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Biological studies with special reference to the influence of certain organic compounds on the growth… Schwangart, Wolfe Michael 1936

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BIOLOGICAL ;STUDIES WITH SPECIAL REFERENCE TO THE INFLUENCE OF CERTAIN ORGANIC COMPOUNDS ON THE GROWTH OF MYCOBACTERIUM TUBERCULOSIS IN VITRO. W. M. Schwangart. B . S . A . Presented i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree of Master o f Sc ience i n A g r i c u l t u r e i n the Department of B a c t e r i o l o g y and P r e v e n t i v e Medic ine U n i v e r s i t y of B r i t i s h Columbia June, 1936 TiBLE OF CONTENTS. INTRODUCTION.. OUTLINE OF WORK . . iL ^}CP.rJj R. IM ILTtf T.A "L * ••••#••#•••••••••••••••••••••••••••••• A. DRUGS . B. MICROORGANISM. , G« MiijDi"oi$« •••«•••»*§•••• •#•••«•••••«•••«« D. THE EFFECT OF DRUGS 4, OBSERVATIONS D IS G XJS S103$ • STJM1MLAKY"• • • • • • • • • • • • • • • * • • « « • • « • • • • • • • • • • » « * * • • • • • • • BIBLIOGRAPHY". INTRODUCTION. 1. At the outset of the work on "The Influence of C e r t a i n Chemicals on the Growth-of My c ob a c te rium Tuberculosis i n V i t r o " which i s a sequel to the i n v e s t i g a t i o n c a r r i e d on u n t i l l a s t year by Mr. Horn(1934)on a r e l a t e d problem i n v i v o , the question of f u r t h e r value of such work came n a t u r a l l y to the f o r e . With the advent of B.C.G. and the p o s s i b i l i t i e s claim-ed by i t s supporters, i n v e s t i g a t i o n s r e l a t i n g to Chemotherapy of Tuberculosis and p a r t i c u l a r l y prophylaxis seem somewhat at cross purposes. However, recent work on the antigenic r e l a t i o n s h i p of s t r a i n s of Mycobacterium t u b e r c u l o s i s seem to throw a c e r t a i n amount of doubt upon the merits claimed f o r B.C.G. Beside the c l a s s i c work of P e t r o f f *and his co-workers on d i s s o c i a t -i o n of the Koch B a c i l l u s the studies of G u i l f o r d Reed and C h r i s t i n e R i c e ' p a r t i c u l a r l y r a i s e a number of questions which w i l l have to be answered before the f i n a l dictum on the much discussed. B .C ,G. can be given. By a quantitative technique these authors show that the S and R change i n human and bovine tubercle b a c i l l i i s accom-panied by a decrease i n s p e c i f i c a n t i g e n i c a c t i v i t y i n com-plement f i x a t i o n r e a c t i o n . R colonies have been shown by the same authors to be g r e a t l y attenuated and a v i r u l e n t to guinea p i g s . • We do not know the r e l a t i o n s h i p of antibodies detected by .complement f i x a t i o n test i n Tuberculosis to the immunity of the infecteS. animal, but the question i s brought up by the * (1930) **{1931) authors how-far such decreasing antigenic values may /effect the immunological p r a c t i c a b i l i t y . v : ?fe must'keep i n mind that s p e c i f i c i t y i s a conception which lends i t s e l f too r e a d i l y to a dogmatic a p p l i c a t i o n . Antigenic s p e c i f i c i t y i s a demonstrable response.of a n t i -bodies aroused by and r e a c t i n g with, the homologous antigen. But such demonstration'has not proven that there i s no.other a l t e r n a t i v e r e a c t i o n f o r such antibodies. For instance, s a l -ine-phenol-alcohol admixture when added to the a l c o h o l i c organ e x t f a c t w i l l increase the sensitiveness o f complement f i x a t i o n , i n the Wassermann test,.but also w i l l cause the l o s s of s p e c i f i c i t y under defined conditions^ Aware; of these f a c t s the f u r t h e r study of problems r e -l a t i n g to chemotherapy appeared to have l o s t none of t h e i r a t t r a c t i o n s despite the slow progress and the accumulation of negative r e s u l t s i n the researches.carried on thus f a r . As f a r bac| as 1884, ( S e h i l l and Fischer)•, we have, upon the suggestion of p r a c t i t i o n e r s , an; i n v e s t i g a t i o n into the a c t i o n of iodine s o l u t i o n s and vapours on t u b e r c u l o s i s sputa. Sputa were;eehtrifuged and treated i n h e r m e t i c a l l y sealed glass i n presence of vapour from subliming iodine or cov-ered f o r an equal time w i t h iodine s o l u t i o n of approximately equal concentration as,the sublimed atmosphere i n the g l a s s . A f t e r subsequent i n j e c t i o n s into guinea p i g s , untreated por-t i o n s of specimens proved f a t a l , but treated portions gave no i n d i c a t i o n of e i t h e r i n f e c t i o n or t o x i c e f f e c t of i o d i n e . •...•TJi-r^ ough'put'. the'years ..that followed spasmodic work was c a r r i e d on and given up again, Pasteur's b i o l o g i c a l approach, c a r r i e d on by Roux, and a f t e r the l a t t e r 1 s death, by Calmette and co-workers, over-shadowed chemotherapy „ As f a r as pro-phy l a x i s and therapeutics was concerned, b i o l o g i c a l approach of the t u b e r c u l o s i s problem had shown more promise of immed-ia t e success:: than the tedious and slow chemical approach. Very extensive contributions have been made by Lydia De Witt"" and references to her research into t u b e r c u l o s i s chemotherapy can be found from 1914 on. U n t i l r e c e n t l y her work has f u r -nished negative information. Few references i n medical pub-l i c a t i o n s can be found to iodine and other chemicals or to combinations with i o d i n e . To c i t e one of them as example: "Hew and n o n - o f f i c i a l remedies" mentions " Iodo-oxy-benzoic-a c i d - s a l t s " f o r intravenous i n j e c t i o n of one gram i n 100 c.c. s a l i n e followed by more s a l i n e , i n treatment of advanced cases of t u b e r c u l o s i s . A p o s s i b l e formula f o r t h i s could be: p a r t i a l success. A few other references of s i m i l a r nature are a v a i l a b l e where drugs, i n a haphazard way, were i n j e c t e d into hopeless cases chancing therapeutic value. These must be regarded as worthless since s t a t i s t i c a l l y they are too i n -complete, and experimentally, they lack proper d e t a i l and foundation. Of i n t e r e s t here i s Calmette Ts view of the matter,—"Con-t r a r y to other d i s i n f e c t a n t s that act on tubercle b a c i l l i bac-* (1914—1928) try The p r a c t i t i o n e r Gainsboro^l i n the "Lancet", reports t e r i c i d a l l y such as dyes (fuchsin and gentian v i o l e t ) or t r i c r e s o l one percent, L y s o l two percent, f o r m a l i n one per-cent , iodine seems to have l i t t l e e f f e c t on c u l t u r e s i n v i t r o , Tested upon cult u r e media i t hardly d i s t u r b s the growth and becomes i n h i b i t i v e only when supplied very massive (5 percent) This i s contrary to the favourable e f f e c t of Iodoform dress-ings on tuberculous abscesses as found by surgeons." Stehego-l e f f under I.Straus's d i r e c t i o n proved t h i s f a c t . He mixed large q u a n t i t i e s of Iodoform with t u b e r c u l o s i s cultures or products which were then inoculated into animals subcutaneous-l y or i n t r a p e r i t o n e a l ! ^ , or even into a n t e r i o r chamber of the eye of r a b b i t s without e v o l u t i o n of the i n f e c t i o n being pre-vented . One must, therefore, conclude that iodoform acts by a l t e r i n g the t i s s u e s immediately about the tuberculous focus which i s being treated and not as an a n t i s e p t i c . A n i l i n e dyes and some Iodine combinations were t r i e d by Renon wi t h no apparent success. Urea added to c u l t u r e media has been found to exert d e f i n i t e i n h i b i t i v e a c t i o n (Rappin 1901). Before r e p o r t i n g ray work, i t i s of i n t e r e s t to mention George Knaysi, 1932. t1 i s known that elementary iodine does not remain i n the animal body as such but i s changed i n t o sodium iodide and p r o t e i n or l i p o i d combination. The speed of the r e a c t i o n i s not known and an i n t e r e s t i n g one to spec-ul a t e upon. Knaysi shows that i n v i t r o tubercle b a c i l l i are unable to grow on n u t r i t i v e s l a n t s a f t e r t h i r t y minutes con-t a c t with as high a d i l u t i o n of elemental iodine as 1:10 4 or to i n f e c t guinea pigs a f t e r exposure of f i f t e e n minutes to iodine i n d i l u t i o n of 1:5 x 10 . He stresses the.point that he did not use clumps (such as one would f i n d i n n e c r o t i c areas), His r a b b i t s survive an intravenous i n j e c t i o n of am-ounts up to 30 rag, of free iodine per kg, of body weight ( e l -emental Iodine i n s o l u t i o n of 1 gram of I i n 100 c.c. of aquT^S percent potassium iodide solution), However, 20 nig, i s f a t a l to r a b b i t s with progressed m i l i a r y t u b e r c u l o s i s . A dose of one rng. per kg. of 'body weight i s equivalent to one 'MACS . . P-aJfct of free I i n one hundred go.pys of blood. He i n j e c t e d f o r a period of two months twice a week, two rug. per kg. of body weight. Apparently t h i s was n e u t r a l i z e d by blood a l -k a l i reserves and f a t absorption. What he c a l l s adequate do r;e--5 mg. of iodine one week a f t e r i n f e c t i o n , 10 rag, second week, 3 mg. t h i r d week, a f t e r end of f o u r t h week 20 mg.