Benjamin White and Oswald T. Avery, Observations on etc. 161 Tafel Iv. i, 1, Photogramm eines Ausstriches einer 22stiindigen Agarkultur. Die wiss- rige ' schwemmung wurde getrocknet, dann durch 10 Minuten mit einer gesiittigten - mkarbonatlésung behandelt, mit Wasser gewaschen und in konzentrierter Jod- awliumlésung eingeschlossen. . == 1000. Big. 2. Lichtbild eines in starker Jodlésung eingeschlossenen Ausstriches einer I4tagigen Agarkultur. V. = 500. Kg. 3. Ientspricht einer anderen Stelle des Praparates von Jig. 2, V. = 1000. Hig. 4, 5, 6. Photogramme von Zopfbildungen und GeiSelverschlingungen aus einer Stitgigen Agarkultur. Der Ausstrich wurde mit lproz. Morphinlésung trocknen xelassen, gewaschen und dann in der starken Jod-Jodkaliumlisung eingeschiossen, V. = 1200, tig. 7. Ausstrich von einer 48stiindigen Agarkultur, in starker Jod-Jodkalium- ldsung photographiert. V. = 1300. Vig. 8, 9. Photogramme eines Ausstriches von einer 48stiindigen Agarkultur, deren Aufschwemmung mit einer Lproz. Morphinlésung gemischt und rasch getrocknet worden war, Nach der Wasserspiilung wurde in der konzentrierten Jod-Jodkaliumlis- ung cingeschlossen. V. = 1300. Nuchdruck vertuten. Observations on certain lactic acid Bacteria of the so-called Bulgaricus type. [Irom the Department of Bacteriology, Hoogland Laboratory, Brooklyn, N.-¥., U. 8. A.J By Benjamin White and Oswald T. Avery. With 2 plates, 4 tables and 1 figure in the text. The attempts to formulate a definite classification of the organisms producing lactic acid fermentations in milk have been rewarded with a more or less incomplete measure of success. There still exists a certain degree of confusion regarding the biological relationships and identities of the more common milk-souring bacteria indigenous to western Europe and America. To the list of organisms already isolated and described there has recently been added those constituting the bacterial flora of the curdled milks of Turkey, Egypt, Bulgaria and other eastern countries. These milks contain bacteria which, at least in so far as their activities in milk are concerned, are unique. A review of the various published descriptions of Kefir, Yoghurt, Mazun, Leben and the less well known Gioddu draws attention to the fact that in the imajority of instances the authors have described the microdreanisis isolated and studied in each case as separate and distinct bacterial entities, Kuntze was the first not only to draw comparisons between the diffe- rent organisms thus described, but also to point out further the possible relationships existing between these particular bacilli and the various species of lactic acid producing organisms already described. During the past two years the bacilli from these milks have received an increased amount of attention from bacteriologists beeause of their almost universal employment in in combating the putrefactive organisms producing intestinal autointoxication. The bacillus isolated from Bulgarian Yoghurt and first described by (rrigoroff of Prof. Massol’s laboratory — the so-called Bacillus bulgaricus or Bacillus of Massol — has been the organism of this type most discussed, and has generally been considered as constitt ling a new aud distinct specics. In this connection however it is of interest Zwelte Abt. Bd. 25. ll 162 ‘Benjamin Withe and Oswaid T. Avery, 1 id: Bacteria of ‘the. eo-called: Bulgaricus type. 163 | ' Bue 8 i a bap to review the descriptions of other bacilli found in soured milks of - -. similan, | ae i i | Jatter “organism, '.when” stained: with’: alkaline methylene ‘blue or with the E’ Neiss‘e r: ‘stain, is: seen‘ to contain: granules’ which are not observed in the cell body of the Bacill us bulgaricus. Although Diggeli men- ions these: granules these authors‘ are the first’ to consider. this feature as ‘an important’ ground for::differentiation.: In addition ‘to this difference, according to:-Luerssen-and-Kuhn , the Kérnchenbacillus is _to be distinguished. om the Bacillus bulgaricus in the following respects; ge ties ee a) By its longer and more slender form and its greater tendency to the rmation of threads and chains. -- sO ‘“b) By the appearance of ‘the ‘colonies of the two bacilli. 6) The Kérnchenbacillus grows at lower temperatures than does the’ Bacillus = b ulgaricus,: good at 37° and °46°, not at all at 22° and only slightly at 50°, while the B ulgaricus’ grows best at 45° to.50°, but develops slowly and feebly at 37° ~d) The former does not grow on potato, while the latter does. ~e) Milk is quickly and firmly coagulated by the Kérnchenbacillus at 37°: to 40°, the Bulgaricus on the other hand produces only a soft curd in milk and then only after. several days incubation. + These characters ascribed to the Bacillus b ulgaricus by the authors’ are not in entire agreement with those described™by others. This matter will be more fully discussed in another part of this paper’), . When one reviews. and compares ‘the original detailed descriptions of he various bacilli mentioned above itis apparent that all'these organisms ¢ possess in common, to a greater or less degree, certain characteristics which would seem to justify their classification in a group separate and distinct irom other more usual organisms producing lactic acid in milk?), It will be ound further that many of these morphological features are included in the ummary of distinctive characters of the group Bacteriu mM cauca- icum (Kern) L. et N., as reviewed by Léhnis, wo distinguishes ix ‘general types, viz. — , I. Milk coagulating, gas producing, Bac. casei Freudenreich. II, Milk coagulating, . non-gas producing, (Bac. casei Leich- tL Milk non-coagulating, gas producing, (Bact. caucasicum). IV. Milk non-coagulating, non-gas producing, (Bac. Delbriicki) LV. Slime producing.-type. ws voOVE »Rankenbildener‘* type. a * ; character. The first published study in this connection is that“of. the microorganisms present in Russian Kefir. Since his observati carried out as early as 1881, the nature of the bacteriological inet: Pe that time renders his results of doubtful value for the purpose of com zi son. It seems probable, as Lohnig suggests, that the bacillus called’) pe Sporacaucasicum by Kern belonged to the Subtilis or Mexe tericus groups. On the other hand it is quite likely that the Bacteri WH I caucasicum isolated from Kefir by Beijerinck was the first indivixg dual of this particular group to come under observation. A similar bacillus from’: | the same source is described by v. Freudenreich; who gives preference ‘# “4 to the name Bacilluscaucasicus. The very thorough investigation % al of the Egyptian Leben raib by Rist and Khou ry revealed the existence « " of two somewhat distinct types of lactic acid bacilli, the Streptobacil lus lebenis andthe Bacillus lebenis. The detailed study of their | characters establishes their general relationship to organisms of the Cauca- sicum group, and furnishes an accurate basis for comparing allied bacteria. ! §, Three years later Grigoroff isolated from Yoghurt two bacilli - which correspond in many respects to the two Leben organisms described by Rist and Khoury. These he designated as Bacillus A and Strepto- bacillus C and it is the former which has been so widely known under: the title of Bacillus bul garicus. Although this bacillus, according | to the author, produces a higher degree of acidity in milk than does the strepto- L bacillus, and although it shows certain other differences in its