; S.fecalis, 16 per cent; pneumococcus, 34 per cent. 
 
 No thorough study of pathogenesis for animals is reported. In 1 of. the tables the S. pyogenes, S. angi- 
 nosus, and pneumococcus groups are indicated as being pathogenic for mice, while 5. salivarius and 5. fecalis 
 are indicated as non-pathogenic. 
 
 Andrewes and Herder 1906. 
 
 A Study of the Streptococci Pathogenic for 
 Man. 
 
 Strains of streptococci and pneu- 
 mococci from 154 throats; 51 normal, 
 75 scarlatinal, 14 measles, 5 tonsilli- 
 tis, 5 pneumonia, 4 larjmgitis. 
 
 Rabbits. 
 
 Intraperitoneal. 
 
 Growth from 1 or 
 2 48 hr. blood agar 
 slants. 
 
 
 "Most cultures of S. pyogenes and some pneumococci isolated from scarlatinal and normal throats have some 
 virulence for rabbits, but S. viridans has practically no virulence for these animals." 
 The lesions produced are not described, merely the number of animals which died or survived being given. 
 
 Ruediger 1906. 
 
 The Streptococci from Scarlatinal and Nor- 
 mal Throats and from Other Sources. 
 
 
 White mice. 
 
 
 
 
 "It is possible to convert some but not all strains possessing the streptococcus cultural type into typical pneumo- 
 cocci by inoculation into white mice." 
 
 Buerger and Ryttenberg 1907. 
 Observations upon Certain Properties Ac- 
 quired by the Pneumococcus in the Human 
 Body. 
 
 1 strain from milk. 
 
 Obtained an aggressin as follows: Washed off the growth from 4 KoUe plates with 
 6 cc. portions of bouUlon. Injected this suspension into the pleural cavity of 4 rabbits 
 which died next day. Found about 15 cc. of clear bloody fluid in the pleural cavities 
 which he pipetted out, sedimented, decanted, and added 0.5 per cent of phenol. Tested 
 sterility and in experiments injected 0.1-2 cc. subcutaneousiy into rabbits. 
 
 Found that a certain amount of immunity could be produced by 3 injections of aggressin. If on the 
 other hand the aggressin was injected simultaneously with live streptococci, the resistance of the animal was 
 considerably lowered. 
 
 Nleter 1907. 
 
 Zur Streptokokkenfrage. 
 
 40 strains of streptococci from vari- 
 ous human infections. 
 
 Mice. (Rabbits, 
 guinea pigs, and mon- 
 keys in some cases.) 
 
 
 
 
 (See Table VIII on General Conclusions, etc.) 
 
 Zangemeister 1909. 
 
 Die Hamolyse der Streptokokken. 
 
TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 Organisms. 
 
 Animals. 
 
 Method. 
 
 "Modified pnmmococci 
 of endocarditis. 
 
 from cases ; Rabbits. 
 
 25 streptococci and 5 pneumo-; Mice, 
 cocci, all isolated from the blood 
 during life. 
 
 Many strains of S. mitior isolated 
 from vaginal secretions of normal 
 puerpera and gravida. 
 
 3 laboratory strains of S. pyogenes 
 for comparison with the above. 
 
 5. lacticus strains isolated from 7.5 
 per cent of 81 samples of milk and 
 from cow dung and skin. 
 
 Mice, rabbits. 
 
 "Modified pneumococci" from cases 
 of endocarditis. 
 
 Rabbits. 
 
 Many strains of 5. pyogenes, S. 
 longissimus, S. conglomeratus, S. bre- 
 nts, S. mucosus, and pneumococcus. 
 
 Rabbits, guinea 
 pigs, mice, dogs, rats. 
 
 Mice. 
 
 Intravenous. 
 
 Amount. 
 
 3-15 cc. of 24 
 hr. bouillon, blood 
 bouillon, or milk cul- 
 tures. 
 
 Intravenous, intra- 
 peritoneal, and "di- 
 rectly into the heart.'" 
 
 Animals often in- 
 jected by 2 or all 3 
 methods simultane- 
 ously. 
 
 Intraperitoneal 
 intrathoracic. 
 
 2-10 cc. of broth, 
 ascites broth, blood 
 broth, or blood cul- 
 ture. 
 
 Growth from 1-4 
 agar or blood agar 
 slants. 
 
 Period of observation. 
 
 (See under Results 
 and Conclusions.) 
 
 Results and conclusions. 
 
 " * * endocarditis developed in five of ten animals inoculated before the special characteristics of the pneu- 
 mococci were lost." 
 
 "Pericarditis developed in six cases," in 2 of which endocardial lesions were absent. 
 
 " * * of 16 rabbits injected with various strains of pneumococci after return to the usual type none developed 
 endocarditis." 
 
 Obtained blood cultures during life in 90 per cent of cases of infective endocarditis. 66 per cent of these 
 cultures were streptococci and 12 per cent were pneumococci (3 strains, S. pyogenes, 9 S. salivarius, 9 S. angino- 
 sus, 8 S. fecalis, 5 pnetumococcus) . 
 
 At autopsy obtained streptococci in 62 per cent of 100 cases, and pneumococci in 19 per cent of cases. 
 
 Concludes that "infective endocarditis is due to the operation of streptococci of low virulence, of the types found 
 in normal feces and in normal saliva." " They are for the most part * * not pathogenic for mice." 
 
 Found strains of 5. mitior pathogenic for mice and raised their virulence by successive passages through 
 mice until they were killed in 24 hrs. after injection. 
 
 "Virulenzprufung an Tieren habe ich ganz unterlassen, da Tier- und Menschenpatho genital durchaus nicht 
 parallel gehen." 
 
 Found 5. lacticus non-pathogenic for mice and rabbits. 
 
 "Injection of the bacteria, cultivated on artificial media for a considerable time, directly into the heart always 
 produced pericarditis and usually also endocarditis." 
 
 " * * the production of endocarditis in animals by pneumococci witlioul injuring the valves^ is closely 
 dependent on special characteristics of the cocci and on the largeness of the dose. It was noted that_ while the first^^ 
 injection rarely caused lesions or death it rendered the animal more susceptible to subsequent inoculations, * * ." 
 
 "In order to produce death promptly after a second injection relatively large doses are necessary. The seemingly 
 increased susceptibility to infection might in reality be an expression of an active immunity and death the result 
 of an overwhelming intoxication from rapid destruction of the organisms injected." 
 
 There is shown "a marked increase in 'virulence' of the bacteria when cultivated in the patient's blood as com- 
 pared with those grown on artificial media, * * ." "Continuous cultivation in fresh normal blood has a similar, 
 but not so marked an effect." 
 
 Gray mice were killed by 0.5- 4.0 cc. of bouillon culture. 
 Rabbits " " " 20 - 30 cc. " •" " . 
 
 Guinea pigs " " " 5 - 10 cc. " " " . 
 
 Dogs " " " 50 -500 cc. " " " . 
 
 Rats (Mus decumanus) were killed by 0.1 cc. or less of bouillon culture of a strain the virulence of which 
 had been raised by passages through guinea pigs. 
 
 Culture filtrates were not toxic for gray mice, rabbits, or guinea pigs. 
 
 In a series of blood cultures taken during life the author found S. mitior seu viridans to be the cause of a 
 characteristic form of endocarditis which he called " schleichende HerzhlappenentzUndnng, Endocarditis tenia." 
 
 S. longissimus was found to be distinctly pathogenic for mice, though less so than 5. pyogenes. 
 
 Reference. 
 
 Rosenow 1909. 
 
 Immunological and Experimental Studies on 
 Pneumococcus and Staphylococcus Endocar- 
 ditis. 
 
 Border 1909. 
 
 Infective Endocarditis: With an Analysis of 
 150 Cases and with Special Reference to the 
 Chronic Form of the Disease. 
 
 Konrad 1909. 
 
 Weitere Beitrage zur Vaginalstreptokokken- 
 frage. 
 
 Sachs 
 
 tjber Streptokokken-Hamolyse. 
 
 1909. 
 
 Bahr 1910. 
 
 Vorkommen und Bedeutung der Strepto- 
 kokken in der Milch. 
 
 Rosenow 1910. 
 
 A Study of Pneumococci from Cases of In- 
 fectious Endocarditis. 
 
 Prleolo 1910. 
 
 Recherches experimentales sur le streptocoque 
 de la gourme. 
 
 Sehottmiiller 1910. 
 
 Endocarditis lenta. Zugleich ein Beitrag 
 zur Artunterscheidung der pathogenen Strepto- 
 kokken. 
 
 Thalmann 1910. 
 
 Streptokokkenerkrankungen in der Armee, 
 Einteilung der Streptokokken und ihre Be- 
 kampfung. 
 
TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 .==——— 
 
 Animals. 
 
 Inoculation. 
 
 Period of observation. 
 
 Results and conclusions. 
 
 Reference. 
 
 Organisms. 
 
 Method. 
 
 Amount. 
 
 
 "V^ains, mostly from mouth, 
 rtroat and oints of cases of rheu- 
 LaSm; a iL from the heart blood 
 Th various local suppurations. 
 
 Rabbits. 
 
 
 
 
 Of the strains tested 15 produced fatal septicemia or pyemia with or without arthritis, 13 produced non- 
 purulent arthritis, and 9 gave negative results. 
 
 Seattle and Yates 1911. 
 
 Sugar Tests and Pathogenicity in the Dif- 
 ferentiation of Streptococci. 
 
 -^"^^^^ from an epidemic of 
 "Grippe" among children. 
 
 Rabbits, white 
 mice, guinea pigs. 
 
 
 
 
 Quite pathogenic for white mice and rabbits, causing sepsis; non-pathogenic for guinea pigs. 
 
 Miiller and Sellgmann 1911. 
 Klinische und bakteriologische Beobach- 
 tungen bei Sauglingsgrippe. II. Bakteriolo- 
 gisches von Seligmann. 
 
 Evidently many strains from vari- 
 ous sources. 
 
 
 
 
 
 Streptococci isolated directly from the blood of human patients were often non-pathogenic for animals. The 
 virulence of most streptococci for animals was raised by passage through animals. 
 
 Roily 1912. 
 Experimentelle bakteriologische Untersuch- 
 ungen von verschiedenen Streptokokkenstam- 
 men. 
 
 6 strains of S. acidi lacttct. 
 4 strains of 5. pyogenes. 
 3 strains of pneumococcus. 
 
 Mice. 
 
 
 - 
 
 
 5. acidi lactici was non-pathogenic for mice; 5. pyogenes and 5. lanceolatus were fatal for mice. 
 
 Saito 1912. 
 Versuche zur Abgrenzung des Streptococcus 
 acidi lactici von Streptococcus pyogenes und 
 Streptococcus lanceolatus. 
 
 26 strains from bovine mastitis; IV 
 from dust, horse saliva, and bovine 
 vaginitis; 1 S. pyogenes, 1 _S. ery- 
 sipdatis, and 1 S. acidi laclict. 
 
 White mice, goat. 
 
 Mice inoculated 
 subcutaneously or in- 
 traperitoneally. 
 
 Goat inoculated 
 into the udder. 
 
 Rather large doses. 
 
 
 5 of the strains from bovine mastitis and the 5. erysipelatis (Fehleisen) killed mice in 2-9 days. Mice sur- 
 vived inoculations with all other strains. 
 
 Inoculation of a goat into the udder with streptococci isolated from bovine vaginitis resulted in a true strep- 
 tococcus mastitis. 
 
 Gminder 1912. 
 Untersuchungen iiber Mastitisstreptokokken 
 und ihre DifEerenzierung von saprophytischen 
 Streptokokken. 
 
 
 Mice. 
 
 
 ■ rfM 
 
 
 (See Table VIII on General Conclusions, etc.) 
 
 Thalmann 1912. 
 Streptococcus viridans im Blut ohne Verander- 
 ung der Herzklappen. Zur Einteilung der Strep- 
 tokokken. 
 
 Streptococci from 20 cows with 
 mastitis compared with several from 
 human sepsis, puerperal fever, and 
 scarlet fever, and 1 strain of S. lacticus. 
 
 Mice. 
 
 
 
 
 Found 2 mastitis strains virulent for mice and points out that streptococci from the human throat (espe- 
 cially from cases of scarlet fever) may or may not be virulent foi mice. 
 
 Salus 1912. 
 Untersuchungen zur Hygiene der Kuhmilch. 
 
 (a) Fresh market milk. 
 lb) Sediment from 10 cc. of milk. 
 (c) Milk after incubation for 3 hrs. 
 Strains isolated from milk, human 
 infections, and infants' feces. 
 
 Guinea pigs. 
 
 Intraperitoneal. 
 
 [a) 5 cc. 
 
 {b) Sediment from 
 10 cc. 
 (c) 5 cc. 
 
 
 14 of 40 guinea pigs inoculated died after 1-11 days. Various organisms were isolated at autopsy but 
 "never a true S. pyogenes in long chains." 
 
 Sterile milk produced a sterile peritonitis in guinea pigs. 
 
 Phagocytic experiments in vitro showed the " Viridenzsahl" (Burgers) of all milk streptococci to be much 
 lower than that of streptococci from various human infections, using human serum and leukocytes. The 
 " Virulenzzahl" of streptococci from infants' feces was also quite low. The " Virulenzzahl" of milk strepto- 
 cocci, using beef blood, was also low but higher than with human blood. 
 
 Puppel 1912. 
 Ueber Streptokokken in der Milch und im 
 Sauglingsstuhl. 
 
 Many strains from sore throats 
 (Chicago epidemic, 1912) and second- 
 ary lesions. 
 
 Rabbits, guinea 
 pigs, mice. 
 
 Intraperitoneal. 
 
 "Small quantities." 
 
 
 Rabbits, guinea pigs, and mice were killed in 12-24 hrs. 
 
 Davis and Rosenow 1912. 
 An Epidemic of Sore Throat Due to a Pecu- 
 liar Streptococcus. 
 
 45 strains from throats and sec- 
 ondary lesions of cases of epidemic 
 sore throat. 
 
 1 strain of ordinary hemolytic 
 streptococci from the udder of a cow. 
 
 Rabbits, guinea 
 pigs. 
 
 Intravenous and 
 intraperitoneal. 
 
 "Relatively small 
 doses." 
 
 
 "These organisms are very highly pathogenic for animals, killing guinea pigs in relatively small doses in from 
 twenty-four to forty-eight hours. They are even more virulent in rabbits. Intravenous injection of small doses 
 causes a generalized infection with death following in from twenty-four to thirty-six hours. If very ^ small doses 
 be given the organisms, like ordinary streptococci, tend to localize in the joints, causing multiple arthritis; also there 
 may be infection of serous cavities generally, and local abscesses in the myocardium. Vegetative endocarditis with 
 hemorrhages may occur but is rare." 
 
 With regard to the strain from the cow's udder: "One blood agar slant growth injected intravenously into a rao- 
 hit caused purulent arthritis of the left knee and death on the fourth day from septicemia. Guinea pigs succumbed 
 usually in twenty-four hours after intraperitoneal injection of tke same amount." 
 
 Davis 1912. 
 
 Bacteriologic Study of Streptococci in Milk 
 in Relation to Epidemic Sore Throat. 
 
TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 Orgauisms. 
 
 Cocci from cases of chronic endo- 
 carditis, pure and mixed with B. 
 fusiformis or streptococci. 
 
 Mixed cultures from the throats of 
 endocarditis cases. 
 
 15 samples of milk "slime" from 
 dairy separators and clarifiers, 24-72 
 hrs. old. 
 
 Mixed cultures of the above in 
 ascites meat broth. 
 
 Strains from milk obtained from 
 individual cows "in sterile form." 
 
 Animals. 
 
 Rabbits. 
 
 1 strain "5. epidemicus" from 
 milk. 
 
 1 strain from throat of carrier. 
 
 26 strains of S. equi (23 laboratory 
 strains of various ages and freshly 
 isolated by the authors). 
 
 27 strains of S. longus isolated by 
 the authors from human pathological 
 material. 
 
 A number of strains of hemolytic 
 streptococci, 5. viridans, S. mucosus, 
 and pneumococcus from various 
 sources. 
 
 14 strains from joint fluid of 14 
 cases of acute rheumatism, and 
 "other strains which have been made 
 to resemble them." 
 
 20 strains from various 
 pathological conditions. 
 
 human 
 
 (o) Rabbits. 
 (6) Guinea pigs. 
 (c) Mice. 
 
 Mice. 
 
 Rabbits, guinea 
 pigs. 
 
 Rabbits, young 
 dogs. (Mice.) 
 
 Mice. 
 
 Rabbits, guinea 
 pigs. 
 
 (a) Rabbits. 
 
 (b) Dog. 
 
 (c) Monkey. 
 
 Inoculation. 
 
 Method. 
 
 Intravenous. 
 
 Subcutaneous. 
 
 Intravenous. 
 
 Subcutaneous. 
 
 Various; rabbits 
 usually intravenous, 
 guinea pigs usually 
 intraperitoneal. 
 
 Intravenous. 
 
 Rabbits. 
 
 Amount. 
 
 Very large doses, 
 in some cases the 
 growth from 4-6 agar 
 slants. 
 
 (a) 6-15 cc. of 
 thick suspension in 
 salt solution. 
 
 ib) 5-7 cc. of the 
 same. 
 
 (c) 0.5-1 cc. 
 
 Probably 2 cc. of 
 serum bouillon cul- 
 ture. 
 
 Various. 
 
 Intravenous. 
 
 (a) Growth from 
 25-75 cc. of 24 hr. 
 ascites dextrose broth 
 culture. 
 
 (6) Growth from 
 250 cc. 
 
 (c) Growth from 
 100 cc. 
 
 Period of observation. 
 
 Up to 15 days. 
 
 " Cultures from Loef- 
 fler's blood serum sus- 
 pended in salt solu- 
 tion." 
 
 "No attempt was 
 made to secure a uni- 
 form dosage." 
 
 Results and conclusions. 
 
 Reference. 
 
 " The results of these experiments show that the endocarditis in the rabbit which follows injection of cocci which I 
 believe to be modified pneumococci, is due to an embolic process. There is first produced valvular hemorrhage from 
 which vegetations develop. The localization in the endocardium of the cocci from chronic infectious endocarditis 
 and of allied cocci from the throat, is due in large part to the presence of fine capillaries in the valves and to the pecu- 
 liar mode of growth of the cocci." 
 
 " The affinity of the endocarditis strains of cocci for the endocardium and of streptococci for joints is shown by 
 the results of injections of mixtures of these organisms, and the exact cause of such affinity is unknown." 
 
 Rosenow 
 
 Experimental Infectious Endocarditis. 
 
 1912. 
 
 "The animals (injected with separator slime) usually died of streptococcemia in from 24 to 72 hours," appar- 
 ently referring to animals of all 3 species. * * "Intravenous injections in the rabbits gave the highest per- 
 centage of positive results. In most cases {24) streptococci in pure culture were obtained, streptococci in predomi- 
 nating numbers but mixed with colon bacillus in nine, and the colon bacillus pure in one. One animal^ yielded a 
 pure culture of a typical pneumococcus. Nine animals survived while three developed streptococcus arthritis later. 
 
 Of the strains isolated from milk obtained in sterile form "two of these when injected into guinea pigs proved 
 virulent. The milk of one cow yielded a typical virulent pneumococcus. These three cows had not had any disease 
 of the udder at any time." * * "hemolyzing streptococci in pure culture" from the milk of "two cows with 
 chronic mastitis" * * "proved only moderately virulent for rabbits, guinea pigs, and white mice." 
 
 A mouse inoculated with the strain from milk died in 24 hrs. of peritonitis and septicemia. 
 The strain from the throat of a carrier was not pathogenic for mice. 
 
 Rosenow 1912. 
 
 A Study of Streptococci from Milk and from 
 Epidemic Sore Throat, and the Effect of Milk 
 on Streptococci. 
 
 (See Table VII on Mutations and Variability.) 
 (See also Table VIII on General Conclusions, etc.) 
 
 With reference to "der Drusecoccus ," — " * * ; bei intravenos infizierten Kaninchen erzeugt er hdufig eine 
 typische starke fibrinbse Perikarditis, die wir beim Streptococcus longus seu erysipelatos nie beobachtet haben." 
 
 "Beide Streptokokkenarten sind auch fUr junge Hunde pathogen. Sowohl mit dem Drusecoccus, als auch mil 
 dem Strept. longus lassen sich auf hdmatogenem Wege typische Gelenkentziindungen und eine ^ akute Enteritis 
 erzeugen, * * . Nach den bisheriges spdrlichen Versuchen mit dem Drusecoccus scheint jedoch dieser filr 
 junge Hunde eine grossere Pathogenitat zu haben, als der S. longus." 
 
 The formation of abscesses in the course of the lymphatics is described as being characteristic for S. equi. 
 
 (See Table VII on Mutations and Variability.) 
 
 Stokes and Haehtel 
 
 Septic Sore Throat, 
 break in Baltimore, Md. 
 of the Outbreak. 
 
 1912. 
 A Milk-Borne Out- 
 Bacteriological Study 
 
 Davis 
 
 Interrelations in the 
 with Special Reference 
 actions. 
 
 1913. 
 Streptococcus Group 
 to Anaphylactic Re- 
 
 Koeh and Poksehlsehewsky 1913. 
 
 Ueber die Artverschiedeaheit des Streptococcus 
 longus seu erysipelatos und des Streptococcus 
 squi (Druse-Streptococcus). 
 
 Bemelmans 1913. 
 
 La specificity des streptocoques de la gourme. 
 
 Rosenow 
 
 Transmutation,s Within 
 Pneumococcus Group. 
 
 the 
 
 1914. 
 Streptococcus- 
 
 The green-producing strains and those not aiiecting the blood agar produced arthritis, endocarditis, and i Rosenow 
 pericarditis. . . . . The Etiology of Acute 
 
 The strains producing slight hemolysis on blood agar produced myositis, myocarditis, endocarditis, peri- ticular and Muscular, 
 carditis, and arthritis; possibly also ulcer of the duodenum (dog) and of the stomach (monkey). [ 
 
 1914. 
 Rheumatism, Ar- 
 
 " Death or recovery only were taken into consideration, 
 severity of the infections from which the cultures came, * 
 
 * * The results obtained are consistent with the 
 
 Floyd and Wolbaeh 1914. 
 
 On the Differentiation of Streptococci. (Pre- 
 liminary notes.) 
 
TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 TABLE Yl— Continued. 
 Pathogenicity and Virulence. 
 
 = — — \ 
 
 Organisms. 
 
 Animals. 
 
 Inoculation. 
 
 Period of observation. 
 
 Results and conclusions. 
 
 Reference. 
 
 
 Method. 
 
 Amoint. 
 
 9 strains from cases of arthritis, 
 septicetnia. scarlet fever, endocardi- 
 tis, and infected finger. 
 
 Rabbits. 
 
 Subcutaneous. 
 
 1-5.5 cc. of 8- 
 19 hr. serun bouillon 
 cultures. 
 
 
 4 of the strains tested were pathogenic. " * * sevaal strains of streptococcus had retained a moderate 
 degree of pathogenicity for animals, although they had been s licuUitred on agar for periods of from nine months 
 to three years." 
 
 * * cultures of streptococci made in the media desc~ibed in this communication (bouillon containing 
 15-30 per cent horse serum or 70-75 per cent rabbit serum) appear to be most virulent at the end of ten to twelve 
 hours' incubation, * ." 
 
 The streptococci were recovered from the heart blood and hemolysis in vivo was noted. 
 
 M'Leod 1914. 
 
 Criteria of Virulence amongst Streptococci, 
 with Some Remarks on Streptococcal Leuco- 
 cidin. 
 
 2 strains of 5. pyogenes. Source 
 not given. 
 
 Rabbits. 
 
 Intravenous. 
 
 Various amounts 
 of 24 hr. bouillon 
 cultures. 
 
 At intervals from 
 5 min. to 30 days 
 after inoculation or 
 until death. 
 
 Withdrew blood samples at various intervals and made plates for counting the streptococci in the circu- 
 lating blood. 
 
 Found that the disease might be acute, producing death in 1-3 days, or chronic, producing death in 5- 
 30 days. In acute cases the number of organisms in the blood is usually much larger than in the chronic 
 cases, and is quite variable from time to time in the latter. 
 
 The bacterial count was always much larger in oxalate blood than in defibrinated samples, thought to be 
 due to mechanical removal of the bacteria by the fibrin. 
 
 Relehstein 1914. 
 
 Ueber den Nachweis der Streptokokken im 
 stromenden Blute. 
 
 6 strains from throats. 
 2 strains from milk slime . 
 
 Rabbits. 
 
 Intravenous. 
 
 
 
 All the strains tried produced' suppurative arthritis or death in rabbits. 
 
 North, White, and Avery 1914. 
 
 A Septic Sore Throat Epidemic in Cortland 
 and Homer, N. Y. 
 
 Milk cultures and streptococcal 
 exudate from tonsillitis case. 
 
 Pure culture of hemolytic strepto- 
 coccus. 
 
 A healthy cow giv- 
 ing about 16 quarts 
 of milk in 24 hrs. 
 
 (a) Milk cultures 
 and tonsillitis exu- 
 date smeared on the 
 healthy teats. 
 
 {b) Culture of he- 
 molytic streptococcus 
 smeared on abraded 
 teat. 
 
 (c) 8 cc. of milk 
 culture of hemolytic 
 streptococcus inject- 
 ed into milk duct by 
 catheter. 
 
 8cc. 
 
 Several wks. 
 
 (a) No infection by this method. 
 
 (6) Mild mastitis without caking of the udder was produced. Streptococci persisted in the milk for over 
 4 wks. 
 
 (c) Mild mastitis confined to the inoculated quarter of tlie udder. Large number of streptococci and leu- 
 kocytes were secreted in the milk for several wks. 
 The authors conclude that: 
 
 "Hemolytic streptococci of human origin may cause mastitis * * in cows." 
 " The streptococci may gain entrance through an abraded or injured surface of the teat." 
 
 " The mastitis may exist without physical evidence. * * though pus and streptococci in large numbers are 
 being secreted in the milk." 
 
 Davis and Capps 1914. 
 
 Experimental Bovine Mastitis Produced with 
 Hemolytic Streptococci of Human Origin. 
 
 Mixture of S. fecalis and pneumo- 
 
 C0CCU3. 
 
 B. coli. 
 
 Guinea pigs. 
 
 Intraperitoneal. 
 
 Various. 
 
 Various. 
 
 The injection of mixed cultures of S. fecalis and pneumococcus was followed by the recovery of only pneu- 
 mococci from the blood. 
 
 The injection of pure cultures of B. coli was followed by the recovery of streptococci from the blood. 
 
 Streptococci were easily isolated from the throats and intestinal contents of normal guinea pigs and from 
 the blood, pleura, peritoneum, etc., of guinea pigs dying "spontaneously." 
 
 (See Table VII on Mutations and Variability.) 
 
 Holman 1914. 
 
 The Relative Longevity of Different Strepto- 
 cocci and Possible Errors in the Isolation and 
 Differentiation of Streptococci. 
 
 12 strains (7 from throats; 3 bo- 
 vine; 1 S. epidemicus from throat, Chi- 
 cago epidemic, 1912; 1 S. epidemicus 
 from milk, Chicago epidemic, 1912). 
 
 Rabbits. 
 
 Probably intrave- 
 nous. 
 
 "1-2 slant agar 
 tubes." 
 
 
 2 strains isolated from the udders of cows produced multiple arthritis in rabbits in 3 or 4 days. 
 All the other strains, except 1 bovine strain, are tabulated as being pathogenic for rabbits without further 
 details. 
 
 Capps and Davis 1914. 
 
 An Epidemic of Streptococcus Sore Throat in 
 Jacksonville, 111., Which Was Traced to the 
 Milk of Cows Affected with Streptococcus 
 Mastitis. 
 
 
 
 
 
 
 (See Table III on The Use of Blood Agar.) 
 (See Table VIII on General Conclusions, etc.) 
 
 Davis 1914. 
 The Growth and Viability of Streptococci of 
 Bovine and Human Origin in Milk and Milk 
 Products. 
 
 28 strains of streptococci from sore 
 throats. 
 
 10 strains from cow's milk, 
 
 Rabbits. 
 
 Intravenous. 
 
 1_ cc. of 24 hr. 
 bouillon culture. 
 
 Up to 3 mos. or 
 unti death of the 
 animal. 
 
 18 of the strains from throats and 4 from milk were pathogenic for rabbits, producing sometimes only a rise 
 in temperature with loss of weight for a few days and sometimes marked polyarthritis or death. 
 (See also Table VIII on General Conclusions, etc.) 
 
 — _— — - — — — _— — . 
 
 Smith and Brown 1915. 
 A Study of Streptococci Isolated from Cer- 
 tain Presumably Milk-Borne Epidemics of 
 Tonsillitis Occurring in Massachusetts in 1913 
 and 1914. 
 
TABLE VI — Continued. 
 Pathogenicity and Virulence. 
 
 TABLE VI— Continued. 
 Pathogenicity and Virulence. 
 
 Organisms. 
 
 2 strains of S. lacticiis from ice 
 cream and from milk. 
 
 Strains of streptococci from the 
 tissues of appendices and from the 
 tonsils of appendicitis patients. 
 
 Protocols of experiments 
 strains from 5 cases are given. 
 
 with 
 
 9 strains from milk sediment. 
 
 2 strains of streptococci easily 
 recognized because of their peculiar 
 characteristics and not like any found 
 in dogs. 
 
 Strains isolated at intervals from 
 the tear sac of a patient with irido- 
 cyclitis. 
 
 Over 109 strains of streptococci 
 (73 from tonsils of normal individuals, 
 4 from tonsils of appendicitis pa- 
 tients, 9 from Steno's duct of paro- 
 titis patients, 23 from dairy products). 
 
 Animals. 
 
 Rabbits, 
 pigs. 
 
 guinea 
 
 Half-grown 
 bits. 
 
 rab- 
 
 Rabbits (800-1,000 
 gm.). 
 
 Half-grown rab- 
 bits, guinea pigs. 
 
 Dogs, cats. 
 
 Rabbits. 
 
 177 rabbits. 
 
 Inoculation. 
 
 Method. 
 
 Subcutaneous. 
 
 Intravenous. 
 
 Intravenous. 
 
 Intravenous; intra- 
 peritoneal. 
 
 Fed. 
 
 Intravenous. 
 
 Amount. 
 
 Growth from 1 24 
 hr. North's medium 
 slant. 
 
 " Very large doses." 
 
 8 cc. of 24 hr. 
 broth culture. 
 
 Growth from 10- 
 45 cc. of broth cul- 
 ture. 
 
 Period of observation. 
 
 Up to 
 longer. 
 
 8 days or 
 
 Nearly 5 mos. 
 
 Results and conclusions. 
 
 (See Table VII on Mutations and Variability.) 
 
 " The elective affinity for the appendix of the streptococci in pure culture or in mixture with fusiform bacilli, iso- 
 lated both from the tonsils at the time of, or soon after, the attack and from the appendix, is striking. Thus, the 
 tonsillar strains produced appendicitis in nineteen of twenty-nine, the appendix strains in twenty-two of thirty, a 
 total of forty-one of fifty-nine (rabbits injected). After cultivation on artificial media for a short time (or 
 after 1-3 animal passages) the elective affinity is soon lost, and strains isolated from the tonsils some time after 
 appendectomy also appear without elective affinity. The results following injections of mixtures of streptococci and 
 colon bacilli are similar {26 of 31), although the tendency to produce lesions in the intestine and gall-bladder is much 
 greater." , . 
 
 "The results of the experiments and observations indicate that appendicitis, in the absence of foreign bodies, com- 
 monly is a hematogenous infection, secondary to some distant focus; that it develops when, for some reason or other, 
 the organisms in the focus, usually streptococci, have acquired an elective affinity for the appendix and at the same 
 time gain entrance into the circulation.' , , / < t 
 
 "The results bear out my theory that a focus of infection is to be looked on, not only as the place of entrance of 
 bacteria, but also as the place where they may acquire the varying affinities necessary to infect distant organs and 
 tissues." 
 
 "We finally adopted 8 cc. of a twenty-four hour broth culture as a standard dose; this amount does not contain 
 enough toxic material to cause immediate symptoms, and usually kills the animal in from two to four weeks." * ^* 
 " The pathogenicity of these streptococci varied greatly, but for the most part they were of relatively low virulence. 
 
 "We inoculated in all twenty-four rabbits. Of these, six lived. Five died showing no gross lesions, nor micro- 
 scopic changes in heart or kidneys. One died of general sepsis and two of acute toxemia. Heart lesions occurred m 
 five, kidney lesions in seven, aortic lesions in three, and joint lesions in two." * * "Endocardial lesions 
 occurred but twice in our rabbits. 
 
 4 strains "were virulent for guinea pigs, producing a fatal peritonitis when injected intraperitoneally." 
 " * * two strains of streptococci isolated from the milk, indistinguishable from each other as to form and 
 cultural characteristics, were nevertheless quite dijferent, as shown by intravenous injection into animals, one local- 
 izing in the muscles and joints, and the other in the gall-bladder." . . . 
 
 Summary; "Virulent streptococci isolated from the milk showed selective preference for certain animal structures, 
 such as joints, muscles, gall-bladder, etc. 
 
 The growth from 
 a blood agar slant or 
 from 5-6 cc. of ascitic 
 dextrose bouillon cul- 
 ture. 
 
 Intravenous. 
 
 Growth from 15- 
 90 cc. of 24 hr. as- 
 cites dextrose broth 
 cultures. 
 
 Animals usually 
 killed in 48 hrs. un- 
 less dead from effects 
 of inoculation. 
 
 The object of the experiments was merely to recover the organifims fed and to note any changes in their 
 characteristics which might have occurred. No pathological effects on the animals are mentioned. 
 (See also Table VII on Mutations and Variability.) 
 
 Hemolytic streptococci isolated from the tear sac of a patient suffering with iridocyclitis produced typical 
 iridocyclitis in 5 of 8 rabbits injected intravenously. ,. . , , , . ., ,.,• • 
 
 " The power of the hemolytic streptococcus in these experiments, to localize m the eye and produce iridocyclitis m 
 rabbits was tost in a relatively short time {!) in the body of the original host {tear sac), {2) in cultures, and {3} dur- 
 ing animal passage. This change in tissue localization seemed not to be associated with any demonstrable decrease 
 
 in lethal power." , , . , ,, • , • r , ^, • ; • ; ; j i , 
 
 • "Attempts to obtain a return of invasive power for the tissues of the eye m strains of streptococci which had lost 
 
 it, by growing the organisms in the living eye, were unsuccessful." 
 
 There is described an epidemic of appendicitis followed soon by an epidemic of parotitis. Among the most 
 significant figures are the following: Soon after the appendicitis epideinic 30 per cent of strams from tonsils 
 of normal individuals 47 per cent of strains from tonsils of appendicitis patients, and 41 per cent of strains 
 from dairy products produced lesions in the appendices of rabbits injected; while only 10 per cent, per cent 
 and 9 per cent respectively produced lesions of the parotid. Dunng and soon after the parotitis epidemic 20 
 per cent of strains from tonsils of normal individuals, 30 per cent of strains from dairy products, and 73 per 
 cent of strains from Steno's duct of patients with parotitis produced parotitis m rabbits; while only 6 per cent, 
 per cent, and 15 per cent respectively produced lesions of the appendix. 
 
 Reference. 
 
 Heinemann 19l5. 
 
 The Variability of Two Strains of Strepto- 
 coccus lacticiis. 
 
 Rosenow 1915. 
 
 The Bacteriology of Appendicitis and Its 
 Production by Intravenous Injection of Strep- 
 tococci and Colon Bacilli. 
 
 Hartzell and Henrlei 1915. 
 
 A Study of Streptococci from Pyorrhea 
 Alveolaris and from Apical Abscesses. 
 
 Rosenow and Moon 1915. 
 
 On an Epidemic of Sore Throat and the 
 
 Virulence of Streptococci Isolated from the 
 
 Milk. 
 
 Broadhurst 1915. 
 
 Environmental Studies of Streptococci with 
 Special Reference to the Fermentative Reac- 
 tions. 
 
 Irons, Brown, and Nadler 1916. 
 
 The Localization of Streptococci in the Eye. 
 A Study of Experimental Iridocyclitis in Rab- 
 bits. 
 
 Rosenow and Dunlap 1916. 
 
 An Epidemic of Appendicitis and Parotitis 
 Probably Due to Streptococci Contained in 
 Dairy Products. 
 
TABLE VI — Concluded. 
 Pathogenicity and Virulence. 
 
 TABLE VI — Concluded. 
 Pathogenicity and Virulence. 
 
 Organisms. 
 
 11 strains of streptococci from 
 tonsils spinal fluid, and pyorrheal 
 Dockets of cases of herpes zoster. ^^ 
 
 Mi.xed cultures and "emulsions 
 from tonsils often used for injection. 
 
 .Animals. 
 
 Rabbits, dogs. (A 
 few guinea pigs.) 
 
 Method. 
 
 Intravenous (Oc- 
 casionally subcuta- 
 neous or intraperito- 
 neal.) 
 
 Amount. 
 
 Growth from 25- 
 90 cc. of 24 hr. as- 
 cites de.xtrose meat 
 broth cultures. 
 
 Period of observation. 
 
 Results and conclusions. 
 
 Chloroformed after 
 1-4 days if not dead 
 from the effects of 
 the inoculation. 
 
 Of 61 animals injected with freshly isolated strains or infectious material 75 per cent developed herpes. 
 "The lesions varied from those very small, just recognizable, to others very large and marked." It is not stated 
 in what percentage of animals other lesions developed, but 29 per cent developed hemorrhage of the stomach, 
 8 per cent ulcer of the duodenum, 10 per cent lesions in the appendix, 16 per cent in the gall-bladder, _ 11 per 
 cent in joints, 11 per cent in pericardium, 11 per cent in muscles, 21 per cent in lungs, 15 per cent in eyes, 
 etc., etc. 
 
 After cultivation for 7-14 days or after 2-5 passages through animals the strains produced herpes no more 
 frequently than various other lesions. 
 
 "It is a noteworthy fact that of the large number of animals injected with cultures from a variety of diseases other 
 than herpes, in which lesions resembling those in man have been produced, none has exhibited typical herpes." 
 
 This paper is largely a review of previous work by the author and others. Apparently no new work on pathogenicity and viru- 
 lence is reported. 
 
 1 strain of human tjqie (5. epi- 
 demicus) isolated by Davis (1912) 
 from bovine mastitis. 
 
 1 strain of S. lacticus. 
 
 1 strain of 5. epidemicus from peri- 
 toneal pus of fatal case of tonsillitis 
 isolated by Davis (1912). 
 
 1 strain of non-pathogenic hemo- 
 lytic streptococcus isolated from nor- 
 mal milk by Davis (1916). 
 
 1 strain of hemolytic human type 
 of streptococcus from milk of cow 
 without gross evidence of mastitis. 
 
 85 strains of hemolytic streptococci 
 from 328 samples of bottle milk. 
 
 Many strains of hemolytic strepto- 
 cocci from cases of tonsiUitis, otitis 
 media, and scarlet fever; also from 
 normal throats and suspected milk. 
 
 51 strains of "Streptococcus hemo- 
 lysans." 
 
 79 strains of "Streptococcus buc- 
 calis." 
 
 8 strains of " Streptococcus fecalis ." 
 
 4 strains of "Streptococcus equinas." 
 
 " * * the streptococci are not specific in their disease production, at least as far as I have been able to deter- 
 mine. There is no evidence to support the view that only one type of streptococcus produces endocarditis or nephri- 
 tis, or gives rise to septicemia in the puerperium, scarlet fever, or other conditions of lowered resistance." 
 
 " The members of the hemolytic group are commonly more virulent and pathogenic, * * than those of the 
 ■ group." 
 
 Healthy cows, rab- 
 bits. 
 
 Half-grown rabbits. 
 
 Into the milk ducts 
 of the cows by means 
 of catheter. 
 
 Rabbits inoculated 
 intravenously, prob- 
 ably. 
 
 Intravenous. 
 
 1-5 cc. of 36 hr. 
 litmus milk cultures 
 injected into milk 
 ducts of cows. 
 
 Growth from 1 
 blood agar slant in- 
 jected into rabbits. 
 
 Growth from 2 
 24 hr. blood agar 
 slants. 
 
 1 cow observed for 
 215 days, the other 
 for 146 days. 
 
 The human type (5. epidemicus) produced in cows very severe garget which persisted in chronic atrophic form 
 for as long as observed (215 and 146 days respectively), and produced in rabbits fatal arthritis. Referring also 
 to the work of Davis and Capps (1914) the author concludes that mastitis produced in cows by the human 
 type of hemolytic streptococci may be severe or the virulent streptococci "may grow and multiply in the milk 
 duels without causing any visible changes in the udder." 
 
 The strains of S. lacticus and the non-pathogenic hemolytic streptococcus from milk produced in the udder 
 acute inflammation of short duration followed by complete recovery. 
 
 The strain of hemolytic streptococci from the milk of the cow without gross evidence of mastitis produced 
 arthritis in rabbits. 
 
 Permitted to live 
 10 days, then killed 
 and autopsied. 
 
 ' The pathogenicity for mice of 0.5 cc. of a 24 hour broth culture intraperitoneally is a fair 
 
 index of its pathogenicity in rabbit- 
 a streptococcus which killed a mous 
 loss of weight in rabbits, followed u 
 injected intravenously. For the pn 
 of the more important cultures rabl 
 
 Comparative results proved that in all the cases studied, 
 in from 3 to 4 days would produce high fever and marked 
 ally by polyarthritis, when 1 cc. of a 24 hour broth was 
 'ninary studies mice were often used. In substantiation 
 
 were used for confirmatory evidence." 
 
 No animal experiments reporte 
 
 "15 strains produced lesions visible at autopsy. These Involved joints and on the whole were mild." 
 "I have gained the impression from many observations that the virulence for rabbits of a given strain of hemo- 
 lytic streptococci runs rather parallel with its virulence for man." 
 
 "Prom these results it is evident that all the strains in this series are relatively avirulent. None could be classed 
 in this respect with the human types of hemolytic streptococci as found in sore throat epidemics." 
 
 From all sources all but a few of the strains producing beta hemolysis and fermenting the substances described 
 in Table V, were pathogenic for mice and rabbits. 
 
 "Streptococcus hemolysans" is "usually parasitic and highly pathogenic, while Streptococcus viridans is either 
 saprophytic or if associated with a pathological process is usually of relatively low virulence." • 
 
 Reference. 
 
 Rosenow and Oftedal 1916. 
 
 The Etiology and Experimental Production of 
 Herpes Zoster. 
 
 Holman 
 
 The Classification of Streptococci. 
 
 1916. 
 
 Mathers 1916. 
 
 Different Types of Streptococci and Their 
 Relation to Bovine Mastitis. 
 
 Davis 1916. 
 
 Hemolytic Streptococci Found in Milk. 
 
 Smillie 1917. 
 
 Studies of the Beta Hemolytic Streptococcus 
 (Smith and Brown). 
 
 Blake 
 
 The Classification of Streptococci. 
 
 1917. 
 
TABLE II. 
 
 A Tabulation of Streptococci with Reference to Type of Appearance in Blood Agar and Fermentation Reactions. 
 
 Mannite Lactose Salicin. 
 
 + 
 
 + 
 
 + 
 
 + 
 
 f + 
 
 f + 
 
 f + 
 
 Groups. 
 
 Type a. 
 S. mitior seu riridans (SchottmuUer). 
 
 S. mitis (Andrewes and Horder). 
 
 S&B 6 
 
 S&B 6 
 
 S&B 
 
 1 
 
 H&H 4 
 
 H&H 7 
 
 
 
 K 1 
 
 K 8 
 
 
 
 Br 50 
 
 Br 18 
 
 Br 
 
 1 
 
 K 1 
 Br 13 
 
 S. salivarius (Andrewes and Horder) . 
 
 S&B 3 
 
 S&B 2 
 
 
 H&H 5 
 
 H&H 5 
 K 3 
 
 
 Br 10 
 
 Br 9 
 
 
 5. equinus (Andrewes and Horder). 
 H&H 1 
 Br 1 
 
 H&H 1 
 Br 3 
 
 S. ignavus (Holman). 
 
 H&H 2 
 Br 2 
 
 Br 1 
 
 S. fecalis (Andrewes and Horder) 
 H&H 1 
 
 K 2 
 
 Br 15 
 
 K 
 Br 
 
 3 
 
 53 
 
 Br 3 
 
 .2 
 
 Br 4 
 
 Br 39 
 
 S&B 1 
 H&H 3 
 
 Br 28 
 
 Br 
 
 Br 
 
 Br 39 
 
 Br 6 
 
 Br 1 
 
 .6 
 
 Br 1 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 Type 0. 
 S. longus pathogenes seu erysipelaios (SchottmuUer). S. htmolylicus (Roily). 
 
 5. pyogenes (Rosenbach). 
 
 S&B 
 
 33 
 
 
 
 K 
 
 22 
 
 
 
 Br 
 
 38 
 
 Br 1 
 
 
 D 
 
 11 
 
 
 
 Sml 
 
 62 
 
 
 
 5. anginosus (Andrewes and Horder). 
 
 S&B 
 
 8 
 
 
 K 
 
 2 
 
 
 Br 
 
 3 
 
 
 Sml 
 
 5 
 
 
 S. equi (Schiltz). 
 
 S&B 1 
 
 Br 
 
 Sml 
 
 5. subaeidus (Holman). 
 
 Br 1 Br 
 
 S. infrequens (Holman). 
 1 
 
 K 3 
 
 Br 7 
 Sml 10 
 
 K 
 
 D 2 
 
 Sml 1 
 
 S.alactqsus (Smith and Brown). 
 
 S&B 4 
 
 
 
 Sml 1 
 
 
 
 .1 
 
 .2 
 
 .3 
 
 .4 
 
 Br 
 D 
 
 .6 
 
 T>-pe T. 
 S. sapraphyl'cus (Mandelbaum). S. anhemolyticus (Zangemeister) . 
 
 K 
 Br 
 
 Br 1 
 
 K 
 Br 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 Subgroups 
 
 Raffinose . . 
 
 Inulin 
 
 Saccharose . 
 The small figures in each square represent the number of strains described by each of the foUowing authors: S&B = Smith and Brown (1915); H&H=HartzeUandHenrici (1915); K=KligIer (1915); Br = Broadhurst (1915); D = Davis (1916); 
 Sml=SmiIlie (1917); B = Brown (1917). 
 
