QR :-NRLF S8K: > B M 577 DOb 1922 A Biochemical Study of Streptococci With Special Emphasis on the Determination of Their Chemical Composition DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science of Columbia University By FRANCES KRASNOW, B.S., A.M. NEW YORK CITY 1922 A Biochemical Study of Streptococci With Special Emphasis on the Determination of Their Chemical Composition DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science of Columbia University By FRANCES KRASNOW, B.S., A.M NEW YORK CITY 1922 CONTENTS I. Introduction 1 . General Statement 2. Historical 3. Object of Research II. Character of Organisms 1 . Source 2. Preparation of media for use in the determination of the biochemical reactions. (a) Sugar-free broth (b) Carbohydrate broth (c) Meat infusion agar (d) Meat infusion gelatin (e) Milk (f) Inulin serum water (g) Bile 3. Preparation of the reagents for the determination of the hydrogen-ion concentration. (a) Standard solutions (b) Indicators 4. Isolation 5. Stock Cultures 6. Seed Cultures 7. Biology and Biochemistry of the organisms (a) Blood reactions (b) Immunity reactions (c) Other biochemical reactions 8. Description of the test organisms III. Preparation of the Mass Cultures I . Medium (a) Choice of the Infusion basis (1) Hemolyzed blood- veal-infusion agar (2) Huntoon agar (3) Hasting gelatin-agar (4) Beef liver agar (5) Beef brain agar (b) Concentration of agar agar (c) Constancy in composition 488835 2. Cultures (a) Preparation of the cultures for mass growing (b) Planting of cultures (c) Removal of growth from agar (1) Washing method (2) Spatula method (d) Drying of growth IV. Analysis of the Bacterial Material 1 . Description of methods employed (a) Nitrogen (b) Total sulphur and phosphorous (c) Ash (d) Moisture (e) Lipins (f) Blanks 2. Records of analysis 3. Summary of the data for the average percentage composition of the streptococci 4. Conclusions from the analytical data V. Analysis of the Lipin Mixture 1 . Outline of the procedure 2. Records of analysis 3. Summary of the data for the average percentages of phosphatide and cholesterol. 4. Conclusions from the data on the cholesterol and phosphatide content VI. Effect of the Lipin Material on Hemolysis 1 . Record of experiments 2. Conclusions from the data on hemolysis VII. Summary of Conclusions VIII. Bibliography. I. INTRODUCTION 1 . GENERAL STATEMENT: The chief purposes pervading researches on the streptococci are two: (i) to get a sound basis for the classification of the different types and (2) to understand the role they play in the general processes of nature. All the results, thus far, are based on the changes which these organisms produce in their environment. Though this in- formation is very valuable we must not forget that in order to compre- hend function we must understand structure and composition . Discovery of the varieties of relation of the actual constituents of the bacterial bodies must lead to a better realization of the very things for which we are seek- ing. With this purpose in view, we have undertaken the work to be de- tailed here. 2. HISTORICAL: This is not the first study on bacterial composition. Thus Brieger as early as 1885 (i) analyzed pneumococcus cultures; Weyl (2) and Hammerschlag in 1891 (3), Hoffman in 1894 (4) De Schweinitz and Dorset in 1895 (5), 1896 (6), 1897 (7), 1898 (8), Ruppel in 1898 (9) and Levene in 1898 (10) and 1901 (n) investigated the chemistry of Baccilus Tuberculosis; Drymount in 1886 (12) and later Vaughan (13) and Wheeler (14) worked with Bacillus Anthrax. More recently Vaughan (15), Leach (16) and Dawson (17) analyzed Bacillus Coli cultures and Nicholle and Alilaire (18) and Bradley (19) used Bacillus Diphtheria as their material for analysis. 3. OBJECT OF RESEARCH: Each of these reports supply interesting additions to our knowledge of the chemical make-up of bacteria. It might, therefore, seem that our problem is a very simple one, differing from previous investigation only in respect to the organism, considered. Such a view cannot be more than superficial. Our aim, here, is not merely to gather data on the chemical composition of the streptococcus it is to gather such data under standardized conditions. Practically nowhere in the literature is there mention of such an attempt, though it is precisely the want of standard methods that has made so many results in bacterio- logical work of little or no value. We have, therefore, made special effort to work out details. This will become apparent with a careful consideration of the methods of procedure described below: II. CHARACTER OF ORGANISMS 1. SOURCE: All the strains of streptococci were obtained from root canal infections in the teeth of man.* 2. PREPARATION OF MEDIA FOR USE IN DETERMINING THE BIOCHEMICAL REACTIONS: (a) Sugar-free broth: 75 grams of Bacto-veal in 1000 cc. of tap water were heated in the Arnold sterilizer for two hours and filtered through filter paper. This infusion was inoculated with B. coli communior (20), incubated at 37 C. for 18 hours and sterilized at "Arnold" temperature for 45 minutes. The hot bouillon easily dissolved the 10 grams of peptone and 5 grams NaCl that were added at this point. An aliquot part (locc.) was cooled and adjusted with 0.05 M NaOH to the hydrogen-ion concen- tration, PH = 7.9 (21). The calculated amount of M NaOH was added *A11 the cultures were obtained from Dr. M. L. Rhein and Dr. J. M. Levy, New York City. to the remainder and set in the Arnold sterilizer for 15 minutes. Such treatment usually results in an increased acidity, the PH changing to a value between 7.7 and 7.5. This preparation had a final PH = 7.7. The medium was filtered and sterilized in the "Arnold" for 30 minutes on three successive days. (b) Carbohydrate broth: The chief point to guard against in this connec- tion is that a sugar in slightly alkaline solution decomposes readily. This occurs to some extent even at room temperature (23 to 25 C.) but very marked decomposition sets in at the temperature of the Arnold sterilizer. It is best, therefore, to sterilize a concentrated (20 per cent) aqueous so- lution of the carbohydrate by boiling (10 to 15 minutes being sufficient) (20). This concentrated solution is then mixed with sterile sugar-free broth in amounts to make the desired dilution. The sterility of the mix- tures was tested by incubating them for 18 hours at 37 C. (c) Meat infusion agar: The basis for this medium was made by in- fusing 75 grams Bacto-veal in 500 cc. tap water in the "Arnold" for two hours. The steps in the procedure for obtaining the finished bouillon product parallelled those for sugar-free broth with the exception that there was no inoculation with B. coli. To this substrate was added an equal quantity of a 4 per cent solution of agar-agar, giving a final con- centration equal to 2 per cent. (d) Meat infusion gelatin: Hiss and Zinsser recipe (22). (e) Milk: Hiss and Zinsser recipe (23). (f) Hiss inulin-serum-ivater : Hiss and Zinsser recipe (24) . (g) Bile: Ox-bile obtained from the slaughter house was sterilized for 20 minutes on three successive days. 3. PREPARATION OF THE REAGENTS USED IN THE DETERMINATION OF HYDROGEN-ION CONCENTRATIONS: In determining hydrogen-ion concen- trations the colorimetric method was chosen. The principles involved need not be reiterated here; they have been thoroughly described by Clark and Lubs (25). The chief sources of error, color of medium and tubidity of culture were obviated by diluting the medium (one part medium to two parts distilled water) and then compensating for the color by Walpole's comparator method of superimposing the color of the medium upon that of the indicator (26, 27). (a) Standard solutions used: i /I5 M disodium hydrogen phosphate I /i 5 M potassium dihydrogen phosphate I /5 M sodium acetate i /5 M acetic acid The salts were recrystallized repeatedly until they satisfied Sorensen's purity standards (28). The acetic acid was obtained by redistilling C.P. glacial acetic acid. The phosphate mixtures were prepared by Sorensen's technique and the acetic acid-sodium acetate mixtures according to Wal- pole's directions (26). The accuracy of these standard mixtures was verified by the hydrogen electrode.