characteristics, | yet the many striking resemblances observed would seem to denote an inti- © mate relationship between these two types. Unfortunately accurate com- .: parisons between these four organisms are rendered impossible by the lack “ f of corresponding details. In 1906 Co hendy described under the name : of Bacillus bulgaricus an organism isolated from Bulgarian milk = { y | sii oe oye ae Hi, which is probably identical with the Baéillus A of Grigoroff. One 4 of the most striking features of this organism was the production in milk .:; of an unusually large amount of lactic acid, 3,23 per cent being found after 4 fo ten days incubation at 86°. In Armenian Mazun D iiggeli demonstrated | i the presence of a somewhat similar bacillus, the protoplasm of which showed . | the presence of more intensely staining granules. It was less active -* oo than Bacillus bulgaricus in the production of acid, yet it exhibited - 2 iF morphological and cultural features peculiar to the Caucasicum group. . i The existence of a related bacterium is suggested by the observations of Gri- xoni, who found but failed to-isolate a long granule-containing bacillus in the Sardinian sour milk Gioddu. Weigmann, Gruber and, Huss described a bacillus repeatedly isolated from Mazun, which is un- doubtedly identical with that studied by Diiggeli. Owing to its origin ' the name Bacillus Mazun was applied to it. : Directly comparable to the observations of Gri goroff, Rist and - Khoury, Diggeli and. Weigmann, Gruber and Huss are the findings of Luerssen and Kuhn who isolated, in addition to a : bacillus obtained from several commercial preparations and called by them og i Bacillus bulgaricus, a second organism which they designate as | § the Kérnchenbacillus, They found these two bacilli to differ from each ~ other in several impurtant characters, and these differences are suffi. 4 cient, they consider, to establish their identity as two distinct species. The © 4 1) Since the completion of the present investigation Heinemann and Hef- eran report the isolation from various sources of numerous cultures of bacilli which they beleive to be identical with the Bacillus bulgaricu s. From their obser- “vations they conolude that’ “the Bacillus bul gearicuas is widely distributed ‘in nature, that it occurs normally in human feces, in tha feces of cows and horses, also in & variety of sour and aromatic foods, in food for cattle, in normal saliva, in normal gastrio juice, and in gastric juioe when hydrochloric acid is absent, in various fermented milks, in ordinary market milk, and in soil both manured and not manured.“ The further corroboration of these findings would, yield much of interest regarding the distribution and occurrence of this organism, . - 4) It is felt that the repetition of the desoriptions of the organisms referred to would be superfluous since they have so recently appeared in the literature, and further, since . these original articles are published in journals easily accessible, 1l* 164 Benjamin Withe and Oswald T. Avery, Among the better known examples of these six types Léhnis men- lions the various varieties of the Bacillus casei Freudenreich, Lactobacillus cauecasiceus Beijerinck, Bacillus cau- casicus Freudenreich, Bacterium casei I—UL Leichman et sazarewski, Bacillus Delbrii¢ki Leichmann, Streptobacillus lebenis and Bacillus lebenis of Rist and Khoury, and the Bacillus Mazun of Dii ggeli, Similar organisms men- tioned by Heinemann are Baeillus panis fermentati and Leptothrix buecalis. Kuntze has recently suggested the probable relationship existing between some of the baeilli of this group and the Oppler-Boas ba- cillus, Bacillus acidophilus, Bacillus gastrophi- lus, and Bacillus bifidus communis, Even more recently Rodella asserts that these five last named organisms are identical, The relationships existing between these various lactic acid producing ba- cilli as might be disclosed by an accurate comparative study of their morphological, cultural, and biochemical characters offers an interesting Hield for further investigation, Such an investigation would undou btedly establish the identity of several of these bacteria which at present are. considered as distinet varieties, and further would yield a more satisfactory basis for a definite classification. By no means the least service erendered would be the abolishment. or change of some of the confusing and in- tppropriate names. According to preeedence the name Bact erium cau- casieuim could be ased te designate the group, but this name as well as the name Bacillus Mazu n, Bacillus bulgaricus, Bacilluslebenis, and S$ treptobacillus lebenis have scant justification when considered upon the ground of their accuracy or descriptiveness. It has been the purpose of the present investigation to observe under identical conditions in a comparative way the salient features of a series of lactic acid producing, milk curdling bacilli originally isolated from oriental milks. In order that the results obtained might serve as a basis of compa- rison in further studies of these and allied organisins the analytical scheme of Lehmann and Neumann, and the plan endorsed by the So- ciety of American Bacteriologists were adopted. Such omissions and varia- tions were made as seemed fo be indicated by the conditions met, but these were of slight. importance. The Organisms, The various cultures chosen for study were of following origins: Bulg, Received from Dr Bes rod ka of the Institue Pasteur, March 1908, under the name ,Bacille bud gare Cohendy.* BOM. An original culture marked ,Baecille bul gare‘ supplied by the Societe Le Ferment of Paris to their American representative, August L908, Led. From the Lederle Laboratories, New York, August 1908, originally from Prof, Metehnikoft. : BK. From Kral, October 1908, marked Bacillus b ulparicus I. oR, From Korat. October 1908, marked Bacillus paralacticus’, Ga] Isolated October 1908 from ,,Bacillac" prepared by the Lactobacilline Co., New York. . Ja-- | Isolated from Lactobacilline Malt obtained in Paris duly 1908. Ta--2 Isolated September 1908 from Lactobacilline Malt prepared by the Franco- American Ferment Co., New York. TX---] Isolated from Bacillac in March 1908, Observations on certain Lactic Acid Bacteria of the so-called Bulgaricus type. {65 XXIV—1 Isolated March 1908 from sour milk obtained from native Syrians in Brooklyn, W. Isolated by and received from Prof. Kuntze of Jeipzig marked ,,Bac t. Yoghurt, Varicetit W“. sba—1 Isolated October 1908 from ,,Zoolak“, a commercial Mazun prepared by Dr. Dadirrian, New York. 36a—1 Isolated October 1908 from a commercial Armenian sour milk. 4la—l Isolated October 1908 from Mazun prepared by native Armenians in New York. 42a—] Isolated October 1908 from sour milk prepared by native Armenians in New York. K. Isolated by and received from Prof. Kuntze of Leipzig marked ,,Kérn- chenbacillus“. For purposes of convenience the cultures from Bulg. to W. will be referred to as ‘Type A, those from 35a—1 to K. inclusive as Type B. While it is quite probable that the above list includes strains having a conuuon origin yet these have been subjected to varying conditions of cultural environment. In order to learn the influence of long continued so- litary cultivation, of symbiosis with yeasts, bacilli and cocci, and of culti- ‘ution in different media the possibly identical strains were therefore in- cluded. These sixteen cultures were cultivated and studied simultaneously and always under identically analogous conditions. In addition to the above some twenty different strains of similar bacilli were isolated from various sources, comprising commereial preparations from Germany, France, Eng- land, Scotland, Switzerland and the United States, also specimens of sour milk obtained from southern Siberia, Sofia, and from the natives of Arme- nia, Syria, and Egypt living in New York City. These isolations were ac- complished during the course of the investigation, and the organisms this isolated were compared as to their essential features with the strains already studied. No new types were found and since none of these organisins showed any significant variations from those listed no mention will be made further than to associate them with the types included in the present study. Detailed Features. I. Morphology. 