 + 
 
 B 
 Br 
 
 -5| 
 41 
 
 Br 1 
 
 B 1 
 
 K 
 Br 
 
 .1 
 
 Br 1 
 
 .3 
 
 Br 7 
 
 Br 6 
 
 Br 2 
 
 Br 5 
 
 Br 1 
 
 Br 3 
 
 Br 4 
 
 .5 
 
 Br 1 
 
 .7 
 
 + 
 
TABLE m. 
 
 The Use of Blood Agar. 
 
 TABLE III. 
 The Use of Blood Agar. 
 
 Organisms. 
 
 40 human and 2 equine 
 strains. 
 
 65 pneumococci. 
 35 streptococci. 
 
 30 strains. 
 
 22 strains from vulval, 
 vaginal, and uterine secre- 
 tions of 10 women during 
 and 4-9 days after labor. 
 
 1 strain from a felon. 
 
 1 strain from throat (an- 
 gina). 
 
 Medium 
 
 Blood. 
 
 Species. 
 
 Rabbit, defibri- 
 nated. 
 
 Human. 
 
 Rabbit or hu- 
 man, defibrinated. 
 
 Human. 
 
 Rabbit, guinea 
 pig, and dog. 
 
 White mice, 
 guinea pig, rabbit, 
 beef, and human. 
 
 Amount. 
 
 Enough to 
 cloud the 
 agar (Nat- 
 vig). 
 
 2 cc. 
 
 0.3-0.5 cc. 
 
 (a) 2 parts. 
 (6) 5 drops. 
 
 3-4 drops. 
 
 Composition. 
 
 Standard agar. 
 
 "Plain agqr." 
 
 12 drops. Standard meat 10 cc. 
 
 infusion agar, neu- 
 tral to phenol- 
 phthalein. 
 
 Tried also blood agar 2:5 for comparisoni. 
 
 Amount 
 
 5 cc. 
 
 (a) 5 pa: s 
 (6) 5 cc. 
 
 Method of inoculation. 
 
 Surface fihns 
 usually, but occa- 
 sionally made deep 
 inoculations. 
 
 Surface and deep 
 inoculation tried. 
 
 Streaks of sev- 
 eral different 
 strains on the same 
 plate. 
 
 Period 
 of incubation. 
 
 Up to 48 
 hrs. or lon- 
 ger. 
 
 18-24 hrs. 
 
 2 days, 
 (possibly 
 longer). 
 
 Results and JEonclusions. 
 
 An " degante aureole d'hemoglohine dissoute" was forced around each colony of 39 of the human and 1 of 
 the equine strains. These are all regarded as of one sriecies. 
 
 Distinct but fainter hemolyzed zones were formed aijound colonics of 1 scarlet fever strain and a " 5(»-e^to- 
 coccus der Druse der Pferde," the former being regarded as a variant of the other human strains, the latter as 
 a distinct species. 
 
 Gave attention to macroscopic appearance only. Stjates that "many if not all of the organisms about to 
 be considered * * produce a certain amount of hemjolysis, not visible macroscopically." 
 
 "Str. longus patho genes seu erysipelalos" forms grky colonies "welche einen durchaus characteristischen 
 kreisrunden hellen Hof urn sick gehUdet haben. Dieser (hirch voUige Resorption des Hdmoglobins entstandene Ho] 
 hat einen Durchmesser von ca. 2-3 mm, je nach dem J\lter der Kultur." 
 
 "Str. mitior seu mridans" forms "feine, fast farblose, \spater gran bis griinschwarze Punkte; amzmeiten Page 
 haben sie sich zum Peil bis zu Kleinstecknadelkopfgrosst entwickeU." * * "Die Hdmolyse des Strept.^ m. ist 
 so gering, dass eine makroskopisch sichtbare Resorption dk Blutfarbstojfes bei Verwendung einer Blutagarmischung 
 von etwa 2: 5 im allgemeinen nicht stattfindet ." 
 
 "Str. mucosus" forms "innerhalb 24 Stunden auf afm Impfstrich ein gldnzender, saftig schleimiger, griin- 
 grwuerBelag,der nach weiteren 48 Stunden eine dunUere fdrbung anzimehmen pflegt,aber der Glanz,das schleimige 
 Aussehen schwindet; der Belag trochnet auch hier. * * Makroskopisch konnte ich bei meinen Stdmmen Hd- 
 molyse erst nach vielen Pagen beobachten." . 
 
 "Der Pneumokokkus einen intensiv dunkel grilnen Fttthstof bildet, dagegen fiihrt er eine makroskopisch sicht- 
 bare Hdmolyse nicht herbei." 
 
 Pneumococci form distinctly green colonies "surrmMded by a narrow zone in which the corpuscles are de- 
 stroyed. Phis zone is never clear and transparent, but at^ays opaque and of a distinct greenish tinge." 
 
 " Phe colonies of streptococci, on the other hand, remain small and grayish, and are surrounded by a perfectly 
 clear transparent zone of hemolysis, the size of which is ii\ direct proportion to their virulence." 
 
 25 strains corresponded in all respects with Schottmi^er's S. longus pathogenes. 
 
 5 strains produced no hemolysis on blood agar (2: 5) [but small clear zones on blood agar containing only 5 
 drops of blood in 5 cc. of agar. The colonies of these $ strains were small dark points, but only 1 of them 
 showed any perceptible greenish tint. The author regards these strains as S. mitior. 
 
 States that surface inoculation was found to be theimore satisfactory because plates inoculated deep {i.e.. 
 poured) became brownish in 12-24 hrs. 
 
 Typical hemolyzed zones with "Resorption des Hdmotlobin" were 2-3 mm. m diameter. 
 Strains non-hemolytic in blood bouillon were also noi-hemolytic in blood agar. 
 
 Appearance with various kinds of blood essentially alike. Describes 3 types of appearances on blood agar: 
 I. (a) Clear colorless zones 2-3 mm. broad. 
 (b) Zones colorless but clouded or hazy. 
 n Small colorless hazy zones surrounded by a greenlrmg and sometmies an outer colorless hazy rmg. 
 in No hemolysis or discoloration. All the anaerobic strains (6 of them) belonged to this group. 
 On blood agar (2 : 5) zones of Type I (a) apparent] but the other types were not distinguishable macro- 
 scopically. 
 
 (See text for quotations.) 
 
 Confirmed and approved SchottmuUer's differentiation of streptococci by means of blood agar plates. 
 (Recommends the use of litmus-lactose-nutrose-agar, according to Drigalski and Conradi for the differentia- 
 tion of pneimiococci, S. viridans, and S. mucosus.) 
 
 Reference. 
 
 Marmorek 1902. 
 
 L'Unit6 des streptocoques pathogtaes pour 
 I'homme. 
 
 (Die Arteinheit der f iir Menschen pathogenen 
 Strep tokokken.) 
 
 Schottmiiller 1903. 
 
 Die Artunterscheidung der fur den Menschen 
 pathogenen Streptokokken durch Blutagar. 
 
 Rosenow 1904. 
 
 Studies in Pneumonia and Pneumococcus In- 
 fections. 
 
 Rieke 1904. 
 
 Beitrage zur Frage der Arteinheit der Strepto- 
 kokken. 
 
 Kemer 1905. 
 
 Experimenteller Beitrag zur Hamolyse und zur 
 Agglutination der Streptokokken. 
 
 Natvig . 1905. 
 
 Bakteriologische Verhaltnisse in weiblichen 
 Genitalsekreten. 
 
 Frankel 1905. 
 
 Ueber menschenpathogene Streptokokken. 
 
TABLE rn — Continued. 
 The Use of Blood Agar. 
 
 TABLE III — Continued. 
 The Use oj Blood Agar. 
 
 ' 
 
 Medium. 
 
 Method of inoculation. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 
 Organisms. 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 [Composition. 
 
 Amount. 
 
 
 Evidently several strains 
 of 5. pyogenes and pneu- 
 mococci. 
 
 "Fresh defibri- 
 nated rabbit blood." 
 
 0,3-0.4 cc. 
 
 Plain agar. 
 
 
 
 Up to 48 
 hrs. 
 
 "/ have found pneumococci which produce hemolyzing colonies similar to those oj S. pyogenes." 
 S. pyogenes produces: 
 
 (a) clear hemolyzed zones on plain agar -|- blood; 
 
 (b) " " " on sugar-free agar -1- blood; 
 
 (c) either no hemolysis or greenish discoloration on glucose agar -|- blood. 
 Pneumococci produce: 
 
 (a) green colonies on plain agar 4- blood; 
 
 lb) may or may not produce slightly greenish colonies on sugar-free agar -|- blood (depending upon the 
 
 kind of blood used — probably dependent upon the amount of sugar in the blood); 
 (c) deep green colonies on sugar-free agar -\- 1 per cent glucose, lactose, or inulin + blood. 
 Conclusion. — "The green coloration of bacterial colonies in blood agar plates is dependent upon the production 
 of acid and the action of this acid {probably lactic acid) on the red corpuscles." 
 (See text for further quotations.) 
 
 Ruediger 1906. 
 
 The Cause of Green Coloration of Bacterial 
 Colonies in Blood- Agar Plates. 
 
 Sugar-free agar 
 (0.5 per cent so- 
 dium chloride, 1 
 per cent Witte's 
 peptone, and 1 . 5 
 per cent agar). 
 
 Strains from 154 throats; 
 51 normal, 75 scarlatinal, 
 14 measles, 5 tonsillitis, S 
 pneumonia, 4 laryngitis. 
 
 Rabbit or hu- 
 man, defibrinated. 
 
 0.5 CO. 
 
 
 
 Deep. 
 
 24 hrs. or 
 longer. 
 
 5. pyogenes produces clear zones on blood agar. 
 
 S. viridans and related varieties produce green colonies with or without partial hemolysis. 
 Pneumococci produce green colonies, sometimes partial hemolysis, and occasionally may be mistaken for 
 S. pyogenes. 
 
 Ruediger 1906. 
 
 The Streptococci from Scarlatinal and Nor- 
 mal Throats and from Other Sources. 
 
 47 streptococci.^ 
 22 pneumococci. 
 
 Human and 
 horse. 
 
 (a) Few drops mixed at 100°C. 
 
 (Voges' medium.) 
 (6) 30 per cent mixed at 100°C. 
 
 (c) Few drops mixed at 45°C. 
 
 (d) 30 per cent mixed at 45°C. 
 
 Surface. 
 
 5 streptococci and 
 5 pneumococci deep 
 also. 
 
 
 Most streptococci (75 per cent) produce more or less hemolysis on blood agar (a few drops of blood added 
 at 45°C.), while only 5 per cent of pneumococci do so. 
 
 All pneumococci (100 per cent) produce a yellow discoloration on blood agar (a few drops of blood added 
 at 100°C.), while only 17 per cent of streptococci do so. 
 
 Did not recognize Schottmiiller's S. mitior seu viridans. No green color was observed in the case of either 
 streptococci or pneumococci. 
 
 Boxer 1906. 
 
 IJeber das Verhalten von Streptokokken und 
 Diplokokken auf Blutnahrboden. 
 
 14 laboratory strains of 
 streptococci from various 
 human lesions. 
 
 1 strain isolated by the 
 author from case of scar- 
 let fever. 
 
 Horse and rab- 
 bit. 
 
 "Bis zum 
 Erzielung ei- 
 ner intensiv 
 blutroten Far- 
 bung." 
 
 
 
 "Stick-" and 
 ' ' AusstrichkuUuren." 
 
 
 "10 Kulturen wiesen hdmolysierende Eigenschaften auf, * " (all from various human lesions). 
 
 "2 Kulturen, * *, wiesen Erscheinungen auf, die mit denjenigen identisch waren, welche ScholtmiiUer der 
 Gruppe des S. viridans zuschreibt" (from the throat in case of rheumatism and from puerperal sepsis). 
 
 "Bei 3 Kulturen waren die Resultate unbestimmt." 
 
 States that he had no strain of S. mucosus. 
 
 The hemolytic strains produced similar appearances on both kinds of blood. The hemolysis was more pro- 
 nounced when larger amounts of blood were employed. Deep colonies developed more hemolysis than sur- 
 face colonies. Addition of glycerol to the agar was accompanied by no change in the appearance of the colonies. 
 
 Colonies of 5. viridans were at first gray, later greenish, with little or no hemolysis. 
 
 Sllberstrom 1906. 
 Ueber die Arteinheit der Streptokokken. 
 
 Tabulation of 700 strains 
 classified according to fer- 
 mentation reactions and 
 with slight reference to 
 hemolysis. 
 
 Sheep (human, 
 rabbit, beef). 
 
 5 per cent. 
 
 
 
 
 
 "It is evident that Schottmiiller's Streptococcus pyogenes corresponds with ours but may include the hemolytic 
 forms we have on other grounds classed as Streptococcus anginosus. His Streptococcus mitior or viridans probably 
 corresponds in general with our Streptococcus salivarius and fcecalis, while his Streptococcus mucosus may be in 
 part identical with the forms we have included under the pneumococci." 
 
 Beyond the above statements and the statements that their 5. pyogenes and 5. anginosus groups are hemo- 
 lytic, while the S. fcecalis group is non-hemolytic, nothing is said regarding hemolysis or appearances on blood 
 agar. 
 
 Andrewes and Herder 1906. 
 A Study of the Streptococci Pathogenic for 
 
 Man. 
 
 Original paper not read. 
 
 Schottmiiller's blood agar. 
 
 
 
 
 Quoted by Nieter (1907) as follows: " Dass der Blutagar nicht den Erwartungen, die ScholtmiiUer daran ge- 
 kniipft hat, entspricht, dass er zwar ein schatzenswertes Hilfsmittel zur kulturellen Diferentialdiagnose zwischen 
 Streptokokken und Pneumokokken darstelle, derart, dass braune bis braun-griinliche Kolonien in diesen Ndhr- 
 boden bei hochstens zweitdgiger Bebriitung fiir Pneumokokken sprechen, hamolytische Hofe dagegen, diese Diagnose 
 ausschliessen. Als Grundlage zur Unterscheidung der verschiedenen Streptokokkenarten sei jedoch das Vorhanden- 
 sein oder Fehlen der blutlosenden Fahigkeit eben so wenig wie alle sonstigen bisher herangezogenen EigentUmlich- 
 keiten geeignet, da sie eine variable Eigenschaft bilde." 
 
 According to Salomon (1908), "Einen Befund von Beitzke und Rosenthal, denen ein Stamm zu verschiedenen 
 Zeiten ungleiche Resultate gab, filhrt Schultze {1907) auf einen Wechsel in der Zusammensetzung der Nahrboden 
 zuriick." 
 
 Beitzke and Rosenthal 1906. 
 Zur Unterscheidung der Streptokokken mit- 
 tels Blutnahrboden. 
 
 
 
 
 
 
 
 
 "Hemolysis in blood agar may be caused by both pneumococci and streptococci." 
 
 "Although many strains [of pneumococci] /aj7 to cause hemolysis in blood agar, the organisms isolated from the 
 circulating blood frequently cause hemolysis when grown in the blood of the host." 
 
 Buerger and Ryttenberg 1907. 
 Observations upon Certain Properties Acquired 
 by the Pneumococcus in the Human Body. 
 
 65 strains (43 from 
 various human pathologi- 
 cal processes; the remain- 
 der from milk, skin, spu- 
 tum, and feces). 
 
 Beef or rabbit. 
 
 1 or 2 cc. 
 
 
 
 Surface. 
 
 24 hrs. 
 
 42 strains of streptococci from pathological processes are tabulated as producing a "heller Hof" on blood 
 agar, the remaining one as producing a "kleiner heller Hof." All other strains produced "kein Hof." No more 
 detailed descriptions of colonies or appearances on blood agar given. 
 
 The above 42 strains are designated as "S. longus seu erysipelatos" and all the others as " S. mitior seu vir- 
 idans." 
 
 Nieter 1907. 
 Zur Streptokokkenfrage. 
 
TABLE III — Continued. 
 The Use of Blood Agar. 
 
 TABLE in — Continued. 
 The Use of Blood Agar. 
 
 ^=""""^ 
 
 Medinm. 
 
 Metiiod of inoculation. 
 
 Period of 
 incubation. 
 
 Results and conclusions. 
 
 
 Organisms . 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 Composition. 
 
 Amount. 
 
 
 
 Human, placen- 
 tal. 
 
 Usually 2 
 pts. 
 
 Tried also 
 0.5 cc. 
 
 / 
 
 Usually 5 
 pts. 
 
 Tried also 
 Sec. 
 
 Surface. 
 
 
 Emphasizes the importance of microscopic examination of the colonies. Found that the zones of S. milior 
 colonies were made up of a collection of discolored non-hemolyzed corpuscles lying against the colony and an 
 outer hemolyzed zone containing only corpuscle shadows. The pneumococcus and 5. mucosus produced simi- 
 lar zones. 5. pathogenes formed clear colorless zones. "Nur zuweilen liegen einige zusammengebalte Reste von 
 roten Blutkorperchen, sogenannte Schatten, der Kolonie auf. Niemals aber enthalten dieselben noch Blutfarhstof . 
 Diese Eigenschaft, die roten Blutkorperchen zur Auflosimg zu bringen und deren Blutjarbstof zu zerstoren, 
 behielt der S. pathogenes trotz monatelanges Fortzuchtung bei" 
 
 Describes "Streptococcus saprophyticus" (nov. sp.?) as being present on all mucoufi membranes (mouth, nose, 
 vagina, intestine) and as forming on blood agar " zarten, feinen, grauweissen Kolonien" without hemolysis or 
 discoloration. 
 
 Concludes that S. mitior produces a toxin which does not diffuse into the medium, as does the hemolysin, 
 but remains bound to the coccus bodies and acts upon the blood corpuscles in contact with the colony, alter- 
 ing them in color and rendering them immune to the hemolysin. 
 
 (See text for quotations.) 
 
 Mandelbaum 1907-08. 
 Zur Streptokokkenfrage. 
 
 38 strains of 5. pyogenes. 
 10 strains of 5. mucosus. 
 13 strains of pneumococci. 
 6 strains of 5. saprophyti- 
 cus. 16 strains of "son- 
 stige Streptokokken," most- 
 ly 5. viridans. 
 
 
 
 
 
 Surface. 
 
 
 Only 16 of the strains of 5. pyogenes were tried on blood agar but all were found to be hemolytic, as were 
 also 4 strains included among "sonstige Streptokokken." 
 
 All strains of pneumococcus and 5. mucosus tested were found to produce more or less greenish colonies. 
 
 S. saprophyticus produced neither hemolysis nor discoloration on blood agar. 
 
 Most of the "sonstige Streptokokken" produced more or less greenish colonies and were evidently 5. mitior 
 seu viridans. 
 
 Salomon 1908. 
 
 Zur Unterscheidung der Streptokokken durch 
 kohlenhydrathaltige Niihrboden. 
 
 Number of strains not 
 given; evidently many 
 strains. 
 
 Human. 
 
 2:pts. 
 
 
 5 pts. 
 
 Surface. 
 
 
 Found streptococci in vaginal secretion of 46 of 136 normal gravida and puerpera (33 per cent), all of S. 
 viridans type. 
 
 Found hemolytic streptococci in cases of severe endometritis (number not given) and in 10 cases of septi- 
 cemia and peritonitis. 
 
 Found an intermediary type of streptococci in puerperal parametritis. 
 
 Fromme 1908. 
 Klinische und bakteriologische Studien zum 
 Puerperalfieber. 
 
 Number of strains not 
 given; evidently many 
 strains. 
 
 Human, placen- 
 tal, defibrinated. 
 Rabbit, for compar- 
 ison in a few cases. 
 
 2 pts. 
 
 
 5 pts. 
 
 
 
 No hemolytic streptococci found in the vaginal secretions of 200 normal gravida, but found hemolytic strep- 
 tococci in 53 of 175 cases of puerperal fever. 
 
 In 8 milder cases only "non-hemolytic" streptococci were found. These strains produced on blood agar 
 more or less greenish discoloration and some produced more or less hemolysis, always clouded and greenish, 
 however. 
 
 Intermediary types were found in convalescent cases. 
 
 Minor differences in appearance on human or rabbit blood agar are noted. 
 
 Heynemann 1908. 
 Die Bedeutung der hamolytischen Strepto- 
 kokken fiir die puerperale Infection. 
 
 From vaginal and uter- 
 ine secretions of 30 nor- 
 mal puerpera. Strains ob- 
 tained with unusual pre- 
 cautions. 
 
 Also 31 strains from 
 various pathological con- 
 ditions. 
 
 Human (proba- 
 bly). 
 
 2 pts. 
 
 
 Spts. 
 
 Surface. 
 
 
 Ante partum only non-hemolytic streptococci were found in the vaginal secretions. Post partum there 
 were found in the uterine cavity no streptococci in 14 cases, hemolytic streptococci in 8 cases, and non-hemo- 
 lytic streptococci in 8 cases. 
 
 They regard 5. mucosus as a distinct type but could observe no constant morphological or cultural differ- 
 ences between 5. longus (erysipelatos) and 5. viridans (mitior) because there were intermediate forms. In gen- 
 eral, however, the former produced on blood agar dear hemolyzed zones several mm. broad, while the latter 
 produced a green discoloration. 
 
 Liidke and Polano 1909. 
 Ueber Hamolyse der Streptokokken. 
 
 Presumably strains from 
 various sources. 
 
 Human, hog, 
 calf, beef, horse, 
 rabbit, hen, and 
 pigeon. 
 
 10 per 
 cent = opti- 
 mum. 
 
 Standard glyce- 
 rol agar. 
 
 10 cc, mak- 
 ing a layer 
 of agar 3-4 
 mm. thick. 
 
 Surface and deep. 
 
 
 Too much blood in the plate or too thick a layer of medium results in slow hemolysis; too little blood, in 
 indefinite zones. 
 
 Describes clear transparent zones (2-3 mm. "im Radius") formed by " gewohnliche Streptokokken" on blood 
 agar plates inoculated either in the depths {i.e., in fluid condition) or by surface streaks. "Der gleiche Hof 
 tritt dann auch auf, wenn man unter LuftabscUuss (iiberschichtet) ziichiet." Microscopically no corpuscle shad- 
 ows were seen within the hemolyzed zones. 
 
 If the plates are sown too thickly vrith streptococci the hemolyzed zones are not characteristic since (1) 
 the individual colonies are hindered in their development, and (2) "eine Sau&rung des Agars sofort die Platte 
 braun verfarbt. 
 
 With regard to the use of different kinds of blood, — "Nie Jiel eine Blutart negativ aus, wenn andere positiv 
 waren." 
 
 As the result of various experiments (see text) the author concludes that "Die Aufhellung ensteht: 1 dadurch, 
 dass Blutfarbsioff aus dem beirofenen Bezirk austritt und sich durch Diffusion im umgebenden Agar verteilt; * ." 
 
 Zangemelster 1909. 
 
 Die Hamolyse der Streptokokken. 
 
TABLE m — Continued. 
 The Use of Blood Agar. 
 
 TABLE Ill—Continued. 
 The Use of Blood Agar. 
 
 t=- 
 
 Medium. 
 
 Method of inoculation. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 
 Organiams. 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 Composition 
 
 Amount. 
 
 
 Several strains of "mod- 
 ified pneumococci" from 
 oases of endocarditis. 
 
 
 
 
 
 
 
 The "modified pneumococci" "always produced more or less greenish discoloration around the colony on blood 
 agar plates but never a zone of hemolysis." 
 
 Rosenow 1909. 
 
 Immunological and Experimental Studies on 
 Pneumococcus and Staphylococcus Endocar- 
 ditis. 
 
 Strains from blood, pus, 
 throats, and blister fluid 
 of 88 cases of scarlet fever, 
 measles, and diphtheria. 
 
 Horse. 
 
 Ice. 
 
 Standard agar 
 and the following 
 modified agar: 
 
 (a) peptone wa- 
 ter agar; 
 
 (6) sugar-free 
 agar {"made from 
 broth with colon 
 bacillus added and 
 sterilized"); 
 
 {c) glucose ag'ar; 
 
 {d) glycerol agar; 
 
 (e) beerwort 
 agar. 
 
 
 Deep (inoculated 
 blood and then 
 mixed with melted 
 agar). 
 
 18-24 hrs. 
 
 By "hemolyzing streptococcus" is meant one "which gives a more or less definite area of hemolysis about each 
 colony when plated out on blood agar." 
 
 "In our laboratory all the hemolyzing strains and also the green ones, when tested and retested in glucose agar 
 blood plates, gave green colonies." 
 
 Anthony 1909. 
 Some Characteristics of the Streptococci 
 Found in Scarlet Fever. 
 
 Many strains of 5. miliar 
 isolated from vaginal se- 
 cretions of normal puer- 
 pera and gravida. 
 
 3 laboratory strains of 
 S. pyogenes for comparison 
 with the above. 
 
 
 
 
 
 " Blutagaraus- 
 strich." 
 
 
 Replating only the most variable fishings repeatedly on standard blood agar and the use of specially modi- 
 fied blood agar revealed "marked variation [in hemolysis] up and down the scale." 
 
 Konrad 1909. 
 Weitere Beitrage zur Vaginal-Streptokokken- 
 frage. 
 
 15 strains of S. longus 
 hemolyticus. 
 
 4 strains of S. brevis 
 non-hemolyticus (mostly) 
 from cases of puerperal 
 sepsis 
 
 1 strain of Staph, aureus. 
 
 Human corpus- 
 cles. 
 
 2.5 CO. 
 
 
 8-10 cc. 
 
 Surface and deep. 
 
 Observa- 
 tions after 
 24, 48, and 
 72 hrs. 
 
 Found streptococci in the vaginal secretions and lochia respectively of 17 of 60 gravida and of 40 of 100 
 normal puerpera. All strains were of the S. viridans type. 
 
 Describes " glashellen Hof" (3-4 mm. broad) formed by the 3 strains of S. longus which he studied for com- 
 parison with his 5. mitior {viridans) strains. 
 
 With regard to the colonies of S. mitior on blood agar: "Nur wenn die Flatten noch auf weitere 6-8 Stunden 
 bebrutete und sie dann bei Zimmertemperatur aufbewahrte, begann nach 48-50 Stunden ein kleiner hamolytischer 
 Hof sich auszubilden, der in weiteren 24 Stunden ziemlich hell und an 2-3 mm. breit wurde. Aber die Farbe und 
 Aussehen der Kolonien war von denen des Streptococcus longus ganz verschieden, graubraun in verschiedenen Schat- 
 tierungen, und wenn man die Flatten mikroskopisch untersuchte, so fand man Mandelbaums Angabe bestatigt: die 
 dunkle Fdrbung der Kolonien rilhrte wirklich von den unter den Kolonien liegenden ungelosten braunrot-grauveaun 
 gefarbten Blutkorperchen her." 
 
 "Bei der Beurteilung der Flatten hdmolyse schloss ich nicht ganz Schottmiiller an und nannte Hdmolyse die glashelle 
 Aufhellung im Umkreis von eiwa 1 bis 2 mm. um die fast stets kleinbleibende, nie konfluierende Kolonie herum. 
 Auf starker bluthaliigen Flatten {3 cc: 8 cc. z. B.) wird die Aufhellung iiberhaupt nicht glashell, sondern mattgelb, 
 
 Most surface colonies of 5. aureus were hemolytic, but the deep colonies were not. 
 
 Sachs 1909. 
 Ueber Streptokokken-Hamolyse. 
 
 Cultures from lochia of 
 44 febrile puerpera and 
 56 afebrile cases. 
 
 Human. 
 
 2pts. 
 
 
 5 pts. 
 
 At first employed 
 only surface inocu- 
 lation, but later in- 
 oculated the blood 
 before mixing it 
 with the fluid agar. 
 
 24 hrs. 
 and longer. 
 
 Similar results were obtained on weakly alkaline blood agar and on dextrose blood agar plates. 
 
 Found deep inoculation {i.e., into fluid agar) more satisfactory than surface streaks or films. 
 
 Found "typisch hdmolysirende Streptohokken" in the lochia of 31 of 44 febrile puerpera and in the lochia of 
 38 of 56 normal puerpera, also in the vaginal secretion of 3 of 20 normal gravida. 
 
 " Unter typischer Hdmolyse verstehe ich nur die vollstdndige Aufhellung der Umgebung der Kolonie, so dass auch 
 mikroskopisch von einer Structur der rothen Blutkorperchen nichts mehr zu sehen ist. In 12-24 Stunden kann die 
 helle Hof, bei nicht zu dicht beschickter Flatte, die Grosse von 2 mm. erreichen {Reaction I nach Natvig)." 
 
 Certain "nicht hamolysirenden Streptokokken" produce no hemolysis in 24 hrs. but after 2 or 3 days incu- 
 bation produce zones which microscopically are seen still to contain some unhemalyzed corpuscles. In the 
 refrigerator the zones of the typical hemolytic streptococci did not change while those of the atypical strains 
 became larger. 
 
 (See text for quotations and further description.) 
 
 Slgwart 1909. 
 Untersuchungen uber die Hamolyse der Strep- 
 tokokken in der Schwangerschaft und im Woch- 
 ensbett. 
 
 5. lacticus found in 75 
 per cent of 81 samples of 
 milk and also in cow dung 
 and on skin. 
 
 
 
 
 
 
 
 S. lacticus produces no hemolysis on blood agar plates. 
 
 Bahr 1910 
 Vorkommen und Bedeutung der Strepto- 
 kokken in der Milch. 
 
TABLE III^ — Continued. 
 The Use of Blood Agar. 
 
 TABLE III — Continued. 
 The Use of Blood Agar. 
 
 
 Medium. 
 
 Method of inoculation. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 
 Organisms. 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 Composition. 
 
 Amount. 
 
 
 Cultures from lochia of 
 febrile and normal puer- 
 pera. 
 
 
 
 
 
 
 
 Found hemolytic streptococci in the lochial secretions of 11-15 per cent of normal afebrilepuerpera, and of 
 32 per ceilt of febrile cases. The streptococci found in both sets of cases were regarded as identical. 
 
 Fabre and Bourret 1910. 
 
 Quelques notions nouvelles sur les strepto- 
 coccies des suites de couches. 
 
 7 strains of 5. erysipd- 
 atos. 
 
 3 " Darmstreptokokkcn." 
 1 5. mitior. 
 
 1 S. mucosus. _ 
 
 4 pneumococci. 
 
 Horse. 
 
 2 pts. 
 
 
 5 pts. 
 
 Surface. 
 
 
 S. erysipelatos produces strong hemolysis in 12-20 hrs., the colonies themselves being gray. 
 " Darmslreptokokken" produce an abundant greenish growth without any trace of hemolysis. 
 Conclusion: The biological and especially the hemolytic characters of streptococci are far too variable to 
 form the basis of differentiation {" Artversckiedenkeit") . 
 (See also Table VH on Mutations and Variability.) 
 
 HoessU 1910. 
 
 Das Verhalten der Streptokokken gegeniiber 
 Plasma und Serum und ihre Umzuchtung. 
 
 5. pyogenes from 74 sup- 
 purations, 37 cases of an- 
 gina, 6 of 15 normal 
 throats, and 7 of 8 scar- 
 latinal throats. 
 
 S. longissimus: 23 strains 
 from normal throats, 41 
 from throats with recent, 
 chronic, or mild lesions. 
 
 S. conglomeralus A : from 
 11 normal throats and 3 
 cases of angina. 
 
 S. conglomeratus B; 14 
 strains, all but 2 from 
 pathological throats. 
 
 S. brevis: strains from 
 intestinal canal, normal 
 throats, heart blood, and 
 lesions in which other 
 bacteria were present also. 
 
 S. mucosus. 
 
 Pneumococci. 
 
 Human or ani- 
 mal (species?). 
 
 5 large 
 drops. 
 
 Meat infusion 
 agar, slightly alka- 
 line to litmus. 
 
 10 cc. 
 
 Surface. 
 
 20-24 hrs. 
 
 5. pyogenes strains isolated from all the sources mentioned were alike and produced round grayish white 
 granular colonies with large, clear, hemolyzed zones. 
 
 S. longissimus formed flat irregular clear to brownish colonies with either no hemolysis or greenish partly 
 hemolyzed zones with collection of corpuscles next to the colony. 
 
 S. conglomeratus A grew on blood agar like S. longissimus. 
 
 S. conglomeratus B formed small, dark, round, discrete, crateriform colonies, often wrinkled; sometunes 
 almost as hemolytic as S. pyogenes and sometimes producing little or no hemolysis; 2 strains produced green 
 colonies. 
 
 S. brevis formed delicate homogeneous or finely granular colonies, usually with more or less green color but 
 non-hemolvtic. 
 
 S. mucosus and pneumococci always produced greenish discoloration and never hemolysis on blood agar. 
 
 The author mentions intermediary forms between S. brevis and 5. conglomeratus B. 
 
 (See quotations in text.) ' 
 
 Thalmann 1910. 
 Streptokokkenerkrankungen in der Armee, 
 Einteilung der Streptokokken und ihre Bekamp- 
 fung. 
 
 
 Horse, rabbit, 
 beef, goat, hog, 
 guinea pig. 
 
 
 
 .1 
 
 
 
 5. equi was found to form "deutliche Resorptionshbfe" around the colonies on blood agar made with any of 
 the bloods tried. 
 
 ! ■' 
 
 Laabs 1910. 
 
 Vergleichende Untersuchungen fiber den 
 Streptococcus equi und andere pathogene 
 Streptokokken. 
 
 
 Schottmiiller's blood agai. 
 
 
 
 On blood agar S. equi produced clear hemolyzed zones 2-4 mm. in diameter. 
 (See also Table VIII on General Conclusions, etc.) 
 
 Prlcolo 1910. 
 
 Recherches exp6rimentales sur le streptocoque 
 de la gourme. 
 
 "Die dabei zur Verwen- 
 dung gelangten Streptokok- 
 kenstdmme {zumeist S. vul- 
 garis) waren sdmtUch 
 menschenpathogener Natur, 
 aus dem Blute von Sepsis- 
 kranken gezUchtet." 
 
 Human, prob- 
 ably. 
 
 
 
 
 
 
 Hemolytic streptococci ("5. vulgaris") produced hemolyzed zones on laked blood agar as well as on the ordi- 
 nary blood agar plates. Hence the author concludes that there is "eine Zerslorung von Blutfarbstojf durch die 
 Streptokokken" as well as a laking of the corpuscles. 
 
 5. viridans produced larger and darker green colonies on laked blood agar than on ordinary blood agar. 
 
 (See quotations in text.) , 
 
 Loening 1910. 
 
 Ueber Unterschiede in der Streptokokken 
 gegenuber zur Geltung kommenden bakterizi- 
 din Wirksamkeit des menschlichen Blutes, in- 
 sonderheit des Zitratblutes. 
 
 8 strains of "modified 
 pneumococci" from cases 
 of endocarditis. 3 strains 
 of pneumococci from pneu- 
 monia cases. 1 5. vir- 
 idans. 1 5. pyogenes. 1 
 M. rheumaticus. 
 
 
 
 
 
 
 
 5. pyogenes produced hemolysis on blood agar plates. 
 
 All other strains produced more or less green color but no hemolysis. " The production of green by the endo- 
 carditis cocci on blood agar when first isolated is always less than in those from pneumonia." 
 
 Rosenow 1910. 
 
 A Study of Pneumococci from Cases of In- 
 fectious Endocarditis. 
 
TABLE III — Continued. 
 The Use of Blood Agar. 
 
 TABLE III — Continued. 
 The Use of Blood Agar. 
 
 Organisms. 
 
 Evidently many strains. 
 
 Streptococci from 100 
 cases of angina and from 
 25 of 100 cases of influ- 
 enza. 
 
 Medium. 
 
 Blood. 
 
 Species. Amount. 
 
 8 strains of streptococci 
 isolated at autopsy during 
 an epidemic of "grippe" 
 among children. 
 
 10 strains of S. patho- 
 genes from throats and 
 joints. 
 
 6 strains of S. mitior from 
 joint, throat, empyema, 
 and bronchitis. 
 
 6 strains of 5. sapro- 
 phyticus from sputum. 
 
 Evidently many strains 
 from various sources; num- 
 ber not given. 
 
 40 hemolytic strepto- 
 cocci (sepsis, scarlet fever, 
 suppurations, and angina). 
 
 10 strains of S. acidi 
 lactici from bovine and 
 human milk. 
 
 6 strains of S. pyogenes 
 from various suppura- 
 tions. 
 
 5 strains of pneumococci 
 from sputum. 
 
 3 strains from 
 
 (1) " Handphlegmone," 
 
 (2) " Zungeninfiltrat," 
 
 (3) lung of guinea pig. 
 
 Rabbit. 
 
 Agar. 
 
 Composition. 
 
 Human, placen- 
 tal. 
 
 Rabbit. 
 
 Rabbit, hog, 
 sheep, beef, calf. 
 
 Sheep, (beef, 
 
 veal, and hog 
 
 blood for com- 
 parison). 
 
 O.S cc. 
 
 2 pts. 
 
 5 per cent. 
 
 Standard agar. 
 
 Meat extract pep- 
 tone agar. 
 
 Method of inoculation. 
 
 3 pts. 
 
 Period 
 of incubation. 
 
 Up to 48 
 hrs. 
 
 Surface. 
 
 Surface. 
 
 24 and 48 
 hrs. 
 
 RMults and conclusions. 
 
 Distinguishes the following three groups of capsule-free streptococci according to their appearances on blood 
 
 agar 
 
 1. Die einen erzeugen in der Umgehung ihrer Kultur einen ausgepragten Aufhellungshof, von dem ich gezeigt 
 dass er ledigUch durch Farbstojfaustritl aus den rolen BlutkSrperchen — nicht oder nar unwesentlich durch 
 Resorption des Hdmoglobins — bedingt wird {' hamolytische' Streptokokken); 
 
 "2. Andere wachsen auf der Bluiagarplatle, ohne den Nahrboden zu verandern; ('5. anhaemolyttcus vui- 
 
 "3. 'Wieder andere bilden zwar ebenfalls keinen Aufhellungshof, verfarben aber das Blulagar in niichsler Nike 
 der Kultur griinlich {'Streptococcus mridans')." 
 
 The author states that the streptococci (5. pyogenes) from cases of angina show considerable diflerence m 
 their hemolytic powers, but as a rule are less hemolytic than those from suppurations of the body surface. 
 
 From 4 of 100 cases of angina he found in large numbers and in almost pure culture streptococci which re- 
 sembled S. pyogenes except that they produced neither hemolysis nor discoloration on blood agar plates. These 
 strains are regarded as varieties of S. pyogenes by the author, however. ^ •■, , „ c 
 
 Found intermediary varieties between 5. longissimus and S. conglomeratus as previously described. Strep- 
 tococcus longissimus und conglomeratus A mil ihren Uebergangsformen gehoren also zu einer Streptokokkenunterart 
 zusammen." 
 
 (See also Table VII on Mutations and Variability.) 
 
 Isolated a streptococcus (" Grippestreptococcus") from 8 cases of "grippe" among children, at autopsy. 
 
 The growth on blood agar is described as being quite characteristic and different from that of other strepto- 
 cocci. It formed rather large flat light brown colonies which appeared dry even on the first day. The me- 
 dium in the neighborhood of the colonies of freshly isolated cultures was discolored brownish. Hemolysis was 
 usually quite weak and not increased by animal passage of the strains. 
 
 Approves Mendelbaum's differentiation of streptococci as (1) S. pyogenes, (2) 5. mitior, and (3) 5. sapro- 
 
 phyticus. , ,, J 1 
 
 "The colonies of S. pathogenes and S. mitior may appear alike macroiscopically on the blood agar plate. 
 Microscopic examination is necessary for their differentiation" (Translation). 
 
 Found classification based upon appearance of cultures in bouillon, length of chain, etc. unsatisfactory be- 
 cause these features were variable, but found character of growth on blood agar quite constant and satisfac- 
 tory for classification as follows: (1) S. hemolyticus, (2) 5. anhemolylicus, (3) S. mridans, (4) 5. mucosus. 
 
 All streptococci which produced hemolysis on blood agar plates were found to produce a filterable hemo- 
 toxin in rabbit serum bouillon. 
 
 The appearance on blood agar was constant for any given species of blood but sornewhat different for dif- 
 ferent species of blood. This was apparent principally among strains of S. acidi lactici, none of which hemo- 
 lyzed rabbit or calf blood but all of which produced slight hemolysis of beef blood, and two of which hemo- 
 lyzed hog blood. S. lanceolalus and S. pyogenes are tabulated as having produced considerable hemolysis of 
 
 all the bloods tried. ... rr r i. i. ■ ; 
 
 "Die schonsten hamolytischen voUkommen hellen, scharf abgegrenzten und mittelmassig bretten Uofe bekam iclt 
 von Streptococcus pyogenes, bei lanceolalus waren die Hofe schmal, aber scharf. Bei acidi lactici feUte eine Hdmo- 
 lyse zuweilen, wenn sie vorhande war, so war sie meist ausgebreitet, di-ffus, aber oft nicht vollstandig. Ich glaube 
 nicht, dass man pyogenes und acidi lactici mit voller Sicherheit durch Hamolyse trennen kann. Scharf war der 
 Unterschied von lanceolalus und acidi lactici." _ 
 
 Noted the appearance of double or multiple concentric rings or zones around some strains of acidi lactici 
 and pneumococci. 
 
 (See quotation in text.) 
 
 When first isolated "Der Blutfarbstoff war nicht nur unter dem Impfstrich, sondern auch noch in breiten Hof 
 gelost." (See Table VII on Mutations and Variability.) 
 
 Concludes that "Die Hamolyse isl als diferentialdiagnostisches Mittel fiir Streptokokken ungeeignel, da sie 
 eine viel zu inkonstante Grosse ist und ohne erkennbare Ursache verschwinden und wieder auftretten kann." 
 
 Reference- 
 
 Zangemeister 1910. 
 
 Ueber die Verbreitung der Streptokokken im 
 Hinblick auf ihre Infektiositat und ihre hamo- 
 lytische Eigenschaft. 
 
 Thalmann 1911. 
 
 Weitere Mitteilungen uber Streptokokken, 
 
 inbesondere fiber pyogene Streptokokken bei 
 
 Erkrankung'en der Atmungsorgane und deren 
 
 Komplikationen. 
 
 Muller and Sellgmann 1911. 
 
 Klinische und bakteriologische Beobach- 
 tungen bei Sauglingsgrippe. 
 II. Bakteriologisches, — von Seligmann. 
 
 LeBlanc 
 
 Zur Artenfrage der Streptokokken. 
 
 1912. 
 
 Roily 1912. 
 
 Experimentelle bakteriologische Untersuchun- 
 gen von verschiedenen Streptokokkenstammen. 
 
 Braun 
 
 Ueber das Streptolysin. 
 
 1912. 
 
 Saito 1912. 
 
 Versuche zur Abgrenzung des Streptococcus, 
 acidi lactici von Streptococcus pyogenes und 
 Streptococcus lanceolalus. 
 
 Jaffe 1912. 
 
 Beobachtunge bei blutlosenden und bei 
 gramnegativen Streptokokken. 
 
TABLE III — Continued. 
 The Use of Blood Agar. 
 
 TABLE III — Continued. 
 The Use of Blood Agar. 
 
 
 Medium. 
 
 Method of inoculation. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 
 Organisms. 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 Composition. 
 
 Amount. 
 
 
 
 
 
 
 
 
 
 Hemolysis was much reduced in blood agar containing sugar. On sugar blood agar hemolytic streptococci 
 produced small hemolyzed zones surrounded by broad grayish green outer zones \vithout hemolysis which 
 the author attributes to the action of the acids formed. He does not think the acids were responsible for the 
 reduction of hemolysis but rather that presence of sugar in the medium resulted in a suppression of the " car- 
 nivorous metabolism" and the adoption of an "herbivorous metabolism." 
 
 Kuhn 1912. 
 
 Einflus von Zucker auf Hamolyse und Viru- 
 lenz. 
 
 36 Strains: 26 from bo- 
 vine mastitis, 3 from 
 stable air, 1 S. acidi lac- 
 tici, 1 from saliva of 
 horse, 3 from bovine va- 
 ginitis, 1 5. pyogenes (Ro- 
 senbach), and 1 S. ery- 
 sipelatos (Fehleisen). 
 
 Beef or goat, 
 fresh, not deiibri- 
 nated. 
 
 2 pts. 
 
 
 5 pts., 7- 
 10 cc. in 
 each Petri 
 dish. 
 
 Surface, probably. 
 
 24 hrs. 
 
 S. pyogenes, S. erysipelatos , and 4 strains from bovine mastitis are indicated as being hemolytic, producing 
 clear colorless zones 2-3 mm. in diameter in 24 hrs. 3 other mastitis strains and the strains from stable 
 dust and horse saliva are marked ±. All other strains were non-hemolytic. 
 
 Colonies on blood agar were described as gray, brown, or grayish brown. 
 
 "Nur die wenigsten Milchstreptokokken zeigen diese hamolytische Eigenschaft. Die meisten bilden kein Hamo- 
 lysin und nehmen eine MiUelstellung zwischen dem S. mitior und dem S. niMosus ein. Die Mehrzakl ist aber 
 dem ersteren beizuordnen." 
 
 Gminder 1912. 
 
 Untersuchungen iiber Mastitisstreptokokken 
 und ihre DiSerenzierung von saprophytischen 
 Streptokokken. 
 
 
 " SchottmiiUer-Agar." 
 
 
 
 Was unsuccessful (in a limited number of experiments) in diffterentiating mastitis streptococci from human 
 strains culturally. Mastitis streptococci usually resembled "S. mitior seu viridans und S. mucosus" on "Schott- 
 midler-Agar." 
 
 Ernst 1912. 
 Eine Entgegnung zu A. Gminders Arbeit. 
 
 
 Human. 
 
 5 large 
 drops. 
 
 Meat infusion 
 agar. 
 
 5-10 cc. 
 