* The actual values obtained prac- tically duplicate those given by these authors. A study of the following table will make this evident. *We are indebted to Dr. Hastings, of the Rockefeller Institute, New York City, for aid in this connection. Author Mixtures in c.c. PH Walpole CHsCOONa CH 3 COOH Given Obtained 5-0 5-0 4.62 4-63 5-0 7-o 4.27 4-34 Sorensen Na 2 HPO4 KH 2 PO 4 9.0 1.0 7.64 7-57 5-0 5-0 6.81 6.78 These figures were chosen as examples because the test materials matched these most often in the colorimetric determinations. (b) Indicators used: The indicators used were those recommended by Clark and Lubs and manufactured by Hynson, Westcott and Dunning, Baltimore. The following table shows the character of the indicator solutions and notes the amounts of the several varieties used: Name Concentra- tion of Solution Solvent Amount used per 10 c.c. Range of PH Tetabromphenolsulphonephtha- lein (Brom phenol blue) 0.04% 95% alcohol 0.3 c.c. 2.8-4.6 Orthocarbobenzeneazodimethyl- anilin (Methyl red) 0.02% water 0.5 c.c. 4.4-6.0 Dibromocresolsulphonephthalein (Brom cresol purple) 0.04% 95% alcohol 0.3 c.c. 5.2-6.8 Phenolsulphonephthalein (Phenol red) 0.02% 95% alcohol 0.3 c.c. 6.8-8.4 4. ISOLATION OF THE STRAINS: The fresh material taken from the root canal was planted on hemolyzed sheep blood-glucose-veal infusion agar slants (0.2 c.c. sterile hemolyzed defibrinated blood plus 0.5 c.c. 12% sterile glucose solution for each 6 c.c. medium) to which o.i c.c. sterile i% glucose-beef-infusion broth had been added after solidification. After 1 8 to 20 hours incubation at 37 C., a loopful was streaked on several whole sheep-blood glucose infusion agar plates. Single colonies fished from these plates were inoculated into glucose infusion broth. If micro- scopic examination proved this a pure streptococcus culture, transplants were made to whole sheep blood infusion agar (0.5 c.c. blood plus 9.5 c.c. agar) and ox bile. The reactions obtained for 70 cultures used as stock strains are given in the following table: Strain 852 853 863 874 877 882 894 904 909 911 922 948 953 956 961 Reaction to Whole Blood I I G I I I I H I I I I G I I Bile Strain O 981 1007 O 1009 IOI2 1015 1028 O 1041 1044 O 1074 1081 O 1094 O 1148 1151 O H54 O 1155 Reaction to Whole Blood I I I G I I I . G G I I I G G I Bile O Bile Strain O 777 802 O 804 830 O 838 898 O 907 910 O 942 O 947 O 948 966 O 978 O 1004 O 1010 O IOII O 1060 O 1097 3 O 4 Reaction to Whole Blood Bile G O I I I I O I O I O I G I O I G I I O H H I O G O I I Reaction to Strain Whole Blood 1159 G 1161 I I2IO H 1220 G 1221 1232 1236 1239 1279 G 1286 I 1293 G 682 I 684 I 688 I 703 G 720 725 I 746 748 770 I O = No solution of organisms by bile. ^ I = Indifferent to blood cells. H =Hemolysis of blood cells. G = Green-producing action on blood cells. 5. STOCK CULTURES: After isolation, each organism was planted in 2 c.c. defibrinated sheep blood, incubated for four hours at 37 C. and then stored in the ice-chest at 10 C. Some of the strains were used after several days isolation, others after several months of cultivation. 6. SEED CULTURE: Two days before a specific culture had to be used, a loopful (platinum loop, 4mm. in diameter) from the stock blood culture was planted in i% glucose broth (9.5 c.c. sugar-free broth plus 0.5 c.c. sterile 20 per cent glucose solution and incubated for 18 hours at 37 C. Then 0.05 c.c. of this subculture was inoculated into plain infusion broth (not made sugar-free) and incubated for the same period. This is desig- nated as a seed culture. In order to insure uniformity in the number of organisms transplanted from such seed cultures to the test media it was necessary to estimate the amount of growth. The cultures were well shaken and diluted to equal opacity by matching in a comparator block (the kind used in the colorimetric determination of hydrogen-ion concentration). Then the accuracy of this procedure was verified by the results obtained from plating definite amounts of the matched cultures and counting the colonies after 18 hours incubation at 37 C. For all comparative tests these matched cultures served as the seeds. 7. BIOCHEMISTRY AND BIOLOGY: (a) Reaction on blood medium: The significance of this reaction may be clearly brought out by a brief review of the literature on the classi- fication of the streptococci. At the very outset investigators attempted to differentiate these organisms according to morphological characters. Thus arose the groups (i) Streptococcus longus and (2) Streptococcus brevis (29, 30) . Such a distinction between types could be nothing more than tentative and therefore was short-lived. Collateral with this grouping, but of more lasting influence, there existed another that based on source of origin. We refer here to such groups as Streptococcus erysipelas (31, 32), Streptococcus equinus, Streptococcus pyrogenes, Streptococcus salivarans, etc. (33, 34, 35). This scheme too, could have but little value for there would be as many 8 groups of streptococci as there are places from which these organisms may be isolated. Then Schottmiiller (36) suggested a new classification into strepto- coccus erysipelas, Streptococcus viridans and Streptococcus mucosus, va- rieties recognized by certain distinctive appearances on blood media. While this method was gaining ground in Germany, English bacteriolo- gists were studying the fermentation reactions of streptococci in order to ascertain whether essential differences could be discovered . In this con- nection the researches of Gordon (33) and Andrews and Horder (34) are significant. Still another basis for classifying the streptococci has been considered that involving the immunity reactions (35, 37-47). Most investigators think these reactions parallel the blood reactions (43, 44, 45) , while Kligler (35) seems to be at variance with such a conclusion. From his experi- ments he finds a closer relationship between strains fermenting the same carbohydrates than between strains having the same hemolytic power. More recently investigations have introduced nothing new so far as fundamental principles of classification are concerned. All of them treat with one, or with a combination of the bases outlined above. Various schemes have been proposed. Most attention has been given to those which make use of the blood reaction. We may mention here the work of Holman (48), Blake (49) and Smith and Brown (45). These authors recognize two main types hemolytic and non-hemolytic, divided into subgroups which depend on the fermentative power of the organisms. And finally, the scheme which has superceded all of these and is now considered as the scheme by some of the most prominent bacteriologists is based primarily on the blood reaction and includes three main groups. Reference is made to the work of Lyall (50), Brown (51) and Park and Williams (52). A thorough consideration of the various suggestions has led also us to conclude that the best basis for a preliminary grouping