--was given too l a t e he claims and h i s animals died due to the com-bined t o x i c e f f e c t of Iodine and traumatic influence of pro-gressed' .tuber culos i s . H, Gideon Wells and O.F. Heidenburg f i n d t h a t compounds of Iodine i n j e c t e d into tuberculous animals enter the glan-dular tubercles with readiness and the proportion of Iodine i n such is much greater than i n most other t i s s u e s except the kidney. Thus the iodine contained i s greater i n the caseous contents of the tubercles than i n the c e l l u l a r periphery. This property does not depend on any s p e c i f i c character of the tubercle for other n e c r o t i c t i s s u e , also.take up more iodine than normal t i s s u e s . The explanation offered i s t h a t : normal c e l l s are only p a r t l y permeable to iodides (except perhaps the kidney c e l l s ) and lose t h i s part-impermeability when i n j e c t e d .or k i l l e d . With i n c r e a s i n g - o r decreasing iodine content by absorption o r e l i m i n a t i o n , /the iodine i n n e c r o t i c areas v a r i e s , I n d i c a t i n g the absence of chemical or p h y s i c a l binding i n .these/areas. The authors thus advance the.hypothesis that :..the\;-tp':a's;s:age. of iodine, into the Tubercle from the blood i s .purely, a p h y s i c a l matter of c r y s t a l l i o d a l iodine compounds . \-d i f f u s i n g through the i n e r t necrotic areas. Easpenberg made an e x t r a c t of Tubercle b a c i l l i i n water : i n presence of chloroform and found i t had a decided a f f i n i t y f o r Iodine and the r e s u l t i h g compound was s p e c i f i c a l l y bac-t e r i c i d a l to t u b e r c u l o s i s organisms* 1 great number of the izo-dyes vfere studied i n experimental, t u b e r c u l o s i s i n guinea pigs by.Be W i t t , ^ e r f i n d i n g s i n connection w i t h drugs such :as mercury t r i p a h e , which are.;vstrongly b a c t e r c i d a l i n t h e l r a c t i o n on t u b e r c l e : b a c | i ^ w e r e : that they also proved very t o x i c to expirimental a n i m a l s I n the case mentioned the pigs succumbed, to mercury poisoning. The f i n d i n g s , however, when examined c l o s e l y suggest f u r t h e r investigation, i n that, ".. or s i m i l a r d i r e c t i o n s . C e r t a i n J i t r o phenyl d e r i v a t i v e s of Mercury had,been found by t h i s author to b r i n g about d e f i n i t e f i b r o s i s , and healing^ of tubercles i n c e r t a i n organs. This phenomenon had not been found i n any of the c o n t r o l animals. 7. OUTLINE OP WORK. From a s e r i e s of compounds at our d i s p o s a l , I . , Meta-iodo benzyl-cinnamate, I I . , 2 Iodo-5 oxy-diphenyl and I I I . , benzyl cinnamate were chosen f o r i n v e s t i g a t i o n . I . Meta-iodo benzyl-cinnamate. o n o — c = CU - cu —^ (Y'^  v  - - - - - " A I I . 2 Iodo-5 oxy-diphenyl. \ I \ 1 I I I . . benzyl cinnamate. _ c u - o — e- -= CU = tw — Compounds I and I I had not h i t h e r t o been prepared and were received newly synthesised from the department of Chemis-t r y , Compound I has been used i n an in_yj-yjo i n v e s t i g a t i o n i n t h i s laboratory and the purpose of our work p a r t l y was to par-a l l e l t h i s i n v i t r o . (Horn, 19-35) . Compound I I had j u s t been prepared by a member of the department of Chemistry of t h i s i n s t i t u t i o n aiming at chemotherapeutic p o s s i b i l i t i e s , ( D a r r a c h , 1935) and had been recommended f o r investigation,, Compound I I I i s a drug on the market at a reasonable p r i c e and was chosen as the non Iodine containing aromatic substance to be tested alongside i t s Iodine isomer compound I i n order to ob-t a i n possible data as to the d i f f e r e n c e i n ge r m i c i d a l power here with and without the Iodine. References as to the chem-i c a l behavior of I and I I p a r t i c u l a r l y were not a v a i l a b l e except f o r the recent accounts of t h e i r synthesis,, (Moore, 1934 and Darrach, 1935). The drugs are w h i t i s h c r y s t a l l i n e substances at room temperature with a melting point above blood temperature and s o l i d i f y i n g point w e l l below blood tem-perature, They were found to be i n s o l u b l e i n water and glycer ine. and I I p a r t i c u l a r l y p r e c i p i t a t e d proteins such as the a l -bumen i n egg? media, when. an attempt was made to disperse i t t h e r e i n . In a d d i t i o n , p a r t i c u l a r l y when warmed to melting tem perature, the drug e x h i b i t e d a decided tendency to come out of emulsion and form an o i l y deposit at the bottom of the mix-ture . In the i n vivo experiment (Horn, 1935) a.culture i s o l a t e d from human p l e u r a l f l u i d and very v i r u l e n t to guinea pigs was used. This organism named X-II proved to be extemely slow growing when cult u r e d on a r t i f i c i a l media and' very exacting i n i t s requirements of accessory growth f a c t o r s and other con-d i t i o n s such as pH and nitrogen and energy sources. In order to get the exact pH during various growth stages and a better i n s i g h t into the metabolic requirements t h i s s t r a i n was grown on Beck and Proskauer's s y n t h e t i c f l u i d and required four months under optimum conditions to form a complete p e l l i c l e over an area of 80 sq. cms., incubated at 38°G. For the f o l l o w i n g reasons i t was decided to pr e f e r t h i s c u l t u r e and accept the time f a c t o r rather than use a f a s t growing avirxxlent s t r a i n such as we have i n stock: 1. The experiment was to be conducted with a view of ob-t a i n i n g data regarding v i r u l e n t s t r a i n s of Mycobacterium tub-e r c u l o s i s « So Our aim was to p a r a l l e l work done with t h i s organism i n vivo and to maintain the i d e n t i c a l metabolic complexity of i t as encountered there. Experiments (J„ W e i n z i r l and F. Knapton, 1927) i n which our own stock s t r a i n among others had been investigated have shown that even the simplest r e a c t i o n such as the pH curve i n f l u i d media vary g r e a t l y i n v i r u l e n t and a v i r u l e n t s t r a i n s of Mycobacterium t u b e r c u l o s i s when cultured, a r t i f i c i a l l y . Such changes w i l l r e s u l t i n reactions producing a wide range of eii&pro ducts with complex biochemical c o n s t i t u e n t s i n v i t r o (o-r- i n vivo) v i s , a l k a l i or a c i d meta p r o t e i n , p o l m e r i z a t i o n or h y d r o l y s i s , f u r t h e r complicating the change from l i v i n g to a r t i f i c i a l substrate. Thus the f i r s t problem encountered was to a r r i v e at an a r t i f i c i a l medium which would provide the f o l l -owing c o n d i t i o n s : 1. To provide a s u i t a b l e a r t i f i c i a l substrate f o r a c c e l -erated growth of v i r u l e n t slow growing s t r a i n s of Mycobacterium 10 t u b e r c u l o s i s . 