1. Whey agar.") ‘The following characteristics are common to all strains; wide variation in length, from 2 2 to 50 p, breadth about I wu Nearly all individuals are in- tensely Gram positive and these show regularity of outline. They are straight to slightly curved, with rounded ends and show no vacuoles or granules, All strains show invo- lution forms exhibiting vacuoles, often appearing aa empty cell membranes. ‘These forms are Gram negative and vary greatly in both dimensions also in form. All show a tendency to chain formation, some being arranged in chains of 6 to 25 segments, The chain may coniain both Gram positive and Gram negative individuals. Gram negative spherical bodies, varying from 0.26 to 1 u are seen adhering directly to the sides of some Gram negative individuals in strains of Type B. 2, Whey. ln their behavior in whey these organisms exhibit some features of decided interest. The tendency toward degeneration and involution is marked. In the early stages of incubation at 37.5° or 44° the bacilli are uni- form in size and intensely Gram positive while in the succeeding stages the irregwar, vacuolated, inflated and ruptured forms predominate. These latter forms invariably decolorize. Between the 18th and 24th hour of in- cubation at 44° the strains of type A develop a morphological peculiarity first noted by Kuntze. This is the formation of oval to kidney-shaped ') The whey, whey agar, and whey gelatin were prepared according to the method of Cohendy. [66 Benjamin Withe and Oswald‘, Avery, nodules attached to a small stem extruding from the cell substance. As the incubation is prolonged these nodules increase in size, often measuring | y. to 2p in length, and their increase always takes place at the expense of the cell protoplasm. It is further observed that without exception these bodies as well as the parent bacillus are Gram negative. Their formation may undoubtedly be considered a true plasmoptosis. Two forms may be distin- guished, In one the stem apparently projects from the cell body, while in the other the nodule is situated at the junetion of two dividing bacilli which form an angle greater than 90° or this juncture may be considered as a point of marked indentation of a single bacillus. (Sec plate 1). While this feature is observed to a slight extent when the bacilli are cultivated on whey agar, and in a very few instances in milk, it appears to be a marked characteri- stic of the growth in whey. Culltres of type B present quite another appearance, No stemmed nodules are present, but instead small spherical bodies are seen which arc tore or Tess securely attached to the cell wall, These may become detached and their presenee fi the medium lead to the suspicion of a coceal eontami- nation, In the case of Che stemmed nodules a siighe bacillus rarely if ever extrades more than one bud, while in the fatter instance the bacterium may’ have a number of these small spheres adherent to it. Te second feature of interest is the tendency shown by the straing of fype A to grow in the form of short bacilli wranged in chains, while the strains of fype B develop to a greater length and exist. almost exclusively as single isolated forms. Forms have been observed in the cultures of type B which exhibit true branehing. These bacilli are Y shaped, the segments of whieh rarely exceed 2 y. in length. Examined in a hanging drop at various stages of developement moti- lity wag never observed. 3. Milk. Since milk is the natural habitat of the bacteria of this parti- cular group, the morphologieal features whieh they manifest when cultivated i this medium may be considered as coustituting a criterion, As in whey the age of the culture has a marked influence upon the form of the bacteria In young cultures the bacilli show regularity of outline although wide vari- ations in theie length are observed. In each of the cultures individuals exist whose length scarcely exceeds their breadth, while others show a decided tendency to thread formation. Threads may be seen which exeecd 50 ve in length. It is therefore difficult to make any definite statements in regard to average length. The breadth is more constant, varying between narrow limits. The average breadth is about 1 #, although the longer forms are more slender and average about 0.7 uw. There is less tendeney to chain for- mation than in whey yet chains of 4 tp 10 segments have been observed in cultures of type A. Type B shows longer and less straight forms. In older cultures longer individuals predominate in all strains. When grown in sym- biosis with yeasts and the more trivial milk organisms the developement of the longer forms is restricted, the length averaging 4 to 8 py. As might be inferred there is little or no degeneracy and very rarely are nodules observed, 4. Mndospores, ‘The presence of spores was observed in no case, In the degencrated forms certain lighter staining areas are seen which have Suir gested the nature of spores to some observers, uthough they are usually considered as vacuoles, Piffard suggests the name ,lucidoles. The fact that these bacteria, when young and most active, exhibit a low degree of Observations on certain Lactic Acid Bakteria of the so-called Bulgaricus type. 167 vitality and resistance would argue against the possibility of reproduction by spore formation. 5. Capsules. Nothing to suggest the presence or format'on of capsules was observed. 6. Staining reactions. All strains are readily stained by the usual anilincs. a) Gram: — Young individuals invariably show intense staining with this method. Older bacilli are more easily decolorized, and both dead and degenerate forms are always negative. In those forms not intensely stained there may be observed the presence of minute, deeply stained granules irregularly distributed throughout the protoplasm. This feature was found to be common to all strains. When chain formation is present not infrequently a segment is seen which is decolorized while the other elements of the chain are deeply stained. Further, single bacilli have been observed which exhihit color gradations from positive at one pole to negative at the other. b) Loeffler’s Methylene blue: — By means of the behavior of the organisms studied to this stain a separation into two types seems possible. ‘The protoplasm of those of Type A is uniformly impregnated while that of Type B shows a distinct differentiation. ‘The cell body is seen to contain a varying number of round to oval bodies or granules having a diameter equal to that of the cell and stained purplish red. ‘These bodies are present whether