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A study on the metabolism of the lactic acid bacteria Wood, Alexander James 1938

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A STUDY O i l THE METABOLISM 01 THE L A C T I C A C I D BACTERIA b y Alexander James Wood A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t of t h e Requirements f o r the Degree of MASTER OF S C I E N C E IN^AGRICULTURE i n the Department of D a i r y i n g The U n i v e r s i t y of B r i t i s h Columbia A p r i l s 1938 s TABLE OF C OSTETTTS. PAR? I; The Rate of Acid Production of C e r t a i n L a c t i c Acid B a c t e r i a i n Mi I k and in Ifilfc Enriched v.< i t h Ye a s t E x t r a c t Page 1 j? S j- X % The Hate of Acid Production of C e r t a i n L a c t i c Acid Bacteria i n the Milk of I n d i v i d u a l Cows on V a r i a b l e Bati ons . . . . . . . . . . . . . a . Pag-e 11 PART l i l t _ . '' The P o s s i b l e S i g n i f i c a n c e of Bios and Vitamin Bp i n the Metabolism of the L a c t i c Acid B a c t e r i a page 59 PAR? IY; Bacteriohhage and B a c t e r i a l Growth Fa c t ors.Page 118 BIBLIOGRAPHY ................................Page 127 XXXXXXXXX A STUDY OK THE METABOLISM OF THE LACTIC AGO 3A0TEEJA The Rate of Acid Production of P e r t a i n  Lact io A c i d Bac te r i a i n ifi 1 k and In Milk Enriched with Yeast E x t r a c t , « ; PAST I. One may assert, without f e a r of c o n t r a d i c t i o n , that the n e c e s s i t y f o r an a d d i t i o n of p l a n t or animal e x t r a c t s to b a c t e r i a l m e d i a was recognized by those who f i r s t engaged i n a s y s t e m a t i c study of m i c r o - o r g a n i s m Even. Antoni van L e s u w e n h o e it, the f a t h e r of the science, a c t u a l l y r e p orts that he found h i s "animalcules' 5 growing most p r o l i f i c a l l y where t h e y had access to complex organic m a t e r i a l though he did not use t h i s s p e c i f i c tens a t t h e t i m e . 1 As f a r as the l a c t i c a c i d b a c t e r i a are concerned O r l a -Jc. n sen (1) wa s probably the f i r s t to observe that the enrichment of ithlfc by yeast extract makes p o s s i b l e th g r o w t h of c e r t a i n species belonging- to t h i s group which o r d i n a r i l y f i n d milk an unsuitable medium. It itay be of f u r t h e r i n t e r e s t before d i s c u s s i n g the l a c t i c acid bacteria i n d e t a i l , to review b r i e f l y some r e s u l t s obtained w i t h various other species. As e a r l y as 1 9 0 4 , Bert rand ( 2 ) , made use of yeast extract as an enrichment to media f o r the growth o f B.» x v l i n a m . tfhile t h i s report i s one of the e a r l i e s t in t h i s f i e l d i t i s well to remember that organic i n f u s i o n s and e x t r a c t s had been used i n the time of Pasteur and t o some extent even before. . Bertrand's pape i s of s i g n i f i c a n c e i n that i t point s out that the growth of Bac t e r ium- j!y_l i num on the yeast medium was much more luxuriant than on the normal Infusion medium used at the time. The attention of workers Soon "became foeussed upon the p o s s i b i l i t y of extend-' ing Bertrand fs technique to other organisms. Hoguchi (3) drew attention to the advantages of t e s t i c u l a r extracts as an enrichment to media for certain Spirochetes. The commencement of the war gave added impetus to the study of "bacterial metabolism, because bacter-i o l o g i c a l laboratories were stripped of t h e i r usual sources of supply. Hie replacement of Witters peptone and Hutrose "became a necessity. The use of purely synthetic media was reported "by Tedder (4) and "by Doryland (5). Attention of bacteriologists was not drawn to the need for growth a c t i -vators or vitamin-like compounds i n b a c t e r i a l media u n t i l the work of Hopkins (6}, Eijkmann (7}t McCollum (8), Funk (9), and many others, on the vitamins* Bainbridge (10) was of the opinion that c e r t a i n organisms could not survive with only protein as a source of nitrogen. Sperry and Eettger (11) obtained evidence confirming t h i s view. She demonstration of an alleged "vitamin" i n tubercle b a c i l l i by Pacini and Russel (12} gave added incentive to studies on the require™ ments of bacteria for accessory growth factors. As a result of evidence of t h i s type Wallis (13) introduced a medium actually selected to contain an optimal quantity of vitamins. Innumerable papers have appeared In addition to those already mentioned, but i t would be impossible to consider them here. The hook ol liexine and Sohoelein (14) affords an excellent reference for those interested i n following the r e s u l t s of t h i s work. Returning to the work of Or la-Jens en (1} we f i n d numerous references to the use of activating extracts* His views are "best expressed i n h i s own words :-"The most t y p i c a l milk bacteria grow "best i n milk, and only with the greatest d i f f i c u l t y i n peptone solutions; there are, however, l a c t i c acid bacteria which, even though they ferment milk sugar i n peptone solutions, thrive poorly i n milk, or require at any x ate to be accustomed to i t , and which w i l l i n consequence rapid l y lose the faculty of so doing i f l e f t for many generations without coming i n contact with milk at a l l . . . . . . . n "We had expected that the pathogenic bacteria would have preferred blood serum or meat extract, and the milk bacteria casein peptone* This, however, d i d not prove to be the case...." "Yeast extract exhibits a more s p e c i f i c action, proving an extremely bad source of nitrogen for a number of pathogenic bacteria, some few micrococci and Mbm. laotioum. whereas i t furthers to a surprising degree the development, and even more the acid formation, of the genera Thermfibacterium and Streptobacterium. Por the genera Betacoocus and Betabacterium, also, yeast extract i s f u l l y as good a source of nitrogen as casein peptone, whereas the reverse i s the case with ordinary saprophytic streptococci " In a later paper Grla-Jensen (15) states that:-"The v i t a l i t y of the l a c t i c acid bacteria does not seem to be enhanced by the use of yeast extract, but the bacteria would rather seem to become pampered, so that when afterwards cultivated i n pure milk they form less acid than previous to the c u l t i v a t i o n with yeast extract." The r e s u l t s of Sadler et a l (16) obtained while studying the s u i t a b i l i t y of various nitrogen sources are of interest. These authors have shown that not only i s the "amount!! of nitrogen but also the "kind" of nitrogen important. Later r e s u l t s from the same laboratory (17) show that the addition of yeast extract to milk for the carrying of mixed starters brings about a profound change i n the numbers and types of organisms i n the starter f l o r a , i t i s known that certain l a c t i c acid bacteria require yeast extract for normal growth but i t i s of fundamental importance to discover the exact amount they require and i f possible the nature of the substance or substances present i n the yeast and bringing about the growth stimulation. With a view to studying t h i s point Sadler and Eagles (unpublished data} used various forage crop extracts as enrichments and determined t h e i r influence on the rate of acid production of certain Streptococci and Betacocci. Continuing t h i s work Sadler et a l (18| made a more intensive study of the effects of forage crop extracts as well as yeast extract. They concluded that:-"The enriching entity has no effect on acid production by Streptococci i n sugar broth. Enrichment with yeast or a l f a l f a extracts causes not only a marked increase i n the acid production of Betacocci, but also a de f i n i t e stimulating effect on the rate of acid production.'! The same authors found that when similar enrichments were added to milk the response was s l i g h t l y d i f f e r e n t . One of the streptococci tested showed sl i g h t stimulation while another appeared to be unaffected. The presence of a factor or factors promoting the growth of these organisms was also shown for a number of other forage crops. It would appear from their r e s u l t s that a l f a l f a i s the "best source of enrich-ment followed successively by clovers and grasses. In View of the results recorded above, i t seemed advisable to investigate further the influence of yeast extract on the rate of acid production of other streptococci to f i n d i f a l l the l a c t i c streptococci respond i n a similar maimer to those studied by Sadler et a i . BXEERIMEJSfgAlL The milk used i n t h i s experiment was obtained from the mixed milk of some 90 cows i n the herd of the Massey Ag r i c u l t u r a l College. The animals were being fed e n t i r e l y on pasture consisting i n the main of clovers and rye grasses. The yeast milk was prepared by adding to the above milk 10$ of yeast extract by volume (prepared by autoclaving 450 grams of bakers yeast i n 750 cos. of water), while the control milk used was obtained from the same bulk sample and contained 10% of added water to compensate for the d i l u t i o n of the yeast milk with the yeast extract. This media was tubed i n 10 cc. quantities and was s t e r i l i z e d by autoclaving at 12 pounds pressure for 25 minutes. The organisms used were as follows:-(1) Streptococcus oremoris - a type culture similar i n a l l c h aracteristics to the Streptococcus cremoris of Orla-Jensen (1919). (2) Streptococcus l a c t i g - a type culture corresponding to the Streptococcus l a c t i s of Orla-Jensen (1919). (3) RWgg,fi21 8 1 1 4 E ~ Streptococcus cremoris strains possessing marked acid producing powers at higher incubation temperatures. Milk and milk yeast tubes to be incubated at 20°, • 30°, and 37° C. were inoculated with one loopful of a clotted milk culture of the respective organisms. The t o t a l t i t r a -table a c i d i t y produced by eaoh organism at the three temper-atures i n milk and yeast milk was determined at regular time in t e r v a l s by t i t r a t i o n of each tube with tenth-normal sodium hydroxide using three drops of phenolphthalein as an indicator. The results of these t i t r a t i o n s are recorded i n Tables I - I I I and figures I-XT. BISGUSSIOff Considering f i r s t , the acid production of the organisms incubated at 20° C., i t i s evident that a l l respond to enrichment of the milk with yeast extract. In the case of the stock cremoris and l a c t i s , the response i s very marked. ••Hot only i s the t o t a l amount of acid produced i n 26 hours greater i n the case of the yeast milk, but also the rate at which the acid i s produced. JJTom the rate curves i n Figures I and I I , i t i s seen that both l a c t i s and cremoris when grown i n milk produce acid f a i r l y slowly, followed by a second increase after a s l i g h t decrease. The second r i s e i n the rate curve i s higher than the f i r s t under the conditions ol o o H • 0 • . "a H H O H • m 01 p! © H 10 O H tO OJ O to to 0 CO 03 OJ t£> O 02 cn tO 0 A: '- • © • © « © - 0 m - « • « 0 H 03 in to to to to 0 id 0 • P H Pi w © W M tO O 03 m 03 02 to ft H to lO tO rH O 0 0> - P id » •» •« • » ' e "« CQ rH to to &~ tO 03 Pi © O O O 10 E» to cn m tO to O cn £~ CO IO cn 03 tO . in O H > e - © 9 * e - 0 • a - » O 01 03 10 tO to e» O 03 - P ^ : P.K <D 03 H CO H to ft H 10 , tO w . to CO H in - p ;d •• • ••« • a •« •» a • * <« to H to in tO to 03 © R 10 o> W to c~ 0 02 03 m 0 cn 0 CO in 03 03 CO O 03 • » «•« - • •••• * « • 0 O ' H H to to in in in O - P m 0 0 4? ££} PfH M O CO tD 02 0 CO CO in to fH Hi H 0 en 02 to cn - P •rj ' « * © « • - • • • & • « ra rH H 03 03 to ra © 03 CO 03 CO tO tO to i> 0 0 to to tD CO 0 in 0 ra H © • • e • • « O *H •H H OJ ^ m tO to O ft O O Pi © •H © ft tO 03 03 03 03 ft G H CO in H tO to in m - P - « * • •» • « • » > • CO N H H 02 03 03 to ra P* © 0 b H to tO CO cn CO in in 0 CO 10 H O lO in CO 0 H • • « > 0 ° ® • 0 ;d H to in in m to 0 • p ; PiM © cn 03 cn in in •. ^ 03 ft H in cn tO to 0 cn H O - p •H 9 - « e « > 0 CO H 03 03 E> • : 03 ft g c • e C ST: f t ( C r c- O to to cn 02 in CO to H rH H H OJ 03 03 o • o to •g O H O H H H EH m •0 P1 O O to 0 to o E - CO cr» N 0 : O r-i - * • * 0 0 O w tO tO to £> o N o -P H Pi G\2 <» w 10 l O cr> CO to co H to cv to •rH •'0 e - « • « " « CO a r-i to to to to o & <T> 00 to m cr> o CO o • • • • -• -.« - « •« o 02 £j tO to tO tO O CV3 • p & MOM ep tn CO ^1 in to u rH CO CO CT» - p • « • « * • • « ' 0 CO LO to t O so to © H 10 H in P* to to 0 .- * • 9 - 9 * » 0 H rH in in. to O 03 O T - i O +=> •P O ' p , Cfl (D H tO CO i-t in F4 H CO HI tO CO -P "Mi • - « • • 0 - • ' • ' a CO "Mi W CO m 83 m Pi <>a tO Hi to to O CO CO O 00 to O 03 H - • ••• * © • « • « 8TJ lO to m to 0 0 -P g • ft d) £- ^ » in to H H 0 0 0 -P •H . .» e - • * • ' « CO to 10 to 03 p 1 ; : 10 er» tO to •0 w to CO to to a HI ": * • • - * ••« 0 0 10 tO to tO to 0 •P M P. © w HI to m H to CO cr> 0 CO - p - • • • e • • • - ft CO H m lO tD to tO 03 o to to a% r-i H 03 O h t ! | i i | i | : T | l | r h r J r : t i l i | t - i | ! : ; l l ! : u | : ! : : | : : : r | i . ' ! - ; l ^ ! of the experiment. I t i s perhaps worthy of note that the sudden drop i n r a t e of a c i d production'in milk i s greater than i n yeast m i l k . The r a t e of increase i n the case of Streptococcus l a c t i s i s much greater than Streptococcus cremoris. Streptococcus K "behaved i n a very s i m i l a r manner t o Streptococcus cremoris w i t h the exception that the ra t e i s However, much greater "both i n milk and yeast m i l k . r£'ga$ns'S: i t may he noted that the yeast m i l k c u l t u r e dropped below the mi lie c u l t u r e between the seventeenth and twenty-second hours. Streptococcus RW22 shows a s l i g h t s t i m u l a t i o n by the yeast m i l k , but not n e a r l y as marked as Streptococcus cremoris, l a c t i s or E. The response of t h i s organism t o yeast e n r i c h -ment might be s a i d to be intermediate between the stock Streptococcus cremoris and Streptococcus K. Streptococcus •^ 21 &oes not appear to be a f f e c t e d t o any appreciable extent by the yeast e x t r a c t . The f a c t o r or f a c t o r s to which yeast extract owes i t s s t i m u l a t i n g p r o p e r t i e s apparently are present i n s u f f i -c i e n t q u a n t i t i e s i n normal cow's m i l k to s a t i s f y the requirements of Streptococcus H^l ©ven i n the e a r l i e s t stages of growth, while Streptococcus cremoris, l a c t i s and E appear to r e q u i r e a d d i t i o n a l stimulus to induce a r a p i d fermentation i n the f i r s t few hours of incubation. I t i s known that a l l three of these organisms, when grown i n mi l k c u l t u r e , produce . a quantity of acid equal to that produced i n yeast milk i f given an additional twenty-four hours of incubation. The results at 30° are very similar to those •obtaining at 20°, with t h e exceptions t h a t t h e yeast extract does not produce as marked a stimulation with Streptococcus cremoris, l a c t i s , E or R W g 2 , hut inoreases very s l i g h t l y t h e rate of acid production of Streptococcus Rgx» It would appear that a temperature o f 37° C. i s beyond the optimum for acid production of Streptococcus / cremoris whereas i t hinders to a lesser degree Streptococcus l a c t i s and E. Cultures R W g 2 and R g l do not seem to be affected by the higher temperature, and, i n fact, Rg^ appears to be stimulated to some extent. The results reported above seemed worthy of further study i n t h a t they confirm t h e results of Whitehead et a l (unpublished data) which have shown t h a t cultures R 2 1 and RWgg a r e very suitable as rapid producers of acid when used i n the manufacture of C h e d d a r cheese. As i t i s the general practice i n Hew Zealand cheese factories t o carry the starter mother cultures i n conical f l a s k s , i t was thought advisable to follow t h e rate of acid formation or the organisms under study i n flasks and to thus obtained recorded contrast the rate aieaser with that cMmMnm i n *est tubes previously. EXTERIMEJN TAJt Again, the milk used was obtained from the Massey College "herd. Six flasks (conical) containing 300 ces. of milk were used. Three were steamed for one hour i n flowing steam and three were autoclaved at 12 pounds pressure for 25 minutes. This was done to compare the rate of acid production i n steamed and autoclaved milk. The organisms used for t h i s experiment were as follows Streptococcus RWI—a fast acid-producing cremoris s t r a i n obtained from So. RWg2 used i n the previonas experiment by immunization with the s p e c i f i c phage. Streptococcus HP--cremoris s t r a i n obtained by the immuni-zation of Sc. R2I' a s above. Streptococcus K—cremoris, the same as used i n the preceding experiment. Each f l a s k was inoculated with 2 drops from a clotted milk culture of the respective organisms. The temperature of incubation was 20° 0. Ten oc. aliquots were drawn at different time intervals by means of a s t e r i l e pipette and the t o t a l t i t r a -table a c i d i t y was determined as described previously. The results are recorded i n Table IV and K.gores XVI-XVIII. DISCUSSION Jrom the results i n Table IV and shown graphically i n figures XVI-XVIII i t would seem that Streptococcus HP i s hindered i n i t s growth by autoclaving of the milk medium as 8 * S H O O EH 5 ol o o 03 B H EH <1 O H O i ro u 03 H LO 03 cn o cn H to cn. 03 LO W " o • o ' 0 • 0 LO tO LO LO cn H ft P" t<3 cn LO o o 02 o H O to LO to • « a • 0 • • l O LO to to t o rH CQ ft 0 OJ o 03 CO cn O to cn l O fc- £> 9 ' • ••• • • LO 03 to 03 H CQ ; ft 02 LO CO tO to O tO 03 03 cn o •- 0 " • -»- - • <'•• ,-44 to , LQ 03 , H to OJ H 03 £j Pi H £- 03 C- to O O CO to H r-i CO 1 o • » •• • • a » ' 9 O l H «-l 03 rW to H TO ft P>' CO 03 : <£) tO o o CO £> 03 03 W 0 - 9 » • -0 •• • • « CO H 63 o o H H H i d © © © !