3. Not to contain proteins i n the accustomed coagulated form such as egg media, i n f u s i o n agar, e t c , 3. Not to contain agar or g e l a t i n nor any other bases , which would tend to decrease v i s c o s i t y with an increase i n temperature but rather the reverse, i n order to prevent the emu l s i f i e d drug from concentrating while i n autoclave. 4 . To provide s o l i d s l a n t s and a smooth inoculable surface and to r e t a i n t h i s c o n d i t i o n when incubated at 38°G. 5. To be s t e r i l i s e a b l e without too much d i f f i c u l t y . 6. To be f a i r l y r e s i s t e n t to d e s s i c a t i o n . 7 . To be comparatively inexpensive. 8. To be thoroughly m i s c i b l e w i t h a suspension of the drug under t e s t . No such medium was a v a i l a b l e and p r a c t i c a l l y a year's wok was expanded i n developing a new s o l i d medium, s t e r i l i s e a b l e by steam under pressure, f u r n i s h i n g the base f o r ready and l u x u r i a n t growth of slow growing s t r a i n s of Mycobacterium tub-e r c u l o s i s from man. 11. EZPER1MEE.TAL. i i . DRUGS . A number of solvents were t r i e d to e f f e c t i n c o r p o r a t i o n of the drugs i n s o l t i t i o n . O i l s were discarded as unsuitable since i n turn they provided the same problem of e m u l s i f i c a t i o n . Am-ino acids then were considered which gave a low enough melting point and were of s u i t a b l e s o l u b i l i t y . • Unfortunately as the melting point of t h i s group of substances decreases so does t h e i r s o l u b i l i t y and the one most nearly'answering our purpose, phenyl-amino-propionic a c i d , was not s u f f i c i e n t l y soluble i n water to give the desired concentrations of the substances to be incorporated.. ' Among the organic solvents none could be found which would take up- the drugs and at the same time i t s e l f prove non-toxic to l i v i n g protoplasm. Grinding the drugs i n a mortar -with molecular equivalents of sodium hydroxide i n water y i e l d e d a more water soluble sodium s a l t with- compound I I only. I t was then decided, to te s t the three compounds by emulsifying them i n t h e i r molten state with a s u i t a b l e medium to as f i n e a d i s -persion as could be obtained mechanically, thus providing un-i f o r m i t y of method and treatment of drugs used during the t e s t . 12. B. MICROORGANISM. My o o"b a o t e r 1 urn tuber c u l os i s (ho/^inls) . r e c e i v e d f o r i n vivo experiments"In 1934 from the T r a n q u i l l e Sanatorium, B.C. where i t was i s o l a t e d from p l e u r a l f l u i d and a t h i r d s u b c u l t u r e when r e c e i v e d h e r e , T h i s i s an extremely slow growing s t r a i n of h i g h v i r u l e n c e i n g u i n e a p i g s . At i n t e r v a l s i t s v i r u l e n c e was t e s t e d and at the c o n c l u s i o n of t h i s work an i n j e c t i o n of •§- c.c. of a s u s p e n s i o n equal to McFarlancL # 4 ' d i l u t e d 10 times i n t o p h y s i o l o g i c a l s a l i n e proved f a t a l to a 500 gram g u i n e a p i g i n 28 days, Post-mortem showed advanced pulmonary Tuber-c u l o s i s , g r e a t l y e n l a r g e d l i v e r and s p l e e n w i t h t y p i c a l l e s i o n s . The organism was r e c o v e r e d . T h i s organism i s r e f e r r e d to as Z - I I . D u r i n g the pr o g r e s s of the work e v o l v i n g the d e s i r e d med-ium d i s s o c i a t i o n was e x p e r i e n c e d , w h i l e the organism was • c u l t u r e d " on l o n g ' s A s p a r a g i n agar. I t was necessary t o sep-a r a t e S and R s t r a i n s by colony p i c k i n g and subsequent animal passage. The R s t r a i n d e s i g n a t e d by X - I I I showed d i s t i n c t l y d i f f e r e n t colony type as w e l l as a d i f f e r e n c e m o r p h o l o g i c a l l y when s t a i n e d a c c o r d i n g to Z i e h l N e i l s e n , (See micro photograph F i g . 1.) This l a t t e r d i f f e r e n c e was observed very pronounced a f t e r a n i mal passage. S a l i n e suspensions equal to McFarland t u r b i d i t y s t a n d a r d §& were made of R and S s t r a i n s , D i l u t i o n s 1 : 20 of these suspensions were used f o r i n t r a p e r i t o n e a l i n -j e c t i o n of guinea p i g s and mice. The amount used was •§- c.c, per a n i m a l . J 13. The" f o l l o w i n g short s e r i e s of t e s t s were made. Guinea pig. No, 115 received an i n j e c t i o n as out l i n e d above made from organism X - I I (S type). Guinea p i g No. 116 received an i n j e c t i o n as outlined above made from organism M ( l o c a l a v i r u l e n t stock) as c o n t r o l , Guinea p i g No. 117 received an i n j e c t i o n as o u t l i n e d above made from organism X - I I I (R type) . The r e s u l t s obtained are shown on Table I . TABLE I. GUINEA -PIG NO. AFTER LIVER SPLEEN L U N G S ORGANISM RECOVERED 115 : 30 days :normal no .-normal, :les i o n s t y p i c a l l e s i o n s , advanced :acid f a s t from pulmonary : a l l organs i n -V : involment.:volved. 116 ; K i l l e d :normal no -normal, :normal, :none : a f t e r 3 :les ions. mo l e s - ;no l e s -• • 0 B « 9 -.months, 117 :61 days -enlarged' :yellow :spots. : ions :ions. : enlarged : f a t t y rdegener-• a t i o n : normal •none R e m a r k T h e r e was l i t t l e doubt l e f t as to the t y p i c a l Tuber-c u l o s i s i n 115. In 116 no signs of i n f e c t i o n could be found. 117 provided us with an i n t e r e s t i n g case. Although despite some 40 s l i d e preparations from ground up t i s s u e of lung, l i v e r and spleen no organism, whatsoever could be demonstrated micro-s c o p i c a l l y . Weight of l i v e r was 67.8 grams (of which ±5$ was' 14. e x t r a e t i b l e by ether) weight of spleen 58 grams i n comparison to a t o t a l weight of pig of 345 grams at time of death. Sub-secuently treated portions of l i v e r and spleen sediment (sod-ium hydroxide' treatment "and c e t r i f u g a t i o n of ground up tis s u e ) were inoculated on Dorset's egg medium and medium P.-11 (des-c r i p t i o n see under B.--Medium) Growth of acid, f a s t organisms a f t e r incubation of 4 weeks, was obtained of the a t y p i c a l R type. On "Dorset's egg" the colonies were moist, smooth and g l i s t e n i n g , somewhat resembling young Staphylococcus culture i n appearance„ On medium R-II the colony type was f l a t , dry and scaly with none of the S t y p i c a l wrinkled or beady appear-ance. (For microscopic examination see F i g . 1.) Another guin-ea p i g i n o c u l a t i o n of m a t e r i a l obtained from treated portions of l i v e r and spleen from 117 was made and a f t e r 2 months, au-topsy revealed caseation of i n g u i n a l gland, tubercles present i n peritoneum and involment of l i v e r . Very few organisms could be detected a f t e r a thorough search of caseous m a t e r i a l from glands. These were d i s t i n c t l y a t y p i c a l when compared with X-II ( F i g . 