 
 
 "Die pyogenen Streptokokken treten durch ihren grossen hellen Hof deuilich hervor; der Streptococcus viridans 
 zeigt Grilnfarbung und Idsst sich vorhandener Hamolyse mikroskopisch dadurch trennen dass die Hdmolyse ringfbr- 
 migen Charakter hat; er halt den Blutfarbstoff der roten Blutkorperchen direkt urn die Kolonie so fest, dass selbst 
 da, wo Viridans-Kolonieen sich im Resorpti'onshofe des Pyogenes entwickeln, diese ihre direkt aufgelagernden roten 
 Blutkorperchen unverandert erhalten. Der Pneumococcus bildet sehr charakteristische Kolonieen, bei denen der 
 Rand wie ein verdickter Ring hervortritt; es zeigt nur sellen und dann geringe ringformige Hamolyse." 
 
 (See also Table VIII on Genera! Conclusions, etc.) 
 
 Thalmann 1912. 
 
 Streptococcus viridans irn Blut ohne Veran- 
 derung der Herzklappen. Zur Einteilung der 
 Streptokokken. 
 
 13 strains of various de- 
 grees of hemolytic activity 
 from vaginal secretion and 
 puerperal sepsis. 
 
 1 strain of S. viridans 
 and a non-hemolytic strep- 
 tococcus for comparison. 
 
 
 
 Agar containing 
 0, 1.5, and 4 per 
 cent dextrose; also 
 glycerol agar. 
 
 
 Surface. 
 
 
 Hemolysis was most extensive on glycerol blood agar, almost as great on blood agar without dextrose, less 
 on 1.5 per cent dextrose bloo^d agar, and least or often negative on 4 per cent dextrose blood agar. 
 
 The S. viridans strain produced less greenish discoloration on 4 per cent dextrose blood agar than on dex- 
 trose-free blooM agar. 
 
 Brownish discoloration accompanied growth on dextrose blood agar in most cases. 
 
 Benthln 1912. 
 
 Beitrage zur Hamolysefrage der Strepto- 
 kokken. 
 
 (a) 20 strains from vari- 
 ous human infections. 
 
 {b) 54 strains from milk 
 of normal cows. 
 
 (c) 18 strains from cows 
 with mastitis. 
 
 (d) 48 strains from mar- 
 ket milk. 
 
 (e) Strains from feces 
 of all but one of 31 nor- 
 mal bottle-fed infants and 
 30 with intestinal dis- 
 orders. 
 
 Goat, also beef, 
 rabbit, and human 
 to some extent. 
 
 
 
 10 cc. 
 
 Surface. 
 
 
 No detailed descriptions of appearances on blood agar are given, but the author evidently uses the term 
 hemolytic to describe even those strains which produce even a trace of hemolysis. 
 
 (a) Of the strains from various human infections 1 hemolyzed human blood only; the others hemolyzed all 
 the bloods tried. 
 
 (6) Of the strains from milk of normal cows almost half produced only a trace of hemolysis of human blood, 
 but a fair amount of hemolysis of beef, goat, and rabbit blood; 9 hemolyzed beef or goat blood only; 16 hemo- 
 lyzed none of the bloods. 
 
 (c) Of the mastitis strains only 1 produced strong hemolysis of human blood. 
 
 (d) Of the strains from market milk 24 were non-hemolytic; 15 showed a tendency to hemolyze; and 9 were 
 green. 
 
 (e) All the streptococci from infants' stools were either non-hemolytic or produced only a trace of hemo- 
 lysis on human blood agar. 
 
 Puppel 1912. 
 Ueber Streptokokken in der Milch und im 
 Sauglingsstuhl. 
 
 35 strains from milk. 
 
 
 
 
 
 
 
 The paper points out that 5. lacticus can be differentiated from S. pyogenes by means of blood agar plates. 
 5. pyogenes produces small colonies surrounded by a large zone of hemolysis, whereas 5. lacticus produces 
 green or grayish Colonies with very little or no hemolysis. 
 
 Ruedlger 1912. 
 A Study of Thirty-Five Strains of Strepto- 
 cocci Isolated from Samples of Milk. 
 
TABLE in — Continued. 
 The Use of Blood Agar. 
 
 TABLE III — Continued. 
 The Use of Blood Agar. 
 
 "^ 
 
 Medium. 
 
 Method of inoculation. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 
 Orgaoisms. 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 Composition. 
 
 Amount. 
 
 
 Strains from throats 
 (septic sore throats) and 
 various complications. 
 
 
 
 
 
 
 
 " The colonies on blood agar plates are larger and more moist than those of the common hemolytic streptococcus 
 {Streptococcus pyogenes); at times there may be a faint greenish tint on transmitted light. The zone of hemolysis 
 is relatively narrow and begins immediately around the colony, and its outer margin is often indistinct. The growth 
 on the surface of blood agar slants is abundant, raised, and moist, but does not present the mucoid appearance of 
 the Streptococcus mucosus." 
 
 "Morphologically in the smears from the throat and the exudates, it occurs in short chains and often in pairs. 
 The cocci, * * , are spherical, but appear in twos in the chain. They are strongly Gram-posilite and are sur- 
 rounded by a definite capsule. The capsidar substance is less abundant than that surrounding the pneumococci, 
 does not indent between the pairs, as is the case in the latter, and is soon lost on artificial cultivation." 
 
 Davis and Rosenow 1912. 
 An Epidemic of Sore Throat Due to a Pe- 
 culiar Streptococcus (Chicago Epidemic 1912). 
 
 45 strains from throats 
 and secondary lesions of 
 cases of epidemic sore 
 throat. 
 
 1 strain of ordinary 
 hemolytic streptococci 
 from udder of cow. 
 
 
 
 
 
 Probably deep 
 since he speaks of 
 growth on the up- 
 per and under sur- 
 faces of plates. 
 No deep colonies 
 described, however. 
 
 
 Practically all the strains from the various complications and some of those from throats had certain pecu- 
 harities by which they were readily differentiated from ordinary streptococci. On blood agar plates "The 
 colonies are larger and more moist than those of the ordinary type (5. pyogenes) . They are raised and tend to spread 
 on the upper or under surface of the media. A distinct zone of hemolysis is always present, which is clear with 
 often a faint greenish tint when viewed by transmitted light. This zone is usually but not always narrower than 
 that prodiiced by the ordinary Streptococcus hemolyticus. It begins immediately about the colony and its outer 
 margin may be indistinct. All the blood-corpuscles in this clear zone have been dissolved." 
 
 "On blood agar slants the growth is abundant, raised and moist but does not have the mucoid appearance of the 
 Streptococcus mucosus. There is much less tendency for the colonies to remain discrete than is the case with ordi- 
 nary Streptococcus pyogenes." 
 
 " * * , hemolytic streptococci, apparently of the ordinary type, were common, especially in the throat and 
 tonsils. These streptococci do not have a capsule; the colonies on blood agar possess wide clear zones of hemolysis; 
 they tend to spread less on the surfaces, and are smaller and less moist than the organism above described." 
 
 Davis 1912. 
 
 .Bacteriologic Study of Streptococci in Milk 
 in Relation to Epidemic Sore Throat (Chicago 
 Epidemic 1912). 
 
 Strains of streptococci 
 from cases of epidemic 
 sore throat and from ''sepa- 
 rator slime." Also strains 
 of 5. pyogenes. 
 
 
 
 \ 
 
 
 
 
 " The diference in the character of the growth of the streptococci (from separator slime) on blood agar plates be- 
 fore and after animal passage was striking, hemolyzing colonies only being present on the plates from the blood of 
 the animals and almost exclusively non-hemolyzing colonies on the plates made directly from the suspensions." 
 
 The ordinary hemolytic streptococcus (S. pyogenes) is described as producing small colonies surrounded by 
 wide zones of hemolysis on blood agar. 
 
 The streptococci obtained from animals inoculated with "separator slime" resembled those obtained from 
 cases of epidemic sore throat and produced a more abundant growth and less hemolysis on blood agar than 
 5. pyogenes. 
 
 Rosenow 1912. 
 .\ Study of Streptococci from Milk and from 
 Epidemic Sore Throat, and the Effect of Milk 
 on Streptococci. 
 
 Strains of 5. epidemicus: 
 1 from milk, 2 from sore 
 throats, 1 from throat of 
 carrier, and 3 from second- 
 ary peritonitis and adeni- 
 tis. 
 
 Rabbit. 
 
 
 
 
 
 
 "On blood agar plates the colonies were moist, somewhat larger than those of Streptococcus pyogenes, had a tend- 
 ency toward spreading, and were surrounded by clear zones of hemolysis with a greenish tinge by transmitted light." 
 "It therefore corresponds in the essential cultural characteristics and in its morphology to the streptococcus epidemicus 
 (Davis)." 
 
 The above description is of the strain isolated from milk, but the other strains are described as being like it. 
 
 Stokes and Hachtel 1912. 
 Septic Sore Throat. A Milk-Borne Epidemic 
 in Baltimore, Md. 
 
 Bacteriological Study of the Outbreak. 
 
 
 
 
 
 
 
 
 State that pneumococci are non-hemolytic on blood agar or blood bouillon but that they produce a dark 
 green discoloration on blood agar. 
 
 Neufeld and Handel 1912. 
 
 Pneumokokken (In KoUe and Wassermann's 
 Handbuch, 2nd edition). 
 
 Strains from vaginal and 
 lochial secretion (before, 
 during, and after labor) 
 of 72 or more normal and 
 24 or more febrile women, 
 ■* of whom died. 
 
 Sheep or guinea 
 pig, defibrinated. 
 
 5 per cent. 
 
 
 
 Surface. 
 
 Anaerobic 
 and aerobic, 
 72 hrs. 
 
 Staphylococci were the most numerous of the organisms found, the streptococci and diphtheroids coming 
 next in order of occurrence. The staphylococci and streptococci are merely tabulated as hemolytic or non- 
 hemolytic. No d'etailed descriptions of appearances on blood agar are given. 
 
 i ., 
 
 Sitzenfrey and Vatnlek 1913. 
 
 Zur Frage der prognostischen und praktischen 
 Verwertung bakteriologischer Befunde bei puer- 
 peralen Prozessen . 
 
 92 strains from milk 
 and the human throat 
 (normal?). 
 
 Rabbit. 
 
 "A few 
 drops." 
 
 
 
 Surface. 
 
 24 hrs. 
 
 17 of the strains were hemolytic, of which 10 strains were from milk and 7 from throats. No detailed de- 
 scriptions of appearances on blood agar are given. 
 
 Stowell, Hilliard, and Sehleslnger 1913. 
 A Statistical Study of the Streptococci from 
 Milk and from the Human Throat. 
 
 
 Human and horse. 
 
 
 
 
 
 
 S. hemolyticus, S. epidemicus, S. mucosus, S. pneumonice, and 5. viridans merely tabulated with reference to 
 amount of hemolysis on blood agar. 
 
 (See Table VIII on General Conclusions, etc., and Table VII on Mutations and Variability.) 
 
 Davis 1913. 
 Interrelations in the Streptococcus Group 
 with Special Reference to Ahaphylactic Reac- 
 tions. 
 
TABLE III — Continued. 
 
 The Use of Blood Agar. 
 
 TABLE in — Continued. 
 The Use of Blood Agar. 
 
 ^ — 
 
 Medium. 
 
 Method of inoculation. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 
 Organisms. 
 
 Blood. 
 
 Agar. 
 
 Reference. 
 
 
 Species. 
 
 Amount. 
 
 Composition. 
 
 Amount. 
 
 
 26 strains of 5. equi (23 
 laboratory strains of vari- 
 ous ages and 3 freshly iso- 
 lated by the author). 
 
 27 strains of S. longus 
 isolated by the authors 
 from human pathological 
 material. 
 
 Rabbit. 2 cc. 
 (Rabbits were inoculated in- 
 travenously with 2 cc. of serum 
 bouillon culture, and in 1 hr. 2 
 cc. of blood were drawn from 
 the ear vein, mixed with agar 
 at 4,S°, and poured into a Petri 
 dish). 
 
 Beef infusion, 
 Witte peptone, agar. 
 
 14 cc. 
 
 Deep. (See un- 
 der Blood.) 
 
 24 hrs. or 
 longer. 
 
 Speaking of 5. equi, "Nach 24 Stunden hat sich als-dann um jede einselne Kolonie innerhalb des Agars die 
 typische, etwa 2 mm. breite glashelle Zone gebildet." 
 
 "Der Rand des hdmolytischen Hofes einer Streptococcus longus-Kolonie zeigt nicht die scharfe Begrenzung wie 
 der des Drusecoccus. Im allgemeinen ist der hdmolytische Hof einer Drusekolonie um ein geringes grosser wie 
 beim Streptococcus longus. Der wichtigste Vnterschied besteht jedoch darin, dass die hdmolytische Zone beim Strep- 
 tococcus longus nach 24 Stunden stationdr bleibt, selbst bei weiterem Verweilen im Brutschrank, wdhrend der HoJ 
 des Drusecoccus sich in den folgenden Tagen sowohl bei Zimmer-. als auch bei Brul-temperatur, langsam vergr assert, 
 wodurch die Hofe benachbarter Kolonien hdufig zusammenfiiessen. Auf diese Weise hellt sich hdufig eine mit 
 dem Druse-Streptococcus bewachsene Blutplatte voUkommen auf. Meist ist auch der Farbenton der Blutplatten 
 beider Arten ein anderer. Der Blutfarbstof erleidet beim Drusecoccus kaum eine Verdnderung, w'ihrend die Farbe 
 der Blutplatte beim Streptococcus longus, zumal beim Vorhandensein zahlreicher Kolonien, einen schmutzig braun- 
 roten Farbenton annimmt {wahrscheinlich durch Bildung von Methdmoglobin)." 
 
 Koeh and Poksehischewsky 1913. 
 Uber die Artverschiedenheit des Strepto- 
 coccus longus seu erysipelatos und des Strepto- 
 coccus equi (Druse-Streptococcus). 
 
 
 
 
 
 
 
 
 The formation of peculiar excresences or droplet-like colonies on coagulated blood serum qr blood agar is 
 described as being characteristic for 5. equi. 
 
 The question of hemolysis is dismissed with the following remark: "On ne reussit pas plus par hSmolyse d 
 Stablir une difference entre les streptocoques de la gourme et d'autres. La propriete de I'hemolyse semble pouvoir 
 Ure acquis e par le milieu de culture." 
 
 Bemelmans 1913. 
 La specificite des streptocoques de la gourme. 
 
 247 strains from a great 
 variety of pathological 
 processes. 
 
 "Freshly prepared blood agar plates were used to demonstrate hemolysis." No other details. 
 
 "In the test for hemolysis about five per cent of the cultures showed the production of a greenish zone about the 
 colonies." 
 
 " * * several cultures secured from the severest general infections have not been hemolytic." 
 In the tables all cultures are simply marked 4- or — with regard to hemolysis. 
 (See also Table VIII on General Conclusions, etc.) 
 
 Floyd and Wolbaeh 1914. 
 On the Differentiation of Streptococci. 
 (Preliminary Notes) . 
 
 Studied many strains 
 from various sources and 
 of various types. 
 
 Human, defibri- 
 nated. 
 
 0.5 cc. 
 
 Standard agar 
 prepared from Lie- 
 big extract and 
 Witte peptone. 
 
 7cc. 
 
 Surface. 
 
 
 Generally describes the colonies on blood agar simply as " hemolyzing," "hazy or slightly hemolyzdng," "green," 
 "grayish," " non-hemolyzing," etc., but in describing the transformations of pneumococci to streptococci he 
 distinguishes between the "clear zone of hemolysis that began immediately outside the colony itself" of the strep- 
 tococcus, and the colony of the partially transformed pneumococcus in which "the hemolysis was outside of a 
 zone in which the corpuscles were intact." 
 
 Rosenow 1914. 
 Transmutations within the Streptococcus- 
 Pneumococcus Group. 
 
 14 strains of strepto- 
 cocci from the joint fluid 
 of 14 cases of acute rheu- 
 matism. 
 
 
 
 
 
 
 
 5 strains "produced green on blood agar." 
 
 6 strains "produced a slight but hazy hemolysis." 
 
 3 strains "produced small grayish colonies * * * without perceptibly affecting the medium." 
 
 Rosenow 1914. 
 The Etiology of Acute Rheumatism, Articu- 
 lar and Muscular. 
 
 9 strains; from cases of 
 arthritis, septicemia, scar- 
 let fever, endocarditis, and 
 infected finger. 
 
 Rabbit, for all 
 strains. 
 
 Human, for 2 
 strains. 
 
 
 
 
 
 
 The amount of hemolysis on blood agar (3 separate tests, interval not stated) is described merely as "marked," 
 "moderate," "distinct," "slight," or "none." As tabulated, the results of the 3 tests for each strain varied from 
 "marked" to "moderate" or between "none" and "slight." All strains produced "marked," "moderate," or 
 "distinct" hemolysis in some of the tests. 
 
 Also determined the "hemolytic index" of each strain in serum bouillon by the special method described by 
 the author in 1912. (See Table IV on The Use of Blood Bouillon and Other Fluid Media.) 
 
 It is concluded " That for the quantitative and qualitative estimation of hemolysin production by streptococci the 
 blood agar plate is unreliable and should give place to titration of the M. H. D. {minimum hemolytic dose) of a cul- 
 ture of streptococci in fluid media." 
 
 M'Leod 1914. 
 
 Criteria of Virulence amongst Streptococci, 
 with Some Remarks on Streptococcal Leuco- 
 cidin. 
 
 2 strains of 5. pyogenes; 
 sources not given. 
 
 Oxalated blood 
 of infected rabbit. 
 
 2 cc. of 
 oxalate solu- 
 tion+blood, 
 3:1. 
 
 4 per cent glyc- 
 erol agar. 
 
 
 Deep, the blood 
 itself being in- 
 fected. 
 
 24 hrs. 
 
 The author gives no descriptions of appearances on blood agar. The results are merely recorded as + or — 
 (hemolytic or non-hemolytic). 
 
 (See also Table VII on Mutations and Variability.) 
 
 Relehsteln 1914. 
 
 Ueber den Nachweis der Streptokokken im 
 striimenden Blute. 
 
 18 strains from throats. 
 1 from cervical gland, and 
 9 from milk slime. 
 
 Rabbit. 
 
 
 
 
 "Conradi meth- 
 od." 
 
 
 On blood agar all strains produced "green" or "faint green" colonies. Only the one from a cervical gland 
 produced any hemolysis. Most of the others produced " methemoglobin brown" zones. 
 
 North, White, and Avery 1914. 
 
 K Septic Sore Throat Epidemic in Cortland 
 and Homer, N. Y. 
 
TABLE III — Continued. 
 The Use of Blood Agar. 
 
 TABLE III — Continued. 
 The Use of Blood Agar. 
 
 Organisms. 
 
 105 strains from various 
 patliological processes, and 
 from milk, normal throats, 
 and human stools. 
 
 164 strains of various 
 organisms (cholera, other 
 vibrios, typhoid, anthrax, 
 froteus, pyocyaneus, etc.) 
 including 27 streptococci 
 and 20 staphylococci. 
 
 6 strains of S hemo- 
 lyiicus. 
 
 5 strains of pneumococci. 
 24 strains of S. viridans. 
 
 12 strains studied, (7 
 from throats, 3 from ud- 
 ders of co\ys, and 1 hu- 
 man and 1 bovine strain 
 from Chicago epidemic, 
 1912, for comparison). 
 
 2 strains studied, 1 
 from blood of fatal case 
 {S. epidemicus) during 
 Chicago epidemic, 1912; 1 
 from cow with inflamed 
 udder, Chicago 1912. 
 
 Medium. 
 
 Blood. 
 
 Species. 
 
 Human. 
 
 Amount. 
 
 Sheep, also hu- 
 man, beef, and 
 rabbit to some ex- 
 tent. 
 
 Human. 
 
 " There were sixteen acute 
 alveolar abscesses, eighteen 
 subacute or chronic ab- 
 scesses, and eight teeth with 
 diseased root-canals from 
 which material was received 
 for bacteriologic examina- 
 tion." 
 
 S3 strains of strepto- 
 cocci from sore throats 
 and secondary lesions in 
 man. 
 
 12 strains from cow's 
 milk. 
 
 5 per cent. 
 
 Agar. 
 
 Composition. 
 
 Method of inoculation. 
 
 Surface. 
 
 Surface. 
 
 Period 
 of incubation. 
 
 1-3 days. 
 
 Up to 
 
 hrs. 
 
 "Blood agar slants." 
 (No other details.) 
 
 Horse. 
 
 Standard veal 
 infusion agar 
 
 (Witte peptone). 
 Reaction = ± 1 
 per cent N acid to 
 phenolphthalein . 
 
 12 cc. 
 
 24 hrs. 
 
 Results and conclusions. 
 
 "Some of the strains, notably the mannite fermenters, produced no lysis. Others obtained from sephcemms pro- 
 duced a wide zone in less than twenty-four hours. Between these two extremes, however, were all gradations. Many 
 of the pneumococci and strains of the 'viridans' type from endocarditis produced a narrow zone m two to three days. 
 The hemolysis depended also on the depth of the medium and the amount of blood tt contained, for tn cin unevenly 
 mixed blood plate it was sometimes observable in the thin, hut not in the thicker portions. It ts difficult, 
 then, to draw a line sharply between hemolyzing and non-hemolyzing types. The property is present to some extent 
 in most streptococci and while in general it is more pronounced in the more virulent type, the differences are essen- 
 tially quantitative and therefore not altogether satisfactory as the basis of classification." 
 
 Recognizes 2 types of hemolysis on blood agar plates; 1) " Hdmopepsie," "Bildung ernes durchsichtigen 
 Hofes urn die einzelnen Kolonieen auf der Platte;" 2) " Hdmoglobinopepsie," " Aufhellung der Blutplalte in der 
 Umgebung der Kolonieen, so dass die Blutplatte transparent und schwach grunlich gefarhl, dagegen nicht durch- 
 sichtig erscheint." . „ 
 
 The streptococci studied were either non-hemolytic or produced ' Hdmopepsie. 
 
 The staphylococci studied all produced "Hdmopepsie." 
 
 Some of the other organisms studied produced "Hamoglobinopepsie." 
 
 (See also Table IV on Blood Bouillon, etc., and discussion in text.) 
 
 "All the strains were tried in blood agar and by this means were separated into three groups, that is, those pro- 
 ducing green pigment, those producing hemolysis, and those producing pneumococci. All were considered pneu- 
 mococci that had well-developed capsules, that fermented inulin, that were green in blood agar, and that were dis- 
 solved by bile." 
 
 No detailed descriptions given. 
 
 Reference. 
 
 Hopkins and Lang 1914. 
 
 Classification of Pathogenic Streptococci by 
 Fermentation Reactions. 
 
 Baerthlein . 1914. 
 
 Ueber Blutveriinderung durch Bakterien. 
 
 Thro 1914. 
 
 Experiments on the Variability of the Fer- 
 mentative Reaction of Bacteria, Especially the 
 Streptococci. 
 
 Of the strains reported all except one (a bovine strain) were hemolytic. 4 of the throat strains and_2 iso- 
 lated from the udders of cows resembled the S. epidemicus from the Chicago epidemic (1912), producing a 
 narrow zone of hemolysis. " The other two (throat strains) resemble the ordinary hemolytic streptococcus.' 
 
 (See also Table VIII on General Conclusions, etc.) 
 
 Surface. 
 
 Deep. 
 
 Aerobic 
 and anaero- 
 bic. 
 
 48 hrs 
 
 Most of the paper is devoted to the subject indicated by the title. . . „ 
 
 "So far as we know, all the streptococci causing epidemics of septic sore throat have been of the hemolytic variety. 
 
 "Those organisms are called hemolytic which cause a distinct clear zone of hemolysis about the colonies when 
 incubated at 37°C. for 24 hours Many streptococci may produce a narrow greenish, grayish, or brownish zone in 
 which, especially after two or more days at times, some clearing of the media may occur. Such strains, however, 
 are not considered true hemolyzers and are referred to as non-hemolytic." ^ . , . . , 
 
 "So far as we know at present, any appreciable number of hemolytic streptococci found in cow's milk obtained 
 carefully in sterile tubes directly from the cow's udder always means a diseased condition." 
 
 Capps and Davis 1914. 
 
 An Epidemic of Streptococcus Sore Throat in 
 Jacksonville, III, Which was Traced to the 
 Milk of Cows Affected with Streptococcus Mas- 
 titis. 
 
 Davis 1914. 
 
 The Growth and Viability of Streptococci of 
 Bovine and Human Origin in Milk and Milk 
 Products. 
 
 "Within twenty-four hours one will usually see a fine granular growth over the surface of the aerobic blood agar 
 slant. If one examines this closer the growth is seen as fine greenish colonies. The water of condensation contains 
 chains of these organisms which are usually Gram-positive. There may also be a similar growth in the anaerobic 
 tube hut one can not distinguish the green color, .it the end of forty-eight hours, however, there is quite a change 
 in the appearance of the anaerobic growth; it is very luxuriant, grayish-white in color and the colonies have a great 
 tendency to fuse. On opening this tube one notices immediately a very offensive odor." 
 
 " * * we found the predominating organism to he a streptococcus. We have obtained many graded ^ varia- 
 tions from a hemolytic streptococcus with a wide zone of hemolysis in the acute abscesses, to a Streptococcus viridans 
 in the chronic, and just recently one in which a Streptococcus mucosus was the predominating organism. t^Ii"'" * 
 will in many instances grow as well anaerobically as they will aerobically." 
 
 These 
 
 The authors distinguish 2 types of appearance in blood agar plates; "Type a") " * * a somewhat 
 greenish discoloration and partial hemolysis of the blood corpuscles immediately surrounding the colony 'arming a 
 rather indefinitely bounded zone one to two millimeters in diameter and surrounded by a second narrow, clearer, not 
 discolored, partially hemolyzed zone. * * Under the microscope many of the corpuscles (in the inner discolored 
 zones) were seen to be present but obviously discolored, the discoloration differing a good deal in intensity for different 
 strains of streptococci. The corpuscles remaining in the outer {clearer) zones were much fewer and never discolored." 
 
 "Type 0") " * * sharply defined, clear, transparent, completely hemolyzed, colorless zones two to four mil- 
 limeters in diameter. Under the microscope no corpuscles were seen to remain within the zones." 
 
 Gilmer and Moody 1914. 
 
 A Study of the Bacteriology of Alveolar Ab- 
 scess and Infected Root Canals. 
 
 Smith and Brown 1915. 
 
 A Study of Streptococci Isolated from Cer- 
 tain Presumably Milk-Borne Epidemics of Ton- 
 sillitis Occurring in Massachusetts in 1913 and 
 1914. 
 
TABLE in — Continued. 
 The Use of Blood Agar. 
 
 TABLE m — Continued. 
 The Use of Blood Agar. 
 
 Medium. 
 
 Organisms. 
 
 Streptococci from 150 
 cases of pyorrhea alveo- 
 laris and apical abscesses. 
 
 2 strains of S. lacticus 
 from ice cream and milk 
 respectively. 
 
 Strains of streptococci 
 isolated from the tissues 
 of the appendix in 17 of 20 
 cases of appendicitis. 
 
 Blood. 
 
 Species. 
 
 Amount. 
 
 Sheep, defibri- 10 per cent, 
 nated, or rabbit, 
 whole. 
 
 Human or goat, 
 defibrinated. 
 
 60 strains from various 
 human infections. 
 
 9 strains from milk 
 sediment. 
 
 A number of strains from 
 sore throats. 
 
 Strains of 5. viridans 
 from various human lesions. 
 
 Rabbit, defibri- 
 nated. 
 
 Agar. 
 
 Method of inoculation. 
 
 Composition. 
 
 Agar plates or 
 slants. 
 
 Amount. 
 
 5 per cent. 
 
 1 cc. 
 
 Beef infusion agar. 
 
 Surface, prob- 
 ably. 
 
 Surface. 
 
 Period 
 of incubation. 
 
 Results and conclusions. 
 
 , the colo- 
 
 In every case but two the colonies on blood agar were surrounded by a green halo. In the two exceptions, 
 
 •^ - ■■ dots without any change in the surrounding blood agar. In no case were hemolytic 
 
 nies appeared as small gray 
 streptococci obtained. 
 
 Both strains, originally non-hemolytic, acquired to some extent the power to hemolyze human and goat 
 blood after animal passages. 
 
 10 cc. 
 
 Human. 
 
 Many strains from vari- 
 ous sources. 
 
 Horse. 
 
 1 cc. to 
 each plate. 
 
 (Presumably 
 meat infusion agar.) 
 
 Surface. 
 
 Surface. 
 
 Hemolytic streptococci 
 from sore throats, 1 strain 
 from secondary peritoni- 
 tis, and 2 from udder of 
 cow. Compared with 
 other hemolytic and non- 
 hemolytic strains. 
 
 Up to 3 
 days. 
 
 "The strains of streptococci which were found to have an affinity for the appendix formed short chains much acid 
 and a diffuse turbidity in ascites dextrose broth, hut no clumps and, with but two exceptions, produced a moderate 
 amount of green on blood agar plates.' 
 
 Reference. 
 
 Hartzell atid Henrlcl 1915. 
 
 A Study of Streptococci from Pyorrhea Alveo- 
 laris and from Apical Abscesses. 
 
 Heinemann 1915. 
 
 The Variability of Two Strains of Strepto- 
 coccus lacticus. 
 
 Rosenow 1915. 
 
 The Bacteriology of Appendicitis and its 
 Production by Intravenous Injection of Strep- 
 tococci. 
 
 "The results were recorded under three heads: H, hemolysis; G, green colonies with or without slight hemolysis; 
 
 and N, gray or brown colonies showing no action on hemoglobin." .,.,.,,,, , ,u, .r.m„„,r^ Ur 
 
 "An interesting observation, the constancy of which was not determined, is the fact that many of the raffinosefer- 
 
 menlers gave green colonies accompanied by a slight zone of hemolysis, while the mannite fermenters usually gave a 
 
 green to a greenish brown colony without any hemolysis. This reaction was merely noted and no significance can 
 
 be claimed for it." ^ ^ _ 
 
 ■lUrrounded 
 organisms 
 
 "The streptococci isolated from the throats produced on blood agar plates rather large moist colonies 
 by narrow, ill-defined, hazy zones of hemolysis, corresponding closely m these characteristics^ with th 
 isolated from the milk, and indistinguishable from the milk "^^''n'-srns after animal passage 
 
 " * * ,the organisms from milk resembled the rheumatic strains (previously studied?) culturaUy and morpno 
 logically." 
 
 Kligler 1915- 
 
 A Study of the Correlation of the Aggluti- 
 nation and the Fermentation Reactions Among 
 the Streptococci. 
 
 Rosenow and Moon 1915. 
 
 On an Epidemic of Sore Throat and the Vir- 
 ulence of Streptococci Isolated from the Milk. 
 
 " When tested in human blood agar, some of the strains were distinctly green without any 'f^lf^Ztththt^Z'te 
 Some grew as minute, green colonies with a narrow clear zone. * * Two strains of the short-chained type, 
 obtained from urine, formed dark brown colonies." , ,. ., ^ ,. ,, ... -^ ,u, j.^„^„m„« r,f hemn- 
 
 "In bood plates if the blood and agar were unevenly distributed, a slight variation '«'*/, ^"^«f;;;j/^7, 
 Ivsis and men -biement took place. In areas of the plale where the agar was of greater depth and there was more 
 bloodX foZi!Ze^egreen,ihile in the areas where the agar was of less thickness, a slight zone of hemolysis was 
 shown." 
 
 Surface 
 deep. 
 
 and 
 
 No descriptions of appearances on blood agar are given except such as are indicated ^Y th^ "se of the W 
 owing terms- "Hemolysis," "Green," "No color," "Haze," " G-hemolys,s:' "G-haze, . 'f^'^^^-^^^'f "' , f " 
 color haze," "Brown," etc. The strains from various sources produced appearances mdicated by the fore- 
 
 't^mrtlro\STt;ains);feces(31);andbloodpath^^^^^^^^^ 
 
 from throat) ; the others = "G-hemolysis," "Haze-hemolysis," "Brown-hemolysis, Haze, Green, G-haze, 
 
 "No color," etc. 
 
 Sr'thSat gS; lpS:fa:d':t^omach'(31); intestine (54); and feces (11). 2 (from throat) = "Haze- 
 h^mnlv^U-" all others = "Green" "Haze," "G-haze," or "No color. , . „ ,. 
 
 'Tog' hroal (80) esophSs 'and stomach (64) ; intestine (37) ; and feces (3S)._, « =1 f-^^^'C "^"Y™ 
 esophagus and stomach, intestine, and throat); others = "Green," " G-hemolysis , G-haze, Haze, No 
 
 '"Piiot'esophagus to intestine (10): 2 = "Hemolysis;" others ^ "Green," "Haze," or ';? color." 
 Hen, stomach and intestine (6): 1 = "Hemolysis ?;" others = "Haze" and No color. 
 Horse feces (6): all = "Green" 
 
 Thro ■...,.^^^^; 
 
 Further Experiments on the Variability of 
 
 the Fermentative Reaction of Bacteria, Espe- 
 cially the Streptococci. 
 
 Broadhurst . 1915. 
 
 Environmental Studies of Streptococci, with 
 Special Reference to the Fermentative Reac- 
 tions. ■ 
 
 Typical hemolysis may not be present on surface streaked blood plates, although it may develop after longer incu- 
 
 '"''""Compared with the streaked plates, the poured plates showed an apparent higher percentage of hemolyzing types 
 and more important, typical wide zones of hemolysis. AUhough the streaked plates are more convenient, it is ..,- 
 dent from this thai they may give confusing results." 
 (See Table VII on Mutations and Variability.) 
 
 Krumwiede and Valentine 1913. 
 
 A Bacteriological Study of an Epidemic of 
 Septic Sore Throat. 
 
TABLE ni — Continued. 
 The Use of Blood Agar. 
 
 TABLE in — Continuea. 
 The Use of Blood Agar. 
 
 Organisms. 
 
 Many strains of strep- 
 tococci; from tonsils of 
 normal individuals and 
 appendicitis patients, 
 
 from Steno's duct in pa- 
 tients with parotitis, from 
 various dairy products, 
 and from removed ap- 
 pendices. 
 
 11 strains of strepto- 
 cocci from tonsils and 
 pyorrheal pockets of cases 
 of herpes zoster. 
 
 Resum6 of work done 
 on man\' strains of vari- 
 ous kinds, studied by the 
 author and others in this 
 and previously reported 
 papers. 
 
 1 strain of hemolytic 
 streptococci (human type) 
 from udder of cow with- 
 out gross evidence of mas- 
 titis. 
 
 1 strain of human type 
 isolated by Davis (1912) 
 from bovine mastitis. 
 
 1 strain of 6". epidemicus 
 isolated by Davis (1912) 
 from peritonitis following 
 tonsillitis. 
 
 8.5 strains of hemo- 
 lytic streptococci from 328 
 samples of bottle milk. 
 
 Medium. 
 
 Blood. 
 
 Species. 
 
 Amount. 
 
 Agar. 
 
 Composition. 
 
 Amount. 
 
 Human, defibri- 
 nated. 
 
 Human. 
 
 Human. 
 
 S per cent. 
 
 "Plain agar {0.6 
 plus)." 
 
 Method of inoculation. 
 
 Period_ 
 of incubation. 
 
 Poured "Surface rubbed." 
 to a depth of 
 about 2mtn."\ 
 
 Results and conclusions. 
 
 (See Table VII on Mutations and Variability.) 
 
 48hrs. 
 
 The authors describe an epidemic of appendicitis followed by an epidemic of parotitis. 
 
 The organisms responsible for the appendicitis were apparently "green-producing streptococci. 
 
 Cultures from samples of butter obtained during the epidemic of parotitis "showed a large number oj d types 
 of colonies oj streptococci: the one produced distinct green colonies on blood agar; the other produced smaller, grayish, 
 elevated, round colonies surrounded by a narrow hazy zone of hemolysis." 
 
 " The cultures from the dairy products showed a preponderance of non-hemolysmg, short-cham-producmg strep- 
 tococci, often in almost pure form and in enormous numbers. Slightly hemolyzing streptococci were found occa- 
 sionally." 
 
 The streptococci believed to cause herpes zoster iormed^'moist green-producing colonies of a non-encapsulated, 
 gram-staining, short-chained, often lanceolate streptococcus. 
 
 " The hemolytic streptococci liberate the hemoglobin, both on solid and in fluid media. * * It appears to me 
 probable that the organism grown on ordinary blood agar liberates the hemoglobin * * and then either destroys 
 ii or uses it as a food material. * * The colonies * * show no evidence of having m them any of the altered 
 forms of hemoglobin. * * the clear zone surrounding the colony appears devoid of any trace of hemoglobin, and 
 if hemolyzed blood be used in the agar, a distinct clear zone develops about the growth." , u -j 
 
 " The non-hemolytic forms growing on blood agar produce either no hemolysis, or more commonly, a pale bluish 
 green to green, followed by brown, and finally a deep dirty brown, almost black color. * * A curious phenomenon 
 of these green-producing streptococci is the apparent storing of some of the altered hemoglobin m the colony itself. 
 * * It is probable that in all these green producers a certain amount of hemolysis actually occurs, although m the 
 vast majority of them it is not visible." . r j, , ,■ 
 
 "Acid production is not necessary for the production of the green color, because m the colonies of B. lactis aero- 
 genes, an actively fermenting non-hemolyzing organism, green is not produced." However, "m the case of the 
 hemolytic streptococci, the hemolysin of which is absent or inactive on dextrose blood agar, the organisms form the 
 green coloration in much the same manner as the non-hemolytic green producers." 
 
 (See text for additional quotations.) 
 
 The streptococci of the human type isolated from one of the cows " gre-ai as small, translucent, slightly moist, 
 grayish-while colonies on blood agar plates with a clear transparent hemolytic zone, from 2 to 4 mm. m diameter; 
 * * >> 
 
 The strains from mastitis (isolated by Davis, 1912) produced "small dry gray colonies with a sharply defined 
 zone of hemolysis from 4 to 6 mm. in diameter." , ,. , ,. r. 
 
 The colonies of the streptococcus from peritoneal pus were "large, moist grayish-white, and spreading, often 
 on the surface almost covering the clear hemolytic areas around them." 
 
 "Only those streptococci were selected the colonies of which were surrounded by a distinct clear zone of hemolysis 
 on human blood agar plates (Type /3, Smith and Brown). The feebly hemolytic streptococci {Type a) were often 
 noted in the milk, but were disregarded, * * ," ,,,..,,., 
 
 " Though all these strains belong to the hemolytic group the degree and character of hemolysis m all instances may 
 not be uniform. The strains, on the basis of hemolysis, have arbitrarily been arranged in 4 groups as follows: 1st, 
 wide clear hemolytic circle, from 2 to 4 or more mm. in diameter; 2nd, clear hemolytic circle from 1 to 3 mm. across 
 with diffuse and indefinite margin; 3rd, small clear hemolytic circle from 1 to 2 mm. across with sharp margin; 4th, 
 gray-green or hazy hemolytic circle from 1 to 1.5 mm. across, hemolysis often incomplete m circle. This 
 
 grouping demonstrates that there is a more or less gradual transition from 1 type to the other, and indicates, as was 
 observed, that a strain may ciiange to some extent its type of hemolysis." 
 
 Reference. 
 
 Irons, Brown, and Nadler 1916. 
 
 The Localization of Streptococci in the Eye. 
 
 Rosenow and Dunlap 1916. 
 
 An Epidemic of Appendicitis and Parotitis 
 
 Probably Due to Streptococci Contained in 
 Dairy Products. 
 
 Rosenow and Oftedal 1916. 
 
 The Etiology and Experimental Production 
 of Herpes Zoster. 
 
 Holman 
 
 The Classification of Streptococci. 
 
 1916. 
 
 Mathers 1916. 
 
 Different Tjqjes of Streptococci and Their 
 Relation to Bovine Mastitis. 
 
 Davis 191 6. 
 
 Hemolytic Streptococci Found in Milk. 
 
TABLE III — Concltcded. 
 The Use of Blood Agar. 
 
 TABLE III — Concltided. 
 The Use of Blood Agar. 
 
 Organisms. 
 
 5 Strains: (1) "A strong- 
 ly hemolytic streptococcus" 
 from a case of erysipelas; 
 (2) "a hemolytic strain 
 from a scarlatinal sore 
 throat;" (3) "a hemolytic 
 streptococcus from the ton- 
 sils in a case of rheumatic 
 fever;" (4) "a non-hemo- 
 lytic streptococcus" from 
 pneumonic sputum; (5) "a 
 green-producing streptococ- 
 cus" from the same source 
 as No. 3. 
 
 Many strains of hemo- 
 lytic streptococci from 
 cases of tonsillitis, otitis 
 media, and scarlet fever; 
 also from normal throats 
 and suspected milk. 
 
 51 strains of "Strepto- 
 coccus hemolysans." 
 
 91 strains of "Strepto- 
 coccus viridans." 
 
 (All from pathological 
 sources.) 
 
 Medium. 
 
 Blood. 
 
 Agar. 
 
 Species. 
 
 Sheep, goat, 
 
 horse, rabbit, and 
 human. 
 
 Horse (guinea 
 pig, rabbit, and 
 dog). 
 
 Human or rab- 
 bit, defibrinated. 
 
 Amount. 
 
 1 cc. de- 
 fibrinated, 
 or 2 cc. cit- 
 rated (blood 
 and citrate 
 solution aa), 
 "unless oth- 
 erwise men- 
 tioned." 3, 6, 
 9, or 12 drops 
 used for 
 special ex- 
 periments. 
 
 1 cc. 
 
 Composition. 
 
 "* all the agar 
 used was of one 
 lot; a portion was 
 made 1 per cent al- 
 kaline with sodium 
 hydrate, another por- 
 tion 1 per cent acid 
 with hydrochloric 
 acid, and a third 
 portion of standard 
 agar was made up 
 with 1 per cent 
 dextrose." 
 
 1.5 cc. 
 
 Veal infusion 
 Witte peptone agar. 
 Reaction "-f 0.8 
 -1.0." 
 
 '•Plainagar{0.3-\- 
 to 0.0-f-)." 
 
 (Probably meat 
 infusion agar.) 
 
 Amount. 
 
 6 CC. 
 
 10 CC. 
 
 IS cc. 
 
 Method of inoculation. 
 
 Surface. 
 
 Deep. 
 
 Deep. 
 
 Period of 
 incubation. 
 
 24, 48, and 
 96 hrs. 
 
 Up 
 hrs. 
 
 to 48 
 
 24 hrs. 
 
 Results and conclusions. 
 
 " * * no hemolysis was produced by any of the organisms in alkaline blood agar." 
 
 "Hemolysis was distinctly inhibited on dextrose blood-agar," and greenish discoloration was enhanced for some 
 
 strains. . , <, ■ j- • 
 
 In acid agar the results were quite different for different strains; e. g., stram No. 1 produced an indistinct 
 zone on the acid medium," while "with Organism No. 3 the results were the reverse." 
 
 With different kinds of blood the results were very different for different strains, some hemolyzing one blood 
 and some another. 
 
 The hemolytic streptococci produced the largest and clearest zones on blood agar contammg the least 
 amounts of blood. . i i. i ■ 
 
 Crowding of colonies on the plate resulted differently for different strams, causmg more or less hemolysis. 
 
 As a standard method it is recommended that streptococci be streaked on defibrinated human blood agar 
 (1 :6) and observed after incubation for 24 hrs. 
 
 The paper is largely epidemiological. Several epidemics of sore throat are studied, the infection being traced 
 to milk in some cases and to contact in others. 
 
 "The beta hemolytic streptococcus of Smith and Brown on horse-hlood-agar plates forms a small lanceolate colony 
 in the depths of the media. Around the colony is a clear-cut zone of hemolysis, from Z to 4 mm. in diameter, at the 
 end of from 24 to 48 hours. Under the low power of the microscope complete disappearance of the outline of the red 
 cells is noted. The border of the hemolytic zone is rather abruptly limited by the dense mass of red cells." 
 
 "It is important to remember that the blood of different animals produces different types of hemolysis. Guinea- 
 pig blood is particularly unsatisfactory. Dog blood produces a brownish discoloration. Washed corpuscles give a 
 different type of hemolysis from that of whole blood, with narrower and less well defined borders." 
 
 (See Table VIII on General Conclusions, etc.) 
 
 Reference. 
 
 Becker 1916. 
 
 The Necessity of a Standard Blood-agar Plate 
 for the Determination of Hemolysis by Strep- 
 tococci. 
 
 SmlUie 1917. 
 
 Studies of the Beta Hemolytic Streptococcus 
 (Smith and Brown). 
 
 Distinguishes hemolytic, green-producing, and indifferent colonies on blood agar, but believes that the latter 
 two should be grouped together as "Streptococcus viridans." 
 (See Table VIII on General Conclusions, etc.) 
 
 Blake 
 
 The Classification of Streptococci. 
 
 1917. 
 
TABLE III — Concltcded. 
 The Use of Blood Agar. 
 
 TABLE III — Concltided. 
 The Use of Blood Agar. 
 
 Organisms. 
 
 5 Strains: (1) "A strong- 
 ly hemolytic streptococcus" 
 from a case of erysipelas; 
 (2) "a hemolytic strain 
 from a scarlatinal sore 
 throat;" (3) "a hemolytic 
 streptococcus from the ton- 
 sils in a case of rheumatic 
 fever;" (4) "a non-hemo- 
 lytic streptococcus" from 
 pneumonic sputum; (5) "a 
 green-producing streptococ- 
 cus" from the same source 
 as No. 3. 
 
 Many strains of hemo- 
 lytic streptococci from 
 cases of tonsillitis, otitis 
 media, and scarlet fever; 
 also from normal throats 
 and suspected milk. 
 
 51 strains of "Strepto- 
 coccus hemolysans." 
 
 91 strains of "Strepto- 
 coccus viridans." 
 
 (All from pathological 
 sources.) 
 
 Medium. 
 
 Blood. 
 
 Agar. 
 
 Species. 
 
 Sheep, goat, 
 
 horse, rabbit, and 
 human. 
 
 Horse (guinea 
 pig, rabbit, and 
 dog). 
 
 Human or rab- 
 bit, defibrinated. 
 
 Amount. 
 
 1 cc. de- 
 fibrinated, 
 or 2 cc. cit- 
 rated (blood 
 and citrate 
 solution aa), 
 "unless oth- 
 erwise men- 
 tioned." 3, 6, 
 9, or 12 drops 
 used for 
 special ex- 
 periments. 
 
 1 cc. 
 
 Composition. 
 