> cd Of © (-1 © H © o O O o 3 o o • 4= © 4= © 4=> © -p Pj pj 4= CO •3 CO •3 CO 1-•H - 1 0 -contraated - w i t h steaming f o r o n e hour, w h e r e a s t h e r e v e r s e " a p p e a r s t o be t h e e a s e with S t r e p t o c o c c u s EV/I a n d I I , The same c h a r a c t e r i s t i c curves f o r r a t e of a c i d produc-t i o n a r e obtained w h e t h e r t h e o r g a n i s m s be grown i n c o n i c a l f l a s k s o f m i l k o r i n t e s t tubes. T h e e x t r e m e s i n r a t e o f a c i d f o r m a t i o n s e e m to be l e s s - m a r k e d i n t h e f l a s k s a s c o n t r a s t e d with the t e s t tubes. The c o m p a r a t i v e value o f steamed and autoclaved m i l k a s m e d i a f o r t h e s e S t r e p t o c o c c i w o u l d a p p e a r to be w o r t h y of f u r t h e r i n v e s t i g a t i o n . T he r e s u l t s c o n f i r m , i n a measure, t h e f i n d i n g s o f O r l a - J e n s e n ( 1 9 ) i n r e g a r d t o t h e i n f l u e n c e o f h e a t t r e a t m e n t o f m i l k o n c e r t a i n l a e t i c a c i d b a c t e r i a , , The q u e s t i o n of rate o f a c i d p r o d u c t i o n b y l a c t i c a c i d S t r e p t o c o c c i i n the m a n u f a c t u r e of c h e e s e i s of s u p r e m e im-portance . A c h e e s e s t a r t e r c u l t u r e m u s t possess the a b i l i t y to p r o d u c e a c i d r a p i d l y a n d a t a c o n s t a n t r a t e . The f i n d i n g s r e p o r t e d u p o n h e r e i n - i n d i c a t e c l e a r l y that, i n s t u d i e s on s t a r t e r organisms, a true p i c t u r e o f . the a c i d p r o d u c i n g a b i l i t y of a p a r t i c u l a r l a c t i c a c i d S t r e p t o -c o c c u s can o n l y be o b t a i n e d w h e n the d e t e r m i n a t i o n of t h e r a t e of a c i d p r o d u c t i o n o f t h e o r g a n i s m i s b e g u n a t t h e commencement o f the l o g a r i t h m i c p h a s e of growth. -8. A STUDY OH THE OF THE LACTIC ACID BACTERIA PART II . The. Rate of Acid Prod u c t i on of_ C e r t a i n  Lact i c Acid B a c t e r i a i n the Mi 1k of Indi v i dual: Cows on Ja r i a ble Rations. - 11 -PAST II In the l i g h t of the' work reported by Sadler e t al (15/ and by Or la-Jensen ( l j , and the r e s u l t s obtained i n P a r t - I of the present paper, i t seemed of i n t e r e s t to ascertain, i f the accessory growth f a c t o r s present i n the cow's di e t pass through the COY; into the milk i n pro p o r t i o n to t h e i r presence or absence in the r a t i o n . Ef f e et of pi e t on Ti tain in Content; Other workers have shown that the vitamin content of the diet has a marked e f f e c t on the vitamin content of the cow's milk. Wendt { 20) has demonstrated that the growth response of ra t s fed on milk obtained from cows r e c e i v i n g green feed or pasture i s f u l l y twice as great as the' response of those fed on milk produced from a hay r a t i o n , The same author showed that the response of rats fed on butter made from the corresponding milks was d e f i n i t e l y lower than the response from d i r e c t " milk, feeding. The r e s u l t s of his extensive and compreh-ensive experiments must be due to f a c t o r s other than the f a t ' soluble vitamins. Other i n v e s t i g a t i o n s described By Wendt {20} show that milft from cows fed on AIT sil a g e contained.} i n the r-mou-nt ingested d a i l y by a r a t , 34 rat units of Yi tarn i n A as compared with 17 units i n milk from cows fed on hay. Milk from cows fed on a hay r a t i o n • - 12 -produced a weight Increase of 102 frame In contrast with 140 grains increase from i l ? milk. This author points out that"the small d i f f e r e n c e In the f a t - s o l u b l e f a c t o r s could not account f o r t h i s d i f f e r e n c e i n growth r a t e , Hoed {21}, discuss i n g the r e s u l t s obtained at the United States Department of A g r i c u l t u r e farm at B e l t s v i l l e during the f i s c a l year 1933, st a t e s ; "The greener the hay fed, the higher i s the content of a s s i m i l a b l e carotene as shown by v a r i a t i o n s i n the yellowness of butter from cows fed hays of d i f f e r e n t q u a l i t i e s - .. ." -— Further, that "the e f f e c t of pasture on the vitamin content of milk p e r s i s t s f o r some time a f t e r dry feeding i s resumed. . . . . . Cumulative data show that c e r t a i n types of roughages are d e f i c i e n t i n f a c t o r s e s s e n t i a l to the normal growth and reps? ID clue t i on of dai ry c a t t l e , The B r i t i s h Medical Research Council Report Sn t i t i e d , " Y i t amine; ii Survey of Present Knowledge"{23} o f f e r s the f o l l o w i n g summary of present knowledge as regards conditions determining the vitamin value of mi lies "The vitamin value of raw cow's milk v a r i e s g r e a t l y according to the diet of the cow. There i s an extensive l i t e r a t u r e on the s u b g e c t, to which only b r i e f reference i s here p o s s i b l e . luce made an important i n v e s t i g a t i o n on- the Influence of sunlight and d i e t on the a n t i r a c h i t i c and growth-promoting p r o p e r t i e s of the cow's milk. The cow was i n successive periods kept in the l i g h t and i n the dark -13-and was fed on fresh green grass and on a dry winter feed. It was found that the Vitamin A oontent of the milk was much greater when the cow was receiving fresh green food than when the diet was one of cereals and roots. The Vitamin D content was found to depend p r i n c i p a l l y on the degree of insolation of the cow9 hut there was some indication that in s o l a t i o n accompanied by a diet of fresh green food, i . e . , when the cow was on pasture i n summer, was more effective than a similar degree of insolation combined with a diet composed only of cereals and roots* A comparison of the value of human and cowis milk (Outhouse et al). when the cows were fed on dry fodder, which, however, included ensilage and a l f a l f a hay, showed that the Vitamin A value of cowt's milk was about the same as human milk; for B vitamins the value of cow's milk was s l i g h t l y better, although the cows had l i t t l e exposure to sunshine. The values found f o r Vitamin £ did not approach those found i n the investigation of OhicK and Hoscoe. ©uthouse et a l give a brief summary of the l i t e r a t u r e pointing out the d i f f e r i n g results of various workers according to the diet and environmental conditions of the cow on which the work was done. It may, however, be accepted that the value of the cow-»s milk i n fat soluble vitamins i s at i t s maximum when the cow i s on pasture i n summer. It has been shown that the Vitamin A and Vitamin D -14- , contents of winter'milk can be enhanced by feeding the cows with cod* l i v e r o i l , and the Vitamin D content by administering i r r a d i a t e d yeast. Differences of opinion exist as to the effect on the a n t i r a c h i t i c potency of i r r a d i a t i n g a cow with u l t r a - v i o l e t rays from an a r t i f i c i a l source. The inference from what has been said i s , however, that the application of u l t r a - v i o l e t rays to the skin of the cow would be expected to r e s u l t i n increasing the Vitamin 3) value of the milk. Such a result has been obtained by many workers, but Steenbock et a l , although finding that the milk of goats could be so affected, were unable to demonstrate an increased a n t i r a -c h i t i c value i n cowTs milk by a r t i f i c i a l i r r a d i a t i o n of the animal. In one of th e i r experiments the milk of cows exposed to sun without change of diet from May 26 to June 16 showed no improvement as the result of insolation, but the milk of the same oows at the end of a summer on pasture was markedly superior. The Vitamin B and Vitamin G contents of cow's milk appear also to vary with the diet of the animal. Kennedy and Dutcher found that 10 cc. of milk from a cow on an adequate ra t i o n would supply an adequate d a i l y provision of B vitamins for a r a t , while 15 cc. from a cow on an inadequate diet would not. The antiscorbutic value was found to vary more widely; i n experiments with guinea pigs, £0 cc. of summer milk were found superior i n n u t r i t i v e and i n antiscorbutic potency to 60 oc* of winter milk. A similar difference was found "by Hess et a l . I t must be recognized that cow,s milk i s a variable food s t u f f , of which tbe vitamin value i n any given instance cannot be assumed. Its use i n infant feeding must be s p e c i a l l y safeguarded, not only i n the use of milk products for the purpose of modification, but the addition to the c h i l d * s diet of the same supplements as are recommended for the breast-fed infant. i ne r a l s i n the ease of the two chief mineral constituents of the milk, the picture i s an e n t i r e l y different one. Many workers, especially Meigs (24) and associates, and Orr (25) and associates, have carried on intensive studies on t3ae mineral metabolism of dairy c a t t l e . In general, i t may be said that the t o t a l calcium and phosphorus i n cow's milk i s not influenced by changes i n d i e t . Evidence has been present! however, -to- show that there may be a variation i n the balance of the different chemical combinations of these two elements as normally found i n milk. li i ^ l X i L i McDowell (26), reviewing the effect of feed and plane of n u t r i t i o n on the butterfat of milk, states: "In spite of a l l t h i s work, however, the issue i s s t i l l not c l e a r l y defined, and the evidence of workers i n different l o c a l i t i e s frequently does not agree.... Many workers have f a i l e d to take note of the natural - 1 6 -f l u c t u a t i o n s i n the f a t content of m i l k from a cow r e c e i v i n g a steady d i e t . " The same author o f f e r s the f o l l o w i n g quota-t t on from the work of Sheehy (27) as a good summary of the .present p o s i t i o n : "Uor have any of the ordinary foods used on the farm any s p e c i f i e d q u a n t i t a t i v e e f f e c t on the milk f a t . i n t h i s respect the r e s u l t s f r a m sugar pulp, mangels, beetroot, t r e a c l e , wet g r a i n s , rice-meal, and green pasture herbage are con c l u s i v e . Roots i n large quantity are reported to depress the percentage of b u t t e r - f a t i n mi l k , and wet g r a i n s , f r e s h pasture and rice-meal are sometimes claimed to have a l i k e e f f e c t . That the e f f e c t i s not due to the agency of these foods per se, i s c l e a r ; but when the foods i n question are . added i n lar g e quantity to the d i e t of a cow which has been r e c e i v i n g an i n s i g n i f i c a n t amount of food, the animal responds w i t h an increased flow of milk which temporarily i s greater than the increased b u t t e r - f a t production, so that the percen-tage of f a t i n the m i l k for, a period a f t e r the change i s reduced below the previous l e v e l . She reduced l e v e l of the f a t percentage i s , however, of a very temporary nature......" The very b r i e f summary presented above must s u f f i c e f o r the present paper. I t i s s u f f i c i e n t , however, to show that c e r t a i n , at l e a s t , of the d i e t a r y constituents w i l l cause changes i n c e r t a i n of the m i l k c o n s t i t u e n t s . Does the same p i c t u r e hold-,true f o r those f a c t o r s normally present i n m i l k and apparently c o n t r o l l i n g the growth of c e r t a i n l a c t i c a c i d b a c t e r i a when m i l k i s used as a substrate? - 17 -\ An e x c e l l e n t opportunity presented I t s e l f to study a small p o r t i o n of t h i s question i n r e s u l t s obatined from f eedi ng*ta?ia 1 s i n progress at the Dairy Research I n s t i t u t e of Few Zealand where i t was the p r i v s l e g e of the author to work under p r o f e s s o r fra. Siddet, During the course of these t r i a l s i t was p o s s i b l e to obtain, d a i l y , skim milk samples from i n d i v i d u a l cows fed on varied r a t i o n s ranging from f u l l complements of concentrates to g r e a t l y reduced complements of concentrates plus pasture. The l a t t e r r a t i o n s might be looked upon as d e f i c i e n t i n some respects. Expc rIment al The milks used and reported upon In t h i s section of the paper were obtained from six cows on c o n t r o l l e d d i e t s The s e r i e s was designed to determine; ( a) w he t he r the i n c l u s -ion of f r e s h pasture to a r a t i o n of meals and hay fed to m i l k i n g cows e x e r c i s e s any i n f l u e n c e on the compostion and b i o l o g i c a l p r o p e r t i e s of the milk and ( b ) i f , on the-other hand, a subnormal plsne of n u t r i t i o n e x e r c i s e s any influence' on the composition of the milk. The various chemical and b a c t e r i o l o g i c a l determ- ' i nat ions made upon these milks were conducted by the s t a f f of the Da i ry Research I n s t i t u t e (!*."„) The d e t a i l s of the experiment were as f o l l o w s ; -Two groups of three cows were used: Group/, comprised? Lexicon - Ayrshire,, 3 years, 247 days o l d , calved 17/5/37 M a p l e b i r d - A y r s h i r e , 2 y e a r s , 272 flays o l d , calved 19/5, L a u r e l - A y r s h i r e , 3 years, 288 days o l d , Calved 14/5/38, -18-Group B comprised: Hiremai--Ayrshire, 6 years 264 days o l d — calved May 5/57; 'Marion—Ayrshire, 2 years, 248 days o l d -calved May 18/57; Juanita--i?re^i/sian, 4 years, 326 days o l d — . A calved A p r i l 4/37. In the f i r s t period, l a u r e l and Juanita were placed on the subnormal ration, i n the second period, lexicon and Heremai, and i n the t h i r d , l a u r e l and Juanita again received the subnormal r a t i o n . A l l cows received for maintenance a ratio n of hay up to a maximum of 16 pounds per day i n the case of ILaurel and Juanita and 14 pounds i n the others, supplemented by a concentrate mixture of equal parts of maize and crushed oats to provide s i x pounds of starch equivalent and 7.5 pounds of digestible protein per 1000 pounds of l i v e weight. The" cows receiving concentrates were fed a mixture made up of bran 1 5 $ , oats 1 5 $ , pollard 1 0 $ , maize meal 1 5 $ , pea meal 2 5 $ , and linseed cake 20$. This was fed at the rate of 5% pounds per gallon of milk produced. This amount provides approximately 2& pounds of starch equivalent per gallon of milk. The cows receiving concentrates and pasture received hal f of their production r a t i o n from the above mixture. The remainder was made up of pasture containing 7 5 $ of rye grass and 25$ of white clover, both cut while s t i l l short. The pasture was fed on the basis of i t s dry matter content, protein and energy value content. The two - 19 -La 11 e;r iwe re determined from the 1 l!--days analyses of the -pasture and Goodman' E ( 2§ ) c onve r s i on f a c t o r s f o r d i g e s t -i b i l i t y nd. starch value- While the maintenance dfee t f o r each animal was kept constant, the production r a t i o n v s r i e d a c c ording to the milk y i e l d . The b a c t e r i o l o g i c a l r e s u l t s , with w hi eh the present paper i s concerned may best be discussed•under two headings section I d e a l i n g with the rate of acid product-ion of- a Streptococcus cremoris s t r a i n (Hp) i n ' samples taken at i n t e r v a l s during the course of the t r i a l and with v i t a l i t y t e s t s using a m o d i f i c a t i o n of the \7hitehead and Cos: (29) method, and s e c t i o n II d e a l i n g wi th the a c i d production of c e r t a i n Betacocci i n the same milks<> SE0T10IT I, EXPEP.ITrFTT T A L For determination of the rate of ac i d production of Streptococcus cremoris (the Hp s t r a i n described fen page 9) composite samples were put up e i t h e r i n 10 cc q u a n t i t i e s in ordinary test tubes or i n 150 cc. q u a n t i t i e s i n 250 cc c o n i c a l f l a s k s . In a l l cases the milks were s u M c 1 ave d at 12 pounds pressure for 25 minutes. The ssme technique f o r the rate of acid production was used as described on page 6. The temperature of incubation,, size of i n i t i a l inoculum, and times of t i t r a t i o n were varied i n and attempt to obtain 'as much information as p o s s i b l e on the rate of acid- product ion of t h i s organism. The i n f l u e n c e of these varyous f a c t o r s such as temperature of incubation, size of Inoculum, . -80- ! method of putting up the medium, ana tie ration of the eow i 1 at the time the samples were taken, are shown in the tables 1 • i 1 * * * whioh follow. In a l l oases the aoid produoed i s expressed in grams per l i t r e of laotio aoid. 1 DETAILE F a l l M a i n t e n a n c e R a t i o n P l u s H a l f P r o d u c t i o n R a t i o n of P a s t u r e and C o n c e n t r a t e s . D KEY TO 3A5I0BS. USTiT) x'T FP VDT" | P u l l M a i n t e n a n c e R a t i o n P l u s H a l f P r o d u c t i o n R a t i o n of C o n c e n t r a t e s , TRIAL F u l l Hai.nter.anee-Ration of C o n c e n t r a t e s - p l u s -' F u l l P r o d u c t ! o h - R a t i o n F u l l M a i n t e n a n c e R a t i o n and P r o d -u c t i o n R a t i o n of Cow ITO. 1 j ' June 15 to J u l y 15 Aug. 16 to Se p t . 1 5 C o n c e n t r a t e s . J u l y 16 to Augvl-5 Cor Ho. 8 June 15 to J u l y 15 Aug. 16 to Sept.15 • • J u l y .16. to. Aug. 15 Cow JTo. 3 J u l y 16 to Aug. 15 Juno 15 to J u l y 15 Aug. -15 to Sept .15 C o\7 17 o . 4 1 J u l y 16 t o Aug. 15 June 15 to J u l y l 5 Aug. 15 to S e p t l 5 1 COB SO, 5 ! 1 June 1-5 to Septl.5 Cow !To. 6 — i j June 15 to. J u l y 16 ! °- r-™9<» '» *»*« e f f e c t and chow t t t ' » » « / « the doV' ' - : _J assumed t h a t s . m i l k v . -21-THE RATE Off AG ID PRODUCTION Off STREPTOCOCCUS  CREMORIS IN THE INDIVIDUAL MEZKS Off SIZ SCOTS  OU VARIAB2E RATIONS. lioop Inoeuluum, temperature £0° C., Test lubes. COW HO. RATION 17 HOURS 20 HOURS 36 HOURS I. •jb"- grass-meal 0.1 0,3 1.2- ' , I I . •jh-meal 0.1 0*4 5.2 I I I . Grass-meal 0*0 0.4 5.6 11. •:• Meal 0.9 2.0 6.0 V. Grass-meal 0.1 0:9 4.7 "71. Meal 0»8 1.9 6.0 TABSSS VI. ' Jane R loop Inoculum. Temperature 20° 0.. Flasks. COW no. RATION 17 20 23 26 HOURS '•I. Grass-meal 1.0 2.1 3.1 4.2 II. 0.8 1.0 2.1 3.1 III. Grass-meal X«*3 2.0 3.0 3.9 •. IY*' Meal 1.3 1.6 2.4 *3 • H V. Grass-meal 1.3 2.2 3.1 4.0 71. Meal 1.6 2.2 3.3 5.4 -23-TABXB YII^ _ Ju.ne Z% laoov inoculum. Temperature 20° Q.. Flasks COW NO. BATIOU 17 20 23 " 26 HOBBS v - .iv' : Grass-meal 1.4 2.8 4.6 5*2 /.IIv • i Meal 0.4 1*6 3.6 4.0 I I I . Grass-meal 1.3 2*6 4.3 5.9 Meal 1.8 8*4; -6*8 . 6.3 ; Y* - ' Grass-meal 1.5 3.1 4,7 5.8 Meal 3.7 6.0 6.4 6.5 -24-TAB1E ¥111. J o ^ e 26- Zooy Inoculum. Temperature 20° 0.. glasks. COW NO. RATION 17 19 22 24 HOURS • I . Grass-meal 2.6 4.3 5.6 6.0 £ Meal 2.7 4.2 5.7 5.9 i l l * . Grass-meal 1.7 3.5 5.7 6.3 IV. Meal 4.6 6.6 6.7 7.2 V. Grass-meal 3.2 5.6 5.7 5.9 Meal 3.7 6.0 6.4 6.5 - 2 5 -TAB1E I X . Oi*.ne, 2 7 Xioop Inoculum. Temperature 20° Q. 7 Flasks. cow no. RAEEOI 17 HOURS 20 HOURS 22 HOURS I . Grass-meal 2.2 5 . 1 6.4 I I . . f Meal' ; 2.6 . v4.t . .'• 5.4 I I I . Grass-meal 3.6 5.3 6.0' /' IV. Meal 4.0 6.3 6.6 ¥. Grass-meal 3 . 1 6 . 1 5*6 S I . Meal 3.4 6,0 6.2 T&31M X * • Juxvc 2?t So op Inoculum. -Temperature 20° G., Alaska. COW 10. RATIO! 17 22 24 26 HOURS X « Grass-meal 1*7-. 3.9 4.7 5.4 I I . t Meal 1«9 4.2 5 & *S 6*6 I I I . Grass-meal 3.0 5.0 5.7 6.5 IV. Meal 1.8 4.0 5.7 6.4 V. Grass-meal 2« 4>2 «3 « 9 6.0 : 71. Meal 2.9 4.5 5.6 6.3 iEABSB II. J uAy S l&oop Inoculum. Temperature 20° 0.. Flasks. cow n o . RATI OH 14 HOURS 16 HOURS 20 HOURS I. II • III a 17. >7. 71. Grass-meal '. Meal . Grass-meal Meal Grass-meal Grass-meal 4*2." 4*0" 5.3 5.0 4.4 5*3 4*8 5.0 5.6 ( .5.4 ' 4.8. 5.6 o« 5*5 6 o 3 6.4 5*8 6.0 TAME X I I . 