THE PREPARATION AND THE CHEMICAL AND BACTERIOLOGICAL ANALYSIS OF ANIMAL AHD VEGETABLE PEPTONES "by ARNOLD EVANS HOOK A THESIS Submitted to the Graduate School of Michigan. State College of Agriculture and Applied Science in p a r t ia l fulfilm ent of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology 19 t o CONTENTS INTRODUCTION........................................................................................................................................................... 1 PREPAKATION OP ANIMAL AND VEGETABLE PEPTONES................................................................... 2 Preparation of Animal Peptones......................- .................................................................. 6 Preparation of Vegetable Peptones.................................................................................... 7 Discussion.......................................................................................................... Summary........................................................................................................................................................... 15 TEE CHEMICAL ANALYSIS OP PEPTONES................................................................................................. l6 Methods of Chemical. Analysis................................................................................................. 19 M aterials......................................................................................... Discussion................................................................................................................................................... 23 Color Reactions of peptones.................................................................................................... 29 Discussion................................................................................................................. Buffering Action of Peptones................................................................................................. 30 Discussion.................................................................. ..................................................... Summary......................................................................................... TEE BACTERIOLOGICAL ANALYSIS OP PREPAREDPEPTONES........................................................ U6 Determination of Sate of Growth ofE. c o l i ............................................................... U6 Plating of Samples of Saw Milk............................................................................................ 5 ^ Grov;th and Gas Production "by Coliform Organisms from Naturally Contaminated Water.......................................................................... 52 Growth of Pathogenic Organisms.............................................................................. Summary........................................................................................................................................................... 70 BIBLIOGRAPHY........................................................................................................................................................... 72 ACKNOW LEDGMENT...................................................................................................................................................... 72 67 INTRODUCTION* IXiring the early days of bacteriology the nitrogen requirements of b ac te ria grown by a r t i f i c i a l means were met by the addition to media of such n atu ra lly occurring substances as blood, urine and other body flu id s . Naegeli ( 3 3 ) 1 SS2 was probably the f i r s t to use egg albu­ min, which he called "peptone” , as a source of nitrogen. Later i t was found that "peptones", derived from the p a r tia l digestion of proteins, would furnish organic nitrogen in a more available form. Since then, peptones and re la te d products have been u tiliz e d on an increasingly g reater scale. In recent times new and b e tte r peptones have been made available to the b a c te rio lo g is t. As a general rule the methods of preparation and the source m aterials of these peptones haw been kept se c re t. Except fo r the several Difco products, th e ir chemical composition is not available. The desire and need for media prepared from chemically pure constituents has created an in te re s t in the chemical composition of peptones. While i t is doubtful that a chemically pure peptone w ill ever be produced by present methods, i t is desirable to know as much as possible of th e ir prepara­ tion and chemical make-up. Therefore peptones were prepared from various animal tissu e s and from samples of hydrolyzed and unhydrolyzed corn gluten. These peptones, as well as samples of twenty-four commercial, brands, were analyzed for various nitrogen fra ctio n s, and were teste d bacteriolog ically for th e ir a b ility to support the growth of b acteria. *This study was aided by a grant from the Com Products Refining Company of Argo, I ll in o i s . PREPARAT I Oil OP ANIMAL AND VEGETABLE PEPTONES The methods of preparing peptones, as found in the lite r a tu r e , f a l l into three general groups as followsi ( l) hydrolysis of proteins "by enzymes; (2) hydrolysis of proteins "by acids and a lk a lies and (3) com­ binations of these two methods. Examples of the preparation of peptones by any one of these methods are numerous. Berthelot (^4-) mixed equal weights of fin ely minced pig pancreas, pig in te s tin a l mucosa and lean beef and allowed these to stand in a 3*100 Na2 C03 solution a t U 0 ° C. u n t i l the digestion was completed. broth was then evaporated to dryness in vacuo below ^5° 0 . Ouiiuiuou wtis xvvLlivl bu uo The f i l t e r e d The peptone suitable fo r the growth of In te stin a l organisms. Soparkar ( 5 1 ) prepared a satisfacto ry sub stitute for W itte's peptone by digesting casein with enzymes obtained from the pancreatic glands of goats. After a few days digestion at hO°C. the enzymes were inactivated by boiling and the ex tract, consisting of a mixture of proteoses, peptones, polypeptides and amino acids, was evaporated to dryness and pulverized. Boez (.9 ) prepared a peptone especially suitable for the growth of Mycobacterium tuberculosis by mixing 500 grams each of lean beef, fresh pig pancreas and pig in te s tin e s with six grams of IIa2 C03 and U o grams of chloroform in two and one— h a lf l i t e r s of water, and allowing the mixture to digest for *46 hours a t 37 ° C. The digestion was a rrested by making the mixture s lig h tly acid with HC1 , a f te r which the solution was f ilte r e d and concentrated. The high amino acid content was responsible fo r the superiority of th is medium over others for the growth of the tubercle b a c illu s. Douglas (lh) heated ground beef heart to "JO to 80 ° C., cooled and added trypsin. The mixture was then digested for two to three hours a f te r which -3 i t was made s lig h tly acid with HC1 and brought to 100 ° C. to p re c ip ita te out unaltered albumins and then f ilte r e d . Douglas obtained very good growth of the typhoid b a c illu s by use of th is solution. Utkin ( 5 7 ) found that jhe by-products obtained from the prepara­ tion of insulin and spermin would y ie ld from 13 to 25 per cent of a h i^ i grade peptone when these by-products were hydrolyzed with pepsin and/or trypsin. Itzioka (26) prepared a "tryptone" from casein by the action of rabbit pancreatic ju ice. M artin's peptone, prepared by the peptic diges­ tion of whole hog stomachs, was found by Mustafa (32} to be superior to W itte's peptone for use in detecting indol production by E. c o ll. Hucker and Carpenter (25) subjected fin e ly ground beef heart to pepsin-HCl digestion fo r a period of eleven hours, a f te r which the pH was adjusted to 7.U, trypsin added and the digestion allowed to continue for 36 hours. Saaples removed at various time in terv als to te s t th e ir a b ility to support b a c te ria l growth were heated to gO°C., dried in vacuo and powder­ ed. Raw lean, beef was also digested by trypsin alone and a fte r digestion treated as was the h eart muscle digest. Grabar (19) obtained a phosphorus rich (2.2 per cent) peptone by the incomplete try p tic digestion of casein. The y ie ld obtained was about five per cent of a peptone containing 13*1 per cent nitrogen. Wallis ( 6 0 ) prepared a s u b stitu te fo r "iTutrose" by digesting casein and peanut flo u r with trypsin in the presence of !JasCo3. The resu ltin g peptone was used fo r making Conradi-Drigalslci medium fo r growth of the colon— typhoid group. The growth promoting properties of th is peptone appeared to be due to the presence of a vitamin associated with the glob­ u lin of the peanut flo u r. -U — .An excellent s u b stitu te for Witte’s peptone was prepared by von Gutfeld (23) by digesting coagulated and washed cakes of horse serum and horse and human blood with pancreatin. Strauss ( 5 2 ) also prepared a su b stitu te for W itte’s peptone by digesting fib rin with trypsin. Leif son and Diamond ( 2 9 ) prepared 115 peptones from beef, beef h eart, beef spleen, beef lung, pork, hog stomach, f is h , ten and soybean flo u r. casein, wheat glu­ These m aterials were digested by pepsin, pancreatin and papain and by combinations of these enzymes. Some of the peptones thus prepared were found to be superior to the commercial brands with which they were compared. Sadikov (U7) prepared peptones by heating various proteins in aqueous solutions of (NH4) 2CO3 or WH4OH in an autoclave for two to twelve hours at 150 to 180 ° 0 . The dried hydrolysates obtained by th is procedure consisted chiefly of peptones. formed. I f the mixture was heated 2 b hours, amino acids were The peptones obtained from f ib r in and casein supported good growth of various types of b ac teria . Grand and Lewis ( 2 0 ) obtained two widely differen t peptones when s ilk was hydrolyzed a t 30 °C. with 70 per cent H2SO4 for 65 to 70 minutes. One of the peptones had a low amino nitrogen content and contained more tyro­ sine than the original silk (12-3 per cent) while the other had a tyrosine content sim ilar to that of many common proteins (2.5 to 5.7 per cent) and had a high amino nitrogen content. Piccioni (38) peptonized commercial gelatin in the presence of HsSO* for SO to 100 hours, a f te r which the solution was heated to 10 0 °C., f il te r e d and concentrated. B acterial and chemical te s ts indicated that this method produced a s a tisfa c to ry peptone. -5 - Brami^c ( 1 0 ) added three l i t e r s of water and 15 ml. of HaSo* to the fib rin obtained from one "p ailfu l" of coagulated blood, and allowed the mixture to stand overnight. I t was then poured into three l i t e r s of water containing IS ml. of H2SO4 .and heated to 50°C. The ex tract of the mucosa of two hogs, prepared a t 35 ° C. , in a l i t e r of s te r ile water, was added and the whole digested a t 37 ° C. for *+8 hours. The resu ltin g liq u id was neutralized with e ith e r NH*0 H or Ba(0 H)2 , c la r if ie d by heating s lig h tly , f i l t e r e d and evaporated in vacuo. Bramigk states th at the peptone thus obtained was id e n tic a l with W itte’s peptone. ^ o lif ^ H s o l i ntnf f V» a cal peptones has received some a tte n tio n . a a ^4 aw V a a 1 a n*4 Snyder ( 1+ 9 ) placed convenient amount of a i r dried plant m aterial in the bottom of a te s t tube, covered these with selenium oxychloride and heated gently over a low flame. When the m aterial was completely peptonized i t was cooled and poured into a l i t e r of s t e r i l e d i s t i l l e d water and allowed to s e t tl e over night. The clear supernatant was then decanted and the p re c ip ita te d material c e n tri­ fuged, washed and taken up in five ml. of s te r ile water. Small q uantities of th is m aterial, when placed in p e tr i p lates as they were poured, increas­ ed the growth of certain plant pathogens. Berthelot and Amoureux (5) prepared a peptone by the action of pepsin and KC1 on peanut press cake. This peptone was ric h in arginine and supported excellent growth of several organisms. These authors, with van Deinse (7 ), prepared a peptone from soybean press cake by a sim ilar method. made i t The high content of soluble carbohydrates present in the peptone especially suitable fo r the growth of certain organisms. Sadikov and Sinitzuin (Us) prepared peptones from yeast by autolysis and by heating in the autoclave a t 150 °C. in the presence of 0 .1 to 0.5 -6 - per cent aqueous H3PO4 . These peptones gave highly v irulent cultures of B. dan.ich. and B. mereshkovskl. Preparation of Animal Peptones The peptones were prepared from various bovine tissu es including lean muscle, h e a rt, liv e r , spleen and brain and from lean pork. The fin e ly ground tissu es were suspended in d i s ti ll e d water in a six l i t e r Pyrex flask and the mixture brought to a pH of 1 . 0 to 1 . 2 by the addition of concentrated HC1 . One-half gram of granular pepsin (Difco, 1 : 1 0 , 0 0 0 ) was s tir r e d in and the mixture allowed to digest in a 52°C. incubator fo r approximately Hs hours, shaking at in terv a ls. The pH was adjusted to 1 . 0 to 1 . 2 every twelve hours by the addition of concentrated HC1 . At the end of the digestion period the supernatant liq u id was siphoned o ff, and the remaining liq u id separated from the undigested residue by f ilte r in g on a Buchner funnel containing a layer of f i l t e r paper pulp. The residue was washed twice with 5^0 nl» of d i s t i l l e d water and the washings added to the peptone solution. The solution was then heated in the autoclave a t 15 pounds pressure fo r 20 minutes to destroy the enzyme and to bring about coagulation of soluble proteins which are heat coagulable, a fte r which i t was cooled and f ilte r e d . The clear solution was concentrated jin vacuo a t a tempera­ ture of 55 ° C. to remove p a rt of the HC1 and to bring the volume approximately to one l i t e r . This solution was then neutralized with *+0 per cent NaOH to pH 7 . 0 0 a t a temperature below 30 °C ., a fte r which i t was placed in a t a l l battery Jar, covered with a layer of toluene and dialyzed in running tap water for Hg to 72 hours to remove the HaCl. was Du Pont Cellophane seamless tubing. The diaJLyzing membrane used After d ialy sis the solution was again brought to pH 7 . 0 0 , axitoclaved as before, cooled, f il te r e d and -7 - poured into shallow Pyrex dishes which were placed in a forced a i r d rie r. After *48 hours in the forced a ir d rier the dishes containing the p a r tia lly dried peptone were placed in a hot a ir oven a t 80 ° C. for com­ p le te drying. Y/hen thoroughly dried, the peptone was scraped from the dishes, ground to a fine powder and placed in "bottles. Table 1 shows the amount of tis su e , pepsin, HC1 and water used in preparing each peptone, together with the grams and per cent yield of peptone obtained. Four "batches of "beef peptone, two "batches each of spleen, liv e r, brain and h eart peptone, and one batch of pork peptone were prepared. The batches of beef peptone wex-e mixed; batches of other peptones were kept separate. These peptones w ill be designated by the source m aterial and batch number, for example, spleen peptone (1); heart peptone(2 ), etc. Preparation of Vegetable Peptones The m aterial used in the preparation of the vegetable peptones was obtained from the Corn Products Refining Company of Argo, I ll in o i s . It consisted of com gluten which had been hydrolyzed by boiling with 20 per cent HC1 for varying lengths of time. follows: The various treatments were as sample 1 was hydrolyzed for two hours; sample 2 for ten hours; sample 3 for l6 hours a f te r which the glutamic acid was removed;and sample U for l6 hours a f te r which the glutamic acid, leucine andtyrosine were removed. The hydrolysates were f i r s t concentrated in vacuo to a thick syrup at a temperature of 55° C. The solution was then d ilu ted to two l i t e r s and brought to pH 7 .0 0 by the addition of H O per cent NaOH, keeping the temperature below 3 0 °C. I t was then dialyzed to remove the HaCl, adjusted -8 - to pH 7 *0 0 , antoclaved, cooled and f il te r e d as were the animal peptones. The resu ltin g solution was black due to the presence of humin nitrogen formed during hydrolysis. Therefore, to prepare a satisfa c to ry peptone i t was necessary to decolorize the solution. This was done by adding 100 grams of Norite “A" per l i t e r of solution, boiling for 10 minutes and f il te r i n g on a Buchner funnel containing a layer of f i l t e r paper pulp. The decolorizing process was repeated i f necessary, and the de­ colorized solution dried in the forced a ir d rier and hot a ir oven as described above fo r animal peptones. Table 2 shows the yields of vege­ table peptones o b t a i n e d f r o m the four hydrolysates. Several batches of peptone were prepared from each sample of hydrolysate, mixed a f te r preparation. the batches being For purposes of id e n tific a tio n these peptones were lab elled according to the hydrolysate sample from which they were prepared; for example: vegetable peptone No. 1 was prepared from hydroly­ sate sample No. 1 , etc. In addition to the above peptones prepared from hydrolysates of com gluten, two additional peptones were prepared from corn gluten before i t was hydrolyzed. A vegetable peptone (la b e lled vegetable peptone No. 5 ) was prepared according to the technique employed for animal peptones and a vegetable tryptone (la b e lle d vegetable tryptone) made by digesting corn gluten with trypsin in the presence of l«aaC0 3 . In both cases i t was necess­ ary to allow the digestion to proceed for three weeks before i t was complete. The procedure fo r preparing the vegetable tryptone was as follows: Nine hundred grams of the dry corn gluten was suspended in three l i t e r s of d i s ti ll e d water by mechanical s ti r r i n g and su ffic ie n t saturated aqueous Ha2 C03 added to bring the pH between 8 . 