1.) The f a i l u r e to observe the presence of organisms i n the ground up t i s s u e s of P i g 117 remains unexplained. There may be a c o n d i t i o n i n the invaded, host which can influence var-i a n t s of organisms to a granular stage i n which i t i s not eas-i l y detected by routine ordinary o i l immersion,, (E.A Walker and M . -Sweeney, 1934) . Another phenomenon, frequently observed, which occurred here was the assymbiosis of Mycobacterium tub e r c u l o s i s with d 15. molds of the' genus -peny-l ium. A f t e r a c c i d e n t a l c o n t a m i n a t i o n on one of the P e t r a g n a n i ' s s l a n t s a mold began to spread cov-e r i n g the young growth of Mycobacterium t u b e r c u l o s i s . T h i s mold, however,' r e s t r i c t e d i t s growth soon to the area above and d i r e c t l y a d j a c e n t to the b a c t e r i a l colonies.. A f t e r a week: a new l u x u r i a n t growth of the b a c t e r i a appeared on top of the mold mycelium to be covered a g a i n by new growth of mold-. This process- could r e p e a t i t s e l f u n t i l stopped by d e s s i c a t i o n . The r e s u l t was a mount of a l t e r n a t i v e l a y e r s of mold growth and b a c t e r i a l • c o l o n i e s of X - I I , The organism r e f e r r e d to as M i s an a c i d - f a s t organism kept in. s t o c k at t h i s i n s t i t u t i o n . I t was r e c e i v e d from the National type c o l l e c t i o n some twelve y e a r s ago and no f u r t h e r d e t a i l s are a v a i l a b l e at t h i s w r i t i n g . I t i s now a very fast-growing organism of t y p i c a l mycobacterium morphology, 'avi r - • u lent to guinea p i g s and an a c t i v e producer of a brownish-pigment oh agar . I t w i l l .grow -on p r a c t i c a l l y any medium. In t h i s paper the term S as applied to type of Myeob-' acterium t u b e r c u l o s i s i s not used i n a s t r i c t l y d e s c r i p t i v e sense. The S form has a colony type as described by P e t r o f f (1930) i of w r i n k l e d , almost granular appearance possesing a smooth margin, i'he R form described here as d i s s o c i a n t on long's medium (no dye added) i s very s i m i l a r to P e t r o f f ' s des-c r i p t i o n of h i s R (Plate 21, Ko. 2, 1930) of very i r r e g u l a r margin and " i s l a n d " appearance on s o l i d and l i q u i d media. I t was t h i s R organism which a f t e r animal passage through G.P. 177 could not be recovered and upon subsequent i n j e c t i o n of ground 16. up n e c r o t i c t i s s u e i n t o another animal was recovered and r e -appeared as a very smooth "moth b a l l " colony i n v i t r o , resem-b l i n g almost i d e n t i c a l l y , what P e t r o f f c a l l s the bovine S type (Plate 19, No. 2, 1930) In t h i s paper the designation R was maintained despite t h i s change (for X - I I I and the term a t y p i c a l ) . Several authors since P e t r o f f (K. Saithburn, 1935, Alex-ander-Jackson B., 1936) have described the S type v a r i a t i o n of Mycobacterium t u b e r c u l o s i s (human) as "smooth" and an extreme S type of g l i s t e n i n g d r o p - l i k e appearance and extremely high i n v i r u l e n c e . I t i s i n t e r e s t i n g to note that G. P. 117 our R ( X - I I I ) was of comparatively low v i r u l e n c e . Upon f u r t h e r an-Imal passage the moth b a l l forms appeared Ato be of very high v i r u l e n c e . (Acute involvement of i n g u i n a l glands, tubercles on peritonems, extreme l e s i o n i n l i v e r and spleen.) 17 C. _ MEDIUM. . • l o obtain, a b e t t e r i n s i g h t into the metabolic requirements of the organism (X-II) some of the media advocated i n the l i t -erature were used f o r c u l t i v a t i o n also with a view of ascer-t a i n i n g f a c t o r s of growth a c c e l e r a t i o n . F i r s t a modified Corper's agar medium was t r i e d . The basis f o r t h i s medium i s : .Sea s a l t .* 5 grams Ammonium phosphate : 3 grams Agar : 30 grams G l y c e r i n : 30 grams Made up to water : 1000 grams This medium was adjusted to^ 7.5, divided into two p a r t s , autoclave! and cooled to 43°C. Then to part I was added ifo of whole egg (obtained as a s e p t i c a l l y as p o s s i b l e ) ; to part I I ifo of d e f i b r i n a t e d Rabbit's blood lafced by an equal volume of water, (Corper, 1919) The l o t s were subsequently i n s p i s s a t e d . A number of "Petragnani's" -slants - were always kept i n stock. A m o d i f i c a t i o n of Petragnani's s e l e c t i v e medium for i s o l a t i o n of Mycobacterium tub e r c u i o s i s from specimens (see media index) had proved very s a t i s f a c t o r y at the Vancouver General H o s p i t a l l a b o r a t o r i e s . This i s a modified coagulated milk--egg medium with-added Malachite green. On i t tra n s -f e r s by loop from a -suspension,equal to McFarland's t u r b i d i t y •|E y i e l d e d w.ell d i s c e r n i b l e growth of X-II a f t e r 14 — 20 days of incubation at -38°C. Throughout -the experiment the same standard loop "was used. Against a l l media under t e s t f o r our s p e c i f i c purpose p a r a l l e l i n o c u l a t i o n s on Petragnani's modif-18. i o a t i o n were c a r r i e d on as a standard of comparison. Table I I and I I I give the r e s u l t of concurrent i n o c u l a t i o n s i n the manner described above onto the media mentioned with the a d d i t i o n of Long's basal asparagin agar with H a r r i s yeast vitamin 1 : 1000 added as a d d i t i o n a l n u t r i e n t . ('. 0Yei, Mao, 19S7) and ordinary Difco Hutrient Agar. A l l media had been adjusted to pH 7.5 before s t e r i l i z a t i o n and were checked a f t e r s t e r i l i t y t e s t s . Table I I I , g i v i n g a comparative s e r i e s of t e s t s with organ-ism M, has been added here to show c e r t a i n d i f f e r e n c e s i n phys-i o l o g i c - m e t a b o l i c reactions of t h i s ,. .". a v i r u l e n t f a s t growing s t r a i n of X - I I , such as l i m i t e d tolerance to massive p r o t e i n and a l k a l i n e r e a c t i o n . (see colour disappearance on Petrag-n a n i ' s ) . 1 ( 3 daysi ihcubatiori 1. Sorper's Agar-. ( no v i s i b l e growth " " 2. Uorper f s Agar ifo blood no v i s i b l e growth -6 days Incubation no v i s i b l e ) growth ^ no v i s i b l e gr'ow th 10 days incub at Ion no v i s i b l e growth no v i s i b l e growth days l n c u | a t l o n no v i s i b l e growth no v i s i b l e growth "SO; days Incubation ';. s l i g h t but w e l l d i s c e r n -i b l e } s l i g h t but w e l l d i s c e r n -i b l e 25 days incubation i n c r e a s i n g growth but medium begins to show too much d e s s i c a t i o n i n c r e a s i n g growth but medium begins to show too much d e s s i c a t i o n 30.days incubation - growth s t a t i o n a r y i n -fluence • of d e s s i c a t i o n growth s t a t i o n a r y i n -fluence of d e s s i c a t i o n ,40 days incubation medium markedly becoming, dried,up, no f u r t h e r growth medium markedly becoming d r i e d up, no f u r t h e r growth 60 days incubation medium and growth both drying up, organisms wher t r a n s f e r r e d s t i l l v i a b l e medium and growth both jdrying up, organisms when t r a n s f e r r e d s t i l l v i a b l e ' -JDable I I (cont.) 3. no v i s i b l e growth 4. no v i s i b l e growth no v i s i b l e growth no v i s i b l e growth no v i s i b l e growth no v i s i b l e growth no v i s i b l e growth • no v i s i b l e growth no v i s i b l e growth no v i s i b l e growth j u s t : v i s i b l e growth no v i s i b l e growth hardly v i s i b l e at point of i n o c u l a t i o n s c a l y , r a p i d l y advancing growth no v i s i b l e growth s t r i n g of wrinkled small heads l u x u r i a n t growth, wrinkled beady no v i s i b l e growth good leaping growth, med-ium s t a r t i n g to dessieate whole of s l a n t becomes eo vered no v i s i b l e growth Increasing d e s s i c a t i o n but s t i l l f u r t h e r growth growth' almost s t a t i o n a r y no v i s i b l e growth growth almost s t a t i o n e r y , medium shows marked d e s s i c -at ion growth s t a t i o n a r y , medium becomes wrinkled from s h r i n k i n g no v i s i b l e growth medium dessicated to l e s s than.half i t s volume growth drying up . • • " • ' medium dessicated to l e s s than h a l f i t s volume growth drying up no v i s i b l e growth ORGANISM M . Medium used . 3 days i n c u b a t i o n TABLES I I I . 1. C o r n e r ' s Agar + Ifo egg growth v i s i b l e • 2. C-or-per's Agar +• l°/o b l o o d . growth v i s i b l e 6 days i n c u b a t i o n growth advanc ing , w h i t -i s h , d r y , obtuse growth advanc ing , w h i t -i s h , d r y , obtuse 10 days i n c u b a t i o n heaping growth, s c a l y , spread ing heads . - < —,-.Tl:-n|l',-heaping growth, s c a l y spreading heads 12 days i n c u b a t i o n growth almost s t a t i o n a r y becoming d i s t i n c t l y deep-er brown •growth- almo.st s t a t l o n a r y ' becoming d i s t i n c t l y deep-er brown 20 days i n c u b a t i o n S ' l o n g ' s B a s a l Asparag-i n Agar + H a r r i s Yeast Yitam'in t -Pet growth s topped, dess] a t i o n . Medium and gi dark brown. :.