2-° Soop Inoculum, Temperature 20° G.. F l a s k s . cow NO. RATION 17 HOURS 18 HOURS 20 HOURS i * I I . XXX o IY* . ' Y l . . Meal Meal-grass •|r Meal •§• Grass-meal Meal Grass-meal 1.26 1.17 2 » 97 1.7' 1.3 2.16 2«£*I3 4.7 4.4 3.0 2.9 4.S 4.23 TAME XIII* Juxy 2.7 loop Inooulum. Temperature 20° C.. Flasks* COW 10. RATION 17 HOURS 18 HOURS 20 HOURS I. Meal 1*8 0*8 4.7 I I . Meal-grass 1*5 2.8 4.5 I l l * 'jt Meal 1.9 3.7 4.8 IV. Meal-grass 1.7 3 . 6 4.9 V* . Meal- 2.9 4.6 4.8 F3iv<5 l b TABSE X E Y . ILoop Inoculum. Temperature 20° 0., Fla s k s COW NO. RATION • : 17 HOURS 18 HOURS 20 HOURS ' . 1 . ' •J- Grass^meal 3*2 3 . 6 I I . •§• Meal 1 . 3 . . I l l * . . Grass-meal 1.1 3.0 • 3.9 ..IY. Meal • ..t. Grass-meal .4.7 4.9 : Meal 1.2 2*9 4.7 -31-TAEIB XV. Ru^-20 Iioop Inoculum. Temperature 20° C., fllasks. cow n o . RATION . 17 HODBS.. ' 19 HOTBS 22 HI3U1S I * % Grass-meal .90 2,7 I I . % Meal .54 .72 1*4 I I I . Grass-meal 1.7 3.6 5.4 IV.. Meal .27 .72 3.8 /'' . V. 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S : .... t s r — i TS:. : x s : Hl S J C O V mma.1 J : S j S W — C — , " cj' : iS is S i • r s i iS S 7 S i iiii Si '1 f j rr i T;T: -'- i \ : • •• 1 Si Si L: S [ • ; IS-.1: n S 4s ; S S J - S L .j... : i r ; r - • V) S 'it :" i • \ V P' •i^ -ssf ttr_i iti ' issi It"-!. !. . 1 TS7T S J ; : ... ' : t | t t 1. - J " T7M£ S T ; : : r r/i Si •1 / Li"-"-! ! r I t . i . f. r*: i / S t S s : i : J ' 1-1 ! .... 1.. _x_LS COV\ 1 • • 1 ; ' i > i S ~ rri i i - i : . \ S ..••|----|. j- . o ':' I - s , • 1 * • s ' | i s •tr .:-• 1 • • ; - : j : : ! . i A '-• i-'jv E -0-1 • • I t : . . . — - "Sis' ' S S / ' , . i . -s t l t i .--J _ _. ' " j i " : ' isiiils - i i i s . i \ • • V i t • & mi • 1 •] . 1 .' M s ir« uxiiai: // -: • 11 -:'a I t •r - ^ /« ;.'p.:-: s i;:: •OW | : ti . • c • ! T : tj . . ' j : : • 1 m e a l ) : 1' 7. 7 S : i. ~ -s s 1 !'••; . •••I- :< :'st : : -j " : : »a -d ; • t i i. S L _ < A ! - . - i S : - s i ; : t : - : - _ . . ; 1" ' . '"SSi''- •T. 1 .•:h:ii • . I ' I I S -36-EXPEBIMEN TAIL ffor the v i t a l i t y t e s t i n g of the milks with Strepto-coccus SB21 the following technique was used;-The milks were pasteurized i n 150 cc. quantities i n glass "beakers at a temperature of 145° G. for 1/2 hour. After cooling to 34° C., ifo of a clot t e d milk culture of the organism was added. The milks were then ripened for one-half hour at 30° G. After the ripening, 0.2 cc. of commercial rennet were added and one hour was allowed for coagulation. At the end of t h i s time the curd was cut i n t o one-quarter inch squares by means of a s t e r i l e spatula, s t i r r e d c a r e f u l l y and placed at 57° G. After the expiration of two hours the whey was drained o f f . The "beakers were then returned to 30° G. for two hours. Mine cc. samples were then withdrawn for the determination of t o t a l t i t r a t a b l e a c i d i t y . This figu r e i s recorded as the f i r s t reading i n the tables which follow. The remainder of the whey was then poured o f f . After a f i n a l incubation period of one hour at 30° C., a second sample was withdrawn for a c i d i t y determination. This figure i s recorded as the second reading. The difference between these two readings i s taken as an index to the v i t a l i t y of the organism i n the respective milks. The results of the v i t a l i t y tests are recorded i n Table XVIII which follows. -37-DIS0¥SSIOH Considering the r e s u l t s as a whole, there appears to "be no d i r e c t r e l a t i o n s h i p between the milks of the s i x cows and t h e i r rations as determinable by means of the v i t a l i t y t e s t . I t must be noted, however, that there appears to be a s l i g h t c o r r e l a t i o n from day to day between each cow Ts milk and the rate of growth of Streptococcus HP i n that milk. That i s to say, the i n d i v i d u a l i t y of the animal appears to have a greater effect on the eowfs milk than does a v a r i a t i o n i n her r a t i o n . Exactly what determines t h i s i n d i v i d u a l i t y was not apparent from the r e s u l t s of t h i s experiment, but • most c e r t a i n l y points to some factor or factors which have not as yet been considered i n the study of the n u t r i t i o n of the dairy cow and the milkds<£ produces. -38-. VITALITY TESTS  Composite samples of evening and morning m i l k Of S i x COWS. Cow Feeding Experiment. TABLE XT! I I . Cow 1st 2nd Increase Cow 1st 2nd Increase 15/6/37 18/6/37 1 .43 ,73 ,3.0 1 .37 .71 .i-34 2 .40 .63 .23 2 » 31 .55 ,24 3 .42 .73 .31 . 3 ,39 .65 .26 4. .43 .80 .37 "', 4 .. .40 .72 «32 .43 .71 ,28 : 5 .42 : .65 »23 6. .48 .76 *28 '6 .45 .70 $. 25 SMP .29 .43 ,14 SMP ,24 .33 .09 16/6/37 21/6/37 1 .41 .67 ,26 1 : .33 . .68 .35 2 ,37 : .62 .25 2 • 28 .50 .22 .36 ,65 .29 3 .38 .65 *27 4 .43 ,69 .26 4 .38 ,69 .31 5 ,42 .63 .21 • 5 .37 «66 .29 6 ,37 .62 .25 6 .59 .69 .30 SMP ,24 .31 .07 SMP *25 .40 .15 S - K - P . = SKtt*\ MILK. P O W D E R Continued on Page 59 ....... -39-TABLE XVIII (cont'd) Cow 1st 2nd Increase Cow | 1st 2nd Increase 17/6/37 22/6/37 1 .34 .72 .38 1 ,32 .57 © 30 2 .28 .56 .28 2 .26 .50 .24 3 .37 .74 .37 3 .30 .43 .13 4 .38 .68 .30 4 .30 .48 .18 5 : .42 .74 .32 5 .30 .48 .18 6 - .41 ,79 ^ v38 6 ,34 .54 .20 ; SIP .25 ,37 .12 SMP .27 .39 «X3 23/6/3? 29/6/37 1 . ;S8 .77 .39 1 .37 .77 .40 2 ;34 ,63 *29 . J 2 . 26 .64 .38 3 .41 .75 '• «34. . 3 .35 .81 .46 4 .41 ,68 .27 4 »36 ;• .75 .39 5 .39 .69 .30 5 »38 .68 .30 6 .46 ,75 .29 6 *39 ,72 ,33 SMP .27 .42 .15 SMP ,29 .46 ,17 24/6/37 30/6/37 1 .36 .68 .32 . .39 , 82 ,43 2 .26 .38 .12 .29 .52 & 33 3 .32 .57 ,25 * .' 3 .76 o 39 .36 ,64 .28 4 .37 . .69 • 33 5 .36 .62 .26 5 ,41 . »«y .32 6 .38 .67 .31 6 ; .44 .76 .32 :SMP . .21 .2.8 .07 SMP .28 .42 .14 Continued on Page 40 -40-COY/ 1st 2nd Increase Oow. 1st. 2nd Increase 25/6/37 * 1/7/37 1 .32 • 63 • 31 1 .42 .71 .29 2 ,56 .31 2 .35 .67 9 32 3 .31 ,67 .36 3 .39 .79 ,40 4 : .35 .63 .28 4 .41 ,67 .26 5 .34 ,65 ,31 5 .39 .61 .22 6 .35 ,69 .34 6 ,42 . 61 *19 SIP . ; .18 .23 *05 SMP ,27 ,42 .15 28/6/37 2/7/37 I'"..' .43 • .79 .36 -1;- .43 ,80 ,37 2 »m': .26 ,34 ,65 .31 3 .40 .  .72 .32 3 ,33 .72 .39 4 ,38 ,70 : .32 ' ' 4 .'40 .78 » 38 5 .41 .74 ,33 ; 5 .41 : .75 .34 6 ,38 ,59 . 21 6 .32 .64 .32 SMP .27 .42 .15 /; SMP 4 24 ; ,39 ,15 5/7/37 12/7/37 1 ,30 .66 ,36 1 .29 ,72 »43 2 ,30 .61 .31 2 6 2 5 .60 © 35 3 • 36 .74 ,38 3 .30 .59 « 2S 4 .45 .67 • .22 4 .34 ,72 .38 5 .45 .67 o 22 5 .39 .70 .31 6 .41 ,65 • 23 6 .30 ,60 • ,30 SMP .25 ,39 SMP »23 .36 0 3.3 Continued on Page 41...... -41-TABLE XVI I I (cont'd) Cow 1st 2nd Increase Gow 1st 2nd Increase 6/7/37 13/7/37 . 1 .35 .69 ,34 1 .35 .57 Q 33 2 « 29 .56 .27 •"; 2 .27 .47 .20 3 .31 .59 ,28 3 .33 .60 .27 4 .37 .69 '•,32' ; 4 ,35 ,55 ,20 5 .38 .72 ;«4 ; 5 ,38 .55 ,17 6 .38 .72 -. ,34 6 .58 .19 S M P .28 .38 .10 S M P »'21 • . .28 .07 14th Feeds changed 8/7/37 15/7/37 1 .39 .64 .25 1 .30 .58 ] o 28 2 .33 .53 A 20 ' 2 ,24 .45 .21 3 .36 .70 .34 3 .27 .63 . 36 4 .42 : » 6 9 : .' .27 ' 4 ,33 .55 .22 5 . ,42 ,69 •] .27 . 5 .,' .37 .60 ,23 6 , i46-- .66 • .20 6 ' 0 3«3 ,57 ,24 S M P .29 : ,43 • .15 ; S E P ^ .24 ,37 ; ,13 9/7/37 16/7/37 ••'1 .39 ,61 : .22 ' ' 1 • 3 9 »63 I ;*34 ' .32 .56 9 3t) 2 .30 ,66 ,36 -3 •.; ,37 .72 ,35 3 .31 .68 m ' -j . 4'. .44 .64 .20 4 .34 .68 ,34 . 5 ' ; i45 .65 .20 5 .68 0 33 6 .46 .69 0 33 6 .37 .64 S M P . . .27 .39 .12 1 S M P .25 $ 39 • <14<. Continued on Page 42 -42-TABLE.XVIII, (cont'd) Gow 1st 2nd Increase 1' "• ' 'Oow . ; 1st 2nd Increase 19/7/37 26/7/3 7 . 1 -- — '• 1 ;32 .78 ,46 2 • 34: • .61 .27 2 .32 .70 ,38 3 .33 .69 • 36 3 & 33 .73 .40 4 .36 • 73 .37 4 .31 .63 $ 32 5 • 34. ; .69 .35 5 ,35 .68 .35 6 .35 ; .63 • 28 6 . 9 35 • 65 i28 SMP -.24 ; .31 ; .07 SMP ; .22 .29 ;07 20/7/37 27/7/37 i .36 • 66 ;50 . 1 • © 35 .72 ,57 2 .34 .70 .36 2 .32 i69 ,57 3 9 32 .74 .. »42 3 .31 • 70 .59 4 .36 .76 .40 ,33 *75 .42 5 .40 .72 •••'.•"••3.8. 5 • e 35 .71 .56 6 .38 .68 •' .30 6 ' • ,36 .60 .24 SMP . — • - - "• . SMP a 23 .33 -.10 22/7/37 29/7/37 1 • 32 .62 .30 1 9 23 .66 .57 2 • 27 .57 • 3d 2 »29 *64 I .35 3 .33 .54 • i2lV ; .28 .61 .53 4 .33 .••ss-' ; 4 ^31 i60 . © 29 5 .33 .56 » 23 5 ,51 • 61 ,30 6 .34 .60 .26 . . j 6 • 32 , 65 i33 SMP .23 .33 SMP • 28 '..40 ,12 j Continued on Page 45. TABLE X V I I I (cont'd) • Cow 1st 2nd Increase Cow 1st 2nd Increase 23/7/37 2/8/37 . 1 .32 .78 ,46 1 • »33 .57 .35 2 .2? .66 ,39 ' " 2 .27 .53 ,26 3 .34 .78 ,44 3 .34 .70 .'36 4 .30 ,75 .,45 V . 4 ,29 .66 .37 5 .36 .67 -i'3i "• 5 ,32 .60 *28 6 \;35 ; .73 ,38 6 ,34 .60 ,26 SMP .25 .36 .11 : SMP ." • 33 a 33 ,10 3/8/37 1 .30 '•7.8 .42 $ o5 .70 & 35 2 «28 *65 ,37 2 .33 .60 .27 3 .35 .75 ,40 :• 2 ,34 ,68 .34 4 .26 $5 V ' • •. «31 ; 4 .36 .66 .30 5 .32 ,64 • 33 5 .35 .69 .34 6 .63 *31 6 $39 .64 ,25 SMP .25 . 33 ,08 SMP —-9/8/37 18/8/37 . 1 .42 .81 . .39 1 «31L • 61 2 ,33 ,64 ,31 :, 2 .24 , 39 9 >L5 3 .40 . .75 «35 3 .39 ,69 .30 4 .33 • 76 •43 4 .36 .65 .29 5 .40 .72 a 33 1 5 ,36 , 65 @ 39 6 » 38 ,66 • 38 1 6 .37 .65 .28 SMP .30 1 ,54 .14 | SMP $ 3 5 .34 ,09 Continued on Page 44 TABLE m i l (cont ' d ) • Oow 1st 2nd Increase Gow 1st 2nd Increase 10/8/37 19/8/37 1 .75 .42 1 .46 ,79 ,33 2 .37 .61 .24 • 2 ,32 .57 a 25 3 .32 .69 .37 .3 _ _ 4 .32 .60 a 28 • 4 ,40 .75 .35 5 • 39 .67 .26 5 .35 .75 ,40 A' .37 .62 .25 .43 .75 e 32 • . S M P ,, — - - S M P __ • ~~< • 11/8/37 20/8/37 : 1 ,35 ;: .77 .42 1 ,38 ,79 ,41 2 .26 .51 2 ,»34 .59 .25 3 .35 .78 .43 3 ,41 .86 .45 4 .37 .70 ,33 4 . .44 .81 .37 5 .37 .70 »33 5 ; .39 ,77 ,38 6 .38 : .63 »25 6 .48 .75 .27 S M P 23/8/37 S M P 30/8/37 .26 ,39 .13 1 .34 ; .6:7 ••: .33 1 ,31 .64 © 33 2 » 22 .54 e 32 2 0 22 ,46 ,24 • 3 .31 ,69 ,38 0 33 .63 •}.;' ,30 .34 ,66 .32 4 ,28 ,59 .31 •'• 5 ,34 .63 .29 5 .30 A 55 .25 6 .34 .62 ,28 6 .34 ,61 .27 S M P ~~ .. . - - ] ; S M P , .21 .30 -09 Continued on Page 45 -46-TABLEXYIII (cont'd) Cow .. 1st 2nd Increase Cow 1st 2nd Increase 24/8/37 31/8/37 - 1 .34 ,67 © 33 1 .28 ,66 .38 2 ,26 .50 .24 2 .24 .60 ,36 3 .38 .69 .31 3 .33 ,70 .37 4 .39 ,66 .27 4 .34 .63 ,29 .34 .64 .30 ' 5 .34 .64 .30 6 • 37 .66 .29 6 ,38 .66 ,28 SMP •20 .42 .17 SMP ,23 •40 . ,17 . 26/8/37 2/9/37 1 .27 ,61 .34 1 • 24 .48 ,24 .27 ^48 ,21 2 .30 • 0 3 ,23 3 .31 . • 64 .33 3 ,28 ,53 • 20 4 ft SO v63V:.; .28 4 i32 .57 .25 5 .35 * 67 #32 5 • 32 .60 .28 6 .36 ,61 • 20 6 •34 .54 ,20 SMP .34 ,34 *io SMP ,24 e 32 .08 27/8/37 1 .36 .64 ,28 2 • 26 • 01 • 20 3 .32 .69 .37 4 •36 .65 .29 5 .33 .63 : .30 6 .35 .63 ,28 SMP ,21 j .30 .09 SEGTIOK I I . EXFERIMEITAX #or the determination of r a t e of a c i d production of two Betacocci i n the m i l k s of these cows the f o l l o w i n g technique was adopted:-Skim samples were tubed i n t e n c c , q u a n t i t i e s and s t e r i l i z e d "by a u t o c l a v i n g at 15 pounds pressure f o r one-half . hour. (The severe s t e r i l i z a t i o n was used because i t was necessary t o avoid a l l chances of contamination as these c u l t u r e s were incubated f o r periods up to 21 days). The two organisms used, C u l t u r e s 173 g and 168g are described i n a l a t e r s e c t i o n ( ) of t h i s paper. One l o o p f u l of a c l o t t e d m i l k c u l t u r e of the r e s p e c t i v e organisms was i n o c u l a t e d i n t o each tube. The c u l t u r e s were incubated at 25° C. and 30° C. The temperature of i n c u b a t i o n , times at which a c i d i t i e s were determined, and t i t r a t a b l e a c i d i t y produced are recorded i n Tables X I I to XXIII. TAB IE XIX. BETACOQOUS 1682— INOCULATES AT 50° C. IE TEST TUBES. 7/7/57. Gow Hov 4 clays ,8; days . 11;. days .1- . 5«5 . ;.7.5 9.2 •• ' • > • 4.6 '' ' 9^. '/ io;i8 3 2*4 6;3 7,6 4 .-. 1*$.. 5.0 8.6 5 2 ,'6 •6.3 8; 5 6 5.5 7.1 8.4 •2. TABLE XX. BETACOOCUS 1 7 3 3 — INOCULATED AT 30° 0. IE- .TEST TUBES. 7/7/57.. Cow Ho*1 5 days 8.days • 11 days 19 days 1 •1*3 1.8 3*3 4.7 2 l a 8 3.3 4,6 7.3 ,: 3 . .0*6 •L © '5 2 » 3 6*4 4 0,7 1*9 2 © 3 4: © 4: 5 0*8 3,0 2.4 4.9 .6 1*3 3.1 2*5 4,-5 -43-T£SIK HI* BETACOOCUS 1732— INOCULATED AT 50°. C,.. 18/7/37 GOWUQ, 2 days . 5 days 14. days 1 0.7 • 4.9 0..4 4,9 0.4 3,0 4 0,6 ';;.4y5... 5 0.7 '••3.6. 6 0V7 4.1 TABLE XXII, BETACOCCUS 1682— INOCULATED AT 20° 0.t - 18/7/57. Oow Hq>! 8 days 14 days 21' days ; '..1 4i0 5.9 2 • 2,0 4i7 6.8 3 '' . iS •• : \ 2 $ 2 5 ,'8 • 4 0.7 : 2®?<§ 5,4 5 1.5 ' -ift- ' 4^ 6 6 0.9 24 6 3 iS TABLE X £ I I I . BETACOCCUS 1752—  INOCULATED at 20° .0 '.•, 18/7/57. Cow Hoi! 8 clays 14 days. 21 days .1 1.7 5.4 5.6 2 2*6 4i-8 7*2 5 1.5 5.9 5*5 4 - .' 1.7 • «3 » 5 5 2i0 3.8 6.4 6 !L9 o 5 o 2 4*5 - 5 4 -D I S C U S S I O N C o n s i d e r i n g the r e s u l t s as a whole t i t i s evident that l i t t l e or no • de tec table variation o ecu red in the milks of the s i x experimental cows, as determined by measurements of the ra t e of-' a c i d production, of Streptococcus cremoris, Betacooous 1732 and Betacoecus 168 2 when these org'an i sms are grown i n the r e s p e c t i v e rai1ks. i s stated p r e v i o u s l y when di scussing t he r e s u l t s of the v i t a l i t y t e s t s i t v? o u 1 d appear that there i s an almost constant a b i l i t y i n the milk of a s p e c i f i c cow t o permit a w e l l defined l e v e l of • i ' a c i d p r o d u c t i o n . For example cow Mo. g, r e g a r d l e s s of the r a t i o n ©he was r e c e i v i n g s appeared to produce a milk which permitted only a slow rate of a c i d p r o d u c t i o n and a comparatively low amount of t o t a l t i t r a t a b l e a c i d i t y . 11 i s of i n t e r e s t to note that t h i s i s the case v/i th wicthe Streptococcus cremoris and with the B e t a c o c c i , Z On the other hand Cow ITo. f produced a milk v/hech was d e f i n i t e l y more s u i t a b l e f o r acid, production than that of any of the other animals. Not only was her milk s u p e r i o r '-when she was r e c e i v i n g a f u l l r a t i o n - 5 5 -of grains or a f u l l r a t i o n of grains plus pastures but a l s o when she was r e c e i v i n g a d e f i c i e n c y d i e t l a c k i n g i n p a s t u re. The f a i l u r e to o b t a i n increased a c i d production ftn the milk of cow' s r e c e i v i n g pasture i s most p e r p l e x i n g when one c o n s i d e r s the r e s u l t s obtained by Wendt (20) and Seed (21) u s i n g s i l a g e and pasture i n r a t i o n s f o r winter milk p r o d u c t i o n . . On the besis of the thexJf r e s u l t s and on the r e s u l t s reported i n the Medical Research Council Report (23) one would expect a marked e f f e c t of. pasture on the vitamin content of the milk. Two explanations 'might be o f f e r e d i n explanation of the r e s u l t s reported here. F i r s t i t i s p o s s i b l e that the length of time the cows were on the s p e c i f i c feeds we s of i n s u f f i c i e n t d u r a t i o n to produce a marked v a r i a t i o n i n the a c t i v a t o r content of the milk. • That such a p o s s i b i l i t y must be considered i s borne out bTJ the f i n d i n g s of Reed (23) i n which he shows that, "the e f f e c t of pasture on the Vitamin. 1 content of milk p e r s i s t s f o r some time a f t e r dry f e e d i n g i s resumed". Since only ten days were allowed as a t r a n s i t i o n p e r i o d from one r a t i o n to another i t i s l i k e l y that the f a i l u r e to obtain the expected r e s u l t s i n t h i s experiment i s due to a " c a r r y over" e f f e c t such as noted by Heed. The second p o s s i b i l i t y which presents i t s e l f , ~ 5 6 -i s that the cow i s able to draw upon her own body supply" of growth f a c t o r s in order to maintain her milk i n p o s i t i v e n u t r i t i v e b a l a n u s . I t i s known that t h i s phenomenon occurs when the cow i s f e d a r a t i o n d e f i c i e n t i n minerals* A STUDY 05T THE METABOLISM OF THE LACTIC ACID BACTERIA. PART I I I . The P o s s i b l e S i g n i f i c a n c e of_ Bios an Vitamin B.^  i_n_ the Me t a b o l i SIB of the L a c t i c Acid B a c t e r i a . -59-PABT I I I . — T h e Possible Significance of Bios and Vitamin Bg i n the Metabolism of Certain l a c t i c Acid Bacteria. The marked effect of yeast extract on the rate of acid production of c e r t a i n l a c t i c acid "bacteria and the great v a r i a t i o n i n d i f f e r e n t milks when used as media for these organisms has heen reviewed and new evidence presented i n Parts I and I I of the present paper* It would he of i n t e r e s t to inquire into the agent or agents responsible for these variations and for the effect of the yeast extraot. A survey of the l i t e r a t u r e on growth factors for plants and animals brought to l i g h t the work which has been progressing for a great number of years on the metabolic requirements of yeasts. I t i s of interest to review t h i s work here as the r e s u l t s obtained i n many eases appear to be quite analagous to the r e s u l t s obtained with the l a c t i c acid bacteria. The work of S^ncke Knudsen ( 3 0 ) points to a symbiotic effect i n K e f i r between yeast and certain l a c t i c acid b acteria. He states that a normal fermentation similar to that produced by K e f i r grains, can be induced i f the l a c t i c acid bacteria i s o l a t e d from K e f i r grains be added to milk containing autolysed yeast (yeast extract). Ho such fermen-t a t i o n i s obtained i f these bacteria are added to milk which does not contain yeast as an enrichment. This work would -60-indioate that the yeast c e l l s provide some factor or factors e s s e n t i a l * f o r the growth of the l a c t i c acid bacteria. Pasteur (31), as a re s u l t of his extensive studies on the metabolic a c t i v i t i e s of yeasts, advanced the hypo-thesis that these c e l l s require only yeast ash, ammonium s a l t s and a fermentable carbohydrate for normal reproduction and fermentation. Duclaux (3E), repeating Pasteur's experi-ments, confirmed the observation that yeast c e l l s may use ammonium'tartrate as a source of nitrogen,and thus refuted the statement of Milion:(331, that P a s t e u r 1 s y e a s t did not u t i l i z e the nitrogen i n the medium, but that t h i s nitrogen evaporated into the a i r . Pasteur *s medium consisted of 100 grams of sucrose, 100 ces. of water, 0.1 gram of ammonium tar t r a t e and 1 gram of yeast ash. This medium,when inoculated with a b i t of yeast the size of a pin-head, produced good growth. A small inoculation induced a very slow fermentation occuring over a long period, while a larger inoculation resulted i n very heavy evolution of gas, the o r i t e r i o n used by Pasteur i n determining the course of fermentation. Very l i t t l e c r i t i c i s m fS Pasteur's hypothesis appeared u n t i l 1871 when i-eibig (34 J, the exponent of the older theory of fermentation, contested the statement that yeast would grow on a medium free of organic material other than the added c r y s t a l l i n e carbohydrate. ILeibig's remarks -61-were prompted by the r e s u l t s of h i s experiments i n whioh he was unable to obtain s a t i s f a c t o r y growth of yeasts, using the conditions of Pasteur*s experiment* The f a i l u r e of i e i b i g ^ s yeast to grow i n the synthetic medium opened wider the breach between the two workers, and introduced a new polemic between t h e i r laboratories. Pasteur's reply to ILeibig i s best stated i n the words of Pasteur himself i n which he offered, "de f a i r e devant ILeibig une quantite aussi considerable q u ' i l pour r a i t raisonnablement l'exiger avec un milieu exclusivement mineral." Unfortunately l e i b i g , who at t h i s time was unable to t r a v e l as a r e s u l t of h i s great age,, did not a v a i l himself of the opportunity presented, with the r e s u l t that Pasteur emerged the v i c t o r . I t was not u n t i l 1901, as a result of the researches of WiIdler (35) that a s a t i s f a c t o r y explanation of these c o n f l i c t i n g views was obtained. Wildier, i n attempting to study the synthesis of various organic phosphorus compounds (phosphates, nuoleins, l e c i t h i n s ) by yeast, was unable to obtain growth ot fermentation when a very small inoculum of yeast was used. The medium consisted of the following constituents:-Water.... 