cultivation has taken place in milk or whey. In the latter case they are more marked in the younger individuals, becoming Jess distinct and fewer in number as developement, progresses, while in milk they are to be found in old cultures. These granules have been observed during varying periods of incubation and their presence noted at the end of 18 hours and after 21 days. Generally they occur only in the minority of forms and are less conspicious in the longer individuals. c) Neisser’s stain: The granules just described ure intensely stained by this procedure. In Type A none of the strains has ever exhibited the least affinity for the polar stain. The granules were noted by Diiggeli in the Bacillus Mazun and under the name ,,Kérnchen“ have been described by Luerssen and Kuhn. More recently this characteristic has been discussed in detail by Kuntze who holds the opinion that their presence is by no means a cou- stant or permanent character, and that their formation is dependant upon conditions of growth which may be lacking at times. He therefore is dis- welined to consider this feature a valid point of differentiation. In the pre- sent study, on the other hand, no variation has ever been noted. Fourteen of the sixteen strains have been under observation during a period of about sixteen months, in which time the influence of solitary cultivation as well as that of symbiosis has been noted. None of the organisms thus observed has been found to either gain or lose this character. On the ground of this evidence the presence of granules is tentatively held as establishing a mark of dissimilarity of type. No inquiry into the nature of these bodies has been attempted. (See plates JII, 1V). Il. Cultural Features. The majority of the cultural features described by Lé hnis as distine- live of the group Bacterium caucasicum are displayed to a conspicuous degree by all of the organisms studied. When freshly isolated from their natural environment — milk — they do not develop on any of the usual nutrient media, even though sugar be present. {solation can only he effected by the employment of whey agar (Cohendy). Kven upon this medium under the optimum conditions as to temperature, ete. the growth is tardy and feeble. This fact, combined with the appearance of the colonies is markedly characteristic. In the structure of the colonies a resemblance is seen to those of Bacillus anthracis, and in a less degree to certain strains of Bacillus subtilis. 168 Benjamin Withe and Oswald T. Avery, 1. Whey agar plates!), The following description is applicable to all the organisms studied with the exceptions as noted: Growth was first noted at the end of 24 hours at 37,5°. Colonies are then minute: a) Surface: at 48 hours about 0.25 to 1.0 mm. diameter, irregular, filamentous, curled, flat. weakly refractive; with low magnification the structure is found to consist of a niass of curled filaments, often in parallel strands, as in Anthrax colonies, often streaming; are translucent, tinged slightly bluish by reflected light and showing bluish white flocks or islands more or lese rectangular in form. ‘The medi is slightly clouded immediately about the point of growth. No tenacity to the surface is shown. b) Subsurface; Minute woolly tufts, delicate not dense, composed of hair-like processes radiating from the center which is yellowish by transmitted light. Exceptions: Strains 6a—l, 7a—l, 7a—-2, IX—I show a distinct, entire, even periphery. Abt times the growth produces no clouding of the inedium. Frequently the colony appears to be nucleated, the center is darker in color and has a slight elevation, being umbonate in character. This feature becomes more apparent as developement progresses. The nucleus may be surrounded by a comparati- vely clear zone and this in turn by a bluish white filamentous border. No chromogenesis is observed, A sour odor is noticeable after 48 hours incubation. (See plates V. VL). While the growth on whey agar furnishes no points of type differentia- tion vet it serves to definitely separate these organisms from all bactera not ineluded in the group Bagterium caucasicum. The resem- blance io Bacillus anthracis, andinaway to Bacillus sub- filis, although borne out\to a stight degree by the morphology is no more | than interesting, Jversgen and Kuln consider that the growth of the Bacillus bulgaricus in the form of round regular colonies haying a smooth periphery is diagnostic of this type. In the present instance if was observed only in those strains whieh had been subjected to cultivation i pure culfure for a period of a year or more. Since it was never observed int the other organisms of type A it might seem that this was a casual or acquired character. 2. Whey agar stabs. ‘The growth first appears at the end of 24 hours in the upper part of the stab as a woolly filament. At the end of 48 hours the developement, progresses downward, delicate hair-like proccesses extend laterally from the line of growth and the medium becomes sligthly clouded. After 3 days incubation the growth is marked along the entire line of inoculation and becomes arborescent with dense clouding of the agar, The surface is scanty with little spreading and is nearly transparent. Further incubation intensifies these characteristics without causing the appearance of further distinguishing features. None of the strains studied showed any marked variation from the above. This growth would seem to constitute a group characteristic. 3. Whey gelatine plates, (10% gelatine.) — Owing to the comparati- vely low incubating temperature necessitated, this medium is poorly adapted for the cultivation and differentiation of this class of bacteria. In several experments the only strains showing growth were Bulg, B. M., 6a—1, 7a—1, va—2 and IX—I, and in each case after long incubation at 25° to 27° the colonies were minute, although a microscopic examination showed their structure to be identical with that of the whey-agar colonies. No growth was observed before six days while all freshly isolated strains failed to develop. 4. Whey gelatine stabs. At first no growth whatever was observed in the case of the organisms of type B, although repeated attempts were made fo cultivate them in this manner, Later, after continued cultivation in milk a slight tendency toward growth was manifested, which never exceeded a ') In this series as in all others unless special mention is made the medium was inoeulated from a 24 hour whey culture planted in turn from a 24 hour milk culture. The medium: contained 2 per cont agar. Observations on certain Lactic Acid Bacteria of the so-called Bulgaricus type. 169 faint tracing in the gelatine. With the strains of Type A, a distinct but va- riable growth was obtained, which may be deseribed thus: At the end of from four to seven days at 25° a faint line is noticed along the track of the stab. ‘This appears as a finely beaded filament which on further incubation extends downward, In the lower balf of the growth single colonies appear which are spherical and whitish in color. The bead-like colonies become mossy and from these mycelioid projections extend laterally. When the colonies are closely segregated the growth ap- dears markedly arborescent, while single beads are beautifully echinate. (See Fig. 1.) The greatest adaptability to this medium was shown by Led, B. K. and P. K.. Sur- face growth. gas production or liquefaction were never observed. The medium was not. clouded. The filamentous character of these colonies is analogous to their appea- rance in agar stabs and is distinctive of the group, although not of the type. The variations noted are probably referable to cultural influences and may be developed by long cultivation. 