0 and S. 5 * (Difco, 1:360) was s ti r r e d in, Three grams of trypsin the mixture covered with an inch layer of Table 1 . Amount of various ingredients used in preparing animal peptones showing the y ield in grams and per cent obtained. Kind of Tissue Lean Lean Lean Lean Beef Beef Beef Eeef Beef Beef Beef Beef Lean beef beef beef beef spleen ( i ; spleen ( 2 ) brain ( 1 ) brain ( 2 ) l iv e r (l) liv e r ( 2 ) heart (l) heart ( 2 ) pork Grams tissue* U5O' 896 5 .4 5 3 5 ,4 8 9 Grams pensin 0 .1 0.1 .-a. cone. HC1 20 32 Li ters water 1 2 1 ,2 4 2 3 . 7 SO 0 .5 0 .5 0 .5 0 .5 0 .5 0 .5 1+13 0.1 20 l 0 .5 0 .5 0 .5 0 .5 31 50 y. ~~ 4,672 4 .1 8 0 5 .1 7 5 3 .4 4 5 1 ,4 0 0 39 25 43 5 5 5 5 4 4 ?6 »«r~\ Hd 1 5 .2 3 6 .7 Per cent y ie ld ’-11 16.9 2 1 § .5 2 0 .4 1 5 .5 1 9 .9 O ( • X O .'J lbO.O 2 0 7 .1 33.4 IO7.2 n 21.9 12-9 5 5 1 0 .7 4 0 3 -0 1 8 2 .0 1 3 .9 1 3 .4 47-7 1 7 .4 5 2 ;7 .0 90.0 3S.8 31.8 1+6 39 Grams pep tone obtained * V .ret weight of tissu e * + Per cent yields figured on dry weight of tissue taken as 20 per cent of wet weight* -1 0 - Table 2 . Yield of vegetable peptones obtained from corn gluten hydrolysates. Hydrolysate number 1 Hours hydrolyzed 2 2 10 16 3* U** 16 Amount of hydrolysate used Treatment of hydrolysate Grams peptone obtained Per cent y ield obtained 1 lite r H lite rs undecolori zed decolorized 7 7 .c 5O5.O 12.6 1 lite r 2 lite rs 1+ l i t e r s undecolori zed decolorized decolori zed 11.0 U i.o 556.0 *X 1 1XXUOi 4 42 lite rs H lite rs V JX V 1 AAOU decolorized decolorized 1 lite r 2 lite rs H lite rs undecolorized decolorized decolorized 7 .7 1.1 2.0 1 3 -3 o)t c 0 tT )i U U6.0 ^9 7 . 0 12. h 2 .3 6.0 12.0 3U.0 0.6 0.6 0.8 * Glutamic acid removed ** Glutamic acid, leucine and tyrosine removed. Table 3. Amount of ingredients used in preparing vegetable peptone A lio. 5 and vegetable tryptone from unhydrolyzed corn gluten. Grams dry corn gluten Li ters water Grams enzyme Yield in grams Per cent yield 900 3 0.1 pepsin 335 3 7 .2 900 3 3-0 trypsin U52 50.2 -1 1 - toluene, corked and placed In the 37°G. incubator for three weeks. The mixture was shaken daily and the pH adjusted to between 8 . 0 and 8.5 every two days. At the end of the digestion period the undigested residue was f i l t e r e d o ff, and the solution brought to pH 7 . 0 0 , autoclaved, cooled and f i l t e r e d . animal peptones. I t was then dialyzed and dried as described for Table 3 shows the amounts of material used in pre­ paring these vegetable peptones, together with the grams and per cent y ield obtained. Discussion The various tissu es were selected for preparing the animal peptones with the idea in mind that certain of the tissues would y ield a peptone especially suitable fo r the growth of certain organisms. gical re s u lts , that this Hie b acteriolo ­ especially the growth of pathogenic organisms, demonstrated theory i s incorrect. The data obtained would indicate that the value of a peptone depends not so much on the original tissue used in i t s preparation as on the treatment accorded the tissu e during the pre­ paration of the peptone. An examination of Table 1 shows that the per cent yield varies from 8.9 fo r one batch of spleen peptone to U7.7 for one batch of liv e r peptone. Table 1 also shows that the per cent y ield fo r two d ifferen t batches of the same peptone v aries considerably. Variation in the per cent y ield between tissu e s might be expected; variation in the per cent y ield when using the same tissu e is not so easily explained. The batches of pep­ tone were prepared xinder as nearly identical conditions as possible. These v ariatio n s may be p a r tia lly explained on the basis of differences in the composition of the tissu es themselves, and some variations may be expected to enter in the preparation of the peptones, especially daring -1 2 - d ia ly sis. Undoubtedly some amino acids and lower peptides as well as other forms of nitrogen were lo s t during d ialy sis. However, i t is un­ lik e ly that th is loss would account for as large a v ariatio n in per cent y ie ld as i s found in the heef liv e r peptone, whereas, i t could account fo r the difference in per cent yield obtained for the beef brain peptone. Certainly the d ialy sis is the one step in the prepara­ tion of the peptones th at is most unpredictable as to i t s the yield of the peptone. influence on Ho attempt was made to determine the amount of the various forms of nitrogen lo s t during d ia ly sis. I t may be great or small, depending upon the tissues used and upon the extent of hydroly­ s is . A one hundred per cent y ie ld could not be obtained because not a l l of the tissu e was digested by the pepsin. While the procedure used for the commercial production of peptone is secret, i t is f e l t that i f the yield of peptones under reasonably controlled laboratory conditions varies with d ifferen t batches, so too could d iffe re n t batches of commercial peptones vary in yield and compo­ s itio n . Since i t is p ra c tic a lly impossible to obtain any two samples of raw m aterials th at are id e n tic a l in e v e r y respect, especially when the samples do not come from the same source, i t w ill be equally im­ possible, even under ideal conditions, to prepare two batches of pep­ tone that w ill be id en tical in composition. An examination of Table 2 shows that the yield of vegetable pep­ tones from corn gluten hydrolysates Ho. 1 , 2 and 3 was larg er when larg er amounts of the hydrolysate was used. i f not clear. The reason fo r th is increased y ie ld Hydrolysate Ho. U - gave a lower y ield because the treatment to remove the amino acids d iluted the protein content, resu ltin g in le ss -1 3 dry matter per l i t e r of solution than was found in the other three hydrolysates. As in the case of the animal peptones, undoubtedly lo s t during d ia ly sis , some n u trie n t m aterials were especially from those hydrolysates that had been hydrolyzed fo r more than ten hours, because here most of the material was in the form of amino acids which were dialyzable to more or le s s extent. The decolorization of the vegetable peptones undoubtedly causes the loss of a small amount of ce rta in essential growth facto rs, as is shown by the growth curves given in Table h and in Figures 1 , 2 and 3 * ( The method of determining the ra te of growth of E. coli is given under Bacter­ iological Analysis.) The organisms grown in decolorized media show a much longer lag phase than do those grown in the undecolorized media, indicating th a t the m aterials removed by decolorization are essen tial in the i n i t i a l stages of growth for E. c o li. - 1 14- T a b le The ra te of growth of S. c o li in decolorized and undecolori zed vegetable peptone broth, with Bacto-peptone as control. (Average of fiv e t r i a l s ) 1*. Peptone 0 hours 2 6 hours hours Lfumber of b acteria per ml. 2h 12 hours hours Ijg hours No. 2 ud. 11 37 27.000 2,000,000 138,000,000 31*2,000,000 No.2 d. 10 12 2 ,8 0 0 £0,000 11,000,000 30,000,000 Ho. 3 ud. 10 k7 31,000 10,000,000 1 8 8 , 0 0 0 ,0 0 0 1*21*. 000, 000 No. 3 d. 10 12 3.100 150,000 i*, 600,000 55,000,000 No.l* ud. . 10 23 i 6 ,c o o 914-0,000 38,500,000 185,000,000 Ho. U d. 10 11 1 , 1*00 175.000 1*50,000 i+,600,000 35 lU , 500 32,000,000 279,000,000 513,000,000 Bacto-peptone 12 1d. ** Decolorized peptone ud. * Undecolorized peptone M M TT3& I wt»W pM0dt4<[ l(«04q U\ o WDi} *0K puB p©ii«too*p *1 i 0 J «»A4U0 tno*d9d-o*OBg iio*uuoo L og o f n n m b ars o f b a c t e r i a w o* CD V Figure s arwrth ©urrei for | . o o ll in broth prepared from dsooloriiod and nndecolorlied vegetable peptone No. 3 Controli Bacto-peptone 9 IE Baoto-peptonn Vegetable peptone No undeoolorlied Vegetable peptone No deoolorlzed 24 Tine in boare 46 Figure 3 Growth curves for 3» o o ll in broth prepared from decolorised and undeoolorlsed vegetable peptone No* 4 9 8 7 3aoto-peptorie 6 Vegetable peptone No. 4 - undeooloriaed Log of numbers of b acteria Control« Baoto-peptone Vegetable peptone No. 4 - decolorised 5 4 3 2 1 2 6 12 24 46 -1 5 - SUMMAB.Y A method is given whereby peptones may be made from various animal tissues by digestion with pepsin in a medium made acid by HC1 . Methods for the preparation of vegetable peptones from hydrolyzed and unhydrolyzed corn gluten a re also given. rIhe re s u lts show th a t: ( l) The per cent y ie ld of peptone obtained varies considerably even when two batches of peptone are made by the same method from the same source m aterial. ( 2 ) The per cent yield of vegetable peptones prepared from samples of hydrolyzed corn gluten was larg e r when larg e r amounts of the hydrolysates were used. ( 3 ) Decolorization of the vegetable peptones prepared from hydrolyzed com gluten re su lte d in the loss of nutrient m aterials as shown by the in fe rio r growth and increased lag phase of E. co li in the decolorized as compared with the undecolorized peptones. -lo THS CHEMIGAL ANALYSIS OF P3 PTONES Consid.era.ble work has been done on the chemical composition of peptones and re la te d substances. However, with a few exceptions, the work has been lim ited to one constituent of the peptones and to a very few d ifferen t brands. The most complete analysis of a large group of peptones may be found in the Difco Manual, published by the Digestive Ferments Company of D etroit, Michigan. McAlpine and Brigham (31) have given a f a ir ly complete nitrogen d istrib u tio n of four peptones, in­ cluding F a irc h ild 's , Witte’ s, Difco Bacto- and proteose-peptones. These authors analyzed the peptones mentioned for to ta l nitrogen, non­ protein nitrogen, ammonia nitrogen and amino nitrogen. The protein and polypeptide nitrogen fractio n s were mathematically determined from these analyses. The to ta l nitrogen content of the peptones was found to be approximately the same, but the content of the nitrogen fractions differed greatly in the various brands. Hucker and Carpenter (25) determined the to ta l nitrogen and amino nitrogen in samples of digested beef heart muscle. Their re su lts showed that as long as the digestion mixture remained acid the to ta l nitrogen content remained f a ir ly constant. However, as soon as the digestion mix­ ture was made alk aline, the t o t a l nitrogen content decreased rapidly, due to loss of ammonia, and, as would be expected from a try p tic digestion, the amino nitrogen content increased rapidly. 3 1 anchetidre (8) determined the diketopiperazine nitrogen in eight peptones by Kjeldahl analysis of the solution which remained a f te r the amino acids and peptides were p re c ip ita te d out by £a(OHja. He found from 1 . 8 2 to J+.35 per cent of th is type of nitrogen to be present in the peptones. -1 7 - T illey ( 5 3 ) sta te d th a t commercial peptones contain unoxidized, p a r t­ ly oxidized and oxidized sulphur compounds. Ho hydrogen sulfide was lib e r ­ ated by b ac te ria from compounds containing oxidized sulphur, but i t was given off freely from those compounds containing p a rtly oxidized or un­ oxidized sulphur. The peptones used were not l is te d as to brands. O'Meara and Macsween (3^) ( 3 5 ) found th at copper and iron present in commercial peptones hindered the growth of a l l Gram positive organisms tested. Again the peptones were not l is te d as to brands. Yaoi ( 6 2 ) showed that commercial peptones d iffered greatly in th e ir cystine content. W itte's contained the greatest amount; followed by Gehe’s, E iedel's and Shiono's peptones. Ba,sle peptone was th ird and Champoteant' s, Tamba's and B i l l a u l t 's were in fourth place with approximately equal amounts. Less cystine was found in Terunchi's, Difco and May-Eaker's peptones than in any of the peptones analyzed. Furth and Deutschberger ( 1 8 ) found Witt£ s peptone and fib rin contained seven per cent arginine. Wherry ( 6 l ) found W itte's and Grubler's peptones contained n i t r i t e s and n itr a te s in su ffic ie n t quantities to in te rfe re with te s ts fo r the pro­ duction of these substances by b acteria. Abel and Geiling ( 1 ) prepared primary and secondary albuminoses from W itte's peptone by repeated s a ltin g out in acid and alkaline mediums with (KH-OsSO* and- p re c ip ita tio n with ethyl alcohol. A more toxic fraction, which was not p re c ip ita te d by (UH-OsSO* in acid-alcohol and which contained histamine, was also prepared. The authors s ta te that the albuminoses thus prepared cannot be regarded as chemical e n titie s . Underhill and Gross (56) separated a solution of W itte's peptone into various fractio n s by e le c tro ly s is . The solution obtained from the -1 8 - anode chamber was acid to litmus; low in ash content; low in tryptophane as compared, with the o riginal solution; and. low in basic nitrogen, ia lly arginine and. lysine. espec­ The solution obtained from the cathode chamber was alkaline to litmus; high in ash content; higher in tryptophane than that of the anode m aterial, but lower than the original solution; and high in basic nitrogen, esp ecially arginine and h istid in e . The solution obtained from the center chamber was neutral to litmus. Kitamura ( 2 8 ) found from 0 . 6 to 1 . 0 per cent of fre e sugar, but no combined sugar in Terunchi's and YJitte's peptones, Treece (55) found that gaswis produced from Difco and Parke, Davis and Company peptones, but not from Armour's and W itte's peptones. Evidence was presented which indicated that th is gas was due to free sugar in the peptone and that the carbohydrate radicals of the peptones were not the source of this gas. Further evidence of the presence of free carbohydrates in peptones was presented by Ander­ son ( 2 ) who found that E. co li would form gas from peptones. Van Slyke and Hart ( 59 ) s^d Eldridge and Rogers (15) found that COs was produced from peptones by the action of certain cheese organisms; Evans (lo ) , and Ayers, Rupp and Mudge (3) found that CO2 was produced by certain strep to ­ cocci from peptones. Gorini ( 2 2 ) found that E. c o li produced indol from W itte's peptone but not from an I ta lia n peptone. Porcher and Penisset ( 3 9 ) showed that the a b ility of four brands of peptones to produce indol when tested with the same s tra in of E. c o l i . d iffered greatly. Ihese re s u lts show that d ifferent peptones vary in th e ir tryptophane content, from which indol is derived. That peptones contain growth promoting substances is indicated by the fact that Ottensooser (37) found W itte's peptone contained group "A" specific substance, while Qrla.