of the strepto- cocci is that last named. The three types recognized are (i) that which hemolyzes the blood corpuscles; (2) that which produces a green colora- tion; (3) that which is indifferent to the presence of blood. Detailed technique for differentiating the "blood types": 0.05 cc. culture from seed tube was dropped on the blood agar (0.5 cc. blood plus 9.5 cc. agar) in a petri dish and spread over the entire surface with a special smooth glass rod. The depth of the agar was 2 mm. Precaution was taken to have the blood evenly distributed. The results recorded were obtained from the examination of colonies widely separated after 24 hours incubation at 37 C. In view of the fact that different laboratories use blood from different animal species we felt it necessary to find out whether the various strains reacted alike on the bloods most commonly used, human, rabbit and sheep. As might have been expected, the response of the organisms to the different kinds of erythrocytes was not the same. The actual ex- perimental findings for 70 cultures are as follows: Strain Reaction to Human Blood Rabbit Blood Sheep Blood H I G H I G H I G 852 853 863 877 874 + + + + + + + + + + + + + + + Strain Reaction to Human Blood Rabbit Blood Sheep Blood H I G H I G H I G 882 + T , 894 + + 4. 904 + 4,- + 909 + 4 r ^ 9" 4- 4- 4. 922 + 4. _i_ 948 -f + 4. 953 956 + + _,_ + + 961 + + i 981 4- 4 r 4, 1007 4- + ^ 1009 4- 4- 4. IOI2 + 4^ i 1015 + ^ + 1028 4- 4^ IO4I + + ^_ 1044 + + i 1074 1 08 1 + + + + + + 1094 4- 4^ + 1148 + + "54 + _|_ + + "55 + 4. i "59 IIDI | t + I2IO 4 r 4^ i I22O ^_ _j_ , 1221 4^ _j_ i 1232 1236 | + + 1239 4- 4,- i 1279 4^ _L i 1286 ^ i i 1293 4 r 4. i 682 4. _j_ i 684 + 4. T 688 4 r i T 703 4^ _)_ i 720 -{- 4^ i 725 4- _j_ i 746 4^ _j_ i 748 + 4. i 770 + 4^ i 777 4- _|_ i 802 4^ i ' 804 4- i 830 4- i i 838 4^ _i_ T 898 4- 4^ i 907 4- _(_ i 910 4- _j_ i 942 4- _|_ i 947 4- _i_ i 948 4^ _j_ T 966 4. _j_ , 978 + 4^ _l_ 1004 -\- ^ i IOIO -\- 4_ i IOII 1060 + | + + + - 1097 4. J. i 3 + + _l_ 4 Note: H=hemolysis. I = indifferent. G = green producing. 10 These results were checked by triplicate determinations. The above detailed tabulation may be summarized as follows:- No. of Strains No. of Strains No. of Source of Blood Producing Producing Indifferent Hemolysis Green Coloration Strains Man 16 7 47 Rabbit 17 7 46 Sheep 4 16 50 No. of "fast" strains 4 7 40 Note: A "fast" strain is one which reacts alike to the blood of all species. This table indicates definitely the probable source for many discrep- ancies. It indicates, too, that a very decided advance in the systematic study of the streptococci may be made by instituting definite standards of procedure. The group of organisms used for analytical material included two "fast 1 ' strains of each type. (b) Immunity reactions: These reactions were studied in order to demonstrate that the chosen organisms were biologically distinct. To prove this point, the agglutinative capacity of each antiserum against each of the antigens was determined. The method of producing the immune sera and the technique for conducting the titrations was that used by Dochez, Avery and Lancefield (53). The results obtained (as may be seen from the table) show no duplica- tion in the strains used. Immune Serum 904 I2IO 863 1074 688 72O 904 1-500 I-IOO o I2IO 1-150 1-450 86 3 1-350 1-250 1074 1-200 1-650 o 688 1-50 1-50 1-30 I-20O I-IOO 720 I-IO I -2O I-2O 1-50 1-550 Note: The numbers under "immune serum" designate the highest dilutions of the antiserum which showed definite specific clumping of the organisms. (c) Other biochemical reactions: Although our choice of the experi- mental strains depended almost entirely on the blood reactions yet a complete characterization of the organisms must include the description of their ability to (i) ferment various carbohydrates and related sub- stances (33, 34); (2) coagulate milk (33, 34); (3) coagulate inulin-serum- water (53, 54); (4) liquefy gelatin (34); (5) dissolve in bile (55). The test substance (10 c.c.) in each series (except 5) was inoculated with 0.05 c.c. culture from the seed tubes. In series (5) 0.3 c.c. bile was mixed with 3 c.c. culture. All except series (3) were incubated at 37 C. for 24 hours. Series (3) was incubated for 6p hours. The follow- ing tabulation lists the reactions given by the experimental cultures: n c Hydrogen-ion Concentration , Reaction in o PH, in Broth B , Strain Blood Reaction imunitj s * 13 o S s : -2 C '2 e-S J^ b * O * % t^ % 5 3 5 1" Control 7-7 7-7 7-7 7-7 7-7 7-7 7-7 7.7 7-7 7-7 o o o o 904 Hemolysis + 7-7 4.8 6.2 4.8 7-7 4-3 4.6 5-9 5-0 7-7 -f o 1210 Hemolysis -j- 7-7 4.6 5-9 4.6 4.6 4-3 6.8 5-9 4.6 7-7 -f- o o 863 Green-producing f- 7-7 4.6 7-7 4.6 4.6 4-3 7-7 6-5 7-7 7-7 o -f- o 1074 Green-producing -j- 7-7 4-3 6-5 4.6 5-3 4-3 5-3 6.2 4.6 7-7 o -(- o 688 Indifferent -f- 7-7 4.6 5-6 4.6 4.6 5-0 7-7 6.2 4-3 7-7 -f- o o 720 Indifferent + 7-7 4-3 6-5 4.6 4.6 4-3 4.8 6-5 4-3 7-7 o + o o Note: In the column under "immunity" the " +" sign designates that the culture is biologically distinct. 8. DESCRIPTION OF THE TEST ORGANISMS: Six test organisms were used: two hemolytic strains, two, green-producing and two, indifferent. The members of each pair were not duplicated individuals of the same type (see carbohydrate reactions). Moreover there was no duplication of strain in the entire series (see agglutination reactions) . III. PREPARATION OF MASS CULTURES i. MEDIUM: (a) Choice of Infusion basis: A comparative study of the different solid media available for streptococcus cultivation shows that the organisms accumulate more rapidly on some than on others. Five media were considered . (1) Hemolyzed blood glucose veal infusion agar: The veal infusion was prepared as described above diluted with an equal volume of 3 per cent agar-agar solution and sterilized. When needed, the agar was melted and cooled slowly to 43-45 C. Sterile hemolyzed defibrinated blood (i volume blood to 2 volumes of distilled water) and sterile 12% glucose solution were then added. The proportion used was o.i c.c. of the diluted blood and 0.25 c.c. of the sugar solution to every 3.0 c.c. agar. (2) Huntoon agar (56) . (3) Hasting gelatin-agar (57): The original directions are somewhat vague. This description is our interpretation: One pound beef meat in loooc.c. water was boiled for one hour, strained through a wire sieve, made up to the original volume, 10 grams peptone and 5 grams NaCl added and set in the Arnold sterilizer for 20 minutes. The infusion thus obtained was used as the solvent for the 15 grams agar-agar and 20 grams gelatin added at this point. Finally, enough NaOH was added to make the mixture neutral to phenolphthalein, the medium being kept at the boiling point during the titration. (4) Beef liver agar: 500 grams fresh hashed beef liver were infused with 500 c.c. tap water for three hours at 55 C., strained through a wire sieve, 10 grams peptone and 5 grams NaCl added and set in the "Arnold" until solution was complete. The hydrogen-ion concentration was adjusted to a PH value = to 7. 9. After being mixed with 500 c.c. 3 per cent agar-agar solution, the infusion was sterilized in the autoclave for 15 minutes at 15 pounds pressure. (5) Beef brain agar: This preparation was made exactly as the beef liver agar except that brain tissue was substituted for liver tissue. Thus prepared, the five media were compared by this procedure: Each medium was tested with each experimental culture. We had, therefore, six series of tests one for each strain. 