5. Whey. At the end of 24 hours at 44° a slight, diffuse clouding of the medium is observed. At 37.5° the appearance of the cloud may be delayed until 48 hours or even tater. Strains of the B type show the heavier growth. Of type A 6a—1l, 7a—l, 7a—2, [X-—-l and XXIV—1 are the most. vigorous. In cultures of this type the cloud is com- posed of a suspension of macroscopic particles while the appearance of those of the second type resembles that seen for example in houillon cultures of B.coli. The most intense clouding appears during the second 24 hours at 44° Directly aft vr the height of the growth ha heen attained the A cul- tures begin to clear. ‘The suspended particles collect in large flocks adhering to the walls of the tubes and some ‘fall to the bottom for- ming a grayish white sediment. Between the sixth and eighth day the medium is en- tirely clear, complete sedimentation of all suspended matter having taken place. In cultures of type B this turbidic’ persists for a Jonger time usually until the twelfth to fourteenth day. No pellicle is fo. med. Although these variations may be ascribed to degrees of vigor, they are constant in each case and tend to emphasize the type differentiation indi- cated by the other described features. 6. Potato. Slants were cut in the usual way. In sume instances the potato was merely washed in distilled water, in others immersed in soda solutions of different di- lutions for varying lengths of time. Therefore both acid, neutral and alkaline strata were provided. The medium was heavily inoculated with cither a 24 hour whey culture or a 24 hour milk culture. In repeated attempts at 25°, 37.5" and 44° no demonstrable growth was obtained with any of the sixteen organisms. The results in this case although confirming the observations of Gri - goroff, Rist and Khoury, and Cohendy, are contrary to those obtained by Luerssen and Kuln, who deseribe the growth on potato as a constant and distinguishing character of the Bacillus bulgaricus, The failure to grow on this medium is in agreement with the behavior of organisms of the Bacterium caucasicum group. 7. Milk. When inoculated with young cultures and incubated at 37.5° to 44° milk is rapidly coagulated into a firm solid curd by all the strains studied. The coagulum produced showed slight variations due to the composition of the milk, and to the amount Fig. 1. 21 day whey gelatine stab cultures Led, B. K., Pp. K.. IX—1. Natural size. 170 By Benjamin Withe and Oswald T. Avery, and nature of the material used for inoculation. The curd was solid, with no shrinkage or cavities, aud was accompanied by the expression of a few drops of clear whey. Thick- ening appears first in the lower part of the tube. The production of gas, or an appreciable peptonisalion of the curd were never observed even after several months preservation. Strains Bulg. and Led. at times prodaced a viseid enrd, offen inereasing to slimi- ness, This character was repeatedly lost and regained, and was peculiar solely to these (wo organisms. When litmus is added to the milk the ehange from blue to red is abe tupl and intense, the curd assuming a deep pink hue. Coagulation takes place in G to 12 hours at 44° to 45°, in & to [FG hours at 37.5° and rarely before 9 to 12 days at 25", At the last named temperature coagulation was noted on lyin type A. cultures. Tuhes inoculated with the strains comprising type B. were observed for 31 days at room temperatave and inno ease had any change taken place. On the thirty-second dav these tibes were incubated at 44 and at the end of 1S hours all showed typical coagulation. Krom these cultures further successful moculations were made, showing that although wrowth was inhibited at 25° vet the bacteria remained viable during this period. Ina siisiler series of trhes embracing the 16 strains inoculated and allowed fo remain for 3todayve at refrigerator temperature Che mitk remained tnehanged. When ineuhadted at the expiration of this period of exposure all strtins produced coagulation within 40 to AS hours. ‘Two of the points of type differentiation mentioned by Luerssen and Kubn are contradicted by the above observations. First, in’ the present instance the organisms of type A. produced cougulation shehtly more rapidly than did those of type BL (eomparable to the kKérn- chenbacillus) while the nature of the curd produced by both was precisely the same. Second, the strains of the former type, though showing a marked preference for higher temperatures, still multiplied and produced coagulation at 25° while those of the latter type remained inactive. The viscosity noted in two strains is a phenomenon previously observed by 8 e- werin. This feature as developed by Bacillus casei has recently been studied by Burri and Thoéni. This bacillus however, exhibits this peculiarity only when grown in symbiosis. & Other media. When freshly isolated from their nattral symbiotic environment fo growth is obtainable on the ordimary media. After a year’s solitary cullivation in tilk these bacteria show an increased vitality and adaplation to foreign environment Recently a feeble erawth has been ob- tained on nutrient agar in the case of strains of dype AL Phe sume agar en- riched with 2°, lactose, strange to say, was less favorable, while the addition of the same amount of dextrose resulted in the development of many large ivpical colonies by all 16 strains. In bouillon, lactose bouillon, and dextrose bouillon ue growth was observed. However, if caleium carbonate be added to the last named medium these organisms grow well). The synthetic medium of Usehinsk y containing 2%, dextrose or lactose remained anehanged. An extract of malt prepared according to the formula of Gohend y fur- nishes an excellent medium for the cultivation of bacilli of this group. Ut may be substituted for whey in the preparation of agar and gelatine. tu lactose bile enriched with peptone a feeble (hough constant growth was ob- served with all strains, This lack of inhibitory power on the part of the bile ts tnferesting in relation to the persistence and growth of these bacilli inthe human intestine following internal administration. The growth in this ameditim is ‘maccompaniod by gas formation, Observed in whey jmalt and milk and on whey agar and whey gelatine vo differences were noted in the growth under aerobic and anaerobic con- (lifions, 1) ‘This medium was suggested by Dr, Garside of New York City. 9 tes eapaeaens Observations on certain Lactic Acid Bacteria of the so-called Buigaricus type. {7{ WI. Physical and biochemical Features. 1, Fermentation. a) Whey in Smith tubes: — Good growth observed in both arms. No production of gas. b) Other media: — Grigoroff states that the Bacillus bul- garicus attacks mannite, saccharose, maltose and lactose, but not rham- nose, dulcite or sorbite, but the nature of the medium he employed could not be ascertained. Co hend y observed the active fermentation of lactose, mal- tose, saccharose, levulose and particularly dextrose. Sinee the completion of the present work Bertrand and Duchacek report that the car- bohydrates fermented by Bacillus bulgaricus are dextrose, man- nose, galactose, levulose and lactose, while arabinose, xylose, sorbose and saccharose are net attacked. Mannite is not transformed into lactie acid. In the present instance many attempts were made to discover a suitable medium with which the action of these bacilli upon the various carbohydrates could be studied. Among the different nutrient substances tested were pep- tone prepared from casein and of other origins, serum waters, the medium of Barsiek ow anda similar solution of Kucasein. In all eases there was a marked acid production from dextrose and lactose, but the results ob- tamed with the other carbohydrates were too variable to warrant any de- finite conclusions. 