200 grams Sucrose 20 " Magnesium sulphate <...). Potassium chloride. ) 0.5 " Ammonium chlorhydrate............) i Calcium carbonate 0.1 - 6 2 -When a sufficiently small inoculum was sown into this medium the yeast cells failed to grow or appear in the normal time. The addition of small quantities of sterile "beer wort to this 'medium, resulted in rapid growth accompanied by fermentation of even, the smallest inoculum used. The ash of yeast cells did not supply the factor necessary for growth, ana thus Wildier concluded that the unknown entity was of an organic nature. To it he assigned the name "Bios" with the remark, "Puisse ce nom bientot faire place a une nom ohimique." To this unknown substance the following properties were assigned:-(1) soluble in water; ( 2 ) insoluble in absolute alcohol and ether; 8 0 $ alcohol permits a good extraction of Bios; (3) not present in Yeast ash; (4) not destroyed by boiling for one-half hour with a dfo solution of sulphuric acid, destroyed by treatment with a 20$ solution; (5) changed by boiling for one-half hour with Vfo solution of sodium hydroxide; (6) not precipitable by lead acetate; phosphotungstio acid; phosphomolybdic acid; or silver nitrate; (7) dialysable through parchment paper; ( 8 ) contained in ideibig^s meat extract, commercial peptone, and in beer wort; (9) Bios is not present in urea, asparagin, analine, tyrosine, nucleinbases, adenine, quanine, thymus nucleic acid, creatine, peptic and tryptic digestion products of chemically pure albumins, edestin, and ovalbumin. Thus the d i f f e r e n c e between Pasteur and ILeibig i s explained by W i l d i e r on the basis that Pasteur used a l a r g e inoculum, w h i l e ILeibig used a smaller b i t of yeast. I n c o n c l u s i o n W i l d i e r s t a t e s : "Ires levures de b i e r e pour se developper et fermenter, doivent etre c u l t i v e e s dans un m i l l i e u q u i , outre de sucre et l e s s p r i n c i p e s mineraux, contienne une substance inconnue en quantite minime, mais indi s p e n s a b l e . ( C o r r e c t i o n a l a l o i de P a s t e u r ) " . While W i l d i e r had apparently s e t t l e d the l e i b i g -Pasteur controversy, he introduced an unknown e n t i t y i n t o the study of yeast metabolism which has r e s u l t e d i n count -- l e s s papers of a h i g h l y c o n t r o v e r s i a l nature. Almost immediately a f t e r the p u b l i c a t i o n of the Bios paper-, Fernbaoh (56} suggested that W i l d i e r *s r e s u l t s were due to the presence of t o x i c m a t e r i a l s i n the medium. He b e l i e v e d that such i m p u r i t i e s might have been introduced w i t h t h e c o n s t i t u e n t s of the medium or might have been due to t r a c e s of copper i n the d i s t i l l e d water. According to Windisch (37), JPernbach s t a t e d that the only organism which i s known t o grow b e t t e r i n m i n e r a l medium than i n a n a t e r a l one, i s A s p e r g i l l u s n i g e r , and that i t may s a f e l y be assumed tha t yeast i s at a d i s t i n c t disadvantage under these c o n d i t i o n s . -Henry (38) was also unable to confirm W i l d i e r rs c o n c l u s i o n . According t o Pringsheim (39), Henry added 5 -64-drops' of wort c u l t u r e s to 500 cos. of W i l d i e r Ts mineral s a l t -sugar medium and secured r i c h growth of yeast. K r e i g e r ( 4 0 ) offered the f i r s t r e a l l y c o n s t r u c t i v e c r i t i c i s m of W i l d i e r s s paper when he s t a t e d that i t is. necessary to take i n t o c o n s i d e r a t i o n the whole physiology of the yeast c e l l . C o n t r a d i c t o r y statements i n W i l d i e r T s r e s u l t s made i t d i f f i c u l t to know just what W i l d i e r intended t o say about the formation of new Bios and i t s disappearance from the medium. Windisch (41) a l s o opposed the views of W i l d i e r and again introduced the question of t o x i c m a t e r i a l s i n the medium. On the other hand, Amand (42) r e p l i e d t o the c r i t i c i s m s of Windisch and other German workers, showing that the water had no i l l e f f e c t s on the yeast c e l l s . I n r e p l y , Windisch (43) suggested that the sugar used by Amand might c o n t a i n the t o x i c i m p u r i t i e s , laumann(44) f u r t h e r c r i t i c i z e d Amand 1s work by s t a t i n g t h a t the r e s u l t s of t h i s worker suggested that the yeast c e l l s consumed Bios without the production of any new B i o s . Uaumann claimed that had Amand prolonged h i s experiments somewhat, he could have shown the presence of " B e a u t i f u l B i o s " i n the form of s o l u b l e p r o t e i n n i t r o g e n . Windisch (45) i n a l a t e r paper, suggested that the a c t i v a t i n g e f f e c t of yeast e x t r a c t was due to i t s p r o t e c t i v e a c t i o n on the t o x i c m a t e r i a l s . This point i s r e a d i l y shown to be i n c o r r e c t , s i n c e the b o i l i n g of p r o t e i n s f o r one-half hour with ifo sulphuric acid destroys their protective action while Bios i s allegedly unaltered by such treatment. Amand (46) showed that yeasts remove Bios; from the 'medium, the amount removed being dependent upon the richness of the medium upon which the c e l l s were growing. Amand also introduced the question as to how the Bios was u t i l i z e d . Is i t destroyed as are the carbohydrates or i s i t used synthet-i c a l l y as are the nitrogenous compounds? lAndner (47J contested the assertion of Wildier / that substances such as urea and peptone did not activate yeast c e l l s . As a result of h i s experiments, he claimed to have shown that urea and peptone were just as active as Bios. Eossowicz (48) i n a series of c a r e f u l l y controlled experiments, found that with a- small inoculum,' a very slow development takes place, that after a month or two the number of c e l l s i s quite large. Carbon dioxide formation was i n d i s c e r n i b l e with the naked eye* His data led him to conclude that the mass of yeast crop seemed to depend some-what on the presence of some organic compounds. He stated that the addition of 1-B ces. of s t e r i l e beer wort accelerated the development of the yeast; without the wort tconditions being equal, a weaker development resulted. She presence of similar activating, substances i n s t e r i l e mold mycelia was also shown by t h i s worker. following the work of Eossowicz, Amand and Ide (49} -66-found that bacteria exert a favourable effect on the growth of yeast. Martinand (50} also sh of PeniciIlium glaucum. Other workers (Muller-Thurgau (51) • and Behrens (52)}, however, showed that t h i s mold may exert an i n h i b i t o r y influence i n a good medium such as grape must. B z i e r z b i c k i (.55)) reported that humus had a favor-able action on the growth and fermentation of small amounts of yeast. Pringsheim (54| introduced another explanation: he stated that yeast may acclimatize themselves to mineral-salt-sugar solutionSfgand develop therein, whether one used a heavy inoculation or Just one c e l l . A greater expenditure of energy was involved "on the part of the yeast when the yeast was supplied with nitrogen, sulphur and phosphorus i n the form of mineral s a l t s than when supplied with organic compounds. According to Pringsheim, Bios was probably protein i n nature, and a protein which was e s p e c i a l l y available to the yeast• Here, as i n the. paper of Uaumann, as well as i n several others, the writers derived incorrect conclusions from t h e i r experiments i n that, had they been f a m i l i a r with the work of Wildier, they would have known that Bios i s not pr e c i p i t a b l e by any of the common protein precipitants, and, therefore, could not possess a proteinaceous nature. Ide (55) f a i l e d to confirm Pringsheim's conclusions i n regard to acclimatization to ammonium s a l t s . -67-The work of Devloo (56) i s extremely i n t e r e s t i n g i n that his researches were carr i e d out i n the l i g h t of a complete understanding of Wildier"s r e s u l t s , and that h i s e f f o r t s were the f i r s t r e a l l y thorough attempts made to obtain active concentrates. Devloo's paper gives evidence of an extremely exhaustive and painstaking search, and may best be reviewed by recording i n t h i s own words the conclusions which he reached:-"Le prinoipe a c t i f du bios de Wildier est une molecule qui se trouve dans les l e c i t h i n e s t e l l e s qu'on les a preparees j u s q u H c i . "G "est une base azotee sans rapports acec l a oholine, e l l e est probablement monoazotee, une amine presentant encore un H l i b r e du r a d i c a l ammonique: c e l a r e s u l t de ses caraoteres indubitables d'etre precipitable par HgGlg plus BafOHJg, et t t r e soluble dans l a solution d'aoide phosphomolybdique• "]Les caraoteres chimiques connus peu vent £tre resumes comme s u i t : (1) E l l e est t r e s soluble dans l'eauj (E) E l l e n'est pas d i s t i l i a b l e ; (3) l e s chlorhyratev, sulfate et oxalate correspondents sont soluble dans l'eau et dans 1'aleool a 75$$ (4) B i l e se l a i s s e p r e c i p i t e r pas le HgClg neutralise par BaOHg sous forme d'un compose mercuriel blanc dont l e mercure est diffiollement enleve au complet -68-: " s i l*on n I a g i t pas sur l e produit assez purif i e . Toutes les autres methodes qui nous l a donnent sous ferine de p r e c i p i t e ne nous l a l i v r e n t pas ous une form acceptable. E l l e se l a i s s e p r e c i p i t e r incompletement par l ' a l c o o l a p a r t i r de 80°, et par un melange des exeipients suivants; 2 volumes a l c o o l 80% , avec 1 volume de ether, d"acetone ou de chloroforme, ou a l c o o l 80% avec l r e t h e r , lacetone ou le chloroforme en grande quantite." Several contemporary papers (57J had appeared with arguments pro and con with, regard to the question of the toxic e f f e c t s of copper on the yeast c e l l s . Irom an exami-nation of these papers i t would appear evident that the f a i l u r e of Wildier 's yeast to grow i n synthetic media was not due to t h i s toxic f a c t o r , since arguments i n favour of such an hypothesis are more than answered "by the c a r e f u l work of 4mand, Ide and Devloo. In a review of t h i s type i t i s necessary to give at least a "brief summary of the vitamin l i t e r a t u r e i f the reader i s to observe any r e l a t i o n s h i p between the growth factors f o r the higher animals and the material which would appear to be e s s e n t i a l f o r the growth of yeast. •For many years p r i o r to the discovery of. the vitamins, i t had been oustomary to estimate the n u t r i t i v e requirements of the animal organism i n terms of what had -69-long been regarded as the four fundamental food units, namely, protein, carbohydrate, f a t and inorganic material, and to underestimate, i f not e n t i r e l y neglect, the possible s i g n i f i -cance, of other l e s s c l e a r l y defined dietary constituents. Many attempts have been made to feed animals ofi various types on diets made up of the four main food consti-tuents i n pure state. Oddly enough i t was through experiments of t h i s nature that the f i r s t suggestion of unknown e n t i t i e s e s s e n t i a l for normal metabolism came. l u n i n (58), i n 1881, while investigating the significance of inorganic s a l t s i n the n u t r i t i o n of the animal, found,as a result of some of his experiments, that whereas adult mice could l i v e for several months i n good health on a diet of milk, they i n v a r i a b l y died within a month i f they received a r a t i o n composed of what he believed t o be the essential ingredients of the milk. In h i s conclusion he remarks: "Mice can l i v e quite w e l l under these conditions when receiving suitable f oods (e.g. milk), but as the above experiments demonstrate that they are unable to l i v e on proteins, f a t s , carbohydrates, s a l t s , and water, i t follows that other substances indispensable for n u t r i t i o n must be present i n milk besides casein, f a t lactose and s a l t s . " Oarl Voit (59),probably the foremost student of t h i s subject of proteins, draws to our attention the necessity fo r not considering proteins as though each were chemically -70-homogenebus and within wide l i m i t s , p h y s i o l o g i c a l l y i n t e r -changeable. A quotation from Voit cautions us to. seek an accurate knowledge of the food intake i n the study of metabolism. He says: "Zu dem Zwecke ware es u n s t r e i t i g am besten, kennte man nur reine, chemische Verbinduggen (die reinen Uahrstoffe) z.B. reines Eiweiss, f e t t , Sucker, Starkemehl, Aschebestandtheile, oder Gemisch deselben geben. Da aber die Mensohen und auch die Thiere nur selten solche geschmacklose Gemenge auf die Bauer aufsunbexmen oder zu ertragen vermogen, so b l e i b t fur die meisten Pa l l e nichts anderes ubrig a l s schon durch die Katur zusammengesetzte < Mischungen (die Hahrungsmittel) zu wahlen. Jedooh ware es wohol moglich und ganx verdeenstvoll, die Grundversuoh, nachdem vorher der Weg mit H i l f e der letzteren Mischugen gefunden worden i s t ^ mit den reinen Soffen zu wiederholen 9 obwohl s i c h dabei s i c h e r l i s h im Wesentlichen keine anderen Resultate ergeben werden• " It was through such studies as Volt's along with Bunge (601 and his co-workers, that a f i r m foundation was set f o r l a t e r work i n the f i e l d of vitamins and growth f a c t o r s . Space does not permit a detailed survey into the early work dealing with the n u t r i t i o n a l e f f e c t s produced by the feeding of pure proteins and the consequent experiments on amino acid metabolism. Information upon this phase of early n u t r i t i o n studies can best be obtained by consulting the reviews of Osborne and Mendel (61), Bunge, Funk (62) and others -71-Many e a r l y experimenters were l e d to conclude that f a i l u r e of l a b o r a t o r y animals t o l i v e and grow on s y n t h e t i c d i e t a r i e s was due to t h e i r monotony or l a c k of f l a v o r i n g . I n the c l a s s i c monograph of Osborne and Mendel (61) are described the r e s u l t s of innumerable c a r e f u l l y c o n t r o l l e d feeding experiments using p u r i f i e d d i e t s . With regard t o these experiments they say: "Although these apparently success-f u l experiments i n d i c a t e d that the combinations of i s o l a t e d food s t u f f s employed s a t i s f i e d the n u t r i t i v e requirements of , the r a t s , and consequently c o n s t i t u t e d a complete food f o r the maintenance of mature animals, a p r o l o n g a t i o n of the obser-v a t i o n s had l e d t o a l e s s f a v o r a b l e outcome. A c o n t i n u a t i o n of the experiments over longer periods has shown th a t i n every case, sooner or l a t e r , the animal R e c l i n e d , and unless a change i n the d i e t was not i n s t i t u t e d w i t h i n a comparatively short time, the animals d i e d . Stepp Ts (63)' work i n d i c a t e s an explanation of these r e s u l t s : Stepp found t h a t whereas mice l i v e s a t i s f a c t o r i l y f o r s e v e r a l months upon c e r t a i n foods, such as wheat bread made w i t h m i l k , they are unable t o l i v e longer than a month when f e d upon the same d i e t a f t e r i t had been subjected t o prolonged e x t r a c t i o n w i t h a l e o h o l and ether. That the e x t r a c t i o n i t s e l f had only lowered the n u t r i t i v e value of the f o o d s t u f f s by removing some e s s e n t i a l component was demonstrated by r e s t o r i n g the ex t r a c t t o the extracted food when i t onoe again became adequate f o r the n u t r i t i o n of mice. -72-He made'numer ous attempts to determine the nature of the unknown factor and concluded that i t was a l i p o i d soluble substance." She c l a s s i c a l experiments of Hopkins (64} i n Great B r i t a i n are of considerable i n t e r e s t . As early as 1906 he wrote as follows: "But further, no animal can l i v e upon a mixture of pure protein f a t and carbohydrate, and even when the necessary i n organic material i s c a r e f u l l y supplied, the animal s t i l l cannot f l o u r i s h . The animal body i s adjusted to l i v e whether upon plant tissues or other animals, and these contain countless substances other than the proteins, carbohydrates, and f a t s . Physiological evolution, I believe, has made some of these w e l l nigh as essential as are the basal constituents of the diet; l e c i t h i n , for instance, has been repeatedly shown to have a marked influence upon n u t r i t i o n , and t h i s just happens to have been t r i e d . The f i e l d i s almost unexplored, only i t i s c e r t a i n that there are many minor factors i n a l l d i e t s , of which the body takes account. In diseases such as r i c k e t s , and p a r t i c u l a r l y i n scurvy, we have had for long years knowledge of a d i e t e t i c f a c t o r , but though we know how to benefit these conditions empirically, the r e a l errors i n the diet to t h i s day are obscure. Hopkins (66J found that young r a t s could not survive on a synthetic diet even when t h i s diet was s u f f i c i e n t to maintain them i n p o s i t i v e n u t r i t i v e balance of a l l necessary -73-food materials. The addition of a very minute quantity o£ milk to t h i s diet permitted normal growth. Hopkins stated: "It i s possible that'what i s absent from a r t i f i c i a l d i ets, and supplied by such addenda as milk and tissue extracts, i s of the nature of an organic complex (or of complexes) which the animal body cannot synthesize. But the amount which seems