• • ' 4 . r agnan i Ts LC— •• D i f e growth s topped, d e s s i c -a t i o n . Medium' and g r . dark brown o N u t r i e n t Agar l u x u r i a n t growth, dry heaped i n p l a c e s f a i r growth not as good as i n 3 no growth growth heap ing , typ-i c a l y e l l o w i s h creamy, co lo r f a i r growth but l e s s than 1, 2, 3. v i s i b l e growth medium t o t a l l y covered turn ing d e c i d e d l y b r -own contoured growth s t a t i o n a r y be-coming brown f a i r growth advancing s lowly medium' d i f f u s e d by . . brown pigment growth smooth beady growth brown, smooth s t a t i o n a r y , medium -be-coming d i s c o l o r e d ad-jacent borders of c o l -onies smooth brown, f l a t , growth pigment d i f f u s i n g i n t o medium same as above, very large y i e l d of growth growth s t a t i o n a r y , dark brown. Return of c o l o r to d i s c o l o r e d areas same as 1 growth more sparse The apparent symbol s i s of Mycobacterium t u b e r c u l o s i s with certain molds as described p r e v i o u s l y i n t h i s paper suggested the use of them to accelerate growth. Though none of the media tes t e d answered our purpose, Long 1s Agar with the necessary added stimulant approached the growth on the egg medium (Petragnani 5 s ) , the former being on the average eight days behind/the l a t t e r . The next table (Table IV) shows a study of the e f f e c t of accessory growth f a c t o r s . A good deal of o l d ground had been covered by now with the aim to evolve a medium f o r i n c o r p o r a t i o n of drugs. The ne c e s s i t y of ferrous s a l t s and c i t r a t e s i n s y n t h e t i c media had been i n v e s t i g a t e d by Reed and Rice (1928) i n d e t a i l , i n connection with the c u l t i v a t i o n of Mycobacterium t u b e r c u l o s i s Proskauer and Bech (1914) and l a t e r Lockeman (1919) have i n v e s t i g a t e d i n d e t a i l the matter of carbohydrates as immediately a v a i l a b l e source of carbon f o r Mycobacterium t u b e r c u l o s i s . P a r t i c u l a r l y , the l a t t e r 1 s work suggested that g l y c e r i n plus the very small amount of monosacharides hydro-l i s e d during the autoclaving process i n medium R - l l would provide ample source of Immediately a v a i l a b l e energy and s t i l l keep the substate w e l l w i t h i n the sugar tolerance of the organism. In t h i s respect and relevant to general metabolic requirements of the organism the extensive studies of Kendall et a l (1914) i n respect to d e s s i c a t i o n and accompanying phenomena i n a r t i f i c i a l media, Harvey's work (1921), and others ( P o i l e and Wasserman, 1912) was taken i n t o account. As supply f o r non-protein nitrogen Perrous Ammonium 20 . c i t r a t e and Ammonium c i t r a t e was chosen (Reed and Rice, 1928). K H P O . l a CO Ivlg SO and Ha 01 were used as inorganic b u f f e r s a l t s and to serve the inorganic requirements of the organism. To provide a wider range of organic nitrogen beside the sodium asparaginate and at the same time secure organic buffer a c t i o n one gram of peptone was added. Asparagin had proved a valuable a d d i t i o n to media intended for the c u l t i v a t i o n of Tubercle b a c i l l i , ( S u l l i v a n M. , 1915), As f a r back as 1908 (Zurewitch 1908) we f i n d pointed put frequent-l y i n the l i t e r a t u r e (Lubinsky, 1895) the s t i m u l a t i n g e f f e c t of potatoes and t h e i r i n f u s i o n i n the c u l t i v a t i o n of t h i s or-ganism. One of the older formulae of t h i s kind (Kolle and Wasssrrnan, 1912) was modified with the view of re p l a c i n g the agar by some other carbohydrate' of s i m i l a r c o l l o i d a l behavior but greater v i s c o s i t y when heated, A f t e r a number of compar-at i v e i n o c u l a t i o n tests' a medium was found equal i n y i e l d to long's Agar under i d e n t i c a l c o n d i t i o n s , Berkefeld f i l t e r e d juiice from f r e s h tomatoes had to be added to both, Long's as w e l l as t h i s new medium c a l l e d E - I ; a f t e r s t e r i l i z a t i o n to accelerate growth-of the organism. The composition of R-I was HgO : 500 grams Agar : 5 grams G l y c o l ; 35 grams Potato f l o u r (Caseo Brand): 9 grams Manufactured by the ' Casco Stock .Co, Montreal. Peptone : IB grams K H 2 P O 4 : 1.5 grams lagCOg : ..1.5 gram s HaCl • : 1 gram MgSO^ : .5 grams F e r r i c Ammonium c i t r a t e : -.05 grams Ammonium c i t r a t e : 2.5 grams Asparagin : 1 gram The accessory f a c t o r s are added a s e p t i c a l l y a f t e r s t e r i l -i z i n g f o r f i f t e e n minutes at 15 pound p r e s s u r e . T h i s new medium was i n c l u d e d i n the t e s t s of acce s sory growth f a c t o r s f o r the growth of our slow growing s t r a i n X - l l i n v i t r o . P r e p a r a t i o n of growth f a c t o r s to he t e s t e d : l o Mold F i l t r a t e . Two 500 c . c . Er lenmeyer f l a s k s r e c e i v e 250 c . c . each of Dii 'co N u t r i e n t b r o t h p l u s Zfo g lucose and are a u t o c l a v e d . A pure c u l t u r e of our mold ( P e n i c i l l i u m ) i s then t r a n s f e r r e d by loop i n t o each and t h i s f o l l o w e d by at l e a s t one weeks i n c u b a t i o n at 3 8 ° C . a . Treatment of Contents of F l a s k A . ' The heavy growth i s t r a n s f e r r e d i n t o a l a rge s t e r i l e mortar and ground up w i t h s t e r i l e p e s t l e i n a sma l l p o r t i o n (10—20 c . c , ) o f the l i q u i d u n t i l w e l l macerated. The whole i s then t r a n s f e r r e d i n t o a s t e r i l e beaker and added to the r e s t of the b r o t h , w e l l mixed and f i l t e r e d twice through washed g l a s s wool or gauze. The r e s u l t i n g l i q u i d should be f ree from coarser p a r t i c l e s and slow uni form t u r b i d i t y . Ad jus t to pH of 7.5 w i t h l a O H . l e t t h i s l i q u i d stand f o r 6 hours covered by s t e r i l e paper or watch g l a s s then de cant i n t o B e r k e f e l d f i l t e r " 1 " and f i l t e r i n t o s t e r i l e s iphon f l a s k (see f i g . 3 . ) . Test sample f o r s t e r i l i t y . b . Treatment of Contents of F l a s k B . The l i q u i d was decanted from the growth and the former f i l t e r e d immediately through B e r k e f e l d candle N i n t o a s t e r i l e s iphon f l a s k a f t e r adjustment to pH 7.5 wi th ITaOH. 23. 2, 3gg F i l t r a t e . I n t o t h r e e 250 c.c. Erlenraeyer f l a s t s two eggs each were broken as a s e p t i c a l l y • a s p o s s i b l e and d i l u t e d to a p p r o x i m a t e l y 200 c ,c, per f l a s k w i t h s t e r i l e , p h y s i o l o g i c a l s a l i n e a d j u s t -ed to pH. 7,6 The f i r s t f l a s k was f i l t e r e d through a " S e i t z " f i l t e r a f t e r the egg mixture was u n i f o r m l y d i s p e r s e d . The second f l a s k was put i n t o the icebox f o r 24 hours and then f i l -t e r e d through " S e i t z , " i n t o a. s t e r i l e s i p h o n f l a s k . The t h i r d fleeek was l e f t 48 hours on i c e b e f o r e p a s s i n g through v. " E e i t s : i f i l t e r . 