— Jensen, Otte and Snog-Kjaer (36) found that -1 9 - bacteriologic peptones contained su ffic ie n t lacto flav in and enough bios to permit the growth of streptococci. On the other hand, Roberts and Bald­ win C^-l) showed that some commercial peptones such as Bacto-peptone may contain a p rin c ip le , removable by certain colloids, which is Inhibiting to sporulation. Hydrophilic colloids such as agar removed more of the principle from peptones than did the hydrophobic ones as exanplified by charcoal. Methods of Chemical Analysis The peptones, both commercial and prepared* were analyzed fo r to ta l nitrogen, to ta l and piumary proteose nitrogen, peptone nitrogen, free amino acid nitrogen, free ammonia nitrogen, amino nitrogen and ash con­ ten t. They were also tested to determine th e ir reactions to various color te s ts . A stock solution of each peptone was prepared by accurately weighing twenty grams, dissolving in approximately 3^0 ml. of d i s ti ll e d water and making up to ^>00 ml. in a volumetric flask. After mixing thoroughly the solution was poured into a s te r il e b o ttle , covered with a layer of toluene and stored in the ice box at All chemical analyses except the ash determinations and certain color te s ts were performed on aliquots of th is stock solution. All determinations were made in tr i p l ic a t e . Total Nitrogen. To determine the to ta l nitrogen content of the peptones, ten ml. aliquots of the stock solution were analyzed according to the Kjeldahl— Gunning method. The indicator used in th is and in other nitrogen determinations was a mixture of methylene olue and methyl red as suggested by Johnson and Green ( 2 7 )* Total Proteose Nitrogen. Small te s t tubes (SS x 13 mm.) were f i l l e d approx­ imately one— h a lf f u l l of C.P. ZnS04 crystals and five ml. of the stock -2 0 - peptone solution added to each tube. The tubes were heated in a water bath to dissolve the ZnSO* and allowed to cool slowly to room temperature. If necessary. more Z11SO4 was added u n til the peptone solution was saturated, as shown by the presence of crystals of ZnSO* in the bottom of the tubes. The pH of the p re c ip ita tio n mixture was not adjusted, but the p recip itatio n was carried out at the pH of saturated ZnSO*, which is approximately 3.5O. The s a lt error was not considered in determining th is pH. The p re c ip ita te of proteose nitrogen was f il te r e d off on a mat of shredded asbestos in a Gooch crucible, and washed several times with saturated ZnSO-i solution to remove the non-proteose nitrogen. The crucible containing the p re c ip ita te was then placed iu a 50 ml. beaker containing approximately fifte e n ml. of dilute ( 1 : 1 ) H2SO4 and the acid heated to dissolve the p re c ip ita te . the p re c ip ita te was dissolved, When the crucible was removed and rinsed with d is ti ll e d water, the washings being added to the acid-proteose solution. The shredded asbestos was removed by f ilte r in g through a second Gooch crucible containing a lay er of shredded asbestos and the f i l t e r washed with d is ti ll e d water. The solution was then placed in a Micro-K jeldahl flask and the amount of proteose nitrogen determined by performing a Micro-Kjeldahl analysis. Primary Proteose Nitrogen. Five ml. aliquots of the stock peptone solution were placed in small te s t tubes and five ml. of saturated aqueous ZnSO* added, giving a fin a l solution one-half saturated with ZnSO*. The tubes were inverted several times to mix the contents and allowed to stand at room teaperature over night to complete p re c ip ita tio n . As in the case of the to ta l proteose nitrogen, the pH of the p recip itatio n mixture was not adjusted, but the p re c ip ita tio n was carried out at the pH of one-half saturated ZnSO* which is approximately 3 -9 °- tubes were then centri- -2 1 - fugalized for one— half hour a t 3^00 r.p.m. , and the p recip itate washed twice with one-half saturated ZnS04 solution, and dissolved in dilute (l:lj H2SO4. The amount of primary proteose nitrogen was determined by Micro-Kjeldahl analysis. Secondary Proteose Nitrogen. The figure for secondary proteose nitrogen was obtained by subtracting the value obtained for primary proteose n itro ­ gen from that obtained for t o t a l proteose nitrogen. Peptone Kjtrogen. Five ml. aliquots of the stock solution were placed in small te s t tubes and five ml. of cold 20 per cent aqueous tannic acid 6 i.nvsr'tsd. SvVsrsl ‘tiin.ss tc mix t haq ccntsiit3 solution fiMiisdLs and placed in the ice box for one-half hour. Preliminary esqaeriments in­ dicated that th is amount of tannic acid was su fficien t to p recip itate a l l the peptone nitrogen in the peptones tested. A large excess was avoided since i t would exert a solvent action on the tannic acid-peptone complex, as shown by Lundln and Schroderheim ( 3 0 )* pH of the p re c ip ita tio n mixture was not adjusted, but performed a t the pH of 20 per cent tannic acid, which is approximately 2 . 8 0 . After completion of p recip itatio n the tubes were centrifugal!zed fo r five minutes a t J>000 r.p.m. and the precip­ ita te washed twice with cold five per cent tannic acid solution. The pre­ c ip ita te was then dissolved in dilute ( 1 : 1 ) H2SO4 by heating in an Arnold steamer. The amount of peptone nitrogen was determined by Ricro-^ jeldahl analysis. Free Amino Acid Nitrogen. Phospho-tungstic acid was chosen as the protein p recipitant in determining the free amino acid nitrogen. used was as follows: The procedure Ten ml. of the stock solution wa9 pipetted into a 50 ml. beaker and 20 ml. of a fiv e per cent aqueous phospho-tungstic acid solution was slowly added by means of a p ipette. The mixture was -2 2 - s tir r e d continuously during the addition of the acid. After standing for 3® minutes at room temperature the p re c ip ita te was f ilte r e d off on a Buchner funnel containing a layer of f i l t e r paper pulp which was covered with a lay er of D icalite f i l t e r - a i d , and washed three times with 25 ml. portions of one per cent phospho-tungstic acid. The f i l t r a t e was te ste d with a few drops of fiv e per cent phospho-tungstic acid to te s t for the complete p re c ip ita tio n of the protein intermediate products, placed in a 200 ml. volumetric flask and diluted to the mark with d is tille d water. F ifty ml. aliquots were concentrated and the amount of free amino acid nitrogen "by the Hicro-£jeldahl method. Ammonia. ITitrogen; Free ammonia present in the peptones was determined "by the aeration method of Bolin ( 1 7 ) as modified by Van Slyke and Cullen (56). Amino Hitrogen: Amino nitrogen was determined by the Van Slyke and Sorenson methods. Ash Content: To determine the amount of inorganic matter present in the peptones, approximately two grams was accurately weighed into porcelain crucibles of a known constant weight. These were then heated by a Meeker burner u n t il the peptone ceased to smoke, and then placed in an e le c tric furnace a t a temperature of approximately 500 °C. for one hour. At the end of th is time the crucibles were placed in a desiccator over calcium chloride for one-half hour to cool and then weighed. This procedure was followed u n til a constant weight of ash was obtained. Materials In addition to the l6 prepared peptones, the following commercial brands were included in the chemical analysis ol peptones. i-fumbers in -2 3 parentheses are the manufacturers hatch numbers. Bacto-peptone (29761U); Bacto-neopeptone (29U9S3); Bacto-proteose-peptone (296S37); Bacto proteosepeptone Nc. 2 (316258); Bacto proteose-peptone No. 3 (312275); Bactotryptone (306619); Bacto-tryptose (3O8O35) and Bacto-protone ( 315785 ). Peptonum Siccrm ( 1 0 0 8 1 7 ) and Armour's Special Peptone ( 111025 ); Baker's Bacteriological Peptone (3239). Cenco Peptone, dry - from meat (9 - 27 39 ). Chaissiang Peptone, a French product. peptone (dry) Albumin peptone (dry) and meat ( 9 0 0 h2 7 ) from Elmer and Amend. Merck’s peptone from meat, dried (33II9). iologic Peptone (3229678). F airch ild ’d peptone ( 360U27 ). Parke, Davis and Company Bacter- Pfsnstieh! Bacteriological Peptone ( 1 0 ^^). Stearn's B acteriological Peptone, N.P., ( 3 ^7 ^— ^)* Witte peptone made p rio r to 1912 and Witte peptone obtained in 19^0 ( 31 ^7 ) from F. Witte, Rostock, Germany. Wilson Peptone "CB" (3IO8O) and Wilson Peptone "C" ( 30732 ). Discussion An examination of Tables 5 and 6 shows that the peptones d if f e r widely in chemical composition. These re su lts are in accord with those of McAlpine and Brigham (31) who found that W itte’s, F a irc h ild 's , Bactopeptone and proteose— peptone v aries considerably in the amounts of various nitrogen fractio n s for which they were tested. With the exception of Bacto-protone, a l l commercial peptones were higher in peptone nitrogen than in any other fractio n for which they were tested . highest in proteose nitrogen. Protone was The amino nitrogen as determined by Van Slyke' s method was, without exception, slig h tly higher tnaii the figure obtained by Sorenson's formol t it r a t io n . T able 5* Chemical A n aly sis o f Commercial Peptones E xpressed in P er Cent. llame of peptone Bacto-peptone Neopeptone Froteose-peptone Proteose-peptone No. 2 F roteose-peptone No. 3 Tryptone Tryptose Fro tone Armour Armour Special11 Baker Cenco Chaissiang E & A Albumin E & A Meat F a irc h ild Merck P fa n s te ih l Parke-Davis Stearn W itte ( 1312) Witte (I9U0) Wilson "CB" Wilson »C" Average * Total n itro g e n Total pro teose n itro g e n Primary p ro teo se n itro g e n Secondary p ro teose n itro g e n 15.72 13*73 13.55 12. Up 12.87 12.77 12.99 1 5 .2U lU.27 13-89 15. U3 15. 3S 13.15 11.21 16.lU lU.UU 15.83 13-56 1U.U2 15. Up O.32 2.10 3.82 6.U0 s . 25 2.69 2-53 13.27 U.37 U.32 3-71 5.U5 0.50 1.U7 1.27 0.50 0.37 l.US 0.63 0.07 0.19 0.19 0.30 3.30 0.16 0.25 5.27 0.6U 0.7U 0.03 0.U1 0.00 0.36 0.01 0.16 0.00 11. Us 8.5O 6.59 1.09 2.32 9.69 S. 83 7-99 3.6U 6.U5 3.16 5.76 1.0U 6.05 3-75 13.92 11.91 5.02 6.58 13.86 U.80 12.53 6.69 7 . SI 10.6s 10.55 10.59 U.U6 2.65 0.07 0.11 0.11 0.17 0.13 0.17 0.05 0.05 0.33 0.12 0.09 0.09 0.07 0.08 0.06 0.?3 C.06 0.17 0.09 0.12 lU.Us 0.02 0.03 1.71 1.07 0.11 0.19 O.Up 1.91 3.63 5.50 U.95 2.53 2.28 8.00 3-73 3. 5S 3.08 5-OU 0.50 1.16 1.2p O.3U O.37 1.U0 0.61 0.50 6.79 5.52 0.9s 2.13 13.90 3.UU 0.66 2.78 7.U1 0.11 13.U5 11.86 l,'ot a b a c te rio lo g ic a l peptone 0.p2 0.0s Pep tone n itro g e n Free Ammonia n itro g e n 0 .0 2 0 .0 0 0.13 0.13 Free amino ac id n itro g e n 1.16 C.99 0.85 1.50 1.06 2.06 l.U l 0.37 1.69 0.57 O.25 Free Amino n itro g e n n itro g e n (Yan Slyke) ( Srenson) 1.58 3-33 2.67 2.72 3.29 2.55 U.56 3.57 I.8 3 3.9U 1.08 1.39 0.S5 3.25 l.U l 1.13 5.21 1.83 3.9U 2.53 1.76 I.8 5 2.5U 3-71 3.90 0.99 2.90 o.Us 1.21 O.U5 0.27 1.92 O.36 1.31 1.12 0.76 0.32 O.52 1 . 5S 3.27 2. pU 2.69 3-11 2.U6 U.53 3. US 1-75 3.0U l.Op 1.36 0.81 3.2U I.3 6 1.05 5 .09 I.7U Ash 2.55 2.75 3. 3S 3.0U 3.2U o.US 5.02 2.U3 2.07 2.23 1.07 1.2b 1.58 3-98 O.7O 1.62 1.18 3.21 2.89 2 . US 1.66 1-79 2 . Us 3.67 1.11 1-57 1.31 U.69 3 .81 0.30 2.59 2.7U i.s6 -25Table 6. The Chemical Analysis of Prepared Peptones Expressed in Per cent. Bane of pentone Beef peptone Spleen peptone ( l ) Spleen peptone ( 2) Liver per t one ( 1 ) L iver peptone ( 2 ) Heart peptone ( 1) Heart peptone ( 2) Brain peptone ( 1 ) Brain peptone ( 2 ) Fork peptone Average Yeg. Yeg. Yeg. Yeg. Yeg. Yeg. peptone Ho. peptone Ho. peptone Ho. peptone i'o. peptone Ho. tryntone*- Average 1* 2* 3! 4* 5** Free amino a c id n itro g e n Total n itro g e n Total proteose n itro g e n Primary p ro teo se n itro g e n Secondary proteose n itro g e n Peptone n itro g e n Free ammonia n itro g e n 14.19 lU.Sk 14.64 16.44 13.44 15.OS 14.20 15.00 11.99 12.58 6.47 4.16 2.88 2 . 3C 5.61 4.76 6.6s 0.77 1.44 3.65 0.22 1.22 0.57 0.75 1.14 0.46 1.40 0.08 O.33 0.90 6.27 2.94 2.31 1.56 4.51 3.90 4.68 O.69 1.11 2.75 10.54 10.94 9.92 2.85 9.23 10.b 7 IO.34 3.12 6.44 8.64 0.21 0.24 0.02 0.19 0.13 0.05 0.14 0.47 0.02 0.14 0.47 0.71 0.67 1.17 O.57 0.50 0.45 1.54 1.00 0.82 I .6 3 2.11 2.39 6.17 1 .6 l 2.06 1.82 4.28 2.94 1.64 1.57 2.07 2.29 5-97 1.52 2.04 1.80 4.26 2.92 1.57 1.19 1-75 2.16 6.04 3.11 1.99 ? Ck 2.88 6.75 4.48 14.24 3-77 0.70 3.07 8.26 0.16 C.79 2.66 2.60 3*52 9-11 7.66 s . 50 12.24 11.45 11.37 0.23 0.13 0.19 0.19 2.38 0.12 0.09 0.09 0.13 0.13 0.41 0.05 0.14 0.04 0.06 0.06 1.98 0.07 C.27 0.15 0.08 0.08 2.52 0 . 4l o.oc 0.00 0.00 o.oc 0.37 0.51 2.87 2-95 3.2b 3*75 1.76 0.00 5.54 7.06 7.16 7.16 2.36 5.24 5.2s 7.00 7.01 7.10 2.30 5.19 2.43 1.47 1.32 2.79 3.94 3-04 10.05 0.54 0.15 O.39 O.73 0.14 2.48 5.76 5.64 2.49 4 Prepared from hnhydrolyzed com gluten. ** Prepared from hydrolysates of com gluten. Amino Amino n itro g e n n itro g e n (Van Slyke) (Srenson) Ash - 26 - In the case of the prepared animal peptones, the results (Tpble 7) show that two hatches of peptone prepared from the same source material vary considerably in chemical composition. This again emphasizes the d if iic u lty of preparing standardized peptones under eith er laboratory or commercial conditions. As in the case of per cent yield, the varia­ tion in chemical composition of two batches of peptone from the same source is probably due either to variations in the original material, or to the treatment of the peptone during preparation. rule, As a general the forms of nitrogen were present in larger amounts in the prepared animal peptones than in the commercial peptones, with the ex­ ception of free amino acid and amino nitrogen. The vegetable peptones prepared from hydrolysates of corn gluten were lower than e ith e r the commercial or animal peptones in a l l forms of nitrogen, with the exception of the free amino acid and amino nitrogen. The low amount of peptone and proteose nitrogen is due to the acid hydrolsis, lib era tin g amino acids. This same hydrolysis would also account for the higher amounts of amino nitrogen found. In the case of the peptone prepared from dry corn gluten, the combination of a f a ir l y high acid content and a temperature of 55°C. f° r three weeks was evidently enough to hydrolyze the peptone nitrogen formed. This resiilted in a lowering of the amount of th is form of nitrogen over that found in other peptones prepared by pepsin— HC1 digestion, and in the production of a larger per­ centage of free amino acid and amino nitrogen. The vegetable tryptone was digested for a sufficient length of time to allow the trypsin to form a comparatively large amount of free amino acids and amino nitrogen. The Tables also show that the free ammonia nitrogen is very low in a ll cases. -27Tables 5 and. 6 show that the sum of the various nitrogen fractions does not equal the figure obtained for to tal nitrogen. This indicates that in some cases one or more forms of nitrogen are being determined by more than one method, and in other cases that not a l l is being determined. the nitrogen The dividing line between proteose nitrogen and peptone nitrogen is undoubtedly very in d istin c t. Thus some proteose nitrogen is probably being determined both by saturating with ZnSO* and oy p re cip ita tio n with tannic acid, nitrogen. Also i t the same being true of peptone is true that some amino acids, especially the basic ones sxich as arginine, lysine and histidine, are precipitated by the addition of phospho-tungstic acid and therefore are not included in the figures for free amino acid nitrogen. The only truly relia b le determin­ ations are those fo r t o t a l nitrogen and free ammonia nitrogen. Lysine i s not determined by Van Slyke* s method unless the deaminization is carried on for one-half hour, so the figures given for amino nitrogen are s lig h tly lover than they would be had this procedure been followed. Thus the figures given in Tables 5 and 6 are most useful for comparative purposes in that they indicate which peptone is higher than another in any one nitrogen fra c tio n . As stated by Ab©l and G-eiling (1), nitrogen fractions such as peptone nitrogen and the several proteose nitrogens cannot be regarded as chemical e n t it ie s . They are defined from th eir reactions under d e fin ite conditions and not by th eir chemical make-up. The fact that the vegetable peptones prepared from l6 hour hydrolysates of com gluten show the presence of small amounts of peptone and proteose nitrogen indicates that free amino acids are precipitated to a slig ht extent by sa.turating with ZnSO* and by the addition of tannic acid. VJhile the amount of heat coagulable nitrogen is not included in -2 5 - Tables 5 a n d 6, it may be men t i o n e d here that none of the peptones contained a significant amount of this type of nitrogen. if the p H of a n y pept o n e However, is a d j usted by the addition of either an acil or a bas e a n d the p e p t o n e solution is then autoclaved, a pre­ cipitate is u s u a l l y formed. /tention snould be made at this time of the work of Rimington and Ka y (U6) and of R i m i n g t o n ( 1+2 ) ( 1+3 ) ( 1+1+) (I+5) on the structure of a p h o s p h o -peptone i s o l a t e d f r o m tryptic digests of casein. peptone h a d an empirical formula of C „ H j O _ P _ , ( j weight of I2H5, wa s strongly levo-rotatory, basic acid. nitrogen. equal to 'j y This a molecular 5 and acted as a nine- One n i n t h of the n i t r o g e n was in the form of amino However, after hydrolysis with H C 1 , the amino nitrogen was the total nitrogen. This indicated that the substance was a peptone c o nsisting of nine amino acids, joined by peptid linkages, all of which were acyclic mono-amino acids. h y d roxyglutamic acid, Further analysis After aci d hydrolysis h ydroxyaminobutyr ic acid and serine were isolated. showed that the peptones was p r o b a b l y made up of three molecules of h y d r o x y glutamic acid, four molecules of hydroxyaminobutyric acid, phosphoric acid. two mol e c u l e s of serine and three molecules of Of the p hosphorus present, two thirds was removed b y the action of b o n e phosphate se, the remaining one— third b e i n g re­ m o v e d b y p h o s p h o r i c esterase of kidney extracts, presence of an ester linkage. indicating the A structural formula, was suggested w hich satisfied all of the experimental findings, although the sequence of the amino acids and the attachment of the phosphorus atoms were not determined. -2 9 - COLOR REACTIONS OF PEPTORSS Various color reactions have heen devised to determine the presence or aoeence of certain components of proteins. The peptones were tested for th e ir reaction to the follov/ing tests! The b iu re t t e s t for peptide linkages; the Rosenheim t e s t for tryptophane, the Killon te s t for tyrosine; the Sakaguchi t e s t for arginine; the ninhydrin reaction for the alpha amino and free carboxyl groups: the xanthoproteic te s t for the benzene nucleus; the Fleitnann te s t for the presence of loosely bound sulphur as found in cystine and cysteine; and the Molish t e s t for the presence of carbohydrates. The r e su lts obtained for these color reactions are shown in Table 7» Discussion Table 7 shows that the commercial peptones are positive to a l l except the Molish reaction. This indicates that a l l peptones are free from carbohydrates. the