s u f f i c i e n t to secure growth i s so small that a c a t a l y t i c or stimulative function seems more l i k e l y . " "The attachment of such indispensable functions to s p e c i f i c accessory constituents of the diet may be foreign to current views upon n u t r i t i o n , so also i s the experimental fact that young animals may f a i l to grow when they are absorbing d a i l y a s u f f i c i e n c y of formative material and energy for the purpose of growth." Osborne and Mendel (66), as a r e s u l t of further experiments, stated: "Whether the deficiency of the purely a r t i f i c i a l diet i s to be attributed to improper proportions of i t s constituents, to improper combinations of these constituents, or to the lack of some essential element, i s at present d i f f i c u l t to define." They do not seem to have regarded t h e i r f a i l u r e as being e n t i r e l y due to deficiency of some essential element from the synthetic diets as they appear to have achieved considerable success i n feeding i n the absence of the hypothetical organic hormones. Hopkins and S e v i l l e (67) challenged these r e s u l t s -74-since they were unahle to maintain t h e i r animals on the diet used by Osborne and Mendel. In complete confirmation of e a r l i e r experiments, i t was found that the addition of a small amount of milk induced normal growth conditions. McGollum and Davis (68) confirmed the i n a b i l i t y of rats to grow on a synthetic diet consisting of casein, lactose, l a r d and inorganic s a l t s . They believed that cessation of growth was due to the absence of some organic complex from the r a t i o n . They found that the ether soluble f r a c t i o n of butter and eggs supplied the missing entity, but that olive o i l and l a r d did not do so. In conclusion they states "Our observation that ether extracts from c e r t a i n sources improve the condition of animals on suoh rations strongly supports the b e l i e f that there are certain accessory a r t i c l e s i n c e r t a i n foodstuffs which are es s e n t i a l for normal growth f o r extended periods. Osborne and Mendel (69), i n a l a t e r paper, concluded that natural milk contained some organic material e s s e n t i a l for growth and reproduction over long periods and not present i n the various synthetic diets they had previously used. Weight declines i n animals on synthetic diets could be arrested by substituting butter for la r d i n these r a t i ens. Krom these,results they concluded that the active p r i n c i p l e resides i n the f a t t y f r a o t i o n of milk. The apparent absence of nitrogen and phosphorus and any ash-yielding or water--75-soluble m a t e r i a l s l e d them t o conclude that the f a c t o r w i t h which they were d e a l i n g was e n t i r e l y d i f f e r e n t from the water-so l u b l e b e r i - b e r i f a c t o r being e x t e n s i v e l y studied by .Funk at the same time. Funk and Maoallum . (70) challenged these r e s u l t s and claimed t o have extracted t r a c e s of n i t r o g e n c o n t a i n i n g m a t e r i a l s from the product prepared a f t e r the manner of Osborne and Mendel (69). Osborne and Mendel showed (72) f u r t h e r that almost any f a t of animal o r i g i n contained the f a t - s o l u b l e f a c t o r but that vegetable o i l s ?;ere n o t i c e a b l y d e f i c i e n t in t h i s r e s p e c t . Attempts to maintain animals on a synthetic d i e t a r y t o which 'the f a t - s o l u b l e f a c t o r had been added were made by a number of workers without success. Thus Funk and Macallum (73} found that young r a t s developed d e f i c i e n c y symptoms s i m i l a r to those of avian b e r i -b e r i when r e c e i v i n g a f a t - s o l u b l e concentrate of the f a c t o r reported by Osborne and Mendel. On the b a s i s of Funk's v i t a m i n theory of b e r i - b e r i , s m a l l q u a n t i t i e s of yeast were added t o the r a t i o n and r e s u l t e d i n very p o s i t i v e growth. The l i t e r a t u r e on the progress made i n studying the agent r e s p o n s i b l e f o r b e r i - b e r i must now be reviewed i n order that i t may be c o r r e l a t e d w i t h s t u d i e s on the f a t -s o l u b l e f a c t o r of McCollum. The d i s e a s e , b e r i - b e r i , had been known f o r hundreds and perhaps thousands of years. I t was, however, coincident -76-with the' introduction of modern r i c e m i l l i n g equipment that b e r i - b e r i came to the attention of the s c i e n t i f i c world.. Wernich and van leent (72) quite properly assumed that there existed a causative r e l a t i o n s h i p between r i c e consumption and b e r i - b e r i * The f i r s t r e a l l y great contribution to our knowledge of t h i s disease came as a r e s u l t of the work of Dutch i n v e s t i -gators i n Java. Yordermann (74), acting on the suggestion of Bijkmann, made a complete s t a t i s t i c a l study of b e r i - b e r i i n Javanese prisons. As a r e s u l t of t h i s study, i t was possible to conclude that b e r i - b e r i came as a r e s u l t of the consumption of polished r i c e - - t h a t the pericarp of r i c e contains a fact or e s s e n t i a l for normal n u t r i t i o n . Braddon (75) confirmed the r e s u l t s of Yordermann working i n the Malay peninsula. Fletcher (76) confirmed Braddon's work using patients i n the Kuala ILumpur insane asylum as experimental subjects. In 1897 Bijkmann and Yordermann (77) gave f i n a l proof to t h e i r previous conclusion when they showed that the s i l v e r v l i s s e n of the r i c e kernel was the protective agent i n the prevention of B e r i - b e r i . They concluded that the s i l v e r s k i n possessed a protective a b i l i t y i n n e u t r a l i z i n g the t o x i c i t y of the r i c e staroh. In addition, Eijkmann made the important observation that the water extract of r i o e bran had therapeutio properties. Phytin was shown to be present i n the r i c e bran, but was -77-without curative powers. It was also observed that the curative agent was dialysable and not precipitable by alcohol. Grij'ns (79J confirmed completely the r e s u l t s of Bi^kmann, and sai d that the disease developed when the diet was lacking c e r t a i n substances which were of importance i n th© metabolism of the peripheral nervous system. She thousands of papers which have appeared since the early r e s u l t s reviewed here are d e f i n i t e l y beyond the scope of the present paper. With the keen interest taken i n the mammalian f a c t ors,renewed in t e r e s t and greater stimulus was given to the studies on yeast and b a c t e r i a l metabolism. After reporting numerous experiments, Williams (80) attempted to correlate the growth substance f o r yeast with the a n t i n e u r i t i c factor f o r animals. She presence of contaminants i n media before s t e r i l i z a t i o n was found to cause stimulation of yeasts inoculated i n t o t h i s medium. Williams (83) f i n a l l y arrived at what he c a l l e d a vitamin number which appeared to permit the estimation of the Vitamin B content of various food s t u f f s . Williams was probably the f i r s t to assume that Bios and Vitamin B were i d e n t i c a l . -Wright (84) freed lemon juice from c i t r i c acid using the method of Harden and Z i l v a (85), and reported that small quantities enabled yeast to grow which would not have grown i n i t s absence. -78-Lindner (86) attempted to explain the Pasteur-ILeibig controversy on the basis of f a t t y degeneration of the yeast c e l l s . He-found that the oxygen content of the medium was of importance i n the c o n t r o l of t h i s phenomenon. Fleming (87} found that the addition of organic nitrogen to yeast media seemed to induce more rapid growth and fermentation. Fleming followed the method of Fulmer et a l (88J and determined the "count". Fleming used a Fleisohmanns yeast preparation i n 0.1$ acetic as a standard Vitamin B preparation* As a r e s u l t of these experiments, the yeast method was discarded as a means of determining Vitamin B potency. Souza and McCollum (89) studied the method of Williams, using a modified technique, and came to the conclusion that the yeast test was> complicated by so many factors that i t was probably of l i t t l e or no value. M n o i s s i e r (90) showed that Oidium l a c t i s requires vitamin-like activators when a small inoculum i s used. lumiere (9.1) reported that the molds do not require vitamins. Euler and Peterson (92) found that the count and carbon dioxide production i s not proportional t o the amount of extract added. Euler and Myrback (93) attempted to d i f f e r e n t i a t e between the f a c t o r s present i n yeast. For the a n t i - n e u r i t i c factor the name Vitamin B was retained. Bioeatalyzer B was -79-s p i i t into three f r a c t i o n s : Biocatalyser I, the growth-promoting factor of yeast; and Biocatalyzers II and I I I , both •2. of which appeared to c o n t r o l fermentation., Harden and Z i l v a (94) attempted to show that yeasts grown i n a medium devoid of Vitamin B are able to synthesize t h i s f a c t o r . Shey concluded that yeast grown on synthetic media contain Vitamin B, but not i n so large quantities as when grown on wort. Innumerable papers (95-99) appeared from 1918 to 1922 discussing the yeast test of Williams* A survey of these papers only leads to the conclusion that t h i s method was e n t i r e l y i n error and consequently was of no p r a c t i o a l s i g n i f i c a n c e . In 1922, Eastcott (100) reported the i s o l a t i o n of one constituent of what might be c a l l e d a Bios complex* Ho t h i s - f a c t o r she assigned the p r o v i s i o n a l appellation Bios I . Later work ( ) from the same laboratory i d e n t i f i e d t h i s factor with the c y c l o - p a r a f f i n i - i n o s i t o l . To the remainder of the Bios complex was assigned the name Bios I I . l a t e r work by the same authors (101) c a r r i e s the f r a c t i o n a t i o n s t i l l further but w i l l be examined l a t e r . Harayannan and Brummond (102) reported a f r a c t i o n -ation procedure by means of which they claimed to have shown that Bios I was of comparatively l i t t l e significanoe as contrasted with the coneentrate they had prepared. -80-As i t appeared p o s s i b l e t o f o l l o w the method of thes •P ro.etiotjC'ut- tc n s workers With the m a t e r i a l s a v a i l a b l e , these f-er-me-m-tati-en-s were pursued. •" - 8 1 ~ 1XPERIMTAL A l f a l f a meal was used as a crude source of Bios f o r a study o f Harayanan 1 s .procedure. I t was of a "brand produced i n C a l i f o r n i a , ' c o n s i s t i n g of leaves and blossoms, 1 and was cured in.such a manner as to preserve the maximum vit a m i n content. I t contained 92,'7% of dry matter. The a l f a l f a meal (567.7 gms.) was extracted w i t h 1570 cos. of 95% (Sp, g. .07525) e t h y l a l c o h o l under r e f l u x at 67 - 70° C. f o r fo u r hours. I t was then f i l t e r e d , washed w i t h 55% (Sp.g. ,9244) a l c o h o l (by volume), pressed dry and re - e x t r a c t e d twice w i t h approximately 900 ces. of 50% a l c o h o l . The combined f i l t r a t e s were then freed of a l c o h o l by d i s t i l l a t i o n i n vacuo. The volume of the l i q u i d obtained a f t e r removal of a l c o h o l was 728 cos. This volume of e x t r a c t was made up to 800 ces. w i t h water. I t contained 113.7 gms. of dry matter and 2.2% o f t o t a l n i t r o g e n . I n order t o determine the s t i m u l a t i n g e f f e c t of t h i s e x t r a c t on the l a c t i c a c i d b a c t e r i a , media were prepared as f o l l o w s : (1) To 60 ocs. of milk,- 3 ces. of e x t r a c t were added. (.0149 gms. o f t o t a l n i t r o g e n per cc. of e x t r a c t ) . (2) To 60 ces, of m i l k , 1.5 ces. o f e x t r a c t plus 1,5 ces. of water were added. (3) To 60 ces, of m i l k , 0,6 ocs. of e x t r a c t p l u s 2.4 ces. of water were added. (4) And as a c o n t r o l , 3 ces. of water were added to 60 ocs. of mi l k . -82-As mentioned elsewhere, the water i s added to make the d i l u t i o n i d e n t i c a l i n each case, and thus make the r e s u l t s i n each tube comparable. In order to determine i f the treatment w i t h a l c o h o l had removed a l l of the s t i m u l a t i n g m a t e r i a l from the a l f a l f a , a water e x t r a c t of the re s i d u e from the a l c o h o l i c treatment was prepared. 100 gms. of the d r i e d r e s i d u e , f r e e of a l c o h o l , were e x t r a c t e d by b o i l i n g w i t h 750 ces. of d i s t i l l e d water f o r one hour. The e x t r a c t thus obtained contained 0.063^ of t o t a l n i t r o g e n . Media were prepared u s i n g the same volumes of e x t r a c t as i n the pr e p a r a t i o n of media u s i n g the a l c o h o l i c e x t r a c t s . The media were tubed i n 10 co^ q u a n t i t i e s , plugged,' and s t e r i l i z e d at twelve pounds pressure f o r twenty-five minutes. The tubes were i n o c u l a t e d as i n the previous work w i t h a 3 mm. :loopful of a v i g o r o u s l y growing casein d i g e s t b r o t h c u l t u r e of organisms EMBgl68 and EMBgl73. The c u l t u r e s were placed at 23° C. and were t i t r a t e d at one, four,; and fourteen days, u s i n g 33/4 HaOH and c a l c u l a t i n g r e s u l t s as grams of l a c t i c a c i d per l i t r e . -84-The r e s u l t s are given i n Table below;-TABLE m r . So Show the E f f e c t s of Alcoholic and Water Extracts of A l f a l f a on the L a c t i c Acid Bacteria. '^ElBg/ie'S'-r x ' 1 EMBg 173 No. 4 Days 10 Days 14 Days No. 4 Days 10 Days 14 Days 1 6*5 '. 1 3.4 '.'•%. Alcoholic Extract 2 3 ; S>4" . '"• %M/.;.'-2 3 :t&' [ 5*4 3.2 ' . -«* Alcoholic Extract 4 !if2 ' ' 0.5 4 v -oW";' 1.1 1 - 1 0.5 0.2 U -o • 2 l . i 2.7 : 2 ; 0.5 0.5 Extract Residue 3 • 4 0.5 0.5 0.9 0*7 ; 4 ; 0.7 0.5 0.0 0.2 From these r e s u l t s i t may be concluded that most of; the stimulating material or "Bios" has been extracted by the alcohol treatment. The next step i n the frac t i o n a t i o n consisted of hydro-l y z i n g the crude alcoholic extract of a l f a l f a with baryta. A solu t i o n of 190 grams of s o l i d barium hydroxide i n 150 ocs. of d i s t i l l e d water was added to the a l c o h o l i c a l f a l f a extract. The mixture was hydrolysed i n the autoclave at 15 pounds pressure for three hours. --85-She hydrolysate was f i l t e r e d and the f i l t r a t e and pr e c i p i t a t e were freed of baryta with sulphuric acid. The baryta p r e c i p i t a t e was brought into suspension i n •water by grinding i n a large mortar before i t was freed of barytai The volume of the baryta f i l t r a t e was 2200 ces., containing 74,2 grams of dry matter and 2.3$ o f nitrogen. The dissolved baryta p r e c i p i t a t e occupied a volume of" 1100 cos* and contained 7.238 grams of dry matter, and 0.291$ of nitrogen. The p r e c i p i t a t e and f i l t r a t e were tested to determine t h e i r stimulating power after the manner outlined previously. Media were prepared from each extract as follows: (1) 6 ces, of extract, plus 60 ces. o f milk. (2) 3 ces. o f extract plus 3 ces. of water, plus - - 60 ces. of milk. (3) 0^6 ces* of" extract, plus 5^4 ces. of water to 60 ces. of milk. (4) 6 ces. of water, plus 60 ces. of milk to serve as - .. a control.' The t i t r a t i o n r e s u l t s from this assay appear i n Table XO" on the following page: -86-TABIE XXY. E f f e c t of Fractionation of A l f a l f a Extract i n * Helationship to i t s Stimulating Power. r | EMBg 168 EMBg 173 ;-jfo:?; 4 Days 10 Days 14 Days Ho. 4 Days 1 10 Days " ' ! ! ' 14 Days l 0,5 1.6 1 0.9 3.2 on *» «: 2 - r — 2 .' ~~ S4 : 4 s H 3 0.7 3 0.5 1,1 •H ' 4 0.2 0,5 . • 4 ; 1*1 — " 1 0.5 0.5 0*7 1*4 o • JL3 • ' ' • a - -p •2 •; •• ;6:.2. •; 0.5 — - 2 0.2 1,1 «H Pi - ' «H 3 0.2 0.5 3 0,2 1,1 O <D 4 0.2 0.5 4 0,2 1.1 f4 : From these results i t i s not possible to state defin-i t e l y whether or not the stimulating substance i s present i n either fraction.» However," i t i s to be observed that maximum amounts of added extract produced measurable stimulation, The i s o l a t i o n procedure was continued, the next step being the p r e c i p i t a t i o n with lead acetate, Th© f i l t r a t e from the baryta treatment2180 ces,, was saturated with a 20$ solution of neutral lead acetate (sugar of lead)• 200 cos. of the solution were required for complete saturation. The s o l u t i o n and preoipi tate were allowed to stand overnight before f i l t e r i n g . Treatment o f lead f i l t r a t e ; The f i l t r a t e from the lead. acetate p r e c i p i t a t i o n (2500 ocs.) was freed o f lead with HgS and the HgS was removed by aerations She solution was then neutralized with 2N HaOH to pH 6*8 and concentrated i n vacuo to a thick syrup, about 450 ocs. The syrup contained 47,11 grams of dry matter and 3«8.9% of t o t a l nitrogen. Treatment of lead p r e c i p i t a t e : The p r e c i p i t a t e from the lead acetate treatment v/as re-dissolved i n d i s t i l l e d water and freed o f lead with HgS and aerated*, The r e s u l t i n g l i q u i d oecupied a volume o f 1250 ces, and contained 8.15 grams o f dry matter,1 and 0.101% of t o t a l nitrogen. In determining the stimulating power o f these two f r a c t i o n s , the following media were prepared: F i l t r a t e : (1) l.B cos, of extract j plus 60 ocs. of milk. (2) 0,94 cc. of extract, plus 0.94 cc. of water to - . 60 ces. o f milk* 5 (3) 0.47 ocs* of extract, plus 1,41 cos. of water to - . 60 ces. of milk. (4) 1.188 ocs* o f water to 60 ocs. of milk** P r e c i p i t a t e : (1) 10 ces. of extract, plus 60 ces. of milk. (2) 5 c c s i o f extraet, plus 5 cos. of water to - . 60 ocs* of milk. (3) 2.5 ces. of extract plus 7.5 cos, of water to 60 ces, o f milk. (4) 10 ces. of water to 60 pes* of milk. The results of the t i t r a t i o n s are given i n Table Zffll on the following page: -88-TABLE Htm. E f f e c t of Lead Fractionation on the Stimulating Power of A l f a l f a Extract EMBg 168 EMBg 173 Ho* 4 Days 10 Days 14 Days Mo. 4 Days 10 Days 14 Days © -p •1 o;i 2,0 1 0,7 4,5 fi-•p • s 0,5 1*6 2 0,7 3,8 H £ • 0.0 3 0*7 . 3-f2 -4 0»Q ; — » 4 0,0 2,3 © •P 0,0 0.7 1 0.5 —«• 1.8 •OS •P *H s 0,0 But m 0.7 2 0,0 — P< •H O © 3 0.0 o*7 / 3 0.0 — • '. 1*8 4 0*0 «.=. .