3. B l o o d and B l o o d serum. Blo o d was o b t a i n e d a s e p t i c a l l y from the r a b b i t by c a r d i a l e s s a n q u i n a t i o n and kept i n s t e r i l e f l a s k w i t h added 10$ Ha-c i t r a t e i n p r o p o r t i o n s 1 t 10, I t was then added t o s o l i d , media at 43°C. j u s t b e f o r e p o u r i n g or s l a n t i n g . The serum was o b t a i n e d by l e t t i n g some of the b l o o d c o a g u l a t e i n s t e r i l e tubes and c a r e f u l l y d r a w i n g o f f serum when needed. • i l l media r e c e i v e d 1 c . c of each substance under t e s t f o r growth promoting a b i l i t y per 9 c.c. of s u b s t a t e . ,J-'he t a b u l -a t i o n of r e s u l t s shows a d i s t i n c t p r e f e r e n c e of the organism f o r s o l i d media when c u l t u r e d i n v i t r o . I t a l s o seems t h a t f i l t r a t e from the macerated mycelium e x e r t s a g r e a t e r s t i m -u l u s than, other f a c t o r s t r i e d here, i l l experiments were done i n d u p l i c a t e and w i t h b l a n k c o n t r o l s and s e v e r a l s l a n t s o f Petragnani's medium were i n o c u l a t e d , as p a r a l l e l s w i t h ' t h e standard t r a n s f e r used throughout the experiment, (J : c.c, of a s u s p e n t i o n e q u a l to McFarland standard .ff4 over 20 with s t a n d -24. ard l o o p ) . Four media were select e d to f u r n i s h the b a s i s f o r t h i s t a b u l a t i o n : two l i q u i d and two s o l i d media i n order to determine whether the organism responds to growth promoting substances d i f f e r e n t l y i n su s t r a t e s of d i f f e r e n t p h y s i o l o g i c a l c o n s t i t u t i o n ; namely: 1. Difco n u t r i e n t broth 2. Basal Asparagin broth (Uyei, 1927) 3. Basal Asparagin agar (Uyei t 1927) 4. ' R-I In the table the name of the medium i s s u b s t i t u t e d by the l e t t e r : B -- Difco n u t r i e n t broth IB — Long's Basal Asparagin broth LA Long's Basal Asparagin agar j>_I j r e w Medium The added substances are i n d i c a t e d by numerals as f o l l o w s : Egg f i l t r a t e — 1 Egg f i l t r a t e (24 hrs. old) --2 Egg f i l t r a t e (48 hrs. old) — 3 Whole Rabbit's Blood — 5 Serum ™-6 Mold f i l t r a t e (decanted) — 7 Mold f i l t r a t e (Macerated) — 8 For example "31" — Difco n u t r i e n t broth plus added egg f i l t r a t e i n proportion 1 i n 10. For l i q u i d media incubation 150 c.c. Erlenmeyer f l a s k s were used c o n t a i n i n g 35 c . c . of the medium, plugged wi th absorbent c o t t o n and p r o t e c t e d by ce l lophane tops aga ins i n f i l t r a t i o n of contaminants . F o r s o l i d media i n c u b a t i o n , s l a n t s were i n o c u l a t e d i : c u l t u r e tubes of the s i z e 6 x f i n c h e s . The l i q u i d media have the great disadvantage of l o s i n g S t >* r a p i d l y i n volume when incubated and are not p r e f e r r e d by the organism apart from being unsuitable f o r incorporating the drug, . Of the s o l i d media R-I provides an e a r l i e r appearance of growth- than Long's agar when Mold f i l t r a t e i s used, as added stimulant, I n t e r e s t i n g to observe i s the influence of serum as com-pared to whole felood. When the former i s added growth seems to appear l a t e r , however i t w i l l soon increase more r a p i d l y and soon surpass i n volume the y i e l d on the media with whole Blood. I t seams e s s e n t i a l to macerate the mold before e x t r a c t i n g i n i t s own substate and subsequent f i l t e r i n g to secure the stimulant effect,, The f a i l u r e to secure growth i n c e r t a i n instances i s prob-ably not due tp any i n h i b i t i n g f a c t o r but i s a phenomenon f r e -quently experienced when working-with exceedingly small inoc-u l a of Mycobacterium tuj3^'j3ul£S_is i n v i t r o . A f u r t h e r short se r i e s of comparative t e s t s , the t a b u l a t i o n of which was found unnecessary here, revealed that the action of a c e r t a i n yeast d i g e s t * ( o r i g i n a l l y prepared by Dr. 0, Jen-sen of Kopenhagen and received here through personal commun-i c a t i o n by the department of Dairy bacteriology) was equal to * This yeast digest i s manufactured now according to o r i g i n a l formula by Digestive Ferments Go, Detroit,Michigan under the name, "Bacto Yeast E x t r a c t . " 27 . that of our mold f i l t r a t e , Since the former requires l e s s time of preparation we su b s t i t u t e d i t f o r the mold e x t r a c t . (see i n s t r u c t ions of preparation of medium R - I I ) , Both yeast or mold e x t r a c t may be added before s t e r i l i z -a t i o n by steam under pressure, which n a t u r a l l y i s not-possible w i t h the other n u t r i e n t s , Apart from becoming p r e c i p i t a t e d out by heat coagulation the l a t t e r seem to lose t h e i r -stim-u l a n t e f f e c t . Unfortunately, due to i t s l i q u i d state when heated to 100° C, the medium R-I permitted water inso l u b l e chemicals to lose t h e i r dispersed state when added and c o l l e c t at the bottome of the c u l t u r e tube. The author observed the phenomenon that i f a mash c o n s i s t i n g of raw potatoes and starch 1 ; 3 with .4$ agar added was heated above 80°G. f o r 10 minutes the mixture would, i f kept at a pH. of 7.5 become suddenly very viscous and pasty and r e t a i n t h i s c o n d i tion throughout autoclaving. I t would be just l i q u i d enough to permit s l a n t i n g d i r e c t l y when removed from the s t e r i l i s e r but a rearrangement of c o l l o i d p a r t i c l e s at any time a f t e r the s e m i - j e l l y stage had been reached was impossible . By combining the R-I formula with the aforementioned pro-cess a medium was evolved which not only proved to be i n v a l -uable f o r our purpose but also an e x c e l l e n t subst Aate f o r l u x -u r i a n t and speedy growth of a slow growing s t r a i n of Mycobac-terium t u b e r c u l o s i s . This f a c t would furthermore suggest i t fo r use i n the production of t u b e r c u l i n B.•E. When compared with the growth obtained on modified Petrag-n a n i ' s medium, t h i s new medium i n some i n s t a n c e s lagged behind somewhat as to the f i r s t appearance of growth, i n a l l cases , however, i t y i e l d e d many t imes the amount of b a c t e r i a l growth 'when sown w i t h -§• c . c . of a suspens ion of X - l l equa l to McFar land s tandard #4 over 20. T h i s medium, des ignated by the i n i t i a l s R - l l , possesses a remarkable power to r e s i s t d e s s i c a t i o n . S l an t s of i t a f t e r hav ing been h e l d at 2 5 ° C . over 3 months, unpro tec ted except f o r a sma l l co t ton p l u g , have shown no s i gns of d e s s i c a t i o n . I t may be r e s t e r i l i z e d f o r 10 minutes at 15 pounds and does not need f u r t h e r n u t r i e n t s s ince the yeast s o l u t i o n i s added before a u t o c l a v i n g . The co lour i s deep brown to b l a c k . To render the medium s e l e c t i v e Ma lach i t e green may be added, 1 p a r t i n 50,000. The author h e s i t a t e s to recommend the i n d i s o r i m i n a n t use of dyes such as M a l a c h i t e green or g e n t i a n v i o l e t r e g a r d l e s s of the b a c t e r i a l s t r a i n and medium u s e d . Ingraham (1933) working w i t h 3. c o l i b r o t h tenders the c o n c l u s i o n that the b a c t e r i o s t a t i c a c t i o n of the l a t t e r dye i s a r e s u l t of i t s a b i l i t y to po i se the o x i d a t i o n - r e d u c t i o n p o t e n t i a l of the med-ium and thus permi t s growth i n such media which are i n f l u e n c e d by i t s presence towards a b a c t e r i o p h y l i c p o t e n t i a l . I found , f o r example, tha t the presence of c r y s t a l v i o l e t 1: 30,000 comple te ly i n h i b i t s growth of R ( a t y p i c a l ) and r e t a r d s growth of S ( t y p i c a l ) s t r a i n s of organism X - l l . on s l a n t s of medium R - l l and on p l a t e s o f P e t r o f f 5 s g e n t i a n v i o l e t — g l y c e r o l — egg medium. P r e p a r a t i o n of Medium R - l l . 1. Into a 500 c.c, beaker weigh out: 29 K H 2 P 0 4 I a 2 C 0 3 l a O l Mg s o 4 F e r r i c Ammonium c i t r a t e Ammonium c i t r a t e Sodium Asparaginate or Asparagin Potato floujr (Casco Brand) Agar Peptone C h o l e s t e r a l 1.5 grams 1.5 grams 1 gram 0.5 grams 0.025 grams 2.5 grams 2 grams 10 grams 4 grams 1 • gram' 0.05 grams Adjust 1000 c.c. of tap water to pH of 7.6 using N/200 NaOH. Add 250 c a c . of the water adjusted to above plus 25 e, of g l y c e r o l . Dissolve .5 grams of yeast digest in.50 c.c. o f adjusted water over f lame, f i l t e r a f t e r d i s s o l v i n g through Whatman's No. 5 paper and' add to so l u t i o n . 