0.05 c.c. of culture from the several seed tubes were evenly distributed over the surfaces of agar slants of the respective media. All the slants were formed by equal amounts of medium. Readings were made after 1 8 hours incubation at 37 C. The data tabulated below indicate the results obtained for one series. They are representative since each series showed the same gradations in the amounts of growth. Medium Estimated Amount of Growth Hemolyzed blood agar Huntoon agar Hasting gelatin-agar Brain agar Liver agar Abundant Abundant Slight Slight Very abundant + + + + + + + + + + + + + + The results are necessarily only comparatively quantitive. They indicate, however, that the liver infusion furnished the optimum material for maximum growth. This may be explained by the fact that the liver normally contains a large store of glycogen, amino acids, various secre- tions, etc., all probably valuable to bacterial- nutrition. (b) Concentration of agar-agar: The more concentrated the agar- agar in a medium, the greater is the insurance against scratching the surface of a solidified layer in a petri dish when removing growth with a smooth spatula. But, on the other hand, too concentrated a preparation will practically inhibit bacterial growth. It therefore, became necessary to find that concentration which permitted the maximum development of colonies and at the same time insured against the possible breaking of the surface. Six series of tests were performed, one for each experimental culture. The infusion subtrate was the same for all; the concentration of the agar-agar was varied for each series. The results show definitely that for the organisms used 2 per cent is the optimum concentration. Percent. || agar-agar || Growth for the Various Organisms Streaking Character Removal of Growth 688 720 863 1074 904 I2IO I.O + + + + + + + + + + + + + + + + + + + + + + + + Unfavor- Impos- able sible i-5 + + + + + + + + + + + + + + + + + + + + + + + + Difficult Difficult 2.O + + + + + + + + + + + + + + + + + + + + Very Very favorable Favorable 2 -5 + + + + + + + + + + + + + + + + + + + + Very Very favorable avorable 3-0 + + + + + + + + + + + + + + Very Very favorable avorable 3-5 + + + + + + + + + + + + + Very Very favorable avorable 4.0 + + + + + + + + Very Very favorable avorable 4-5 + + + + + + Very Very favorable avorable 5- + + + + + Very Very favorable avorable (c) Constancy in composition of medium: To insure absolute constancy in the composition of the medium a stock infusion was prepared from one large shipment of fresh hashed liver (200 pounds). This was estimated as the quantity large enough to cover the needs of the entire investigation. 2. CULTURES: (a) Preparation of cultures for mass growing: Jablons liver peptone broth (58) was used for the cultivation of the test organisms preliminary to mass growing. This medium attracted our attention because it contained the same ingredients as the solid medium selected, thus favoring exceptional uniformity in the metabolic reactions of the bacteria throughout the entire experimental period. That the bouillon supported hardy development may be proven by the following experiments com- paring it with other good liquid media: (i) Huntoon broth (56) and (2) Rosenow broth (59). The test media were inoculated with one loopful of culture from the seed tubes, kept at room temperature for 60 hours and then incubated for 24 hours at 37 C. Tests were made on 50 strains of streptococci. A comparison of the amounts of growth in the different preparations will give convincing proof that Jablons medium favors rapid growth of the organisms under consideration. The results tabulated represent the reactions given by all the organisms. Medium Amount of Growth Estimate Description Jablons Broth Rosenow Broth Huntoon Broth + + + + + + + + + + + Very abundant Very abundant Quite abundant At regular intervals transplants to glucose blood agar were made, incubated for 24 hours at 37 C. and examined for growth. If growth was slight or doubtful, subcultures were made to confirm the results. By this procedure further evidence was obtained regarding the quality of the liver broth. A mere glance at the following summary shows that, in addition to supporting very rapid growth, Jablons medium also supports very prolonged viability. Medium No. of Cultures Dead in Specified No. of Weeks After Inoculation Per cent. Viable After 4 6 IO 10 Weeks Jablons Broth o o o IOO Rosenow Broth o i 2 96 Huntoon Broth o i 3 94 These results place each medium in the order of preference for the cultivation of streptococci, if longevity without transplant is taken as the criterion. With four weeks as the limiting period no differences are noticed; the three preparations match each other exactly. But, more prolonged viability necessarily infers less interference with normal metabolism. This makes Jablons medium the most efficient since it is so constructed as to give the organisms the greatest chance for continued vitality. After growing the organisms in this broth for one month, 0.05 c.c. was transplanted to liver agar plates and grown at 37 C. for 18 hours. From this two successive subcultures to solid liver medium were made on two consecutive days. The last culture served as the seed culture for mass planting. 14 The mass cultures were grown for 16-18 hours at 37 C., thus producing cultures that contained a minimum of degenerated cells. Of course, this procedure did not supply the maximum amount of growth from each planting, but it did supply the optimum material for the chemical analysis of the normal organisms. This point seems to have been entirely over- looked in previous investigations on bacterial composition. In these, the chief interest was to procure the maximum amount of material from each planting. No attention was, therefore, paid to the metabolic plane of the organism. Thus Brieger (i) used growth four weeks old, Leach (60) worked with cultures one to two weeks old, and Vaughan (15), stating no definite period, gathered the growth when it had reached the maximum. (b) Planting of Cultures: The agar was allowed to cool slowly to 43- 45 C., thus insuring a minimum volume of water of condensation in the petri dishes after solidification. This is a necessary precaution since in too moist a chamber the growth becomes saturated with moisture. Such a condition is very unfavorable for the purpose at hand, because (i) it is impossible to remove any significant part of the bacterial masses from the agar surface and (2) the small fraction that is obtained is contami- nated with diffused substance from the medium. The depth of the agar layer in the plates was 4mm. Transplants were made from the special seed plates described above by means o a bent smooth glass rod. (c) Removal of the growth from the agar: Two possible procedures sug- gested themselves. One method involves the detachment of the growth from the subjacent agar with bent glass rods, simplifying its removal by washing with physiological salt solution and pipetting off the bacterial suspension (15, 16). It is evident that a maximum amount of material may be obtained in this way but it is not optimum material for the present study. Much of the soluble substances in the medium are washed into the salt solution. Then, too, some substances diffuse from the cell bodies into the outer liquid. Such material may be removed by several washings and centrifugations, each time discarding the supernatant fluid. By doing so, however, the diffusible bacterial constituents are continually being drawn upon. The other-method, although not as economical regarding the collection of material, is far superior in providing a pure mass