••;o;!7-' j 4 0.0 — 1*6 From these results," i t may he seen that a d e f i n i t e stimulating e f f e c t i s produced by the lead f i l t r a t e . This may or may not be due to the addition of nitrogenous material to the medium, or may be due to a " B i o s - l i k e " substance. In the next step, 5 the lead f i l t r a t e was evaporated nearly to dryness and extracted with 80$ alcohol f o r the removal o f any rinorganio material present. No p r e c i p i t a t e was obtained. The aloohol was removed by d i s t i l l a t i o n i n vacuo and the extract was treated with phosphotungstic acid. A large excess of dry phosphotungstic acid was added to the extract and the mixture was allowed to stand overnight• It was then f i l t e r e d . Treatment of Phosphotungstic p r e c i p i t a t e : The preoipitate was ground with s o l i d baryta and water -89*; i n a mortar to remove the phosphotungstic a c i d / and the excess of^ barium removed with HgSO^. Treatment of Phosphotungstio F i l t r a t e : The f i l t r a t e was freed of phosphotungstic acid with baryta, the baryta removed with sulphuric acid and the -solution was.concentrated i n vacuo• 162 ces. of solution were obtained containing 42,18 grams of dry matter and 0.166% t o t a l nitrogen. As the stimulating substance i s said to be p r e c i p i -tated by phosphotungstic aoid (according to Narayanan), i t was assumed that the "Bios" was present i n the p r e c i p i t a t e . The f i l t r a t e of the phosphotungstic p r e c i p i t a t i o n was tested f o r "Bios" a c t i v i t y * Media were prepared as follows: (1) 10 ocs, of extract,^ plus 100 ocs, of milkv (2) 5 ocs, of extract," plus 5 ces, of water to 100 cos; of milk. (3) 2,5 ocs, of extract, 1 plus 7.5 ces; of water , - to 100 ocs, of milk. (4) 10 cos. of water tp 100 ocs, of milk. The r e s u l t s of t i t r a t i o n s are given i n Table X K Y I I , on the following page: -90-TABLE XXYII » T i t r a t i o n Be suits of Media Containing the Phospho-tungstio Acid F i l t r a t e Fraction of the •* , Extract • -© 4= cd , f-i , -P ,H' . vi EMB2 168 EMBg 173 4 Bays 10 Bays 14 Days l o . 4 Days 10 Days 1 (IS 3 4 Shis mc 0,9 o;]5 sdia d o -•o;s 0.5 > .. •0.$' ;ted on a 0.7 • o;o 0.7 a too' • . 2 3 ; 4 laving 0.7 0.7 0.7 not usee 1.4 1.1 0.9 L) £.0 2.0 0.7 I t would appear from these r e s u l t s that a s l i g h t stimu-l a t i o n i s obtained when the phosphotungstic f i l t r a t e i s employed as an enriching agent; although Narayanan states that the stimulating substance i s p r e c i p i t a t e d . The s o l u t i o n of the phosphotungstic pr e c i p i t a t e was then reduced i n volume j i n vacuo. To this concentrated solution was added 10 ces. of a concentrated solution of s i l v e r n i t r a t e . Following saturation with baryta more s i l v e r n i t r a t e was added u n t i l p r e c i p i t a t i o n appeared to be complete. The mixture was f i l t e r e d . P r e cipitate and f i l t r a t e were freed of barium and s i l v e r with sulphuric acid and hydrogen sulphide. The f i l t r a t e was concentrated i n vacuo to 574 ces. I t contained 10.012 grams of dry matter. The s i l v e r f i l t r a t e was tested for "Bios" a c t i v i t y . Media were prepared as-follows: -91-(1) 5 ces« of extract^ plus 100 ces. of milk. (2) 2.5 ces• of extract, 1 plus 2,5 ocs. of water to * 100 ocs, of milk. (3) 1.25:cos, of extract, plus 3,75 ces. of water plus 100 cos• of milk; (4) J5 .ces«. of water to 100 ocs., of milk. T h e ' t i t r a t i o n . r e s u l t s follow: TABES XXYIII* : T i t r a t i o n Be suits of Media Containing the S i l v e r F i l t r a t e Fraction of the Extract j Filtrate EMBg 168 3SMBg 173 j Filtrate 4 Davs 10 Days 14 Days No. |4 Days 110 Davs 14 Days j Filtrate 1 2 i 3 This n -'-.•©•is.. •' 0.;l5; iedia cl< 0.5. 0i7 i sited on 0.2 0.5 auto< 2, '; 3 ' .4 \ slaving 0.2 0.2 -•042-: and was 0,5 o# 0.9 not used. 0«;7 0.7 •  l i l . The r e s u l t s obtained cannot be considered as s i g n i -f i c a n t . The f a i l u r e to obtain stimulation may be due to traces of s i l v e r i n the f i l t r a t e , or possibly due to some chemical change i n the solution caused by the severe treatment with barium,- lead^ 1 e t c I t might also indicate that Bios i s of a multiple nature (this question has been reopened to investigation by the work of Lash M i l l e r et a l (1933)'. No further b i o l o g i c a l tests were performed although the p re c i p i tat ion with p l a t i n i c chloride had-been carried out befjore ;the above r e s u l t s were obtained. • -92« I t i a not p o s s i b l e to state d e f i n i t e l y that Narayanans procedure i s at. fault,: as,* the d i f f i c u l t y involved i n removing the various p r e c i p i t a t i n g agents introduces the p o s s i b i l i t y .of toxic e f f e c t s , due t© the presence of traces of silver,' barium, and phosphotungstic acid i n the various f r a c t i o n s . In view of the re s u l t s reported above attempts were made to follow the procedure reported by M i l l e r et a l (10B) f o r the i s o l a t i o n of Bios concentrates. This procedure was followed i n d e t a i l using a l f a l f a meal as the raw material. Minor departures from the f r a c t i o n a t i o n method des-cribed by M i l l e r were found necessary as the work progressed, SXPERIMffif T AL One pound of a l f a l f a meal was extracted with 2jQG0 pes» of d i s t i l l e d water i n flowing steam for two hours and autoclaved f o r one hour. I t was then f i l t e r e d . The residue was re-extracted with IjDOO cos, of d i s t i l l e d water f o r one-h a l f hour. The re-extraction was apparently unnecessary as the second f i l t r a t e appeared to contain very l i t t l e organic matter,1 She f i l t r a t e containing about 1,600 cos, was d i s t i l l e d i n vacuo to approximately 175 ocs • So the reduced f i l t r a t e were added 350 cos, of alcohol (95$ by volume), She precip-i t a t e obtained was f i l t e r e d o f f end washed with 50 ces, of 66$ alcohol (by volume), F i l t r a t e and washings were combined and then reduced to dryness by d i s t i l l a t i o n i n vacuo, She residue was dissolved i n d i s t i l l e d water,' neutralized with normal HaOH and then made up to a volume of 180 ces, 10 6c. portions of thi s solution were placed i n 50 ce» Erlenmeyer flasks f o r hydrolysis with 2 gram portions of powdered barium hydroxide, and the whole heated to b o i l i n g i n a water bath for three hours* To the hot hydrolysate was added 20 ces* of 96% alcohol (by volume), and the mixture was thoroughly shaken * After standing f o r some time,i the hydrolysate was f i l t e r e d . The f i l t r a t e , p r e c i p i t a t e , and washings were kept separate. Treatment of P r e c i p i t a t e : The p r e c i p i t a t e was extracted with 15 ocs. of hot water and f i l t e r e d . The solutio n obtained by t h i s treatment was then freed of barium with sulphuric a c i d . This solution;'' a f t e r n e u t r a l i z i n g ^ corresponds to the crude Bios " I " solution of Mi l l e r * 1 Treatment of the F i l t r a t e : The f i l t r a t e of the barium hydroxide hydrolysate was freed of barium with sulphuric acid. The s o l u t i o n was then evaporated repeatedly i n vacuo to remove v o l a t i l e acids. The residue thus formed was taken up with d i s t i l l e d water;! neutralized,' and made up to a volume of 25 cos, Tliis s o l u t i o n now corresponds to the crude Bios " I I " s o l u t i o n of Miller** Fractionation of Bios " I I " : One gram of Dare© char-coal was shaken for 15 minutes with 30 ces, of a solution of orude Bios " I I " prepared a f t e r the manner previously outlined, and 1 cc. o f 2N sulphuric acid. This mixture was then -94-f i l t e r e d * a n d washed with 15 ces, of d i s t i l l e d water, The f i l t r a t e was then shaken f o r an additional f i f t e e n minutes, with a half-gram portion of charcoal, The f i l t r a t e s and Washings were a l l combined as were the charcoals from the two operations. The combined f i l t r a t e and washings from the charcoal treatment were freed of sulphuric acid by baryta;'' evaporated i n vacuo,' and made up to 80 ces. This solution corresponds to the crude Bios "Sa*1 solution of lash M i l l e r * The charcoal from the two operations was shaken for 30 minutes with 40 ces, of fre s h l y prepared "ace tone-ammonia" reagent,! prepared as follows: 5 ces, concentrated ammonia/ 35 ces. of d i s t i l l e d water/I Acetone to BOO ces. The mixture was f i l t e r e d , and the charcoal washed with 10 cos. of the acetone-ammonia. I t was again shaken for 30 minutes with 25 ces, of ace tone-ammonia/ f i l t e r e d and washed as before. Th© extracts and washings were combined, and evapor-ated repeatedly i n vacuo to remove the acetone-ammonia. The residue was then dissolved i n d i s t i l l e d water, neutralized with 1/4 H. KaOH and made up to 25 ces, Shis solution corres-ponds to the crude Bios "2b" solution of Lash M i l l e r , In the preparation of media to be used i n testing the stimulating power of the "Bios" extracts,; milk enriched with the various f r a c t i o n s was employed. According to Lash. Miller,-i t i s necessary to have a l l three "Bios" fractions present i n the media i n order to secure stimulation, So check the -95-results bbtained i n h i s work,i media were prepared using various combinations of two "Bios" fractions—media containing one "Bios" only;' and media containing a l l three "Bios" concen-tra t e s . Since Lash M i l l e r has i d e n t i f i e d the Bios " I " f r a c t i o n with the c y c l o p a r a f f i n " I n o s i t o l " , media were prepared, using I n o s i t o l solution as a substitute f o r Bios " I " . The solution used., was prepared by .dissolving. O.lv or;.0-';4:\gramS''.^:.iinosi.te/' i n 50 cos * of watery . Media were prepared, 1 as i n Table XXIX. TABLE: XXIX. . Table of Media 1 P l a i n milk 2 Milk • 24 C C S »i Hg0: ': Milfc ...0*5 cos* o r i g i n a l extract 4 •M l i * • 12 ocs, 0. E. +. .3 ocs, i n o s i t e 5.. • ; M i i f c ,12 ces. i n o s i t e 6 Milk + .12 cos. Bios " I " 7 Miik .12 ocs. Bios ?IIa" 8 Milk + .24 ces. Bios '.'lib? 9 Milk .48 ces. Bga.4>.12-ces. Bgb 10 Milk •f .6 ces. Bios " l i b " * .12 ces. Bios "1" 11 Milk. + .12 ces. Bios-"IIa" + .6 ces. i n o s i t e 12 Miik *• <j6 ces* Bgb +-.6 ces. i n o s i t e 13 M i l K * .12 ces. Bios " I f 4- .48 ces. Bga 4 .24 ccs.BgB 14 Milk ,6 ces.1 i n o s i t e + ,48 ces. Bga> .24 ces. BgB The above figures r e f e r to the concentration of the added substance i n 10 cos. of culture media. The concen-trations given appeared to give the maximal stimulus to growth*' -96-The media were inoculated and treated as i n previous experiments. Results of t i t r a t i o n s are given i n Table XXX. From a study of the data obtained, i t may be observed that the cultures enriched with the o r i g i n a l extract, and with the "Bios" f r a c t i o n s , give a higher production of acid than the milk controls, even where only one, or two, of the "Bios" concentrates are present. I t i s evident that i n the media containing a l l three fractions there i s a stimulation,as also i n the media i n which i n o s i t o l replaces Bios " I " . I t i s also evident that' i n the media containing Bios " I " and Bios 2b", there i s a stimulation. This i s explained on the basis of Lash M i l l e r 1 s data as being due to the fact that some Bios "2a" i s present i n the crude Bios "2b" solu t i o n . -The results obtained are possibly not s u f f i c i e n t l y clear-cut to be absolutely outside the l i m i t s of error/' but there are d e f i n i t e indications that the method of f r a c t i o n -ation of Bios, as advanced by Lash Miller,' can be checked by using l a c t i c acid cultures. <-»97~» wm zzx a C u l t u r e EMBg 173 4 Days 14 Days 0.9 1.6 . M'.V HgO I 0/9 0,9 3.8 5,9 7,4 G. E. +. i n o s i t o l 1.8 5 & 2 I n o s i t o l 0.9 1,6 2 © 3 1.1 2 © 0 3 © 2 0.5 1,8 2 © 3 BgB 0.;9 : : . ; " ' ; i ; i " ' 2.0-Bga + B]_ 1.1 3 ffl 2 3,4 Bgb B 1 1.1 3„2 • Bga *. i n o s i t o l 5 0.7 . 2/0 2,0 Bgb 4 i n o s i t o l 0.9 1.8 2,3 B]_ . 4 Bga > Bgb 0,9 '.' 2,0 ' ^  3.2 I n o s i t o l + Bga + Bgb 0,7 2.0 2.8 Through the courtesy o f Dr, Lash M i l l e r / i t was possible to obtain samples of his "Bios" concentrates,prepared from tomato extract, and a portion of ash-free " i n o s i t e " , prepared by Eastman, She Bios fractions obtained from the Toronto Laboratories were added to casein digest broth as shown i n Table XXXI.. (DSB a Dougle Strength Broth). The results obtained are. also recorded i n the table following. Culture EMBg 173g was used,' -98-TABLE XXXI. I n o s i t o l .75 cc. bios 4- 10.5 cc. DSB 4 9.75 cc. HgO Days 4 • 14 0.4 1.1 Bga 3.2 Bga 4 10.5 cc. D.S.B. 4 7.3 cc. HgO 1.6 Bga 4 10.5 cc. D.S.B. 4 8.9 cc. HgO 1.4 1,1 3.2 3,2 0.5 BgB 4 10.5 ce. D.S.B. 4 10.0. cc. HgO 0.25 BgB 4 10o5 cc. D.S.B. 4 10^25 cc* HgO 0.9 0.9 3®S ; 2*7 ' B ga 4 Bgb 3.2 Bga 4 0.5 Bgb 4 10.5 cc. D.S.B. 4 6.80 C C . HgO 1.6 Bga 4 0.25-B2b 4 10.5 cc. D.S.B. 4 8.65 cc. HgO 1.8 1*6 -.4,1. 4*1 Bga 4 i n o s i t o l 3.2 Bga 4 0.75 inbs, 4 10 o5 cc. DSB 4 6.55 cc. HgO 1.6 Bga 4 0.75 inos. 4 10.5 c c DSB 4 8.15 cc. HgO ,,1*4 : 3.9 3.9 Bgb 4 i n o s i t o l 0.5 Bga 4 0.75 inos* 4 ' 10.5 cc. D.S.B. 4 9,25 cc. HgO . 0.25 Bga f 0.75 inos. 4 10.5 cc. DSB 4 9.50 cc. HgO 1.3 1 « 1 3.6 3.4 B 2a 4 2 b 4 inos. 3.2 cc. Bga 4 0.5 cc. 2^ 4 • 0,75 inos. 4 10.5 ec. DBS 4 6*05 cc, HgO 1,6 cc. Bga 4 0,25" cc. 2 B $ 0.75- inos. 4 10*5 cc. DSB,.4,7*9 cc. HgO .3*4^ .. 3,2 7,2 6 «'8 Yeast 0.375 YE 4 . 10*|5 cc. DSB 4 10*125 cc. HgO 3.1 6.4 -99-From the foregoing results, i t i s evident that the Bios fractions a f t e r M i l l e r et a l , prepared from a l f a l f a and those prepared i n the Toronto Laboratories .> give almost i d e n t i c a l r e s u l t s • Further,; i t would appear evident that the stimulating substance f o r the l a c t i c acid bacteria shown to be present i n a l f a l f a , ' yeast extract/ and several common forage crops i s i d e n t i c a l with the Bios of M i l l e r , In order to e s t a b l i s h even more concretely the r e l a t i o n -ship between M i l l e r ' s Bios preparations and the growth factors apparently es s e n t i a l f o r the Betacoooi, several additional fractionations were performed, using a l f a l f a as '.a crude source. The results obtained on these repetitions confirmed i n entirety the e a r l i e r r e s u l t s * Considerable d i f f i c u l t y was encountered using the f r a c t i o n s prepared a f t e r the manner of M i l l e r et a l . In many cases,' e s p e c i a l l y using a l f a l f a as a crude source, i t was found d i f f i c u l t to enrich milk with these fractions without causing a tough,- leathery c l o t to form on autoclaving. As a result,' i t was found extremely d i f f i c u l t to observe growth changes i n the milk cultures. I t must be noted, however, that the c l o t t i n g of the milk on autoclaving did not appear to fsgduce i n any way i t s s u i t a b i l i t y as a medium fo r the Betacoooi.. I t i s possible that the c l o t t i n g of milk enriched with Bios concentrates, af t e r autoclaving/ i s due to the preserve of various oxy-acids formed during the hydrolysis of the extracts with barium hydroxide• -100-In view of the d i f f i c u l t i e s mentioned above, an attempt was made to so a l t e r the procedure that they might be overcome. •• The modified technique developed was b r i e f l y as follows. The o r i g i n a l extract from a l f a l f a , tomatoes, or any other suitable source is treated with tannic acid after auto-claving f o r 1§ hours at 15 lbs* pressure* The mixture i s then allowed to stand overnight and the pre c i p i t a t e then centifuged off* Saturated Lead acetate (neutral) is then added u n t i l p r e c i p i t a t i o n ceases, and the r e s u l t i n g p r e c i p i t a t e is removed by f i l t r a t i o n . S u f f i c i e n t Basic Lead (after Hawk and Bergeim (103))is added to the f i l t r a t e to complete p r e c i p i t a t i o n * The f i l t r a t e obtained from the Basic Lead treatment is then treated with Saturated Ba(0H)2 u n t i l p r e c i p i t a t i o n ceases. A f t e r removal of the Ba(0H)g p r e c i p i t a t e , the excess Barium and Lead are removed quantitatively with 12N sulphuric acid. The so l u t i o n thus obtained i s treated with Daroo (Eastman) char-coal at the rate of 2.5 grams per 100 cc. for one and one-half hours with constant agi t a t i o n . The charcoal i s then f i l t e r e d o f f and the f i l t r a t e treated with a si m i l a r amount of charcoal f o r one-half hour. This charcoal i s then f i l t e r e d off and combined with the carbon from the f i r s t treatment. The Bios II B i s recovered from the charcoal by elution with acetone and ammonia, as i n the procedure of M i l l e r et a l * The eluant thus obtained i s freed of ace tone and ammonia by repeated d i s t i l l a t i o n i n vacuo. The volume i s then made up to 50 ces. with d i s t i l l e d water. The f i l t r a t e from the charcoal treat-„ meat containing the Bios II A i s then reduced i n volume by -101-dis t i l l a t i o n i n vacuo and i s then made up to 50 ecs, with d i s t i l l e d water. In Table XXXII is recorded the results of a fraction-ation conducted using the above procedure, using white clover, rye grass,' and a mixture of white clover and rye grass. -102-TABIE m i l . ' White Clover Rye Grass Clover & Rye 388 gms. 227 gms. 219 gms. Volume of H20 used 1200 co. 1200 cc. 1200 oc. Volume o f extract ,1200.cc* 1070 oc. 1250 cc. Per cent, D.M, of 14.31% 21.51% 16.78% Per oent. t o t a l E£ 4.70% 4.89% 4.60% Per cent, t o t a l ash 2.75% • •-- ...... 2.74% 2.11% Volume of PbAo2 added (0.5 g/oc.)... 102 cc. 55 O G e 23 cc; Volume of f i l t r a t e 1280 i©5o- ; 1300 , Volume of B a s i c Lead (,25 gms.FbO/oo.) 142 co. 87 oo. 130 oc. Volume o f f i l t r a t e . , 1375 co. 1350 oc. 1500 cc. Volume of Saturated 130 cc. 170 cc. 125 Oc* ' .25;Vgms* 25 gms. 25 gms. • Dare©"''':.. • i Dare© 4.6 :-:4;; 10 gms. 10 gms. 10 gms. Dareo Daroo Dare® ) 400 cc. 400 cc. 400 cc. Acetone- Acetone- Acetone-'.•. ••'/•••/••'••' '. '-• .. < - - .<-ammonia ammonia ammonia -103-MecLia were prepared from the o r i g i n a l extracts obtained i n commencing the fractionations reported i n Sable XXXII, She concentrations used were as follows:-.30 oc* of o r i g i n a l extract plus 150 cc* milk = 20$ enrichment 20 " " " K n " 150 " " = 13$ " 10 " " 15 n 150 1 1 " s 6.6$ " 5 " « « n -n 2.5Q « " „ g # 3^ n .2 " " n " , ! 150 " M ~ 1.3$ " 1 » " - " « * 150 n " -s ,66$ " A control set of media was< prepared with water as an enrichment i n the same manner as above. A l l media was tubed i n 10 pc, quantities and auto-claved at 12 l b s , for l/2 hour. Shis media was then . inoculated with a Streptobacterium and a Betacoccus and incubated at 30° C, She amount of acid produced by Betacoccus 1732 i s recorded i n Sable XXXIII and that produced by Streptobacterium 4A7 i n Sable XXXIV. -104-TABLE m i l l . Betacoccus 1732—Incubation Temperature 30° C. \ PERCENTAGE ENRICHMENT. 20$ 13$ 6.6$ . 3,3$ 1.3$ .66$ . I* 48 hours Incubat: tons Water 0.0 0.0 0.0 0,0 0,0 0.0 Yeast 2.4 6.5 1,8 1.7 3,3 0.6 Clover 3*9 2.2 1.8 0,8 2,0 1.3 Bye 0.5 0.7 -« 0.8 0,3 0,4 Mixed 0.0 0,0 0,4 0.5 0.8 0.7 I I . 