commercial The positive bixxret test Indicates th a t hydrolysis has not been completed. The positive Rosenheim t e s t indicates th at none of the peptones tested are prepared by acid hydrolysis, or i f they are, i t is only a very mild treatment, since a more d ra s tic hydrolysis would destroy the identity of the tryptophane present. Table 7» Peptone RosenBiuret Millon heim Test test te s t A. Commercial Bacto-peptone Heopeptone Proteosepeptone Proteosepeptone lio. 2 Protaosepeptone No. 3 Tryptone Tryptose Protone Wilson "C3« Wilson "C1 Witte (1912) Witte (19U0) Armour Armour Special Pfansteihl Baker1s Cenco Albumin B & A Meat Fai rchild Chaissiang Merck Steam Parke-Davis & Co. B. Animal Beef Spleen (1) Spleen (2) Liver ( l) Liver (2) Heart (1) Heart (2) Brain ( l) Brain (2) Pork C. Vegetable Number 1 Humber 2 iTomber 3 Humber b Kumber 5 Tryptone The Color Reactions of Peptones * + • + + + + -r + + *r “T f T ■ * -f + + T "t* + -i+ + -r -t*r + + -r — — -t Sakguchi Hinhydrin te s t test T ■+ + + Xantho- F le itproteic mann Molisch te s t te s t test *r *r T + T + -i+ T *r *r -t-t*r + •r T + + T + T + + + + + *r -r -r + + + + -r + + + + -r + -t ~r ~r -t + + T + 1 + •+ ■ + + •+ -* » + + + + ■+ + T + + + + + -r + + -r -r + + -i- + T + ■ f- •+ ■ ■ + • + + •* + T I" + -t+ + -t* -r •t- -t* + -t *r + -t 1- + + •t* 1* — + ■f -t + T T -r •>' + + + + + + + + ■+■ + + + + + + + + -j-* • -*I' 1+ + T + + •+• + 1- + f. + T + + + -f + f T + + T + -r + -v + t J- + T -t T -3 0 - The prepared animal peptones were positive to a l l te s ts except the Molish reaction, with the exception of the two liv e r and two spleen peptones, which were positive to this t e s t . In the case of the liv e r peptones th is may he explained by the presence of glycogen in the l iv e r . The presence of carbohydrates in the spleen peptone is not so easily explained. The four vegetable peptones prepared from hydrolyzed corn gluten gave positive Sakaguchi, ninhydrin, xanthoproteic, reactions and negative b iu ret and Rosenheim te s ts . positive to Millon’s t e s t , while the fourth, been removed, was negative. Fleitmann end Molish The f i r s t three were from which the tyrosine had The positive Molish te s t indicates that not a ll the corn starch had been removed from the protein. The positive xanthoproteic t e s t was probably due to the presence of phenylalanine and/or tyrosine, since the tryptophane was destroyed by the acid hydrolysis, as indicated by the negative Rosenheim te s t. The peptone prepared from the sample of corn gluten that had been hydrolyzed only two hours (Vege­ table peptone No. 1) gave a f a in tly positive biuret t e s t , indicating that not a l l the peptide linkages were broken, as they were in the remain­ ing vegetable peptones. Vegetable peptone No. 5 fiJ^d vegetable tryptone were positive to a ll t e s t s . This again indicates the presence of carbohydrates in the original protein, and also shows that the hydrolysis was not as complete as in the case of the other vegetable peptones. BUFFERING ACTION OF FRPTOI'IES To determine the buffering action a one per cent solution of each peptone was made up by dissolving exactly two grams m 150 ml. of dis­ t i l l e d water. When dissolved, the solution was placed in a 200 ml. volu­ -3 1 - metric flask, d iluted to the mark with d i s t i l l e d water and thoroughly Twenty ml. of this solution was pipetted into a 50 ml. beaker mixed. and the pH value taken before and a f te r the addition of various amounts of 0.01H HG1 and 0.01U NaOH. on each peptone. A duplicate set of determinations was run The pH readings were taken using a Beckman pH meter. A second set of buffer determinations was made on one per cent peptone solutions whose i n i t i a l pH had been adjusted to exactly 7 .00. To do ta is two grams of the peptone were weighed and dissolved in aporox- imately 190 ml. of d i s t i l l e d water. The pH was adjusted to 7 .00 by the addition of e ith e r 0.01N IIC1 or 0.011T NaOH, and the solution diluted to 200 ml. in a volumetric flask. The pH value was then determined a fte r various amounts of 0.0111 HC1 and 0.01N NaOH were added to 20 ml. of the neutral solution. Table S gives the pH values obtained a fte r adding various amounts of 0.01K HC1 to the unadjusted peptone solutions and Table 9 the pH values afte r adding 0.01N HaOH. Tables 10 and 11 give the pH values obtained a f t e r adding HC1 and HaOK, respectively, to the adjusted peptone solutions. Discussion The peptones are l i s t e d in Tables S and 10 in the order of their increasing i n i t i a l acid ity. as follows: (l) They may be roughly divided into four groups those that are alkalin in reaction, pH 7 .00 to 7*60; (2) those th at are neutral in reaction, pH J . 0 0 ; (3) those that are s lig h t­ ly acid, pH 6.00 to 7 .00; and ( U) those that are strongly acid, pH h . S S to 6.00. This method of grouping the peptones shows that twelve are alkaline, four are neutral, strongly acid. twelve are slightly acid and twelve are - T ahle 8 . a> d cc id O a • d rp* O.OC l.OC 2 . CO 3. CO 4.00 5. CO 6 . co 7.00 S. CO 3 . CO 10.0 12.0 i 4 .o 16.0 18.0 2 0 .0 © p o p ft^ © OJ ft H !T \ ©H P w p ft 7.60 7 .U1 7-24 7.02 6.83 6.53 6.23 6.00 5-71 5-51 S 3i 5. 00 U.71 4. 5 3 4.37 4.19 © 0} O ft P< s p ft 1 o o -p c£ sq 7o l 7-^3 7 .29 7.12 6.95 6.76 6.51 6.23 6.01 5*71 5.U9 5-24 4. 98 4 .8 2 4.64 4. 5 1 i p< ~ © 7ft ft ( • © o « ft o © p o p ft © P o p 7.2s 7.05 6 .7 3 6.55 6. 2 9 6.05 5.8 0 5 - 5S 5.3U 5.11 U.92 U.63 4 . 42 4.25 U.os 3-93 32 - Change i n pH o f a one p e r c e n t p e p to n e s o lu tio n C u n a d ju s te d ) a f t e r a d d in g v a r io u s am ounts o f 0 .0 1 H HC1. © p o +» o p ft 1 o p p ro ft 7 . 1S 7 .0 0 6.83 0. 6 3 6.50 6. 27 6.01 3. s i 5-57 5.31 5.10 H.76 4.51 M o 4.h 3-97 . X m X X .2 a L S' 5 Q -9 _X m ?n 5 "Sri S?2 7.14 6 .8 9 6.61 6.32 6 .0 6 5 .89 5-72 5.5s 5.46 5.36 5-23 5-11 M 9 4.88 b. 78 4.67 I 2 X n $ 7 ’lb 6 .S 5 6.07 6.50 6.33 6 .1 7 6 .0 1 5 .85 5.69 5 .5 2 5.U1 5.17 u.9 5 b.Jb 4*55 4.37 . a; A, c p. ,* A o ^ n, Q> o P mo h p._y CO 03 © 2 2 p4--^ © O p p. © ft--* ft 2 ft p 7.13 6.91 6.6s 6.44 6.12 5 . Si 5-57 5-39 5. 22 5 .0 1 4. 8 2 7-12 6 .9 6 6.74 6 .5 3 6.33 6.15 5-97 5. 82 5. 69 5 .5 6 5.4 8 5. 30 5. 16 5.01 4. 90 4 .8 0 7 .1 2 b.63 4.4 5 H.3O U.l6 4 .o 4 1 u, ©ft" id . © O H ft ® ft © ©O JiJ tfl ft © 7.1c 6 .80 7 . Q3 0.9b 6. 59 6 . 4o b.83 6 . 7 3 6 .2 7 6 .6 1 6 .1 1 6 .49 5.97 6 .39 5 .8 5 6 . 2 4 5. 72 6. 09 5.6 1 5-93 5.51 5.65 5-37 5.1+0 5.2 1 5-19 5 . 0 s 5 . CO 4.36 '4.85 4. 8 2 © fl 0 p P cJ © P P W dp< ft P o 7.09 6.QU 6 .7 9 6.6l 6 . U2 6.24 6.06 5 .9 1 5.74 5.6 0 5. 4 2 5.24 5 .0 4 4.89 4 .71 4.59 0 P , © P. c l !; OO ©S H ft 7.05 6 .9 7 6 .81 6. 68 6. :3 6 .3 7 6 .2 2 6.04 5 -S7 5.7c 5.55 5.29 5.02 4.87 4.69 5.45 8 © t W f, © . H O ,Qft ®i P© I ft ft © tfl t.-i ,Q ®N P,IA ft | . © . © O ft O o ©© c3 P © © ©p 7.0c 6.94 6 .8 2 6 .6 3 6.42 6.20 5-98 5-78 5.6 1 5 .4 5 5-35 5.16 4.97 4.78 4.61 4.47 O p O p 7 .0 0 6.75 6.48 6 .1 9 5. 9 0 5.6c 9.31 4. 10 4. 95 4-79 4. 6 6 4.37 4 .12 4. 0 0 3.90 3.81 ^0 O © -p 7.CC 6 .6 2 6 .3 2 6 .0 6 5.81 5.61 5 .4 4 5.31 5.19 5.O6 4.99 4.82 4.67 4.54 4.43 4.33 © aC ©O ft © ft © ft CJ | a, © . © . - > H O P, PJ ( j\ ©H i O —* P C\J © P © © © p pftt P ft p4 a 7 .0 0 6 . 3 7 6. 89 6 . 4o 6.7 1 5*97 6 . 5 1 5 .6 1 6. s i 5 . 3s 6. 09 5 .2 1 5-87 5. 0 7 5 .6 7 4. 97 5. 45 4. 8 5 5.29 4 . 7 4 5. 1 5 4.6 3 4.91 4 . 4l 4.72 4. 26 4. 52 4. 12 4 . 3 s 4.0 1 4.22 3. 9 0 aO I ft O © f l f t d © p P ^ P *0 O © ft C hv-> © 6.92 6.82 6.69 6.51 5.53 6.15 5-97 5-77 5.60 5.46 5-31 5.09 4.90 4.71 4.54 4. si © £ ^' ^ d © © ®a i D O H O © -p 6.32 6.55 6.21 -.9 6 5.75 5.60 5-48 3-37 5*27 5.17 5.10 4.98 4.88 ■*•77 4.67 4.58 ftp 6.82 6.55 6.37 o .l 6 3-93 5.71 5.48 5*31 5.14 5.00 4.87 4.66 4.44 4.31 4. 1S 3*39 auoqdad ^oaaj'i euoqdad euoqdsd q«9H VS® a u o q d a d X t fx g q .s u B j j anoqced s tA -a a ‘ >=' d - i a j 9U0ql9d jaatBg; QUO% - d a d PTTqo.iTBd i— 3 S \ g . £ f D p J S f c f ~ -J rH ^ no rA A A A rA A IA N o ,CT .=* J C T *2 '\C P !» rn WO (\| U) o IAf\i J ^ n CO J AA AA 0 1 ^ 0 ^ 5 Avd UO j -m A A A A A A A, A, ,A A A A A A ° ^ LT^ _=i ^}^ 1a A A ; A A Uli VD A A A o o u q q (2 ) eu o q d a d a a A p i a u o q d a d p © xoad s u n oraay i no no i auoqdad u u u a q s O NO NO LCO r — o CM UO rH CM ■=}- rToCO i n CM O OOCO N -N -C U O ^ A A A -, S " - 8 m I___ NO A C — A A A A ^ A A A A A NO o S)iS A~A . A Ti A1 00 '-° lOoMnn o NO CM rH O QQ O'— ICO NO CM I I rH i . A ^O UO LCO-CT -M ~--l NO m n o :< . NONONO H rpCOCD J- rH CMrH CMNo ICOr-, , _ _ I' A A ° A w r '~ '~ ° L(0 - ::^ n n j o o o co mo no LCO UOLCO-ZJ- jf _zt ,=J- JM" n o NO no cm o o cvi c o c o c o r-— r-— o m o c o rH CO MO -CT CM I—I o CO N—MO -=T CM CM uo uo i i o j - J j- j- .Oi ul't! 4 nononoA A A 9 )0 o o rt H NO OM O ITi MD U O -^ t CM r-1 o o co to r— VO o n o cm r— do uouo lcouo lco A iroA A A A ^i* A A A A CM CM g o O MO ICO NOMO MO N - O UO C— rH CO r— CO I -H NO <—I O CO 60 t"-MO MD J (\l rH CO CO uo lco uo uo A AA A A A A A A A A A rH rH U O ^ t UO rH CTO 6 0 0 0 CO O MQ rH OOUOLCO O O l l O H HO r— IX) -^1 NO CM iH rH o o CO VO U O -3- od -rt NOt'ONONON"n UO UO IXO^J- .=*• t t J=t CO O (— O LCO r l I— MD UO UO 6 0 NO I— rH NOMO OOVO NO CM C J CTOt— MO U O ^ i N"\ CM O OO t— UO auoq O -or I ro ICO CM 6 0 U 0 CM CM NO O I— 6 0 CM MO rH t'— LCO NO rH O 6 0 C—MO U O ^ t NO rH O O OO auoqdad jiuomav (I) auoqdad uxtjjg; it ffOn auoqdad u o 3Xf.M ( C o n t in u e d ) OJ CTO • ico uo^f 4 j * * • (X) auoqdad qxeajj auoqdad je© £ auoq -da do am -o q o er CD suoqaacT ua a p a s pappa I0H HTO'O *TW • • • « » * '.O • • • • • no no no LCO CMCTOCD CM 6 0 UO OJ O NO f— CM CTO C— OO COMDU O ^ t CV rH O CTO t— U O ^ t CM rH • MO MO • -zt UO UO UO UO UO^J- ,=1- ^=f • • ^ u o u o ir o u o uo_=fr rH -= t NO rH 8. -CT t o h i o n ai o o < 0 U O U 0 .T * l ' M \ | rH O cm attoqdad upmxqxV W a/Caq axqeqaSs/^ Table 1 No No no n o n o AAAAAAAAAAAAAAA A r O ^ vi ? sCO NO O eu o q d a d 1 cro CO f — O O A S 'o * * • * * • * « * • * • UO ICO UO UO LCO ICO UO UO-d" x t X X J - OO m o (M t o J NO rH CTO CO MD J f NO « • * # • « • V D CO UO UO U O ICO UO • ♦ X tX t ( \ I H (\l 6 i M ) W N H O CM rH O O O N - C O C t N \C M • • • • • • • • * UO UO U O ^ t X t S t x t x t N O -^ ' MD VO CTO OO 6 0 O u o rH 6 0 C— rH NO 6 0 p t QO r— u o N o rH CTO N -M O t t NO rH CJO CD MO t t • V O MO • ** * * * ** • • .* .* * -=T J MO MO'■O UO UO UO UO LCOLCO^T -^t t t NO rH NO NO N O ^ t N— iH O 6 0 CTO CTO rH MO O CM NO rH GO r— UO N o CM iH CTO 6 0 MD UO NO CM O * * • • * • • * • • • • • • V.O VO VO LCA \ C \ IC \ (CA LCN ^ ^ ^ * * *ZT MO CM 6 0 ^ CM ^ t ICO CTOC-O NO NO N - Q NO g j -=S- N o O 6 0 C .Q -=1‘ CMO 0 0 6 0 I— UO-=T CM rH OO V Q V O V-O LOs UA\ i r ^ L£> a }At" a J" a 1* f^> ICO N , 6 0 v o NO N - CM MO rH DO rH UO OJ CTO I— co NO <0| O CTO I— N— ICO NO CM O v .o v .o loi i r \ Li’. lcotco uo-^t LCMMD T\ jH N O'x tt (0.1I—c-NOWHHCJOMNijOwJ; C T O j-— o 60 MO ti CMO OON"''® J A . to MO CO MD u o UO UOUOUO-CT t t t t t t t t NO O O O O O O o rH A O O O O O O O O O O O O O O O ^ O O O O ^ UOMO N C O O o H r l H H H O O <\J -34T ab le 9 . 1 *d © rH O w S T) H 0) O . O * 0) d 0 -p A 0) Pi 1 Pi 0) Pi C O •H 0) pt rH 2 ‘ ci 0 p ;-i ID ttO Q © d *■ 0 0) P id d cS Ph 7. CO 6.82 6. 70 6.59 O.50 6.38 6.29 6 . 1 S 6. 12 6.00 5.96 5.90 5.83 5.76 5*72 5.61+ 5.62 3.5U 5.55 5.45 5.46 5.29 5-31 14.0 5.13 > 1 6 lb.O 5.00 5.00 1S .0 4.87 4.85 20.0 4 .71 4.70 c.cc 1.00 2.00 3*00 4 . CO 5. CO 6 . GO 7.0c S.OC 9.0C 10.0 12.0 7. 00 -pH oj >— x 0) d O P O, 9 Ph © d 0 •p Pi © Pi d © t> •H a •rH © d m pi 7.00 6.7s 6.5U 6.32 6.11 5*95 5.80 5.66 5.55 5.U7 5*39 5.21 5.08 4-97 4.83 4.70 7.0 0 6.95 6.5U 6.76 6.65 6.55 6.1a 6.31 6.16 6.03 5.90 5.60 5.35 5.10 U.S9 U.69 Change in pH o f a one p e r c e n t p e p to n e s o l u tio n ( a d j u s t e d to pH v a r io u s am ounts o f 0.01ft HC1. © a 0 -p & Pi P ■ 7.0 0 6.b8 6.3 5 6.06 5.S5 5.6U 5-Ub 5.33 5.22 5.13 5.05 4.90 U.80 U.70 k.d' U.bl CVI '—1 © 0 O p Pi 7. 00 6.90 6.72 6.55 6.38 6.19 6.00 5.82 5,66 5.51 5o 9 7.00 6 . 3^ 5-17 5.16 U.97 it. 78 it. 61 it. H7 M 7 U.60 H.63 It. 50 6.82 6.61 6.te 6.20 5.9s 5.78 5.61 5.U5 5-35 _ b © P O P Pi a> Pi « •© S © d 0 p Pi © Pi a 0 co © a 0 p /- • 1 © Pi cu © pH • ■3 P p © pH p to 7.00 6.61 6.27 5*95 5.6s 5,i£ 5-27 5.15 5.00 U.92 4.85 U.75 U.63 it. 56 it. 50 it.i)6 •rt 7.00 6.89 6.72 6.58 6. Ho 6.21 6. 03 5.88 5.71 5.52 5.38 5.16 it.q6 it. 78 it.60 it.iti CD 7.00 6.7b 6.37 6.01 5.77 5.56 5.H1 5.27 5.15 5.06 it.93 U.SU it. 72 it. 61 U. 3i it. 41 © d 0 p Pi © Pi © d 0 rH ^—' © d O H (D > C) © p; p © dc pH a> P Pi © Pi r*< t~ iH a) d 7.0C 0.8S 6.66 5 . 1+2 5-9 5 6.20 5.75 5.96 5-57 5.75 5. He 5.5s 5-28 5 .it 2 5.16 5.29 5.06 5.19 it.90 it. 99 H.7U it. 81 it. 61 it. 65 it. 50 U. UO it. Ho 7.00 o.sH 6.56 6.27 p Pi © P. CD •• T3 H •rH rP O Pi •H © Pi 7.00 6.89 6.72 b. 53 b. 32 6.08 5*89 5.67 5 -it9 5.3U 5.20 5.00 U. 80 it.oH it. 50 it. 36 © d 0 p Pi © Pi 1—i .d •rl © P C D O © 7.00 6.62 6.32 6.06 5.81 5.61 5 -ltit 5*31 5.19 5.06 it.99 it. 82 it.67 H. Sit U.it3 ^•33 7.00 6.79 6.60 6.it2 6.26 OJ •> —• © a 0 p Pi © Pi d © •rt id 7.00 6.82 pH ■3 p 0 © Pi C O 9 d 0 p m » d d 0 g d < Pi 9 Pi 7.00 6.91 © d 0 p a © Pi P d © © K 7-00 6.88 6.0I 6.79 6.71 6.39 6.55 b .i li 6.10 5.90 6.5C 6.29 6. lit 5.92 6.18 5-97 5.81 5.52 5.65 5-39 5.50 5*33 5.22 5*11 5.12 it. 90 I+.70 it.60 it. 31 it.91 U.7U it. 60 H. i+5 it. 30 5*70 6.08 5.88 5.67 5*70 5 o l 5*55 5*38 5.34 >22 5.01 5.20 4.99 4.81 4.66 4.46 4.30 4.80 4 .b 0 4.43 4.29 - T able 9* o W si ^5 rH 0) O rd • rj O< tf © rt o p Pi © ft u rt o n © rt o p Pi © c\j rt v-r © © H £< rn © rt o •p ft © Pl = m rt o o = M rH ;H h" * a 0. 00 1.00 2.CC 3.00 b.00 5.00 7.00 6.89 6 .7 3 0 .o0 6.^5 6.29 6.10 7 .0 0 5-91 8.00 5.72 9.00 5.56 1 0 . 0 5.1+1 1 2 . 0 5.12 l b . o b.59 16. C b.Ss 18.0 b.b7 20.0 b.27 S .00 7. 0 0 6.88 6.71 6. 53 6. 33 6.11 5.31 5.69 5.5O 5-33 3.19 b.95 U.7S b.59 b.bo b .?6 7.00 6.8 5 6.66 6.b6 6.23 6.00 5.76 5.56 5. bo 5.25 5.11 1+.39. b.70 b.52 u.39 b.?5 35- Change in pH o f a one p e r c e n t p e p to n e s o lu ti o n ( a d ju s te d to pH am ounts o f 0 . O il' KOI. © rt o ■p i— I © Pi © © n 7 .0 0 6.85 6.63 6.b2 6.20 5 . 9S 5*53 5.3s 5.2b 5.12 b.91 b.70 '4.52 4 .38 b.22 © rt o -p !-i © o ©.rt" p cn P H •H 1 Pi © Pi hO rt 3 © •h rt C OO c» p •H rt H rH O 7 .0 0 6.S9 6.71 6.51 6.31 5.09 5 -S7 5.63 5A5 5.29 5-15 b.91 b.72 b. 51 b.3S b.22 7.0 0 © cn o p pi r"i rt P 1 o p o rt m 7.00 0.S3 6 .9 0 6 . 6 s 6.75 6. 51 6.59 6.31 6.39 6.09 6.18 5.8b 5.91 5.60 5.85 5. UO 5-b2 5.2 0 5 .2 b 5.08 5.09 b.S5 b .s 6 b.65 b.6b b.10 b.b7 U.32 b.30 U.19 b.18 Pi © p, rt •H Q 3 p © 3 g p **i M 7.00 6.79 6.57 6.29 6.01 5.78 5-59 5 . bo 5.22 5.11 5.00 b .s o b.6o b.bi b.27 b.10 © rt o p o rt P, 1 o +3 q n 7 .0 0 6.87 0. 71 6 . 5b 6. 36 6.15 5-93 5 *& 5.15 5.2b 3 . 08 b .si b.59 b.bo b.26 b.10 © rt o p Pi © Pi f5 rH ©^ © rH Pi o ©' 3 prt rt o P r © ft o •p o rt a f t e r a d d in g v a rio u s ].Q) i © r<-\ U! O • © o P ft o rt © Pi rt o O -P p ft q © Cv Pi n n R 7 .0 0 7.00 6 .So 6. 55 b.15 5.7s 5.52 5.30 5.1b 7 .0 0 6.7O 6.b5 6 .2 0 5-97 5-75 5-55 5-35 5 .1 b b.97 b.so ■»-53 b.30 b.ib 3 -9S 3. S3 6.55 6.31 5.99 5-75 5-53 5-33 5-13 b.96 b .80 b.56 b.35 b.is b.02 3.09 b.99 b .s b b.75 b.62 b.b2 b.26 b.12 b.oo 3* 88 I © OJ © o ■ ©o P ft o rt © ft rt o o -p p Pi o © rt Pi Pi 7.00 6. 75 6.bs 6.19 5-90 3.60 5.31 5*10 b.35 b.79 b.66 ^•37 b.12 b.oo 3. 90 3.81 © rt -e~ rt o © rt © P p Pi p Pi m — 11 o rt i-H 7 .0 0 6. 8 3 6 .b9 6 .0 0 5 .5 6 5. 2b 5 .01 b.S2 b.69 b.55 b.b2 b.21 b.01 3.S9 3-73 3.6s o o p p © p © P o o p d k© -H rt •'jj i*4 rq © o 7.0 0 6 .b s 6 .0 b 5.6 5 5.3s 5.16 5.00 b.S3 b.70 b. 56 b.b2 b.23 b.02 3.88 3.72 3.5S 7.00 6. bo 5.91 5. 5 0 5.21 5 .0 0 b .82 b.67 b.52 b.bo b.28 b.07 3.88 3-72 3 .56 3-b2 i Pi © p co rt © u © ft © fl O +» 7.0c 6.bi 5-85 5 -b2 5.13 b.33 b.75 b.6c b.b6 b.31 b.13 3-96 3-78 3.60 3. b 5 3*32 - Table 10. Ml Q cfl M F; -d h a) ° . T) £ O ci . rl © d o P ' 'x pHC\J © H PiCr\ rH © —' P P •H o . c o 7 .6 0 1 . 0 0 7. 67 2. 0 0 7 .S 5 3.00 5.00 H.00 S. 18 5 .00 g . 3H 6 .0 0 s M 7. co 8 .6 2 s .o c 3.7s 9 .0 0 8 .9 1 1 0 .0 9 .0 5 12 .0 9*35 1 4 .0 9-57 1 6 .0 9 . 8 5 1S .0 9*95 2 0 .0 10.13 © 0! o p u p 1 o o c' m -p 7-51 7 .6 2 7.74 7 7*14 7-22 7 .4 1 7 .5 5 7.69 7-21 7. 9 2 S . 01 S . 10 £. 19 s . 25 8.39 S . 49 8.59 8.69 S . 79 © d o p & P P 1 O P o tfl rq 7- 1 4 7 .2 5 7-37 7 .4 9 7 .6 1 7. 73 7-85 7.9 7 8 .1 1 s . 25 s . 34 8.54 8 .7 2 S . 93 9 .0 7 9.16 © d o p Pi © Ph 1 © 02 O © P o p Pi 7 .1 3 7 .3 2 7 .4 3 7.54 7.64 7 .7 6 7-87 7.9 7 8.0 8 8 .2 0 8.3O 8.9 1 8 .74 8 .9 5 9.13 9. 2 8 © d o p Oi © rH P h- h © > •rl id 7. 12 7.44 7.64 7-78 7. 96 8.11 8. 22 s . 32 8.40 8.5O 8.60 8-73 8 .8 3 S. 97 9.10 9 .2 0 © d o p Pi © pH."-* OJ d •H d p W 7. 12 7 .28 7o 7 7. 45 7 .5 0 7.55 7.59 7.68 7. 76 7.88 7.96 s . 13 S. 22 8. 4 1 3.56 S . 77 i Pird- © Pi • O © id H o © «d d P © W © p* o p 7.10 7 .27 7 .4 8 7.70 7-85 7-99 8 .0 8 s . 17 8.23 8.3I 8. 3 8 s . 49 s . 59 s.Gs s . 75 8 .8 0 © d o p Pi © Pi © d o p r~l © Pl_ d b O s: K H 1 Pi © LT\ Pi « © O H Id A ci © p a © 0 o Pi •H 3: W) p © > 7. 09 7 .2 7 7-41 7 .5 2 7.64 7-73 7-83 7-93 8 .0 3 8 .1 2 8.22 8. 4 9 8.65 8 .8 9 9-04 9 .2 9 7.08 7.3 2 7. 42 7.54 7.65 7.74 7-85 7. 93 8. 02 8 .1 0 8.18 8. 36 s . 52 8 .67 8.80 8 .9 0 7-00 7.25 7 .4 3 7.59 7 .7 0 7.S3 7*32 8.00 8.10 s . 