2. Indol, Whey cultures of different ages were tested by several me- thods. Negative results were constant. 3. Toleration of acids. Since the members of types A and B produce exceptionally large amounts of lactic acid, their toleration seems to be limi- ted only by the maximum acidity produced in each case, beyond which they are unable to utilize further the residuum of unaltered sugar in the medium. This acid toleration may be made use of in isolating these organisms from milerial confaining other bacteria, Such a separation may be accomplished by adding to the medium a quantity of acid sufficient to inhibit the deve- lopement of foreign species. Leva found that the addition of 0.35 per cent jactic acid to Cohendy agar facilitated the isolation of Bacillus bulgaricus from the feces of patients after therapeutic administration. More recently Hein»“ann and Hefferan report the successful iso- lation of this organism som various soucres by adding 0,5 per cent glacial acetic acid to glucose bouillon. 4. Vitality. In addition to the observations on the behavior of these bacilli in milk it made be further stated that their vitality varies inversely with the period of incubation temperature, but directly with the length of time of special cullivation. Cultures in malt and whey display a greater degree of longevity than do those in milk. In the former case viable cultures have been obtained at the end of three months. 5. Thermal death-poits. Methods: a) 48 hour whey cultures were immersed ina water bath for the designated period and transplants were immediately made into milk, b) Tubes of whey were inoculated with a definite amount of an active whey cul- ture and after subjection to the various temperatures were then incubated. A minimum exposure of [5 minutes to 60° was necessary to kill all strains. 6. Dessication, In a dry condition the vitality is greater than in milk, Whey and milk cultures dessicated over sulphuric acid in a vacuum were found to be still viable after a lapse of four months. Considering their short 172 By Benjamin Withe and Oswald Tf, Avery, survival in milk and the absence of a capsule or spores, their resistance in this ease would seem to present an anomaly, %. Inzvmes, The addition of calcium carbonate, calcium chloride, and ane clioride in excess of Che amount required to neutralize {he acid pro- duced, falied to prevent congulation of the milk. This mieht argue for the presence of an enzyme. It is hoped to continue studies in (his direction, 8, Acid production. a) Milk: Methods: ‘Tubes contaming 1 ce, each of decreamed milk were steri« lized and then ineuhated for 48 hours at 37.59 and allowed to stand for several days at room temperiture to insure sterility. All Cubes in each series were filled from the same lof of milk. ‘These tubes were inoculated with 0,05 cc. of a 24 hour milk culture and in- cubated for the determined periods in a inoist atmosphere, At the end of these periods See, ol the milk was diluted with about 45 ee. of water, then brought to the boiling point and titrated with o/jg NaOH. with phenolphthalein as indicator, Hach series included {wo or more control tubes of the same sample of milk ineubated and titrated under si- milay conditions, Phe titre of the control in each ease was sulitracted fram the titre ol the corresponding inoculated tubes. Whey cultures were similarly inoculated, controtled wd titted. "The figures in the tables represent. the quantify of ™/,, NaOH required to neutralize {0 ee. of inoculated milk. Table bo Acidity produced in milk at 57,5°. 7 bony tae ee 6 hrs. | 12 hrs. 18 hrs,! 24 ‘hrs.| 36 hrs.) 48 hes.'72 hrs, 5 days|7 days 10 days Type AL) 0.660) Kt | M29 | 18.00 | 92.77 | 27.27 | 29.98 [ner ga77) aaa Type O69) | 354 wR | BOR | ORL | TBE | 952) 113881 O74) TEAR Table Il Acidity produced in milk at 44°. Series {. 6 hrs. | 12 hrs! 18 hrs.! 24 his. 36 brs.| 48 hvs./72 hes.!5 days:7 days LO days 25.70 | 25.01 27.86) 28.47! 97.84 5.56 | 644 | 680) 9.49 ' 9.46) 8.88 13.72 | 12.04 ! Type A. | 2.62 | 19 | i778 | 21.79 | 24.37 Type B. | 289 | 3.98 | Table Wh. Acidity produced in milk at 44° Series 2, 36 heal 48 hea./72 hrsis days:7 day: 10 days Type A. | o.71 i [4 hrs. | 12 hrs. | 18 hrs.) 24 hrs. | Type BL | 0.86 | 6.96 | 16.59 | 19.90 | 22.42 | 24.37 | 23.96; 26.09 i2 B12 | 10.5% 5 12.60; 160 | 1214 | 12451 1406 | | Table IV. Acidity produced in whey at 44° 72 5 7 \ i : , | | 6 hrs, 12 hrs.| 18 hrs 24 hrs,| 36 hrs.; 48 lirs.'72 hrs,[5 days,7 days! 10 days . oe i ee | To - Fype A. 0.66 | 2.07 | 4.07 | 4.25 | 4.58 | we BTL 5.58 | 6.901 6.0; Mvpe BO} 0465 | 200} 320 1 so | 5.89 KBR 602) BBN) 57K The accompanying charts offer a more graphic representation of the acil production in milk and whey, An examination of these tables and charts would seem to permit, the Following inferences: AL dn both types the acid: production in milk is slower at 37,0" than al LM B. ON higher deeree of acidity is attained at 37.5% C. The acid production is most active in the first 24 hours, then writ dually rises, reaching a maximum about the seventh daw, D. The bacilli of type A without exception elaborate an amount of i i Observations on certain Lactic Adid Bacteria of the so-called Bulgaricus type. 173 acid equal to about twice the ameunt produced by the strains of Type B. This would seem to constitute an important type difference hitherto unnoted, K. No significant: differences between the types or strains are shown in the acidity produced in whey. The small amout of acid formed in this medi is in marked contrast to the quantity found under analogous con- ditions in milk. 0. Acids produced, A. Lactic acid: The statements of various authors regarding the nature of the lactic acid produced by this group of organisms show a discordance referable partly to the methods of analysis employed as well as to inherent differences exhibited by separate species. Grigoroff reports that both tle Bacillus A and the Stre ptobacillus C produce inactive avid, but the method of determination is not given. From a 5 day eulture of the Bacillus bulgaricus Be rtrand and Weisweiller isolated the lactic acid by means of the zine salts and conclude that the acid produced was a mixture of the laevo and dextro modifications with a pre- dominance of the latter. In quoting from the work of Luerssen and Kulin certain authors have erred in stiting that. they found only the dextro- rofatory modification in cultures of the Bacillus bul garieus and Kérnchonbacillus . In the original publication it is specifically mentioned that “the whey turns the plane of palarized light to the right". This ean seareely he interpreted as indicating the presence of dextro-rota- tory lactic acid. Bertrand and Duch acek find that the Bul- faricus produces exactly equal amounts of both the right and left acids, wd that this action is due to an intracellular