78 hours Incubation 1.5 0.4 1.3 " 2 * 1 . ; 0,1 0,3 • Yeast 5.0 6.4 5.3 3.8 5.1 4.3 '--Clover' ; 5.0 1.6 6.4 3.2 2,0 :', • Bye • . • -i 1.9 0.9 0.8 0*8 1.4 0.0. : o*^ j 0.7 0.7 0*6 I I I . 96 hours Incubation : Water 1.6 2.3 2.7 2.6 2.0 .' Yeast 7.2 -4* 5' ; 2*5 : 4.6 3.7 ; .6.1- ' ; Clover 0,9 7.2 6,0 2.1 • Bye • •••••xi'o'i "1/3 r • — — " 1,4 0.9 1.2 ' Mixed 2.5 0.8 0,8 0,8 : 0.5 0*8 IV. 120 hours Incubation : Water 2.4 6.6 3,4 0.7 i.: 2,4 • Yeast 7.2 4,8 6,5 4,0 Clover 6.4 ;•• 6*3 ••; ;4v7: ; 7*'4'-'r 2,7 Bye 1.8 ™~» 1.6 •: : i . l •'. 1.4 Mixed 0.9 0,9 ••: 0,9 0.9 0.7 0,9 -105-TABEB X X X I V , , T i t r a t i o n restate, Streptobacterium 4A7 (I.R.S.) 20$ 1 13$ . 3.3$ 1.3$ * 66$ . I* 24 hours Incubat riOtt Water 0,0 0.0 o-i'i"- 0.1 0*1 Yeast .•iW2 0.9 0.7 0,4 0,2 : o.:i Glover .0,5' 0.6 0*7 : 0.6 0*4 0.3 Rye 0.4= 0.5 «»-» 0,2 0.2 0.0 Mixed 0.2 0.0 • 0*1.; 0.0 0.0 0*0 . I I . 48 hours Incubation Water 0.4 0.4 • 0.3 0,3 0.3 0.4 Yeast 6.0 2*8 • life . : 1.6 0.4 0.6 Glover 1*5 1.7 ' 1*3 ; 0.8 0.7 0.5 Rye 1.1 ; 0.8 0.4 0.4 0.8 , Mixed 0.7 0.5 0.6 ' 0.9 0.8 0,6 I I I . 72 hours Incub at ion i •'..' • Water 0.5 0.3 0,4 0,4 0.3 0,3 Yeast 6,3 ;4.4 3.0 1.6 0.9 : 0.7 Glover 2,0 1.9 1.6 1.0 0.9 0,6 Rye 1.2 1.2 mm *m+ 0.4 0.4 '• 0.8 Mixed 1.0 0.7 f;.©a5. 0.7 0,4 0,6 IV. 96 hours Inoubation Water 0.':8 0,5 0.4 0.4 0,4 0.4 Yeast 7.2 5.6 M $ 2,1 . 1.0 0,9 Clover 2.8 2.1 ' 1^ 7-' / 0,9 0.9 1,0 Rye l.:3 1.3 • - ™ ovs 0.7 0,9 Mixed 1.3 0.8 0.7 . 0.5 0,7 -106-DISU0SSI0H * . . . . From these results i t i s evident that yeast extract i s a more suitable form of enrichment for the Streptobacterium than clover extract followed by rye grass and the mixture of clover and rye grass. On the other hand, the white clover extract i s d e f i n i t e l y more potent when added as an enrichment f o r the growth of the Betacocous, The yeast extract appears to be f a i r l y active i n higher oonoentrations, whereas the clover appears to possess toxic properties when the enrichment becomes too large, EXPERIMENTAL She Bias f r a c t i o n s prepared from the extracts reported upon above were added to milk i n l / 2 oc. and 1 oo. quantities and the rate of acid production of Strep tobacterium 4A7 at 30° C, i n these milks was determined, These results are recorded i n Table XXV. -107-TABL1 XX&V* * B l G a Assay of Clover. Rye and Mixed Strep tobacterium 4A7, Temperature 30° C. 3 davs 4 days 6 days Clover 2a l/2 oc. :: 0^3 ' • . ; 2.0 4,9 " 2a 1 co. 0.4 0,'3 2,3 4,9 Clover 2B l/2 cc. 1,0 : o;6 1.3 .2*5 : a . • 2B i.-oo., - Q>4 • 2.1 2,7 Clover 2A + 2B l/2 oc. 0,4 0,5 2*5 6.7 " 2A 4 2B 1 cc. 1.2 • 1& 1 3,9 7,4 Milk 4" Water Kontrel 0.4 0.6 :'. 1*2 ' 1*4 Rye 2a 1/2 cc. 0,5 0,6 2,5 .1 5.7 Rye 2a 1 co. 0,7 1.2 \'' -'3.6 ; 5*8 Rye 2B l/2 oc. 1.2 0.4 0,8 : 5*8 Rye 2B 1 cc* 1.3 1.1 2*0 ; 2.6 Rye 2a 4:2b l/2 cc. 1.2 2.3 :' ' 5,0 8*7 Rye 2a 4 2b 1 cc. 1.8 1.3 ' 4,7 ; 6*6 Mixed 2a l/2 cc. 0.4 0,4 . . 0*9 • 4.7 Mixed 2a 1 oc. : 0,4 0.5 : Mixed 2b 1/2 oc. 0.4 0,5 - ; 1*9 • 1.2 Mixed 2b 1 0 0 . 1.1 1.0 2.2 4.9 Mixed 2a 4 2b 1/2 cc. 0.9 - 2.2 4*8 7.1 Mixed 2a 4 2b 1 cc. 1.1 2.7 4*0;. 8.4 -108-DISCUSSIOH An examination of these results shows that the modified f r a c t i o n a t i o n procedure i s suooessful i n p u r i f y i n g • and segregating the two Bios f a c t o r s . That the factors have been concentrated considerably i s evidenced by the marked stimulation obtained here as contrasted with that recorded i n Table XXXIV using the o r i g i n a l extracts. Again i t i s evident that the Bios I I A and II B factors act as separate stimulating e n t i t i e s and at the same time give a s t i l l more marked e f f e c t when added i n combination as enrichments to milk. The work of Orla-Jensen et a l (104) on the "Vitamin Requirements of the L a c t i c Acid Bacteria" came to hand a f t e r the work on the M i l l e r method of Bios fr a c t i o n a t i o n had been completed. The conclusions reached by this author are of i n t e r e s t i n that they confirm i n a measure the re s u l t s of the work recorded above. He concludes; " ( l ) The true l a c t i o acid bacteria require a thermostable -> a l k a l i - M a t substance. This i s absorbed by activated char oo a l at a l l hydrogen ion ooncentrations within the acid range, and s i m i l a r l y to l a o t a f l a v i n i t i s e a s i l y eluated by means of p y r i d i n and methyl alcohol. This substance influences favorably the growth of yeast, and may thus be considered i d e n t i c a l with pantothenic acid (Williams (105)) which i s the main constituent of Bios . Many facts seem to indicate that this activator oo cur ring i n milk i s also i d e n t i o a l with B6, which means that Vitamin B2 must consist o f Bios and Laotaflavin, (2) The l a c t i c aoid baoteria require l a o t a f l a v i n i n varying amounts i n addition to Bios. The rod shaped forms need 0.5 mg. l a o t a f l a v i n per l i t r e , while the Streptococci are s a t i s f i e d with a smaller amount. -109-"(3) Glutathione cannot replace lact@flavin as activator for the l a c t i c acid "bacteria. (4) I t i s possible to measure the content of Bios and lact©flavin i n various materials, adding them to carbon-treated milk and after inoculation with a suitable l a c t i c acid bacterium* tabulating the acid formed. If i t i s desired to detect Bios, l a c t e f l a v i n must be added to the carbon-treated milk. On the other hand, i f the lactal'lavin content i s to be tested, the treated milk without the addition of either Bios or l a c t e f l a v i n must contain both activators. The l a t t e r i s the case with substances such as autolyzed yeast, tomato paste and malt extract." As i t seemed d i f f i c u l t to reconcile the properties of the "Pantothenio a c i d " of Williams (105) with the Bios of M i l l e r , i t was considered advisable to test the factor of Williams with a view to confirming, or otherwise, the r e s u l t s of Orla-Jensen et a l (104). Through the courtesy of Dr. Williams (106), i t was possible to obtain a small quantity of Pantothenic acid for t h i s purpose. In addition to the work of Or la-Jensen, a further paper by M i l l e r (107) suggests the possible i d e n t i t y of B-alanine and leucine with Bios II A. Again through the courtesy of Dr. Williams (106) a small quantity of B-alanine was obtained to check at the same time the r e s u l t s of M i l l e r et a l (107). . EXPEEIMSH TAIL Media were prepared containing leucine, alanine, leucine and alanine i n combination, pantothenic acid, carnosine, Bios I I B and combinations of alanine and leucine with Bios II B. These enrichments were added to concentrations of .05, 0.1, -110-0*15, 0*2, 0,25 and 1.0 mgs* per oc* The amount of acid produoed "by culture EMBgl73 i f i v e days i n the enriched milks was determined and i s .recorded i n Table XXXVI below; TABLE XXXVI. Amount added .05. 0.1 0.15 0.2 0.25 ' 1*0 Leucine 0.5 0,5 0.5 0.5 0.5 Alanine 0 o9 0,5 0.5 0,5 0.5 — _ Leucine * Alanine ; 0*5 0,5 0.5 0,5 0,5 Pantothenic acid 0.5 0*5 0.5 0*5 ; 0,5 : = .0*5 Carmosine 0,5 0,5 0.5 • '0,5 ; 0.5 — — Bios II B 0,9 1.8 — — Alanine 4 Bios IIB 1.6 2.3 2.7 3.6 : '4«3:;;: * 5.2 Leucine 4 Bios TLB 1.6 2.5 3.6 4.3 : — — • Milk control 'i ' 1 i i " 1' 0.5 — • — — DISCUSSION From these r e s u l t s i t i s evident that Pantothenic acid cannot replace the Bi©s I I B of M i l l e r i n the metabolism of t h i s organism. There appears to be some stimulation by B-alanine and 1-leucine i n the presence of Bios. I I B, although the e f f e c t s are so small that no d e f i n i t e conclusions can be drawn ^ ! The other alternative i n explanation of Orla-Jensen's (104) r e s u l t may be that the Bias of milk as' i s o l a t e d by this - I l l - . worker is not the same as the Bios I I B of M i l l e r . This hardly seems l i k e l y , ' however, i n that their i s o l a t i o n , and hence chemical properties/ appear to he similar-, EXPERIMENTAL In an e f f o r t to prove or disprove t h i s point./ Bias II B, prepared a f t e r the manner of" Miller/and the. Pantothenic acid of Williams were added t o milk .'Which, had "been, .freed o f Bios, "by treatment with charcoal as described "by Orla-Jensen (104). The Bias I I B concentrate was added i n quantities of •1; .25, .50/ ,75 and 1 per cent to the charcoaled milk and Pantothenic acid i n concentrations o f ,1, ,5 and 1 mg. ,per cc. Again Culture EMBgl75 was used. After 5 days growth, the results were as follows?-TABLE XXXVII. CONCENTRATION . 1 • 25 .50 .75 1 Bios II B per cent 0*8 0,8 1.1 2.4 2.7 Pantothenic acid mgs/co 0.0 0,0 0*0 Charcoaled milk ...... .1 0,0 — .. 0,0 DISCUSSION I t i s evident from the data presented i n Table XXXVII that Panto thenic acid, cannot replace the factor or factors removed from milk by treatment with charcoal. From these results i t i s a l s o apparent that. Orla-Jensen T's hypothesis -112-as to the probable nature of Bi,os becomes untenable. The f a c t that Bi:os II B i s able to replace at l e a s t a po r t i o n of th© activators removed, by the carbon would suggest the possibl e i d e n t i t y of. Orla-Jensen's milk Bias with the Bias II B factor of M i l l e r et a l . In the l i g h t of the res u l t s obtained above, i t seemed advisable to study further the e f f e c t of Bias II B when added to carbon-treated milk/ EXEERIMM TAL Media were prepared using o r i g i n a l milk without charcoal treatment,' charcoal treated milk and charcoal treated milk to which had been added Bi.oa II B as an enrich-ment to the extent ot 1 per oent. Betacoccus culture 1MB2173 and Streptococcus culture EMB.jl73 were inoculated int© these media. She amount of acid produced was deter-mined at the end of 10 days inoculation at 230° C. The res u l t s are recorded i n Table XXVIII below:-TABLE XXXVIII. Control Charcoaled Charcoaled milk milk milk 4 Bias Betacoccus EMB2173 1.3 0.0 3.7 Streptococcus EMB-jl73 3.8 0/0 2.0 -113-DISCUSSION The addition of Bios II B to oharcpaled milk appears to replace f u l l y the activating material removed by the char-.coal insofar as the Betacoccal s t r a i n i s concerned. The Streptococcus, on the other hand, appears to require additional enriclament i f i t i s to produce acid to the same extent as i n normal milk. Orla-Jensen has shown that the Streptococci require l a c t a f l a v i n i n addition to Bios f o r their most intensive acid production. I t i s possible then that Culture EMB^173 would have produced an amount of acid equal to that produced i n normal milk i f l a c t a f l a v i n had been added* EJPEBIMm.TAI. In an e f f o r t to .gain information on the influence of the other Bios factors when added to charcoaled milk on the acid production of the l a c t i c Streptococci, the following media were prepared. The growth factors v/ere added to 10 cc. of milk i n the following quantities: .25 co. of Bios I (0.4 mgs/cc.) .25 cc. of Bios II A, .25 cc, of Bios II B, ;25 cc, of Bios I * .25 cc. Bios II A,; .25 cc. of Bios I 4 .25 cc, Bios II B, •35 cc* of Bios I 4 .25 cc. Bios II A 4 .25 cc. Bios II B. Streptoooocal Cultures EMBgll4, EMB24, B B 2 i , E l B i l 7 3 , EMB214 and EMBg219 were inoculated i n t o charcoaled milk con-taining the above enrichments. The following results were -114-obtained af t e r growth for 24 hours at 230 C, TABLE XXXIX. .CULTURE NO. EMB2114 ; iEiB 24 EMBglj EI ©3173 EMBgl4 EMBg219 Bios I 0,9 i;j8 0.9 0.7 0,9 1.4 1.1 1.6 . 0.9 0.5 ; 0.9 : 0,9 Bios,11 B 2 .3 3,4 1.8 0.9 •1,4. >; 2,3 Bios''I # I I A 0.9. 1,6 .•'o/e 0,5 1*1 1*6 Bias I 4 II B 1,6 3,4 ' ; 1.3..-; 1.1 ' 1,6 1.4 Bias II A * I I B 2 4.5 2,3 0,9 2*0 2 $ 3 Normal milk 9,0 5.9 4,1 1 04 5 & 2 5.0 Charcoaled milk 0,0 :- 0*0 • . '• 0.0 0,0 0/0 0.0 DISCUSSION That Bio,s I, II A and II B are esse n t i a l factors f o r the growth of certain, l a c t i c acid streptococci i s evident from the r e s u l t s recorded i n Table XXXIX. I t i s also apparent that some additional growth promoter i s e s s e n t i a l i f these organisms are to produce afildlto the maximum extent. I t i s of interest to note that the Bios fractions apparently are of minor importance for the growth of Culture EMBgll4, while they almost e n t i r e l y s a t i s f y the requirements of Culture EMBg4* The data presented also serve to indicate that there is a great v a r i a t i o n i n the need for growth activators by d i f f e r e n t streptococci. -115u ' BXPERIMS1TAL The e f f e c t of the three Bio.s f r a c t i o n s and a number of other compounds, which i t was expected might possess a c t i v a t i n g properties,was determined by adding them as enrichments t o a s y n t h e t i c m i l k and u s i n g Culture EMB2195 as the t e s t organism* The s y n t h e t i c milk used was prepared as f o l l o w s ; 30 gms. l i g h t white s o l u b l e casein was suspended i n approximately 500 cc. of d i s t i l l e d water. When a good suspension had been obtained, 0.80 gms, Ca(0H) 2 were added s l o w l y . T0£ the Ca caseinate obtained above, the f o l l o w i n g compounds were added:-£0.6 cc. o f 5% MgH 4P 20 8 44*0 cc* o f 5% Na 3C 6H 50 7 lOi'5 cc. of 5% KgCgB^Oy 46*0 cc. o f 5fo % E P 0 4 20*0 cc* of 5fo HaCl 0.2 cc. o f 5fo FeClg The hydrogen i o n c o n c e n t r a t i o n of the s o l u t i o n thus obtained was adjusted to pH. 6.7 by the a d d i t i o n of H/4 NaOH. T h i r t y grams of l a c t o s e were then added and the t o t a l volume was made up to 1000 cc. T h i r t y grams of l a c t o s e were added to serve as a source of carbon. The media prepared and the a c i d produced by Culture BMB]_195 a f t e r i n c u b a t i o n f o r 4 days at 23° C. i s recorded i n Table XL, -116-TABLE XL. MEDIUM ACIDITY Control .3 cc. A l f a l f a extract to 10 ec. synthetic milk .3 cc. Bios I ,3 cc, Bios II A ,3 co,i Bios II B .3 c c Bios I f ,3 co. Bios I I A ,3 c c Bios I 4 ,3 cc. Bios II B .3 cc. Bios II A 4 .3 cc. Bios I I B ,3 cc. Bios I 4 .3 cc. Bios II A 4 .3 cc.Bios IIB ,3 cc. Tryptophane .3 oc. Glutamic acid .3 oc, 1-aspartic acid ,3 cc. Pantothenic acid 1*0 cc. Carnosine 0,3 cc, 1-leucine 0,3 c c Indol 0.3 cc. Quanine HC1 0,3 cc, Arginine HC1 0,3 c c B-indol acetic 0.3 cc. Adenine 0,3 cc. Tyrosine 0,3 cc. H i s t i d i n e 0.3 cc. Ergothionine 0.3 o c B-alanine 0.2 oc, B-alanine, leucine, Bios I I B * Bios I , 0.0 1,4 0.0 1 08 0 e0 1,8 0.0 2.0 0,0 0.0 0,0 0.0 0.0 0,5 0,0 0.2 0 .0 0,0 0.0 0,0 0,0 0,0 0.0 0,0/: -117-BISCUSSIOIT She p o s i t i v e growth of Streptococcus 195^ i n the synthetic milk to which Bios has been added / confirms e a r l i e r results as to the necessity of these factors f o r the growth of the l a c t i c Streptococci, She f a i l u r e of Pantothenic a c i d to permit the i n i t i a t i o n of growth gives added support to the view expressed previously that this compound i s not i d e n t i c a l with the milk Bios o f Orla-Jensen. The f a i l u r e of the various other compounds tested to support the growth of this organism reduces at l e a s t t o a degree the number of possible compounds which w i l l at some time be found to be present as the active p r i n c i p l e s i n the Bios concentrates and to show that they are probably not essential f o r growth of Culture EMB-jl95. A STUDY OH THE METABOLISM OP THE • LA0T 10 ACID B i C T E R I A . PiRT 17. Bjjctc r l pyhure nn Bacte rj al growth Pact or s . -118-PART- l¥.-BAGTimiOPHAGE M S BAOTERIAI, ••GROWTH FACTORS The phenomenon of bacteriophage on transmissable l y s e s was f i r s t recorded by Tw&rt (108) in.1915. This obvervation ' was confirmed independently i n 1917 by d'Herelle (109). The mode of a c t i o n and nature of the l y t i c agent were d i s -cussed very f u l l y by t h i s worker. Even now, a f t e r some 22 years of i n t e n s i v e study, the i s s u e i s f a r from c l e a r . An e x c e l l e n t general d e s c r i p t i o n of l y t i c a c t i o n i s provided by Whitehead and Cox (110); "but simply to say that phage acts i n most respects as though i t were a v i r u s p a r a s i t i c upon the b a c t e r i a l species or s t r a i n which shows the c h a r a c t e r i s t i c r e a c t i o n . The phage m u l t i p l i e s w i t h the organisms and at a c e r t a i n stage induces an almost complete and r a p i d l y s i s on d i s s o l u t i o n of the b a c t e r i a l c e l l s , A very s m a l l amount of a l y s e d c u l t u r e s u f f i c e s to r e s t a r t the chain of events i n a f r e s h c u l t u r e , and thus the phage i s transmissable i n s e r i e s , apparently ''ad i n f i n i t u m " . While many other S t r e p t o c o c c i , e s p e c i a l l y haemalytic ones,' have long been known to undergo spontaneous l y s i s , the f i r s t r eport of t h i s phenomenon i n c u l t u r e s of l a c t i c S t r e p t o c o c c i i s to be a t t r i b u t e d to Hodley and Dobney (111). Whitehead and Cox (112) appear to be the f i r s t to study the commercial aspects of the l a c t i c bacteriophage. These workers, w h i l e studying the " f a i l u r e " of s t a r t e r s i n the Cheddar cheese i n d u s t r y of lev/ Zealand, were able to show that at l e a s t a p o r t i o n of these f a i l u r e s was due to the -119-spontaneous " f l a r i n g up" of bacteriophage. Before discussing further the work of these authors, i t i s necessary f i r s t to outline b r i e f l y the more p r a c t i c a l aspects of the sta r t e r " f a i l u r e " question with which they were confronted* large cheese f a c t o r i e s , many of them handling 80,000 l b s . of milk during the spring and early summer months, were encountering spasmodic f a i l u r e s of t h e i r starters to produce acid. 1 In some eases the f a i l u r e oocurred i n the starter mother culture, i n others i t occurred i n the bulk starter, and i n many cases i n the cheese vat during the manufacturing process. When lyses occurs i n the sta r t e r s , i t i s usual to f i n d the starter unclotted aft e r the usual setting period. I t may, however, occur after the starter has set, and con-sequently the f a i l u r e w i l l not be detected u n t i l the starter has been added to the cheese vat. When the f a i l u r e occurs during the make process, i t may arise at almost any point from setting of the cheese milk to ou^'l^ag of the curd. Naturally the f a i l u r e s i n the star t e r s and during the e a r l i e r stages (up to an a c i d i t y of .8$ l a c t i c acid) of manufacture are of the greatest economic importance. The method used i n the carrying of cheese starters under Hew Zealand conditions i s ofi interest to the following discussion. Almost a l l f a c t o r i e s carry a mother culture, consisting of either a mixed starter or of a single l a c t i o DAIRY RESEARCH I INSTITUTE (¥. Z ? i THe f o l l o w i n g procedure i s recommended Y?ith s i n g l e s t r a i n s t a r t e r c u l t u r e s HP. , R"'I . , or Y.. , {1) On Receipt of the tuberjs put the whole of the contents into a f l a s k c o n t a i n i n g not more than one p i n t of m i l k which has been p a s t e u r i s e d at 190 to 200 F. f o r 45 minutes and cooled to 70-72°F. The f l a s k should • be of such a si z e , that one p i n t of milk about h a l f f i l l s i t . {2} Carry on the mother cu l t u r e d a i l y by i n o c u l a t i o n of 10 ces. of the previous day's t h i c k c u l t u r e i n t o one p i n t of milk p a s t e u r i s e d as de-scribed above, {3) P a s t e u r i s e the bulk s t a r t e r milk at 190-200°?. f o r one hour, cool to 7 2 0 F. ( u s i n g s t e r i l i z e d .thermometer to determine the temperature of the milk) and i n o c u l a t e at the rate of 1 J p i n t s of mother c u l t u r e to 20 g a l l o n s of milk.