19 s . 27 5. 41 8.5S 8. 7 2 8 .8 5 8 .9 5 P © d o > a> d © Pi 1 CM © © • O O © P O Pi P o I p Pi • O © Id H rO © ci d P o © p fAfl © i> © d o P r N PhQ ©JPi 0~i rH ©w P P ■rl 3: © d © d o o p Pi © Pi & © p 1 O P t) eJ w Pl = m d o o = © 7.0 0 7 . 0 0 7 .0 0 6 . 9 7 6-93 7-12 7.19 7.19 7 .3 2 7.15 7 . 2 3 7 .4 2 7. 31 7. 5 2 7 .2 9 7.33 7-59 7 . 4 4 7 . 7 0 7 .4 2 7 .4 5 7-76 7 .5 3 7 .8 5 7.5 1 7-57 7 .8 9 7 .59 7. 99 7 .6 1 7 - 6 ? 7. 99 7.66 8 . 1 0 7-71 7 .7 7 £ .0 8 7-77 8 . 2 0 7.81 7-87 8 .1 5 7. 8 9 s . 31 7 .9 1 7 *94 8 .2 1 8 .0 1 8 . 4 0 8 . 0 0 8 .0 2 8. 29 8 .1 2 s . 45 8 .0 8 8 . 2 0 8. 4 1 8 .4 1 8 . 6 2 8. 2 6 s . 39 8.5O 8 .6 1 8 .7 8 8 . 4 2 8 .5 3 8-59 8 . 8 4 s . 91 S . 59 s . 70 8 .6 8 9 .1 2 9 . 0 4 8. 7 1 8.8 5 S . 73 9 .3 7 9 . 1 7 7. 86 '“ i •M o Pi *P o p D i a> ft ri P © H< J) cJ o p +> a> W © © p c/i > 6.10 6.53 6.90 7.17 7.38 7.57 7.71 7.31+ 7.96 8.01+ s . 13 8.30 8.1+2 8.55 8.69 s . 73 5.98 6.37 6.65 6.S9 7.10 7.27 7.1+7 7.62 7.79 7.97 s . 16 8.58 8.95 9.18 9.I+3 9.65 rH 05 •H o a) © P. fl to o ■p p P. p < u OP, © o p Pi Q ) Ph(\) Sh © > 5.62 6.15 6.1+0 6.63 6.85 7.01 7*19 7*37 7*55 7.67 7.82 8.12 S.lsO 8.68 8.89 9.12 I p O Pi © Pi Pt *d H -P © Pi o o p © o < 5.91 5.82 6.30 6.01 0.71 6.33 6.98 6.57 7.18 6.79 7*39 7.0c 7*51 7.20 7.67 7.38 7.81 7*59 7*95 7 . SO s . 05 7.98 s . 31 8.37 8.59 8.68 8.86 8.95 9*11 9*17 0.36 9*35 © 5*53 6.18 6.7s 7.21 7.62 7*99 8 29 8, ;6 s . 79 8.95 9.12 9.1+3 9*71 9.91 10.13 IO.32 •H © Si P o o p -p •H ft © P TU o p Pi © Pi l © a o ft -p © ft © % ft 5*51 5.21 5*73 5 . s i 5*93 6.1+s 6.22 6.97 6.1+9 7 . l+l 6.71+ 7»7° 6.96 s . c i 7.16 8.28 7*32 s . 1+9 7 . 1+7 s .6 s 7 .6 l s .s 6 7 -81+ 9.20 S. 10 3-1+8 8-28 9.70 8.1+1 9.91 s . 60 10.10 ! pi 5.20 5.59 5.92 6.1s 6 .U5 6.65 ft © ft rH Pi Si *ri P ©© p a W0 d P tu 7. 0 0 7-19 7.H2 7.59 7.76 7.S9 7-39 8. 08 8 .1 5 8. 21 8.29 S. Hi s . 50 s . 59 8.68 S. 73 Change in pH of a one p e r cent peptone s o lu tio n (ad ju ste d ) a f t e r adding vario u s amounts of 0.01 N NaOE © r. M o p t ft © Pi s d P 1 o • © 0 H O f t ft ft p © to © > ft p © © > 7 .00 7 .0 0 *7 7. 2 5 7 o ° 7 *H3 7 . 6 0 7.59 7 .8 1 7 .7 0 8 . 0 0 7 .8 3 8 . 1 1 7 .9 2 8 . 2 2 £.00 S o l £ . 1 0 S.HO s . 19 7.H7 8. 2 7 S o l S. Hi s . 62 S . 58 8. 73 s . 72 8.31 8 .8 5 8 . 0 0 s . 95 s . 96 © d © d o 4-> CU © FP ftO d ~ 0 n H ft 7. 00 7.30 7.H2 7. 52 7 .6 3 o p ft © ft OJ © H • ft O E ft P © y) © > rl © d o p ftT S ft i o p o.a FP 7-00 7 . 0 0 7-37 7 .2 1 7 .6 0 7 . HO 7-79 7. 57 7-95 7 .71 h i s 8 .0 9 7 .3 3 7.88 8 . 2 1 7.9H 7 . 9S s . 30 s . 05 s . 07 8 . 3 s s . 15 8. 16 8.H7 s . 23 8.2H 8 . 5 2 s . 31 S.H3 8. 65 8.H9 S.5S 8 . 7 s S.oH 8 .7 3 £ .8 6 8 . 7 6 8.89 8.9H S . 89 9.00 9 . 0 0 9 .0 1 rH _✓ —" © d © d o p ft © ft d •rl ft d rq 7 .0 0 7 .19 7-35 7 .5 0 7. 6 5 h is 7.91 8 .0 1 8 .1 2 8 .2 2 8.33 s . 51 o.6S 8. 8 1 8.96 0.0 9 o p ft © ft d © > •rl ft © d o p ft © ft o © d i o p o ft PP 7.0C 7 .0 0 7 .2 3 7.15 7.H6 7 .3 0 7. 65 7.HH 7 .8 1 7 .5 6 7*93 7. 69 8 . 1 0 7 .8 2 8 .2 0 7.9H 3 .3 0 8.0H S.Ho s . 16 s . 50 s . 27 S.6H S.HS 3 . 7 s S . 70 £.89 8. 85 9.00 9.01 9-10 9 . 1 0 - Table 11. 1 w o cfi ft* (H 'd D r-i C r— 1ft o• ft cfi o t ri C.00 1.00 2.00 3.0C 1+.0C 5.00 6.00 7.00 s . 00 9.00 10 .0 12.0 1U.0 16.0 18.0 20.0 £ u p © rH P © d a P o ©p © > 7.00 7.^1 7.65 7 . so 7.95 s . 09 8. 22 S . S3 SM 8.56 8.65 8. SO s . 90 9.00 9.0s 9 . lit © d o p ft © ft i © m o © p o d ft 7.00 7.1 s 7.31 7.1+1+ 7-95 7.6s • © d o p ft © ft ua cfi © p in © d o p ft © © CT\ P ft Pp •ri 7 . CO 7.00 7.30 7.57 7 . 7S 7.97 8.15 7.79 8 .3 I 7.92 £ .% 8.01 8.5U s . 13 8.63 8.23 S . 73 8 . 1+7 8.88 £.68 9.03 S. 37 9.15 9.05 9.2b 9.19 9 o 7 39 - Change in pH of a one p er cent peptone so lu tio n (adjusted) a f t e r adding v ario u s amount of O.Olh HaOH. 1 Pi © ft © •«H r» f$ 8 * O ©P u d cfi ft 7.00 7.19 7.27 7.31 7.33 7 . 1+1+ 7.75 7.93 7.93 7-39 8.13 7.66 8.31 7.77 8.1+8 7.89 8.61 8.01 8 . 7 1* 8.12 8.8b S.Sl 9.11 s . 61 9.29 s . z h 9 . 1+2 9.12 9. SO 9.37 9.55 © d o p ft © ft •H ft 7.00 © d o p —s ft O J ©H Pi 00 H ©^ P P rl ^ © p d o o ©p ft O (Q ©n Pi d d O C 5: 7.00 7.00 7-12 7-15 7.21 7.31 7.30 7.1+3 7.I+9 7.1+1+ 7.65 7.69 7.60 7.88 7.81 7.73 8.10 7.97 7-89 S . 3C 8.17 8.06 8.S0 s . 32 8.22 8 . 6 s 8 . 1*9 8. 3s 8.88 8.66 8.56 9 . 0C s . 97 8.90 9 . 1S 9 . 18 9.2 0 9.3S 9 . 1+2 9.1+9 9-33 9-55 9.70 9.70 9.76 9. 80 9.S3 ft ft >-/ a c © d o o p ft © ft p d © © p © Pi P d C D © ft ft 7-00 7.12 7 00 © d d •H 0 s -P d ft ,□ © 3 Pc *sj Pi 7.00 7.12 7: i 5 7-30 7.31 7.1+2 7.I+5 7 . 1+9 7 . 5 ^ 7-59 7.68 7.69 7.72 7.85 7.83 7-89 7-98 7-98 8.01 8. 13 7.29 8.11 8.28 o p G. © ft o o i Pi © ft © © p r p. o p © d o p Pi © Pi d © pi Vo ft p •© -l- d © o u 7-00 7.00 7 . 1+5 7.1+6 7.00 7.1+1+ 7.82 £.19 7.82 7-82 S . 11 8.36 S.bC s . 13 8.1+8 8-78 S . 98 9.13 £.83 S.Q8 8.39 S. 61 8.82 8.99 s . 32 9.30 9.11 9.12 s . 51 9 . 1+0 9.26 9.26 s . 70 9.33 9.36 9.35 9 . 0 s 9-78 9-58 3 .S0 9.37 9.98 9 ; 79 9.65 9. 61 10.13 9.35 9 . SO 10.13 9.93 9.67 9.82 9.85 1 C.00 10. ll+ s . 17 5.31 8.1+2 S . 1+8 S.SO 8.85 9.13 9.18 9.1+2 9 . 1+5 9.67 9.81+ © d -l+oThese results agree to some extent with those of Chamot and Georgia (12), who d i v i d e d the p e p tones w h i c h they tested into three groups as follows: slightly alkaline, slightly a c i d and highly acid. These authors found Witte's (1912 ), B a c t o - p e p t o n e a n d Proteose-pep tone to h e slightly alkaline. The p r e ­ sent w o r k shows Witte's (I9I2) a n d Froteose-peptone to h e slightly alkaline, hut the sample of Bacto-peptone tested was v e r y slightly acid, p H 6.97. Chamot a n d G e o r g i a f ound Armour's and Parke, to he only slightly aci d (between p H " J . 0 0 Davis and Company's peptones and 5 *SO) while the present re­ sults show A r m o u r ' s in this group at a p H of b . 3 1 a n d Parke, Company's in the strongly a c i d group w i t h a p H of 5. 20. third group (highly a c i d - p H 5 .00 a n d above) a n d S t e a r n ’s (pH ^+.7 5 ) • Davis and Chamot and Georgia's includes Fairchild's (pH U.7 S) ^h© p r e s e n t w ork places these two peptones in the same general grouping, hut the p H values o b t ained were slightly higher, pH 5*51 a*111 5 *9 1 * respectively. The r e l ative b u f f e r i n g ability of the peptones is best shown in Tables 9 11, where the p e p tones w e r e a d j u s t e d to p H 1 . 0 0 be f o r e the addition of aci d or alkali. The peptones are l i s t e d in b o t h tables in the order of their and 20 the p H reached after H C 1 or of the a d j usted peptone solution, or 0 .01 K N a O H was added to 20 ml. b y the V a n Slyke b u f f e r index c o m p u t e d from the results. of b u f f e r i n g v alue p l a c e s Vegetable peptone Ko. H- first, Conmany's second. Liver peptone ( 1 ) third, ml. of 0 .01N decreasing b u f f e r ability as shown b y This criterion Parke, Dpjvis and etc. , when acid was added. 10 also shows that the p r e p a r e d pepto n e s are, against acid than are the commercial peptones. Table as a group, better buffers The leading ten peptones include seven p r e p a r e d peptones a n d three commercial brands. n o t e d that a few of the highly aci d peptones such as Parke, It is to be David and Company's. S t e a r n ’s, F a i r c h i l d ' b and P f anstiehl appear to be good buffers against acid, while other h i g h l y acid peptones such as Baker's, Merck's and Cenco are -U l- re la tiv e ly poor buffers against acid. VJhen a lk a li is added to the adjusted peptone solutions the results (Table l l ) show that Bacto-tryptose is the best buffer, and Vegetable peptone No. followed by Armour* s The commercial peptones appear to be much b ette r buffers against a lk a li than are the prepared peptones as shown by the fact that seven of the leading ten peptones are commercial products. Here again some of the more acid peptones such as Fairchild’s and Ghaissiang appear to have a much b e tte r buffer action against a lk a li than do others in the same group, such as Baker's and Cenco. The fact that such highly acid peptones as Baker's, Cenco, and Merck's show no buffering action against either acid or a lk a li a f te r they have been adjusted to pH 7*00 indicates that neutralization does not affect th eir buffering capacity. This in turn would seem to indicate that the neutralization of peptones, as is practiced in preparing media for bacteriological use, has l i t t l e effect on the buffering a b i l i t y of a peptone. as would be expected, Thus i t may be concluded th at, the buffering action of a peptone is due to inherent properties of the peptone, rather than to the acid or a lk a li used in neutralizing the peptone. As a general rule, when unadjusted, the pep­ tones having a high i n i t i a l pH are b e tte r buffers against acid than those having a low i n i t i a l pH, and vice versa. However, when the peptones are placed on an equal basis by adjusting the solution to pH 7*G0, this relationship i s l o s t . These resu lts (Tables 8,9.10 and- H) show that, in general, the i n i t i a l pH of a peptone has no relation to i t s a b ility as a buffer, e ith e r against acid or base, if the peptone has been neutral­ ized. Figure U shows the buffer curves obtained when the pH is plotted against the ml. of acid or a lk a li added tc the peptone solution. Since 20f Figaro 4 ul B u f f s r a a r r t t o f on* p * r o * n t pop* on* * o l n t Io n * 161 B ra in p epton* (2 ) B aot o - t r y p t 0 * * V e g e t a b l e p e p t o n e N o. 2 L iv e r peptone (2 ) F a i r c h i l d ’ * pepton* a s '14 16 18 20- 3 .5 4 .0 4 .5 5 .0 5 .5 6.0 7 .0 8. 0 i 9 . 5 1 ' -1+2tne curves for a l l peptones, with one exception, show the same general trend, only a few typical examples are presented. The exception is brain peptone ( 2 ), which is included in Figure H. These curves indicate that the peptones have the le a s t buffering action between pH 5 *5 snd 7*5* This is unfortunate, since i t is in this range that buffering action in oacteriological media is most desirable. Bronfenbrenner, DeBord and Orr (11) studied the buffer capacity of Difco, Proteose, V/itte, Aminoid, Fairchild, Roche and Armour peptones. These authors found the greatest buffer action between pH 8 and 9 and the least between pH h and 5, which is somewhat lower than the resu lts obtained in the present work. When used in bacteriologies,! media, the a b ility of a peptone to buffer a solution against the production of acid is more important than i t s a b i li ty to buffer against the production of a lk a li, since the presence of carbohydrates in many media usually results in the formation of acids. Also the buffering a b ility of a peptone between pH 7*00 and 6.00 i s more important than i t s buffering a b i l i t y between pH 5-00 and b.CO, because i t is desirable to maintain n eu trality for as long a period as possible- Y/ith these facts in mind, the approximate amount of 0.01H HC1 needed to change a one per cent peptone solution from pH J . O Q from Table 9. to pH 6.00 was determined The results are given in Table 12. This table shows that under the conditions of the experiment, Brain peptone (2 ) best buffer against acid, followed by Armour's peptone. that, while a prepared peptone is best, is by far the I t also shows the buffer values of the prepared peptones as a group is slightly less than that of commercial peptones, since six of the leading ten peptones are commercial products. When the amount of acid needed to change a one per cent peptone solution from pH 7 .00 to 6.00 i s compared with the amount needed to change -113Table 12. Peptone 1. 2. 3H. 5. s. 7. s. 9. 10. li. 12. 13. lH . 15. 16. 17. IS. 19. 20. 21. 22. 23. 2b . 2526. 27. 28. 29. 30. 3132. 33. 3U. 35* 36. 37. 3 s. 39. Uo. Approximate amount of 0.01N HC1 needed to bring a one oer cent peptone solution (adjusted to pH 7 . 0 ) to pH 6.00 and 5*00. ML. to bring to pH 6 . 00 _____ Brain peptone (2) Armour peptone Bacto-tryptone Wilson peptone **G” Vegetable peptone No. 5 Heart peptone (2) Pork peptone Bacto-tryptose Pfansteihl peptone Bacto-pro tone Spleen peptone ( 2 ) Armour* s special peptone Heart peptone ( l ) Vegetable peptone No. H Wilson peptone nCBn Beef peptone F a irc h ild 's peptone Witte peptone ( I 9 U0 ) Chaissiang peptone Witte peptone (1912) Brain peptone ( l ) Parke, Davis peptone Liver peptone ( l) Liver peptone ( 2 ) Bacto-neopep tone 3 & A Albumin peptone Spleen peptone ( l) Pro teose-peptone Proteose-peptone No. 3 Vegetable peptone No. 1 Proteose-peptone No. 2 Vegetable tryptone Vegetable peptone No. 2 Vegetable peptone No. 3 Merck peptone Steam 's peptone E & A Meat peptone B aker's peptone Bacto-peptone Cenco peptone 9 . 5O 6 .50 6 .00 6 . co 6 . co 6 .0 0 6 .0 0 5 .90 5 .6 0 5 .6 0 5.5 0 5 .50 5 . Ho 5 .0 0 5 .00 5 .0 0 H. 9 5 U. 9 0 U. 9 0 U. 9 0 U .so U. 7 5 U. 6 0 u. 60 U. 20 U. 0 0 U. 0 0 U .0 0 3-95 3-90 3-75 3 .0 0 3 .0 0 3 .0 0 3.00 2 .90 2 .8 0 2.75 2 .5 0 2 .0 0 1. 2. 3. U. 5. 6. 7. 8. 910. 11. 12. 13. lU. 15. l6 . 17. 18. 19. 20. 21. 22. 23. 2U. 25. 2o27. 28. 29* 30. 31. 32. 33. 3 U. 35* 36. 37. 38. 39. HO. Pep tone M L. to bring to pH 5 .00 Brain peptone ( 2 ) Vegetable peptone No. U Liver peptone (1) Parke, Davis peptone Pork peptone Heart peptone (2) Vegetable peptone No. 5 Wilson peptone "C" Bacto-tryptone Armour peptone Brain peptone (1) Fairchild's peptone Pfansteihl peptone Armour's special peptone Heart peptone ( l ) Spleen peptone (2) Vegetable tryptone Witte peptone ( I 9 U0 ) Liver peptone (2) Wilson peptone "CB" Beef peptone Vegetable peptone No. 1 Chaissiang peptone Bacto-protone Bacto-tryptose Vegetable peptone No. 2 Vegetable peptone No. 3 3 & A Albumin peptone Witte peptone (1912) Spleen peptone (l) Proteose peptone No. 3 Proteose peptone Bacto-neopeptone Stearn's peptone Proteose-peptone No. 2 Bacto-peptone Merck's peptone Cenco peptone E & A Meat peptone Baker's peptone 17.00 16.00 16.00 16.00 1 U. 0 0 1 U. 0 0 1 U. 0 0 13.80 13.00 1 3.00 12.00 1 2 . 00 12.00 12.00 12.00 11.60 11.30 11.20 11.00 11.00 11.00 10.60 10.60 10.60 1 0 . Uo 1 0 .0 0 1 0 .0 0 10.00 9.5 0 9 .00 9 .00 S. 75 0.60 8 .00 7 .6 0 7r . 0_0_ 0 .0 0 6 .0 0 5 .0 0 u .6 0 the pH from 6.00 to 5 .00. the resta te (Table 1 3 ) show that Brain peptone (2) maintains i t s position as the best Buffer against acid. This table also shows that a peptone which is a good buffer between pH 6.00 and 5.00 is not necessarily a good buffer between pH 7.00 and 6.00, and vice versa. Thus the re su lts show that peptones vary considerably in th e ir buffer action between any two pH values, and that there is no correlation between th e ir buffering a b i li ty at two different sets of pH units. These resu lts support the findings of Bronf enbrenner, DeBord and Orr ( l l ) . Attempts to correlate the buffering a b ility of the peptones with their chemical composition f a ile d to show any relationship between the buffer index and the amount of any of the various nitrogen fractions. This indicates tha.t the buffering capacity of a peptone is not due to any one nitrogenous component, but rather i t is due to some component not analyzed or, more lik e ly , to the way the various components are combined in the peptone molecule. The ash content of a peptone should have some influence on the buffering a b ility , of calcium or sodium. especially i f i t contains phosphates The presence in a peptone of a small amount of such s a lts as these would resu lt in a b ette r buffer than i f a large amount of ash consisting of neutral s a lts , such as llaCl, was present. Summary Chemical analysis was made of large numbers of peptones for total nitrogen, t o t a l , primary and secondary proteose nitrogen, peptone nitrogen, free ammonia nitrogen, content. free amino acid nitrogen, amino nitrogen and ash The r e s u lts obtained show that: (l) The commercial and prepared peptones are f a i r l y uniform as to to ta l nitrogen content. table peptones are, as a group, (2) The vege­ lower than the commercial peptones in i - 1+ 5 - ( 3 ) The peptones, "both commercial and prepared, vary to ta l nitrogen. widely in th eir content of the various nitrogen fractions. (U) The commercial and prepared animal peptones, are, with the exception of Bacto-protone, higher in peptone nitrogen than any other nitrogen frac­ tion. (5) The amino nitrogen as determined by Sorenson's foriaol t i t r a t i o n consistently gave s lig h tly lower re su lts than the same fraction as deter­ mined "by Van Slyke's method. (6; The vegetable peptones prepared from hydrolyzed com gluten are higher in free amino acid and amino nitrogen than are the commercial and prepared peptones. ammonia nitrogen i s low in a l l peptones. (7) The content of free (S) The sum of the nitrogen fractions does not equal the figure for to ta l nitrogen, indicating that more than one nitrogen fraction was determined "by the same analysis, or that not a l l of certain forms of nitrogen is "being determined. ( 9 ) The commercial peptones gave positive "biuret, Mi11on, Bosenheim, Sakaguchi, ninhydrin, xanthoproteic and Bleitmann te s ts . They were a l l negative to the Molish reaction. (10) 'The prejjared animal peptones, with the exception of the two spleen andtwo liv e r peptones, were positive to a l l except the Molish reaction. The four exceptions were positive to the Molish reactions. (ll) The four vegetable peptones prepared from hydrolyzed corn gluten gave positive Sakaguchi, ninhydrin, xanthoproteic, Fleitmann and Molish reactions, and negative Rosenheim and biuret tests. peptones number 1,2 and 3 were positive to Millon's te s t, Vegetaole while number 1+ was negative. Vegetable peptone No. 5 and vegetable tryptone were positive to a l l t e s t s . (12) The i n i t i a l pH of a one per cent peptone solution may be used as the basis for roughly dividing the peptones into four groups as follows; alkaline, pH J . 