consisting only of "intact" bacterial structures. This process is simpler and involves fewer manipulations than the first. Thus, the smoothened end of a microscope slide is held lightly but firmly on the agar surface and rotated slowly clockwise. At the same time the dish is turned slowly counter- clockwise. The growth is thereby gathered on the slide and very easily tapped off into a thoroughly dried shallow glass dish. In order to determine definitely the method best suited for our purposes, tests were made on: (1) The supernatant NaCl solution after centrifugati on of thebacter- suspension, obtained by removing the growth from the agar with NaCl solution. (2) The supernatant fluid after centrifugation of the bacterial suspen- sion in distilled water, obtained as in (i) with distilled water substituted for the NaCl solution. (3) The NaCl solution after being applied to a fresh agar surface and manipulated as though growth was actually being removed. (4) The distilled water wash under the same conditions as in (3). is (5) The supernatant fluid from a bacterial suspension in a NaCl solu- tion, the bacteria having been removed by the spatula method. (6) The supernatant fluid from a bacterial suspension in distilled water, the growth having been removed as in (5). (7) The NaCl solution used to wash a glass slide manipulated over a fresh agar surface as though growth was being removed. (8) The distilled water wash obtained as in (7) . The tabulation below gives the results of the qualitative tests for phosphate, chloride, protein and protein split products, and dextrose. The numbers in parentheses refer to the several descriptions listed above. Tests Performed Test Material Protein or Chloride Phosphate Protein Split Products Dextrose (i) + + + + + + + + o (2) + + + + -j--J--p- + + + + + o 1 (5) ++++ o o (6) (7) o o o o o (8) o o o Note: With medium containing I per cent, dextrose (i) to (4) gave for sugar. result These experiments show that: (1) Substances are transferred from the medium to the wash solution . (2) Bacteria suspended in distilled water or NaCl solution may give up some of their substance to the surrounding liquid. (3) Nothing is removed from a medium by the mere application of a smooth spatula (70). This evidence points definitely to the second method as the superior one. (d) Drying of growth: The technic was that recommended by Shackell (61) with one exception: the bacteria were not frozen before drying. Layers I to 2 mm. thick in shallow plates dried to a scaly, porous mass in 24 to 36 hours. The temperature during the process varied between 21 and 22 C., the pressure between o and 10 mm. Hg. The material was then pulverized with glass mortar and pestle to a very fine powder, spread in thin layers on glazed paper plates and dried to constant weight. The dried substance was transferred to weighing bottles fitted with ground glass stoppers and stored in a dessiccator over concentrated H 2 SO 4 . IV. ANALYSIS OF THE BACTERIAL SUBSTANCE i. METHODS: (a) Nitrogen: Nitrogen was determined by the Kjeldahl process (Gunning-Arnold Dyer modification) (62) . Digestion of the substance in concentrated sulphuric acid was completed with small quantities of metallic mercury. Before distillation, the mercury was precipitated with potassium sulphide. The titrations were made with 0.02 M NaOH. 16 (b) Total Sulphur and Phosphorus: Sulphur was determined by the method adopted by the the Association of Official Agricultural Chem- ists (63). The filtrate from the barium chloride precipitation was used to determine phosphorus (64). (c) Ash: Inorganic matter was estimated by direct incineration of the dried substance in a platinum crucible over a very low flame. (d) Moisture: The medium was cooled slowly to 43 C. The agar for the entire series of determinations was obtained from the same flask and the necessary number of petri dishes filled at the same time. The organisms were grown and collected as described above. About o.i gram of growth was rapidly transferred to a rubber stoppered glass vial weigh- ing about 0.3 gram. Two sets of determinations of water content were made: (i) by drying in vacuo (o-iomm. Hgand 22 C.) and (2) by drying in an air bath (ioo-uo). (e) Lipins (material extracted with ether) : The substance was weighed in cone-shaped filter paper thimbles. The thimbles were suspended by means of wires to reflux condensers. These were fitted with flasks con- taining ether. After 24 hours of hot extraction the thimbles containing the extracted bacteria were dried in vacuo to constant weight. The difference in the weight of the thimble before and after extraction rep- presents the weight of lipins. (f) Blanks: Blank determinations on the reagents used were made in all cases. In the analyses for N the accuracy of the method for the small quantities obtained was checked by estimating the N recovered from known quantities of pure salt (ammonium sulphate) . 2. RECORDS OF ANALYSIS: (a) Organism No. 688: Nitrogen: 0.0199 gram material gave 0.0019 gram N = 9.55 per cent N; 0.0217 gram material gave 0.0022 gram N = 10.13 per cent N. Sulphur: 0.5336 gram material gave 0.0247 gram BaSO 4 = 0.63 per cent S; 0.5769 gram material gave .0259 gram BaSO 4 = 0.61 per cent S. Phosphorus: 0.5336 gram material gave 0.0393 gram Mg 2 P 2 O7 = 2.05 per cent P; 0.5769 gram material gave 0.0432 gram Mg 2 P 2 O 7 = 2.08 per cent P. Ash: 0.5354 gram material gave 0.0526 gram Ash = 9.82 per cent Ash; 0.4694 gram material gave 0.0458 gram Ash = 9.75 per cent Ash. Moisture: A. Determined by drying the bacterial material in vacuo: 0.066 1 gram material gave 0.0485 gram H 2 O = 73.37 per cent; 0.0866 gram material gave 0.0636 gram H 2 O = 73.44 per cent. B. Determined by drying the bacterial material at 1 00 C: 0.0899 gram material gave 0.0671 gram H 2 O = 74.63 per cent; 0.0769 gram material gave 0.0573 gram H 2 O = 74.52 per cent. Lipins: 0.3073 gram bacterial material gave 0.0047 gram Lipins = 1.53 per cent; 0.3343 gram bacterial material gave 0.0055 gram Lipins = 1.64 per cent. (b) Organism No. 720: Nitrogen: 0.0257 gram material gave 0.0026 gram N = io.ii per cent N; 0.0243 gram material gave 0.0025 gram N = 10.28 per cent N. 17 Sulphur: 0.5741 gram material gave 0.0253 gram BaSO 4 = 0.60 per cent S; 0.5320 gram material gave 0.0234 gram BaSO 4 = 0.60 per cent S. Phosphorus: 0.5741 gram material gave 0.0429 gram Mg 2 P2O 7 = 2.08 per cent P; 0.5320 gram material gave 0.0390 gram Mg 2 P2O 7 = 2.O4 per cent P. Ash: 0.2910 gram material gave 0.0301 gram Ash = 10.34 P er cent Ash; 0.3011 gram material gave 0.0311 gram Ash = 10.33 P er cent Ash. Moisture: A. Determined by drying the bacterial material in vacuo: 0.0638 gram material gave 0.0452 gram H 2 O = 7O.85 per cent; 0.0864 gram material gave 0.0611 gram H 2 O = 70.72 per cent. B. Determined by drying the substance at 100 C: 0.0781 gram material gave 0.0560 gram H 2 O = 7i .70 per cent; 0.0897 gram material gave 0.0643 gram H 2 O = 7i.68 per cent. Lipins: 0.3377 gram bacterial material gave 0.0035 gram Lipins=i.O3 per cent; 0.3131 gram bacterial material gave 0.0033 gram Lipins = 1.05 per cent. (c) Organism No. 904: Nitrogen: 0.0235 gram material gave 0.0025 gram N = 10.64 per cent N; 0.0213 gram material gave 0.0022 gram N = 10.33 per cent N. Sulphur: 0.6347 gram material gave 0.0265 gram BaSO 4 = o.57 per cent S; 0.5096 gram material gave 0.0202 gram BaSO 4 = 0.54 per cent S. Phosphorus: 0.6347 gram material gave 0.0494 gram Mg 2 P 2 O 7 = 2.17 per cent P; 0.5096 gram material gave 0.0405 gram Mg 2 P 2 O 7 = 2.2i per cent P. Ash: 0.5208 gram material gave 0.0538 gram Ash = 10.33 P er cent Ash; 0.4933 gram material gave 0.0508 gram Ash = 10.30 per cent Ash. Moisture: A. Determined by drying the substance in vacuo: 0.0475 gram material gave 0.0347 gram H 2 O = 73.O5; 0.0620 gram material gave 0.0453 gram H 2 O = 73.O7 per cent. B. Determined by drying the bacterial material at 100: 0.0530 gram material gave 0.0401 gram H 2 O = 75-66 per cent; 0.0668 gram material gave 0.0506 gram H 2 O = 75-75 per cent. Lipins: 0.3390 gram bacterial material gave 0.0122 gram Lipins = 3. 