lactase. The strains studied by Heinemann all produced the inactive type without a trace of ac- tive acid. In the present. investigation the following method was adopted; The whey obtained from 500 cc. of the milk culture was aciclified with phosphoric acid 5 volumes of water free ether were added, then thoroughly agitated in a shaking apparatus, This step was repeated five times and, after an exact separation from the whey, the ether extracts were united and eva porated to dryness in vacuo at 45° ‘The residue was taken up in water, the solution clarified hy filtration through animal chareoal. aud after the addition of an excess of gine carhonnte it was boiled and filtered, After roncentralion on a water bath the solution was allowed to stand. The crystals formed were removed by filtration, Further crops of crystals forming in the successive filtrates were presorved and examined separately. ‘The determination of the water of crystallisation was accomplished in the usual manner. The analyses of the zine salts obtained from the whey of 24 hour milk cultures are as forlows; I oe Per cent | | | Culture | Per cent | Caleulated for Culture Calculated for { Type A, | water | 4n(C,H503), + 3 HO water | 2n(C3H,04), 1! 2H, I NXIV—1] 18.21 | 18.18 35a—I] 12.77 | 12.90 Bulg | 18.21 | 36a—I 12.91 mM.) a8 | 4ta—1 | 13.04 | Pad | 18.60 | 42a-~-] 13.05 ROK. 18.40) | K 12.88 MeoK, 17.93 | tin | 18.04 re 18.20 Ta--2 18.12 | IX. -] 18.02 | W 18.10 | l74 Benjamin Withe and Oswald T. Avery, Concentrated solutions of these salts were prepared and their rotatory power determined. In the ease of all the strains of type A the solutions exhibited rotations foo slight to be of any significance. The solutions from type B, on the other hand, showed a marked rotation to. the tight, therefore demonstrating the ‘production of the laevo-rofatery acid. The crystals separated) rom subsequent fractions from the 24 hour cultures of strains of both types had the same composition and rotatory power as the initial erystals. AU series of observations was made on cultures inemhated 7 days, Id days, 2t days and 30 divs respectively at 44°, An Unvarving production of the inactive acid alone was noled in all {he strains of type AL The type B organisms, 24 hour cultures, showed a production of the active acid. Tu 1. 14. 21. and 30 day cultires of strains of this type, the erystals obtained represented approximately equal amounts of the inactive and active acids, The variations in this feature of the activity of the bacilli of the two fypes are tentatively considered as constituting a further point of type differ entiation. ; I. Other acids: The aeids already demonstrated in cultures of the Ba- cillus bulgarieus are acetic, formie and suecinie acids (Bertrand and Weisweiller, Bertrand and Duchacek) lWeinemann finds that the volatile acids constitute 5, 8 to 6. f per cent of the total acidity. Alt the straits observed in the present instanee produced a small amount of volatile acid. The exact amount and the nature of the acids were not determined, Although the bacilli of these types have little or no action on the casein and fat in milk, yet they produce a bitter, acrid taste and odor whieh may be due to minute quantities of products formed in the cleavage of these sub- stances, "This peculiarity was laeking in cultures af type B organisms. HH. Other products. Grigoroft reports the formation of a trace of alcolol by the Bacillus A and the Stre ptobacillus (This substance was also found by Gourbet, while Luers senand Kuhn note that 24 hour whey cultures of the Bul garicus and Kérnchenbacillus gave positive jodoform reactions. A series of experiments included the distillation of the whey (after faint alkalization with sodium tarbonate) from 10 day milk cultures of the strains of types A and B, These distillates all contained small but appreciable amounts of alcoho! but no acetone or aldehyde, 12. Pathogenicity. These organisms are non-pathogenic to man and the wsual laboratory animals. No untoward effects in man have been observed following the ingestion of large amounts of whey and milk cultures. Summary. The principal charaeters eommon fo all the strains studied ag compared lo the echaraeteors dis- tinetive of the group Bacterium caueasieum as noted by Loéhnis are as follows; Group Bacterium cauca- steum L. ef N, Great variability of cedl form. Strains of types A aud B, Great variability of eell foran, Observations on certain Lactic Acid Bacteria of the so-called Bulgaricus tyqe. 175 Length=2y to 0 yor more. Breadth = generally about 1p Chain formation marked iu some straing, Longer and slenderer forms secninoldereul- tures in milk, Noun-motile, now-sporu- lating Viable bacilli are Gram positive, dead and in- volution forms Gram negative. Difficult to growth in feeble. cultivate, most media Freshly isolated, growth is obtainable only on media containing wh cy or malt, and in milk. Grow equally well under aerobic and anaerobie conditions, Optimum temperature for growth is 44% 450 Pajy growth at 30% slight at 255 none at 20° Colonies on whey agarare round to irregular, grayish white, 0,5—1,5 mm, curled, filamen- tous strueture, peri- phery mostly filamen- tous, often streaming, in a few cases smooth and even, Gelatine not liquefied, No surface growth on ge - latine stab cultures, Along the stab the Length =2yuto Dwor more. Breadth = 08 B10 1.20 y, Chain formation notunu- sual, Favorable conditions re- sult in the appearance of more slender form &, The ahsenece of motility and spore formation is épparently constant. With the exception ol the dead and involution forms all bacilli of the Sroup are Gram posi- tive. The intensity of growth is mostly slight and is dependant on the natur of the medium, Ontheusual meat pepton media, as well as on potato, particularly in thecase of freshly iso- lated strains no growth takes place, The disinelination to Srowinthe presence of free oxygen is marked. For many forms the opti- mum lies between 40 and 50° the minimum at 25°, while other members ofthe group are able to stow tolerably well at 20° and under. Colonies are white ot yellowish white, most- ly smaller than head of a pin. Periphery is cither eir- cular, even or sinuate, lobed at times with root-like ramifica- tions. So far liquefaction of gelatine has never been observed, The gelatine tures show no surface stab eul- typically growth. 