0 0 to 7 »60; neutral, pH 7*^0; slightly acid, -4 6 pH 6.00 to 7.00 and. strongly acid, pH 4.89 to 6.00. of a l l peptones varies widely. ( 1 3 ) The i n i t i a l pH (l4) When the buffer index, obtained a fte r adding 20 ml. of 0.01K HC1 or NaOH to a neutralized peptone solution is used as the c r ite rio n of buffering a b ility , the prepared peptones, as a group, are shown to be b ette r buffers against acids and the commercial peptones to be b e t te r buffers against bases. When the amount of 0 .C11T KC1 needed to change a peptone solution from pH S.CO to 7 .00 is used as the c rite rio n , the commercial peptones are shown to be sligh tly superior to the prepared peptones as buffers against acids. is the best buffer against acid. ( 1 3 ) Brain peptone (2) (l4) The peptones, as a general rule, show the le a s t buffering action between pH 5 . 5 and 7 . 9 . BACTERIOLOGICAL ANALYSIS O F PREPARED FEPTOiJSS The methods of determining the efficacy of bacteriological peptones to support the growth of bacteria are legion. solve each problem as i t aris e s. A new te s t may be devised to The tests used to determine the a b i li ty of the prepared peptones to support the growth of bacteria include the determination of the rate of growth of E. c o li; plating of samples of raw milk; growth and gas production by coliform organisms found in naturally contaminated water and the testin g for the growth of certain pathogenic organisms. Whenever possible standard or recommended media were used as controls in these experiments. Determination of Rate of Growth of E. coll. The medium used for the determination of the rate of growth of S. coli consisted of five-tenths per cent peptone end five-tenths per cent HaCl a t a pH of 7 .00. Fifty ml. of this medium was inoculated with 4 -U 7one ml. of a. 1 : 1 , 0 0 0 , 0 0 0 dilution, of a twelve hour broth culture of S. coll and incubated at 37°0. After inoculation the medium was shaken to distribute the organisms and plated out immediately and at two hour intervals fo r the f i r s t hours. twelve hours, and again at the end of 2 k and Us Explicate p la te s were poured for each peptone tested. The plating medium used was as follows: Bacto-peptone................................... 0 - 5* 1 Bacto Beef-extract......................0*3/^ UaCl . «• »• «». • • • • • • • • • . . *. • . 0. 3/0 Agar............................................. 1-5/d Water..................................................1000. 0 ml. pH.................................................................7 .OO The p lates were incubated for Us hours at 37°C- and the colonies counted with the aid of a Qpebec Colony Counter. The agerage of the results of five experiments using four vegetable peptones prepared from hydrolysates of corn gluten in comparison with Bacto-tryptone, tryptose and peptone is given in Table 1 3 . Figure lU shows the curves obtained when the log of the numbers of b ac teria was plotted against the time in hours. The vegetable peptones used in these experiments were decolorized with Iforite "A". An examination of the results given in Table 1 3 shows that vegetable peptones number 1,2 and 3 give b e tte r growth of E. coli during the f i r s t six hours than do the Difco products. After six hours Bacto-tryptose is b e tte r than any of the vegetable peptones, and at the end of 12 hours the Difco peptones gave b e tte r growth than did the vegetable peptones- Reference to Tables 5 and 6 shows that the vegetable peptones are higher in free amino acid nitrogen than are any of the Difco products. This seems to indicate that a peptone containing a large amount of this type of nitrogen will i n i t i a t e a fa s te r rate of growth, resulting in a shorter lag phase, will a peptone which contains a smaller amount of free amino acids. other hand, than On the the presence of proteose and/or peptone nitrogen in a peptone makes i t capable ox carrying out a more extended growth, as is shown by ^ -b s- Time in hours Bactotry p to s e Bactotryp tone Bactopeptone B a c te ria p e r ml. Vegetable Vegetable peptone No. 1 peptone No. 2 Vegetable peptone No. 3 13 23 19 20 19 19 18 2 51 5S 6b 69 7^ 79 1+5 u 590 810 1355 I5U9 1636 i960 393 6 ib,6oo 19,300 2C,bOC 20,800 21,500 23,200 8,800 s 283,000 503,000 1,550,000 53b,000 536,000 1, 1*87.000 63,000 10 2 , 280,000 3,770,000 u,h70,000 2,5^0,000 0 00 0 b, COO, 000 58 b, 000 12 5b,COO, 000 66,000,000 77,000,000 33,000,000 U3,000,000 1*7,000,000 7,000,000 2b 233, 000,000 39^,000,000 675,000,000 189,000,000 2U0,000,000 25b,coo,000 b2,000,000 iis 229,000,000 333,000,000 351,000,000 102,000,000 185.000,000 205,000,000 76,000,000 l-N, 0 Taole 13* Comparison of the r a t e of growth of 3 . c o li in broth prepared from fo u r vegetable peptones and three Difco p e p to n e s .• Average of fiv e t r i a l s . j l Vegetable peptone No. b -119the Difco products. These re su lts support the findings of Hettger, Bernian and Sturge (UO), who found that the presence in a medium of free amino acids is desi able or even necessary before many organisms can i t i t i a t e growth. Some organisms cannot u t i l i z e the more complex nitrogen fractions u n til growth has been i n i t i a t e d by u t i l i z a t i o n of free amino acids. However, once growth has s ta rte d the bacteria will produce enzymes to break down these more complex fractio ns. rule, Hartley (2^) has pointed out that, as a general the more complete the digestion of the protein, growth of certain organisms. amino acids, the b ette r the Gordon and M ’Leod (21) found that certain tryptophane in p a rtic u la r, are toxic to bacteria. Since the tryptophane in the sanples of hydrolyzed corn gluten has been destroy ed by the hydrolyzed corn gluten has been destroyed by the hydrolysis treatment, the peptones prepared from these sartples may be less toxic to S. coli than the commercial peptones tested, in which tryptophane is present as shown by the positive Kosenheim te s t. The positive Molish te s t shows the presence of carbohydrates, probably glucose, in the vegetable peptones, which would also aid in the growth of the organisms during the early stages. Figure lM - shows that the growth rates of the three vegetable peptones and the three Difco peptones are very similar, table peptone ho. U is much in fe rio r to the others. and that Vege­ Soth Table lp and Figure lH show that the vegetable peptones Ho. 1,2 and 3 and the Difco pe'otones gave maximum numbers of ID . coli in 2 hours, and then started to recede, while vegetable peptone iio. U did not show maximum growth u n t il US hours or l a t e r . This was doubtless due to the removal of leucine and tyrosine as previously stated. This would also indicate - t f .■ . ... |. ■i: . i q - : -;: ; r F : : . : i :.: i . ■• t .. .-.-.t-.t Flgur# 5 Growth carras for £ . o flll In broth prtpartd from Ytgatabla and Difoo ptptooes Ltgtndi Baoto-poptont Baoto-tryptons Baoto-tryptos# Ttgttablt ptpfcona Ho* 1 Vegatablt paptont Ho. 2 Vtgatablt poptont Ho. 5 Vagatabla poptont Ho. 4 2 4 6 6 10 12 24 Tlat In honrt 49 -5 0 that one or "both of these amino acids were necessary for growth during the i n i t i a l period. Plating of Samples of Haw Milk. The medium used in te stin g the a b ility of the various prepared peptones to grow the organisms found in raw milk was Standard J-hlk Agar in which the tryptone was replaced with one of the prepared peptones. Standard r-iilk Agar (51) was used as the control. The method was as follows; A 1:1,000 dilution of the raw milk sample was prepared and shaken for at least five minutes in a shaking machine to break up clumps of bacteria and to thoroughly distribute the organisms in the d ilutio n water. Duplicate plates of this dilution were poured for each peptone to be tested. The plates were incubated at 37° C* for HS hours and the colonies were then counted with the aid of a Quebec Colony Counter. Samples of milk showing more than J>C 0 or less than 3© colonies per p la te were discarded. Table 1*4- gives the resu lts obtained from 100 samples of raw milk tested with vegetable peptones No. 1,2 and 3» as compared with Bactotryptone- Table 1 5 gives the re su lts obtained with undecolorized vegetable peptones No. 1, 3 5 and vegetable tryptone as compared with Bacto-tryptone and tryptose. Table l6 gives the results obtained with 5® samples of raw milk tested with vegetable peptone No. 3 and 5 and vegetable tryptone as compared with Bacto-tryptose. Tables 17 and IS give the re su lts obtained with 100 samples of raw milk tested with eight animal peptones as compared with Bacto— tryptone. Discussion The average resu lts obtained when comparing the vegetable pep­ tones with Bacto-tryptone and try p to se (Tables lU, and 15 and lo ) show -5 1 - Coraparison o f t h r e e v e g e t a b l e p e p t o n e s and B a c t o - t r y p t o n e f o r grow th o r o rg a n ism s i n samples of raw m ilk. Vegetable peptone No.l 1 2 I 5 6 7 s 9 10 n 12 13 lU15 16 17 IS 19 20 21 22 23 21+ 25 26 27 28 29 30, 31 32 33 3 *+ £ 3° 37 38 39 Uo 1+1 hz 12 U5 U6 h i Us U9 RO 3l+,000 U 0 .0 0 0 21+5 ,0 0 0 105.000 Ui+,000 78,000 98 ,0 0 0 UU.ooo U-U.ooo 50.000 1 0 2 .0 0 0 57.000 33,000 77.000 70 000 37;000 37.000 18 U,000 58.000 33.000 53.000 31.000 53.000 21.000 120,000 U2.000 Ul+,000 19.000 1 0 2 ,0 0 0 113.000 107.000 lUS.OOO 1 8 6 .0 0 0 1+1,000 s6 ,o o o U o .o o o 8 1 .0 0 0 68.000 72.000 2 0 .0 0 0 30.000 52.000 31.000 37.000 1+1,000 50.000 30.000 251+.COO 272,000 31.000 75 .0 0 0 Bacteria per ml Vegetable Vegetable peptone Ho.2 peptone IJ0.3 3U,000 1+1,000 1+1,000 1+3,000 112,000 11+5,000 98,000 112.000 8l+,000 63.000 70.000 8 0 .0 0 0 59.000 69,000 1+9 , 0 0 0 1+1,000 '4 1 ,0 0 0 1+1,000 32.000 36.000 78,000 61.000 93,000 1 0 2 .0 0 0 1+0,000 3 U ,o o o 65.000 87.000 r a A n n • jy f vw W (y w v V 1+5,000 9Uf ooo 25.000 59.000 1 1 2 ,0 0 0 lUU,000 5 8 . 000 62.000 1+1+,000 21.000 8 1 . 000 52.000 61.000 1+1,000 8 8 .000 55.000 39.000 30.000 1 2 8 ,0 0 0 12l+,000 1 + 6 ,0 0 0 1+6,000 60.000 1+2 ,0 0 0 30.000 22.000 1 0 8 ,0 0 0 5 U ,o o o 1 2 0 ,0 0 0 ui+,000 130,000 1 2 8 ,0 0 0 178,000 1 8 2 ,0 0 0 67,000 90,000 3 U ,o o o 78.000 94.000 78.000 2 1 .0 0 0 31.000 6 U ,o o o 3 0 .0 0 0 40 .0 0 0 51.000 1+9,000 3 7 .0 0 0 258.000 301.000 36.000 77. o 01OO 2 1 1 ,0 0 0 1 9 7 .0 0 0 8 2 .0 0 0 97.000 1+3,000 87.000 Bactotryptone 1+7 , 0 0 0 l+l+,000 1 2 6 .0 0 0 1 0 9 , coo 6 5 . 000 5 0 .0 0 0 5l+,000 1+2 , 0 0 0 1+1 , 0 0 0 2 6 .0 0 0 1 1 9 .0 0 0 7 6 .0 0 0 3 3 .0 0 0 ll+u, 000 6 3 .0 0 0 U 3 ,0 0 0 2 3 .0 0 0 9 1 .0 0 0 1+3,000 6 6 .0 0 0 5 5 .0 0 0 1+ 3 , 0 0 0 5 3 .0 0 0 33. 0°0 128,000 1+3,000 1+0,000 28.000 7 0 .0 0 0 1 1 6 .0 0 0 1 1 9 .0 0 0 2 2 1 .0 0 0 i 6 U ,o o o 81 .0 0 0 9 1 .0 0 0 83.000 8U .000 95.000 a ll, 000 8 3 .0 0 0 8 9 .0 0 0 3 6 .0 0 0 22,000 3 U ,o o o 6s,000 3 6 .0 0 0 60.000 6 U ,o o o 53.000 39.OCO 2 8 2 ,0 0 0 3 7 7 ,0 0 0 3 9 ,oco 8 2 , ICO 33.000 6 s ,o c o 3 8 .0 0 0 u s , 000 57.000 3 2 .0 0 0 1+6.000 2 7 8 .0 0 0 3 2 7 .0 0 0 3 5 .0 0 0 78.UOO OOOOo o OOo o o o o o o o a r— o o o o o o o o o pirf 2 s is H H s i H is $ s R g g f t H H HCJCU ^ ^ r l iv ^ rOtu wVrnijoHOBOin &vo mto t* to 3 ^ o ss 5 ^ ^ r* -^ ^ ^ cn ^ p O O O O Q O O O O O O O ^ ^ ^ O O O O Q O o o o o o o o o ; ci d o © -P hO p, u Q) H H rl ^ *g ^ C J ^ K S^ VO to o o o in Jo 0) -P o o n fp o < d O to &O § M H fO H ^ ^ ^ ^ ^ H O J ^ t ^ ^ ^ ^ ^ " O? H ^ CJ ^ ^ ^ ^ CD CD > PJ P- u © 4> OJ O d m o o poooo8oooooooo§oocooo8oooo?oogoooog oO oO oO oO oO O Oo Oo Oo Oo Oo Oo oO oO oO <* o o o o o o o o o o o o 6 6 6 d J >o ^ ^ 8 p >8 § p >8 ^ >o o 8 o B ^ i % oj ir\ 0) K H n © © P +» o Table l£ S ftw '& fS J? R S 'k w to S 'S w H H rl OJ H H H OJ OJ O O O S o OOo OOOOOoOooQooOooOo oOo o Q O O O O O O Q OJ O O O O Q O O ^ vo to O O O O O O O Q O O O p O Q O O O O O O O O O O g O O oogooooooooogygggggoooo6oo8oooo8ooooooooo ri H H H H '•O o’ to rl rl a Vi © OH P KVd- ITAVO I-CO CTNO H OJ t ^ t to a oj h ^ H v S ' o P ^ O ^ H OJ cvih^ 5 w ^ ' - n w f«0 P- a) © f> PJ ^ lvJr^ CD +> lU. ( Continued) i OJ to £ \0 J-M CTc O H CM J£vO £ - CO £ O © © ha f © CQ u u © ©o ►o Si < *!H $ - Thble l p . Sample -Humber 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Comparison, o i f o u r u n d e c o l o r i z e d v e g e t a b l e p e p t o n e s w i t h B a c t o t r y p t o n e a n d t r y p t o s e f o r g r o w t h o f o r g a n i s m s i n s a m p l e s o f raw m ilk. Vegetable Vegetable pept one Hp. 1 peptone Ho. 3 23,000 44,000 67,000 258,000 29,000 73,000 30,000 60,000 27,000 lb , 000 38,000 4 2 ,0 0 0 51,000 45,000 1 4 ,0 0 0 29,000 17,000 43,000 4 6 ,0 0 0 21,000 69,000 4 1 .0 0 0 4 ,000 1,000 6,000 2b 2 4 ,0 0 0 27 28 29 30 31 32 33 34 35 36 37 4,00c 5,000 36,000 107,000 3,000 15,000 1,000 3,000 38 39 4o 4i 42 43 4 b- 4 ,0 0 0 45,000 33,000 37.000 4 ,0 0 0 7,000 19,000 10,000 5,000 6,000 3,000 6,000 1 9 ,0 0 0 37,000 88,000 2 5 s , 000 1+7,000 173.000 3 0 .0 0 0 66, oqc 1 6 2 .0 0 0 2 7 .0 0 0 5 2 .0 0 0 24.000 46.000 35 *so o 14.000 3 1 .0 0 0 20.000 4 3 .0 0 0 5 0 .0 0 0 2 4 , 000 73.000 U3 ,000 1.000 5 .0 0 0 18,000 4 .0 0 0 6 .0 0 0 12,000 1+3,000 1 7 0 ,0 0 0 1+1+, 000 31+, 000 1.000 9,000 11,000 1+1,000 7 3 .0 0 0 6 5 .0 0 0 s.ooc 1 7 .0 0 0 21.000 21,000 2,000 4 1 .0 0 0 50 6,000 8,000 32,000 5 .0 0 0 2 3 .0 0 0 9 .0 0 0 7.000 1 3 .0 0 0 57.000 Average 30,400 1+7,360 45 46 47 4s 49 53 - 4 ,0 0 0 Bacteria per ml. Vegetable ’age table Bactopeptone ho, t ryptone____tryptone 21+.000 3 8 .0 0 0 7 4 .0 0 0 3 6 7 .0 0 0 5 9 . 0G0 3 0 4 .0 0 0 ’ 5 2 ,0 0 0 6 6 .0 0 0 5 1 , o co 7.000 7 3 .0 0 0 3 1 .0 0 0 5 4 .0 0 0 yj , www 7 5 .0 0 0 3 6 .0 0 0 5 1 .0 0 0 5 2 .0 0 0 4 8 .0 0 0 31,0*00 8 3 .0 0 0 4 2 .0 0 0 66. 0 0 0 3 2 .0 0 0 2 4 .0 0 0 3,000 1 7 .0 0 0 3 1 .0 0 0 5 2 .0 0 0 221,000 4 6 .0 0 0 9 7 .0 0 0 2 8 .0 0 0 3 0 .0 0 0 4 2 .0 0 0 4 1 , oco 87.000 1 0 2 , OCO 2 5 .0 0 0 2 4 .0 0 0 3 6 .0 0 0 6 5 .0 0 0 2 6 .0 0 0 1 5 4 ,0 0 0 2 9 , coo 4 7 .0 0 0 2 6 .0 0 0 20.000 5 7 , 00 c 5 i,o c o 6 2 ,0 0 0 23,000 42.000 87.000 209,000 36,000 155,000 27,000 56,000 135,000 21,000 102,000 3s, oco 51.000 — » / >A A 9V' —W 21.000 32,000 23,000 57.000 57.000 41.000 71.000 43.000 52.000 30.000 80 .0 0 0 3s,000 14.000 91.000 51.000 198,000 58.000 99.000 21.000 44.000 38.000 55.000 £7,000 108 ,000 33.000 29.000 34.000 8 2 .0 0 0 25.000 41 .000 12 , CCC 34.000 27.000 27.000 12.000 7 r\ Oa a 58,100 Bactotryptose 30,000 45,000 96,000 369,000 63,000 315,000 43,000 60,000 113,000 29,000 1+6,000 86,000 321,000 4 6 ,0 0 0 278,000 74,000 67,000 156,000 28,000 3 7 ,0 0 0 110,000 45,000 51,000 cfJf o/wv* -rc 23,000 39,000 51,000 4 6 ,ooo 47,000 35.000 4 4 ,000 121,0a 46 COC l+s, 000 )~ 1-f79W T w 27,000 43,000 62,000 52,000 58.000 42,000 28,000 26,000 30,000 76,000 25,000 75,000 46,000 136.000 4 6 ,0 0 0 74,000 4 4 ,000 53,000 25,000 8 2 ,0 0 0 35 ,000 47,000 91,000 51,000 1 8 2 ,0 0 0 24,000 60,000 98,000 17.000 23,000 35,000 51,000 75,000 9 7 ,0 0 0 64,000 28,000 1 13,000 20,000 37,000 33,000 4 4 ,0 0 0 55,000 92,000 34,000 37,000 23,000 3 7 ,0 0 0 30,00c 25,000 86,000 39 ,coo 104 ,0 0 0 33.COO 30,000 36,000 42.000 8,000 29,00c 21,000 2 0 . OCO 17,000 74,000 64,000 43,000 76,000 57.200 70,000 - Table lo. Sample number 1 2 5 6 7 8 9 10 n 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 $ 35 36 37 38. 39 40 41 42 ^3 44 45 46 ^7 48 49 50 Average 54 - uoraparison oi t h r e e v e g e t a b l e p e p to n e s and B a c to -try p to n e ao r g r o w t h o f o r g a n i s m s i n s a m p l e s o f r a w m i l k . Bacteria per ml. V eg eta b le D e n t o n e H a. ^ 6 6 ,0 0 0 3 3 .0 0 0 3 7 .0 0 0 1 2 9 ,0 0 0 1 2 7 ,0 0 0 1 1 9 .0 0 0 1 1 8 ,0 0 0 1 2 4 , 000 38 , 000 1 4 4 ,0 0 0 3 2 ,0 0 0 4 2 ,0 0 0 2 5 7 ,0 0 0 1 5 7 ,0 0 0 3 5 .0 0 0 8 7 .0 0 0 2 7 ,0 0 0 1 7 ,0 0 0 1 9 6 ,0 0 0 1 1 9 ,0 0 0 1 2 5 ,0 0 0 1 0 6 ,0 0 0 4 2 ,0 0 0 1 1 4 ,0 0 0 1 5 ,0 0 0 2 4 7 ,0 0 0 1 9 1 ,0 0 0 9 3 .0 0 0 1 4 9 ,0 0 0 2 4 0 ,0 0 0 4 2 ,0 0 0 4 2 ,0 0 0 4 6 ,0 0 0 4 3 ,0 0 0 4 7 ,0 0 0 3 2 ,0 0 0 6 1 ,0 0 0 2 4 8 ,0 0 0 4 2 ,0 0 0 1 8 ,0 0 0 9 8 .0 0 0 1 2 4 ,0 0 0 9 9 ,0 0 0 2 4 5 , OCO 1 5 2 ,0 0 0 3 2 ,0 0 0 1 1 6 ,0 0 0 1 1 9 ,0 0 0 3 0 .0 0 0 8 2 ,0 0 0 9 3 ,6 0 0 V eg eta b le ----- p e n t o n e llo. R 2 1 ,0 0 0 6 6 .0 0 0 41 .0 0 0 1 3 9 .0 0 0 2 1 8 ,0 0 0 1 6 9 ,0 0 0 1 2 4 , 000 1 1 8 ,0 0 0 2 5 ,0 0 0 1 9 0 ,0 0 0 9 5 .0 0 0 48 ,0 0 0 3 0 6 .0 0 0 1 4 4 ,0 0 0 8 3 ,0 0 0 9 5 ,0 0 0 3 0 ,0 0 0 3 6 ,0 0 0 2 7 4 ,0 0 0 1 2 2 ,0 0 0 1 2 5 ,0 0 0 1 2 2 ,0 0 0 8 2 ,0 0 0 1 1 2 ,0 0 0 1 0 ,0 0 0 2 3 9 ,0 0 0 1 9 0 ,0 0 0 9 4 ,0 0 0 1 7 2 ,0 0 0 2 1 0 ,0 0 0 4 r .0 0 0 5 6 ,0 0 0 4 4 , 000 4 3 ,0 0 0 5 3 .0 0 0 4 7 ,0 0 0 9 2 ,0 0 0 2 6 8 ,0 0 0 4 6 ,0 0 0 3 0 ,0 0 0 1 0 9 ,0 0 0 1 3 1 ,0 0 0 9 4 ,0 0 0 2 4 6 ,0 0 0 2 1 4 ,0 0 0 4 5 , 000 1 1 2 ,0 0 0 5 9 ,0 0 0 1 2 , 000 6 5 . coo 1 1 1 ,2 0 0 V e g e t a b le 4 4 ,0 0 0 1 9 .0 0 0 3 2 .0 0 0 8 2 ,0 0 0 1 0 8 ,0 0 0 9 7 .0 0 0 1 2 3 ,0 0 0 1 1 6 ,0 0 0 3 1 ,0 0 0 1 9 5 . o co 3 3 ,0 0 0 4 l ,0 0 0 3 5 3 .0 0 0 1 2 2 ,0 0 0 7 0 ,0 0 0 8 0 ,0 0 0 3 2 ,0 0 0 1 6 , OCO 2 2 0 ,0 0 0 1 0 0 , 000 1 2 1 ,0 0 0 ? 