 "* all the agar 
 used was of one 
 lot; a portion was 
 made 1 per cent al- 
 kaline with sodium 
 hydrate, another por- 
 tion 1 per cent acid 
 with hydrochloric 
 acid, and a third 
 portion of standard 
 agar was made up 
 with 1 per cent 
 dextrose." 
 
 1.5 cc. 
 
 Veal infusion 
 Witte peptone agar. 
 Reaction "-f 0.8 
 -1.0." 
 
 '•Plainagar{0.3-\- 
 to 0.0-f-)." 
 
 (Probably meat 
 infusion agar.) 
 
 Amount. 
 
 6 cc. 
 
 10 CC. 
 
 IS cc. 
 
 Method of inoculation. 
 
 Surface. 
 
 Deep. 
 
 Deep. 
 
 Period of 
 incubation. 
 
 24, 48, and 
 96 hrs. 
 
 Up 
 hrs. 
 
 to 48 
 
 24 hrs. 
 
 Results and conclusions. 
 
 " * * no hemolysis was produced by any of the organisms in alkaline blood agar." 
 
 "Hemolysis was distinctly inhibited on dextrose blood-agar," and greenish discoloration was enhanced for some 
 
 strains. . , <, ■ j- • 
 
 In acid agar the results were quite different for different strains; e. g., stram No. 1 produced an indistinct 
 zone on the acid medium," while "with Organism No. 3 the results were the reverse." 
 
 With different kinds of blood the results were very different for different strains, some hemolyzing one blood 
 and some another. 
 
 The hemolytic streptococci produced the largest and clearest zones on blood agar contammg the least 
 amounts of blood. . i i. i ■ 
 
 Crowding of colonies on the plate resulted differently for different strams, causmg more or less hemolysis. 
 
 As a standard method it is recommended that streptococci be streaked on defibrinated human blood agar 
 (1 :6) and observed after incubation for 24 hrs. 
 
 The paper is largely epidemiological. Several epidemics of sore throat are studied, the infection being traced 
 to milk in some cases and to contact in others. 
 
 "The beta hemolytic streptococcus of Smith and Brown on horse-hlood-agar plates forms a small lanceolate colony 
 in the depths of the media. Around the colony is a clear-cut zone of hemolysis, from Z to 4 mm. in diameter, at the 
 end of from 24 to 48 hours. Under the low power of the microscope complete disappearance of the outline of the red 
 cells is noted. The border of the hemolytic zone is rather abruptly limited by the dense mass of red cells." 
 
 "It is important to remember that the blood of different animals produces different types of hemolysis. Guinea- 
 pig blood is particularly unsatisfactory. Dog blood produces a brownish discoloration. Washed corpuscles give a 
 different type of hemolysis from that of whole blood, with narrower and less well defined borders." 
 
 (See Table VIII on General Conclusions, etc.) 
 
 Reference. 
 
 Becker 1916. 
 
 The Necessity of a Standard Blood-agar Plate 
 for the Determination of Hemolysis by Strep- 
 tococci. 
 
 SmlUie 1917. 
 
 Studies of the Beta Hemolytic Streptococcus 
 (Smith and Brown). 
 
 Distinguishes hemolytic, green-producing, and indifferent colonies on blood agar, but believes that the latter 
 two should be grouped together as "Streptococcus viridans." 
 (See Table VIII on General Conclusions, etc.) 
 
 Blake 
 
 The Classification of Streptococci. 
 
 1917. 
 
TABLE IV — Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 TABLE IV — Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 Organisms. 
 
 30 strains, of which 25 were 
 5. longus pathogenes. The other 
 ,^ were thought to be S. mitior 
 jy the author. 
 
 "Zur Zeit steht leider kein 
 Streptokohkenstamm zur Ver- 
 'iigung, der ah S. mitior ange- 
 sehen werden kann, um seine 
 Wirkung auf BlutbouiUon mil 
 der des S. longus zu wrgleichen." 
 
 16 strains from various hu- 
 man lesions, normal mouths, 
 and intestines. 
 
 . 24 strains: 22 from vulval, 
 vaginal, and uterine discharges 
 of 10 women during and 4r-9 
 days after labor; 1 from a felon; 
 1 from a sore throat. 
 
 6 streptococci. 
 6 pneumococci. 
 
 14 laboratory strains of 
 streptococci from various hu- 
 man lesions. 
 
 1 strain from the blood of a 
 
 scarlet fever case. 
 
 65 -trains (43 from various 
 human pathological processes; 
 the remainder from milk, skin, 
 sputum, and feces). 
 
 Blood. 
 
 Species. 
 
 Human. 
 
 Rabbit. (Human, 
 guinea pig, dog, beef, 
 and frog for compari- 
 son.) 
 
 Used only washed 
 corpuscles. 
 
 Rabbit, beef, hu- 
 man. 
 
 Human (possibly 
 others) . 
 
 Horse, rabbit. 
 
 Amount. 
 
 10 drops. 
 
 A few drops. 
 
 12 drops. 
 
 Fluid media. 
 
 Composition. 
 
 (o) "Weakly alkaline 
 meat infusion peptone 
 bouillon." 
 
 (b) Heated (i hr. at 
 55°) rabbit, dog, or 
 beef serum plus a 
 couple of drops of 
 whole blood. 
 
 Amount. 
 
 10 cc. 
 
 10 cc. 
 
 10 cc. 
 
 Period of incubation. 
 
 Several days. 
 
 (a) Up to 3-4 days 
 without shaking. 
 (6) 24hrs. 
 
 Results and conclusions. 
 
 All strains of S. longus pathogenes produced at first laking and a burgundy red color, but later (after sev- 
 eral days) the color changed to brownish red. Occasional shaking of the tubes or incubation of the cultures 
 in shallow layers in Erlenmeyer flasks hastened the appearance of the brownish color. Killed bouillon cul- 
 tures (60° for 1 hr.) or sterile filtrates caused no apparent change in blood added to them. 
 
 Spectroscopically the change from burgundy red to brownish red was found to be a change from oxyhemo- 
 globin to methemoglobin. 
 
 The author concludes that "Diese Umwandlung des Oxyhamoglobins in neutrales Methdmoglobin ist eine Folge 
 der physiologischen Funktionen der Streptokokken, sie ist gekniipft an den Lebensprozess derselben." 
 
 The author points out that the brownish red discoloration of blood bouillon described by Schottmiiller as 
 characteristic of S. mitior is produced also by 5. longus pathogenes, and therefore "Es kan deshalb von der Schott- 
 miiller angegebene Unterschied in der Funktion des Streptococcus longus und Streptococcus mitior nicht bestdligl 
 werden." 
 
 The author measured the amount of hemolysis by the height to which the layer of released hemoglobin rose 
 in the undisturbed tube of blood bouillon. 
 
 Strains pathogenic for animals were generally hemolytic. Hemolysis began in 6-8 hrs. after inoculation 
 of the medium, producing a burgundy red layer beginning at the bottom of the tube. After 3-4 days the 
 hemoglobin became brownish. 
 
 Dog blood was most easily hemolyzed; human and frog blood were least readily hemolyzed. 
 
 The addition of 2 per cent instead of 0.5 per cent sodium chloride to the bouillon inhibited hemolysis to 
 some extent. 
 
 The hemolytic activity of bouillon cultures was destroyed by heating for 30 min. at 55°C. 
 
 Hemolytic filtrates were obtained from cultures in medium {b). These filtrates were more weakly hemolytic 
 than the cultures and were inactivated in 15 min. at 55°C. 
 
 Blood bouillon heat- 
 ed at 100°C. ^ 
 
 Blood bouillon, un- 
 heated. 
 
 The method of procuring the hemolytic filtrates and of testing them was supposedly the same 
 as described by the author in 1903. 
 
 Beef or rabbit. 
 
 In "alkaline bouillon" (neutral to phenolphthalein) only the most hemolytic strains (type la on blood agar) 
 produced hemolysis. In "neutral bouillon" (2 per cent N acid to phenolphthalein) all aerobic strains pro- 
 duced complete hemolysis in 2-5 days, with burgundy red color, later turning to brown. 
 
 The anaerobic strains produced hemolysis with burgundy red discoloration but no brownish discoloration, 
 though they produced good amounts of acid. 
 
 Finding the brownish discoloration of blood to be produced by free acid and oxygen the author concludes 
 that, since acid production and hemolysis occur together in cultures in "neutral" bouillon, there is probably 
 a causal connection between them. Being unable to obtain hemolytic filtrates from neutral bouillon cultures, 
 however, he concludes that the acid is much more hemolytic in a nascent state. 
 
 Heated blood bouillon was unaltered by either streptococci or pneumococci. 
 
 Unheated blood bouillon was laked and turned burgundy red by the streptococci; under the influence of 
 the pneumococci the blood became at first dark red and then green (precipitate). 
 
 Spectroscopic examination showed the burgundy red color to be due to oxyhemoglobin, brown discoloration 
 due to methemoglobin, and the greenish precipitate was found to be an albuminous precipitate the color of 
 which was due not to pigment but to an optical efiiect. 
 
 Either kind of blood in bouillon was completely laked by the strams which were hemolytic on blood agar, 
 leaving a sediment of corpuscle shadows. 
 
 The strains which were non-hemolytic on blood agar produced no laking of blood in bouillon, the blood cor- 
 puscles settling on the bottom of the tube unaltered. 
 
 In addition to the results described in 1903 the author gives the following: 
 
 Streptolysin "is intimately associated with the globulins of the serum in which it is produced and has not yet been 
 separated from them. It can be separated quantitatively from the serum albumin hy_ saturation with magnesium 
 sulphate at 35°C. or by half-saturation with ammonium stdphate, and it seems not unlikely that it is a globulin." 
 
 "It cannot be extracted from the serum with alcohol, ether, or chloroform." 
 
 "Attempts to produce a specific antistreptolysin have not been successful." \ 
 
 The 43 strains from pathological processes are all recorded as being hemolytic in blood bouillon; aU others 
 were non-hemolytic. 
 
 Reference. 
 
 Rleke 1904. 
 
 Beitrage zur Frage der Arteinheit der Strepto- 
 kokken. 
 
 Kerner 1905. 
 
 Experimenteller Beitrag zur Hamolyse und 
 zur Agglutination der Streptokokken. 
 
 Natvlg . 1905. 
 
 Bakteriologische Verhaltnisse in weiblichen 
 Genitalsekreten. 
 
 Boxer 1906. 
 
 Ueber das Verhalten von Streptokokken und 
 Diplokokken auf Blutnahrboden. 
 
 Silberstrom 1906 
 
 Ueber die Arteinheit der Streptokokken. 
 
 Ruedlger 
 
 A Study of the Nature of Streptolysin. 
 
 1907. 
 
 Nleter 
 
 Zur Streptokokkenfrage. 
 
 1907. 
 
TABLE IV— Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 TABLE IV — Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 Organisms. 
 
 Many strains from vaginal 
 and uterine secretions, and 
 from various pathological proc- 
 esses. 
 
 Presumably strains 
 various sources. 
 
 from 
 
 15 strains of 5. longus hemo- 
 lylicus. 
 
 4 strains of S. hrevis non- 
 hemolyticus . 
 
 (Mostly from cases of puer- 
 peral sepsis.) 
 
 40 strains of hemolytic strep- 
 tococci from various himian 
 pathological conditions. 
 
 Blood. 
 
 Species. 
 
 Human. 
 
 Amount. 
 
 "Several 
 drops." 
 
 Fluid media- 
 
 Composition. 
 
 Amount. 
 
 Used blood bouillon according to the method of Kerner. 
 
 Human corpuscles, 
 
 washed. 
 
 1 cc. of 5 
 per cent sus- 
 pension. 
 
 Various amounts of bouillon 
 cultures were diluted to 1 cc. 
 with salt solution. 
 
 Period of incubation. 
 
 24 hrs. 
 
 2 hrs. at 37° and 
 20-22 hrs. in the re 
 frigerator. 
 
 Fresh normal rabbit serum was diluted 1 : 10 with standard bouillon, filtered, and heated 30 
 min. at 60°C. It was heavily inoculated with a fresh streptococcus culture and incubated 8-10 
 hrs. 
 
 The culture Was filtered through a Reichel or Chamberland filter and the hemolytic activity of 
 the filtrate tested by mixing 0.5 cc. of 5 per cent suspension of rabbit corpuscles with 1.5 cc. of 
 various dilutions of the filtrate. 
 
 Results and conclusions. 
 
 5. pathogenes was found to lake the blood and change the color to burgundy red, becoming brownish in 
 8-10 days. Corpuscle shadows and bacterial cells were found m the sediment. ,. , , , 
 
 5. mitior produced no laking but a brownish discoloration of the corpuscles. If these discolored corpuscles 
 
 were sedimented and placed m fresh bouillon inoculated with 5. pathogenes or hemolytic staphylococci, they 
 
 were readily laked, but not by S. mitior. . 
 
 S. saprophyticus darkened the blood in bouillon but did not lake it. _ 
 
 The author concludes that in blood bouillon cultures of 5. miliar all the corpuscles are m contact with the 
 
 streptococci and are therefore "immunized" against hemolysis by S. mihor, but, since they can still be hemo- 
 
 lyzed by 5. pathogenes or staphylococci the hemolysin of S. mitior must be different from that of S. pathogenes. 
 
 No special results were recorded and so they were presumably in agreement with those obtained on blood 
 agar. 
 
 "Impft man die Streptokokken in Bluthouillon, so sieht man nach 24 Stunden den unteren Teil der Bouillon 
 durchsichtig weinrot, wahrend in KontroUrohrchen {oder bei nicht hamolytischen Streptokokken) die Blutkorperchen 
 in der Kuppe des Reagenzrohrchens als kleiner, schwartzroter Bodensatz liegen und die Bouillon dariiber vollig unge- 
 farbt bleibt." 
 
 The wine-red color was found to be due to reduced hemoglobin (spectroscopic analysis) formed by the strepto- 
 cocci. Sterile blood bouillon controls remained blood-red. 
 
 The author states that it is important to note that (1) the bouillon must be isotonic with the blood, and 
 "Es ist * * zu achten, dass (3) wird die Sdnerimg d^r Bouillon verhindert (z. B. durch Zusatz pulverisierter 
 Kreide, Marmorstaub); sonst findet eine unler Umstanden ttorende Braunjarbung statt (Methdmoglobin)." 
 
 All strains of S. longus hemolyticus produced hemolysis in strongly alkaline bouillon, weakly alkaline bouil- 
 lon, calcium carbonate bouillon, sugar bouillon, and lactic acid bouillon. 
 
 The author concludes that hemolysis is not a function of the acid produced since hemolysis is not inhibited by 
 growth in media containing calcium carbonate and is not augmented by growth in dextrose bouillon nor by acidi- 
 fication of the bouillon (lactic acid). Since acid formation may arrest the growth of the culture, however, hemo- 
 lysis is to this extent dependent upon it. 
 
 No hemolysin was found in filtrates or centrifugates. The author concludes that hemolysis by streptococci is 
 dependent upon growth in presence of the blood and that heat or acid production inhibits hemolysis only by 
 killing the streptococci or preventing their growth. Schlesinger's " Hamolysoid" experiment could not be 
 confirmed. 
 
 (See also Table VHI on General Conclusions, etc.) 
 
 Streptococci which produce hemolysis on blood agar plates produce in rabbit serum bouillon in 8-10 hrs. an 
 abundant hemotoxin which is filterable. It is destroyed usually in 30 min. at 60°C. or in 6 hrs. at 37°. It is 
 very resistant to strong acid (HCl) and alkali. The results of anaerobic cultivation do not differ from those 
 of aerobic cultivation. 
 
 Acid formation and hemolysin production are not identical since a large amount of acid may be accompanied 
 by no hemolytic activity. 
 
 The filtrates of 10 hr. cultures of some streptococci are toxic for rabbits but not for mice or guinea pigs. The 
 toxin is not identical with the hemolysin. 
 
 The blood corpuscles from animals of different species have different susceptibilities towards the streptoly- 
 sin. Most susceptible are the corpuscles from those animals which are most susceptible to streptococcus infec- 
 tion (rabbit, mouse, man). Sheep and guinea pigs are least susceptible. 
 
 Normal rabbit, guinea pig, horse, and human sera contain antilysin. The immunization of rabbits by the in- 
 jection of streptolysin has resulted in no increase in the antilysin in their sera. 
 
 The hemolytic activity was much reduced in blood bouillon containing sugar. 
 
 (See Table VII on Mutations and Variability.) (See also Table III on The Use of Blood Agar.) 
 
 Reference. 
 
 Mandelbaum 
 
 Zur Streptokokkenfrage. 
 
 1907-08. 
 
 Liidke and Polano 
 
 Uebei: Hamotyse der Streptokokken. 
 
 1909. 
 
 Zangemeistep 
 
 Die Hamolyse der Streptokokken. 
 
 1909. 
 
 Sachs 
 
 tjber Streptokokken-Hamolyse. 
 
 1909. 
 
 Braun 
 
 Ueber das Streptolysin. 
 
 1912. 
 
 Kuhn 1912. 
 
 Einfluss von Zucker auf Hamolyse und Viru- 
 lenz. 
 
TABLE IV — Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 The Use of 
 
 TABLE IV — Continued. 
 Blood Bouillon and Other Fluid Media. 
 
 Organisms. 
 
 =t= 
 
 Blood. 
 
 Fluid medial 
 
 26 strains from bovine mas- 
 tit'^- ■ r • ^l, 
 10 strains from various other 
 
 sources. 
 
 Species. 
 
 Beef or goat, defibri- 
 nated. 
 
 Amount. 
 
 Composition. 
 
 Amount. 
 
 8 drops. 
 
 Determined the "M. if. £>._" 
 of several strains of hemolytic 
 streptococci and selected one 
 for further experiment. 
 
 ilost experiments were made 
 with 2 "strongly hemolytic 
 strains," though others were 
 tested also. 
 
 Ox blood corpuscles, 
 washed. 
 
 1 cc. of 5 
 per cent sus- 
 pension. 
 
 Ordinary weakly aj 10 cc. 
 kaline bouillon, prJ 
 sumably meat infusic' 
 bouillon. 
 
 To 1 cc. portions the cor- 
 puscle suspension -Ire added 
 graduated amounts i culture 
 in distinctly alkaline a litmus) 
 bouillon plus 15-2( per cent 
 of horse serum (prev isly heat- 
 ed to 56° for 1 hr.). 
 
 Period of incubation. 
 
 12-24 hrs. 
 
 Results and conclusions. 
 
 2 hrs. at 37°C. 
 
 The best medium for hemolysin production was found to Ifetandard bouillon (weakly alkahne 
 to litmus) plus 40-50 per cent of horse serum (inactivated 56° for 30 mm.). Next best was 
 bouillon plus 33 per cent of inactivated ascitic fluid. Less eitent was bouillon plus 10 per cent 
 of inactivated rabbit serum. Standard bouillon alone procjed very httle hemolysin. Incon- 
 stant results were obtained with 1 per cent dextrose bouillon. . , , j 
 
 The hemolytic activity was tested by adding 8 cc. of l-/ler cent suspension of washed cor- 
 puscles to 2 cc. of culture or filtrate in various dilutions, '(e mixtures were mcubated 2 hrs. at 
 37° and refrigerated over night. Horse blood corpuscles weref5ed for most experiments, though 
 those of many other animals were also tried. 
 
 The methods were the same as those employed by the authin 1912. 
 
 164 strains of various organ- 
 isms (cholera, other vibrios, 
 typhoid, anthrax, proteus, pyo- 
 cyaneus, etc.) including 27 
 strains of streptococci and 20 
 staphylococci. 
 
 Sheep corpuscles, 
 presumably washed. 
 
 18 throat strains. 
 
 1 strain from cervical adeni- 
 
 tis. 
 
 9 strains from milk slime. 
 
 Sheep and human 
 corpuscles, washed. 
 
 5 cc. of 5 
 per cent sus- 
 pension. 
 
 1 cc. of 5 
 per cent sus- 
 pension. 
 
 The corpuscle suaision was 
 mixed with 1 cc. of It solution 
 containing \ loop oftlture ma- 
 terial. 
 
 Added to the coiscle sus- 
 pension 0.5 cc. of Ur. ascitic 
 broth culture. 
 
 48 hrs. at 37°C. 
 
 2 hrs. at 37° and 
 over night in the re- 
 frigerator. 
 
 (1) Strongly hemolytic strains rendered the bouUion almost clear and burgundy red. 
 
 (2) Weakly hemolytic strains left a brownish red sediment of corpuscles covered by a clear burgundy red 
 layer with colorless bouillon above. j- i ,• c ^i. 
 
 (3) Non-hemolytic strains left a sediment of brownish or reddish corpuscles with no discoloration ot the 
 supernatant bouillon. 
 
 Measured hemolytic activity by determining the "minimum hemolytic dose" {"M. H.D. ); t.e., the smallest 
 amount of culture or filtrate which produced complete hemolysis of 1 cc. of 5 per cent suspension of washed 
 ox blood corpuscles in 2 hrs. at 37°C. The "hemolytic index" is defined as the reciprocal of the M H. D.; 
 i.e., the number of cubic centimeters of 5 per cent corpuscle suspension which would be completely hemo- 
 Ivzed by 1 cc. of culture in 2 hrs. at 37°C. . \. ■ >. 
 ' The author concludes that virulence and hemolytic power are closely associated amongst pathogenic strep- 
 Filtrates were obtained which had about one-third the hemolj-tic activity of the cultures. The hemolysin 
 entered into combination with the corpuscles in 2 hrs. at 0°C. but did not produce hemolysis at this tempera- 
 ture. It was inactivated in 30 min. at 55°C. Schlesinger's "lUmolysoid" experiment could not be verifaed. 
 The normal sera of man, horse, rabbit, and guinea pig were found to be antihemolytic to a certain extent, 
 but no immune antihemolysin was produced by the injection of rabbits with hemolytic filtrates. 
 
 The author us°d cultures for most of his experiments, though hemolytic filtrates also were obtained. 
 
 The hemolytic activity of cultures reached a maximum in 7-18 hrs., more rapidly m aerobic than m anaerobic 
 cultures. 
 
 The hemolysin could be extracted by ether. , -, ■ ^^r^ Ar^ t^ t 
 
 A hemolytic filtrate was inactivated in 6 min. at 75°C., 30 min. at 67°, 70 mm. at 60 , and in 20-40 hrs. at 
 37°. The hemolytic ether extract resisted 100°C. for 10 min. . . „ ., , ^ ■ . 
 
 The addition of 5 per cent of peptone or 30 per cent inactivated serum or ascitic fluid to the test mixture 
 (hemolytic culture and corpuscles) resulted in a considerable (sometimes 300 per cent) increase in hemolytic ac- 
 tivity. The author adopts the explanation of Walbum (working with staphylolysm) that a prolysm is lomied 
 by the bacterial cell and is activated (forming hemolysin) by something m the peptone, serum, or ascitic fluid 
 added to the medium. . , , v • 
 
 Antistreptolysin was not detected in normal human, horse, beef, sheep, goat, dog, pig, rabbit, guinea pig, or 
 pigeon serum. The author was unable to produce an immune antistreptolysin by injection of the goat or gui- 
 nea pig. _^^^ 
 
 (See Table VIII on General Conclusions, etc.) 
 
 In addition to "Hamopepsie" and "HdmogloUnopepsie" observed in blood agar or blood gelatin only, the 
 author describes "true hemolysis;" i.e., the release of unaltered hemoglobin from the corpuscles, the stromata 
 remaining intact, a phenomenon observed in fluid media only. • , -n i. 
 
 The author states that he employed bacterial suspensions in salt solution rather than in bouillon because 
 different lots of bouillon were apt to vary and because some bouillons were in themr elves hemo/.tic. 
 
 (See also Table III on The Use of Blood Agar.) 
 
 --/'"-■ 
 
 action iha '-t ',hat obtained 
 
 " This method is considered by the authors as yielding more definite evidence of hei: 
 by observing the colonies on blood agar." • t* u 
 
 Only 1 strain, that from a cervical gland, produced hemolysis in the washed corpuscle suspension, it ^be- 
 longed to Group C (see Table V on Fermentation Reactions). 3 other strains of Group C produced no 
 color" in the corpuscle suspension; all the other strains produced "methemoglobm brown. No differences are 
 mentioned in the results obtained by the use of the 2 kincis of blood corpuscles used in these suspensions. 
 
 Reference. 
 
 Gmlnder 1912. 
 
 Untersuchungen liber Mastitis streptokokken 
 und ihre Differenzierung von saprophytischen 
 Streptokokken. 
 
 M'Leod 1912. 
 
 On the Hemolysin Produced by Pathogenic 
 Streptococci, and on the Existence of Antihemo- 
 lysin in the Sera of Normal and Immunized 
 Animals. 
 
 von Hellens 
 
 Untersuchungen iiber Streptolysin. 
 
 1913. 
 
 M'Leod 1914. 
 
 Criteria of Virulence amongst Streptococci, 
 with Some Remarks on Streptococcal Leucoci- 
 din. 
 
 Baerthleln . 1914. 
 
 Ueber Blutveranderung durch Bakterien, 
 
 North, White, and Avery 1914. 
 
 A Septic Sore Throat Epidemic in Cortland 
 and Homer, N. Y. 
 
TABLE IV — Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 TABLE VJ —Continued. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 Organisms. 
 
 263 strains from various 
 sources, mostly pathological 
 conditions. 
 
 Blood. 
 
 Species. 
 
 Sheep 
 washed. 
 
 corpuscles, 
 
 Amount. 
 
 Evidently a number of 
 strains of pneumococci; no de- 
 tails regarding them. 
 
 cc. of 5 
 cent sus- 
 pension in salt 
 solution. 
 
 1 
 
 per 
 
 Fluid medj 
 
 r 
 
 Composition. 
 
 Amount. 
 
 Added to the cffpuscle sus- 
 pension "usually" 15 cc. of 18 
 hr. calcium carbmate ascitic 
 broth culture (ascitic iiuid 1 
 part, peptone bcuillon, -|- 3, 
 5 parts). 
 
 "Solutions of the bodies of pneumococci obtained by dissoi, tg them in 
 dilute solutions of sodium chelate, by permitting them to unde, i autolysis, 
 or by freezing, drying, and then grinding in salt solution, are 6 ively hemo- 
 lytic for rabbit, sheep, guinea pig, and human red blood corpi' ies." 
 
 "Toxins, as above described, * * , usually produce ac e death in 
 guinea pigs in doses of three to four cubic centimeters, and molysis of 
 0.5 of a cubic centimeter of sheep corpuscles in doses of 0.0 'of a cubic 
 centimeter or less, the whole mixture of toxin and corpuscles be \g made up 
 to 2.5 cubic centimeters with salt solution." 
 
 Period of incubation. 
 
 1 hr. at 37.5°C. 
 
 From 5 min. 
 hrs. at 37°C. 
 
 to 3 
 
 Results and conclusions. 
 
 The author describes three distinct reactions with the blood corpuscles: (1) "Hemolytic," a complete lakmg 
 of the corpuscles; (2) "Melhemoglobin producers," producing a change in color of the unlaked corpuscles from 
 bright red to dark brown due to the transformation of oxyhemoglobin to methemoglobin; (3) Indifferent, no 
 change in the corpuscles. . ,. . , „ ^, ^ n • -u 
 
 On the basis of the above reactions and the fermentation of muhn, salicm, anti raffinose the foUowmg scheme 
 
 of classification is proposed: 
 
 -| ■ Inulin — 
 
 Methemoglobin 
 
 Bile-soluble 
 Salicin -f- 
 Raffinose 4- 
 
 Pneumococcus 
 and Pneumo- 
 coccus mucosus 
 
 Hemolysis 
 
 I 
 Salicm -|- 
 
 5. pyogenes 
 
 Methemoglobin 
 Raffinose -1- 
 
 Salicin -f- 
 
 Salicin — 
 
 I 
 Indifferent 
 
 I 
 Salicin + 
 Raffinose ± 
 
 S. fecalis and 
 other strep- 
 tococci 
 
 Streptococcus viri- 
 dans and variants 
 
 Cultures in 2 per cent peptone bouillon were more hemolytic than those in 1 per cent peptone bouillon. 
 The hemolytic activity was more or less inhibited by 1 per cent fermentable sugar or by over 2 per cent of 
 
 salt 
 
 Centrifugates were less hemolytic than whole cultures. Hemolytic filtrates could not be obtained through 
 
 a Berkefeld N filter. 
 The hemolytic activity of cultures was destroyed in 30 min. at 56 C. 
 Normal sera of man, guinea pig, rabbit, sheep, and ox were more or less antihemolytic. 
 
 "While the power to produce hemolysis in culture medium is not possessed by pneumococci, or, if so, to a very 
 slight degree, observations which we have made indicate that the bodies of pneumococci contain a substance or sub- 
 stances, which when set free are actively hemolytic, and that the serum of animals immunized to the bacterta or to 
 the bacterial substance has increased power of neutralizing this lytic poison." .... 
 
 "The substance on which this hemolytic property depends is very labile, much of its activity is lost on passing 
 through a filter, and it is destroyed by the action of trypsin." , . , , • r j j,; j 
 
 "It may therefore be concluded that the bodies of pneumococci contain a toxin that ts hemolytic J or red blood cor- 
 puscles. This substance is not simply a product of autolysis but undoubtedly exists preformed in the bacterial 
 cell. However, it is not given up to the surrounding fluid as long as the bodies of the bacteria are intact. It may 
 therefore be considered a hemolytic endotoxin." 
 
 Reference. 
 
 Lyall 1914. 
 
 On the Classification of the Streptococci. 
 Observations on Hemolysin Production by the 
 Streptococci. 
 
 Cole 
 
 Pneumococcus Hemotoxin. 
 
 1914. 
 
TABLE IV — Concluded. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 TABLE IV — Concluded. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 Organisms. 
 
 Blood. 
 
 Fluid media. 
 
 Period of incubation. 
 
 Results and conclusions. 
 
 Reference. 
 
 
 Kind. Amount. 
 
 Composition. Amount. 
 
 
 Evidently a number of 
 strains of pneumococci; no de- 
 tails regarding them. 
 
 To 1 cc. of various dilutions of the bouillon culture, bacterial emul- 
 sion, sterile bouillon, filtrate, or other material to be tested was added 1 
 cc. of salt solution (plain or containing sugars, etc.) and 0.5 cc. of sheep 
 blood corpuscle suspension or hemoglobin solution. 
 
 From a few min. to 
 24 hrs. at 37°C. 
 
 "When pneumococci are grown in media containing blood or hemoglobin, the red color of the latter is changed 
 to a greenish brown, * * " (on blood agar or in blood bouillon). "This change is undoubtedly due to the 
 formation of methemoglobin." 
 
 " This reaction occurs only when the pneumococci are living; it is not induced by the culture fluid or by extracts 
 of the bacteria." 
 
 "Boiling a broth culture, or even heating it to 56''C. for one half hour, before addition of blood prevents the reac- 
 tion from occurring." 
 
 " The reaction does not occur when hemoglobin is added to an emulsion of washed pneumococci in sail solution. 
 However, if minute traces of dextrose be added to such a mixture, the reaction quickly occurs. The dextrose may 
 be replaced by any one of a number of other sugars, and also by certain other organic substances (peptone, egg albu- 
 min), if the latter are added in large amounts." 
 
 " The reaction does not occur in the absence of oxygen." 
 
 "From the work of others it is probable that the formation of methemoglobin is always a reaction of ^ oxidation. 
 In the formation of methemoglobin by reducing agents, the latter are first oxidized, this occurring better in the pres- 
 ence of oxyhemoglobin." 
 
 "According to this interpretation the formation of methemoglobin by pneumococci occurs as a result of reduction 
 and oxidative processes occurring in the neighborhood of the bacteria * * , and without producing substances 
 capable of isolation." 
 
 Cole 1914. 
 The Production of Methemoglobin by Pneu- 
 mococci. 
 
 29 strains of streptococci 
 from various human infections 
 (endocarditis, tonsillitis, alveo- 
 lar abscess, scarlet fever, rheu- 
 matism, pneumonia, pyemia, 
 diphtheria). 
 
 Graduated amounts of 12 hr. ascitic bouillon cultures were made up 
 to 1 cc. with salt solution and then mixed with 1 cc. of washed sheep 
 corpuscles. 
 
 " The smallest amount of culture which caused complete transformation 
 of oxyhemoglobin to methemoglohin was taken as a measure of the activity 
 of a given strain and may be spoken of as the methemoglobin litre of that 
 . strain." 
 
 Up to 2 hrs. at 
 
 37°C. 
 
 This paper is practically a repetition of Cole's paper on "The Production of Methemoglobin by Pneumo- 
 cocci," except that strains of S. viridans are used instead of pneumococci. The same results as those of Cole 
 are obtained throughout. In addition, however, the following observation is significant: " * there seems to 
 be no relationship between the carbohydrate cleavage power of a strain of streptococcus and its ability^ to form methemo- 
 globin in the presence of various carbohydrates. Of six strains which formed methemoglobin in the presence of 
 inulin, three fermented the sugar, three did not; and of five strains causing methemoglobin formation in the presence 
 of mannite, only one fermented that substance." 
 
 "Since methemoglobin may be formed by streptococci in the presence of sugars which the organisms do not fer- 
 ment, it seems improbable that the reaction depends upon intermediate or end products formed by the action of the 
 streptococci on the nutrient substance present" {e.g., carbon dioxide or acids). 
 
 Blake 1916. 
 The Formation of Methemoglobin by Strep- 
 tococcus viridans. 
 
 51 strains of "Streptococcus 
 hemolysans." 
 
 91 strains of "Streptococcus 
 viridans." 
 
 (All from pathological 
 sources.) 
 
 " * * about one hundred strains were tested by adding one cubic centimeter of eighteen hour ascitic- 
 broth cultures to one cubic centimeter of a five per cent suspension of washed sheep corpuscles in 
 small test-tubes and incubating for two hours at 37° C." 
 
 "Under these conditions (with 5. viridans) methemoglobin formation has always occurred, and no trace of hemo- 
 lysis has been demonstrable. On the other hand, hemolytic streptococci under the same conditions have consistently 
 caused complete hemolysis without methemoglobin formation in from ten to thirty minutes. 
 
 Blake 1917. 
 
 The Classification of Streptococci. 
 
TABLE V. 
 Fermentation Reactions. 
 
 TABLE V. 
 
 Fermentation Reactions. 
 
 Organisms. 
 
 Strains of streptococci 
 isolated from the milk of 
 cows with chronic mastitis. 
 
 28 strains of streptococci. 
 9 strains of pneumococci. 
 
 21 strains; 19 of them 
 from vulval, vaginal, and 
 uterine secretions of 10 
 women during and 4-9 days 
 after labor; 1 from a felon; 
 1 from angina. 3 of the 
 strains from the women were 
 anaerobic. 
 
 300 strains from normal 
 sputum. 
 
 300 strains from normal 
 human stools. 
 
 20 strains from various 
 lesions in man. 
 
 2 strains from outside 
 sources. 
 
 Tabulation of 701 strains 
 including those of Gordon 
 and Houston (from horse 
 dung, air, human feces, sa- 
 liva, throats, various lesions, 
 etc.) 
 
 Media. 
 
 Composition. 
 
 Test substances. 
 
 Meat infusion bouillon, 
 with and without peptone, 
 salt, or calcium carbonate. 
 
 Serum water medium, (1 
 pt. beef serum; 2 pts. wa- 
 ter; + 1 per cent of test 
 substance), sterilized at 
 100°C. for 10 min. 3 suc- 
 cessive days. 
 
 Standard meat infusion 
 bouillon. 
 
 "Ordinary beef broth f reed 
 from sugar by cultivating B. 
 coli therein for 3 days at 
 37°C. and then steritized, 
 jittered, rendered slightly alka- 
 line and tinted with litmus." 
 
 Gordon's sugar-free broth 
 or Liebig extract broth (?). 
 
 124 strains of 5. pyogenes 
 from various lesions. 
 
 65 strains (43 from various 
 human pathological proc- 
 esses; remainder from milk, 
 sputum, skin, and feces). 
 
 Dextrose, lactose, sac- 
 charose, mannite, glycerol. 
 2-5 per cent. 
 
 Dextrose, levulose, galac- 
 tose, maltose, saccharose, 
 lactose, dextrin, starch, gly- 
 cogen, and inulin. 
 
 Dextrose (sterilized in the 
 autoclave after being added 
 to the bouillon) . 
 
 1 per cent of saccharose, 
 lactose, raffinose, inulin, sali- 
 cin, coniferin, or mannite. 
 
 Milk. 
 
 Saccharose, lactose, raffi- 
 nose, inulin, salicin, coni- 
 ferin, or mannite. 
 
 Milk. 
 
 Modified Hiss serum wa- 
 ter medium, containing 2 
 per cent peptone, 2 per cent 
 carbohydrate, and 25 per 
 cent beef serum or ascitic 
 fluid. 
 
 Sterilized fractionally before adding serum. Serum 
 heated 30 min. at 6S-70°C. and then filtered. 
 
 Dextrin, inulin, lactose, 
 saccharose, maltose, raffi- 
 nose, rhamnose, salicin, 
 mannite, and dulcite. 
 
 Indicator or titration. 
 
 Period o{ incubation. 
 
 Fermentation of 
 the substance is in- 
 dicated by coagu- 
 lation of the serum. 
 
 Titration (phe- 
 nolphthalein). 
 
 Litmus. 
 
 Litmus. 
 
 Bouillon (no details) -|- 1 
 per cent test substance. 
 
 Used also dextrose- and 
 lactose-Barsiekow media. 
 
 Dextrose, levulose, mal- 
 tose, lactose, saccharose, 
 raffinose, and salicin. 
 
 Milk. 
 
 No mention of 
 acid production or 
 any means of de- 
 tecting it except in 
 case of litmus dex- 
 trose bouillon, lit- 
 mus lactose bouil- 
 lon, and the Bat- 
 siekow media. 
 
 3-8 days. 
 
 3 days. 
 
 Results and conclusions. 
 
 In calcium carbonate bouillon cultures the sugars added were rapidly consumed. Saccharose, like dex- 
 trose, lactose, and mannite, was rapidly fermented but at no time during the fermentation of saccharose could 
 a positive reaction with Fehling's solution be obtained. j j j u 
 
 Noting that cultures in sugar broth died out rather quickly whereas if calcium carbonate was added they 
 remained viable often for several months, the authors conclude that the death of the cultures is brought about 
 by the acids formed. 
 
 Streptococci ferment dextrose, levulose, and galactose readily; saccharose, lactose, and maltose less readily; 
 dextrin, starch, and glycogen after several days or not at a.11; and inulin not at all. 
 Pneumococci fermented all the substances including inulin. 
 
 In standard bouillon (without dextrose) the acid production of different strains varied within quite narrow 
 
 limits. - 4.1, c 1 
 
 In dextrose bouillon the acid production appeared to be limited by the acid tolerance of the strains, tiie bnal 
 
 titrations varying from 3.3-5.9 per cent n acid. . . ^ , j ..i, • 
 
 The author states that acid production begins very early and reaches a maximum^ m 2 days, and that m 
 
 dextrose bouillon the bacteria die in 1-3 days after the maximum acidity has been attained. 
 
 Reference. 
 
 Noeard and Mollereau 
 
 Sur une mammite 
 laitieres. 
 
 contagieuse 
 
 1887. 
 des vaches 
 
 Hiss 1902. 
 
 A Contribution to the Physiological Differen- 
 tiation of Pneumococcus and Streptococcus, 
 and to Methods of Staining Capsules. 
 
 Natvig 1905. 
 
 Bakteriologische Verhaltnisse in weiblichen 
 Genitalsekreten. 
 
 Tabulated the strains with regard to source and to their reaction towards the 9 test substances (includ- 
 ing also the reduction of neutral red in bouillon anaerobically) . 
 
 Believes these tests afford a definite basis on which to classify the streptococci, and hopes it may be use- 
 ful in connection with serum therapy by enabling one to choose a suitable antistreptococcus serum for the 
 case in hand. 
 
 Of the 701 strains tabulated about 200 represent the "Type Forms" characterized in the foUowing table; 
 all the other strains were "Variants" with regard to action on one or more of the test substances. 
 (For table of " Type Forms" see Table VHl oh General Conclusions, etc.) 
 
 "All strains fermented dextrin, lactose, maltose, saccharose, and salicin, bu' none fermented inulin, raffinose, 
 rhamnose, and dulcit." 63 strains fermented mannite; 61 did not. The action on mannite had no relation to 
 the type of infection but all strains isolated from the same individual behaved alike towards mannite. 
 
 " Von meinen samtli hen Stammm, welche ich in Gdrungsrohrch n mi diesen verschiedenen Zuctierarten zusam- 
 men b achte, seigte nicht ein einziger auch nur eine Spur von Garung." _ 
 
 All strains produced add (red) in litmus dextrose bouillon and dextrose Barsiekow medium, but showed 
 negative or late positivs reactions in litmus lactose bouillon or lactose Barsiekow medium. 
 
 Concludes that he has been unable to confirm Gordon's method of differentiation {i.e., "Garungsvermogen 
 in Bouillon zu 1 Proaent mit Saccharose, Laktose, etc." . 
 
 Gordon 1905. 
 
 A Ready Method of Differentiating Strepto- 
 cocci and Some Results Already Obtained by 
 its Application. 
 
 Andrewes and Border 1906. 
 
 A Study of the Streptococci Pathogenic for 
 Man. 
 
 Ruedlger 1906 
 
 The Streptococci from Scarlatinal and Nor- 
 mal Throats and from Other Sources. 
 
 Nieter 
 
 Zur Streptokokkenfrage. 
 
 1907. 
 
TABLE V — Continued. 
 Fermentation Reactions. 
 
 TABLE V — Continued. 
 Fermentation Reactions. 
 
 Organisms. 
 
 s of streptococci 
 uus pathological 
 
 "l strain from milk, 
 go strains of pneumococci. 
 8 strains of S. mucosus. 
 
 38 strains of S. pyogenes-. 
 10 strains of S. mucosus. 
 13 strains of pneumococ- 
 
 cus. 
 
 strains of 5. saprophyti- 
 cus. 
 
 16 strains of "sonstige 
 Streptokokken," mostly S. 
 mitior evidently. 
 
 "Modified pneumococci" 
 from cases of endocarditis. 
 
 15 strains of S. longus he- 
 moiyticus. 
 
 4 strains of S. brevis non- 
 hemolyticus . 
 
 (Most strains from cases 
 of puerperal sepsis.) 
 
 302 strains of streptococci 
 from 49 samples of fresh hu- 
 man, bovine, and equine 
 feces. 
 
 Media- 
 
 Composition. 
 
 (a) Sugar-free broth of 
 the following reaction 0.8 
 per cent acid, 0.5 per cent 
 acid, neutral, 0.5 per cent 
 alkaline. 
 
 (6) Sugar-free broth (0.5 
 per cent acid) -\- | volume 
 of ascitic fluid. 
 
 (c) Beef serum and water. 
 
 (No further details.) 
 
 To the above media added 
 1 per cent of test substances. 
 
 Sterilized fractionally, 3 
 days. 
 
 Litmus ascites agar + 1 
 per cent of test substance, 
 streaked with culture. 
 
 A few strains were also 
 tested in bouillon -|- 1 per 
 cent glycerol, dextrose, 
 starch, mannite, and raffi- 
 nose. 
 
 1 per cent dextrose bouil- 
 lon; reaction = 1 per cent 
 N acid to litmus. 
 
 Liebig extract, Witte 
 peptone, bouillon -{- 1 per 
 cent of test substance. 
 
 Test substances. 
 
 Dextrose, levulose, galac- 
 tose, arabinose, rhamnose, 
 saccharose, lactose, dextrin, 
 inulin, mannite, dulcite. 
 
 Dextrose, levulose, galac- 
 tose, arabinose, mannose, 
 maltose, saccharose, lactose, 
 raffinose, glycerol, mannite, 
 isodulcite, dulcite, adonite, 
 erythrite, dextrin, starch, 
 inulin. 
 
 (Sterilized in 10 per cent 
 solutions in water and then 
 added to agar or bouillon.) 
 
 Dextrose only. 
 
 Dextrose, lactose, raffi- 
 nose, mannite. 
 
 Indicatoi or titration. 
 
 Litmus. 
 
 Litmus in agar. 
 
 Bouillon cultures 
 titrated (phenol- 
 phthalein). 
 
 Titrated against 
 0.01 N acid or alka- 
 li using litmus 
 paper as indicator. 
 
 Titrated cold 
 (phenolphthalein) . 
 
 Period of incubation. 
 
 4 days. 
 
 Agar observed 3 
 days or longer. 
 
 Bouillon cultures 
 incubated 40 hrs. 
 
 Various. 
 
 72 hrs. 
 
 Results and corclusions. 
 
 Sugar-free broth (0.5 per cent acid) plus \ volume of asitic fluid was found to be the most suitable medium 
 for fermentation tests. "Beef semm water {Hanna, 1898; Hiss, 1904) although a good culture medium, was found 
 unreliable. The results in it were variable * * ." The other media tried also gave less satisfactory results. 
 In broth without ascitic fluid a number of strains failed to attack mannite, lactose, saccharose, or dextrin. 
 
 Of the 34 strains of streptococci (not including S. mucosus) all fermented dextrose,^ levulose, galactose, mal- 
 tose, and dextrin, but varied with respect to saccharose, lactose, mannite, and inulin, the largest group (19 
 strains) fermenting saccharose and lactose but not mannite and inulin. 
 
 "The serum (ascitic) media are even more important for ihe pneumococcus than for streptococci." 
 
 15 strains of pneumococcus and 8 strains of S. mucosus gave identicalfermentation reactions, as follows: 
 (-[-) dextrose, levulose, galactose, maltose, saccharose, lactose, inulin, and dextrin. 
 (— ) mannite, dulcite, arabinose, and rhamnose. 
 
 Of 65 strains of pneumococcus tested with inulin only, "it was found that a riumber of them failed to produce 
 acid in certain generations. All the organisms, however, fermented inulin at some time or other of their life history." 
 
 "Chemical tests of this kind should be made only in the media which are most favorable for the growth of the 
 organisms." 
 
 The authors accept the capsule stain described by one of them (Buerger, 1907) as diagnostic for the dis- 
 tinguishing of pneumococci from streptococci. 
 
 " The fermentation of inulin is of limited value in the differential diagnosis between pneumococci and strepto- 
 cocci. Pneumococci may lose the power to produce acid in this carbohydrate either temporarily or permanently." 
 