59 per cent; 0.2981 gram bacterial material gave 0.0108 gram Lipins = 3. 62 per cent. (d) Organism No. 1210: Nitrogen: 0.0209 gram material gave 0.0020 gram N = 9-57 per cent N; 0.0238 gram material gave 0.0023 gram N = 9-66 per cent N. Sulphur: 0.5301 gram material gave 0.0230 gram BaSO 4 = o.6o per cent S; 0.5007 gram material gave 0.0217 gram BaSO 4 = 0.60 per cent S. Phosphorus: 0.5301 gram material gave 0.0410 gram Mg 2 P 2 O 7 = 2.15 per cent P; 0.5007 gram material gave 0.0399 gram Mg 2 P 2 O 7 = 2.22 per cent P. Ash: 0.2687 gram material gave 0.0266 gram Ash = 9. 90 per cent Ash; 0.2087 gram material gave 0.0206 gram Ash = 9. 87 per cent Ash. 18 Moisture: A. Determined by drying the bacterial material in vacuo: 0.0745 gram material gave 0.0544 gram H 2 O = 73.O2 per cent; 0.1033 gram material gave 0.0753 gram H 2 O = 72.89 per cent. B. Determined by drying the bacterial material at 100: 0.0466 gram material gave 0.0349 gram H 2 O = 74-9O per cent; 0.0739 gram material gave 0.0554 gram H 2 O = 74.97 per cent. Lipins: 0.2051 gram bacterial material gave 0.0083 gram Lipins = 4.04 per cent; 0.2251 gram bacterial material gave 0.0086 gram Lipins = 3. 82 per cent. (e) Organism No. 863: Nitrogen: 0.0265 gram material gave 0.0026 gram N = 9.8i per cent N; 0.0224 gram material gave 0.0023 gram N = 10.27 per cent N. Sulphur: 0.6206 gram material gave 0.0254 gram BaSO 4 = o.56 per cent S; 0.7039 gram material gave 0.0283 gram BaSO 4 = 0.55 per cent S. Phosphorus: 0.6206 gram material gave 0.0444 gram Mg 2 P 2 O7 = 1.99 per cent P; 0.7039 gram material gave 0.0498 gram Mg 2 P 2 O 7 =i.97 per cent P. Ash: 0.4887 gram material gave 0.0457 gram Ash = 9.35 per cent Ash; 0.5881 gram material gave 0.0552 gram Ash = 9. 38 per cent Ash. Moisture: A. Determined by drying the bacterial material in vacuo: 0.0665 gram material gave 0.0465 gram ^0 = 69.93 per cent; o. 0743 gram material gave 0.0521 gram H 2 O = 7O.I2 per cent. B. Determined by drying the substance at 100: -577 gram material gave 0.0432 gram H 2 O = 74.87 per cent. Lipins: 0.4098 gram bacterial material gave 0.0226 gram Lipins = 5.51 per cent; 0.3933 gram material gave 0.0208 gram Lipins = 5.29 per cent. (f) Organism No. 1074: Nitrogen: 0.0209 gram material gave 0.0021 gram N= 10.05 per cent N; 0.0198 gram material gave 0.0019 gram N = 9-59 per cent N. Sulphur: 0.5037 gram material gave 0.0221 gram BaSO 4 = o.6o per cent S; 0.4791 gram material gave 0.0199 gram BaSC>4 = 0.57 per cent S. Phosphorus: 0.5037 gram material gave 0.0358 gram Mg 2 P 2 O7 = 1.98 per cent P; 0.4791 gram material gave 0.0338 gram Mg 2 P 2 O 7 = 1.96 per cent P. Moisture: A. Determined by drying the bacterial material in vacuo: 0.1070 gram material gave 0.0765 gram H 2 O = 7i-5O per cent; 0.0896 gram material gave 0.0642 gram H 2 O = 7i.65 per cent. B. Determined by drying the substance at 100: 0.0668 gram material gave 0.0506 gram H 2 O = 75.60 per cent; 0.0552 gram material gave 0.0416 gram H 2 O = 75-36 per cent. Lipins: 0.3080 gram bacterial material gave 0.0166 gram Lipins = 5-39 P er cent; 0.2995 gram bacterial material gave 0.0162 gram Lipins = 5. 40 per cent. Ash: 0.3024 gram material gave 0.0301 gram Ash = 9. 95 per cent Ash; 0.4024 gram material gave 0.0398 gram Ash = 9. 89 per cent Ash. 19 3. SUMMARY OF THE DATA FOR THE AVERAGE PERCENTAGE COMPOSI- TION OF THE STREPTOCOCCI. Description of Organism Composition Moisture Nu mber Type N S P Ash Lipins Vacuo IOO-1 10 688 Indifferent 9.8 0.62 2.06 9.78 1.58 7340 74-57 720 Indifferent IO.I 0.60 2.06 10.33 1.04 70.78 71.69 904 Hemolytic 10.4 0-55 2.19 10.31 3.60 73.06 75-70 I2IO Hemolytic 9.6 0.60 2.18 9.88 3-93 72.95 74-93 86 3 Green producing IQ.O 0-55 1.98 9-36 5-40 70.02 74-87 1074 Green producing 9.8 0.58 1.97 9.92 5-39 71-57 7548 4. CONCLUSIONS FROM THE ABOVE ANALYTICAL DATA: The above summary indicates the average results of the various analyses. It also brings into comparison the figures for the composition of the different streptococci and points out these facts: (a) There are no marked graded differences in either the nitrogen or the sulphur content. It becomes apparent, therefore, that the total amounts of protein material in the different organisms varies but slightly, if at all. (b) The phosphorus content is practically constant. This indicates no striking variations in the quantity of nucleoprotein (the phosphorus due to phosphatide being very small in amount) . (c) Regarding the results for the moisture determinations only slight deviations are noticed. The percentages obtained by drying the sub- stance at iooC. are higher than those obtained by drying the material in vacuo. Such increases in the values were expected because of the greater disintegration at the higher temperature. (d) Most interesting, however, are the figures obtained for the amounts of Lipin material. These vary greatly for the different types of strep- tococci. The percentage of lipins is lowest in the indifferent type and highest in the green producing type, the hemolytic type taking a posi- tion between these two. The characteristic relations described above may be expressed by the ratio: 10 : 29 : 41 ; or briefly : Indifferent: Hemolytic: Green-producing 3:4 These results separate the streptococci into three groups corre- sponding to those obtained when the reaction on blood media is used as the criterion. Because of the apparent significance of this relationship, we repeated this part of the investigation. Fresh cultures were amassed and dried as described above. A series of nine organisms were used, three from each representative group, the strains 688, 720, 904, 1210, 863 and 1074 being included . The three new strains were chosen at random from our stock cultures of indifferent, green-producing and hemolytic streptococci. These results were obtained: 20 Designation of Organism Number Type Average Percentage Lipins 688 Indifferent 1.46 720 Indifferent 1 .54 725 Indifferent 1.16 904 Hemolytic 3.16 1210 Hemolytic 3.73 1012 Hemolytic 3-79 863 Green-producing 5.31 1074 Green-producing 5.34 953 Green-producing 5.68 A mere glance at the table brings to light the possible ratio: 1:3:4 This similates the data gathered from our first group of determinations. V. ANALYSIS OF THE LIPIN MIXTURE 1. OUTLINE FOR THE WORK: The very striking regularity in the amounts of Lipin material and the fact that the groups of streptococci parallel those obtained by the blood media reaction suggested the possible relation between the nature of the Lipin mixture and the hemolyzing capacity of the organisms. In an investigation of this character the chief factors to be considered are (i) the phosphatide content, (2) the choles- terol content and (3) the ratio between the phosphatide and cholesterol. A preliminary qualitative examination of the material showed that all the extracts contained both cholesterol and phosphatide. These sub- stances were then determined quantitatively by nephelometric methods (65,66,67,68). 