2 S P e e l three potatoes'of the si z e of a large egg and f i n e l y mash through meat grinder* Avoid coarse p a r t i c l e s and. cover mash with 300 c , c . of adjusted water. Mix thoroughly and adjust to pH. of 7.8. Let mash s e t t l e to bottom and wait t i l l deep rust or brown to black i n colour, 3, Now mix 1 and 2 and heat i n water bath with constant s t i r r i n g and adding another 150 c.c, of adjusted water. As soon as coagulation has occurred take away flame and disperse Into l a r g e tubes (18 a. 1.50 mm.) a p p r o x i m a t e l y 6--7 e,c, p e r ^ ' tube,' The f i n a l pH s h o u l d be 7,4; i t can be a d j u s t e d at t h i s stage to any d e s i r e d pH-. I t i s ad'vistable to use s l a r g e t o r e f u n n e l t i p • nd d i s p e n -s i n g hose (£- i n c h i f p o s s i b l e ) . Tubes are plugged vyith non-absorbent c o t t o n . l u t o c l a v e f o r 10 mi mites at 15 pounds of p r e s s u r e and take care to l e t p r e s s u r e down s l o w l y . S l a n t tubes i n d i v i d u a l l y tippin£ them b e f o r e p u t t i n g i n t o permanent s l a n t i n g p o s i t i o n . L e t r e s t o v e r n i g h t . I t i s recommended to l e t the - s l a n t s remain i n s l a n t i n g p o s i t i o n f o r a day or two b e f o r e s t o r i n g i n i c e box or c o o l p l a c e » The c o s t of m a t e r i a l f o r t h i s medium i s very moderate and has been c a l c u l a t e d a t 22 cents per 100 s l a n t s ( i n c u l t u r e tubes 18 x 150 mm.} a c c o r d i n g to the p r i c e s p a i d here per s m a l l ' l o t f o r the v a r i o u s c o n s t i t u e n t s , T h i s compares f a v -o r a b l y w i t h the cost' of the customery co a g u l a t e d egg media which even at a very low p r i c e of egg c o s t s none-than three times as much and l e f r a ^ n a a - i 1 a which i s more than .twice as e x p e n s i v e 5 not c o u n t i n g the a d d i t i o n a l time and work expended i n p r e p a r i n g and s t e r i l i s i n g the l a t t e r . The medium' i s at p r e s e n t under t e s t f o r i t s s u i t a b i l i t y f o r i s o l a t i o n of Fy cob act or 3. urn tuber c u i o s i s from s out urn and other specimens, A r e p o r t i n r e s p e c t to t h i s cannot be had f o r some months. 31 o Two f u r t h e r media constituents w i l l be described b r i e f l y which were used i n combination with medium E - l l i n the ex-periment o u t l i n e d i n the next paragraph. ."Bios" E x t r a c t , I t had been shown (Wood, 1936) that Molds and yeasts furnished an e x c e l l e n t source of B i o s , a strong growth stim-ulant f o r c e r t a i n Streptococci and Rhizobia. (Eagles et a l , 1936), (West, 1936). Bios i s a nitrogenous extract u s u a l l y obtained from A l f a l f a or Tomatoes which can be f r a c t i o n a t e d i n t o three f r a c t i o n s showing d i s t i n c t p r o p e r t i e s each. When tes t e d b i o l o g i c a l l y they were shown (Eagles et a l , 1936) to be i d e n t i c a l with those of M i l l e r et a l . The extract used here had been obtained from the Department of Dairy Chemistry, I t had been prepared from A l f a l f a by the Tannic a c i d method (wrood, 1935). The e x t r a c t , free from inorganic s a l t s , i s dark brown i n colour and has a pH of 6.8 which corresponds to the pH of both mold and yeast e x t r a c t s prepared and described p r e v i o u s l y i n t h i s paper. . To get a s t i m u l a t i n g e f f e c t i t had to be d i l u t e d 1: 1000. When added i n higher concentrations growth of organisms was i n h i b i t e d . Veal I n f u s i o n (double st r e n g t h ) . (Modified Connaught Laboratory method). Fresh boneless v e a l i s minced a f t e r removal of surplus f a t . 500 c.c. of water i s added to 500 grams of meat. S t i r thor-oughly and skim o f f f a t . Repeat several times. Let stand i n ice box overnight. Mix w e l l i n l i t r e beaker. In water bath slowly r a i s e temperature to 65°C. S t i r frequently and skim o f f f a t . How q u i c k l y rais.e temperature to 90°C.• and h o l d f o r 30 m i n u t e s . D r a i n as a s e p t i c - a l l y as p o s s i b l e and f i l t e r t hrough s t e r i l e gauze, A d j u s t pH to 7,9 w i t h l a O H . Then; add: D i f c o p r o t e o s e .-- S$ E a C l . —..5$. M a l t o s e . --.4$ Readjust to pH 7.5, f i l t e r t hrough s t e r i l e Whatman's Ho. 5 i n t o s t e r i l e 500 c.c. f l a s k . Put through Per k e f e l d "S" Into s t e r i l e 500 .c.c. f l a s k . J u s t b e f o r e r a i s e temperature to 80°G. Prom the syphon f l a s k , t h i s "double s t r e n g t h i n f u s i o n " can e a s i l y be dispensed as desired, and kept i n d e f i n i t e l y . I f b r o t h j e l l i e s , warm to . 6 5 ° C . b e f o r e a t t e m p t i n g t o syphon. (See Pig,3) K . B . In p r e p a r i n g the medium ^-11 the author found i t most con-v e n i e n t to weigh out a l l the i n g r e d i e n t s f o r s o l u t i o n at the c lo se of the day and to l e t them s tand overn ight to d i s s o l v e i n a covered beaker . Then g r i n d the potatoes the next morn-i n g , add yeast s o l u t i o n and mix-the whole. W i t h i n an hour the medium was ready fo r d i s p e n s i p n and the a u t o c l a v e . With age, p a r t i c u l a r l y when incubated f o r very long p e r i o d s , the medium somewhat l i g h t e n s i n co lour wi th no e f f e c t on i t s q u a l i t y . , 33 o D. THE EFFECT OF DRUGS. Before examining the e f f e c t of the drugs on the growth of Mycobacterium t u b e r c u l o s i s i n v i t r o i t was deemed advisable to take i n t o account: 1. The p o s s i b i l i t y that i n a medium of high p r o t e i n con- • tent the a c t i o n of the emulsoid drugs may be n e u t r a l i s e d due to amphoteric nature of the pr o t e i n s or be diminished through c o l l o i d coating by the former. 2. The batch of R - l l had to receive c r y s t a l - v i o l e t ( l : 30,000) to keep down contamination since p e t r i p l a t e s were be-ing used f o r c u l t u r i n g . By a subsequent a d d i t i o n of mold f i l -t r a t e or Bio s , the l a t t e r being i n the nature of an amino a c i d (Wood, 1936), the OR may be influenced to give d i f f e r e n t growth r e s u l t s . Object: In the f i n a l t e s t of the drugs under examination these f a c t o r s were accounted f o r i n the f o l l o w i n g mariner: Procedure: Si x l o t s of R - l l of 300 c.c. each were prepared and la b -e l l e d A,B,G,D,E and F. Before heating, each l o t received the a l l o t t e d amount of compound 1. B ~ 7 $ t 0 - - l#, D--2$ , e t c . A was kept as blank. As soon as the desired v i s c o s i t y was reached a l l were dispensed i n t o large tubes, 13 c.c. per tube ,(at l e a s t 24 tubes per l o t ) plugged, the batches kept sep-a r a t e l y and s t e r i l i s e d i n autoclave f o r 10 minutes at 15 pounds. Mold f i l t r a t e , Bios and Yeal i n f u s i o n had pre v i o u s l y been pre-pared and held ready i n siphon f l a s k s . A f t e r cooling to 50°C. 34... •• A f t e r (1 bo l i n g to 50°C. four tubes of Batch A received 7 c.c. uf veal i n f u s i o n a s e p t i c a l l y (See ^ i g . 3)'by a s p e c i a l l y con-s t r u c t e d dispensing apparatus and were marked V. A. f u r t h e r four r e c e ived i n the same manner 7 c.c. of Mold f i l t r a t e and were marked M. This was repeated w i t h Bios and then with ' mixtures' of the three added substances. The batches were s t i l l kept separately and by now the 24 tubes i n container A, had been, marked as f o l l o w s : 4 tubes............. M — 7 c.c. Mold f i l t r a t e 4 tubes............. V --7 c.c. Veal i n f u s i o n : 4 tubes............. B --7 c.c. Bios extract d i l u t e d 1:1000 A tubes............-MB -- 3.5 c.c. M and 3.5 c.c. B 4 tubes.. MV -- 3.5 c.c. M and 3.5 c.c. V 4 tubes BV — 3.5 c.c. B and 3.5 c.c. V " T h i s process was repeated w i t h a l l batches from A to F, which were ke j t at constant temperature (50°C). The tubes were poured Into previously matched P e t r i plates immediately and allowed to harden, i'he plat e s were marked accordingly. Sal i n e suspensions of -X-H' and of i t s d i s s o c i a n t X - I I I were made equal to.McFarland T u r b i d i t y standard #4. Of each s e r i e s of four of each batch two received 1 c.c. each of X-II suspension and two received 1 c.c. of &-II suspension. This gave the f o l l o w i n g arrangement: • . : (See f o l l o w i n g page.) ; TABLE IV&-. —-— ^ . AGE -•-' B I . • B 2. 