 " The precipitation of glucose serum agar although more frequently a property of streptococci, may also be produced 
 by the action of pneumococci." 
 
 Glycerol, starch, mannite, raffinose, and arabinose in agar were found to be of differential value. 
 
 Members of the S. pyogenes group fermented starch almost always, and arabinose rarely. They varied 
 with respect to the other substances. Starch was fermented by none of the other groups. 
 
 Pneumoco(f i and many strains in the S. mucosus group fermented none of the substances in agar. 
 
 The S. saprophyticus group had the same fermentation reactions as the S. pyogenes group. 
 
 There was nothing characteristic about the fermentation reactions of the group of " Sonnstige Streptokokken," 
 evidently a miscellaneous group. 
 
 In carbohydrate bouillon some of the pneumococci did produce acid from, dextrose, starch, raffinose, and 
 in one or two cases from mannite and glycerol. Most of those tested and also the S. mucosus were capable 
 of producing alkali when the test substances were not fermented. In no case did S. pyogenes produce any 
 alkali. 
 
 "Most of the strains soon after isolation fermented inulin to a mild degree, a property which all soon lost." 
 
 Considers only the question of the relation of acid production to hemolysis. 
 (See Table VIII on General Conclusions, etc.) 
 
 " There are three points of difference which seem to deserve investigation. First, the presence of streptococci 
 forming over 3.5 per cent of acid in dextrose broth would seem in general to be characteristic of human stools. Second, 
 raffinose-fermenting forms (S. salivarius) appear to be more abundant in bovine than in human feces. Third, and of 
 most importance, mannit-fermenling streptococci (S. fecalis) , which make up about one-quarter of the human strep- 
 tococci, are very rare in the feces of the horse and cow." .According to the authors' tables most equine strains 
 attack dextrose only. 
 
 In material polluted with streptococci, therefore, the number of lactose and raffinose fermenters should help 
 to distinguish between human or bovine strains and equine strains; the number of mannite fermenters should 
 help to distinguish between human strains and equine or bovine strains. 
 
 Reference. 
 
 Buerger 1907. 
 
 The Differentiation of StreptocoGci by Means 
 of Fermentative Tests. 
 
 Buerger and Ryttenberg 1907. 
 
 Observations upon Certain Properties Ac- 
 quired by the Pneumococcus in the Human 
 Body. 
 
 Salomon 1908. 
 
 Zur UnterscheifiuDg der Streptokokken durch 
 kohlenhydrathaltige Nahrboden. 
 
 Rosenow 1909. 
 
 Immunological and Experimental Studies on 
 Pneumococcus and Staphylococcus Endocarditis. 
 
 Sachs 
 
 Uber Streptokokken-Hamolyse. 
 
 1909. 
 
 Winslow and Palmer 1910. 
 
 A Comparative Study of Intestinal Strepto- 
 cocci from the Horse, the Cow, and Man. 
 
TABLE V — Continued. 
 Fermentation Reactions. 
 
 TABLE V' — Continued. 
 Fermentation Reactions. 
 
 Organisms. 
 
 S. lacticus from 75 per cent 
 of 81 milk samples and from 
 co'.v dung and skin. 
 
 5. pyogenes found in 2 
 samples, 1 from cow with 
 mastitis, the other from mar- 
 ket milk. 
 
 8 strains of 
 pnemnococci" from cases of 
 endocarditis. 
 
 3 strains of pneumococci 
 from pneumonia. 
 
 1 S. viridans. 
 
 1 S, pyogenes. 
 
 1 M. rheumaticiis. 
 
 42 strains, mostly^ from 
 mouth, throat, and joints of 
 cases of rheumatism, a few 
 from the heart blood and lo- 
 cal lesions. 
 
 8 strains of streptococci 
 isolated from human sub- 
 jects. 
 
 1 strain from horse dung. 
 
 Media. 
 
 Composition. 
 
 Bouillon. 
 Gelatin. 
 
 Houston's medium (dis- 
 tilled water containing 1 
 per cent lemco, 1 per cent 
 peptone, 0.1 per cent so- 
 dium bicarbonate and lit- 
 mus). 
 
 10 strains of S. acidi lac- 
 lici from human and bovine 
 milk. 
 
 6 strains of 5. pyogenes 
 from various suppurations. 
 
 5 strains of pneumococci 
 from sputum. 
 
 26 strains of streptococci 
 from bovine mastitis. 
 
 10 strains from other 
 sources. 
 
 Gordon's sugar-free broth 
 was used at first, but 
 later and for most experi- 
 ments used media prepared 
 from lemco. 
 
 Meat extract bouillon. 
 
 (1) "BouiUon" (not stated 
 whether sugar-free or not). 
 
 (2) Barsiekow media. 
 
 Test substances. 
 
 Mannite, lactose, levulose, 
 inulin, salicin, 1 per cent. 
 
 Saccharose, lactose, raffi- 
 nose, inulin, salicin, man- 
 nite. 
 
 Milk. 
 
 Lactose, saccharose, man- 
 nite, salicin, raf&nose, inu- 
 lin. 
 
 Milk. 
 
 Salicin, lactose, saccha- 
 rose, rafifinose, mannite, inu- 
 lin. 
 
 Milk. 
 
 Dextrose, lactose, man- 
 nite, inulin (a few with raf- 
 iinose and arabinose). 
 
 Dextrose, lactose, man- 
 nite. 
 Milk. 
 
 Indicator or titration. 
 
 Litmus. 
 
 Litmus, proba- 
 bly. 
 
 Litmus added to 
 cultures; also ti- 
 trated. 
 
 Period of incubation. 
 
 Litmus. 
 
 Results and cOfClusions. 
 
 The author states that he was wiable to differentiate 5. equi from other streptococci by means of sugar 
 bouillon. 
 
 Milk was never coagulated by S. equi. 
 
 No gas was formed from any of the test substances in bouillon or in gelatin. 
 
 Acid production is not mentioned by the author. 
 
 S. lacticus produces a large amount of acid and coagulates milk in 24 hrs., even in the presence of calcium 
 carbonate. 
 (See also Table VIII on General Conclusions, etc.) 
 
 "Pneumococci from the sputum in pneumonia ferment inulin more strongly than those isolated from the blood, 
 while pneumococci from cases of endocarditis ferment this carbohydrate still less and sometimes not at all" 
 
 "The author's tables show that the fermentation reactions of the various strains of endocarditis cocci and 
 pneumococci differed among themselves with regard to most of the test substances. 
 
 Strains classified according to Andrewes and Horder: 
 
 3 S. pyogenes and its variants. 23 5. salivarius and its variants. 6 S. fecaiis and its variants. 4 5. 
 
 anginosus and its variants. 
 6 not corresponding to any of these tj'pes or variants. 
 3 strains tested at intervals during subculture varied rith respect to fermentation of inulin. Of 5 passed 
 through rabbits 3 varied with respect to one or more substmces. Many strains which showed no fermentation 
 of inulin in 3 or 3 days did ferment it in 8 days and could be trained to ferment it readily. 
 
 "Prom this work we can not stiggest any definite method for difercntiating between the various strains of strep- 
 tococci we have used in our experiments, and in our hands 'Gordons tests' have proved quite unreliable for this 
 purpose." 
 
 Strains were tested at intervals of a few mos. to 2 yrs. Found fermentation reactions inconstant. 
 
 (See Table VII on Mutations and Variability.) 
 
 "It follows that the method of identifying varieties of streplocoeci by means of the series of test media which have 
 been employed in these experiments rests on no fixed or specific differences in the organisms themselves. The differ- 
 ences observed cannot in any sense le regarded as permanent, hut would seem to be due to merely temporary changes 
 in the metabolism of the organisms concerned." 
 
 All strains fermented dextrose and lactose, most acid btmg produced by S. acidi lactici and least by S. pyo- 
 genes. Mannite was fermented by about J of the S. acili lactici strains and 1 of the S. lanceolatus strains, 
 the others producing only 0.2-0.3 per cent of acid. No :icid was produced by 5. pyogenes in mannite. In 
 inulin S. acidi lactici produced up to 0.7 per cent acid (except 1 strain which in its 2nd test produced 6 per 
 cent acid). 5. lanceolatus produced up to 0.6 per cent acid, and S. pyogenes not more than 0.1 per cent acid 
 from inulin. 
 
 The author apparently regards even the smallest amount of acid production as a positive test, however. 
 
 Milk was coagulated by all strains except 5 from stable (kist and bovine vaginitis. 
 
 Dextrose was fermented by all strains. 
 
 Mannite was fermented by none. 
 
 Lactose was fermented by all strains except 6 from horse saliva, stable dust, and bovine vaginitis. 
 
 Reference. 
 
 Laabs 1910. 
 
 Vergleichende Untersuchungen iiber den 
 Streptococcus equi und andere pathogene Strep- 
 tokokken. 
 
 Prlcolo 1910. 
 
 Recherches experimentales sur le streptocoque 
 de la gourme. 
 
 Bahr 1910. 
 
 Vorkommeu und Bedeutung der Strepto- 
 kokken in der Milch. 
 
 Rosenow 
 
 A Study of Pneumococci 
 fectious Endocarditis. 
 
 1910. 
 from Cases of la- 
 
 Beattie and Yates 1911. 
 
 Sugar Tests and Pathogenicity in the Differ- 
 entiation of Streptococci. 
 
 Walker 1911. 
 
 On Variatioii and Adaptation in Bacteria, 
 Illustrated by Observations upon Streptococci, 
 with Special Reference to the Value of Fer- 
 mentation Tests as Applied to These Organis.-QS, 
 
 Saito 1912. 
 
 Versuche zur Abgrenzung des Streptococcus 
 acidi la£tici von Streptococcus pyogenes und 
 Streptococcus lanceolatus. 
 
 Gmlader 1912. 
 
 Untersuchungen iiber Mastitisstreptokokken 
 und ihre Differenzierung von saprophytischen 
 Streptokokken. 
 
Organisms. 
 
 Many strains from sore 
 throats and complications. 
 
 100 strains of streptococci 
 isolated from 100 samples 
 of supposedly normal dairy 
 milk. 
 
 202 strains from human 
 and bavine feces (Winslow 
 and Palmer). 
 
 101 strains from milk 
 (Broadhurst). 
 
 55 strains from normal 
 and diseased throats (Hil- 
 liard) . 
 
 17 strains from various 
 sources (Ballinger) . 
 
 55 strains of streptococci 
 from normal throats, and 15 
 from diseased throats. 
 
 16 strains from milk. 
 
 92 strains from horse, hu- 
 man, rabbit, and guinea pig 
 feces, dust, milk, throats, 
 and pathological conditions. 
 
 45 strains from throats 
 and secondary lesions of 
 cases of epidemic sore throat. 
 
 1 strain of ordinary hemo- 
 lytic streptococcus from the 
 udder of a cow. 
 
 1 strain of S. epidemicus 
 from milk. 
 
 2 strains of 5. epidemicus 
 from sore throats. 
 
 1 strain from throat of 
 carrier. 
 
 3 strains from secondary 
 peritonitis and adenitis. 
 
 TABLE V — Continued. 
 Fermentation Reactions. 
 
 TABLE Y— Continued. 
 Fermentation Reactions. 
 
 Media. 
 
 Composition. 
 
 "Sugar-free broth," (meth- 
 od of preparation not given) 
 -f- 1 per cent of test sub- 
 stance. Average reaction of 
 controls = 1.6 per cent n 
 acid to phenolphthalein. 
 
 Method of sterilization not given 
 
 Test substances. 
 
 Indicator or titration. 
 
 Inulin 
 Milk. 
 
 Saccharose, lactose, salicin, 
 raffinose, mannite, inulin. 
 Milk. 
 
 Titrated, (phe- 
 nolphthalein). Bi- 
 ometric method. 
 Intermodal point 
 = 1.5 per cent N 
 acid above control. 
 
 Period of incubation. 
 
 3 days. 
 
 This paper is a summarization of previous work by the author and others, and involves no new 
 experimental work. 
 
 Results and conclusions. 
 
 'Milk is always acidified and sometimes coagulated. Inulin is ?iot fermented." 
 
 Excluding coagulation of milk, reduction of neutral red, and morphology from the differential criteria, the 
 100 strains fell into 20 fermentative groups, not more than 15 belonging to any one group. The groups are 
 not named. The tests excluded were found unsatisfactory and inconstant. 
 
 "The milk streptococci are characterized by unusually high fermentative powers" and "are most like the human 
 strains; there is less likeness between the milk and the bovine strains; they show practically no resemblance to tlie 
 equine strains." 
 
 Attaches soecial importance to the titration of the reaction of cultures as carried out by American observers 
 as opposed to the qualitative results obtained by the English observers by the use of litmus media. 
 
 Notes an order of availability {"Metabolic Gradient"— Howe) among the test substances used, the order 
 being- dextrose— lactose— salicin— saccharose— mannite— inulin— raffinose. "// any member of the series ts 
 fermented, the chances are that those ahead of it mil be fermenied also. If any member ts not attacked, the chances 
 are that those behind it will not be attacked either." " * * the size of the molecule appears to be the mam factor 
 involved." 
 
 "1 per cent carbohydrate 
 broth." No other details. 
 
 j Dextrose, lactose, maltose, 
 I saccharose, raffinose, man- 
 I nite. 
 Milk. 
 
 Titrated. 
 
 72 hrs. 
 
 Hiss litmus serum water 
 and "peptone solution col- 
 ored with litmus." • 
 
 Saccharose, lactose, ralfi- 
 nose, inulin, salicin, sorbite. 
 
 'Bouillon." 
 
 Dextrose, maltose, lac- 
 tose, saccharose, dextrin, raf- 
 finose, mannite, inulin. 
 
 Litmus. 
 
 Also titrated acid 
 formed in some 
 
 "Streptococci from the human throat and from fresh mdlk very generally ferment one or more of the sugars dex- 
 trose, lactose, maltose, and saccharose, attacking them most readily in the order named, ney do not generaly fer- 
 ment raffinose or mannite." 
 
 (See also Table VIII on General Conclusions, etc.) 
 
 Experienced difTiculty in keeping strains alive. 
 
 Found the titration of acid formed in cultures of no particular value or advantage over use ot litmus as 
 
 Dextrose, levulose, galactose, and dextrin were fermented by all the streptococci and are therefore consid- 
 ered of no differential value. 
 
 Most of the grouDS of Andrewes and Border were found. ,c- r^r x.^ ^-tt 
 
 Found fermentation reactions usually constant, but observed some irregularities. (See Table Vli on Mu- 
 tations and Variability.) ,• c ^ 
 
 Concludes that the fermentation reactions, like other methods that have been suggested, are unsatisfactory 
 as a means of classification of the streptococci. 
 
 "On sugars, milk, and otiter mediums these two varieties of streptococci grow practically alike" {i.e., the pacu- 
 liar type of streptococcus here described and considered responsible for the epidemic, later (1913) called 5. 
 epidemicus; and the ordinary S. hemolyticus) . . 
 
 "Milk is always acidified and usually coagulated. These cocci ferment glucose, lactose, saccharose, dexinn, 
 and maltose, but not inulin, mannite or raffinose." 
 
 "When first isolated this streptococcus fermented dextrose, maltose, lactose, saccharose, and dextrin bouillon, 
 hit failed to ferment inulin, raffinose, and mannite bouillon; however, after several months of laboratory existence 
 the organism now ferments raffinose broth. It was not very soluble in bile. ^ It therefore corresponds m the essen- 
 tial cultural characteristics and in Us morphology to the streptococcus epidemicus." 
 
 The above description applies to the strain from milk, but the others are described as being like it except 
 that the strain from the carrier and one strain from adenitis failed to ferment raffinose. 
 
 Reference. 
 
 Davis and Rosenow 1912. 
 An Epidemic of Sore Throat Due to a Pecu- 
 liar Streptococcus. 
 
 Broadhurst 1912. 
 
 A Biometrical Study of ililk Streptococci. 
 
 Winslow 1912. 
 
 The Classification of the Streptococci by their 
 Action upon Carbohydrates and Related Or- 
 ganic Media. (A Summary.) 
 
 Stowell and Hilllard 1912. 
 
 i\ Comparison of the Streptococci from Milk 
 and from the Human Throat. 
 
 Bergey 1912. 
 
 Differentiation of Cultures of Streptococcus. 
 
 Davis . 1912. 
 
 Bacteriologic Study of Streptococci m Milk 
 in Relation to Epidemic Sore Throat. 
 
 Stokes and Haehtel 1912. 
 
 Septic Sore Throat. A Milk-Bome Outbreak 
 in Baltimore, Md. Bacteriological Study of the 
 Outbreak. 
 
TABLE V — Continued. 
 Fermentation Reactions. 
 
 TABLE V — Continued. 
 Fermentation Reactions. 
 
 Organisms. 
 
 240 Strains of streptococci 
 from milk and the human 
 throat (normal ?). 
 
 12 strains. 
 
 Fecal streptococci; 123 
 human, 129 horse, and 97 
 bovine. 
 
 26 strains of 5. equi (23 
 laboratory strains of various 
 ages, and 3 freshly isolated 
 by the authors) . 
 
 27 strains of S. longus iso- 
 lated by the authors from 
 human pathological material. 
 
 Media. 
 
 Composition. 
 
 Test substances. 
 
 Liebig extract bouillon + 1 
 per cent of test substance. 
 Sterilized at 100°C. on 3 
 successive days. 
 
 Comparison of results 
 with: 
 
 (1) Sugar-free meat infu- 
 sion bouillon (fermented by 
 B. coli 7 days at 37°C. and 
 then filtered through Berke- 
 feld). 
 
 (2) Meat extract bouil- 
 lon. 
 
 Both sterilized at 100°C. 
 30 min. on 2 days after 
 adding 1 per cent sugar. 
 
 Beef extract bouillon. 
 Reaction = 0.5 per cent N 
 acid to phenolphthalein. 
 Added 1 per cent of test 
 substance. 
 
 (a) Horse serum bouillon, 
 the serum being sterilized 
 by heating 1 hr. at 60° on 
 3 successive days. 
 
 {b) " Chapoteaut-Aszites- 
 agar." 
 
 To 9 cc. of either medium 
 there was added 1 cc. of a 
 10 per cent sugar solution. 
 
 Dextrose, lactose, saccha- 
 rose, salicin, raflinose, inu- 
 lin, mannite. 
 
 Inulin. 
 
 Saccharose, lactose, sal- 
 icin, raffmose, mannite, inu- 
 lin. 
 
 Indi.-ator or titration. 
 
 Period of incubation. 
 
 Titrated (phe- 
 nolphthalein) . 
 
 Dextrose, lactose, saccha- 
 rose, rattinose, mannite, inu- 
 lin, salicin. 
 
 Rafnnose, saccharose, lac- 
 tose, maltose, galactose, 
 mannose, levulose, dextrose, 
 mannite, dulcite, sorbite. 
 
 (10 per cent in litmus so- 
 lution, sterilized by steam- 
 ing 2 or 3 mins. on 3 suc- 
 cessive days.) 
 
 Titrated cold 
 (phenolphthalein) . 
 
 Litmus. 
 
 Titrations also 
 made with litmus 
 as an indicator. 
 
 3 days. 
 
 3 days. 
 
 1-10 days. 
 
 Results and conclusions. 
 
 Mannite is concluded to be of no differential signiilcance since it was fermented by only 2 "aberrant" 
 strains. 
 
 The streptococci are arranged into 9 groups according to their action on the remaming 6 substances. 
 (See also Table VIII on General Conclusions, etc.) 
 
 Reference. 
 
 Stowell, Hilliard, and Sehlesinger 1913. 
 
 A Statistical Study of the Streptococci from 
 ?ililk and from the Human Throat. 
 
 " Sugar fermentation with the streptococcus group has on the whole not been trustworthy or satisfactory on account 
 of inconsistency of results." r. • 
 
 By consideration of a number of characters 5 groups of streptococci are differentiated: 5. hemolyttcus, S. epi- 
 demicus, S. mucosus, S. pneumonia, and S. viridans. The only fermentation considered is that with mulm, 
 S. hemolyticus, and S. epidemicus being non-fermenters of inulin. 
 
 (See Table VIII on General Conclusions, etc.) 
 
 " The fermentative activities of streptococci vary greatly tvith the use of meat or of meat extract in making the 
 special media. * * the differences are not only most marked (often reaching 200 to 300 per cent), but they are 
 remarkably uniform in amount and direction." 
 
 "Qualitative results, estimated with litmus as an indicator, are not comparable with quantitative ones, with 
 phenolphthalein as an indicator, if meat extract is used in making the special media. * * in meat extract media, 
 where the final acidity record often drops down to 0.7-1.0, litmus with its higher neutral point gives no indication 
 of the possible powers of the strain under investigation." 
 
 Results are tabulated and compared with those of Winslow and of Houston; in general agreement. 
 
 "Streptococci that ferment mannite are found abundantly only in human feces." 65 per cent of streptococci 
 from human feces fermented mannite, as opposed to only 2 or 3 per cent from equine and bovine feces. 
 
 "No strains which fermented rajfinose were isolated from human feces and only 12 per cent of those obtained 
 from horse manure attacked this sugar. On the other hand, 73 per cent of the streptococci from cow dung fermented 
 rafmose." ^^ 
 
 " Streptococci fermenting lactose are comparatively rare in horse dung. 
 
 4 strains are reported which fermented lactose and raflinose or lactose only, but not dextrose. 
 
 10 strains of S. equi and 10 of 5. longus were tested against all the substances. Dextrose, levulose, man- 
 nose, galactose, maltose, and saccharose were fermented by all of them, raflinose by none. The remaining 
 4 substances were fermented by the number of strains indicated in the table. 
 
 Lactose. Mannite. Dulcite. Sorbite 
 
 -1- - + - + - + 
 
 5. equi. 
 S. longus. 
 
 10 
 
 10 
 10 
 
 All strains (53) were titrated after growth in mannite, dextrose, and levulose serum bouillon. 
 mum acidity was attained in 48 hrs. ,,.,,,,, 
 
 "Der Streptococcus longus bildet starke Sdure im Mannit und rotet blaue Lakmus-Mannit-nahrboden, wakrend 
 der Streptococcus equi die blaue Farbe sehr wenig oder gar nicht verdndert; dieser Unterschied lasst sich praktisch 
 zur Differentialdiagnosis beider Arten verwenden." , ,,. , 
 
 "Der Druse-Streptococcus ist also gegen die von ihm selbst gebildete Skure viel empfindhcher als der Streptococcus 
 longus; der erstere besitzt uberhaupt eine geringere Widerstandsfahigkeit gegen schddigende Einfliisse, ah 
 letztere." 
 
 The maxi- 
 
 der 
 
 Davis 1913. 
 
 Interrelations in the Streptococcus Group 
 with Special Reference to Anaphylactic Reac- 
 tions. 
 
 Broadhurst 
 
 Effect of I\Ieat and 
 upon the Fermentative 
 cocci. 
 
 1913. 
 
 Meat Extract Media 
 Activity of Strepto- 
 
 FuUer and Armstrong 1913. 
 
 The FJifferentiation of Fecal Streptococci by 
 their Fermentative Reactions in Carbohydrate 
 Media. , > ; 
 
 Koch and Poksehisehewsky 1913. 
 
 Ueber die Artverschiedenheit des Streptococcus 
 longus sen erysipelatos und des Streptococcus 
 equi (Druse-streptococcus). 
 
TABLE IV. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 TABLE IV. 
 The Use of Blood Bouillon and Other Fluid Media. 
 
 
 Blood. 
 
 Fluid media. 
 
 Period of incubation. 
 
 Results and conclusions. 
 
 Reference. 
 
 
 Species. 1 Amount. 
 
 Composition. Amount. 
 
 
 
 Pure rabbit serum heated 30 min. at 55°C. was inoculated with 2 or 3 drops of heart blood from 
 a rabbit killed by inoculation with streptococci. The culture was incubated for about 10 hrs., 
 diluted with an equal amount of salt solution, and then filtered through a porcelain filter. 
 
 To various amounts of filtrate 1 drop of defibrinated rabbit blood was added. Results were 
 read after incubation for 2 hrs. at 37°. (Beef, sheep, guinea pig, dog, human, and horse blood 
 were also tried.) 
 
 Hemolytic filtrates were obtained. 
 
 The hemolysin was non-dialyzable and was inactivated by heating at 70°C. for 2 hrs. or 55° for 10 hrs. 
 Hemolysis was retarded by the addition of salt to the filtrate. 
 
 The filtrates were non-toxic for rabbits or sheep but injection of rabbits failed to produce an immune anti- 
 hemolysin. 
 
 Besredka 1901. 
 De I'hemolysine streptococcique. 
 
 17 strains of streptococci (5 
 from "Druseerkrankung von 
 Pferde," others from various 
 human infections. 
 
 Rabbit, defibrinated. 
 
 A small 
 amount. 
 
 Horse meat bouillon 
 containing 0.5 per cent 
 salt, 0.5 per cent pep- 
 tone, and 0.1 per cent 
 dextrose. Reaction 
 alkaline to litmus but 
 acid to phenolphtha- 
 lein. 
 
 
 24 hrs. 
 
 The blood was completely laked by cultures of virulent streptococci in 12-24 hrs. 
 
 The author was unable to obtain hemolytic filtrates though they are said to have been toxic. 
 
 Aronson 1901. 
 Untersuchungen fiber Streptokokken und 
 Antistreptokokken-serum. 
 
 40 strains of human origin. 
 2 equine strains. 
 
 Rabbit, defibrinated. 
 
 "A little." 
 
 "Peptone bouillon." 
 
 
 
 All strains produced laking and burgundy red color in blood bouillon. A scarlet fever strain and one equine 
 strain {"Streptococcus der Druse") were less hemolytic than the others. 
 (See also Table VIII on General Conclusions, etc.) 
 
 Marmorek 1902. 
 L'Unite des streptocoques pathogSnes pour 
 I'homme. 
 
 .\ number of pathogenic and 
 non-pathogenic strains from 
 various sources. 
 
 A series of tubes of ordinary weakly alkaline bouillon was inoculated with streptococci. A tube 
 was removed each day for a hemolysin test. 
 
 Hemolytic activity was tested by adding 1 drop of defibrinated rabbit blood to 5 cc. of culture. 
 The mixture was incubated 2 hrs. and refrigerated over night. 
 
 ■ 1 
 
 Filtrates gave inconstant results, though centrifugates of hemolytic cultures were hemolytic. 
 
 The hemolytic streptococci were generally those from pathological sources. 
 
 The hemolytic activity of cultures was destroyed in 15 min. at 60°C. or in 8 days at 37°. 
 
 In 2 hrs. at 0°C. no hemolysis was produced by cultures, but after such an exposure the washed corpuscles 
 from the mixture underwent hemolysis in 1 hr. at 37°C. The author concludes that at 0° there was a com- 
 bination of hemolysin with corpuscles. 
 
 A drop of blood exposed (2 hrs. at 37°) to an old (8 days) hemolytically inactive culture was washed in salt 
 solution and found to be resistant to hemolysis by a young active culture. The author concludes that the cor- 
 puscles were saturated wth "Hamolysoid" and hence incapable of hemolysis. 
 
 Sehlesinger 1903. 
 Experimentelle Untersuchungen fiber das 
 Hemolysin der Streptokokken. 
 
 The strain employed was is- 
 olated from a retroperitoneal 
 abscess in a guinea pig which 
 had been inoculated intraperi- 
 toneally with a fungus. 
 
 "Mix 1 part of human serum with 2 parts of rabbit serum, heat the mixture to 56°C. for 30 min- 
 utes, inoculate this with a young virulent culture of streptococci, incubate for 20 to 22 hours, dilute 
 with an equal volume of physiologic salt solution, and pass through a porcelain filler." 
 
 Tested the action of filtrates on corpuscles of man, rabbit, dog, goat, sheep, hog, guinea pig, 
 chicken, ox, and horse. 
 
 •1 
 
 "One drop of a thick suspension of these corpuscles, washed, is completely hemolysed by 1 cc. of streptocolysin in 
 1\ hours" at 37°. 
 
 Washed corpuscles were more easily hemolyzed than unwashed because of the presence of " antihemolysin" 
 in the serum. 
 
 The corpuscles of man and the rabbit were found least resistant while those of the chicken, ox, and horse 
 were most resistant. 
 
 The hemolysin is said to be an organic substance, destroyed in 2 hrs. at 70°C., deteriorating rapidly at 37° 
 and gradually at room temperature, but keeping for a much longer time in the refrigerator. 
 
 "It is destroyed by peptic digestion." "It is non-dialysable." 
 
 "It is composed of a haptophore and a toxophore group, which are firmly bound together. The haptophore group 
 may be neutralized with chicken serum and the toxophore group is destroyed by zinc chlorid." 
 
 "The sera of some animals contain antistreptocolysin." 
 
 "A weak solution of formaldehyde has antihemolytic properties." 
 
 " The filtered cultures of a virulent streptococcus, in heated serum, are toxic for rabbits." 
 
 Ruedlger 1903. 
 The Production and Nature of Streptoco- 
 lysin. 
 
 
 Human. 
 
 
 
 
 
 "Wahrend meist der S. longus dem Blutfarbstof allmahlich eine karminrote Nuancierung verleiht, * * wird 
 Blutbouillon, in der sich das Blut bald nach dem Zusatz sliarf am Boden von der klaren Bouillon abgesetzt, durch 
 den S. mitior in eine gleichmdssig braunrot FlUssigkeit verwandelt." 
 
 With regard to the pneumococcus, "Blutbouillon zeigl ebenfalls schon nach 24 Stunden Aufenthalt bei37°eine 
 deutliche GrUnfdrbung." 
 
 Sohottmiiller 1903. 
 Die Artunterscheidung der fiir Menschen 
 pathogenen Streptokokken durch Blutagar. 
 
TABLE vn. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 Thought he had changed 5. longus into 5. hrevis 
 by passages through mice (intraperitoneal injections). 
 
 The hemolytic activity of stock cultures remained 
 unaltered for several mos. 
 
 Successive passages of strains through series of 
 white mice resulted in increased virulence and hemo- 
 lytic activity. 
 
 " * * it has been found that * * the pnea- 
 mococcus when grown in the serum, both natural and 
 pneumonic, soon loses its hemolytic power." 
 
 3 of the 5 strains which the author regarded as 
 5. mitior (see Table III on The Use of Blood Agar) 
 were soon lost and the other 2 after 5 rrios. 
 cultivation acquired the characters of typical 
 5. longus pathogenes. 
 
 The hemolytic activity of pathogenic strains of 
 streptococci was increased by animal passage (rab- 
 bits). The hemolytic activity was decreased by 
 passage through sugar bouillon. 
 
 Reports that 1 strain of streptococci during sev- 
 eral passages through mice showed variations as to 
 virulence and hemolytic activity, being originally- 
 hemolytic and virulent but finally losing both char- 
 acters. 
 
 Found no change in his test reactions during cul- 
 tivation for periods up to a fortnight. Passed 11 
 strains through mice; 9 were unchanged, 1 acquired 
 the ability to ferment salicin, 1 lost the power to 
 produce green fluorescence in neutral red broth. 
 
 With regard to the constancy of the fermentation 
 reactions of 5. pyogenes, "Five strains were tested 3 
 times, at intervals tf 2 to 3 months, and the results 
 were always the same." 
 
 " * * the secretion of hemolysin by bacteria is 
 a variable character. It is very pronounced in some 
 strains of streptococcus, rather feeble in others, and 
 may be absent entirely in others. I have two strains 
 of streptococcus which have lost this property entirely 
 by being grown in glucose broth for about two years. 
 They now produce brownish colonies'in plain blood 
 agar plates and greenish colonies in plates of glucose 
 agar and blood." 
 
 Cited by Mandelbaum (1907-08) as having 
 claimed to transform S. pathogenes into S. mitior by 
 prolonged cultivation, and S. mitior into 5. patho- 
 genes by animal passage and cultivation. 
 
 Reference. 
 
 Waldvogel 1894. 
 
 Ueber das Wachsthum des 
 Streptococcus longus in Bouil- 
 lon. 
 
 Sehleslnger 1903. 
 
 Experimentelle Untersuch- 
 ungen ttber das Hamolysin der 
 Streptokokken. 
 
 Rosenow 1904. 
 
 Studies in Pneumonia and 
 Pneumococcus Infections. 
 
 Rleke 1904. 
 
 Beitrage zur Frage der Ar- 
 teinheit der Streptokokken. 
 
 Kerner 1905. 
 
 Experimenteller Beitrag zur 
 Hamolyse und zur Agglutina- 
 tion der Streptokokken. 
 
 Natvlg 1905. 
 
 Bakteriologische Verhalt- 
 
 nisse in weiblichen Genitalse- 
 kreten. 
 
 Gordon 1905. 
 
 A Ready Method of Differen- 
 tiating Streptococci and Some 
 Results Already Obtained by 
 Its Application. 
 
 Ruedlger 1906. 
 
 The Streptococci from Scar- 
 latinal and Normal Throats 
 and from Other Sources. 
 
 Beltzke and Rosenthal 1906. 
 
 Zur Unterscheidung der 
 Streptokokken mittels Blut- 
 nahrboden. 
 
TABLE vn — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 According to Salomon (1908), "Einen Befund von 
 Beitzke und Rosenthal, denen ein Stamm zu verschiede- 
 nen Zeilen ungleiche Residtate gab, ftihrt Schultze 
 (1907) auf einen Wechsel in der Zusammensetzung 
 der Ndhrboden zurilck." 
 
 "It is important to note that the fermentative prop- 
 erties described suffered no change from generation to 
 generation" over a period of from 6-8 mos. 
 
 The authors accepted the capsule stain described 
 by Buerger as diagnostic for the diflferentiation of 
 pneumococci from streptococci. 
 
 "Although not a single cultural property is differen- 
 tial between the streptococcus and the pneumococcus, 
 we may speak of a streptococcus and a pneumococcus 
 cultural type, in order to facilitate further discussions. 
 The streptococcus cultural type would include the fol- 
 lowing: non-fermentation of inulin, precipitation of 
 glucose serum agar, and hemolysis in blood media; 
 the pneumococcus cultural type, fermentation of inu- 
 lin, non-precipitation, and failure to produce hemo- 
 lysis." 
 
 "Pneumococci may acquire certain unusual cultural 
 properties in human blood or in human' exudates (in 
 vivo). The power to ferment inulin may become lost, 
 and the ability to cause precipitation in glucose serum 
 agar may be gained. In other words a substitution of 
 the common cultural features of pyogenic streptococci 
 may take place; the organisms then resemble the 'strep- 
 tococcus cultural type.' " 
 
 "It is possible to convert some but not all strains 
 possessing the streptococcus cultural type into typical 
 pneumococci by inoculation into white mice." 
 
 "The tendency of pneumococci of the streptococcus 
 cultural type as well as those which have been con- 
 verted to the normal variety, seems to be toward a 
 gradual degeneration which manifests itself in the 
 assumption of permanent streptococcic features. Such 
 organisms can then no longer be differentiated from 
 streptococci." 
 
 " Two months after isolation all the atypical organ- 
 isms had apparently acquired fixed streptococcus cul- 
 tural characteristics and repeated animal inoculation 
 failed to bring about a change." 
 
 Found the hemolytic characters of streptococci 
 unchanged by heat or by cultivation on plain agar 
 or blood agar for 3-6 mos. By prolonged cul- 
 tivation on blood agar strains of 5. mitior which 
 originally produced visible zones in 3-S days were 
 trained to produce them in 2-3 days and might be 
 visible even in 24 hrs., but these zones did not 
 equal those of 5. pathogenes in size or clearness. 
 
 Reference. 
 
 Beitzke and 1906. 
 
 Rosenthal — Continued. 
 
 Buerger 1907. 
 
 The Differentiation of Strep- 
 tococci by Means of Fermenta- 
 tive Tests. 
 
 Buerger and Ryttenberg 1907. 
 
 Observations upon Certain 
 
 Properties Acquired by the 
 
 Pneumococcus in the Human 
 
 Body. 
 
 Mandelbaum 1907-08. 
 
 Zur Streptokokkenfrage. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 The author had never witnessed the transforma- 
 tion of one of the following fundamental types to 
 another: S. pathogenes, S. mitior, S. saprophyticus. 
 
 Says that the hemolytic character of streptococci 
 isolated from cases of severe endometritis was soon 
 lost in artificial cultivation. He regarded the 
 pathogenic and hemolytic characters of streptococci 
 as extremely labile and easily modified by artificial 
 cultivation and therefore emphasized the impor- 
 tance of investigating strains bacteriologically when 
 freshly isolated. 
 
 The streptococci of moderate hemolytic power 
 found in cases of parametritis were regarded as hemo- 
 lytic streptococci modified within the body. 
 
 The author was unable to change hemolytic to 
 non-hemolytic or green-producing streptococci or 
 vice versa by cultural methods. He suspects, how- 
 ever, that some of the saprophytic non-hemolytic 
 streptococci normally present in the vagina may, 
 in their struggle with the living body, acquire viru- 
 lence and hemolytic power. 
 
 The hemolytic character of streptococci was re- 
 duced by artificial cultivation and increased by 
 animal passage, but in only 1 case did the au- 
 thors observe an actual change from the longus to 
 the mitior type or vice versa. They give no further 
 details regarding this observation. 
 
 Although some of the strains of S. mitior became 
 more hemolytic as a result of animal passages, yet 
 "Eine Hamolysierungsfdhigkeit kann man ihm wohl 
 anztlchten, aber diesdbe geht stets langsam vor sich 
 und erreichl selbst in 48 Stunden nicht diesen Grad 
 der Hamolyse, den der Streptococcus longus in der 
 ersten Kultur innerhaib von 16-18 Stunden erzeugt." 
 
 The author states that the hemolytic characters 
 of streptococci are generally quite constant and 
 "Fortzuchten auf kiinstlicken hdmoglobinfreien Ndhr- 
 boden veranderte an hamolytischen Stdmmen eben- 
 sowenig, wie das Zuchten nickthdmolytischer Strepto- 
 kokken auf Blutndhrboden." 
 
 The author does, however, describe the following 
 variations as having been observed by him: 
 
 (aj " * * ein Stamm — der hochvirulent fiir 
 MWuse war — wShrend fortgesetzter Tierpassagen 
 seine hdmolyUsche Kraft reduziertel" 
 
 (6) "Ferner habe ich * * den Uebergang 
 eines nichthamolytiscken Streptococcus in einen 
 exquisit hamolytischen an zwei W'ochnerinnen ein- 
 wandfrei beobachtet." 
 
 "Auf der andern Seite besteht auf kiinstlicken 
 Ndhrboden fiir den einzelnen Stamm selbst graduell 
 
 Reference. 
 
 Mandelbaum — 
 
 Continued. 
 
 1907-08. 
 
 Fromme 1908. 
 
 Klinische und bakteriolo- 
 gische Studien zum Puerperal- 
 fieber. 
 
 Heynemann 1908. 
 
 Die Bedeutung der hamo- 
 lytischen Strep tokokken fOr 
 die puerperale Infection. 
 
 Ludke and Polano 1909. 
 
 Ueber Hamolyse der Strepto- 
 kokken. 
 
 Konrad 1909. 
 
 Weitere Beitrage zur Vagi- 
 nalstreptokokkenfrage. 
 
 Zangemelster 1909. 
 
 Die Hamolyse der Strepto- 
 kokken. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 eine Konstanz, welche so weit geht, dass man einzdne 
 StWmme aus der Art und GrSsse Hires hamolytischen 
 Hofes ohne weiteres erkennen und tenter- anderen 
 hamolytischen Streptokokkeri' gelegentlich heraus- 
 finden kann." 
 
 "Jedenfalls kann ich nach meinen Beobachtungen 
 mil Bestimmtkeit sagen, dass die einmal vorhandene 
 Fahigkeit eines Streptococcus, eine iypische Hamolyse 
 hervorzurufen, eine filr den betreffenden Streptococcus 
 constante Eigenschaft ist, die sich nicht cdteriren Idsst 
 durch kleine AbSnderungen der Untersuchungs- 
 methoden, sondern die Hamolyse tritt ein, wie und 
 wo der Streptococcus auf Blutagar angeht, sei es im 
 Ausstrich oder in der Aussaat, sei es in der Tiefe 
 Oder an den Oberfldsche. Auch das Alter der Strepto- 
 coccus dndert an der Hamolyse nichts; ich hdbe Stdmme 
 wochenlang fortgeziichtet, sie haben ihr Wackstum in 
 Bouillon geSndert, sie wucksen Anfangs in klarer 
 Bouillon in grossen Flocken, dann iipig und diffus die 
 Bouillon triibend, zuletzt ganz hummerlich mit leichter 
 Triibung nur noch bei tSglicher Ueberimpfung, sie 
 konnten im Brutschrank wettergezilchtet sein oder 
 wochenlang auf Bis slehen, sie konnten von einer tSd- 
 lichen Peritonitis stammen oder von einer gesunden 
 Wochnerin: auf Blutagar gebracht — zeigten sie wieder 
 dieselbe Hamolyse, wie wenn sie frisch vom Menschen 
 gewonnen wdren." 
 
 "Hemolysis is a constant property of some strains 
 of streptococci." (Translation.) 
 
 The author's strains underwent no change in 
 hemolytic or other cultural characters during 6 
 mos. of artificial cultivation. 
 
 With regard to so called "modified pneumococci" 
 from cases of endocarditis, "Most of the strains soon 
 after isolation fermented inulinto a mild degree, a prop- 
 erty which all soon lost." 
 
 " The stained specimen gave diplococcus forms and 
 chains." 
 
 "By cultivation on artificial media all the strains 
 change gradually into typical lanceolate diplococci, 
 often encapsulated, growing as typical pneumococci 
 in broth and on blood agar slants. On animal inocu- 
 lation this modification occurred abruptly. It has 
 been impossible to so modify strains of S. viridans 
 which these organisms much resemble." 
 
 "In subsequent platings, as in the case of fishings 
 from the original throat cultures, there was a marked 
 variation up and down the scale. Hemolyzing colo- 
 nies varied in hemolysis, became slightly hemolyzing, 
 doubtful, green, or showed no green nor hemolysis. 
 Slightly hemolyzing colonies became hemMyzing, re- 
 mained slightly hemolyzing, or were doubtful, green. 
 
 Reference. 
 
 Zangemeister — Continued. 1909. 
 
 Slgwart 1909. 
 
 Untersuchungen iiber die 
 
 Haniolyse der Streptokokken 
 
 in der Schwangerschaf t und im 
 
 Wochenbett. 
 
 Sachs 1909. 
 
 Ueber Streptokokkeu-Hamo- 
 lyse. 
 
 Rosenow 1909. 
 
 Immunological and Experi- 
 mental Studies on Pneumo- 
 coccus and Staphylococcus En- 
 docarditis. 
 
 Anthony 1909. 
 
 Some Characteristics of the 
 Streptococci Found in Scarlet 
 Fever. 
 
TABJLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 or showed no green nor hemolysis. Green colonies 
 remained green for the most part, but a few returned to 
 hemolyzing, slightly hemolyzing colonies, or were 
 doubtful." 
 
 "Although the power of hemolyzing streptococci to 
 cause hemolysis seems to be a slightly variable quality 
 * * , yet there is a distinct tendency in this 
 power to continue, * * for in the retest the ma- 
 jority of colonies were hemolyzing." 
 
 The author conducted experiments with 3 hemo- 
 lytic and 2 green-producing non-hemolytic strains 
 of "5. mitis:' 
 
 Dilute vaginal secretion and undiluted saliva 
 were sterilized b,y heating for 3-15 hrs. at 58° or 
 S6°C. respectively. Milk was treated with "Per- 
 hydrol {Merck)" which was then inactivated by the 
 addition of the "Katalase, Hepin" said to have been 
 recently discovered by Much and Romer. There 
 resulted "eine vollkommen bakterien- und perhydrol- 
 freie Milch mil Rohmilcheigenschaften." 
 
 By soaking for 24 hrs. in the vaginal secretion, 1 
 hemolytic strain was changed into a non-hemolytic 
 green-producer; 1 strain of 5. mitis became strongly 
 hemolytic. 
 
 After 24 hrs. in the saliva, 1 hemolytic strain 
 formed green colonies but remained hemolytic; 
 another hemolytic strain came tofoim"breite, lehm- 
 farbene Rasen ohne HSmolyse." 
 
 After 24 hrs. soaking in the milk, 1 mitis strain 
 became strongly hemolytic; 1 hemolytic strain pro- 
 duced large greenish colonies but remained hemo- 
 lytic; another hemolytic strain produced large white 
 colonies but remained hemolytic. 
 
 " Zusammenfassung: 1. Es geldng durch Ein- 
 bringen von StreptokokkenstHmmen in Vaginalsekret, 
 Milch oder Speichel, hdmolytische in nichthdmolyt- 
 ische umzuwandeln und umgekehrt." 
 
 With regard to the claims of others that the vari- 
 ous groups of streptococci described by him (1903) 
 are not constant in their properties, he states that 
 except for quantitative variations (e.g., in the amount 
 of hemolysis) he has never seen such variations. 
 
 "WoU sind mir ausnakmsweise Stimme von 
 Erysipelstreptokokken, welche unter gewissen Um- 
 stSnden wenig oder gar nicht hamolysierten, vorge- 
 kommen, aber Farbstoff bildeten sie nicht. Und 
 umgekehrt trifft man gdegentlich aiuf einm Strepto- 
 coccus mitior, der zu Zeilen hSmolysiert, aber trotzdem 
 erzeugt er Farbstoff, der erkennbar ist, so lang der hWmo- 
 lytische Hof nicht vorhanden ist." 
 
 He admits that there may be intermediary forms 
 but believes that they can nevertheless be differen- 
 tiated culturally especially by reference to their 
 pathological effects. 
 
 Reference. 
 
 Anthony — Continued. 
 
 1909. 
 
 Zopprltz 
 
 Ueber 
 suche. 
 
 1909. 
 Streptokokkenver- 
 
 Sehottmiiller 1910. 
 
 Endocarditis lenta. Zug- 
 
 leich ein Beitrag zur Artunter- 
 
 scheidung der pathogenen 
 
 Streptokokken. 
 
TABLE va^Contmued. 
 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 Reference. 
 
 Hemolytic streptococci carried along on artificial 
 media gradually, though slowly, Idst hemolytic 
 power. 
 