2. RECORDS OF ANALYSIS OF THE LIPIN MIXTURE. (a) Organism No. 688: Phosphatide: 2.3560 gram bacterial material gave 0.0400 gram Lipins which yielded 0.0000125 gram H 3 PO 4 = 0.25 per cent Phosphatide; 2.4960 gram bacterial material gave 0.0380 gram Lipins which yielded 0.0000125 gram H 3 PO4= 0.25 per cent Phosphatide. Cholesterol: 2.5360 gram bacterial material gave 0.0400 gram Lipins which yielded 0.00064 gram Cholesterol =1.6 percent Cholesterol; 2. 4960 gram bacterial material gave 0.0380 gram Lipins which yielded 0.00064 gram cholesterol = 1.7 per cent cholesterol. (b) Organism No. 720: Phosphatide: 2.4411 gram bacterial material gave 0.0290 gram Lipins which yielded 0.0000087 gram H 3 PO 4 = 0.24 per cent Phosphatide; 2.4301 gram bacterial material gave 0.0280 gram Lipins which yielded 0.0000087 gram H 3 PO 4 = 0.24 per cent Phosphatide. Cholesterol: 2.4411 gram bacterial material gave 0.0290 gram Lipins which yielded 0.00046 gram Cholesterol = 1.5 per cent 2.4302 gram bacterial material gave 0.0280 gram Lipins which yielded 0.00046 gram Cholesterol =1.5 per cent. (c) Organism No. 904: Phosphatide: 1.7090 gram bacterial material gave 0.0710 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.064 per cent Phosphatide; i .6980 gram bacterial material gave 0.0640 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.072 per cent Phosphatide. Cholesterol: 1.7090 gram bacterial material gave 0.0710 gram Lipins which yielded 0.00078 gram Cholesterol = 1.09 per cent; 1.6980 gram bacterial material gave 0.0640 gram Lipins which yielded 0.0007 gram Cholesterol = 1.08 per cent. (d) Organism No. 1210: Phosphatide: 1.7091 gram bacterial material gave 0.0700 gram Lipins which yielded 0.000006 gram HsPO 4 = 0.064 per cent Phosphatide; 1.7100 gram bacterial material gave 0.0710 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.064 per cent Phosphatide. Cholesterol: 1.7091 gram bacterial material gave 0.0700 gram Lipins which yielded 0.0007 gram Cholesterol = i.o per cent Cholesterol; 1.7100 gram bacterial ma'terial gave 0.0710 gram Lipins which yielded 0.00074 gram Cholesterol = 1.02 per cent. (e) Organism No. 86 j: Phosphatide: 2.5725 gram bacterial material gave 0.1366 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.035 P er cent Phosphatide; 2.5728 gram bacterial material gave 0.1362 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.035 per cent " Phosphatide. Cholesterol: 2.5725 gram bacterial material gave 0.1366 gram Lipins which yielded o.ooio gram Cholesterol = 0.73 per cent; 2.5728 gram bacterial material gave 0.1362 gram Lipins which yielded o.ooio gram Cholesterol = 0.73 per cent. (f) Organism No. 1074: Phosphatide: 2.5361 gram bacterial material gave 0.1321 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.036 per cent Phosphatide; 2.5360 gram bacterial material gave 0.1325 gram Lipins which yielded 0.000006 gram H 3 PO 4 = 0.036 per cent Phosphatide. Cholesterol: 2.5361 gram bacterial material *~ ~ """ Lipins which yielded o.ooio gram Cholester 3. SUMMARY OF THE DATA FOR THE AVERAGE PERCENTAGES OF PHOS- PHATIDE AND CHOLESTEROL. Organism No. 688 720 904 1210 863 1074 Phosphatide 0.25 0.24 0.068 0.064 0.035 0.036 Cholesterol l.6 5 1.50 1. 08 I.OI 0.73 0.75 Ratio C:P 6:1 6:1 16:1 16:1 21:1 21:1 4. CONCLUSIONS FROM THE DATA FOR THE CHOLESTEROL AND PHOS- PHATIDE CONTENT. (a) The content of phosphatide decreases in the different types in the order named: indifferent, hemolytic, green-producing. (b) The same relation is noticed in the amounts of cholesterol contained in the different organisms. (c) The ratio C:P increases when the types are listed in the same order. VI. EFFECT OF THE LIPIN MATERIAL ON HEMOLYSIS i. RECORD OP EXPERIMENTS: Since the ratios of cholesterol to phos- phatide are different for the different types of streptococci it was interest- ing to investigate the effects which the lipin mixtures would have on the hemolytic action of saponin.* A 10 per cent washed rabbit cell suspen- sion was used in the experiments. The method used was essentially that of Ranson (69). Preliminary titrations were made to obtain the dose of saponin that will hemolyze I .o c.c. of cell suspension in the presence and absence of cholesterol (the amount of cholesterol used approximated that in the lipin mixture) . That amount of saponin which produced hemolysis in the absence of cholesterol but which failed to do so in those tubes which contained cholesterol was taken as the test dose. This dose was then added to graded amounts of lipin material dissolved in ether. The pro- tocols below show in detail the experiments performed and the results obtained. Experiment (a): To determine the largest amount of saponin that produces hemolysis in the presence of cholesterol. Tube no. i 2 3 4 5 6 7 8 9 Saponin in c.c. 0.2% in 0.9% NaCl I.O 0.5 0.2$ O.IS O.IO O.08 0.06 0.04 o.o* Cholesterol in c.c. 0.01% in Ether O. I 0. I O. I O. I O. I O. I O.I O. I 0. I 0.9% NaCl inc. c. 1.90 2.40 2.63 2.7S 2. SO 2.82 2.84 a. 86 s.88 Incubation at 25 C. for 15 Minutes Cell suspension in c.c. I.O I.O I.O I.O I.O I.O I.O I.O I.O Readings: 8 minutes + + + + + + + + ++++ +++ +++ o o 15 minutes + + + + + + + + ++++ +++ +++ + + o o 60 minutes + + + + + + + + +++ + +++ + ++++ + + + + + o o 12 hours + + + + + + + + ++++ +++ + +++ + + + + + + + + + ++++ o This experiment shows that in the presence of cholesterol 0.08 cc. of a 0.2% saponin solution is the largest amount that does not hemolyze i.o c.c. cell suspension in 8 minutes. Experiment (b): To determine the smallest amount of saponin that produces hemolysis in the absence of cholesterol . Tube no. i 2 3 4 5 6 7 8 Saponin I.O 0.5 0.25 0.15 0. I 0.08 0.06 0.04 o. 02 Ether O. I O. I O. I O. I O. I O. I O. I 0. I 0. I Salt Solution 1.90 2.40 2.65 2.75 2.80 2.82 2. 84 2.8. 2.8* Incubation at 25 C. for 15 Minutes Cell Suspension i.o i.o i.o i.o i.o i.o i.o i.o i.o Readings: 8 minutes 15 minutes 60 minutes 12 hours *The saponin was obtained from Eimer and Amend, New York City. 23 The results of this experiment indicate that in the absence of choles- terol 0.08 c.c. of a 0.2% saponin solution is the smallest amount that produces hemolysis with i.o c.c. cell suspension in 8 minutes. This is, therefore, the test dose and was used in determining the effect of the ether extract on the action of saponin. The lipins extracted from the bacteria were dissolved in definite volumes of ether: three-fifths of this solution was evaporated to a volume of 3cc. and then emulsified with 27 cc. of 0.9% NaCl solution. Graded amounts of this emulsion were added to the various tubes. Experiment (c): To determine the effect of the extract from organism no. 688 on the action of saponin. Tube No. I 2 3 4 Extract in c.c. I.O i-5 2.0 2-5 Saponin in c.c. 0.08 0.08 0.08 0.08 Salt solution in c.c. 1.92 1.42 O.92 0.42 Incubation at 25 for 15 Minutes Cell suspension in c.c. I.O I.O I.O I.O Readings: 10 minutes o o 20 minutes o o o o I hour -j_ -j_ -)_ .f. 12 hours + + + 4- Experiments (d), (e), (f), (g), (h) were performed respectively with extracts from organisms 720, 904, 1210, 863, 1074. The results parallelled exactly those of experiment (c). As checks on the validity of the above experiments controls were run on the various ingredients: No. i 2 3 4 5 6 7 8 9 10 Combination Salt solution and cell suspension Lecithin and cell suspension Cholesterol and cell suspension Saponin and cell suspension Extract 688 and cell suspension Extract 720 and cell suspension Extract 904 and cell suspension Extract 1210 and cell suspension Extract 863 and cell suspension Extract 1074 and cell suspension Hemolysis o o o 4-4-4- + o o o o o o 2. CONCLUSIONS FROM DATA ON HEMOLYSIS. (a) The lipin mixtures from the various organisms show the same effect on the action of saponin they inhibit hemolysis. (b) The differences, mentioned above, in the ratios of cholesterol to phosphatide cannot be correlated with differences in the action of the ether extracts on the hemolytic power of saponin. VII. SUMMARY OF CONCLUSIONS. 