4 000 3 3 ,0 0 0 1 0 9 ,0 0 0 5 .0 0 0 2 3 3 ,0 0 0 1 8 4 ,0 0 0 8 9 ,0 0 0 1 8 0 ,0 0 0 2 9 5 ,0 0 0 4 1 ,0 0 0 3 8 ,0 0 0 3 1 ,0 0 0 4 0 ,0 0 0 4 4 ,o o o 3 2 ,0 0 0 9 3 . o co 2 4 8 ,0 0 0 4 2 ,0 0 0 2 7 ,0 0 0 1 1 4 ,0 0 0 1 2 2 ,0 0 0 i o 4 ,o o o 2 1 5 ,0 0 0 1 6 0 , oco 4 4 ,000 1 1 9 ,0 0 0 8 2 ,0 0 0 2 3 ,0 0 0 9 4 ,0 0 0 9 9 ,2 0 0 B-jctot, rvn f nria 3 4 ,0 0 0 2 9 .0 0 0 3 5 .0 0 0 1 1 4 ,0 0 0 1 2 3 ,0 0 0 1 1 9 ,0 0 0 1 0 7 ,0 0 0 1 1 8 ,0 0 0 1 3 ,0 0 0 1 2 5 ,0 0 0 2 0 ,0 0 0 3 7 .0 0 0 2 6 1 ,0 0 0 1 3 8 ,0 0 0 8 7 ,0 0 0 6 6 ,0 0 0 2 1 ,0 0 0 1 4 ,0 0 0 1 2 8 ,0 0 0 1 1 1 ,0 0 0 1 2 0 ,0 0 0 9 2 ,0 0 0 3 5 .0 0 0 1 1 8 ,0 0 0 3 8 ,0 0 0 2 9 5 ,0 0 0 1 7 2 ,0 0 0 8 5 ,0 0 0 1 9 1 ,0 0 0 2 3 2 ,0 0 0 4 4 ,0 0 0 3 8 ,0 0 0 3 5 .0 0 0 4 4 ,0 0 0 4 2 ,0 0 0 3 5 .0 0 0 9 0 ,0 0 0 2 1 5 ,0 0 0 4 8 ,0 0 0 1 6 ,0 0 0 9 4 ,0 0 0 1 1 7 ,0 0 0 8 3 ,0 0 0 2 7 4 ,0 0 0 1 9 2 ,0 0 0 2 0 ,0 0 0 1 1 2 ,0 0 0 1 1 7 ,0 0 0 4 4 ,0 0 0 7 5 .0 0 0 9 5 ,3 0 0 - Table 1 7 . Sample number 1 2 3 U 5 6 7 8 9 10 11 12 1? 14 15 16 17 18 19 20 21 22 23 2b 25 26 27 28 29 30 31 32 33 3*+ 35 36 37 3s 39 bo Ul 1+2 U3 1+1+ 1+5 U6 1+7 1+8 1+9 50 Average 55- Comparison of prepared animal peptones v/ith Bacto-tryptone for growth of organisms in raw milk. Beef Spleen peptone________peptone ( l) 3 6 ,0 0 0 1+0,000 3 5 .0 0 0 3 2 ,0 0 0 2 2 ,0 0 0 31+, 0 0 0 1+1+, 0 0 0 1+7,0 00 33.000 37,000 38,000 1+3,000 8 8 ,0 0 0 1 3 9 ,0 0 0 1 2 1 ,0 0 0 1 5 0 ,0 0 0 U 2 ,0 0 0 3 9 ,0 0 0 1 3 6 ,0 0 0 1 0 7 ,0 0 0 3 5 ,0 0 0 1+1,000 2 1 ,0 0 0 3b ,000 3 5 .0 0 0 1+1+.000 33.000 5 1 ,0 0 0 5 1 ,0 0 0 514,000 6 s ,0 0 0 5 6 ,0 0 0 3 7 ,0 0 0 2 7 ,0 0 0 9 8 .0 0 0 8 6 ,0 0 0 3 1 ,0 0 0 1+3,000 121+, 0 0 0 1 1 1 ,0 0 0 1 0 2 ,0 0 0 1 1 2 ,0 0 0 1+3,000 5 1 ,0 0 0 28,000 3 0 ,0 0 0 6 8 ,0 0 0 6 5 ,0 0 0 3 6 ,0 0 0 35,000 1 2 7 ,0 0 0 1 2 5 , OCD 1 1 6 ,0 0 0 121+.000 1 1 2 ,0 0 0 1 1 2 ,0 0 0 82,000 7 5 ,0 0 0 148,000 1+8,000 5 5 ,0 0 0 6i+, 0 00 1 1 5 .0 0 0 lll+.OOO 118,000 n i+ .o o o 11+9,000 13!+. 0 0 0 4 6 ,0 0 0 5 0 ,0 0 0 HU,0 0 0 4-1+, 0 0 0 9 6 ,0 0 0 1+5,000 3 7 ,0 0 0 1+6,000 6i+, 0 0 0 1+2,000 1 1 0 ,0 0 0 1 0 1 ,0 0 0 108, OCO 1 0 5 ,0 0 0 1+3,000 3I+, 00 0 1+5,000 48,000 2 1+.000 2 3 ,0 0 0 3 5 ,0 0 0 3 2 ,0 0 0 5 5 ,0 0 0 1+0,000 1+0,000 1+1,000 6 6 ,0 0 0 6 2 ,0 0 0 2 7 ,0 0 0 2 0 ,0 0 0 5 2 ,0 0 0 3 9 ,0 0 0 6 2 ,1 0 c 6 6 ,0 0 0 Spleen jpeptone _(2 ) 40,000 5 5 ,0 0 0 37.000 1+6,000 1+3,000 3 s ,0 0 0 1 2 3 ,0 0 0 1 5 3 ,0 0 0 1+5,000 1 1 3 ,0 0 0 1+7,000 1+1,000 5 1 ,0 0 0 5 7 ,0 0 0 5 5 ,0 0 0 6 1 ,0 0 0 2 0 ,0 0 0 8 3 ,0 0 0 5 2 ,0 0 0 1 5 5 ,0 0 0 1 0 6 ,0 0 0 5 1 ,0 0 0 1+5,000 6 0 ,0 0 0 1+9,000 1 2 7 ,0 0 0 1 1 7 ,0 0 0 1 1 5 ,0 0 0 9 1 ,0 0 0 5 0 ,0 0 0 5 1 ,0 0 0 1 1 5 ,0 0 0 1 1 3 ,0 0 0 1 7 6 ,0 0 0 1+2,000 1+5,000 8 7 ,0 0 0 1+9,000 3 8 ,0 0 0 1 0 9 ,0 0 0 lOl+.OOO 1+5,000 5 1 ,0 0 0 2 6 ,0 0 0 i+ 6 ,o co 7 1 ,0 0 0 U s ,0 0 0 7 8 ,0 0 0 3 1 ,0 0 0 3 6 ,0 0 0 6 9 ,7 0 0 Liver peptone (2) Bactotryptone U 2 .0 0 0 3 2 ,0 0 0 2 U .0 0 0 3 8 ,0 0 0 5 3 ,0 0 0 40,000 1 3 3 ,0 0 0 1 0 2 ,0 0 0 U 5 ,o o o i3 U ,o o o 3 1 ,0 0 0 51,000 3 7 ,0 0 0 3 3 .0 0 0 5 1 ,0 0 0 5 8 ,0 0 0 5 1 .0 0 c 1 3 7 ,0 0 0 5 2 ,0 0 0 1 1 3 ,0 0 0 9 1 ,0 0 0 1+3,000 3 5 .0 0 0 6 6 ,0 0 0 3 8 ,0 0 0 118,000 1 1 6 ,0 0 0 1 1 3 ,0 0 0 9 3 .0 0 0 1+7,000 5 2 ,0 0 0 lOU.OOO 1 0 6 ,0 0 0 1 7 8 ,0 0 0 5 5 .0 0 0 1+8,000 S U ,0 0 0 3 U ,0 0 c U 9, 0 0 0 1 1 1 ,0 0 0 1 0 6 ,0 0 0 3 9 ,0 0 0 5 1 ,0 0 0 1+3,000 1+5,000 5 2 ,0 0 c 3 9 ,0 0 0 6 9 ,0 0 0 3 2 ,0 0 0 3 7 .oco 6 6 ,5 0 0 l+c,000 1+1,000 3 8 ,0 0 0 U 2 .0 0 0 3 5 ,0 0 0 40,000 1 2 0 ,0 0 0 11+8,000 1+1,000 1 5 2 ,0 0 0 5 0 ,0 0 0 3 6 ,0 0 0 6 3 ,0 0 0 1+6,000 l+u,000 U9, 0 0 0 6 5 ,0 0 0 9 7 ,0 0 0 6 7 ,0 0 0 1 2 2 ,0 0 0 9 3 .0 0 0 1+1,000 1+2,000 6 3 ,0 0 0 1+1,000 1 2 1 ,0 0 0 1 2 3 ,0 0 0 1 1 5 ,0 0 0 89,000 UU.000 1+3,000 1 1 7 .0 0 0 1 1 2 ,0 0 0 2 1 6 .0 0 0 U 2 .0 0 0 2 5 ,0 0 0 7 5 ,0 0 0 2 7 ,0 0 0 7 0 ,0 0 0 1 1 0 ,0 0 0 1 1 0 ,0 0 0 U 7 .0 0 0 6 3 .0 0 c 2 6 ,0 0 0 U7,0 0 0 6 s .0 0 0 1+7, o c o 51,000 H i ,0 0 0 2 1 ,0 0 0 6 s ,5 0 0 - Table IS. Sample number Comparison of p r e p a r e d animal peptones with Bacto-tryptone for growth of organisms in raw milk. Pork pepton6 k 2 ,0 0 0 3 8 ,0 0 0 U2, 0 0 0 3 2 .0 0 0 HO,000 5 3 s ,0 0 0 6 3 2 ,coo 7 2 3 1 ,0 0 0 8 1 0 3 ,0 0 0 9 H i,0 0 0 10 k g ,0 0 0 11 3 2 ,0 0 0 12 7 6 ,0 0 0 13 3 7 ,0 0 0 lk 3 2 ,0 0 0 3 1 ,0 0 0 lb HO,000 17 2 5 7 ,0 0 0 18 6 5 ,0 0 0 19 9 6 ,0 0 0 20 H p .o o o 21 5 9 ,0 0 0 22 2 k ,000 23 1 1 7 ,0 0 0 2k 3 2 ,0 0 0 . 25 5 5 ,0 0 0 26 H3.000 27 3 9 ,0 0 0 28 5 1 ,0 0 0 29 6 k , 000 30 1 0 7 .0 0 0 31 5 2 ,0 0 0 32 37,000 33 1 3 0 ,0 0 0 3H 2 1 ,0 0 0 35 HS, 0 0 0 36 H 2 .0 0 0 37 9 0 ,0 0 0 38 39,000 39 H o ,0 0 0 Ho H s .o o o Hi 5 8 ,0 0 0 k2 62,000 k3 H 2 .0 0 0 HH 5 5 ,0 0 0 k5 U 2 ,00© H6 ilk ,o c o k7 9 0 ,0 0 0 Hs 1 1 2 ,0 0 0 kg 2 9 6 ,0 0 0 50 Aver; a g e 6 7 , 1 0 0 1 2 3 H 56- Brain p e p t o n e (2) H i , 000 35. 000 H i , 000 3 3 .0 0 0 H2.000 37.0 00 3 6 .0 0 0 1 9 3 .0 0 0 1 0 6 .0 0 0 H3, c o o k 8 ,0 0 0 3 5 .0 0 0 3 5 .0 0 0 H 6 .00 0 3 3 .0 0 0 p d . , uuu H2, 0 0 0 2 7 3 .0 0 0 5 5 .0 0 0 7 2 .0 0 0 H 5 ,o o o 2 6 .0 0 0 3 7 .0 0 0 1 0 1 .0 0 0 3 k , 000 k 2 ,0 0 0 H o,000 H o ,0 0 0 2 3 .0 0 0 H 3 ,o o o 75 . 000 5 1 .0 0 0 37.000 1 3 5 .0 0 0 2 1 ,0 0 0 H9, 0 0 0 H o ,0 0 c 98,000 3 k ,000 37.000 H-5,000 5 1 .0 0 0 k 3 ,0 0 0 H i,0 0 0 5 6 .0 0 0 HU,000 1 0 6 .0 0 0 9 1 .0 0 0 1 2 1 .0 0 0 2 1 2 ,0 0 0 6 2 .0 0 0 B a c t e r i a p e r ml. Heart peptone (1) U2, 0 0 0 3 6 ,0 0 0 39.000 3H, 00 0 HH.000 H o,00 0 3 5 .0 0 0 2 3 6 .0 0 0 1 2 9 .0 0 0 k 2 ,0 0 0 HU,COO 3 k ,o o o 3 3 .0 0 0 3 9 .0 0 0 3 3 .0 0 0 , VW H5, c o o 2 1 5 ,0 0 0 5 6 .0 0 0 6 H ,o o o H3.000 Hs.OOO 2 9 .0 0 0 1 1 H , 000 H3.000 k s , 000 H5, 0 0 0 H i ,000 2 2 .0 0 0 HH.000 9 k , 000 5 2 .0 0 0 3 5 .0 0 0 12H, 0 0 0 2 1 .0 0 0 H 3 ,000 H o ,000 9 2 .0 0 0 3 7 .0 0 0 H 3 ,o o o H2.000 5 2 .0 0 0 Up,000 H2.0 0 0 5 6 , ooc H5, 0 0 0 ioH.ooo 95»000 1 0 2 ,0 0 0 2 3 k ,000 6 2 ,9 0 0 Heart peptone (2) k2 ,OCO 3 7 .0 0 0 H i, 0 0 0 35.000 U6,ooo H i , 000 38 .0 0 0 2 k l,0 0 0 1 2 6 ,0 0 0 k k ,o o c 5 3 .0 0 0 3 7 .0 0 0 3 9 .0 0 0 H 5 ,000 3 7 .0 0 0 7<2 AAr w HH,0 0 0 2 6 8 .0 0 0 6 H ,o o o 85.000 HH.000 5 6 ,0 0 0 3k, 0 0 0 1 2 1 .0 0 0 H i,0 0 0 H9, 0 0 0 H i , 000 HO, 000 3 1 ,0 0 0 5 k ,000 1 1 3 .0 0 0 53 , 0 0 0 '5 6 ,0 0 0 1 3 6 .0 0 0 21,000 5 3 .0 0 0 H2.00C 113.000 3 ! , coo Hp.OOO H2, 0 0 0 5 5 .0 0 0 6 3 .0 0 0 k3,ooc 6 1 .0 0 0 H7,0 0 0 1 1 1 .0 0 0 9 2 .0 0 0 118,000 222,000 6 8 ,180 Bactotryptone 3 2 .0 0 0 k 2 ,0 0 0 3 9 .0 0 0 31.000 5+7,000 3 9 .0 0 0 3 7 .0 0 0 1 9 9 .0 0 0 111.000 H 2 .0 0 0 5 2 .0 0 0 3 7 .0 0 0 HH.000 37.000 38.000 AQC. 3s!000 2 3 1 .0 0 0 62,000 6 7 .0 0 0 H 6 ,o o o 3 3 .0 0 0 3 k ,000 1 1 6 .0 0 0 3 0 .0 0 0 3 9 .0 0 0 3 9 .0 0 0 3 7 .0 0 0 3 1 .0 0 0 5 k , 000 1 0 5 .0 0 0 k 2 ,0 0 0 H i , 000 1 3 k , 000 3 1 .0 0 0 H6.000 H 2 .0 0 0 1 0 9 .0 0 0 3 0 .0 0 0 5 1 .0 0 0 HE, 000 5 1 .0 0 0 5 9 .0 0 0 H2,0 0 0 5 1 .0 0 0 k 5 ,c o o 1 0 6 ,0 0 c 10.000 1 1 9 ,0 0 0 2H 5, o c o 6 H .1 0 0 -5 7 th at vegetable peptone No. 3, vegetable peptone No. 5 and vegetable tryptone are s lig h tly superior to Bacto-tryptone but in fe rio r to Bacto— tryp to se in th e ir a b ility to grow organisms found in samples of raw milk. However, the averages obtained for vegetable peptone No. 1 and vegetable peptone No. 2 are not s ig n ific a n tly lower than the average for 3 acto -try p to n e, indicating that these peptones are approxiirately on a par with Bacto-tryptone in th e ir a b ility to grow organisms found in raw milk. Further comparative te s ts showed vegetable peptone No. 5 and vegetable tryptone to be superior to vegetable peptone No. 3 * This ind icates that peptones prepared from unhydrolyzed corn gluten are superior to those prepared from hydrolyzed corn gluten. iabla lb shows that the undecolorized peptones are much in fe rio r to vegetable peptone No. 5 vegetable tryptone, and are probably in fe rio r to the decolorized peptones prepared from the same hydrolysate sample. The dark color of the media prepared from these undecolorized peptones accounts for th e ir in f e r io r ity , since the p la te s poured v/ith these media were very hard to count. The average re s u lts given in Tables 17 ana. 18 show that the spleen peptone (l). heart peptone ( 2 ) and pork peptone are superior to Bactotryptone for growing organisms found in samples of raw milk. lhe averages fo r the other prepared animal peptones are only very slig h tly lower than those for Bacto-tryptone, ind icating th at a l l these peptones are approx­ imately as e f f ic ie n t as Bacto-tryptone for th is purpose. -5 8 - Growth and Gas Production by Coliform Organisms from Naturally Contaminated Water. The medium used in determining the usefulness of the prepared pep­ tones for growth and gas production "by coliform organisms found in naturally contaminated water was as follows: KaHrO* U.O KH2PO*..............................................................1.5 HaCl................................................................5.O Lactose............................................................5*0 Tryptose or a prepared peptone................................20.0 D is tille d water 1 0 0 0 .0 pK........................................................................f .0 grams grams grams grams grams ml. The above medium, with tryptose, as recommended by Darby and Mallmann (13) was used as the control. The medium was tubed in 10 ml. amounts with Durham fermentation tubes, and s te r il iz e d for 20 minutes at fifte e n pounds pressure. The tubes were inoculated with one ml. of contaminated riv e r water and incubated a t 37 ° 0 . f ° r 36 hours. The number of organisms of the coliform type present in the inoculum was determined by p la tin g out one ml. on Violet Red Bile Agar. The inoculated tubes were observed for the time of f i r s t v isib le growth and for f i r s t gas production and for the amount of gas at the end of 3b hours. The re su lts are shown in Tables 19 to 26. Discussion When the prepared animal and vegetaDxe peptones are compared with Bacto-tryptose fo r th e ir a b ility to grow coliform organisms in a lactosebuffered broth the re s u lts (Table 19 to 26j show that tryptose is much superior to any of the prepared peptones, both in a b ility f i r s t v is ib le growth and the f i r s t v isib le gas. to produce the The amount of gas pro- T a b l e 19* Growth, a n d g a s p r o d u c t i o n b y c o l i f o r m o r g a n i s m s f r o m n a t u r a l l y c o n tam in ated w a te r in la c t o s e b u f f e r e d b ro th p re p a re d w ith v e g e t a b l e p e p to n e s and B a c t o - t r y p t o s e . Sample No. 1 Inoculum = 6 E. coli per tube Hours Peptone Vegetable Peptone H O P5 Vegetable Peptone No*. 2 Tube Ko • 1. 2. 3* h. 5* - 10 + T + T + 11 + + + + -r 12 + T + 1- 1. — + 2. )• - b. 5* 9 Vegetable Peptone No. 3 2. 3H. 5* 1 2 Tryptose 3 5 17 32 25 * 25 50 9 + -t+ + + + + + + + 9 9 9 9 9 9 9 9 9 9 1-2 1 1 1 1 2 fc -t- 9 9 9 9 9 9 9 9 9 9 1% 1 1 1 1 2* 10 2 5 5 25*; 50 30 23 5 'p 5 5 10* 10 25 # 25 70 75 55 8o* — •t-t- + T + -fr- *r ■ t 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 1* 1 1 1 1 * i*r T + 13 © 9 © © + + + — - lb 9 9 9 9 9 — “W — l. 13 0 1 3 H 2 10 5 1£ 1 1 2 5 23 23 50 Legend: - = no v isib le growth + = v isib le growth 9 = v isib le growth and slig h t amount of gas 1 $ ,e tc . ■ one per cent gas in Dunham fermentation tubes. i i 2 10 10 10 2 2 2 2 50 25 10# 10 10 10 20 50 Ho 75 80 90 80 - 60 - TcJble 2 0 . G r o w t h and. g a s p r o d u c t i o n b y c o l i f o r m o r g a n i s m s f r o m n a t u r a l l y c o n t a m i n a t e d w a t e r i n l a c t o s e “b u f f e r e d b r o t h p r e p a r e d w i t h v e g e t a b l e p e p to n e and B a c t o - t r y p t o s e . Sample Ho. 2. Peptone Vegetable Pentone ITo*. 1 Vo ohl O Peatone Ho*. 2 Vegetable P e n tone Ho*. 3 Tryptose Tube Ho. 9 10 + 32 bob 20* + -tT -* ■ + T T 1 * + + © © © © ±yo 1 1 1 1 IO56 2 © © © © © © T •i + T + T T -r *r © © © © © © © © © © © © T © © © l ‘,S 1 1 1 1 2* 2 2 1 1 50^ 50 25 30 10 -*■ + * T * 9 Q i-;o i i © i 3 i 2 80 $ 75 75 75 75 30$ 90 -r 2/a 2 2 5®r> ■i1 + m. ?. - 3* — + + T + + 5- IT 10ft 1 1 1 1 1. U. 13 © © © © © + + + + l. 2. 3* 14 © + © © © •* l. 2. 3* b. 5- Hours 12. .. 13 * + -T *r ■ t* 1. 2. 3b. o • b. 5- 11 Inoculum = 17 3 . coli per tube • — — — ▼ T T + 5 5 5 5 Legend: 1 y!> , - = no visible growth + = visible growth $ sc visible growth and slight amount ot &ns e tc.= one per cent gas in D u n h a m fermentation tubes 25 50 50 50 2 30 1 1 1 10 20 10 2 5 5 25^ 10 10 25 10 75 90 90 - 61 - Table 21. Growth a n d g a s p r o d u c t i o n by c o l i f o r m o r g a n is m s fro m n a t u r a l l y c o n t a m in a t e d w a t e r i n l a c t o s e - o u f f e r e d broth, p re p a re d , w ith v e g e t a b l e p e p t o n e s e n d B a c t o —t r y p t o s e . Sample Ho. 3. Peptone Tube Ho. Vegetable Pep to ne Ho . 1 . Vegetable Peptone No. 2 1. 2. 3U. 5* Peptone No. 3* . 9 -t- + - -r © + ■ t-i- © © "1 O © © © © © © — © © © 5 ch 1 1 1 - + T 4. — + t 1. -i 2. 34. 5. - "T + + — 2. -t- 3- T 4. 5* + + 11 T + -t•t1- - - 11 -t~r T -h *r 2. 3- l. Tryptose 10 1 . 5* Vegetable Inoculum = 2S 3 . coli per tube © © © © © © © © 1 Legend; - = no v is ib le growth t * v isib le growth $ = v isib le growth and slig h t amount of gas l^o, etc. ** one per cent gas in Dunham fermentation tubes © © © 2% 5 R 5 5 20 31 10% 10 10 10 10 20% 10# 10 10 10 20 20% 20 20 25 25 10£ 5 15 5 5 255$ 20 50 20 25 80 £p 80 15 15 15 8 5^ 85 15 25 20 20 20 so 75 - 62 - T ab le 22. Growth a n d g a s p r o d u c t i o n b y c o l i f o r m o r g a n i s m s from n a t u r a l l y co n tam in ated w ater in la c t o s e - b u f f e r e d b r o th p re p a re d w ith v e g e t a b l e p e p t o n e s a n d 3 a c t o —t r y p t o s e . Sample No. U. Peptone Tube No. Vegetable Peptone llo. 1. Vegetable Peotone No. 2 . Vegetable Pept o n e Ho- Tryptose 3 1. 2. 3. U. 5* 1. 2. 3* k. 5. - - 1- 10 11 13 20 “T © © © © © 10% 10 10 10 10 © © © © © 10jb 20 10 © Q © © © 5p _ - + -t+ -r 1" * T © © + _ - - 1. 2. 3. h. — 5. + 1. 2. 3- f + 5* Inoculum = 169 E. coli per tube T f f + * ■ “t © © © © © © © © *r *r -r + © © © © © l l l 1 Legend: - ■ ■t- = © = l/'i, e t c •r no visible growth visible growth visible growth and slight amount of gas one p e r cent gas in liunham fermentation tubes. 5 ;5 5 5 5 15 10 25 20 70 ^ P 05 75 70 - -- 31 2Op 25 20 20 20 20% 25 20 20 20 25 p 25 Ho 50 U5 75 ;< = 75 75 75 75 - Table 23. 63- P r o d l ?-c t J on c o lif o r m o rg a n ism s from n a t u r a l l y contam inated, w a te r in l a c t o s e - b u f f e r e d b r o th p re p a re d w ith a n i m a l p e p t o n e s a n d B a c t o —t r y p t o s e . Sample Number 1, Tube Liumber Peptone Beef peptone 1 2 3 U 5 Spleen peptone ( 2 ) Liver peptone ( 2 ) __ + + "r — T + 10 ?5 40 50 75 75 80 50 60 80 80 © © 2 2 © 80 30 O + © © 50 50 UO 50 1*5 1 1 1 1 1 15 5 5 60 50 so 55 75 75 10 60 50 80 SO 10 10 10 10 10 20 75 75 SO 60 20 ho 50 15 75 85 T T + + + 5 - 1 2 _ 3 1 2 3 T - + + - - t - + © © © © © © © © © © 2 2 2 2 2 - + •t* + h 5 + t- + T 5$ 5 1 2 3 15 15 Legend: - = * ®= 1/3, etc. « 70 $ so 60 70 ... 1 l l 1 - 301? 70 25 © © © © © 1 2 U 36 l 1 1 -t+ - 22 © © © + •i- 3 12 lf° 5 3 11 © © © © © + h 5 Bacto-tryptose 9 + + h lh E. coli per tube. Hours s — — 1 2 Spleen peptone ( l ) Inoculum no v is ib le growth v is ib le growth v isib le growth and slig h t amount of gas one per cent gas in Dunham fermentation tubes ho i+5 80 75 70 70 85 75 -6 > t- T a b l e 2l+. Growth a n d g a s p r o d u c t i o n b y c o l i f o r m o rg a n is m s p r e p a r e d fr o m n a t u r a l l y c o n t a m i n a t e d w a t e r i n l a c t o s e - b u f f e r e d "broth p r e p a r e d w i t h a n im a l p e p t o n e s an 1 B a c t o - t r y p t o s e . Sample Number 2 Peptone l\ibe lumber 1 o Beef peptone 'H Inoculum 9 - 3 - U - 5 - 10 •tT *r -t-t 11 Hours 12 “T T 1 1 Spleen peptone ( l) Spleen peptone ( 2 ) 2 3 - + "T h - *r -t* 5 - + -r 1 2 T T — -t- -f +• *r T “t 3 - h - 5 - l Liver peptone ( 2 ) Bacto-tryptose + + 2 3 — h .. 