 Many observations led the author to believe that 
 in_ their struggle with the living tissues streptococci 
 might become hemolytic and again become non- 
 hemolytic when growing as saprophytes. The fol- 
 lowing appear especially significant: 
 
 Wounds becoming accidentally infected were 
 found to harbor at first only nton-hemolytic strepto- 
 cocci, then weakly hemolytic, and finally strongly 
 hemolytic strains in pure culture. "Ich bemerke 
 dazu, dass es sich in solchen Fallen nicht etwa urn 
 die Verdriingung, einen nichthamolyHschen handelt — 
 (since in such cases at some time during the infec- 
 tion both hemolytic and non-hemolytic forms would 
 be found) — , sondern dass sich im Sekret dann stets 
 eine ReinkuUur mn Streptokokken findet, die in 
 gleicher Weise zunachst gar nicht, dann schwach und 
 dann ausgesprochen hdmolysieren." 
 
 "Die vollige Beschrankung der hamolytischen Strep- 
 tokokken auf Infektionsgebiete und deren Umgebung 
 •war nun aber nur dann erklSrlich, wenn die h moly- 
 tischen Streptokokken, ehe sie ins Wundgebiet I ommen, 
 in der Regel anhamolytisch sind, und wenn sie avr- 
 dererseits ihre HUmolyse wieder verlieren, sobald sie 
 aus dem Infektionsbereich herauskommen." 
 
 Hemolytic streptococci were smeared between the 
 shoulders of normal women. In some cases the 
 streptococci were all dead in about 3 days; in others 
 only non-hemolytic streptococci were recovered. 
 Referring also to the results of Zoppritz (1909) who 
 altered the hemolytic characters of streptococci by 
 soaking them in vaginal secretion, milk, and saliva, 
 he says: "Es scheinen also besonders gewisse normale 
 Korpersekrete die Fahigkeit zu haben, den Strepto- 
 kokken die Hamolyse zu nehmen." 
 
 Zangemelster 1910. 
 
 Ueber die Verbreitung der 
 
 Streptokokken im Hinblidc auf 
 
 ihre Infektiositat und ihre ha- 
 
 molytische Eigenschaft. 
 
 Quoted by Hoessli (1910) as believing that S. 
 mitis could be changed into S. erysipelatos and vice 
 versa in the living body. 
 
 Much ? 
 
 By various passages of strains through horse 
 blood plasma and serum, (1) 1 of the strains of 
 " Dairmkokken" became hemolytic and in all other 
 respects like 5. erysipelatos; (2) there was obtained 
 from 5. mitior a variant which produced neither 
 hemolysis nor greenish discoloration and also a 
 variant which produced in 48 hrs. a "beginnende 
 Hamolyse" around the green colonies and from 1 
 of these colonies a strain which produced "starke 
 Hamolyse" in 16-24 hrs.; (3) the S . miicosus stmin 
 after prolonged cultivation on horse blood agar 
 plates acquired the ability to produce a small amount 
 of hemolysis; (4) a pneumococcus after 6 trans- 
 plants on blood agar showed "ziemlich starke Hamo- 
 lyse" apparent after 48 hrs. incubation, but by 
 passage through serum the hemolytic character was 
 again lost. 
 
 Hoessli 1910. 
 Das Verhalten der Strepto- 
 kokken gegenuber Plasma und 
 Serum and ihre Umzflchtung. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 Reference. 
 
 The author emphasizes the importance of classi- 
 fying streptococci when first isolated since, he states, 
 prolonged cultivation on artificial media brings 
 about changes in the characters of pathogenic germs 
 (e.g., the gonococcus, meningococcus, and cholera 
 vibrios). 
 
 Thalmann 1910. 
 
 Streptokokkenerkrankungen 
 
 in der Armee, Einteilung der 
 
 Streptokokken und ihre Be- 
 
 kampfung. 
 
 "The endocarditis cocci usually ferment inulin 
 slowly when first isolated; a short time later this 
 power may be increased, only to disappear entirely 
 after prolonged cultivation on blood agar. The pneu- 
 mococci from pneumonia at first ferment irmlin 
 strongly, but also lose it on long cultivation." 
 
 "Prolonged cultivation on artificial media, a short 
 residence in normal serum, and animal passage, as 
 already pointed out, have a pronounced effect on the 
 morphology and cultural characteristics of these organ- 
 isms. * * reversion in morphology to the normal 
 type occurs with the return of original pathogenic 
 properties." 
 
 Rosenow 1910. 
 A Study of Pneumococci 
 from Cases of Infectious En- 
 docarditis. 
 
 (See Table V on Fermentation Reactions.) 
 
 Seattle and Yates 1911. 
 Sugar Tests and Pathoge- 
 nicity in the Diflferentiation of 
 Streptococci. 
 
 "Der S. conglomeratus A verliert auch bei Vort- 
 zuchtung sehr bald das Vermijgen, in Bouillon feste 
 Konglomerate zu bilden; in Bouillon wiichst er dann 
 in lockeren Flocken ahnlich den Uebergangsformen." 
 
 Thalmann 1911. 
 Weitere Mitteilungen uber 
 Streptokokken, inbesondere 
 iiber pyogene Streptokokken 
 bei Erkrankungen der At- 
 mungsorgane und deren Kom- 
 plikationen. 
 
 "As regards the streptococci, * * -. If cul- 
 tures of these organisms are examined at considerable 
 intervals of time their reactions in the test media are 
 by no means constant, but on the contrary are found 
 to undergo remarkable changes. Further, * * 
 continued culture in a particular medium may en- 
 tirely alter the reactions of these organisms not only 
 to that medium, itself, but {it may be) to others also of 
 the carbohydrate mMia." 
 
 "The differences observed cannot in any sense be 
 regarded as permanent, but would seem to be due to 
 merely temporary changes in the metabolism of the 
 organisms concerned." 
 
 Walker 1911. 
 On Variation and Adapta- 
 tion in Bacteria, Illustrated 
 by Observations upon Strepto- 
 cocci, with Special Reference 
 to the Value of Fermentation 
 Tests as Applied to these 
 Organisms. 
 
 Employing the microscopic criteria of Mandel- 
 baum the author attempted to repeat the work of 
 Zoppritz in exposing streptococci to the action of 
 saliva, milk, horse serum, and horse blood plasma. 
 
 He concludes that: "Transmutation of one type 
 of streptococcus into another through cultivation on 
 blood agar, agar, in bouillon, saliva, miUii, or horse 
 
 LeBlane 191-2. 
 
 Zur Artenfrage der Strepto- 
 kokken. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 serum does not occur. The transformation of 5. 
 mitior into 5. pathogenes can not be admitted on the 
 basis of their similar macroscopic appearance on 
 the blood agar plate. The microscopic study of 
 their colonies is absolutely necessary." (Free 
 translation.) 
 
 The author tried cultivation of various strepto- 
 cocci for many mos. in horse serum, horse blood 
 plasma, human and animal blood agar plates, hu- 
 man blood plasma, and various standard media of 
 various reactions. He obtained no variations. 
 
 Having in view the conclusions of Zangemeister 
 (1910) the author made sterile wounds on animals, 
 infected them with pure strains of hemolytic, green- 
 producing, and non-hemolytic streptococci, protected 
 the wounds from accidental infection by glass caps, 
 and always recovered from these wounds only the 
 organism inoculated. 
 
 Retested the fermentation reactions of his strains 
 2 or 3 times at intervals of several mos. and found 
 no changes except slight quantitative variations in 
 the amount of acid produced. 
 
 The appearance oh blood agar was constant for 
 any given species of blood but was somewhat dif- 
 ferent for different species. 
 
 3 strains, originally hemolytic, became like "mri- 
 dis" after prolonged cultivation on blood agar. 
 By 'cultivation in bouillon the hemolytic character 
 was again restored in filU vigor. 
 
 Variation in the form, size, and consistency of 
 colonies was also noted.. 
 
 The author found that by prolonged cultivation 
 of streptococci in sugar bouillon the hemolytic 
 power might be more or less permanently reduced. 
 
 The morphological characters of mastitis strep- 
 tococci {" Staketenform" and paired arrangement 
 within the chain) became greatly modified by arti- 
 ficial cultivation. The characteristics of the growth 
 in bouillon were very variable. The hemolytic 
 characters in blood agar and blood bouillon were 
 found inconstant and therefore not a reliable means 
 of differentiation. 
 
 The author points out that he had previously 
 recognized that the. peculiar morphological char- 
 acters of mastitis streptococci were not constant 
 during artificial cultivation and that other, per- 
 haps saprophytic, streptococci might exhibit them 
 under some conditions; e.g., cultivation in raw milk 
 or serum. 
 
 (See Ernst, 1910, in Table Vm on General Con- 
 clusions, etc.) 
 
 Reference. 
 
 LeBlanc — Continued. 
 
 1912. 
 
 Roily 1912. 
 
 Experimentelle bakteriolog- 
 
 ische XJntersuchungen von ver- 
 
 schiedenen Streptokokken- 
 
 stanmien. 
 
 Salto 1912. 
 
 Versuche zur Abgrenzung des 
 
 Streptococcus acidi lactici von 
 
 Streptococcus pyogenes und 
 
 Streptococcus lanceolatus. 
 
 JaB€ 
 
 1912. 
 
 Beobachtungen bei blutlo- 
 senden und bei gramnegativen 
 Streptokokken. 
 
 Kuhn 1912. 
 
 Einfluss von Zucker auf 
 Haraolyse und Virulenz. 
 
 Gmlnder 1912. 
 
 Untersuchungen iiber Masti- 
 
 tisstreptokokken und ihre Dif- 
 
 ferenzierung von saprophy- 
 
 tischen Streptokokken. 
 
 Ernst 1912. 
 
 Eine Entgegnung zu A. 
 Gminders Arbeit: "Untersuch- 
 ungen fiber Mastitisstreptokok- 
 ken u. s. w." 
 
TABLE Vll— Continued. 
 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 The author has come to the conclusion that 5. 
 conglomeratus A and B are only degeneration forms 
 of other streptococci (5. pyogenes and 5. viridans), 
 brought about by cultivation or growth under 
 unfavorable conditions — ^possibly poor oxygen sup- 
 ply. "Ich fund die Form des Conglomeratus B be- 
 sonders in der Tiefe der Lakunen exterpierter Mandeln 
 — und zwar hier mitunter in enormen Mengen—und 
 bei chronischer Entzilndung des Zahnfleisches. Die 
 kleinen unregelrnHssigen, hScherigen, in den Ndhr- 
 boden, eingewacksenen Kolonieen zeigen mitunter 
 ausgesprochene HSmolyse, ein Zeichen, wie diese 
 Eigenschaft der pyogenen Streptokokken auch unter 
 ungiinstigen Bedingungen festgehalten wird." 
 
 "Die Keime verUeren ihre HUmolyse nur temporar. 
 Die Starke der Hdmolyse isl abhdngig von den Sub- 
 straten, auj denen die Bakterien zu wachsen ge- 
 zwungen werden. Aus dem Verhaltendes temporaren 
 V erschwinden des Hamolyse lasst sich keineswegs der 
 Schlms Ziehen, doss durch den Traubenzucker eine 
 Verwandlung der hSmolytischen Streptokokken in 
 ahdmolytische statthat. Der Verlust der HSmolyse ist 
 als nichts anderes aufzufassen, als die Hemmung 
 einer Eigenschaft eines Streptococcus, der Hdmolyse, 
 bedingt durch das N dhrsuhstrat." 
 
 The coagulation of milk, reduction of neutral 
 red, and morphology were excluded from the tests 
 employed because found to give inconstant results. 
 
 The fermentation reactions were usually con- 
 stant but showed some irregularities. "On repeat- 
 ing the tests with a particular culture it is not un- 
 usual to find irregularity in the results, * *. The 
 characters which an organism manifests when first 
 isolated or when growing in full vigor can usually be 
 brought out repeatedly in subsequent tests, so that the 
 evidence seems to be in favor of the idea that their char- 
 acters are fixed, and that the irregularities in the results 
 are to be attributed to lack of sufficient growth." 
 
 With regard to the peculiar streptococcus found 
 in the secondary lesions and sometimes in the 
 throats and believed to be responsible for the epi- 
 demic, the author says: "When grown on ordinary 
 mediums the. encapsulated coccus — at least some 
 strains — loses its capsule and the growth on slants 
 is less profuse and moist. By animal passage the 
 capsule arid the other properties mentioned return." 
 
 With regard to the strain resembling the "ordi- 
 nary hemolytic streptococcus," isolated from the 
 gargety cow, he says: " * * after three animal 
 passages the cocci in the peritoneal exudate of the 
 guinea pigs acquired distinct capsules and their growth 
 
 Reference. 
 
 Thalmann 1912. 
 
 Streptococcus viridans im 
 
 Blut ohne Veranderung der 
 
 Herzklappen. Zur Einteilung 
 
 der Streptokokken. 
 
 Benthln 1912. 
 
 Beitrage zur Hamolysefrage 
 der Streptokokken. 
 
 Broadhurst 1912. 
 
 A Biometrical Study of Milk 
 Streptococci. 
 
 Bergey 1912. 
 
 Differentiation of Cultures 
 of Streptococcus. 
 
 Davis 1912. 
 
 Bacteriologic Study of Strep- 
 tococci in Milk in Relation to 
 Epidemic Sore Throat. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 Reference. 
 
 on blood agar slants and plates became more watery 
 and mticoid. These properties were again readily 
 lost on ordinary media. In other words, after ani- 
 mal passage it simulated in every respect the encapsu- 
 lated streptococci described above in the exitdates of the 
 epidemic cases. ' Before animal passage it simulated 
 the hemolytic streptococci commonly found in the sore 
 throats." 
 
 B&vis— Continued. 1912. 
 
 "The conclusion drawn in my earlier papers that 
 the cocci in question are modified and attenuated 
 pneumococci has received ■additional support be- 
 cause growths from single cocci from strains iso- 
 lated originally from the blood of cases of endocar- 
 ditis, as well as from throats, when passed through 
 animals, take on cultural and pathogenic properties 
 indistinguishable from those of typical pneumococci." 
 
 "I know that the idea that these strains from endocar- 
 ditis are modified pneumococci is not generally held, 
 but in view of the facts cited, the conclusion is forced 
 on me that the organisms which are isolated from this 
 type of endocarditis, the organisms designated by 
 Schottmiiller as Streptococcus viridans and by Harder 
 as 'saprophytic streptococci,' are in reality pneumo- 
 cocci that have become attenuated and peculiarly 
 modified as the result of environmental conditions." 
 
 Rosenow 1912. 
 Experimental Infectious En- 
 docarditis. 
 
 The peculiar streptococci from cases of epidemic 
 sore throat "sooner or later assume the characteristics 
 of streptococcus pyogenes" in artificial cultivation. 
 
 "By placing Strepttcoccus pyogenes in unheated 
 sterile milk it becomes modifi^ so as to correspond 
 to the streptococci of epidemic sore throat. The 
 modifications may be accentuated by passage through 
 guinea pigs, and in some cases cultures like those of 
 streptococcus mucosus may restdt." 
 
 Tubes of unheated sterile milk inoculated with 
 ordinary hemolytic streptococci "placed at 37° C, 
 and in which growth, with acid production, occurred 
 did not appreciably change the streptococci, whereas 
 the streptococci in the milk kept outside the incubator 
 were modified perceptibly." 
 
 " * * subcultures on blood agar plates from the 
 upper and much dried portion of old blood agar tube 
 cultures {72 days) of streptococci from fatal cases for 
 several generations produced dry fine colonies sur- 
 rounded by a well defined zone of hemolysis, while 
 cultures from the lower and moist part of the same old 
 tubes gave moist large colonies with little hemolysis." 
 
 Rosenow 1912. 
 
 A Study of Streptococci from 
 
 Milk and from Epidemic Sore 
 
 Throat, and the Effect of Milk 
 
 on Streptococci. 
 
 (See Table V on Fermentation Reactions.) 
 
 Stokes and Haehtel 1912. 
 Septic Sore Throat. A Milk- 
 Borne Epidemic in Baltunore, 
 Md. Bacteriological Study of 
 the Outbreak. 
 
TABLE vn — Continued. 
 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 The authors believe that the hemolytic charac- 
 ters of the streptococci are more or less variable, 
 being influenced probably by the media on which 
 they are cultivated prior to inoculation onto blood 
 agar plate^, and also by growth in and on the ani- 
 mal body. 
 
 Very few changes in fermentation reactions were 
 observed after cultivation of the strains for several 
 wks. 
 
 Reports the transformation of 5. hemolyticus into 
 5. mucosus by passages through guinea pigs, and 
 the transformation of S. mucosus into 5. hemolyticus 
 by the injection of the former intravenously into a 
 rabbit and recovery of the latter from a joint 
 exudate. 
 
 The depression of the fermentative activity was 
 not permanent and was evidently due only to the 
 immediate use of the extract bouillon. Growth in 
 extract bouillon effected no change in the fer- 
 mentative powers of the strains which persisted 
 after they were transferred to more favorable 
 media. 
 
 "While most of the organisms have retained their 
 hemolytic power for many weeks, a number of cul- 
 tures lost this property after several months' cultiva- 
 tion on blood serum." "In no instance did the cul- 
 tures, which were non-hemolytic, acquire the property 
 of hemolysis while under cultivation." 
 
 The fermentative "properties persist after long 
 cultivation on ordinary media." 
 
 By exposing the organisms to various more or 
 less adverse conditions and to repeated animal 
 passages the author reports the following trans- 
 mutations: (1) change of hemolytic streptococci 
 into 5. viridans Qr npn-hemolytic streptococci by 
 drying on blood agar slants, growth on blood agar 
 near colonies of 5. subtilis, growth on ascites dextrose 
 agar in an atmosphere of oxygen, grojvth in hypo- 
 tonic media, and passage through rabbits; (2) 
 change of S. viridans into pneumococci by passage 
 through guinea pigs; (3) change of pneumococci into 
 S. mutosus by passage through guinea pigs; (4) 
 chanj^e of S. mucosus into 5. viridans by passage 
 through guinea pigs followed by prolonged cultiva- 
 tion on blood agar; (5) change of pneumococci into 
 S. viridans by growth on ascites dextrose agar in 
 oxygen atmosphere and by growth in hypertonic 
 
 Reference. 
 
 Sitzenfrey and Vatniek 1913. 
 
 Zur Frage der prognostischen 
 
 Verwertung bakteriologischer 
 
 Befunde bei puerperalen Pro- 
 
 Stowell, HilUard, and 
 
 Sehlesinger 1913, 
 
 A Statistical Study of the 
 Streptococci from Milk and 
 from the Human Throat. 
 
 Davis 1913. 
 
 Interrelations in the Strep- 
 tococcus Group with Special 
 Reference to Anaphylactic Re- 
 actions. 
 
 Broadhurst 1913. 
 
 Effect of Meat and Meat 
 Extract Media upon the Fer- 
 mentative Activity of Strepto- 
 cocci. 
 
 Floyd and Wolbach 1914. 
 
 On the Differentiation of 
 
 Streptococci. (Preliminary 
 
 Notes.) 
 
 Rosenow 1914. 
 
 Transmutations within the 
 Streptococcus - Pneumococcus 
 Group. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions.. 
 
 dialyzed beef serum; (6) change of pneumococci 
 into hemolytic streptococci by drying on blood 
 agar slant, sealing infected blood in tube for 5 
 yrs., growth on ascites dextrose agar in oxygen 
 atmosphere, growth in defibrinated blood in oxygen 
 atmosphere, and growth on blood agar near colonies 
 of B. coli (hemolytic); (7) change of 5. mridans into 
 hemolytic streptococci by growth on ascites dex- 
 trose agar in oxygen atmosphere, by growth on 
 blood agar for 1 yr., and by suspension in distilled 
 water for 1 month. By successive steps hemolytic 
 streptococci were changed into 5. mucosiis. 
 
 The primary criterion of change was the appear- 
 ance of growth on blood agar plates but these 
 changes were found to be accompanied by appropri- 
 ate changes in fementation reactions (especially 
 inulin), solubility in bile, agglutination reactions, 
 morphology, pathogenicity, ability to grow, in broth 
 culture filtrates (Marmorek's test), etc. 
 
 "By appropriate means strains of the three varie- 
 ties (of streptococci from rheumatism) have been 
 converted each one into the other." 
 
 A green-producing strain was changed into one 
 producing a narrow zone of hemolysis by "soaking" 
 in distilled water for 3 wks.; its morphology and 
 pathogenicity changed correspondingly. "After pro- 
 longed cultivation the strains which prodiiced green 
 at first come to_ resemble S. viridans in morphology, 
 in ctdtural, and pathogenic properties. The strains 
 which, produced a slight, hazy hemolysis at first now 
 ustially produced a wide zone of hemolysis, and when 
 injected produced arthritis but no endocarditis and 
 pericarditis." "By means of animal passage strains 
 of each group have been converted into typical pneu- 
 
 " Amongst fifty different strains of streptococci 
 frequently tested (by the authpr's special method ?) 
 as to their hemolytic powers during the period that 
 they were in subculture — a period varying from six 
 months to three and one-half years — I have never 
 met a hemolytic strain which lost the power of produc- 
 ing hemolysis. In one instance a streptococcus which 
 at the outset was non-hemolytic gradually developed 
 the power of hemolysin production, and ultimately was 
 only slightly less hemolytic on an artificial media than 
 some of the strains which were distinctly hemolytic 
 from the outset. This was strain P. M. 8488." The 
 author later admits that this result may possibly 
 have been due to mixed culture. 
 
 Both strains of streptococci (originally hemo- 
 lytic) had inconstant hemolytic properties when in- 
 jected into rabbits. From some rabbits only hem- 
 olytic cultures were recovered; from others only 
 non-hemolytic; while from others both hemolytic 
 and non-hemolytic colonies developed at the same 
 or different times. 
 
 Reference. 
 
 Rosenow^Contimied. 
 
 1914. 
 
 Rosenow . 1914. 
 
 The Etiology of Acute Rheu- 
 matism, Articular and Muscu- 
 lar. 
 
 M'Leod 1914. 
 
 Criteria of Virulence amongst 
 Streptococci, with Some Re- 
 marks on Streptococcal Leu- 
 cocidin. 
 
 Relehsteln 1914. 
 
 Ueber den Nachweis der 
 Streptokokken im stromenden 
 Blute. 
 
TABLE VII — Continued. 
 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 The fennentation reactions were in general quite 
 constant, the few exceptions being probably due to 
 poor growth at times. 
 
 "No change was noted in the cultural or patho- 
 genic properties of the streptococci after growing in 
 the udder of the cow for four weeks." 
 
 (See Table V on Fermentation Reactions.) 
 Though recognizing that the variability in the 
 action of bacteria on various substances may be 
 due to differences in technical procedure the author 
 regards "the exceptional variability of the group of 
 bacteria" as also a true cause. 
 
 "The constancy of carbohydrate reactions is evi- 
 denced by the fact that less than five per cent of the 
 strains tested showed any change on subsequent inocu- 
 lation." 
 
 "The growing of hemolytic streptococci with cul- 
 tures of the Bacillus suUilis for long periods has no 
 efect in altering their hemolytic properties." 
 
 "Hemolytic streptococci, growing on blood agar in 
 mixed culture with a green-prodtwing form, show an 
 apparent predominance, * * ." 
 
 " * * the streptococcus predominates on cul- 
 ture media while the pneumococcus gains the ascend- 
 ency in the animal body." 
 
 "Isolation from animals of streptococci, differing 
 in important characteristics from those injected, can- 
 not be taken as proof of a change in the characteristics 
 of the injected streptococcus within the animal body, 
 owin-; to the ease with which streptococci are able to 
 enter the tissues from the intestines 'or respiratory 
 tract of animals as well as man." 
 
 In regard to 2 strains of streptococci from the 
 Chicago epidemic (1912), 1 from the blood of a 
 fatal case and the other from the udder of a cow, 
 he says: "These organisms have maintained their 
 properties and their virulence for animals unimpaired 
 for over two years." 
 
 No variations were noted as a result of treating 
 the streptococci with milk or milk products. 
 
 "Some * * characters were inconstant, notably 
 the sedimentation or clouding of bouillon." 
 
 " The hemolytic activity of the ff types has remained 
 fairly constant. In no case has it disappeared. 
 Strain B-15 from a cow forms an apparent excep- 
 tion, but this strain has in the course of our studies 
 split up into a series of forms differing in their taking 
 capacity, som£ being non-hemolytic at present." 
 
 Reference. 
 
 Hopkins and Lang 1914. 
 
 Classification of Pathogenic 
 Streptococci by Fennentation 
 Reactions. 
 
 Davis and Capps 1914. 
 
 Experimental Bovine Masti- 
 tis Produced with Hemolytic 
 Streptococci of Human Origin. 
 
 Tliro ■ 1914. 
 
 Experiments on the Varia- 
 bility of the Fermentative Re- 
 action of Bacteria, Especially 
 the Streptococci. 
 
 Lyall 1914. 
 
 On the Classification of the 
 Streptococci. 
 
 Holman 1914. 
 
 The Relative Longevity 'of 
 Different Streptococci and Pos- 
 sible Errors in the Isolation 
 and Differentiation of Strepto- 
 cocci. 
 
 Davis 1914. 
 
 The Growth and Viability of 
 Streptococci of Bovine and 
 Himian - Origin in Milk and 
 Milk Products. 
 
 Smith and Brown 1915. 
 
 A Study of Streptococci Iso- 
 lated from Certain Presum- 
 ably Milk-Borne Epidemics of 
 Tonsillitis Occurring in Massa- 
 chusetts in 1913 and 1914. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 "When we, however, observe these races over a long 
 period of time we are struck by the persistence of ap- 
 parently minor cultural and other characters under 
 laboratory conditions. In our hands none of the 
 strains studied have lost or gained any capacity to 
 ferment certain sugars or alcohols." 
 
 "In our own repeated tests of the various races of 
 streptococci epctending over a period of fourteen months, 
 we have observed some loss of hemolytic activity in a 
 certain group and perliaps some decline of pathogenic 
 power, but no loss of identity when all characters were 
 considered." 
 
 "Spontaneous changes in cultural characters do not 
 proceed rapidly enough if they go on at all, to inter- 
 fere with current bacteriological methods." 
 
 The virulence of 1 strain was greatly raised 
 by inoculation into the anterior chamber of the 
 eye of a rabbit. Recovered from the eye 3 days 
 later, 5 cc. kiUed a rabbit in 24 hours (intraperi- 
 toneally). 1 cc. of culture from this last rabbit 
 killed a third in 3 wks. "Although this strepto- 
 coccus was raised in virulence from nil to the maxi- 
 mum, it showed no changes in its cultural characters." 
 
 Virulence was markedly increased by animal 
 passage. 
 
 "Chain formaiion of the Streptococcus lacticus 
 is favored by animal passage and by cultivation in 
 media containing blood serum, without the addition 
 of carbohydrate." 
 
 Both strains originally non-Kemolytic acquired 
 to some extent the power to hemolyze human and 
 goat blood after animal passage. 
 
 "By animal passage, the amount of acid produced 
 by the original strain progressively decreased, and 
 fermentation of some of the substances was inhibited." 
 (Notably salicin and mannite.) 
 
 " The fermentative ability is not changed materially 
 by repeated transfers through litmus milk." 
 
 With regard to growth on blood agar plates: 
 "The results were not always constant and in two 
 or three cases it was uncertain in which group the 
 organism belonged." 
 
 With regard to fermentation reactions: "The re- 
 sults of the second series of titrations (2 weeks 
 after the first and in 2 per cent instea:d of 1 per cent 
 peptone broth) were similar to those of the first, except 
 that in a number of instances salicin was fermented in 
 the second series only. This is in all probability due 
 to the more favorable character of the former (2 per 
 cent peptone) medium." 
 
 Reports variations with regard to the fermenta- 
 tion of salicin and rafSnose by individual colonies 
 from plates of supposedly pure cultures of strepto- 
 cocci. 
 
 Reference. 
 
 Smith and Brown 
 
 — Continued. 
 
 19IS. 
 
 Hartzell and Henricl 1915. 
 A. Study of Streptococci from 
 Pyorrhea Alveolaris and from 
 Apical Abscesses. 
 
 Heinemann 1915. 
 
 The Variability of Two 
 
 Strains of Streptococcus lacticus. 
 
 KUgler 1915. 
 
 A Study of the Correlation 
 
 of the Agglutination and the 
 
 Fermentation Reactions among 
 
 the Streptococci. 
 
 Thro 1915. 
 
 Further Experiments on the 
 Variability of the Fennenta- 
 tive Reaction of Bacteria, Es- 
 pecially the Streptococci. 
 
TABLE VII — Continued. 
 Mutations and Variability. 
 
 Results and conclusions. 
 
 Of 134 representative strains retested in carbo- 
 hydrate media at intervals for 1 yr., 63-77 per cent 
 remained constant. The variation which did occur 
 took place along certain well defined paths from 
 which tie author constructs a "family-tree" con- 
 cerning which she says: "A glance * * will show 
 (i) tluit these 134 strains included several stable 
 groups of considerable size; (2) that between these 
 groups there are usually well-worn paths; (J) that the 
 variation is more often progressive; and {4) that these 
 combinations fall into three main groups, each prob- 
 ably sharing a different ancestral history, through 
 lactose, through saccharose, or through salicin." 
 
 Variations of temperature and of media were at- 
 tended by only temporary variations in the charac- 
 teristics of the organisms. 
 
 The eji:posure of strains to the action of saliva, 
 intestinal extract, and to the products of growth of 
 other strains caused changes in fermentation reac- 
 tions and minor changes in appearances on blood 
 agar. Exposure to raw or heated milk caused no 
 change. 
 
 Strains recovered after being fed (within or with- 
 out celloidin capsules) to puppies showed changes 
 in fermentation reactions and in hemolytic prop- 
 erties. 
 
 "The hemolytic properties and fermentative reac- 
 tions in various sugar media of the streptococci from 
 the tear sac (of the patient) were studied from time 
 to time after growth on culture media and after stic- 
 cessive passages through animals. The zone of 
 hemolysis on blood agar plates became somewhat 
 smaller after 2 months growth on culture media {blood 
 agar). After 6 month the 11th subculture showed 
 no further decrease in the zone of hemolysis." 
 
 "When first isolated, cultures formed acid in media 
 containing dextrose, saccharose, lactose, or salicin, 
 but not in media containing raffinose, munnite, or 
 inulin. In later cultures (2 months), mannite and 
 rafinose were inconstantly fermented. After passage 
 through a rabbit [intraocular injection), the strepto- 
 coccus, being recovered in pure culture, when plcu:ed in 
 sugar media again gave the same reactions as when 
 first isolated from the patient." 
 
 The author believes that most reported mutations 
 or variations were due to (1) mixed cultures; (2) 
 unfavorable culture media; (3) insufficient incuba-, 
 tion; and (4) the recovery from animals of othfer 
 strains than the one injected. 
 
 ' *■ * no noteworthy changes in the morphology 
 or cultural characteristics of the invading organisms 
 were observed in frequent examinations of the milk 
 throughout the course of the infections." 
 
 Reference. 
 
 Broadhurst 1915. 
 
 Environmental Studies of 
 Streptococci with Special Ref- 
 erence to the Fermentative 
 Reactions. 
 
 Irons, Brown, and Nadler 
 
 1916. 
 The Localization of Strepto- 
 cocci in the Eye. A Study of 
 Experimental Iridocyclitis in 
 Rabbits. 
 
 Holman 1916. 
 
 The Classification of Strep- 
 tococci. 
 
 Mathers 1916. 
 
 Different Ty^t% of Strepto- 
 cocci and Their Relation to 
 Bovine Mastitis. 
 
TABLE VII — Concluded. 
 Mutations and Variability. 
 
 Kesults and conclusions. 
 
 During 9 mos. "only slight variations in cer- 
 tain properties, as for example changes in sugar reac- 
 tions, were occasionally noted. No radical change 
 was observed in the character of hemolysis on human 
 blood agar plates in any of the strains. I have, how- 
 ever, observed slight loss or gain in hemolytic power in 
 some of the strains, in others slight alterations in the 
 character of hemolysis, * * . That virulence of 
 streptococci can be altered by repeated transfer from 
 animal to animal, or by growth on artificial media, is 
 a well known phenomenon. * * Any alterations 
 suggesting the origin of mutants were not observed." 
 
 "My experience has been that under similar condi- 
 tions, a given organism, if freshly isolated (within 
 from 2 to 3 weeks), will produce a definite and uniform 
 type of hemolysis, and that the diameter of the hemo- 
 lytic zone will be constant within a moderate latitude.* * 
 These hemolytic characteristics change, however, in 
 some instances after prolonged cultivation on artificial 
 media." 
 
 Reference. 
 
 Davis 1916. 
 
 Hemolytic Streptococci 
 
 Found in Milfc. 
 
 Smlllie. 1917. 
 
 Studies of the Beta Hemo- 
 lytic Streptococcus (Smith and 
 Brown). 
 
TABLE VIII 
 
 General Conclusions with reference to the Correlation of 
 
 Source, Pathogenicity, Hemolysis, Fermentation 
 
 Reactions, etc. 
 
TABLE Vm. 
 
 General Conclusions with Reference to the Correlation of Source, 
 
 Pathogenicity, Hemolysis, Fermentation, etc. 
 
 Results and conclusions. 
 
 The authors regard the prevalent form of mild 
 infectious chronic mastitis in dairy cows as due to 
 the streptococcus described and transmitted from 
 cow to cow by the hands of the milkers. 
 
 The organisms were streptococci forming long 
 chains and an abundant soft deposit in bouillon 
 without clouding it. 
 
 The authors describe a small diplococcus growing 
 ai small chains in fluid media (mixture of equal 
 parts of milk and bouillon). 1% was Gram-positive. 
 Small transparent granular colonies were formed on 
 blood agar tno other particulars). No growth took 
 place on plain agar or gelatin. 
 
 Found that filtrates from bouillon cultures of 
 streptococci of human origin would no longer serve 
 as media for the growth of other strains, while sta- 
 phylococci and pneumococci grew well. He regards 
 this as a specific reaction indicating that all human 
 streptococci are of the same species. 
 
 One of the scarlet fever strains grew slightly in a 
 streptococcus filtrate and the "Streptococcus der 
 Druse" grew well. The former is regarded as a 
 variant and the latter as a dbtinct species. 
 
 The hemolytic activity and virulence of strep- 
 tococci were fotmd to be generally parallel. 
 
 " * * it has been found that highly virulent 
 pneumococci (such as have been passed through a 
 series of rabbits) possess a greater ptmer of hemolysis 
 than do those of a lower degree of virulence, * * ." 
 
 In general the strains pathogenic 'for animals 
 were hemolytic (few exceptions). 
 
 Strains. 
 
 Source. 
 
 Hemolysis. 
 
 Pathogenesis. 
 
 4 
 2 
 2 
 
 1 
 1 
 1 
 
 ? 
 Scarlet fever. 
 Normal Throats. 
 Intestine. 
 Cholecystitis. 
 Endocarditis. 
 
 Distinct. 
 
 Moderate. 
 
 None. 
 <t 
 
 tt 
 
 (t 
 
 Hi^. 
 
 Negative. 
 it 
 
 ti 
 
 tt 
 
 It 
 
 (The pathogenesis indicated is for rabbits.) 
 
 From work on agglutination the author concludes 
 that this is not a very reliable or delicate method 
 of determining relationship among streptococci. 
 
 He could find no quantitative relation between 
 hemolysis and virulence though "it seems that a 
 
 Reference. 
 
 Nocard and Mollereau 1887. 
 Sur une mammite contagi- 
 euse des vaches laitiSres. 
 
 Poynton and Paine 1902. 
 
 Eine kurze Zusammenfas- 
 sung der Resultate einer Un- 
 tersuchung betre£Eend die Path- 
 ogenesis des akuten Rheuma- 
 tismus. 
 
 Marmorek 1902. 
 
 LlJnit^ des streptocoques 
 pathongdnes pour I'homme. 
 
 (Die Artenheit der fur 
 Menschen pathogenen Strep- 
 tokokken.) 
 
 Schleslnger 1903. 
 
 Experimentelle Untersuch- 
 ungen tiber das Hamoljrsin der 
 Streptokokken. 
 
 Rosenow 1904. 
 
 Studies in Pneumonia and 
 Pneumococcus Infections. 
 
 Kerner 190S. 
 
 Experimenteller Beitrag zur 
 Hamolyse und zur Agglutina- 
 tion der Streptokokken. 
 
 Natvlg 1905. 
 
 Bakteriologische Verhalt- 
 
 nisse in weiblichen Genital- 
 sekreten. 
 
TABLE Vin — Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 streptococcus which has the power to produce a 
 considerable amount of hemolysis in alkaline media, 
 is in general virulent for the ordinary laboratory 
 animals." (Translation.) 
 
 "Man sieht, dass zwischen den 'longus'-dhnlichen 
 und den 'mitior'-dhnUchen Stiimmen keine scharfen 
 Grenzen zu Ziehen sind. Nach dem ohigen diirfte 
 die betreffende Eintheilung Schottmiiller's nur wenig 
 Berechtigung.haher.." . 
 
 The morphological, cultural, biochemical, and 
 pathogenic properties of the facultative anaerobic 
 streptococci were considered too variable to afford 
 a safe basis for differentiation. 
 
 A general relation between source, fermentative 
 characters, hemolytic powers, and virulence (with 
 numerous exceptions), is indicated in the authors' 
 tables. The following are the type forms. 
 
 Reference. 
 
 Natvig — Continued. 
 
 1905. 
 
 Andrewes and Herder 1906. 
 
 A Study of the Streptococci 
 Pathogenic for Man. 
 
 Species. 
 
 S. equinus. . . 
 
 S. mitis 
 
 S. pyogenes. . 
 S. salivarius. 
 S. anginosus. 
 
 S.fecaiis. . . . 
 
 Pneumo- 
 coccus . 
 
 + 
 
 + 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 + 
 
 ^■1 
 
 g 
 
 Medius. 
 
 Brevis. 
 
 Longus. 
 
 Brevis. 
 
 Longus. 
 
 Brevis. 
 Brevis. 
 
 
 ++ 
 
 ++ 
 
 Source. 
 
 + 
 
 Horse dung, air, 
 dust. 
 
 Human saliva 
 and feces. 
 
 Pathological con- 
 ditions. 
 
 Normal mouth 
 and intestine. 
 
 Pathological con- 
 ditions; sapro- 
 phytic. 
 
 Pathological con- 
 ditions; sapro- 
 phytic. 
 
 Pathological con- 
 ditions; sapro- 
 phytic. 
 
 " Scarlatina.— All the blood agar plates inocu- 
 lated with material from scarlatinal throats con- 
 tained many S. pyogenes colonies and varying num- 
 bers of green colonies." 
 
 "Normal throats. — All blood agar plates inocu- 
 lated with material from normal throats contained 
 many green and slightly hemolyzing greenish colo- 
 nies. S. pyogenes colonies were found in 30 of 51 
 throats which were studied, but were never present in 
 large numbers and were entirely absent in 21 cases 
 {41.2 per cent)." 
 
 Ruedlger 1906. 
 
 The Streptococci from Scar- 
 latinal and iJormal Throats 
 and from Other Sources. 
 
TABLE Via— Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 "Measles throats. — The findings in the throats 
 from measles patients corresponded closely to those of 
 normal throats.^' 
 
 "Tonsillitis. — S. pyogenes colonies always pre- 
 dominated in these plates, but green colonies also were 
 present." 
 
 "Pharyngitis." — In 4 cases no colonies of S. 
 pyogenes were found, but many green or brown colo- 
 nies were found. 
 
 "Pneumonia. — Three of the five sets of plates in- 
 oculated with material from the tonsils of pnemnonia 
 patients contained, very few S. pyogenes colonies, and 
 the other two sets did not contain o»y-. * * Typ- 
 ical pneumococci were easily isolated from all plates." 
 
 " * * green colonies were found in all blood 
 agar plates except two sets which were inoculated with 
 m'sterial from scarlatinal throats. * * Some of 
 these organisms seem to correspond to Schottmidler' s 
 S. viridans, but others are. very closely related to the 
 pneumococcus. In addition to the green colonies 
 there are often found slightly hemolyzing colonies 
 which may sometimes have a green tint. These colo- 
 nies, which are more abundant in 'plates inoculated 
 with material from normal throats and throats of 
 measles patients than in plates inoculated with ma- 
 terial from scarlatinal or tonsillitis throats, must not 
 be confounded with the S. pyogenes colonies." 
 
 Used human blood agar plates. Found non- 
 hemolytic streptococci in 16 per cent of normal 
 gravida and in a small percentage of normal puer- 
 pera, but hemolytic streptococci in those with puer- 
 peral fever. No hemolytic streptococci were found 
 in normal gravida or puerpera. 
 
 Could obtain no trustworthy results by aggluti- 
 nation because of spontaneous agglutination of 
 controls. 
 
 The clouding of bouillon and length of chain 
 bore no apparent relation to the source or other 
 characteristics of the strains studied. 
 
 Did not find the size of the hemolyzed zone to be 
 an index of virulence. 
 
 (See Table VII on Mutations and Variability.) 
 
 While prepared to admit that decidedly hemo- 
 lytic streptococci without a high degree of virulence 
 may be found, the author maintains that the find- 
 ing of hemolytic streptococci in puerperal cases is 
 always a grave indication. 
 
 Reference. 
 
 Ruedlger — Continued. 
 
 1906. 
 
 Gonnet 1907. 
 
 Streptocoque pyogSne et in- 
 fection puer^rale. 
 
 Nleter 
 
 1907. 
 
 Zur Streptokokkenfrage. 
 
 Mandelbaum 1907-08. 
 
 Zur Streptokokkenfrage. 
 
 Fromme 1908. 
 
 Klinische und bakteriolo- 
 gische Studien zum Puerperal- 
 fieber . 
 
 Heynemann 1908. 
 
 Die Bedeutung der hamo- 
 lytischen Streptokokken fur 
 die puerperale Infection. 
 
TABLE vni — Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 The authors could attach no prognostic value to 
 the hemolytic character and could note no relation 
 between hemolysis and virulence as judged either 
 by the condition of the patient or by inoculation of 
 the strains into mice. 
 
 "Es geht daraus mil voller Klarheit hervor, dass 
 sich zwar unter den hdmolytischen Streptokokken 
 tier- und voraussichtUch menschenrirulente haufiger 
 finden als unter den nichthamolytischen, dass diese 
 Eigenschaften aber nicht vollig parallel gehen, dass 
 sie nicht voneinander abhdngen." 
 
 "Ich halte das hSmolytische VermSgen bis zum 
 gewissen Grade fiir einen Energiebeweis, da die nicht- 
 hamolytischen Streptokokken sich im aUgemeinen 
 schwerer fortzUchten lassen, langsamer wachsen, vie- 
 niger Saure bilden etc." 
 
 " * * Of the 30 scarlet fever throats, 8 cases 
 showed no hemolyzing colonies at all; 4 showed no 
 green colonies, and 18 showed both green and hemolyz- 
 ing colonies. Of the 24 measles throats, all showed 
 green colonies and 4 showed no hemolyzing colonies." 
 
 "In contrast to the throat cultures, these strains 
 from blood and pus {Z7 strains), when tested in pure 
 culture in blood plates each gave good hemolyzing 
 colonies with one exception." 
 
 Concludes that: Hemolysis and virulence do not 
 run parallel since after long cultivation strains 
 may still be hemolytic but may have lost their 
 virulence. On the other hand, hemolysis is a prop- 
 erty of strains which under proper conditions may 
 be very pathogenic. 
 
 Hemolysis and acid production are both depend- 
 ent upon the ability of the streptococci to mul- 
 tiply. HeAolysis, however, is not a function of the 
 acid produced in cultures. Dextrose bouillon di- 
 minishes the hemolytic activity only by checking 
 the growth of the culture because of a development 
 of acid. 
 
 The author concludes that although hemolysis 
 on blood agar is no index of virulence, yet this does 
 not contradict Schottmiiller's conclusion that the 
 streptococci of severe infections are hemolytic. 
 
 Found streptococci from cases of severe puer- 
 peral sepsis always hemolytic, and considers the 
 finding of hemolytic streptococci in such cases 
 always a grave prognostic sign. 
 
 Graduated amounts of 2 per cent lecithin emul- 
 sion and 24 hr. bouillon cultures of hemolytic strep- 
 tococci were mixed and incubated. The growth of 
 strains isolated from the more severe pathological 
 
 Reference. 
 
 Ludke and Polano 1909. 
 
 Ueber Hamolyse der Strep- 
 tokokken. < 
 
 Zangemelster 1909. 
 
 Die Hamolyse der Strepto- 
 kokken. 
 
 Anthony 1909. 
 
 Some Characteristics of the 
 Streptococci Found in Scarlet 
 Fever. 
 
 Sachs 1909. 
 
 Ueber Streptokokken-Hamo- 
 lyse. 
 
 Slgwart 1909. 
 
 Untersuchungen fiber die 
 
 Hamolyse der Streptokokken 
 
 in der Schwangerschaft und im 
 
 Wochehbett. 
 
 Fromme 1909. 
 
 Die Streptokokken in dem 
 Genitalsekret von Schwangeren 
 und Wochnerinnen. 
 
 Fromme 1909. 
 
 Neue Untersuchungen uber 
 die Differenzierung der hamo- 
 lytischen Streptokokken. 
 
TABLE "Vlll — Continued. 
 General Conclusions. 
 
 Kssults and conclusions. 
 
 conditions was inhibited less by the lecithin than 
 that of those from mild infections. 
 
 Claims that in stained films of milk sediment 
 one can recognize certain streptococci as "para- 
 sitic" by their paired arrangement within the 
 chains and the broad flattened form {" Staketten- 
 form") of the elements. "In bestimmten morpho- 
 logischen Merkmalen, z. B. Querstelhmg der Veil- 
 gUeder, kapselahnUcher UmhuUung und anderem, 
 haben wir ein Mittd, aus dem Eider stammende 
 Streptokokken von nackkagUch in die Milch gelangien 
 zu unterscheiden." These observations refer to the 
 appearance of the streptococci in the milk only, 
 not in cultures. 
 
 Claims that S. lacticus can be distinguished from 
 5. pyogenes by the larger amount of acid formed, 
 growth at lower temperature, lanceolate mor- 
 phology, formation of mvolution forms, and the 
 appearance of colonies on agar. 
 