1 . There are no marked graded differences in either the N or S content. It becomes apparent, therefore, that the total amounts of protein material in the different organism varies but slightly. 2. The P content is practically constant. This indicates no striking variations in the quantity of nucleoprotein (the P from the phosphatide being very small in amount). 3. Regarding the results for the moisture determinations only slight deviations are noticed. The percentages obtained by drying the material at 100 C. are higher than those obtained by drying the substances in vacuo. Such increases in the values were expected because of the greater disintegration at the higher temperature. 4. Most interesting are the figures obtained for the amount of lipin material. These vary greatly for the different types of streptococci. The percentage of lipins is lowest in the indifferent type and highest in the green-producing type, the hemolytic type taking a position between these two. The characteristic relations may be expressed by the ratio: Indifferent: Hemolytic: Green Producing:: 1:3:4. These results seem to seperate the streptococci into three groups corresponding to those obtained when the reaction on blood is used as the criterion. 5. The content of phosphatide in the lipin mixture decreases for the different types in the order named: indifferent, hemolytic, green-pro- ducing. 6. The same relation is given by the amounts of cholesterol contained in the different organisms. 7. The ratio C:P increases when the types are listed as above. 8 . The lipin mixtures from the various organisms show the same effect on the action of saponin: they inhibit hemolysis. 9. The above mentioned differences in the ratios of cholesterol to phosphatide cannot be correlated with differences in the action of the ether extracts on the hemolytic power of saponin. 25 BIBLIOGRAPHY (1) Brieger, Zt.f. phys. Chem., 1885, ix, I. (2) Weyl, Deutsch. med. Wochschr., 1891, xvii, 256. (3) Hammerschlag, Centralbl,f. klin. Med., 1891, xii, 9. (4) Hoffman, Wien. klin. Wochschr., 1894, 712. (5) De Schweinitz and Dorset, J. Am. Chem. Soc., 1895, xvn . 6<>5- (6) J. Am. Chem. Soc., 1896, xviii, 449. (7) J. Am. Chem. Soc., 1897, xix, 782. (8) J. Am. Chem. Soc., 1898, xx, 618. (9) Ruppel.Z/./. phys. Chem., 1898, xxvi, 218. (10) Levene, Med. Record, 1898, liv., 873. (11) J. Med. Research, 1901, vi, 135. (12) Drymount, Arch.f. exp. Path. u. Pharm., 1886, xxi, 309. (13) Vaughan, Trans. Ass. Am. Phys., 1902, xvii, 313. (14) Wheeler, J. Am. Med. Ass., 1905, xliv, 1271. (15) V. C. Vaughan, V. C. Vaughan, Jr., J.W. Vaughan, Protein Split Products in Relation to Immunity and Disease, 1913. (Lea & Febiger, Philadelphia and New York). (16) Leach, J.Biol. Chem., 1906, i, 463. (17) Dawson, J.Bact., 1919, iv, 133. (18) Nicolle and Alilaire, Ann. de. I'Institute Pasteur, 1909, xxiii, 547. (19) Bradley, J.Biol. Chem., 1918, xxxiii, 525. (20) Avery & Cullen, J.Exp. Med., 1919, xxx, 359. (21) /. Exp. Med., 1919, xxix, 215. (22) Hiss and Zinsser, Text Book of Bact., 1916, 126, (Appleton & Co., New York.) (23) Text Book of Bact., 1916, 130. (24) Text Book of Bact., 1916, 132. (25) Clark and Lubs, J.Bact., 1917, ii, i, 109, 191. (26) Walpole, Biochem. Jour., 1914, viii, 628. (27) Dernby and Avery, J.Exp. Med., 1918, xxviJi, 345. (28) Sorensen, Biochem. Zt., 1909, xxv, 131. (29) Von Lingelsheim, Zt. Hyg., 1891, x, 331. (30) Kurth, Quoted by Kolle and von Wassermann, Handbuch der path. Microorg. 2" Ed. 1912, 14, 456. (31) Fehleisen, Die Aetiologie des Erysipels, Berlin, 1883. (32) Rosenbach, Microorg. bei d. Wundinfections des Menchen, Weisboden, 1891. (33) Gordon, La ncet, 1905, ii, 1400. (34) Andrews and Horder, Lancet, 1906, ii, 708. (35) Kligler, J. Inf. Dis., 1915, xvi, 327. (36) Schottmiiller, Munch, med Wochschr., 1903, 1, 849. (37) Von de Welde, Centr.Bakt., 1898, xxiii, 481. (38) Baginsky and Sommerfeld, Ber. klin. Wochschr., 1900, xxvii, 588. (39) De Waele and Sugg, Archiv. internat. pharm. et therap. 1904, xii, 205. (40) Meyer, Deutsch. med. Wochschr., 1902, xxviii, 751. (41) Aronson, Deutsch. med. Wochschr., 1903, xxix, 439. (42) Reudiger, J. Inf. Dis., 1906, iii, 663, 775. (43) Reudiger, Science, 1912, xxxv, 223. (44) Davis, J. Inf. Dis., 1913, xii, 386. (45) Smith and Brown, J. Med. Res., 1915, xxxi, 445. (46) Besredka and Dopter, Ann. de. Vlnstitut Pasteur, 1904, xviii, 37. (47) Swift and Thro, Arch. Int. Med., 1911, vii, 24. 26 (48) Holman, /. Med. Res., 1916, xxxiv, 377. (49) Blake, /. Med. Res., 1917, xxxvi, 99. (50) Lyall, J. Med. Res., 1914, xxx, 487, 415. (51) Brown, Monograph, Rock., Inst. Med. Res., no. 9, 1919. (52) Park and Williams, Path. Microorg., 1920 (Lea & Febiger, Philadelphia and New York) . (53) Dpchez, Avery and Lancefield, J.Exp. Med., 1919, xxx, 179. (54) Hiss, J.Exp. Med., 1905, vi, 317. (55) Neufeld.Zisc&r./. Hyg., 1900, xxxiv, 454. (56) Huntoon, J. Inf. Dis., 1918, xxiii, 169. (57) Dr. Hasting's formula obtained from Dr. M. L. Rhein, New York City. (58) Jablons and Pease, C. r. Soc. de biol., 1919, i, 205. (59) Rosenow, J. Dental Res., 1919, i, 205. (60) Leach, J.Biol. Chem., 1907, iii, 443. (61) Shackell, Am. J. Physiol., 1909, xxiv, 325. (62) Sherman, Organic Analysis, 1919, 291 (Macmillan & Co., New York). (63) Assoc. of Official Agricultural Chemists, U. S. Dept. Agr. Bur. Chem. Bui. 107 (Revised) p. 23. (64) Koch, Hygienic Laboratory Bui. 103, p. 79. (65) Bloor, J.Biol. Chem., 1915, xxii, 133. (66) J.Biol. Chem., 1916, xxiv, 447. (67) Kober and Egerer, /. Am. Chem. Soc., 1915, xxxvii, 2373. (68) Bloor, J. Biol. Chem., 1916, xxiv, 227. (69) Ranson, Deutsch. med. Wochschr., 1901, xxvii, 194. (70) Nishimura, Archiv.f. Hyg., 1893, xviii, 318. 27 ACKNOWLEDGMENT Throughout the course of these researches I have received many important suggestions from Professor W. J. Gies, Dr. E. G. Miller, Jr., and the several members of the Depart- ment of Biological Chemistry. It is, therefore, a pleasure to express my extreme indebtedness for the valuable criticism and helpful guidance which were so willingly given me. PUBLICATION Is there a Parallelism Between Normal Agglutinins and Hemolysins of Human Blood (with Reuben Ottenberg) Proceedings of the New York Pathological Society, 1919, xix, 83 VITA Frances Krasnow was born in New York, October 16, 1894. She received her preliminary and secondary education in the Public Schools of New York City. In December 1912 she was elected to free member- ship in the Brooklyn Institute of Arts and Sciences. From February to June 1913 she pursued studies at this Institute and at Hunter College. Receiving a University Scholarship in August 1913 she entered Barnard College, Columbia University, and was graduated with the degree Bache- lor of Science, magna cum laude, in February 1917. She continued her work in the Graduate Schools and received the degree of Master of Arts in June 1917. At the same time she was awarded the Kohn prize for Mathematics, given honors for Zoology and Physics and elected member of the Phi Beta Kappa Society. In July 1918 she was appointed assistant in Bacteriology at Columbia University, and in the Fall of 1919 received a similar appointment in the Department of Biological Chemistry, which position she now holds. Gaylord Bros. Makers Syracuse, N. Y. PAT. JAN. 21, 1908 488835 UNIVERSITY OF CALIFORNIA LIBRARY