176 erowh is filiform, headed, laterwith hori- zontally projecting ra- mifieations. Mediuain clear, lnoagar stabs the growth is the same as above, but heavier. The medium is clouded The growth in whev pro- duecs elouding which disappears in 5 to 14 days forming a grayish white sediment, No growth on potato, Milk coagulated in 8--I8 hours at 44°, and is the most favorable medium for growth Most rapid coagulationtakes place al 44° The lactic acid formed is criherinactive or Laevo- rotatory. Asmall quantity of vola- tile aeid is produced. No appreciable peptoni- zation of the curd Non-pathogenic liwillhbeseen that, with a few exceptions, aostriking resemblance between the various or- those of Is studied and growp. eanuisos easieum Benjamin Withe and Oswald T. Avery. Along the stab the growth is cuneiform, threadlike or beaded, rarely showiug horti- zontal projections, Agar stab,as arule, weak ly developed. Narrow ribbonor dotted growth along stab. The pre- sence of sugar in the agarorgelatine causes clouding, In bouillon, when growth takes place, only a whi- tish sediment is obser- ved in the clear fluid, while the addition of dextrose or Lactose pro- duces a weak to strong. clouding as well as an increase in the sedi- men t. On potato as arule there is no development, at best it is slight. Milk in generalis coagu- lated more slowly than by streptoeucci. Some individuals grow only slightly in this medium. The optimum for coa- gulation lies mostly between 40° and 50% for some 30° The Jactie acid formed is mostly laevo-lactie acid, more vareley the inactive or dextro mo- dification. Only a trace of volatile acids are produced, il at all. Several organisms of the group peptonize casein Pathogenicity has never been observed. there Gia the Baeterium Observations on certain Lactic Acid Bacteria of the so-called Bulgaricus type. 177 Variations in the characteristics of the various strains pointing to type differentiation are — Type A. With Loeffler’s methy- lene blue or with Neis- ser’s stain the proto- Type B. With these stains the presence of intensely staining granules may be demonstrated inthe protoplasm, Produce 12 to 16 per cent lactic acid in milk. The lactic acid formed is always the laevo-rota- tory modification. plasm is homogencous- ly stained. . Produce 27 to 3.7 per cent lactic acid in milk. The Lactic acid formed is the inactive modifi- cation. Resume, l. A review of the morphological, cultural and biochemical features of the lactic acid producing bacilli from Yoghurt, Mazun, and Leben appears to justify their classification as a single group. 2 This group would seem to be identical with the group Bacterium caucasicum (Kern) — Lehmann and Neumann, 3. The significant variations exhibited by these bacilli in regard to the presence or absence of gra- nules demonstrable by differential stains, the de- gree of lactic acid production, and the nature of the lactic acid formed suggests a further differ- eutiation into two distinet types. These types might be designated as the true type (Type the para type (Type B). The authors wish to express their thanks to Prof. Dr. W. Kuntze of the Land- wirtschaftliches Institut, der Universitit Leipzig and to Dr. Bearedka of the In- stitut Pasteur, Paris for cultures, and to acknowledge their indebtedness to Dr. fHlenry G. Piffard of New York for his deep interest and many valuable sug- gestions, as well as for the various cultures and samples of foreign sour milks which he hes so graciously procured for this investigation. A) and Bibliography. Kuntze: Studien iiber fermentierte Milch. (Centralb]. {. Bakt. Abt. LE 1908. Bd. 20, p. 737.) Grigoroff: tude sur un lait fermenté comestible, le ,,Kisselo mléko“ de Bulgare. (Revue médicale de la Suisse Romande. ‘I’. 25. 1905. p. 714.) Kern: (Bulletin de la Societé impériale des naturalistes de Moscou, 1881. No. 3.) quoted by v Freudenreich, Bakteriologische Untersuchungen iiber den Kefir. (Cen- tralbl. f. Bakt. Abt. II. Bd. 3. 1897. p. 47, 87, 135.) Léhnis: Versuch einer Gruppierung der Milchsiurebakterien, Abt. IL. Bd. 18. 1907. p. 97.) —: Die Benennung der Milchsiurebakterien. (Centralbl. f. Bakt. Abt. 1. 1909. p. 553.) Beijerinek: Sur le Kefir. (Archives neérlandaises des sciences exactes et naturelles. 'T, 23. 1889. p. 428.) v. Freudenreich, see Kern. Rist and Khoury: Etudes sur un lait fermonté comestible — Le Leben’ di hgypte. {Annal. do PInst. Pasteur. JL 16. 1902. p. 68.) Cohondy: Mssais d'acclimatation microbienne persistante dans la cavilé intestinale. (Compt. rend. de la Soc. de Biol. T. 1. 1906. p. 364 ) Zweite Abt. Bd, 25. 12 (Centralbl. f. Bakt. {7s Carl Vahte, Cohendy: Doseription d'un ferment. lactique puissant: capable de sacclimater dans Fintestin de lhomine, (Compt. Rend. de la Soe. de Biol, Te TE. WOG. p. 558.) Mssai de traitement de lenterite nuico-membranense aigue prr Vacelimatation Mun ferment lactique dans le gros intestin, (Compt. Rend. de ja Soc. de Biol. 'T. 1. 1906, p. 872.) Ditggeli: Bakteriologische Untersuchungen iiber das armenische Mazun. (Cen. tralbl. f Bakt. AbLL Bd. Lb. Mt. yt. 577.) (rixoni: Tl Gioddp. (Annali della medie. navale. Vol. 2. 1905.) Ref. (Contralhl, fh. Bakt. Abt UN Bd £5. E906, p. 750.) Weiemann, Gruber and Huss: Uber armenischos Mayan. | (Centralbl £. Baki Abt TL Bde to. 1907. p. 70.) ° Lucrssen and Kuhn: Yoghurt, die bulgarisehe Saucrmileh. (CentralbL [ Bakt, Abt UL Bd. 20. 1907-08, p. 234.) Rodelba: Mageneareinon und Milchsiiurebakterien. (Centralbl. {. Balt. Abt. 1, Bd. 47. 1908. p, 445.) Piffard: A study of sour milks. (N.Y. Med. Jour, Jan. 4. 1908.) Sewerin: UWinige Kreebnisce und Bomerkungen iiber den sogenannien Bacillus bul. garicus und das Milehsiiurepriparat ..Laktobacillines. (Centralbl. f. Bakt. Abt. UL, Bd. 22. 1008, p. 8.) Burri and Thoéni: Uberfiihrung von normalen, echten Milehsiurebakterien in {adenziehonde Rassen, (Centralbh f Bakt. Abt. IT. Bd. 23. 1909. p. 82.) Bertrand and Duchacek: Action du ferment bulgare sur les principaux sueres, (Annal. de inst. Pastetr T. 23. 1909. p. 402.) Leva: Zar Beurteilung der Wirkung des Laktohaeillins und der Yoghurunileh. (Berl. Klin. Wehnsehr. 1968. p. 922.) Heinemann and Hefferan: A study of Bacillus bulgaricus. (Jour. Inf. Di- seases, Vol. G. 1900) p. 304.) Bertrand and Weiswei lfer: Action du ferment bulgare sur le lait. (Anmnal. de (Inst, Pastenr EL 20, (906 p. 977.) Lehmann and Neumann: Atlas und GrundriB der Bakteriologie. 4. Aufl. 1907. Gourbet: Notes sur la fermentation du Yoghourt. (Compt. Rend. de la Soc. de Biol. T. 60. 1906. p. 495.) Explanation of plates. Tafel I, Fig. I. 48 hour (44°) whey culture, IX——1, Gram stain, showing nodules. b+ L5Q0. Wie, 2. 24 hour (44°) whey culture, XXIV—1, Gram stain, showing chain tor. mation. 1: 1500. Fig. 8 48 hour (44°) milk culture, 35a. metachromatic granules. 1 + 1500, Wig. 4. 48 hour (449) mitk culture, 36a—-1, Neisser stain, showing metacliro- yuutie granules, 1 + 1500, 1, Loeffler methylene blue, showing Tafel LL Fig. 5. Colony of XX1V—1, whey agar plate, 7 days (44°). 1:70. Hig. 6. Same showing margin of colony. 1 : 350. Nachdruck verhoten. Vergleichende Untersuchungen tiber die Myxobakteriazeen und Bakteriazeen, sowie die Rhodobakteriazeen und Spirillazeen. JAus dem Botanischen Institut der Universitat Marburg. | Von Carl Vahle, Mit 2 ‘Tafeln. Vorliegende Arbeit ist im Laufe der Jahre 1906—1909 unter Leitime des Herr Prof. Arthur Me yor ja Botaniselen tastitut der Universitat Marburg enistanden. Sie hat in ihrer Gesaintheil die Aufeabe, die Verwandt- [r ° a . spe ye ee Cntralblatt pf. Bakterwingte Abt. G Bd 27 Whee a. dere, Lactic, deut fiacterva. Tih f uction , While Avery : aoa Verlag von Gustav Fischer in Jena. . ~ P.Weise, Lith Jena. Gniralilalt ( Bakterivogie Abt. H. Ba. 25 White a. deere, Lactic, dent Iuetaria, Tab 2 TL 4 be P.Weise, Lith, Jena White & Avery, . Verlag von Gustav Fischer in Jena Centralblat. f Bakteriologie Abt. I Bd. 25 While wu. deere Lactic, dew Hacheren. fith, tt. O days Wante & Avery. Verlag von Gustav Fischer in Jena. P.Weise, Lith. Jana. Qntralilatt f Bakteriologie Abt. Bd, 2 ‘White s Avery. Tero ° - : White uo dvere, Lutetic, dent Bacteria, Thy. i PWeise, Lith. Jena. Verlag von Gustav Fischer ia Jenn ee While Very a ON ee Pee 4k iM AEs ia 1 i i ee. SST. JRF SE es — MRE, HEE SSK Ute. eR. WTAE 9 WHopsiem a eR. TE TSESRSSIUAD OS sryton FONTS ee Sa TT, ot Se re eee a ‘ce -¢ “Big ay usoys Auiojoo jo wisse]C “9 BIT ‘og K cary awBe Soya T—ATXX Suojoo GFF) dep 2 “GM Taf. U1. re White u. Avery, Lactic Acid Bactersa. 5. _ Centralblatt f. Bakteriologie. Abt. II. Ba. 2.