 Regards the organisms isolated by him from 
 cases of endocarditis as modified pneumococci, 
 which, however, when isolated come to resemble 
 S. viridans. "{!) The chains are made up of dis- 
 tinct elongated diplococci, often lanceolate in shape, 
 * * . (2) They always produce a variable amount 
 of green on blood agar, but usually much less than the 
 typical pneumococci, and never hemolysis. {3) All 
 the strains ferment inulin." 
 
 (See also Table vn on Mutations and Variability.) 
 
 Thinks that the amount of hemolytic activity 
 may have some relation to virulence but states 
 that differences between S. vulgaris and 5. viridans 
 can not be explained as being due merely to differ- 
 ence in virulence since "Eine lange fortgesetzte 
 kilnstliche Zilchtung der Stdmme, viodurch ivohl 
 sicher atich die Virulenz dem Menschen gegen-tiber 
 beeintrachtigt wird, verSndert die W achstumseigen- 
 tiimlichkeiten nicht." 
 
 "Le streptocoque de la gourme posside les mimes 
 caraclhres morphologiqties el culturels des strepto- 
 coques pyogenes et de I'erysipile. Dans les cultures 
 en agar-sang et dans le bouillon-soMg, il monlre de 
 possider les proprietis himolytiques du Strepto- 
 coccus longus seu erysipelatos." 
 
 The streptococci found on the hands of mid- 
 wives, in normal secretions, and on furniture, walls, 
 and other surroundings (in the hospital ?) were all 
 non-hemolytic, while almost all of those found, in 
 wounds and septic processes, especially puerperal 
 sepsis, were hemolytic. 
 
 H^ference. 
 
 Fromme — Continued. 
 
 1909. 
 
 Ernst 1909. 
 
 Ueber Milchstreptokokken 
 und Streptokokkenmastitis. 
 
 Bahr 1910. 
 
 Vorkommen und Bedeutung 
 der Streptokokken in der 
 Milch. 
 
 Rosenow 1910. 
 
 A Study of Pneumococci from 
 Cases of Infectious Endocar- 
 ditis. 
 
 Schottmuller 1910. 
 
 Endocarditis lenta. Zug- 
 
 leich ein Beitrag zur Artunter- 
 
 scheidung der pathogenen 
 
 Streptokokken. 
 
 Prlcolo 1910. 
 
 Recherches exp^rimentales 
 sur le streptocoque de la 
 gourme. 
 
 Zangemeister 1910. 
 
 Ueber die Verbreitung der 
 
 Streptokokken im Hinblick auf 
 
 ihre Infektiositat und ihre hi- 
 
 molytische Eigenschaft. 
 
TABLE Yin— Contimied. 
 
 General Conclusions. 
 
 Results and conclusions. 
 
 The author concludes that: "Die hamolytischen 
 Streptokokken sind stels als infektios anzusehen; 
 denn sie verdanken ja ihre Hamolyse niir die F'dhig- 
 keit, kiirzlich infiziert zu haben." 
 
 "Die anhamolytischen Streptokokken sind keines- 
 wegs amsckliesslich harmlose Schmarotzer," since some 
 of them at least may become virulent. 
 
 "Die Nichthamolyse bietet also keine Garantie fUr 
 eine Amrtdenz." 
 
 Conducted experiments with Marmorek's test 
 and concludes that "Day Verhalten der Streptokok- 
 ken im Bouillonfiltrat spricht fiir ihre Einheit," but 
 classifies the various varieties as follows: 5. pyo- 
 genes, S. longissimus, S. conglomeratus A and B, 
 and 5. brevis — according to their growth on gly- 
 cerol agar, blood agar, and in bouillon. 
 
 Regards all but S. pyogenes as common inhabit- 
 ants of normal throats and regards angina as the 
 common means of dissemination of S. pyogenes. 
 
 Cited by E. W. A. Walker (1911) as follows: 
 Investigations carried out by Jensen "and Ms co- 
 worker Holth on a number oj microorganisms, in- 
 cluding streptococci, by the application of long series 
 of chemical and biological tests: Jensen states that 
 Holth has examined 150 different strains of strepto- 
 cocci in relation to all the carbohydrates and poly- 
 valent alcohols which he could obtain, and to a number 
 of glucosides in addition. The conclusion arrived at 
 is that culture in bouillon containing these substances, 
 followed by titration of the reaction affords a valuable 
 means of identification, differentiation and classifica- 
 tion of these organisms. These observers regard lac- 
 tose, trehalose, cellobiose, and gentobiose as of great 
 value for the purpose of classification, and find that, 
 further differentiation is possible by the use of vari- 
 ous polyvalent alcohols and glucosides." 
 
 " Using these methods Holth and Jensen find, even- 
 in 'closely similar conditions of disease,' that the 
 strains of streptococci present in different cases are 
 different, * * . Jensen states that Holth' s ex- 
 periments {not yet published) lead to the conclusion 
 that the differences are actually fixed." 
 
 " * * Jensen next proceeds, to discuss the 
 special streptococcal disease of horses, namely, 
 strangles. After pointing out the fact that the course 
 and pathology of this disease varies remarkably in 
 different cases, he states that Holth, * * , has 
 definitely proved them all to he identical (i.e., the 
 streptococci). And the constancy of the reactions 
 thus found is not attributable to the residence of the 
 organisms in the horse, since this animal harbours 
 many other streptococci which present quite different 
 
 Reference. 
 
 Zangemelster — Continued. 1910. 
 
 Thalmann 1910. 
 
 Streptokokkenerkrankungen 
 in der Armee. Einteilung der 
 Streptokokken und ihre Be- 
 kampfung. 
 
 Jensen 1910. 
 
 Adkillelse og gruppering of 
 
 naerstaaende bakterieformer 
 
 ved forhold til kemiske for- 
 
 bindelser. 
 
TABLE vm — Continued. 
 
 General Conclusions. 
 
 Results and conclusions. 
 
 series 0} reactions in the same media." Holth 
 studied about 40 different strains of strangles 
 streptococci from Danish, German. English, and 
 Swedish horses. 
 
 The authors point out that although hemolysis 
 on blood agar is not an index of virulence, yet 
 they have found the rapidity with which hemoly- 
 sis occurs to be significant, the virulent strains 
 producing clear zones in less than 24 hrs. The 
 number of colonies of hemolytic streptococci found 
 on plates inoculated directly with blood (septi- 
 cemia) or with lochial secretion was also significant. 
 
 Appljdng the " Blutschvamm" and "lecithin" 
 tests of Fromme the authors found virulence indi- 
 cated for hemolytic streptococci from both severe 
 cases of streptococcus infection and from normal 
 afebrile puerpera. 
 
 They conclude that there are "porteurs sains" 
 of hemolytic streptococci which may be sources of 
 infection for other individuals. 
 
 Referring largely to the work of Fromme (1909) 
 the author says: " Zusammenfassend mochie ich 
 behauplen, dass dem Lecithin auf pathogene Strep- 
 tokokken besonders wirksame Eigenschajten nicht 
 zukommen. Die wachstumhemmende Kraft des-Leci- 
 thins isl zum grSssten Teil an den Grad seiner 
 Aciditat gebunden und ist daher nicht spezifisch. 
 Eine Diferenzierung der hamolytischen Strepto- 
 kokken in verschiedene Arten ist vermittelst Lecithins 
 nicht mogUch." 
 
 Finding septicemia produced by organisms from 
 all the fermentative groups, the authors could dis- 
 cover no coordination between source, fermentation 
 reactions, and pathogenicity for rabbits. 
 
 Using Fromme's technique the author found no 
 relation between virulence (as judged by the sources 
 of the strains) and growth in lecithin bouillon. 
 
 Did not regard hemolysis as an index of relative 
 virulence of strains. 
 (See Table VII on Mutations and Variability.) 
 
 Reference. 
 
 Jensen — Continued. 
 
 1910. 
 
 Fabre and Bourret 1910. 
 
 Quelques notions nouvelles 
 sur les streptococcies des suites 
 de couches. 
 
 Sachs 1910. 
 
 Ueber die Bedeutung des 
 
 Lecithins fiir die Unterscheidung 
 
 verschiedener Arten unter den 
 
 hamolytischen Streptokokken. 
 
 Beattle and Yates 1911. 
 
 Sugar Tests and Pathoge- 
 nicity in the Differentiation of 
 Streptococci. 
 
 Maehtle 1911. 
 
 Zur Differenzierung der ha- 
 molytischen Streptokokken mit- 
 tels Lezithinbouillon.' 
 
 Thalmann 1911. 
 
 Weitere Mitteilungen uber 
 Streptokokken, inbesondere 
 iiber pyogene Streptokokken 
 bei Erkrankungen der At- 
 mungsorgane und deren Kom- 
 plikationen. 
 
TABLE VIII — Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 "The conclusion, therefore, is apparently un- 
 avoidable that, in spite of the very extensive observa- 
 tions of Gordon, Houston, and Andrewes and Border 
 on the streptococci, there is still no evidence of the 
 existence in the human subject of more than one 
 microorganism, Streptococcus, though this may vary 
 as greatty in its chemical reactions in different cases 
 as it is known to do in virulence. This view agrees 
 with those consistently advanced by Marmorek and 
 others." 
 
 Found hemolysis on blood agar to be no index 
 of virulence. Found hemolytic streptococci which 
 were non-virulent for animals, and a strain of S. 
 anhemolyticus vulgaris (septicemia) which was 
 quite virulent for mice. Isolated S. viridans 13 
 times and 5. anhemolyticus vulgaris twice from the 
 blood in cases of s^sis. 
 
 S. acidi lactici was found to resemble S. lanceo- 
 latus (the pneumococcus) culturally and morpho- 
 logically and these two species are considered by 
 the autiior to be nearly related. 
 
 The author does not find that hemolysis and 
 virulence run perfectly parallel but believes them 
 both associated with a parasitic ("carnivorous 
 metabolism") mode of life. The presence of sugar 
 in the medium results in a suppression of the car- 
 nivorous metabolism and the adoption of an her- 
 bivorous metabolism — saprophytic existence. Ex- 
 perimentally he finds hemolysis in blood bouillon 
 or blood agar much reduced by the presence of sugar 
 in the medium. 
 
 (See Table VII on Mutations and Variability.) 
 
 Pathogenesis and hemolytic activity were found 
 to bear no relation to each other. 
 
 "The difierentiation of mastitis streptococci 
 among themselves as well as from saprophytic 
 streptococci, is not possible from the standpoint of 
 cultural methods only." (Translation.) 
 
 Bouillon was somewhat clouded by only 5 of the 
 mastitis strains. Of the 26 mastitis strains 11 
 formed chains of less than 50 elements and 9 or 
 possibly 11 formed chains of over 400 elements, 
 leaving only 4 of intermediary length. 
 
 Does not believe that the morphological peculi- 
 arity described by Ernst (1909) can be relied upon 
 for the differentiation of mastitis streptococci from 
 others. 
 
 Reference. 
 
 Walker 1911. 
 
 On Variation and Adaptation 
 in Bacteria, Illustrated by Ob- 
 servations upon Streptococci, 
 with Special Reference to the 
 Value of Fermentation Tests as 
 Applied to these Organisms. 
 
 Roily 1912. 
 
 Experimentelle bakteriolo- 
 
 gische Untersuchungen von 
 
 verschiedenen Streptokokken- 
 
 stammen. 
 
 Saito 1912. 
 
 Versuche zur Abgrenzung 
 
 des Streptococcus acidi lactici 
 
 von Streptococcus pyogenes und 
 
 Streptococcus lanceolatus. 
 
 Kuhn 1912. 
 
 Einfiuss von Zucker auf 
 Hamolyse und Virulenz. 
 
 Ominder 1912. 
 
 Untersuchungen uber Mas- 
 
 titisstreptokokken und ihre 
 . Differenzierung von saprophy- 
 
 tischen Streptokokken. 
 
TABLE VHI — Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 In reply to Gminder the author states that he 
 had not maintained that the differential characters 
 previously described by him (1909) persisted during 
 cultivation on artificial media. 
 
 Concludes that bovine milk or mastitis strepto- 
 cocci are harmless as regards summer diarrhea of 
 children, that there is no difference in the strepto- 
 coccal flora of the intestines of normal children and 
 of those with diarrhea, and that there is no reason 
 to believe that streptococci are the cause of sum- 
 mer diarrhea in infants. 
 
 In reply to Puppel the author points out that he 
 maintained only- that the finding of "Staketen- 
 form" streptococci in milk was diagnostic of mas- 
 titis in the cows giving the milk, but did not main- 
 tain that all bovine mastitis was caused by such 
 streptococci. 
 
 Considers Schottmiiller's differentiation of S. 
 hemolyticus and S. mitior fundamental. 
 
 States that pyogenic streptococci from the 
 mouth are often less hemolytic than those from 
 suppurations: "Es finden sich alle Abstufungen," 
 however. 
 
 Found comparative uniformity in the cultural 
 characteristics of the S. pyogenes (hemolyticus) 
 group but regarded the S. mitior group as made up 
 of a number of varieties. 
 
 The streptococci found in human feces and in 
 pathological processes related to the intestine, 
 e.g., appendicitis and peritonitis, produced green 
 colonies on blood agar, were non-pathogenic for 
 mice, and clouded bouillon. The green-producing 
 streptococci from the mouth, on the other hand, 
 were pathogenic for mice. S. longiisimus formed 
 large moist green colonies on blood agar, produced 
 very long chains of ovoid elements without cloud- 
 ing the bouillon, and was quite pathogenic for 
 mice. The author proposes for S. longissimus a 
 special place in the S. viridans {mitior) group. 
 
 "Virulence and hemolytic power are closely asso- 
 ciated amongst pathogenic streptococci," provided 
 the hemolytic power is measured by the special 
 method used by the author. 
 
 (See Table IV on The Use of Blood Bouillon and 
 Other Fluid Media.) 
 
 Did not employ blood agar or fermentation re- 
 actions, but from a study of the various strains 
 in bouillon, on agar, in milk, and injected into 
 mice, the author is unable to differentiate viru- 
 lent from avirulent streptococci or bovine mastitis 
 strains from those of human origin. 
 
 Reference, 
 
 Ernst 1912. 
 
 Eine Entgegnung zu A. 
 
 Gminders Arbeit: "Untersuch- 
 
 ungen uber Mastitisstrepto- 
 
 kokken u. s. w." 
 
 Puppel 1912. 
 
 Ueber Streptokokken in der 
 Milch und im Sauglingsstuhl. 
 
 Ernst 1912. 
 
 Eine Berichtigung zu R. 
 Puppels Arbeit: "Ueber Strep- 
 tokokken irL der Milch und in 
 SSuglingsstuhl." 
 
 Thalmann 1912. 
 
 Streptococcus viridans im 
 Blut ohne Veranderung der 
 Heizklappen. Zur Einteilung 
 der Streptokokken. 
 
 M'Leod 1912. 
 
 On the Hemolysin Produced 
 by Pathogenic Streptococci, 
 and on the Existence of Anti- 
 hemolysin in the Sera of Nor- 
 mal and Immunized Animals. 
 
 Salus 1912. 
 
 Untersuchungen zur Hygiene 
 der Kuhmilch. 
 
TABLE Vin — Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 "Streptococcus lacticus has no sanitary signifi- 
 cance, as it is found in nearly all samples of clean, 
 soured or fresh milk, and very often in the healthy 
 milk ducts. Streptococctis pyogenes, on the other 
 hand, seems to occur but rarely in milk and is in- 
 dicative of the existence of an inflamed condition 
 of the udder of the cow furnishing the milk." 
 (Quoted from abstract in Science.) 
 
 The author states that the differential diagno- 
 sis of streptococcus mastitis must depend upon the 
 finding of " Mastitisstreptokokken" in the milk 
 sediment. He agrees with Ernst that this strepto- 
 coccus can be differentiated microscopically. (See 
 Ernst, 1909.) 
 
 The author divides streptococci into four main 
 groups: 5. longus, S. lacticus, S. mucosus, and pneu- 
 mococcus. 
 
 Under 5. longus {pyogenes) he includes strepto- 
 cocci forming chains of six or more elem'ents and 
 describes as belonging to this group: (1) 5. pyogenes 
 (hemolytic), (2) the obligate anaerobic streptococci, 
 (3) 5. conglomeraius, (4) 5. longissimus, (5) '5. mitis 
 sell viridans. " * * halte ich den S. longissimus, 
 S. conglomerattts {ebenso wie den viridans) nur fUr 
 Modifikationen des S. longus, die ihre EigentUmr- 
 lichkeiten in Reaktion auf die Einwirkung der heson- 
 deren Oertlickheit gebildet kaben, aber kein 'Arten' 
 darstellen." 
 
 With regard to S. viridans found in the blood in 
 cases of endocarditis he says,^"/cA nehme hieran, 
 dass sich die Hdmolyse hier unter der Einwirhung des 
 erkrankten Organismus verloren hat." 
 
 "Infektionen des Darmes." — "The streptococci 
 found in the normal. intestine are the same as those 
 found in the mouth, throat, and sputum — the 
 non-hemolytic 5. longus and especially 5. lacticUs 
 (Kruse)." 
 
 "Infektionen im Puerperium." — ^The author found 
 streptococci resembling 5. longus pathogenes in every 
 respect in most vaginas examined. He states that 
 hemolytic streptococci are common in normal vag- 
 inal secretions and lochial discharge, and are of no 
 pathogenic significance. 
 
 In fihns from throats and exudates the cocci 
 occurred in pairs and short chains, often paired 
 within the chain, were strongly Gram-positive and 
 had definite capsules which were soon lost during 
 artificial cultivation. 
 
 ■ "Tke organism isolated, is an encapsulated hemo- 
 lytic streptococcus of high virulence. It appears to 
 occupy a position between the ordinary hemolytic 
 streptococcus and the Streptococcus mucosus." 
 
 Reference. 
 
 Ruedlger 1912. 
 
 A Study of Thirty-Five 
 Strains of Streptococci Iso- 
 lated from Samples of Milk. 
 
 Trommsdorfl 1912, 
 
 Ueber den gegenwartigen 
 Stand der Mastitisfrage in 
 ihrer Beziehung zur Milch- 
 hygiene. 
 
 von Llngelshelm 
 
 Streptokokken. 
 
 1912. 
 
 Davis and Rosenow 1912. 
 
 An Epidemic of Sore Throat 
 Due to- a Peculiar Strepto- 
 coccus. 
 
TABLE VIII — Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 Reference. 
 
 "Tke streptococci of the sore throat and the nor- 
 mal throat show no cultural differentiation in rela- 
 tion to the carbohydrates used. Virulence tests might 
 have separated the two' groups." 
 
 " The throat streptococci do not readily ferment at 
 20°C. any of the sugars used, while the milk organisms 
 attack the same sugars and to the same extent at this 
 temperature as at 37°C. This is, perhaps, the most 
 valuable differential feature between chained cocci from 
 these two sources." 
 
 Stowell and HlUiard 1912. 
 A Comparison of the Strep- 
 tococci from Milk and from 
 the Human Throat. 
 
 "The epidemic of sore throat in Chicago in the 
 winter of 1911-12 was caused by an organism be- 
 longing to the streptococcus group. In the exu- 
 dates and in the body it was usually encapsulated, 
 but not infrequently in the throats a capsule was not 
 present. It was highly pathogenic for animals, 
 readily producing arthritis in rabbits, and occasionally 
 endocarditis. * * From a typical case of mastitis 
 in a cow from a farm in this region a streptococcus 
 was obtained pure which * on animal passage 
 agreed in all essential respects to the human epidemic 
 streptococcus. A coccus identical in morphology, in 
 culture, and in pathogenicity was obtained from a 
 human case of tonsillitis and arthritis on the same 
 farm. The relation of these streptococci to the com- 
 mon hemolytic variety. Streptococcus pyogenes, is 
 certainly very close. They may be identical, the dif- 
 ferences noted being caused by environmental factors." 
 
 Davis 1912. 
 Bacteriologic Study of Strep- 
 tococci in Milk in Relation to 
 Epidemic Sore Throat. 
 
 (See Table VII on Mutations and Variability.) 
 
 Rosenow 1912. 
 Experimental Infectious En- 
 docarditis. 
 
 "Streptococci, virulent for animals, but which 
 differ from typical Streptococcus pyogenes in a more 
 abundant growth, in being encapsulated and not 
 forming chains, and in causing but little hemolysis, 
 occur in predominating numbers in epidemic sore 
 throat of milk-borne origin." 
 
 Rosenow 1912. 
 
 A Study of Streptococci 
 
 from Milk and from Epidemic 
 
 Sore Throat and the Effect of 
 
 Milk on Streptococci. 
 
 There was no apparent relation ,between the 
 hemolytic power of the organisms and the condition 
 of the patients from which they were isolated. 
 
 The cocci and diphtheroid organisms were found 
 to constitute the 'chief flora of the vagina, as they 
 do of other mucous membranes (nose, throat, 
 mouth). 
 
 The author does not regard hemolysis as of great 
 jrognostic or pathogenic significance since hemo- 
 ytic cocci were found in normal cases. Furthermore 
 the hemolytic powfer of one and the same strain of 
 streptococcus was found variable. 
 
 Sitzenfrey and Vatniek 1913. 
 
 Zur Frage der prognostischen 
 
 und praktischen Verwertung 
 
 bakteriologischer Befunde bei 
 
 puerperalen Prozessen. 
 
TABLE VUl— Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 No correlation between hemolytic activity and 
 fermentation reactions was observed. 
 
 "We believe that the foUowing features are sufficient 
 to separate milk from the throat streptococci: (/) they 
 yield over 2.5 per cent acid in lactose and saccharose at 
 37''C.; (2) they seldom ferment a substance higher than 
 saccharose in the metaboUc series (Dextrose, Lactose, 
 Saccharose, Salicin, Raffinose, Inulin, Mannite); 
 (J) they readily grow in dextrose, lactose, and saccha- 
 rose at 20°C. Throat streptococci, on the other hand, 
 (i) seldom yield over 2.5 per cent acid in any substance 
 at any temperature; (2) over 40 per cent yield 1.2 
 per cent acid in either salicin or raffinose or in both 
 at 37°C.; (J) at 20°C. they almost never attack any 
 of the test substances." 
 
 Reference. 
 
 Stowell, Hllllard, and 
 
 Schlesinger 1913. 
 
 A Statistical Study of the 
 Streptococci from Milk and 
 from the Human Throat. 
 
 
 
 Relations of Streptococci. ' 
 
 
 
 
 Blood agar. 
 
 
 s 
 
 t 
 
 A 
 
 k 
 
 
 
 } 
 
 .s 
 
 1 
 
 o 
 
 1/3 
 
 s 
 
 1 
 
 .3' 
 1 
 
 1 
 
 ■&I 
 
 
 Organisms. 
 
 1 
 
 u 
 
 si 
 
 o 
 
 5. hemolyticus . 
 
 +++ 
 
 
 
 
 
 
 
 
 
 + + + 
 
 ± 
 
 S. epidemicus. 
 
 ++ 
 
 
 
 + 
 
 ± 
 
 
 
 + + + 
 
 • + 
 
 S. mucosus . . . 
 
 ± 
 
 ++ 
 
 +++ 
 
 +++ 
 
 ++ 
 
 + + 
 
 =b ' 
 
 S. pneumoniae. 
 
 
 
 +++ 
 
 + 
 
 ++ 
 
 ++ 
 
 ± 
 
 +? 
 
 S. viridans . . . 
 
 
 
 +++ 
 
 
 
 
 
 + 
 
 =fc 
 
 +++ 
 
 Davis 1913. 
 
 Interrelations in 
 the Streptococcus 
 Group with Spe- 
 cial Reference to 
 Anaphylactic Re- 
 actions. 
 
 In addition to the differences noted in the tables 
 on Fermentation Reactions and on Pathogenicity 
 and Virulence the authors state that in horse serum 
 bouillon 5. equi usually produces less clouding than 
 S. longus. 
 
 "Die zwischen beiden Streptokokkenarten von 
 friiheren Untersuchern und von uns festgestdlten 
 neuen Unterschiede beweisen, dass der Streptococcus 
 longus seu erysipdatos und der Druse-Streptococcus 
 zwar nahe verwandte, aber verschiedene Arten sind." 
 
 The author believes that the pneumonia following 
 "la gourme" in horses is secondary and due to 5. 
 pyogenes, a different streptococcus from that caus- 
 ing "la gourme," having no Capsule and different 
 fermentation reactions. 
 
 "On the whole we fed that our work supports the 
 validity of Gordon's tests." 
 
 The results "would indicate in general that the 
 more active the fermentative powers, the less is the 
 virulence. * * the more virulent cultures 
 less profusely." 
 
 Koch and Poksehlschewsky 
 
 1913. 
 tJber die Artverschiedenheit 
 des Streptococcus longus seu 
 erysipdatos und des Strepto- 
 coccus equi (Druse-streptococ- 
 cus). 
 
 Bemelmans 1913. 
 
 La specificity des strepto- 
 coques de la gourme. 
 
 Floyd and Wolbaeh 1914. 
 
 On the Differentiation of 
 Streptococci. (Preliminary 
 
 Notes.) 
 
TABLE Yin— Continued. 
 
 General Conclusions. 
 
 Results and conclusions. 
 
 "While the properties of hemolysis and formation 
 of long chains in general'go with virulence, neither of 
 these properties is characteristically associated with 
 virulence, or grouping according to fermentative prop- 
 erties." 
 
 "The degree of hemolysis which the streptococci 
 tested produced in vivo was intimately related to their 
 virulence." 
 
 Regarding hemolysis in blood agar and in heated 
 serum bouillon cultures, " * * amongst the 
 strains of streptococci tested there is no intimate re- 
 lationship between the degree of hemolytic power as 
 observed in these artificial media and viridence for the 
 rabbit. Nor is there much relationship between the 
 amount of hemolysis on one medium and that on 
 another; and, lastly, there is no constancy in the amount 
 of hemolysis produced by any strain of streptococcus 
 when tested repeatedly on the same medium." 
 
 If, however, the streptococci are grown for 12- 
 18 iirs. in fresh unheated and undiluted serum 
 "of the species of animal for which it is desired to 
 determine the virulence of the streptococcus" and the 
 "hemolytic index" then determined, "it is seen at 
 once thai the last method affords a means of differenti- 
 ating very sharply between the virulent and non- 
 virulent streptococci, which under more artificial con- 
 ditions of cidture appear to he equal in hemolytic 
 power." 
 
 "Amongst hemolytic streptococci the power to pro- 
 duce hemolysin in the animal body, or in cultural 
 conditions closely resembling those met with in the 
 animal-body, is closely allied to virulence." 
 
 Notwithstanding the lack of hemolysis, the 
 authors think most of their strains agree quite well 
 with Davis' description of S. epidemicus. 
 
 "Bacteriological examination showed that cul- 
 tures from the throats of 4 patients contained strep- 
 tococci apparently identical with strains of strepto- 
 cocci obtained from the milk slime from the 2 cows 
 suffering with garget (Group D in Table V on Fer- 
 mentation Reactions). Cultures from the throats 
 of 8 other patients contained streptococci of this same 
 type, but differing, by slight variations only, in their 
 carbohydrate fermentations." 
 
 "Some of the strains, notably the mannite fer- 
 m'enters, produced no lysis. Others obtained from 
 septicemias produced a wide zone in less than 24 
 hrs. '* * The property is present to some ex- 
 tent in most streptococci, and while in general it is 
 more pronounced in the more virulent type, the dif- 
 ferences are essentially quantitative * * ." 
 
 Reference. 
 
 Floyd and Wolbach — 
 
 Continued. 
 
 1914. 
 
 M'Leod 1914. 
 
 Criteria of Virulence amongst 
 Streptococci, with Some Re- 
 marks on Streptococcal Leuco- 
 cidin. 
 
 North, White, and Avery 1914. 
 A Septic Sore Throat Epi- 
 demic in Cortland and Homer, 
 
 N. Y. 
 
 Hopkins and Lang 1914. 
 
 Classification of Pathogenic 
 Streptococci by Fermentation 
 Reactions. 
 
TABLE VIII — Coniinued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 Although "in general, the strains which coagulated 
 milk were of low pathogenicity, and vice versa; yet 
 there were many exceptions * * ." 
 
 "We found that most of the virulent pyogenic strains 
 formed long chains and that most -of those found in 
 mild infections of the mucous membranes formed short 
 chains. There were many exceptions, however. We 
 do not think, therefore, that morphology can he used as 
 a basis for classification, * * ." 
 
 "Serum broth is the most favorable medium for 
 the isolation and growth of streptococci. It is so 
 well suited to the growth of streptococci that in mixed 
 cultures, even vigorous organisms such as the bacillus 
 coli are overgrown in 24 hours." 
 
 "Cultures containing different forms of strepto- 
 cocci should be planted in serum broth for at least 24 
 hours before plating on blood agar. If this is not 
 done, many of the more pathogenic forms are liable to 
 be overlooked." 
 
 "Of the six strains isolated from the throats four 
 grow like the streptococcus isolated during the Chicago 
 epidemic two years ago. The other two * * re- 
 semble the ordinary hemolytic streptococcus. * * 
 The organisms from the cows and from the throats 
 agree in practically every detail." 
 
 The above mentioned organisms are tabulated as 
 being hemolytic, having capsules, being pathogenic 
 for rabbits, and fermenting dextrose, lactose, sac- 
 charose, and maltose. 
 
 "So far as we know at present, any appreciable 
 number of hemolytic streptococci found in cow's 
 milk obtained carefully in sterile tubes directly from 
 the cow's udder always means a diseased condition. 
 * * It is, of course, true that udder disease is often 
 caused by non-hemolytic streptococci, but as already 
 stated, so far as we know, these bear no relation to 
 sore throat. I have examined many of these organ- 
 isms and they have little or no virulence for animals. 
 On the other hand, the hemolytic streptococci, that I 
 have examined from the udder, are always pathogenic 
 to some degree for animals and usually quite highly 
 virulent." 
 
 (See also Table III on The Use of Blood Agar.) 
 
 Bases a system of classification on fermenta- 
 tion of salicin, raffinose, mannite, and inulin, and 
 the hemolytic character as determined by a special 
 method. 
 
 "The hemolytic titre does not afford any absolute 
 tilre of virulence." 
 
 "Hemolysin production is inhibited by the addition 
 of sugar. It does not appear to be associated with the 
 amount of acid produced." 
 
 (See Table IV on The Use of Blood BouiUon.) 
 
 Reference. 
 
 Hopkins and Lang — 
 
 Continued. 
 
 1914. 
 
 Holman 1914. 
 
 A Method for Making Car- 
 bohydrate Serum Broth of 
 Constant Composition for Use 
 in the Study of Streptococci. 
 
 Capps and Davis 1914. 
 
 An Epidemic of Streptococ- 
 cus Sore Throat in Jackson- 
 ville, 111., Which Was Traced 
 to the Milk of Cows Affected 
 with Streptococcus Mastitis. 
 
 Davis 1914. 
 
 The Growth and Viability 
 
 of Streptococci of Bovine and 
 
 Human Origin in Milk and 
 
 Milk Products. 
 
 Lyall 1914. 
 
 On the Classification of the 
 Streptococci. 
 
TABLE vni— Continued. 
 General Concliisions. 
 
 Results and conclusions. 
 
 "Hellmuth describes in detail a series of experi- 
 ments undertaken to show whether hemolysis does 
 occur with the vaginal bacteria during menstrua- 
 tion, and he finds that it does not. Even in two 
 cases in which metorrhagia had persisted for weeks 
 no hemolytic bacteria could be demonstrated. In 
 the three cases in which hemolytic bacteria were 
 found he thinks that they did not develop during 
 menstruation from non-hemolytic ones but reached 
 the vagina through invasion or inoculation. He 
 does not feel that he has proved that the faculty of 
 hemolysis could not develop under long continued 
 hemorrhage, as for example in cases of myoma, 
 but he thinks his experiments were sufficient to 
 prove that hemolysis is not merely the result of the 
 blood content of the nutrient medium of the bac- 
 teria." (Review from /. Am. Med. Assn.) 
 
 All the strains found to be pathogenic for rab- 
 bits were of the hemolytic type and belonged to the 
 following two fermentative groups. 
 
 I 
 S 
 
 S 
 i-l 
 + 
 
 .a 
 
 S 
 
 1. + + + + --- + 
 
 2. + + + -- + - + 
 (See also Table V on Fermentation Reactions.) 
 All but 2 of the strains in these 2 groups injected 
 
 into rabbits were pathogenic. 
 
 " The streptococci of cow mastitis or garget are dif- 
 ferent from the streptococci of human tonsillitis." 
 
 Epidemic milk-borne tonsillitis is regarded as 
 being probably due to streptococci of human origin 
 inoculated into the udders of cows during milking, 
 multiplying in the milk ducts and being shed into 
 the milk for some time after. 
 
 (See Table III on The Use of Blood Agar.) 
 
 "To summarize, we have shown that streptococci 
 of the viridans group are constantly present in peri- 
 dental suppurations. * * These streptococci are 
 normally of low virulence hut are at times able to 
 produce lesions of the heart, aorta, kidneys, and 
 joints, * * . We can not claim from the evi- 
 dence so far obtained, that these streptococci bear an 
 etiologic relationship to dental abscesses and pyorrhea, 
 but from the point of view of metastatic infections it is 
 important to know that such organisms are constantly 
 
 Reference. 
 
 Hellmuth 1914. 
 
 Does Menstruation Influ- 
 ence Hemolysis by Vaginal 
 Bacteria? 
 
 Smith and Brown 1915. 
 
 A Study of Streptococci Iso- 
 lated from Certain Presumably 
 Milk-Borne Epidemics of Ton- 
 sillitis Occurring in Massachu- 
 setts in 1913 and 1914. 
 
 Rosenow 1915. 
 
 The Bacteriology of Appen- 
 dicitis and Its Production by 
 Intravenous Injection of Strep- 
 tococci and Colon Bacilli. 
 
 Hartzell and Henriel 1915. 
 
 A Study of Streptococci 
 from Pyorrhea Alveolaris and 
 from Apical Abscesses. 
 
TABLE Ylll— Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 present in lesions presenting a large ulcerated surface, 
 through which they may, and probably do, frequently 
 pcLss into the deeper tissues and the blood stream." 
 
 " The agglutination reaction was not found to 
 separate the streptococci into large groups. How- 
 ever, by its correlation with the fermentation reac- 
 tions, the probable relationship of these types is indi- 
 cated." 
 
 "The agglutination tests tend to show that a divi- 
 sion of the streptococci on the basis of hemolysis is 
 not warranted, whereas a separation according to the 
 fermentation reactions appears to coincide more closely 
 with their natural relationship." 
 
 The author suggests the following classification: 
 
 "5. pyogenes. — SaUcin fermenters, which do not 
 ferment raffinose or mannite, are generally hemolytic, 
 and strongly pathogenic." 
 
 "S. salivarius.— Raffinose fermenters, ustially fer- 
 ment salicin, but do not ferment mannite, generally 
 produce a green colony on blood agar, and usually 
 coMse subacute and chronic infections. 
 
 "S. fecalis. — Mannite fermenters, generally fer- 
 ment salicin, rarely ferment raffinose, and are vari- 
 able in their reaction to blood and in their pathogenicity." 
 
 (See also Table III on The Use of Blood Agar.) 
 
 "Pathologic samples yield the greatest proportion 
 of hemolyzing strains. * * Hemolysis does not 
 seem to be correlated with the results in litmus milk, 
 gelatin, or with any of the Gordon reactions or com- 
 plexes." 
 
 "Strains from pathologic conditions and from 
 throats vften fail to develop in gelatin; strains from 
 other sources usually grow readily at room tem- 
 
 " Saprophytic strains are usually high fermenters." 
 "Morphologic characteristics are not, independently 
 or in connection with other characteristics, definitely 
 
 The authors endorse the conclusions of Smith and 
 Brown (1915) regarding the spread of tonsillitis 
 by the infection of cows with streptococci of hu- 
 man origin and the necessity of studying the strains 
 isolated for "cultural identity in every detail or im- 
 munological identity." 
 
 The strains isolated from throat and peritoneal 
 cavity of patients, from persons in the suspected 
 dairy, and from the cow "were the same in their 
 cultural characteristics." 
 
 "It would appear, then, that these epidemics of 
 appendicitis arid parotitis were due to streptococci 
 contained in, dairy products." 
 
 "The cultures from the dairy products showed a 
 preponderance of non-hemolyzing, short-chain-pro- 
 
 Keference. 
 
 Hartzell and 
 
 Henriel — Continued. 
 
 1915. 
 
 Kllgler 1915. 
 
 A Study of the Correlation 
 
 of the Agglutination and the 
 
 Fermentation Reactions among 
 
 the Streptococci. 
 
 Broadhurst 1915. 
 
 Environmental Studies of 
 Streptococci. 
 
 Krumwlede and Valentine 
 
 1915. 
 A Bacteriological Study of 
 an Epidemic of Septic Sore 
 Throat. 
 
 Rosenow and Dunlap 1916. 
 An Epidemic of Appendici- 
 tis and Parotitis Probably Due 
 to Streptococci Contained in 
 Dairy Products. 
 
TABLE VIII- 
 
 General Conclusions. 
 
 Results and conclusions. 
 
 ducing streptococci, often in almost pure form and 
 in enormous numbers. Slightly hemolyzing strepto- 
 cocci were found occasionally. All were of a relatively 
 low grade of virulence, but those producing parotitis 
 caused death (in rabbits) more frequently than did 
 those producing appendicitis." 
 
 "It would appear, then, that herpes zoster is due 
 to a streptococcus having elective affinity for the ganglia 
 and the posterior roots. The possibility, however, 
 that the disease in some instances may be due to other 
 bacteria having a similar affinity must be admitted." 
 
 "Since the streptococci lose the characteristic affinity 
 after cultivation on artificial media, after animal 
 passage, and apparently in the focus of infection after 
 recovery, the conclusion seems warranted that the 
 atrium of infection is not only the place of entrance, 
 but the place where the streptococci, by growth in 
 symbiosis with other bacteria and under varying 
 grades of oxygen pressure, may acquire the peculiar 
 properties necessary to infect in this particular 
 manner." 
 
 "The classification which has proved of practical 
 value during several years of routine use, and which 
 I wish here to report, is based on the reactions on 
 blood agar and the fermentation or non-fermentation 
 of the carbohydrates, lactose, mannite, saUcin, and 
 inulin in serum broth" 
 
 Streptococci, Gram-Positive Cocci in Chains, No Capsules. 
 H Hemolysis ' 
 
 Reference. 
 
 Rosenow and 
 
 TyxmlSiVr— Continued. 
 
 1916. 
 
 Rosenow and Oftedal 1916. 
 The Etiology and Experi- 
 mental Production of Herpes 
 Zoster. 
 
 Holman 1916. 
 
 The Classification of Strep- 
 tococci. 
 
 -| ^Lactose 
 
 ' I I. 
 
 -f-Mannite— H-Manmte- 
 
 -| ^Lactose 
 
 I ■ 1 
 
 +Manmte— -fMamute— 
 
 SaUcin Salicin Salicin Salicin SaUcin SaUcin SaUcin SaUcin 
 
 -t- -+ -+'-+ -+ -+ -+ -+ - 
 
 
 S -5 ■* 
 
 Co' Co Co 
 
 CO CO 
 
 Co Co 
 
 CO Co 
 
 
 8 
 8 
 
 ■§> •§• 
 
 
 (» 
 
 
 Co Co Co Co Co Co Co 
 
 The above scheme does not include inulin; in the 
 text, however, inulin-fermenting and non-ferment- 
 ing varieties of nearly all the non-hemolytic organ- 
 isms are mentioned, but no hemolytic inulin ferment- 
 ers are mentioned. 
 
TABLE Yin— Continued. 
 General Conclusions. 
 
 Results and conclusions. 
 
 "Hemolytic streptococci having a wide clear zone 
 occur commonly in both pasteurized and unpasteur- 
 ized {certified) milk. These strains vary among 
 themselves. They are more resistant to heat than 
 human strains of hemolytic streptococci, and possess 
 little or no virulence for rabbits, therefore in all proba- 
 bility none for man. They rapidly acidify and co- 
 agulate milk and grow well at 20°C. They form 
 short or long chains, but as seen in milk often appear 
 in pairs or a chain of few elements. "While they are 
 definitely hemolytic {Type /3, Smith and Brown), the 
 characteristics of the hemolytic zone on plates may 
 vary in certain respects." 
 
 " The milk strains are different from certain strains 
 of hemolytic streptococci found at times in diseased 
 udders of cows. These latter resemble the strains of 
 hemolytic streptococci from human sources, and are 
 virulent for rabbits." 
 
 "Between hemolytic area and pathogenicity there 
 exists no constant parallelism." 
 
 In 2 epidemics of septic sore throat streptococci 
 isolated from the throats of patients were identical 
 in cultural characteristics and agglutination reac- 
 tions with streptococci isolated from the milk sup- 
 plies and in 1 case from the udder of a cow in the 
 dairy. 
 
 " The types of streptococci isolated" from the above 
 sources "are identical in their cultural characteristics 
 and virulence for animals with the type of streptococci 
 described by Smith and Brown in their studies of the 
 milk epidemics of Massachusetts of 1914-1915. This 
 type of streptococcus is a human strain, with a group 
 of definite characteristics** ." 
 
 " The streptococcus of this type is seldom found in 
 normal throats {1% of the cases studied.)" 
 
 "The throats of individuals who have had tonsillitis 
 may harbor the beta hemolytic streptococcus for 3 
 months or more after acute symptoms of the disease 
 have subsided." 
 
 " This streptococcus is found in large numbers in 
 the throats of a certain proportion of cases of sporadic 
 tonsillitis {5 of 20 studied)." 
 
 " The beta streptococcus is frequently found in the 
 throats of acute cases of scarlet fever, and may remain 
 or months in the purulent discharges of the cases com- 
 plicated by otitis media, etc." 
 
 "These facts explain how the discharges from a case 
 of scarlet fever may contaminate a milk supply and 
 produce a milk-borne epidemic not of scarlet fever, but 
 of septic sore throat." 
 
 Reference. 
 
 Davis 1916. 
 
 Hemolytic Streptococci 
 
 Found in Milk. 
 
 Smlllle 1917. 
 
 Studies of the Beta Hemolytic 
 Streptococcus (Smith and 
 Brown). 
 
TABLE VIII — Concluded. 
 General Conclusions. 
 
 Results and Conclusions. 
 
 The following scheme of classification "for clinical 
 purposes" is advocated: 
 
 Streptococci, 
 Gram-positive, bile insoluble cocci in chains. 
 Hemolysis — ^Blood agar — Methemoglobin and 
 Indifferent 
 
 5. viridans 
 
 S. hemolysans 
 
 Lactose+ Lactose + Lactose— 
 Mannite— Mannite-h Mannite — 
 
 S. buccalis 5. fecalis S. equinus 
 "While it is fully recognized that more extensive 
 study of the growth-characteristics, pathogenicity, and 
 metabolic activities of various strains of streptococci 
 are of much value in experimental and epidemiological 
 work, such investigations are too cumbersome and time- 
 consuming, and of insufficient value for purposes of 
 routine identification. It is believed that adoption of 
 the proposed method of classification will serve to obvi- 
 ate the confusion that has hitherto existed and to pro- 
 vide an adequate means of identification of streptococci 
 for clinical purposes until such time as an immuno- 
 logical basis for classification shall be developed." 
 
 "A series of agglutination experiments by the author 
 though somewhat limited in number, lends weight to 
 the valtie of differentiation by the blood agar plate 
 method." 
 
 Reference. 
 
 Blake 1917. 
 
 The Classification of Strep- 
 tococci. 
 
Plate 1. 
 
 Fig. 1. (Page 7.) The method of allowing an agar plate to "dry" or 
 "harden" before incubation. 
 
 Fig. 2. (Page 28.) (a) The typical growth of Streptococcus epidemicus or of 
 Streptococcus mucosus on an agar slant. (6) The growth of Streptococcus epi- 
 demicus or of Streptococcus mucosus on an agar slant the upper part of which 
 has become somewhat "dried out." (c) The growth of Streptococcus pyogenes 
 and most other streptococci and pneumococci on an agar slant inoculated as 
 described. ., 
 
 Fig. 3. (Page 20.) Below the line: the appearance of three deep colonies as 
 viewed from the surface of anagarplate. Above the line : the same three colonies 
 as they would appear if viewed laterally. 
 
MONOGRAPH NO. 9. 
 
 PLATE 1. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 2. 
 Streptococcus D-ly. 
 
 Fig. 1. March, 1915. (Pages 17, 80.) Horse blood, fee; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 17.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Figs, la and 2a. Photomicrographs of a typical deep colony indicated by 
 arrows in Figs. 1 and 2. 
 
 Fig. 3. March, 1916. (Page 80.) Horse blood, | cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
MONOGRAPH NO. 9. 
 
 PLATE 2. 
 
 (Brown: Blood agar for study of streptococci.) 
 
•,,.v^;'y Plate 3. > 
 
 A; . Streptococcus D-ly. 
 
 Fig. 1. April, 1915. (Page 50.) Horse blood, f cc; standard veal Witte 
 peptone agar, 12 cc. ; dextrose, 1 per cent. Incubated 48 hours. 
 
 Fig. 2. April, 1915. (Page 50.) Horse blood, fee; standard Liebig extract 
 Witte peptone agar, 12 cc; dextrose, 1 per cent. Incubated 48 hours. 
 
 Fig. 3. (Page 50.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc. 
 Incubated 48 hours. 
 
 Fig. 4. (Page 50.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc; 
 dextrose, 0.1 per cent. Incubated 48 hours. 
 
 Fig. 5. .(Page 50.) Rabbit blood, | cc; Ruediger sugar-free agar, 12 cc; 
 dextrose, 1 per cent. Incubated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, and 5a. Photomicrographs of typical deep colonies indi- 
 cated by arrows in Figs. 1,2,3,4, and 5. 
 
MONOGRAPH NO. 9. 
 
 PLATE 3. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 4. 
 Streptococcus A-18 {Cow)p. 
 
 Fig. 1. March 16, 1915. (Page 30.) Horse blood, f cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 !Fig. 2. The same plate as shown in Fig. 1. Incubated 48 hours; refrigerated 
 48 hburs. ~ ' 
 
 Fig. 3. March 22, 191S. (Pages 29, 80.) Horse blood, f cc. ; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours: j. 
 