* I f necessary keep more than one f l a s k of mother c u l t u r e i n order to have the amount necessary f o r i n o c u l a t i n g the bulk s t a r t e r milk. (4) Do not dip into the bulk s t a r t e r m i l k or the mother c u l t u r e a n y t h i n g which has not been th ouro ughly steamed or b o i l e d i n water, This r e f e r s to p i p e t t e s f o r i n o c u l a t i n g , thermometers, and can s t i r r e r s . (Kote; the above procedure i s copied from a c i r c u l a r sent out by the Dairy Research I n s t i t u t e to a l l f a c t o r i e s u sing s i n g l e s t r a i n s t a r t e r s . ) -120-Streptococous. The use of single s t r a i n starters i s , however, a very recent development, due to the a c t i v i t i e s of the Dairy Research I n s t i t u t e (N.Z.) ,and the Dairy D i v i s i o n of the Hew Zealand Department of Agriculture factory operators now use what might he termed se m i - s c i e n t i f i c apparatus and methods i n the care and handling of t h e i r starter cultures. The normal procedure used is outlined below and is that recommended by the Dairy Research I n s t i t u t e . The pro-cedure for mixed sta r t e r s i s i d e n t i c a l with the exception that a smaller inocumum i s used. The l a c t i c phage has only occurred once under laboratory conditions. This case has been reported upon by Whitehead and Cox. Evidence obtained at t h i s time suggested that the organism was i n a "sensitive state" due to aeration of the milk medium i n which i t was growing. In view of the extremely e r r a t i c nature of the phage phenomenon and i t s appearance most often i n certain s p e c i f i c f a c t o r i e s , i t appeared possible that the l y s i s may have been due to a n u t r i t i v e misbalance of the milk used as a medium from the c u l t i v a t i o n of the s t a r t e r . Since i t seemed impossible to obtain spontaneous phaging under laboratory conditions, i t was necessary to add an active phage f i l t r a t e to cultures used i n the following study. Consequently, the r e s u l t s reported can only be interpreted with t h i s thought i n mind. -121-EXPERIMMTAX In order to f a m i l i a r i z e the writer with the various forms of phaging and with the appearance of organisms exposed .to an active phage, milk cultures of Streptococcus cremoris (H.P.) were treated with an active f i l t r a t e obtained from a culture whioh had lysed. The technique follows: seven drops from a c l o t t e d milk culture of t h i s organism were inoculated into several tubes of milk. Phage was added i n d i l u t i o n s from the neat phage up to 1 to 10 m i l l i o n . A 1/2 cc. of a 1:20,000 of methylene blue was also added to each tube. The cultures were then incubated at 37° C. i n the water bath. Slides were prepared at the end of a four-hour incuba-t i o n and then at more frequent inte r v a l s u n t i l l y s i s had oocurred. •PISOITSSIOH Considering f i r s t the methylene blue reaction of the cultures, i t was of i n t e r e s t to note that i n every oase the organism reduced the methylene blue i n about three and one-h a l f hours. In the ease of tubes to which phage had been added the leuco methylene blue very quickly returned to the chromo compound. An examination of the microscopic prepara-tions showed that the l y t i c action occurred "just a f t e r the reduction of the methylene blue and consequently i t would appear that l y s i s caused a return of the milk to i t s normal oxygen tension, i f the methylene blue can be r e l i e d upon as an accurate measure of oxidation reduction p o t e n t i a l . -122-The organism appeared i n long chains of w e l l shaped de p l o c o c c i j u s t p r i o r t o phaging. Just a f t e r l y s i s , consider-able q u a n t i t i e s of what appeared to be c e l l debris could be ' observed. EXPERIMENTAL I n an e f f o r t to a s c e r t a i n the i n f l u e n c e of Bios. I I B on the phaging phenomenon, m i l k media was prepared containing .5$ Bi o s I I B and enriched m i l k from the same bulk sample. Shis media was i n o c u l a t e d w i t h one drop of a c l o t t e d m i l k c u l t u r e of Streptococcus EP. One drop of a l/lOOO d i l u t i o n of phage was added t o a l t e r n a t e tubes. These c u l t u r e s were incubated at SO 0 and the amount of a c i d produced at the end of 10 hours was determined. S l i d e s were prepared at the time of t i t r a t i o n to determine the degree of l y s i s . The r e s u l t s are recorded i n Table X M . TABES- XEE. MEDIUM ACID AT 10 DEGREE OF HOURS ' EYSIS 1 Charcoaled M i l k 2.0 gms. 2 " " * Bios I I B 3.2 " 3 " " t Bios I I B * Phage 0.7 " * * 4 " " * Phage 0.0 " + 4 5 M i l k 6.5 . 6 M i l k t Bios I I B 6.5 7 M i l k * Bios I I B 4 Phage 2.1 8 M i l k 4 Phage . 3.0 • 4 4 4 4 -123-DISCUSSIQN From these r e s u l t s i t i s evident that Bios I I B does not appear to a l t e r the phage r e a c t i o n of t h i s organism. I t ' i s p o s s i b l e that the phaging occurred sooner i n the tube ( 7 ) t o which Bios I I B had been added, since the organism produced a smaller q u a n t i t y of a c i d under these c o n d i t i o n s . EXPERIMENTAL I f phage a r i s e s i n c u l t u r e s of l a c t i c s t r e p t o c o c c i due t o an i n s u f f i c i e n c y of Bios I I B i n the m i l k medium, then i t should be p o s s i b l e t o induce the phenomenon under l a b o r a t o r y c o n d i t i o n s by t r e a t i n g m i l k w i t h c h a r c o a l t o remove the Bios I I B present, then adding t h i s f a c t o r to the m i l k i n v a r y i n g q u a n t i t i e s . Table X I I I presents data obtained by i n o c u l a t i n g Streptococcus ^ S t r e p t o c o c c u s RWgg, and Streptococcus R g l i n media of the above nature. These organisms were s e l e c t e d because they had a l l , on a number of occasions, phaged under d a i r y f a c t o r y c o n d i t i o n s . - 1 2 4 -H M OJ a o -P o •P CQ O W H O a o <d -P fl-PK «J O ft . EH ft O EH O 4S O H O in to to to o W in to feoj m oj few o w OS te 02 W OJ o • • o o to to to .cr» ' # • • to to to oj OJ • - « OJ tO to ' 9 to • o to &» to • • • - • to -st) LO ' « CQ • * to cn to • 9 9 CO -sH PS oi - • o o " » H 03 OJ « . » 03 OJ OJ e OJ IN ?N • © OJ OJ OJ to 10 o w OJ fe OJ K OJ H M o o OJ to OJ in MO ••a , 9 to to en 9 OJ in H O • 9 OJ c*j in OJ OJ OJ 03 • 9 tO "o~ - • i 3 H « oj o o tD 03 « feOI K oj H M p9 o W o W 03 o © o ' # o in o to © Ht to OJ « OJ o - ••• o o ' o o o OJ 4 H M H H rd rQ SOJ ffl fd •d <D © . HI r-i O td ro o o o o O OJ SH <s Ctf O id o o M r-i v • a oj ti <D :» HJ # o y o o r-l 1—J S OJ ^ . w <d 0 • 03 H O H cd o cd o o O tD o ?H • PH cd o cd Xi o o iaf OJ cd o o O O PH • «J H Si o 3 •H H cd o -.126-PISOUSSIQH Since these streptococci a l l produced,a©*wA even under conditions of low Bios I I B concentrations, i t seems d i f f i c u l t to expect the development of phage under f i e l d conditions, due to a deficiency of t h i s f a c t o r i n natural milk. I t i s recognized, of course, that lactaflavin,which has been shown to he essential (107) f o r the l a c t i c streptococci, may possibly be the responsible agent. However, t h i s hardly seems l i k e l y , since the majority of the f l a v i n present i n the o r i g i n a l .milk i s removed by the charcoal under the conditions used f o r the preparation of the media recorded i n Table XMI. Various other enrichments such as 1-leucine, B-alanine, indol-aoetic acid, and oarnosine, were added to milk, but i n no case did phaging of Streptococci K, RWI, R21 occur. Since the milks of the cows on experimental d i e t s (Part II) became available at t h i s time;, further work on the e f f e c t of added activators was not pursued, The f a c t that Streptococcus HPgi did not f a i l to produoe acid on any occasion when, inoculated, d a i l y into the milks from the exper-imental feeding t r i a l was accepted as prima f a c i e evidence that v a r i a t i o n i n the growth factor content of normal milk does not affect to an appreciable extent i t s a b i l i t y to induce phaging of l a c t i c streptococci. On the other hand, i t i s of interest to note that no occurrence of phage was recorded i n Part I of the present -126-paper under what might he termed hypo concentrations of activators supplied by enrichment of the milk medium with yeast extract. Hence i t would appear to be l o g i c a l to state that a v a r i a t i o n i n the amounts of growth-promoting substances present i n normal milk does not affect i n any way the s u s c e p t i b i l i t y of l a c t i c streptococci to bacteriophage. This statement must, however, be q u a l i f i e d by recording that a markedly disturbed balance of the various activator present i n normal milk may have an effect on the appearanc of phage. - 127 BIBLIOGRAPHY I (1) GrlE-Jonsen s 5 ., 3. Z g l . Baaske Yi dens k, S e-1 3 k. Sf c r l f t e r , . F eturv. Og Mathemat i sfc AFP, , 8,, Sao kke , Y V Z . (1919) (2/ Bert rand, G-. Ann. de Chem, et de-PJjjrs, (8),3, 121, (1904) (3) ITogKchI, H. Jour. Exp. Ked. 1±. 99, (1911) ( 4 ) Vedder, K. Jour, B n f s c t , D i s , 16.- 33 5«, (1915) (5) Dory land,H. From G, P a n k , The Vitamins, (6) Hopkins, F, G , Analyst 31., 39 5, (1906) (7) Eijkraann, 0. Geneesk. T i d j c h r , ITed-Ind, 30., 295 , 1890), i b i d 36, 214, (1896) (8) i'cCollcJr., E.V. Amer. Journ, P h y s i o l . 29., 210,(1911) • (9) Funk, C. Jour, P h y s i o l . 43, 395,(1911) (|,0) E a i n b r l d g e , F.A, Jour. Hyg. , Si., 341, 1911 (11) Sperry, L»F« and Rettger, 5 » J » , Jour. B i o l . . C h e is . , 20., 44 5, ( 1915) (12) Pacini,, H. and R u s s e l l , E„ J» Jour. Bio, 0 h c- m. , 54, 4 3, ( 1918) (13) '.Vallis, M. A g r i c . Jour . of I n d i a , 12., 621,, (1917) (14) l e T i n e , M.and Schoenlein,, H.T7*, A Compilation of Culture K c d i a , Williams and THlkins» (1930) Baltimore, U.S.A.. (15) Crla-Jensen, S. » 'Orli-Jensen, A.D. , and Spur, B., Jour. Bact. 12, 3 33, (19 2 6) (16) Sadler, W. , E a g l e s , B»A * , and Pendray,, 5,, Can, Jour,, of Research. ''7. ? , 37 ( 1932) 128 (17) Eagles, B„A, , O k u l i t o h , 0. T dad Campbell, A.G. n. Jour, Research, B14„ 311 . (1936 (IS) S a d l e r , , Eag l e s , B.A,„ Bowen, J.F., and T,7oofis A * J Can. ffour. Res. B14. 1 3 9 , ( 1 2 3 6 ) (19) Orl a - Jenae n,, S., Jour, Bact„ FART II (20) Wendt, G ., T i d s k r , Lantman, March, (1935) • (21) Seed, TJ.S.D.i., Washington, (1933) ( 2 3 ) Med, Res, C o u n c i l Report,, f i t a c i f n s , A Survey of Presence Knowledge, London, (1932) (24) Meigs, G, TT.S.DA., B c l t s v i l l e , Washington, D.C. (25) Orr, J, (26) McDowell, P.H. , IT.So Jour, of S o l , and Tech, 18, 137 (19 36) (27) Sheeny, 1. J . , Jour, Dept. Lands and A g r i c , , I r e l a n d 52, 18, (19 33). (28) Woodman' J . J . , Conversion Tab 1 e s , M i n i s t r y of F i s h e r i e s and A g r i c u l t u r e , London. (29) Whitehead H.R., and Cox, G. , IT. 2. Jour. 3ci= and Tech, 1 3 , 5-/ 304, ( 19 32), ( 3 0 ) Soncke Knudsen, (31) Pasteur, L. , Ann, de C'him. et Phys., 3'rd Series., 58., 323, ( 18 60 ) (32) Duclaus, II, , Comp. Rend, Acad, S c i , 58., 1114, (18 64). (33) H i l l o n , (34) L e i b i g , J . von. , Ann. Ds Chim et Phys. 2 3 . , 5, (1871). (35) T J i l d i e r , E. , La C e l l u l e , IS., 313, (1901). (36) Fernback. A., Ann, de l a B r a s s i e r e et de l a D i f e t i l l i e r e , 510, (1901) - 1 2 9 -(37) W i n d i s o h , T7. , W o c h e n s c h r . B r a u e r e i , .19., 5 2 ? , ( 1 9 0 1 } . (38) Henry, J, , Ann,, I r a s s e r e i et D i s t i l l e r I e, 129 . $1902). (39/ Pringsheim, H. , Cent. Bakt,,, .16 111, (40) I r e l g e r , Amsriksnischcr Bierbauer, 712 (1901)* (41) ^ i n d i s c h , W, , Wochenschr., f . B r a u e r i , 19., 5 2 7 , (1902). (42) A s i a n d, A. , .La Cellule,, 20, 2 2 5 , ( 1 9 0 2 ) . (43) Windssch, W. , Wochenschr, P., i r a u e r i , 19;, Z, (1902), (44) lautnann, H. , S e i t . Tech, B i o l . , 7 , 1, (1919) (45) W i n d i s c h , W. , Wochenschr. f . Braaeri , .19.., 27., ( 1 9 0 2 ) . (46) A.-aand, La C e l l u l e , j>l, 329, (1904), (47) Lindner, P., Wochenschr* f . B r a a e r i , 22., 258 , (1905) , (48) Eossowicz, A . $ 3. L s n d w . Ve r s u b hswe s s en , 6., 27, ( 1 9 0 3 ) * (49) Amand, A and I do.-, M. , Cent, B a k t . „ II 7 18., 193,(1907} , (50) E a r t i n a n d , Y;, He r u e de V i t i c u l t u r e , 29., 2 9 , (1908), (51) K a i l e r - T h u r g a u s E . , J a h r e s b . d - S c h w . , Y e r g c u & h s t a t . , i n Wodenswei1, 1 1 , ( 1892). (52) Behrens, J» , B e r i c h t e d. G r o s s h e r z Lands,, V e r s u c h s a n s t s 1 t A a g u s t e b e r g „ ( 1 9 0 2 ) . (53) D z i e r z b i o k i , A»$ A n z i e g e r der Akoa., d, , Wisfsensch 5 i n Drakau. , 651 , ( 1909) (54) P r i n g s h e i m , H., Cent, 3 a k t . , 16.» 1 1 1 , ( 1 9 0 6 $ . (55) Ide, K. , Cent, B a k t . , I I , 18., 193,( 1907), ( 56) . B e v l o o , H. , La C e l l u l e , 2j5, 3 61 , ( 1906), (57) Tanner, F.W. , C h e m i c a l Reviews, i , 4, 4 1 3 ? ( 1925). (58) L u a i n , F.qa H o p p e . - S e y l , 3., 5., 31, (1881), ( 5 9 ) V o l t , C , ( 6 0 ) Bungej G*, L c h r b u c h d e r p h y s i o l o g i s c h e n und P a t h o l o g i s c h e n Chemie. (61) Osborne, T. 3. , and Mendel. L,B. f Feeding experiments with i s o l a t e d food substances. P u b l i c , Carnegie I n s t c . Io. 156(1911) (62) Funk, 0. f The VI taraines, Wi-lliaud and tfilfcins, 3a 1 ti.tore ,. U.S.A. , (1922). • (63) Stepp, \7, , Biochem. 2. 22., 452, ( 1908), (64) Hopkins, F„G» . Analyst 0 31. (, ( 190 6), (65) Hopkins, F,G., Jour, P h y s i o l . , 44* 425,. (1912), (66) Osborne T, B* * sad Mendel, LoB., Jour. 3 i o l . CJhero., 13, 233, 1912, (67) Hopkins 8 F-.G, and H e i r l l l e , A.* Biochem, Journ. , 7, 97, (1913). (68) McOollum, B..V. and 2 a ? i s . M., Journ. B i o l . Chem. , 15.. 17 6* (1913), (69) Osborne, T . 3., and 2.1c nd c 1 , L. 3 . , Journ , B i o l . Chem,-, 15, 311, (1913). ( 70 ) Funk, 0. and MacOal lum, A . 3 . , Biochem, «Joura. , 7., 356, (1913), (71) Osborne f . B.., and Mendel,, L. B. ,* Jour. B i o l . Chem. „ 1 5 , 423, (1913), (72) Funk, aG , and I,'a o Gal 1 nm, A . B . , Jour, B i o l , Che ra. ., 23, 413, (1915), (73) Wo m i ch: Geogrphl "sch-raedi z i n i sche Stud i en. B e r l i n ( 1878 ) . van Leent j Gen. T i j d s r . voor Ned. Indi e , 48, { 18 98 } . (74) Yordermann^ Gen, T i j d s r * voor Wed. In d i e , 4 3 . , (18 98 )., (75) Braddon, 7.L. t Spt. H.M. Sec. of State for C o l o n i c s , Kuala Lumpur (1905). • (76) Fletcher,, l a n c e t - June 29'th, ( 1907). (77) Ei: .j k m a n n C » s and Yordermann, (79) G r i j n s , G., Geneesk, TIddschr. led-Ind,„ 40., 3 (1909 ). (80) W i l l i a m s , R.J. , Jour. B i o l , Ohoa. 42., 859 „ (1920)• - 131-p (80) W i l l i a m s , R. J,, Ibid, 465, (1919), (84) 'Yripht, O.K. , Bioohein. Journ. 16* 1 3 7 ? ( *?22) . (85) *Harden» A, ., and. Silva, S.S., Biohhem. Journ, 12. ,259,'{1918)'. (8 6) L i n d n e r , p.. , Bent, E s s i g s i n d , 24, 103,( 1920) . (87) Fleming, W.D., Journ, B i o l , Chem., 49, 119, (1921)• (88) Pulmer, E.I* , and Felson, 7.1., ffaid, 51., 77 , (1922} • (89) Soiisa G,P«, and McOollum, E. V. , Eblfi, 44., 113 s ( 19 20 ) (90) Linnoisseir, G. C.R. Soc, Biol. , 83, 34 6,, (19 20). (91) Lurnl e re s A . , Ann ». I n s t . Pa st . .35. 102, (1921).. (92) Euler, H, von. and Pederson, 1 . , Exp, S t a t i o n Sec, 4J>, 8 66s, (1922 ), (93) Euler, H. von and Kyrback, X. I b i d , 46., 866, (1922) * (94) Harden, A. A. and S i l v a , S.S, Bio, Journ. 15., 438 . (95) Ho soya, 5, and Kuroya, M., Inst, for Infect. Dia, 2, 265 5 (19 23), (96) Levene,. P.l,, and Yande rhoe ven» B. J,C. , Science, 59 , 27 6,( 19 24 ) . (97') Lobeck, A-, Dissert. Unlv-. of Geneva, (1922). (98) MacDonald, K.B. , Jour, Biol.-Oherc. 54., 243, (.99) M i l l e r , V»L* * Science, 5£, 197, (1924) . (100) Eastcott, E.Y., Trans. Hoy. Soc* Canada, Sec.3, 3'rd s e r i e s , 17., 157 , (1924), (101) M i l l e r 17,L., E a s t c o t t , E» V» , and Maoonachie, J.E. , J , Am. Che a, Soc. 5.5...7. 502 (1933). (102) Haryanan, B.T., Bio, Jour. 24* 6 (1930), (103) Ha-^ k and Bergeia, P r a c t i c a l P h y s i o l o g i c a l Chemistry. (104) Orle-Jensen, S., Otte, IT.C. , and Snog-Kjaer, A., The Vitamin and Hi trogen Requirements of the L a c t i c Acid B a c t e r i a , Copen-hagen , (1936)• - 132 -(105') W i l l i a m s , B.J, , and Saunders,, 3«H. Bio, Jours, 28 , 180 7, (1934). (1061 W i l l i a m s , R.J. Dr,, Professor of Chemistry, Oregon State C o l l e g e , Pergonal c omrnuni ca t i on, 1107) f i l l e r , W.L. , Traa s.. Roy. Soc, Canada, (1935) , (108) Two r t , Lance t , i i , 1241? (1915). (109) d ' B s r e l l e , Co nipt. Rendu,load. S c i , 165., 173,( 1917) . (110) Whitehead, H.R, and C o x , G. I\T»Z» Jo arm, Sci,. and Tech, 1_6, 319,(19 35). (111) Hadley • and Babney, Proc. Soc, Exp. Ifed„'24s 13,( 1926) (112) Same a s(110), X X - 133 -The writ er wishes to acknowledge the i n s p i r a t i and help so k i n d l y given at a l l times by Dr. B * i . Eagles under whom, t h i s work was conducted. For permission to use the data presented on pages 31 to 99 the author expresses h i s si n c e r e s t thanks to Mr, J.F. Bow en with whom t h i s work was c a r d e d on. Thanks are due to the Department of S c i e n t i f i c and I n d u s t r i a l Research of the Government of the Dominion of Hew Zealand f o r the p r o v i s i o n of l a b o r a t o r y f a c i l i t i e s and for f i n a n c i a l aid which made p o s s i b l e the work recorded In Parts I, II and IV. For advice and c r i t i c i s m the author i s deeply indebted to P r o f e s s o r Wm, Riddet and Dr. H,H. Whitehead Of the Dairy Research I n s t i t u t e of lie w Seal an d , and to Mr. I , R. Sherwood and G . J. E » Hunter of the same l a b o r a t o r y a p p r e c i a t i o n i s expressed f o r permission to use c e r t a i n of t h e i r experimental findings-. The a i d of P r o f e s s o r P . l , Bo vi ng of the Department of Agronomy i n the p r e p a r a t i o n of the manuscript was much a p p r e c i a t e d . 

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