5 - *r * t* l 2 ** ■ t* + © © e + ® G 3 h 5 — T . © © © 20 36 © © © © © 10B 20B 5 1C 15 5 75 70 © © © © © 20 10 10 75 70 15 15 70 70 © © © © © 25 70 70 75 30 25 so 5 1+5 75 70 70 75 20 25 l 5 1 © 5 2 10 20 5 2 2 5 coli per tub e - 2* 15 25 1 ° Uo 35 U5 5C 50 6o 50 i+5 Legend: 1 - * no v i s i b l e growth. •t ~ v i s i b l e grov/th © = v i s i b l e g r o w t h a n d s l i g h t amount oi g a s e t c . = one p e r c e n t g a s i n Dunham f e r m e n t a t i o n t u b e s . so 70 65 75 so 80 S5 90 so sc 65- - Ta b l e 2 5 . Growth a n d g a s p r o d u c t i o n b y c o l i f o r m o rg a n is m s from n a t u r a l l y co n tam in ated w a ter in la c t o s e - b u f f e r e d b r o th p re p a red w ith anim al p e n to n e s and B a c to - tr y p to s e . Sample Number 1 Peptone ' Heart peptone ( l) Tube Number 1 2 3 . — T 1- - *T' 5 — -t- - X 1 2 Pork peptone 10 - 4 5 Brain peptone ( 2 ) 9 h 1 2 Heart peptone ( 2 ) Inocmlum — - + *r T -r — U * + 5 l _ 2 _ 3 - + - + 4 5 1 2 Bacto-tryptose 3 1+ 5 T + -r t -1 © *r 13 14 36 10 §o S0 }b SC © © © © © 2% ©' 10 2 10 T + f © © © © © 5 2 5 K 15 5 U5 © © © © © 5 2 5 5 5 25 15 © © © © © 2 10 5 5 5 15 40 4o 30 so 70 2 20 30 45 75 75 © © © © © 3 45 90 P 10 23 30 20 50 45 60 53 55 85 ~r -*• ■t -r + + - 12 •t-t•» T + t - 3 11 21 E. coli per tube 10 15 r— 5 ✓ Legend: - * no v isib le growth + *= v isib le growth © = v isib le growth and sligh t amount of gas 1 (~, etc. » one per cent gas in Dunham fermentation tubes 30 10 30 10 Uo 5 20 40 20 30 25 80 75 80 75 75 7R 80 80 75 70 75 so 75 75 so 90 so Tp.ble 2 o . G r o w t h a n d g a s p r o d u c t i o n "by c o l i f o r m o r g a n i s m s f r o m n a t u r a l l y c o n ta m in a te d v;ater i n l a c t o s e - b u f f e r e d b r o t h p r e p a r e d w ith a n im a l p e p to n e s and B a c to - t- r y p to s e . Sample Humber 2 Peptone Tube Humber 1 2 Heart peptone ( l ) Heart peptone ( 2 ) 3 o 1C 11 - -i+ + + © © - -* ■ -i- 36 10# 10 SCd 9 2p 2 10 10 - n- - -t- © © 1 2 — © © © 5 8 - *v )« *T — •*- «♦- 5 * + T + C . - -r -t* 3 H 5 - T + T T f 9 5 l T 9 2 25 c. + 9 5 5 5 5 30 15 15 30 10 Ho 15 15 30 35 50 30 3 _ - 3 - T b — + + l 2 3 H 5 © + 9 + © e © + © ■t © © © 2 1 3 2 5 © © © © 20 10 Legend: - * •*- = © = l/of etc. = 2 -fr* + + *r T 5 Bac to-tryptose lH 5 <*> Pork peptone 13 12 H l Brain peptone ( 2 ) 29 E . coli oer tub e Inoculum no visible growth visible growth visible growth and sli^it amount of gas one p e r cent gas in Hinham fermentation tubes 50 10 HO 75 70 75 SO 2 15 15 5 k 20 Ho 5 15 65 5 25 10 30 20 25 25 85 75 5 10 50 H5 Ho Ho 75 75 75 7 *> 1 so 80 75 80 so 70 50 80 75 70 35 6c 70 80 90 90 6o £0 So - 67- duced at any given time is larg er in tryptose broth than in vegetable peptone broth. As in the case of the ra te of growth of E. c o li. (Table 1 3 )» these re su lts show Vegetable peptone IJo. 3 to give the best growth of coliform organisms of any of the peptones prepared from hydrolyzed corn gluten. Bacto-tryptose is also shown to be b etter than any of the prepared animal peptones, of which the pork peptone was the best. In contrast to the vegetable peptones, the animal peptones have the a b ility to support growth long enough for a large amount of gas to be produced, whereas the vegetable peptones do not support growth long enough to allow the maximum amount of gas which these organisms are capable of producing from the lactose present. At the end of y S hours the coliform organisms in the animal peptone broth produced approximately the seme amount of gas as they do in tryptose broth. animal peptone broth. However, they produce i t more slowly in the Thus, to give a positive presumptive te s t for B. c o l i , the animal peptone broth would require a longer incubation period than would tryptose broth. Growth of Pathogenic Organisms. In this experiment the following organisms were employed: four u nidentified hemolytic streptococci recently isolated from milk powder; an hemolytic streptococci which had been on a r t i f i c i a l media for some time; Brucella abortus, Brucella m elitensis, and Brucella su is; Eberthella typhosa, Salmonella e n t e r it id is , salmonella paratyphi and Salmonella schottm ulleri. t I■ '■ «■ I W - —— ■■ IIPasteurella ■.11I ■ I *' ■■— avicida; ' ■“ Shigella dysenter ia e ; Pseudomonas aeruginosa; Erysipelothrix rhusiopathiae; and aureus. S tapnyl o coccu b Prepared p e p tone-glucose agar slants were inoculated w i t h the above organisms and. incubated for two weeks at 3 before calling -6 8 tham negative. Tryptose-glucose age.r was used as the control. The re s u lts for this experiment are given in Table 27. Discussion The re su lts show that the prepared peptones have the a b ility to support the growth of nearly a l l the pathogenic organisms tested. In th is respect the animal peptones were superior to the vegetable peptones, since the hemolytic streptococci would not grow on media prepared from the la tte r. G-rov/th on the animal peptones was equal to that fouui on tryotose, both as to amount and time of appearance. The fa ilu re of the vegetable peptones to support the growth of hemolytic streptococci probably lie s in the fact that the nutrients obtained from blood are not present as they are in the animal peptones. .Berthelot, Amoureux and P e tit ( 6 ) prepared peptones by digestion of peanut meal with pepsin and pancreatin. growth of Cl. te t a n i . 'Both peptones supported good It is in tere stin g to note that this organism pro­ duced toxin from the pepsin peptone, but fa ile d to produce toxin when grown in the pancreatic peptone. These re su lts emphasize the importance of the effect of the method of preparation of a peptone upon i ts efficiency as a b acteriological medium. While the use of vegetable proteins for the preparation of bacter­ io lo g ical media has received some attention, the re su lts obtained indicate th at these proteins may be more generally useful for th is purpose than is generally supposed. Iheir a b ility to replace peptones in many bacter­ iological te s ts has not as yet been determined, but there is reason to believe that if further work is done on these products, more uses for them may be found. Complete amino acid analysis of corn gluten shows that i t contains a l l of the essential amino acids in amounts necessary to support -69Tahle 27- Comparison of the growth of pathogenic organisms on prepared peptones and 3nc to - tr y p to s e O o i--- Yr Hemolytic streptococci A Hemolytic streptococci 3 Hemolytic streptococci C Hemolytic streptococci D Hemolytic streptococci Brucella abortus Brucella melitensis Brucella suie Sal. enteritidis SaL paratyphi Sal. schottmulleri Shigella dysenteriae Pasteurella avicida Pseudomonas aeruginosa Ersipelothrix rhusiopathiae Staphylococcus aureus A t a> © pi a PI P| o O o -p -p -p ft Pi ft ft P. a> © © © f t ft Pi ft (M K'l ^ i O © 0> C © ft H rH ft ft A •3 tc 15 •3 ft +» -p p © © 0) © f< D r-\D Hi © ® © £ J» > > ® Organism o r -1 Y-i © o r © Si o ft © ft in © rH r=> +> © f© © > © G G ft o< >i Pi ft © H P © ft © *iD © t=- H © Pi G ft ft © ft ■ft © © T - - t - - - - - - - - "T ‘i + T T 1 1- r -r* — • © a o -p Pi © Pi Pi © © H — s OJ S-' © M o ft Pi © Pi a • '’*■ OJ © rt o -p ft © Pi Pi © > *rf & © © ft ft to T T T • t- • OJ —* © P o ft ft © ft G ft Bf Pi m + + •> OJ ® PI O ft ft ® Pi ft Pi g3 ® W © to ® Pi o ft ft ® ft ft Pi nJ ® ® Pi o pG & ft ® 1 ft o ft o a) o m ft ft t + i- T • T 0 ft T f + - t T 1 T i •t- T + - * T *i T T + *t + + T *r t T T 1 *r * T + t + T T T T + + i* T t t -t + t T *t- + + + + ■* i T T T T T T T + i- T 1 T T •t -t- f + + i- + -T + T T *r + T T t + r -r + T T 1 T + t + *T T -v T T -r i T 1- 1 1 T T ■t f T *r n -t T *t T T T T T T 1 T 1 T T r t + T T T i T -r i* + -r T T i+ T T t T + + -r T 1 T T 1- T + t T t T T f ■ t + T -70grovth. The use of several proteins, as is the case in animal peptones, instead, of a single p ro tein, may re su lt in the formation of peptones that wUll Ue b e tte r suited to meet the nitrogen requirements of bacteria. It is doubtful that they w ill ever be able to replace the animal peptones for growth of the more fastid io us pathogens, out, since, as has been pointed they do not possess even the small amount of blood nutrients "nresent in animal peptones. They are to be recommended as a c h e a t -; source of n itro ­ gen for growing the more common saphrophytic organisms. Summary Methods fo r the comparative analysis of prepared animal and vegeta^ble peptones with certain commercial brands as controls are given, which include the determination of the rate of growth of 2. c o ll; the growth of organi sms in raw mi 11c; the growth and gas production of coliform organisms in naturally contaminated water; and the grov;th of certain pathogenic organisms. The re su lts obtained show that: ( l) Vegetable peptones number 1 , 2 and 3 support growth of E. coli during the lag phase and for the f i r s t six hours of growth b e tte r than do Bacto-pep tone, tryptone and tryptose. After six hours Bacto-tryptose is b etter than these vegetable peptones and a f te r twelve hours the Diico products support a, larg er and more rapid growth than do the vegetable peptones. ( 2 , Vege­ table peptone Ho. 3 supports growth of 2 . coli b ette r than do the other vegetable peptones tested. any of the (3) Vegetable peptone i.o. U is in fe rio r to other peptones tested . increasing numbers o f E . (U) Vegetable peptone Ho. U shows coli for US hours or longer, while the other -7 1 peptones tested show maximum growth at 2U hours, and then the numbers of coli decrease. (5) An average of the re su lts obtained by plating out 100 samples of raw milk shows Vegetable pep tone ho. 3 to he very s lig h tly superior to Bacto— tryptone in i t s a b ility to grow the organism found in raw milk. The difference is not large enough to be sig nificant. (6) Undecolorized vegetable peptones are less e ffic ie n t in growing the organisms found in raw milk than are the same peptones when decolorized. ( 7 ) Vegetable peptone ho. 3 is significantly b ette r than vegetable tryp,tone, vegetable peptone No. 5 or Bacto-tryptone, but in fe rio r to Bacto-tryptose fo r-th e same purpose. (S) Cf the prepared animal peptones, the two spleen peptones and the pork peptone are shown tc be superior to Bactotryptone; the remaining animal peptones are in fe rio r. ( 9 ) 2he prepared animal and vegetable peptones are inferio r to Bacto-tryptose for growth and gas production by coliform organism* found in naturally contaminated water. Vegetable peptone IT0. 3 pork peptone are the best of the vegetable and animal peptones, respectively. ( 1 0 ) The vegetaDle peptones did not support the growth of hemolytic streptococci, but the growth of a l l other pathogens tested. heart peptone (2), tested . did support ( 1 1 ) Brain pertone ( 2 ;, and pork peptone supported the growth of a l l pathogens Liver peptone ( 2 ) and heart peptone ( 2 ) supported growtn of a ll pathogens except hemolytic streptococcus C from milk powder; beef peptone, spleen peptone (1) and (2) supported growth of a l l pathogens except hemoly­ tic streptococci B and C from milk powder. BI3LI0GBAPHY and Geiling, 3 .M.K. Abel, J .J . Some hitherto undescribed properties of the constituents of W itte's peptone. J. Pharmacology 2^: 1-27. 192U. Anderson, B. Gaseous metabolism of some anaerobic b acteria. 2 *04- 2 8 1 . Dis. Ayers, Inf. 192U. S.H.; Hupp, P. and 'Pudge, G.S. The production of ammonia and carbon dioxide by streptococci. Berthelot, A. J. J. His. 2j _ \ Inf. 235 - 2 oC. 1921 . Applications d'une peptone proteolytique de viane et de muqueuse in te s tin a le a l a preparation des milieux de cil-ture. r.nrrvry x- - . y a n A -.- ~ v w ** * w—- le r th e lo t, A. and Amoureux, B. ^ •— • TOT17* — Preparation of culture media using a peptone prepared by the action of pepsin on peanut press cake. Bull. soc. chim. b io l. 1 6 : A.bs. 2^: lW . 15 .I-I56U. 193 **. Cited from Chem. 1935. B erthelot, A., Amoureux, G. end P e tit, D. Remarks on the composition of peanut meal peptone and i t s use for the culture of pathogenic b ac teria . 3 u l l . soc. chim. b io l. from Chem. Abs. 2^1 1 So5 - 12: 1023 — IC30. 1 9 3 0 * Cited 193 ^* Berthelot, A., Amoureux, G. and Van Diense, P. Advantages of a peptone prepared by peptic digestion of soybean press cake in the oreparation of culture media. 1 9 3 ^* Cited from Chem. Blanchetiere, A. £6: Boez, L. Bull. Abs. 2 9 : soc. chim b io l. lb*4o. 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Ind. & Eng. Chem. (Analytical. Edition) 1930. 2 8 . Kitamura, K. Combined sugar in serum albumin, egg white and peptone solution. 20 - 2 *+. J. Kyoto Frefectural Med. College j>: Cited from Chem. Abs. 2 3 : 29. Leif son, 3 . and Diamond, S. (A bstract). U237• 1927 * 1929 * Preparation of bacteriological peptones J. 3 act. 3 8 : 111 - 1 1 2 . ti tt " Uber die Pallung von Eiweissstoffen 3 0 . Lundin, H. and Schroderheim, J. 1939* und ih rer Spaltungsprodukte mit Tannin. Biochem. Zeit. 258: 1- 2 3 * W i ­ ll. McAlpine, J.A. and Brigham, C.D. b acteriological peptones. 3 2 . Mustafa, A. Some chemical studies of commercial J. 3 act. l6 : 25I-256. 192 S. La production de l'in d o l par des germes microbiens indoli- genes; ses raoports aver la peptone u tilis e e . b io l. 12bi U5O-U51. 1937* Conrpt rend. soc. 3 3 * Kaegeli, Rrnahrung der niederen Pilze durch Xohlenstoff und S tickstoffverbindunger Untersuchungen uber niedere P ilze. Cited from Eicker and Carpenter. 3 *+. 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Rettger, L .P .; Berman, IT, and Sturges, V .r. S. Ihrther studies on b ac teria l n u tritio n . The u tiliz a tio n of proteid and non-protein nitrogen. J. Bact. 1^: l p - 3 3 * k l. Roberts, J.L. 1916. and Baldwin, I.L. The effects of certain colloids endospores formation by Bacillus s u b ti ll s . J. Bact. on 37 “ 38 » 1937. 14.2. Rimington, C. Some phosphorus compounds of milk. caseinogen. 21: 2 CU-2 0 7 . III. The action of a lk a li upon caseinogen. Dephosphorized Biochem. J. 1927. 14.3. Rimington, C. Phosphylation of proteins- Biochem. J. I4J4.. Rimington, C. The phosphorus of caseinogen. I. 21: 2 / 2- 2 8 1 . 1927* Isolation of a phosphours- c o n t a i n i n g p e p to n e from t r y p t i c J. 21: 117 9-1186. d ig e sts of caeinogen. 1927. H5. Rimington, G. The phosphorus of caseinogen. Biochem. J. 2 1 : peptone. Biochem. U6 . Rimington, C. and Hay, H.D. II. IIE7-II93. Constitution of phospho- 1927. Some phosphorus compounds of milk. II. The lib e ra tio n of phosphorus from caseinogen by enzymes and other 3 iochem. J. agents. 2 0 : 777 - 7 9 0 ; 1926. it 1+7 . SacLikov, V.S. Truck. 205: Uber die Spaltung von Eiweissstoffen durch Ammoniak unter (Verfehren zur Darstellung von Peptonen) . 360-36S. I929. US. Sadikov, V.S. and Sinitzuin, N.P. Proc. Sci. In st. Vitamin 19 3 6 . 6U67 * 193 °* The peptonization of plant m aterials and i t s application in bacteriology. Abs. A study of methods for u tiliz in g yeasts. Research (USSR) 1.:IU2-I69. Cited from Chem. Abs. U9. Snyder, R.M. Biochem. Zeit. 12 .: 5 0 . Sopaiker, M.3 . 3511 . Abs. Bact. 2 .! lUO. Cited from Chem. 1925. Prepe.ration of peptones. Laboratory, pp. 1925* 9-10. Report Bombay Bacteriological Cited from Chem. Abs. 1 ^: 2891 * 191 S. 5 1 . Standard Methods for the Bxamination of Hairy Products. American Public Health Association. Seventh Bdition. 1939* 5 2 . Strauss, W. Ueber die Verwendbarkeit des Peptonum sicc. Riedel fur bakteriologische Zwecke. Centr. f . Bakt.I Abt. IU2-IU3 192U. 5 3 . T illey , P. W. Variations in hydrogen sulphide production by bacteria. J. Bact. 8: 5U. T illey, F.W. 115 - 1 2 0 . 1923- The re la tio n between the chemical composition of peptones and hydrogen sulphide production by b acteria. 1923. J. Bact. 8: 287 - 29 5 * 55* T r e e c e , 3. j,. cteg production from commercial peptones Tay A. aerogenes and E. c o li. J. Inf. Dis. ]+2 : 56. Underhills, P.P. pud Gross, E.G. 1928. H95-500 Pharmacological action and chemical c h a ra c te ristic s of products produced from W itte's peptone by hydrolysis. 5 7 * Utkin, L.M. J. Pharmacology Preparing peptones. Farm In s t. 11. 1930* Bull. ITauch. J. Biol. Chem. 5 9 * Van Slyke, L.L. and Hart, Z.B. 2U: 117 - 12 2 . Wherry, U.S. 2 590 * 1916. The relatio n of carbon dioxide to proteoly­ 1T.Y. Agr. Uxpt. Sta. Bull. Improvements in Bacteriological media. e ffic ie n t su b stitu te for "nutrose". 61. IChim- 1903. 6 0 . VZallis, H.l.M. 1917* Issledovatel. The determination of urea "by the urease sis in the ripening of Cheddar cheese. No. 2 3 1 . 1928. Cited from Chem. Abe. j>0 : 5 S. Van Slyke, D.D. and Cullen, G.E. method. 6 9 -S O . Cited from Chem. Abs. 12: Agr. Jour. ^9 3 * I. India A new and 12: 62I-632. 1918. A search into the n itra te and n i t r i t e content of Witte's peptone with special reference to i t s influence on the demonstration of the ir.dol and cherry red reactions. J. Inf. Dis. 2_: H30-UU5. 1905. 6 2 . Yaoi, H. The cystine content of peptones for bacteriological use. Japan Med. World 21: 59 S• 1927. 6: 11 lb-llb. I92S. Cited from Chem. Abs. The author wishes to express his grateful appreciation to hr. P. Y 7. Pat>ian, Professor of Bacteriology, under whose able guidance th is work was done, for his unfailing in tere st throughout the course of the work and for his assistance and criticism s during the preparation of this manuscript. The author also wishes to express his sincere gratitude to Professor C. D. Ball of the department of Chemistiy for many helpful suggestions made throughout the course of the experiment. ACkhO'rLBDGhZd.T The author wishes to express his grateful appreciation to hr. P. W. Pahian, Pro f e s s o r of Bacteriology, u n d e r whose able guidance this w o r k was throughout done, for his u n f a i l i n g interest the course of the work an d for his assistance and criticisms during the p r e p a r a t i o n of this manuscript. The author also wishes to ei-cpress his sincere gratitude to Pr o f e s s o r C. D. Ball of the I^partraent of Chemistry m a n y helpfo.l suggestions made throughout the course of the e:q?eriment •