 Fig. 4. The same plate as shown in Fig. 3. Incubated 48 hours; refrigeratel 
 48 hours. ^ 
 
 Fig, 5. March, 1916. (Page 80.) Horse blood, f cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 6. (Page 30.) Washed corpuscles from f cc. of horse blood; fermentef 
 vea'l-infusion Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Figs, la, 2a, Sa, and 6a. Photomicrographs of typical deep colonies indicated 
 by arrows in Figs. 1, 2, 5, and 6. 
 
MONOGRAPH NO. 9. 
 
 PLATE 4. 
 
 i i 
 
 (Brown: Blood agar for study of streptococci.) 
 
•Tr . Plate 5. 
 
 ,.\ Streptococcus B-18 {Cow)0. 
 
 Fig. 1. Marcli, 1915. (Page^ 29, 80.) Horse blood, f cc; standard veal 
 ■Witte peptone agar, 12 cc. Incubated 48 hours. -.''■ 
 
 Fig. 2. The same plate as shown in Fjg. 1. Incubated 48 hours; refrigerated 
 48 hours. 
 
 Fig. 3. March, 1916. (Page 80.) Horse blood, f cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours: ' 
 
 Fig. 4. (Page 30.). Washed, corpuscles from f cc. of horse blood; fermented 
 veal infusion Witte peptone agar, 12 cc. Incubated 48 hours. 
 .' Figs, la, 2a, 3a, and 4a. Photomicrographs of typical deep colonies indicated- 
 . by arrows in Figs. 1,2, 3, and 4. , . 
 
MONOGRAPH NO. 9. 
 
 PLATE 5. 
 
 (Brown: Blood a?ac for study of streptococci.) 
 
Plate 6. 
 Streptococcus A-STp. 
 
 Fig. 1. March, 1915. (Pages 14, 80.) Horse blood, f cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 15.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 3. March, 1916. (Pages 32, .44, 80.) Horse blood, f cc.-; standard 
 veal Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 44.) Horse blood, J cc; standard LieWg extract Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 5. (Page 32.) Horse blood, 4 cc; standard veal Witte peptone agairj 
 12 cc Incubated 48 hours. 
 
 Fig. 6. (Page 32.) Horse blood, | cc; standard veal Witte peptone agar, 
 24 cc. Incubated 48 hours. 
 
 Fig. 7. (Page 32.) Horse blood. If cc; standard veal -Witte peptone agar, 
 2^ cc. Incubated 48 hours. 
 
 Fig. 8. (Page 48.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc. 
 Iijjpubated, 48 hours. 
 
 Fig. 9. (Page 49.) Rabbit blood, | cc; Ruediger sugar-free agar, 12 cc; 
 dextrose, 0.1 per cent. Incubated 48 hours. 
 
 ■ Fig. 10. (Page 49.) Rabbit blood, f cc; Ruediger sugar^free agar, 12 cc; 
 dextrose, 1 per cent: Incubated 48 hours. 
 
 Figs, la, 2a, 8a, and 9a. Photomicrographs of typical deep colonies indicated 
 by arrows in Figs. 1, 2, 8. and 9. 
 
MONOGRAPH NO. 9. 
 
 PLATE 6- 
 
 (Brown: Blood agar for study of streptococci.) '^^ 
 
Elate 7. 
 Streptococcus D-AD4 /3. 
 
 Fig. 1. March, 1915. . (Pages 26, 80.). Horse blood, | cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 26.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. • i- . 
 
 Fig. 3. (Page 38.) Rabbit blood, f cc; standard veal Witte peptoneragar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 38.) The same plate as shown in Fig. 3. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. S. (Page 38.)" Human, blood, f cc; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 6. (Page, 38.) The same, plate as shown in Fig. 5. Incubate'd 48 
 hours;- refrigerated 48 hours.- 
 
 Figs, la, 2a, 3a, 4a, 5a, and 6a. Photomicrographs of typical deep colonies 
 fndicated by arrows in Figs. 1, 2, 3, 4, 5, and 6. 
 
MONOGRAPH NO. 9. 
 
 PLATE 7. 
 
 
 
 * 
 
 •l ^ 
 
 
 
 o 
 
 o^ ^ 
 
 ii 
 
 i 
 
 -Jr.: . 
 
 
 
 \1 .0 .„;■■ 
 
 D i 
 
 !>«<) 
 
 i^ ■'-:.- 
 
 "J 
 
 4 ■ .:-» 
 
 <>i> 
 
 V 
 
 • • ' ' 
 
 ^v-* 
 
 ""*. 
 
 ■•K 
 
 
 
 ■Si^ .*V--'* 
 
 ..^m 
 
 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 8. , 
 Streptococcus D-AD4 p. 
 
 Fig. 1. March, 1916. (Pages 48, 80.) Horse blood, f cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. ' 
 
 . *;. F[G. 2. (Page 48.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc. 
 Incubated 48 hours. . ' 
 
 Fig. 3. (Page 48.) Rabbit blood, f cc. ; Ruediger sugar-free agar, 12 cc.; 
 dextrose, 0.1 per cent. Incubated 48 hours. 
 
 Fig. 4. (Page 48.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc; 
 dextrose, 1 per cent. Incubated 48 hours. 
 
 Figs. 5, 6, and 7. (Page 45.) Streptococci D-AD4 /3, C-64/3, and X-38 p re- 
 spectively. Horse blood, f cc; standard beef Digestive Ferments Co. peptone 
 agar, 12 cc. Incubated 48 hdurs. 
 
 .. Figs, la, 2a, 3a, and 4a. Photomicrographs of typical deep colonies indicated 
 by arrow's in Figs. 1, 2, 3, and 4. 
 
MONOGRAPH NO. 9. 
 
 PLATE 8. 
 
 (Brown : Blood agar for study of streptococci.) 
 
Plate 9. 
 Streptococcus B-2b {Cow) j3. 
 
 Fig. 1. March, 1915. (Pages 27, 80.) Horse blood, f cc; standard veaJ 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 27.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 3. March, 1916. (Pages 44, 80.) Horse blood, f cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 4. March, 1916. (Page 44.) Horse blood f cc; standard veal Diges- 
 tive Ferments Co. peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 5. (Page 49.) Rabbit blood, 1 cc; Ruediger sugar-free agar, 12 cc. 
 Incubated 48 hours. 
 
 Fig. 6. (Page 49.) Rabbit blood, 1 cc; Ruediger sugar-free agar, 12 cc; 
 dextrose, 1 per cent. Incubated 48 hours. 
 
 Figs, la, 2a, Sa, and 6a. Photomicrographs of typical deep colonies indicated 
 by arrows in Figs. 1, 2, 5, and 6. 
 
 Figs. 3a and 4a. Photomicrograpihs of top surface colonies indicated by arrows 
 in Figs. 3 and 4. 
 
MONOGRAPH NO. 9. 
 
 PLATE 9. 
 
 » 
 
 m 
 
 
 ^^1 
 
 m 
 
 > < 
 
 
 
 
 (Brownr Blood agar for study of streptococci.) 
 
Plate 10. 
 Streptococcus B-6a'. 
 
 Fig. 1. March, 1915. (Page 16.) Horse blood, | cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 17.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 3. (Pages 17, 34.) Rabbit blood, f cc; standard veal Witte peptone 
 agar, 12 cc. Incubated 48 hours. , 
 
 Fig. 4. (Pages 17, 34.) The same plate as shown in Fig. 3. Incubated 48 
 hours; refrigerated 48 hours. 
 
 Fig. 5. (Page 35.) Human blood, f cc; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 6. (Page 35.) The same plate as shown in Fig. 5. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 7. (Page 49.) Horse blood, f cc; standard veal Witte peptone agar, 
 12 cc.; dextrose, 1 per cent. Incubated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, Sa, 6a, and 7a. Photomicrograph? of typical deep colonies 
 indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, and 7. 
 
MONOGRAPH NO. 9. 
 
 PLATE 10. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 11. ■ 
 
 Streptococcus F-2a'. 
 
 Fig. 1. March, 1915. (Page 26.) Horse blood, f cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page"26.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 ■ refrigerated 48 hours. 
 
 Fig. 3. (Page 34.) Rabbit blood, | cc; standard veal Witte pept6ne,'agar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 34.) The same plate as shown in Fig. 3. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. S. (Page 35.) Human blood, f cc; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hours. . 
 
 Fig. 6. (Page 35.) The same plate as shown in Fig. 5. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, Sa, and 6a. Photomicrographs of typical deep colonies 
 indicated by arrows in Figs. 1, 2, 3, 4, 5, and 6. . 
 
MONOGRAPH NO. 9. 
 
 PLATE 11. 
 
 o 
 
 
 o 
 O 
 
 O. ''o-, 
 4 
 
 ■■■- i 
 o 
 
 o 
 
 (Brown : Blood agar for study of streptococci. ) 
 
Plate 12. 
 Streptococcus A-BLa. 
 
 Fig. 1. March, 1915. (Pages 19, 80.) Horse blood, i cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Pages 19, 80.) The same plate as shown in Fig. 1. Incubated 48 
 hours; refrigerated 48 hours. 
 
 Fig. 3. (Page 19.) The same plate as shown in Fig. 1 . Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours. 
 
 Fig. 4. March, 1916. (Pages 32, 80.) Horse blood, f cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. S. (Pages 32, 80.) The same plate as shown in Fig. 4. Incubated 
 48 hours; refrigerated 48 hours. 
 
 Fig. 6. March, 1916. (Page 32.) Horse blood, 1| cc; standard veal Witte 
 peptone agar, 12 cc. fiicubated 48 hours. 
 
 Fig. 7. (Page 32.) The same plate as shown in Fig. 6. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, 6a, and 7a. ■ Photomicrographs of typical deep colonies 
 indicated by arrows in Figs. 1, 2, 3, 4, 6, and 7. 
 
MONOGRAPH NO. 9. 
 
 PLATE 12. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 13. 
 Streptococcus A-BLa. 
 
 Fig. 1. (Page 56.) Horse blood, f cc; standard beef Digestive Ferments 
 Co. peptone agar, 12 cc. Incubated 48 hours anaerobically; refrigerated 72 hours 
 aerobically. 
 
 Fig. 2. (Page 56.) The sanie plate as shown in Fig. 1. Incubated 48 hours 
 anaerobically; refrigerated 72 hours aerobically; incubated 24 hours aerobically. 
 
 Fig. 3. (Page 56.) The same plate as shown in Fig. 1. Incubated 48 hours 
 anaerobically; refrigerated 72 hours aerobically; incubated 24 hours aerobi- 
 cally; refrigerated 24 hours aerobically. 
 
 Fig. 4. (Page 56.) Rabbit blood, | cc; standard beef Digestive Ferments 
 Go. peptone agar, 12 cc. Incubated 48 hours anaerobically; refrigerated 72 
 hours aerobically. - - 
 
 Fig. 5. (Page 56.) The same plate as shown in Fig. 4. Incubated 48 hours 
 anaerobically; refrigerated 72 hours aerobically; incubated 24 hours aerobically. 
 
 Fig. 6. (Page 56.) The same plate as shown in Fig- 4. Incubated 48 hours 
 anaerobically; refrigerated 72 hours aerobically; incubated 24 hours aerobically; 
 refrigerated 24 hours aerobically. 
 
 Figs, la, 2a, 3a, 4a, 5a, and 6a. Photomicrographs of typical deep colonies 
 indicated by arrows in Figs. 1, 2, 3, 4, 5, and 6. 
 
MONOGRAPH NO. 9. 
 
 PLATE 13. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 14. 
 Streptococcus D-4a. ; 
 
 Fig. 1. March, .1915. (Page 19.) Horse blood, f cc; standard veal Witte 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 19.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 3. (Page 19.) The same plate as shown in Fig. 1. Incubated 48 hours: 
 refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours. 
 
 Fig. 4. (Page 36.) Rabbit blood, f- cc; standard veal Witte peptone a,gar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 5. (Page 36.) The same plate as shown in Fig. 4. Incubated 48 hours; 
 refrigerated 48 hoiirs. 
 
 Fig. 6. (Page 36.) The same plate as shown in Fig. i. ' Incubated 48 
 hours; refrigerated 48 hours; incubated 48 hours. 
 
 .Fig. 7. (Page 36.) Human blood, f cc.; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hour^. ■'■ ■ . ,-.. - 
 
 Fig. 8. (Page 360 The same plate as shown in Fig. 7. Incubated 48 
 .hours; refrigerated 48 hours;^' " . 
 
 Fig. 9. (Page 36.) The same plate as shown in Fig. 7. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours. 
 
 Fig. JO. (Page 36.) The same plate as shown in Fig. 7. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours. 
 
 Figs, la, 2a, 3a, 3b, 4a, Sa, 6a, 7a, 8a, 9a', and 10a. Photomicrographs of typical 
 deep colonies indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 
 
MONOGRAPH NO. 9. 
 
 {Brown: Blood agar for study of streptococci.) 
 
Plate 15. 
 Streptococcus B-7a. 
 
 Fig. 1. March, 1915. (Pages 8, 35, 80.) Horse blood, | cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Pages 10, 35, 80.) The same plate as shown in Fig. 1. Incubated 
 48 hours; refrigerated 48 hours. 
 
 Fig. 3.- (Page 11.) The same plate as shown in Fig. 1. Incubated 48 hours; , 
 refrigerated 48 hours; incubated 48 hours. 
 
 Fig. 4. (Page 11.) The same plate as shown in Fig. 1.. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours; 
 
 Fig. 5. (Page 35.) Rabbit blood, f cc; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 6. (Page 35.) The same plate as shown in Fig. S. Incubate^ 48 
 hours; refrigerated 48 hours. 
 
 Fig. 7. (Page 36.) The same plate as shown in Fig. 5. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours. 
 
 Fig. 8. (Page 36.) The same plate as shown in Fig. 5. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours. 
 
 Fig. 9. (Page 35.) Human blood, f cc. ; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 10. (Page 35.) The same plate as shown in Fig. 9. Incubated 48 
 hours; refrigerated 48 hours. 
 
 Fig. 11. (Page 35.) The same plate as shown in Fig. 9. Incubated 48 
 hours; refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, and 11a. Photomicrographs of 
 typical deep colonies indicated by arrows in Figs. 1, 2, 3,4, 5, 6, 7,8„9, 10, and 11. 
 
MONOGRAPH NOl 9. 
 
 PLATE 15. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 16. 
 Streptococcus B-7a. 
 
 Fig. 1. March, 1916. (Pages 42, 80.) Horse blood, f cc; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 80.) The same plate as shown in Fig. 1 . Incubated 48 hours 
 refrigerated 48 hours. 
 
 Fiis. 3. (Page 42.) Horse blood, f cc; standard Liebig extract Witte pep 
 tone agar, 12 cc. Incubated 48 hours. 
 
 , Fig. 4. (Page 42.) The same plate as shown in Fig. 3. Incubated 48 hours 
 refrigerated 48 hours. ] 
 
 Fig. 5. (Page S3.) Rabbit blood, f cc; Rue'diger sugar-free agar, 12 cc, 
 Incubated 48 hours. . 
 
 Fig. 6. (Page 53.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc. 
 dextrose, 0.1 per cent. Incubated 48 hours. 
 
 Fig. 7. (Page 53.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc, 
 dextrose, 1 percent. Incubated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, 5a, 6a, and'7a. Photomicrographs of typical deep colo' 
 nies indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, and 7. 
 
MONOGRAPH NO. 9. 
 
 PLATE 16. 
 
 {Brown: Blood agar forstudy of streptococci.) 
 
Plate 17. 
 Streptococcus E-4 a. 
 
 Fig. 1.; March, 1915. (Pages 19, 80.) Horse blood, fee; standard veal 
 Witte peptone agar, 12 cc. Incubated 48 hours. 
 
 . Fig. 2. (Pages 19,-80.) The same_plate as shown in Fig. 1. Incubated 48 
 hours; refrigerated 48 hours. - 
 
 Fig. 3. (Page 19.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours. 
 
 FiG.,4, (Page 19.) The same plate as shown in Fig 1. Incubated 48 hours; 
 refrigerated 48 hours; incubated 48 hours; refrigerated 48 hours. 
 
 Fig. 5. March,- 1916. (Pages 42, 80.) Horse blood, f cc; standard veal 
 Witte peptone agar, 12 cc._ Incubated 48 hours. 
 
 Fig. 6. (Page 80.) The same plate as shown in Fig. 5. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 7 . (Page 42.) Horse blood, | cc; standard Liebig extract Witte pep-, 
 tone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 8. (Page 42.) The same plate as shown in Fig. 7. Incubated 48 hours; 
 tbfrigerated 48 hours. 
 
 Figs, la, 2a,' 3a, 4a, Sa, 6a, 7a, and 8a. Photomicrographs of typical deep 
 colonies indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, 7, and 8. 
 
MONOGRAPH NO. 9. 
 
 PLATE 17. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 18. 
 Streptococcus D-AD82 a. 
 
 Fig. 1. (Page 22.) Horse blood, | ccl; standard beef Witte peptone agar, 12 
 cc. Incubated 48 hours; 
 
 FiG. 2. (Page 22.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 3. (Page 37.) Rabbit. blood, f cc; standard beef Witte peptonfe ag^r, 
 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 37.) The saine plate as shown in Fjg. 3. Incubated 48 hours; 
 refrigerated 48 hours. - - 
 
 Fig. S. (Pages 8, it.) , Human blood, f cc ;. standard beef Witte peplpiie' •• 
 agar, 1 2 cc. Incubated 48 hours. 
 
 Fig. 6. (Pages 8, 37.) The same plate as shown in Fig. 5. Incubg,ted 48 
 hours; refrigerated 48 hours. - . 
 
 Figs, la, 2a, 3a, 4a, Sa, and 6a. Photomicrographs of typical deep ftoloriieS 
 indicated by arrows marked (D) in Figs. 1, 2, 3, 4, 5, and 6. 
 
 "Figs, lb, 2b, 3b, 4b, Sb, and 6b. Photomicrographs of typical surf a!ce colonies 
 ihdicated by arrows marked (S) in Figs. 1, 2, 3, 4, 5, and 6. 
 
 Fig. 7; (Page 22.) ..Standard beef Witte peptone agar, 12 cc. Incubated 48 
 hours. ' 
 
 Figs. 7a and 7b. Photomicrographs of colonies within the encircled area of Fig. 
 7. . ■ 
 
MONOGRAPH NO. 9. 
 
 PLATE 18. 
 
 (Brown: Blood agar for study of streptococci.) 
 
' ■ . Plate 19. 
 
 Streptococcus F-2 a. 
 
 Fig. 1. (Page 21.) Horse blood, f cc; standard beef Digestive Ferments Co. 
 peptone agar, 12 cc. Incubated 48 hours. 
 
 fiG. 3. (Page 21.) Standard beef Digestive Ferments Co. peptone agar, 12 
 cc. Incubated 48 hours'. 
 
 Streptococcus D-1 a. 
 
 Fig. 2. (Page 21.) Horse blood, f cc; standard beef Digestive Ferments 
 Co. peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 21.) Standard beef Digestive Ferments Co. peptone agar, 12 
 cc. Incubated 48 hours. 
 
 Figs, la, 2a, 3a, 3b, 3c, 4a, 4b, 4c, 4d, and 4e. Photomicrographs of typical 
 deep colonies indicated by arrows in Figs. 1, 2, 3, and 4. 
 
MONOGRAPH NO. 9. 
 
 PLATE 19. 
 
 ■:'i'f^.a«!*J»V'.. 
 
 a 
 
 4? 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 20. 
 Streptococcus Brig.-IIa,a. 
 
 Fig. 1. (Page 24.) Horse blood, | cc; standard beef Digestive Ferments 
 Co. peptone agar, 12 cc. Incubated 48 hours aerobically. 
 
 Fig. 2. (Page 24.) The same plate as shown in Fig. 1. Incubated 48 hours 
 aerobicaUy; refrigerated 48 hours aerobically. 
 
 -Fig. 3. (Page 24.) The same plate as shown in Fig. 1. Incubated 48 hours 
 aerobically; refrigerated 48 hours aerobically; incubated 24 hours aerobically; re- 
 frigerated 48 hours aerobically. 
 
 Fig. 4. (Page 57.) Horse blood, f cc; standard beef Digestive Ferments Co. 
 peptone agar, 12 cc. Incubated 48 hours anaerobicaUy. 
 
 Fig. S. (Page 57.) The same plate as shown in Fig. 4, Incubated 48 hours 
 ^naerobically ; refrigerated 48 hours aerobically. 
 
 Fig. 6. (Page 57.) The same plate as shown in Fig. 4. Incubated 48 hours 
 anaerobicaUy; refrigerated 48 hours aerobically; incubated 24 hours aerobically; 
 refrigerated 48 hours aerobically. 
 
 Figs, la, 2a, 3a, 4a, Sa, and 6a. Photomicrographs of typical deep colonies 
 indicated by arrows in Figs. 1, 2, 3, 4, 5, and 6. 
 
MONOGRAPH NO. 9. 
 
 PLATE 20. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 21. 
 Pneumococcus Cole I 109. 
 
 Fig. 1. (Pages 25, 43.) Horse blood, f cc; standard beef Digestive Fer- 
 ments Co. peptone agar, 12 cc. Incubated 48 hours aerobically. 
 
 Fig. 2\. (Pages 25, 43.) The same plate as shown in Fig. 1. Incubated 48 
 hours aerobically; refrigerated 72 hours aerobicaUy- 
 
 FiG. 3. (Page 25.) " The same plate as shown in Fig. 1. Incubated 48 hours 
 aerobically I refrigerated 72 hours aerobically; incubated 34 hours aerobically. 
 
 Fig. 4. (Page 25.) The same plate as shown in Fig. 1. Incubated 48 hours 
 aerobically; refrigerated 72 hours aerobically; incubated 24 hours aerobically; 
 refrigerated 24 hours aerobically. 
 
 Fig. 5. (Page 57.) Hoirse bldod, fee; standard teef Digestive Ferments Go; 
 peptone agar, 12 cc. Incubated 48 hours anaerobically. 
 
 Fig. 6. (Page 58.) The same plate as shown in Fig. '5. Incubated 48 hours 
 anaerobically; refrigerated 72 hours aerobically. 
 
 ' r ■ Fig. 7. (Page 58.)- The same plate as shown in Fig. 5. Incubated 48 hours 
 anaerobically; refrigerated .72 hours aerobically; incubated 24 hours aerobically. 
 
 Fig. 8. (Page 58.) The same plate as shown in Fig. 5. IncUbated 48 hours 
 anaerobically; refrigerated 72 hours aerobically; incubated 24 hours aerobically; 
 refrigerated 24 hours aerobically. 
 
 Figs, ia- 2a, 3a, 4a, 4", S^, 5", 6\ 6", 1\ 7", 7™, 8', 8^, and 8™. Photomicro- 
 graphs of tjrpical deep colonies indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, 7, and 
 8. 
 
 Figs, ^'^^and 4'-^. Photomicrographs of bottom and top surface colonies indi- 
 cated by arrows (III) and (IV) in Fig- 4. 
 
MONOGRAPH NO. 9. 
 
 PLATE 21. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 22. 
 Pneumococcus Cole I 109. 
 
 Fig. ir (Page 37.) Rabbit blood, f cc; standard beef Digestive Ferments Co. 
 peptone agar, 12 cc, Incubated 48 hours aerobicaUy, . 
 
 Fig. 2. (Page 37.) The same plate as shown in Fig. 1, Incubated 48 hQui;s 
 aerobicaUy; refrigerated 72 hours aerobicaUy, . ■", 
 
 Fig. 3. (Page 37.) The sa,me plate as shown in Fig. 1 , Incubated 48 hours 
 aerobicaUy; refrigerated 72 hours aerobicaUy; incubated 24 hours aerobifcally, 
 
 Fig. 4, (Page 57.) Rabbit blood, f cc; standard beef Digestive Ferments 
 ,.Co. peptone agar, 12 cc, Incubated 48 hours anaerobically, 
 ''■ Fig. 5. (Page 58.) The sarne plate as shown in Fig. 4.- Incubated 48 hours 
 anaerobically; refrigerated 72 hours aerobicaUy. 
 
 Fig. 6. (F'age 58.) The same plate as shown in Fig. 4, Incubated 48 
 hours anaerobically; refrigerated 72 hours aerobicaUy; incubated' 24 hours 
 aerobicaUy. 
 
 - Tigs, la, 2a, 3a,_ 4a, Sa, and 6a. Photomicrographs of typical deep' colonies 
 indicated by arrows in Figs. 1, 2, 3, 4, 5, and 6. 
 
MONOGRAPH NO. 9. 
 
 PLATE 22. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 23. 
 Pneumococcus Cole I 109. 
 
 Fig. 1. (Page 43.) Horse blood, | cc; standard veal Witte peptone , agar, 
 12, cc; Incubated 48 hours. 
 
 Fi&v2. (Page 43.) The same plate as shown in Fig. 1. Incubated 48 hours; 
 tefrigerated 48 hours. 
 
 Fig. 3. (Page 43.) Horse blood, §■ cc, ; standard Liebig extract Digestive Fer- 
 ments Co. peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 43.) The same plate as shown in Fig. 3. Incubated 48 hours; 
 refrigerated 48 hours. ... 
 
 Fig. S. (Pagers.) Human blood, f cc; standard veal Witte peptone agar, 
 12 cc. Incubated 48 hours. 
 
 Fig. 6. (Page 38.) - The same plate as shown in Fig. S. Incubated 48 hours; 
 refrigerated 48 hours. ... 
 
 ;— Figs, la, 2a, 3a, 4a, Sa, and 6a. . Photomicrographs of typical deep colonies in- 
 dicated by arrows in Figs. 1, 2, 3j 4, S, and 6. 
 
MONOGRAPH NO. 9. 
 
 PLATE 23. 
 
 {Brown: Blood agar for study of streptococci.) 
 
Plate 24. 
 Pneumococcus Cole II 45. 
 
 Fig. 1. (Pages 25, 44.) Horse blood, f cc; standard veal Witte peptone 
 agar, 12 cc. Incubated 48 hours. 
 
 Fig. 2. (Page 25.) The same plate as -shown in Fig. 1. Incubated 48 
 hours; refrigerated 48 hours. 
 
 Fig. 3. (Page 44.) Horse blood, | cc; standard veal ■ Digestive Ferments 
 Co. peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 4. (Page 44.) The same plate as shown in Fig, 3. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 5. (Page 43.) ; Horse blood, f cc; standard Liebig extract Witte peptone 
 agar, 12 cc. Incubated 48 hours. 
 
 Fig. 6. (Page 43.) The same plate as shown in Fig. 5. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Fig. 7. (Page 43.) Horse blood, f cc.; standard Liebig extract Digestive 
 Ferments Co! peptone agar, 12 cc. Incubated 48 hours. 
 
 Fig. 8. (PjVGE 43.) The same plate as shown in Fig. 7. Incubated 48 hours; 
 refrigerated 48 hours. 
 
 Figs, la, 2a, 3d, 4d, 5a, 6a, 7a, and 8a. Photomicrographs of typical deep 
 ■ colonies indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, 7, and 8. 
 
 Figs. 3t and 4t. Photomicrographs of a typical top surface colony indicated by 
 arrows (T) in Figs. 3 and 4." 
 
 Figs. 3b and 4b. Photomicrographs of a tvoical bottom surface colony indi- 
 cated by arrows (B) in Figs. 3 and 4. 
 
MONOGRAPH NO. 9. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 25. 
 ': ;• Pneumococcus Cole II 45. 
 
 Fig. 1; (Page 58.) Horse blood, f cc; standard beef Digestive Ferments 
 Co. p.eptone agar, 12 cc;. Incubated 48 hours anaerobically. . " 
 
 Fig. 2. .(Page 58.) The same plate as shown in Pig. 1. Incubated 48 hours 
 anaerpbicaUy; refrigerated 48 hours aerobically. • 
 
 FIG. 3. (Page 58.) The sa;me plate as shown in Fig. 1. Incuba,ted 48 hours 
 anaerobically; refrigerated ,48 hours aerobically; incubated 24 hours aerobically ^ 
 
 Fig. 4. (Page 58.) Rabbit blood, f cc; standard beef Digestive Ferments 
 Co.'pepfone"agar, 12 cc. Iftcuba,ted'48 hours anaerobically. 
 
 Fig. 5'. (Page 58.) The same plate as shown .in Fig. 4. Incubated 48 hours 
 aiiaerbbically; refrigerated 48 hours aerobically. ''-.-,'■ 
 
 FiG. 6. (Page 58;) The same plate as shown in Fig, 4. Incubated 48 hours'- 
 'anaerobically; refrigerated 48 hours aerobically; incubated 24 hours aerobically.;. 
 
 Fig. 7.' (Page 53.) Rabbit blood, | cc; Ruediger sugar-free agar, 12 cc. 
 inciibited 48' hours. 
 
 Fig. .8. (Page 53.) Rabbit blood, f cc; Ruediger sugar-free agar, 12 cc^; 
 dextrose, 1 per cent. Incubated 48 hours. 
 
 Figs, la, 2a, 3a, 4a, Sa, 6*''j6'^", 7a, and 8a. Photomicrographs of typical 
 deep colonies indicated by arrows in Figs. 1, 2, 3, 4, 5, 6, 7, aiid 8. 
 
 Pneumococcus Cole 11-37. "- -- 
 
 Fig. 9. (Page 59".) Horse blood, f cc. ; standard beef Digestive Ferments to. 
 peptone agar, 12 cc. Incubated 48 hours anaerobically. 
 
MONOGRAPH NO. 9. 
 
 PLATE 25. 
 
 (Brown : Blood agar for study of streptococci.) 
 
Plate 26. 
 - ^ - Streptococcus X-32 /3. 
 
 Fig. 1- (Page 47. J Horse blood, | cc; standard veal Witte peptone agar, 
 12 cc; dextrose, 1 per cent. Incubated 24 hours. 
 
 Fig. 2. (Page 47.) _ Horse blood, f cc; standard Liebig extract Witte peptone 
 agar, 12 cc; dextrose, 1 per cent. Incubated 24 hours. 
 
 Fig. 3. (Page 46.) Horse blood, f cc; standard Liebig extract Witte pep- 
 tone agar, 12 cc. Incubated Zi'hours. 
 
 Streptococcus C-64 p. 
 
 Fig. 4. (Page 47.) Horse blood, f cc; standard veal Witte peptone agar, 
 12 cc; dextrose, 1 per cent. Incubated 24 hours. 
 
 Fig. 5. (Page '47.) -Horse blood, f cc; standard Liebig extract Witte pep- 
 5 tone agar, 12 cc; dextrose,- 1 per. cent. Incubated 24 hours. 
 
 Fig. 6. (Page 46.) Ho'rse blood, f, cc; standard Liebig extract Witte pep- 
 tone agar, 12 cc Incubated' 24 hours. 
 
 ^ Streptococcus AST p. 
 
 Fig. 7. (Page 47.) Horse blood, f cc; standard veal Witte peptone agar, 
 12 cc; dextrose, 1 per cent. Incubated 24 hours. 
 
 Fig.' 8-. (Page 47.) Horse blood, f cc; standard Liebig extract Witte pep- 
 tone agar, 12 cc; dextrose, 1 per cent. Incubated 24 hours. 
 
 Figs, la^ 4a, and 7a. Photomicrographs of typical deep colonies indicated by 
 arrows in Figs. 1,- 4, and 7. 
 
MONOGRAPH NO. 9. 
 
 PLATE 26. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 27. 
 
 ■ Streptococcus B-7a. 
 
 Fig. 1. (Page 73.) Horse bipod, f cc; standard beef Digestive Ferments Co. 
 peptone agar,- 12 cc. Streajk I = streptococcus B-Ta. Streak Ila = strep- 
 tocQGCUs D-AD4/3. Streak Ilia = streptococcus A-ST/3.* Incubated 24 hours... 
 . FXG- 2. (Page 73.) The same plate as shown in Fig. 1. Streaked as follows 
 after the photograph shown- in Fig. 1 had been taken. . Streak lib = strepto- 
 coccus D-AD4^. Streak Illb = streptococcus A-ST;8. Incubated 24 hours 
 longer. 
 
 Pneumococcus Cole II 45. 
 
 ■ Fig. 3. (Page 74.) Horse blood, f cc. ; -standard beef Digestive Ferments 
 Co. peptone agar, 12 cc. • .Streak I = pneumococcus Cole II 45. Streak Ila 
 - streptococcus D-AD4|8. Streak Illa = streptococcus A-ST/3. Incubated 
 24 hours. ■ • " 
 
 Fig. 4. (Page 74.) The same plate as shown in Fig. 3. Streaked as follows 
 after the: photograph shown in Fig^ 3 had been taken. Streak lib = strepto- 
 coccus D-AD4/3. Streak lllb = streptococcus A-ST|8. Incubated 24 hours. 
 lo;iger. ,• .* 
 
MONOGRAPH NO. 9. 
 
 PLATE 27. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 28. 
 (Pages 61 to 63.) 
 
 The medium in all plates is standard beef Digestive Ferments Co. peptone 
 agar, 12 cc; streaked as follows: Streak 1 = pneumococcus Cole II 109. 
 StsEAK 2 ?= streptococcus A-BLa. Streak 3 = streptococcus B-2b (cow)|8. 
 Streak 4 = streptococcus D-AD4/3. Streak 5 = streptococcus A-18 (cow);8. 
 
 Fig. 1. Metabolized 48 hours by pneumococcus Cole II 109, then streaked 
 and intubated. 48 hours. 
 
 Fig. 2. Metabolized 48 hours by streptococcus A-BLa, then streaked and in- 
 cubated 48 hours. 
 
 Fig. 3. Metabolized 48 hours by streptococcus B-2b (cow)j8, then streaked and 
 incubated 48 hours. 
 
 ' Fig. 4. Metabolized 48 hours by streptococcus D-AD4^, then streaked and 
 incubated 48 hours. .' 
 
 Fig. 5. Metabolized 48 hours by streptococcus A-18 (cow)/3, then streaked and 
 incubated 48 hours i 
 
 Fig. 6. Control plate. Incubated 48 hours sterile, then streaked and incubated 
 '48 hours. > 
 
MONOGRAPH NO. 9 
 
 
 C^.J 
 
 m. ...^ 
 
 ,. .. . vr ■/,:■. ■ y.7: ,-:'. ;«>»»^^Am^ 
 
 r^iiSirfi^sftijiitBiiiiiKriiii 
 
 - .7^- 
 
 
 ©c '' 
 
 
 % 
 
 
 «*» 1 
 
 M 
 
 
 WIKMKtKlkiMk,.' ■ 
 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 29. 
 
 Fig. 1. (Page 63.) Horse blood, | cc; standard beef Digestive Ferments 
 Co. peptone agar, 12 cc. Incubated sterile for 48 hours as control for plates 
 shown in Figs. 2 and 3, then streaked and reincubated 48 hours. • Streak I = 
 streptococcus A-18 (cow). Streak II = streptococcus B-2b (cow);8. Streak 
 in = streptococcus D-AD4/3. Streak IV = streptococcus A-ST;8. 
 
 Fig. 2.. (Pages 63, 73.) Horse blood, i cc; standard beef Digestive Fermeuts 
 Co. peptone agar, 12 cc. Metabolized 48 hours by streptococcus B-7a, then 
 streaked and reincubated 48 hours. Streaks = the same as on the plate shown 
 ihFig.l. 
 
 ' ;FiG. 3. (Pages 63,73.^ Horse blood, ^ ccf; standard beef Digestive Ferments 
 Go.'peptone agar, 12 cc. Metabolized 48 hours by pneumococcus Cole II 4S, 
 then streaked and reincubated 48 hours. Streaks = the same as on the plate 
 shown in Fig. 1. . ' * • 
 
 'Fig. 4. (Page 64.) Horse, blddd, fee; standard veal Witte peptone agar, 
 12. GG. streaked as follows:' Streak II = streptococcus D-1 7. Streak III =' 
 streptococci D-I7 and D-AD4/3 mixed. Streak IV = streptococcus D-AD4/3. 
 Incubated 24 hours. 
 
 Fig. S. (Page 64.) Horse blood, f cc; standard beef Digestive Ferments 
 Co. peptone agar, 12 cc. streaked as follows: Streak 1 = streptococcus A-BLa. 
 Streak 2 = streptococci A-BLa and D-AD4/3 mixed. Streak 3 = strepto- 
 coccus D-AD4/3. Streak 4 = streptococcus D-AD4i8 and pneumococcus Cole 
 II 45 mixed. Streak 5 = pneumococcus Cole II 45. Incubated 24 hours. 
 
MONOGRAPH NO. 9. 
 
 PLATE 29. 
 
 (Brown: Blood agar for study of streptococci. 
 
Plate 30. 
 
 Fig. 1. (Page 66.) Standard beef Digestive Ferments Co. peptone agar, 
 12 cc. Sterile. 
 
 Fig. 2. (Page 66.) Horse bjcrod, i cc, laked by 2 volumes of distilled water ;- 
 standard beef Digestive Ferments Co. peptone agar, 12 cc. Heavily inoculated 
 with 'Str£pipcoccus pyogenes G-33p. Incubated 24 hours. ■ - 
 
 i Fig. 3. (Page 66.) A sterile plate similar to that shown in Fig'. 2.- Incubated. 
 2,4 hours. ' -. ^- 
 
 EiG. 4. (Page 66.) Streptococcus B-2b (cow)|8; Horse blood, f cc, laked 
 by 2 volumes of distilled water and centrifuged 30 minutes at 3,000 revolutions 
 per minute; standard beef Digestive Ferments Co. jjeptone agar, 12 cc Incubatejd , 
 24 hours. , '. 
 
 Fig. S. (Page 66.). Streptococcus D-AD4/?.- The same medium as used iii 
 ,_ the plate, shown in Fig.^ 4. Incubated 24 hours. ' 
 
 Fig. 6. (Page 68.) Streptococcus D-AD4/3. Medium similar to that used iii , 
 the 'plate shown in Fig. 5 except that the' corpuscle shadows were incompletely 
 •sedimented by centrifugation. Incubated 24 hours. 
 
 Fig. 7. (Page 66.) Streptococcus A-ST^. The same medium as used in 
 the plate shgwn in Fig. 4. llicubated 24 hours. . , ,,■ 
 
 Fig. 8. (Page 69.) Horse blood, f cc; standard veal Witte peptone' agar, 
 12 cc. Holes sucked in the mediuiri by means of a glass tube and then filled with 
 sterile laked blood agar. Phqjtographed before incubation. 
 
 Fig. 9. (Page 69.) "The sanie plate as shown in Fig. 8. Photographed' after 
 incubation for 24 hours. 
 
MONOGRAPH NO . 9. 
 
 PLATE 30. 
 
 (Brown: Elood agar for study of streptococci.) 
 
•;;,,■, Plate 31. 
 
 "'' ': '■ ■ (Page 84.) 
 
 Medium used throughout = horse blood, | cc. ; standard veal Witte peptone 
 agar, 12 cc. ■ 
 
 Photomicrographs of typical deep colonies, taken after incubation for 48 hours. 
 (Figs. 1, 3, S, 7, 9, 11, and 13) and after refrigeration for 48 hours longer (Figs.' 2, 
 4, 6, 8, 10, 12, and 14). 
 
 Fig. 1. Streptococcus D-AD4/5(. 
 
 Fig. 2. The same colony as shown in Fig. 1. 
 
 Fig. 3. Streptococcus F-2q:'. 
 : Fig. 4. The same colony as shown in Fig. 3. 
 
 Fig. 5. Streptococcus B-6a'. 
 
 Fig. 6. The same colony as shown in Fig. 5. 
 
 Fig. 7. Streptococcus A-BLa. 
 
 FiG. 8. The same colony as shown in Fig. 7. 
 
 Fig. 9. Streptococcus B-7a. 
 
 Fig. 10. The same colony as shown in Fig. 9. 
 
 Fig. 11. Streptococcus D-4a. 
 
 Fig. 12. The same colony as shown in Fig. 11. 
 
 Fig. 13. Streptococcus D-It. 
 
 Fig. 14. The sarne colony as shown in Fig. 13. 
 
MONOGRAPH NO. 9. 
 
 PLATE 31. 
 
 (Brown: Blood agar for study of streptococci.) 
 
Plate 32. 
 (Page 83.) 
 
 Figs. 1, 2, and 3. Horse blood, f cc; standard veal Digestive Ferments Co. 
 peptone agar, 12 cc. Incubated 24 hours. (Three characteristic plates from 
 convalescent or chronic sore throats.) 
 
 Figs. 2a, 2b, 2c, 2d, 3a, 3b, 3c, and 3d. Photomicrographs of deep colonies 
 indicated by arrows in Figs. 2 and 3. 
 
MONOGRAPH NO 
 
 PLATE 32. 
 
 (Brown: Blood agar for study of streptococci.) 
 
' . Plate 33. 
 
 (Page 103.) 
 The apparatus used for photographing the agar plates and colonies shown in 
 the preceding plates.. 
 
MONOGRAPH NO. 9. 
 
 PLATE 33. 
 
 (Rrown: Blood agar for study of streptococci.) 
 
- ,. ^ • Plate 34. 
 
 ■■■ 'i:;*'' ;^" "7 ; ' (Page 98.) 
 
 ' ,_. ■ ^ ' ; , ■"•■ ^ -•, ...:.■ ''5^ 
 
 i^-:;,'.' 4; 'System of notes for.'prtserving the genealogy and other details in the study 
 <k; of. bacterial cultures; , , ■ 
 
RAPH NO. 9. 
 
 PLATE 34. 
 
 
 
 
 
 P 
 
 - I 
 
 
 
 
 
 
 
 
 
 
 
 R»'' 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 OK j^m 
 
 
 « 
 
 !r 
 
 «ti- 
 
 : 
 
 
 
 
 
 
 
 £ U 
 
 ? 
 
 
 
 " 
 
 
 (Brown: Blood afiar for study of streptococci.) 
 
MONOGItALPHS OF THE ROCKEFELl^R INSTITUTE FOR MEDlCAI. RESEARCH 
 
 ^ Under tlie head:of. Mpnbgmphof The JSiack^eflef ImtiWef<frM'^iialS^tiearch'p:ep\xHUheA (lom 
 time to'time idientifie papets <Mmb are so extensive, 6i ^require sad) ^^borate illustrations, as .to 
 reMer'tliem unsuitab|le 'for current petKidiad isatiea. ' 'Mottograpks will be sent post paiid on appli* 
 cationj ftt'W.W eatli; p|(3*ble in advance by eheck or moh^yoM^,^ 
 
 Monograph N<^l.;(Issue!i Jim| 30, IMO)' a^ i:^ 
 
 S0QK FLEXNER and J. W,. JOBtlNQ. Studies lipon k transplantable rat tumor, '^atjsi I'-lS. 
 Jw#, JOBLING., The biology of a mixed tumor of the rat. MAUD LI l^ENTBN. ' Experi- 
 mcfita on the bifiuence of radiuim bromide on a jrarcmpmtttoti^; tumctr of the rat. : J. W. 
 JOBLING. Spontaneous tumors of the m<!>nse.: Plates lJ-^28, ; J. W, JOBtllfG. Tiansplaii- 
 tatibn aperimen'b in ilfo^ocM r^.4u^,with a carcmoJiiatous te^^ 
 
 Monograph No. 2. (Issued: febjiiauylSi 19^^ 
 
 WnXIAM TRAVIS HOWARD and GSGAR T. SCHULTZ. Studies in the biblogy of twnor i*lls. 
 
 ;.iPhitei 1-6, - / ■ ," V,.-- ',:'::■' ^^.^•'' '.'■'''■'. '^ '''■'' ' 
 
 .'- KAonograph No. 3. ■ (Issued Mardi IS, 1911.) ' 
 
 BENJAMIi? T; TERRY. Chemo-tlieiipentic ta^qpaiSosbme studies with special reference to the 
 
 "/■' ■, ■jmmiiBHy,((i^Owiag''Cure. """"A'' ;'"■""'';';"'"'• ■' ■'' ■',',.''"-' 
 
 Monbgrai* No, 4. (ftsiied Ju^ ' 
 
 FRAiitdifS W. PEABODY, GEORGE DRAPER,jind A. R, DOCHEZ. A clinical Study of acute, 
 ,pi&myelitis. Plates US,- . V 
 
 f. ' Monograph . No, 5. (Issued September 27i 1915.) ' , 
 
 J. P., EIIM0NDS. Studies in BaciUus wtkhii. With tpedal reference to ClkssiGcafion and to its tela- 
 , tion/toflianJieai.'' ';,;,:■ ', / ,■.-■, ■■.;•,'. ,V'''".\,- j. . '■''■''^'''-■'r-.i ■■ 
 
 jMoiiograph No. 6. (Issued JTanuaiySii 1916.) 
 
 JAMES; Ll STODDARD ai^ fetLlOTT C. CIJTLER. Torula infection in man. Plates 1-9, ; 
 
 Monograph No. 7. Issued October 16, 1917.) ^ ^, 
 
 OSWALD T. AVES,Y, H. T. CBliFKER^INGi RUFUS COtE,, and A, R, DOCHEZ; A9iite 
 lobar pneiumonia. prevention and senim treatment, Plati^ i-3, >'^ 
 
 Monograph N(v«i: (Issued Jiay 31, 191^ 
 
 ERIi^ST C. DICKSON, Botulism, A ctoicbl and eiliefi^ental study. Places 1-10. 
 
 , Monograph No. ?. .^issued Jaauaiy 21, 1919.) p^^- 
 
 ' jiyifiS HOWARp BE.OWN| Tjieuseof blbod agar fca the study, of streptococci Plates 1-34. 
 
 ■■',/'•/";:' "";,,; i,^'. '.Mbnogrftph 'No. ID.' :(Bi press.) ' ..\\ ,.'' ' -'C;'-'; 
 WILLIAM G. &A(5<5^S££UM. The pathHogy of tl(e'pneumQiiias in the United States Army 
 camps during tbe •j&iter of 1917-18; : I^^^ 
 
 ■'/J ,'■■•" Mo«ograiJi.'No.-H., (Inp«reS9.),. ■:'.,'.;, ,,,: / ^ i ;". 'l': yy. y^ r' 
 
 FREDERICK ^.>ALLteN, iEPGAB STILLJIAN, '^pd REGINALD FITZ,' %otal ^ietar^^'^^^^^ 
 lation In'^the ti*atment of diabetes. - , i ; , ., 
 
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