4 days no growth no growth no grow th 10 days no grow th no growth no growth 15 days no growth no growth no grow t h 20 days no growth no growth s.ubsurfae.e growth 25 days s l i g h t growth on surface and sub-surface pud small amount of subsurface growth increased subsur-face growth" 30 days no increase i n sur-face growth; no sed-iment; more subsur-face growth no increase i n •growth medium c l e a r but heavy growth mid- ' ?i/ay between bottom and surface 40 days same as above; med-ium dessieated 50$ more than 60$ dessieated same as above; dess-i c a t i o n 50$ 50 days no increase i n growth* medium dess-i c a t i n g to syrup almost dry * TABLE 17b. dessieated to syrupy l i q u e r AGE LB I . "LB 2. LB 3. 4 days no growth ' no grow th no growth 10 days no grow th no growth no growth 15 days no growth no grow th no growth a o days one small p e l l i c l e • fl a k e one s m a l l p e l l i c l e f l a k e Dne small p e l l i c l e f l a k e : 25 days very slow increase - very slow increase very slow increase 30 days the small p e l l i c l e hardly increased the small p e l l i c l e hardly increased the small p e l l i c l e l a r d l y Increased 1 40 days development stopped d e s s i c a t i o n over 50$ development stopped d e s s i c a t i o n over 50$: • • • s levelopment stopped^ l e s s i c a t i o n over 50$ | 50 days I N.B. "subsurface grow th"-—a granular growth of the organisms somewhat below the surface of l i q u i d not rendering the same t u r b i d not forming a sediment, , • m i -no ^growth -no growth B 6. (ho growth no growth no growth Hi-no growth BJ3. no grow th . no growth no growth no growth no growth no growth no growth no growth no growth s l i g h t p e l l i c l e subsurface growth no growth no growth s l i g h t growth on surface and sub-surface no " v i s i b l e i\n^ crease i n growth no growth ho growth [medium c l e a r but heavy growth mid^ |way between bott om and surface d e s s i c a t i o n over 50$ almost dry d e s s i c a t i o n over so$ d e s s i c a t i o n over 60$ de s s i c a t i o n over 60$ almost dry dry condensed l i q u i d few p e l l i c l e Iflakes TABLE I?b« (eont.) no growth no growth no growth" nu growthr somewha t~b e tt"e r" no growth no growth no growth no growth no growth no growth no growth no growth one small peIL^~ i c l e f l a k e p e l l i c l e about s i z e of l i t t l e f i n g e r small p e l l i c l e . f l a k e j u s t appear- crease Ing very slow i n -" p e l l i c l e — slowly; over 50$ volume lose! r a p i d increase same as LB 5 hardly increased growth " p e l l i c l e slowly:; over 50$ volume l o s t growth stopped i e s s i c a t i o n over 50$ no growth no growth one small p e l l -i c l e f l a k e more r a p i d i n -crease p e l l i c l e s i z e of thumb n a i l and subj surface growth almost dry almost dry f a i r l y good p e l l i c j s t i l l growing over 50$ dessicated almos t dry , almost d$y | p e l l i e l e almost icomplete AGE. 4 days LA 1. no growth TABLE; I V C . [ :. . LA 2. no growth LA 3. no growth 10 days no growth no growth no growth 15 days no growth no growth no growth 20 days very s l i g h t growth very s l i g h t growth very s l i g h t growth 25 days one colony v i s i b l e one colony V i s i b l e one colony v i s i b l e 30 days s e v e r a l colonies growth i n c r e a s i n g s e v e r a l colonies growth Increasing no.growth 40 days growth heaped wrink-l e d f a i r y i e l d growth, leaped wrink-l e d f a i r y i e l d no grow th 50 days growth s topped d e s s i c a t i o n growth stopped d e s s i c a t i o n grow th stopped d e s s i c a t i o n AGE, 4 days R l 1. J no growth TABLE IVd. R l 2. no growth R l 3. : no growth 10 days no pgrowth 1 no growth no growth 15 days no growth . • no growth no growth 20 days growth very s l i g h t growth very s l i g h t growth very s l i g h t 25 days growth increased s e v e r a l colonies growth increased s e v e r a l colonies growth increased sev-' e r a l colonies 30 days growth very l i k e LA 1 growth very l i k e LA 1 good growth very l i k e ! LA 1 40 days f a i r y i e l d growth stops much dess-i c a t i o n f a i r y i e l d much d e s s i c a t i o n f a i r y i e l d much dess-' i c a t l o n 50 days• medium shr i n k i n g slowly medium s h r i n k i n g slowly medium s h r i n k i n g slowly i LA 5. no growth ; TABLE rste :"... LAB. V. • no growth (cent . ) " LA. 7. no growth LA 8. no growth no growth no .growth no growth no growth no growth no growth . no growth no growth 1 very s l i g h t g r . very s l i g h t g r . very s l i g h t g r . s l i g h t but d i s t i n c t 1 colony v i s i b l e 1 c olony v i s i b l e very s l i g h t g r . r a p i d grow th s e v e r a l c o l o n i e s w r i n k l e d heaped good grow th good growth, good growth l u x u r i a n t growth as i n LA 1 q u i t e good y i e l d b e t t e r than l-?-7 f a i r y i e l d e x c e l l e n t heavy growth surpa s s ing a l l others growth stopped d e s s i c a t i o n growth stopped d e s s i c a t i o n growth s topped d e s s i c a t i o n growth stopped d e s s i c a t i o n no growth ' : SABLE; i v d ( , RL e,: ] no growth * cont . ) R 1 7 . no growth R l 8. ,• no growth no growth no growth no growth no growth no growth very s l i g h t g r ^ : , no growth 1 colony v i s i b l e '. very:;v s l i g h t g r . 1 colony v i s i b l e and other marks of growth no growth s e v e r a l c o l o n i e s good growth : grQWj-th'ih creased s e v e r a l c o l o n i e s good grow t h ; no growth ' e x c e l l e n t growth very heavy y i e l d . good growth very l i k e LA 1 growth very much stage, of Rl. 8 at 25 days -no growth • .• growth at l i m i t .: whole medium cov-ered f a i r growth y i e l d ; . stops much dess-i c a t i o n good y i e l d growth, stops much dess-i c a t i o n no. growth . i e x c e l l e n t y i e l d liuch b e t t e r than L—7 med ium sh r i n k i h g t Qedium s h r i n k i n g i slowly ne d ium s hr ink ing i s lowly i nedium s h r i n k i n g s lowly X - I I I X - I I ' -X-IIT x-II G O 0 0 0 0 0 0 8 Y e 3 i> c a e >s & s © c e a © o 0 0 0 O O 0 0 c « © a « 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 " 0 0 no drug no drug , 7 $ drug , 7 $ drug X - I I I X - I I . - X--1II X - I I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 , 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 , 0 0 0 0 0 0 ' 0 0 0 0 0 0 8 MB 8 MY 8 BY « a a f o a o e s us -fee. M Y B MB MY # Ifo Zfo 2% 0M X - I I I X - I I X - I I I X - I I 0 0 0 0 0 0 C O 0 . 0 0 0 0 0 0 0 . . . ....... Y 0 0 0 0 0 0 0 0- .............. B 0 0 0 0 0 0 , 0 0 ............... i Li 0 0 0 0 0 0 0 0 • . . . . . . . . . . . . . . MY 0 0 0 0 0 0 .0 0 ......... BY of of *kf 4.f This experiment was repeated with compound TTo. 2 and com-pound No. 3. Each experiment required 144 p e t r i p l a t e s . To prevent d e s s i c a t i o n of the p l a t e s during incubation •they were sealed w i t h Gutta Percha rubber s p l i c i n g tape. The tape was n e i t h e r treated nor s t e r i l i s e d p r e v i o u s l y . When put-t i n g i t on, i t was stretched to the extent that approximately 25 centimetres would meet end over end ( See P i g . 4.) The i n -cubation temperature was 38° C. The p l a t e s were kept i n the incubator and a f t e r two weeks examined p e r i o d i c a l l y . During the pouring of some of the high er concentrations part of the drug p a r t i c u l a r l y i n the 3% and 4$ dispersions concentrated and caused large globules to stand out on the p l a t e . The ,7% drug mixture, however, i n every instance retained an even d i s p e r s i o n . A f t e r 3 months a l l p l a t e s were opened and regardless of sign of growth t h e i r surfaces were washed thoroughly with s t e r i l e s a l i n e by spraying and rubbing over with pasteur pip-e t t e s . These s a l i n e washings were t r a n s f e r r e d a s e p t i c a l l y i n -to s t e r i l e t e s t tubes and evaporated at 37°G to a volume of 3 c.c. and incubated f o r 24 hours. A f t e r t h i s they were w e l l shaken and sub cultured by means of t r a n s f e r s of -§- c.c. with pasteur p i p e t t e s onto R-II s l a n t s (no dye added). Control i n -oculations were made i_n vivo from the washings of blanks and drug p l a t e s containing .7$ d i l u t i o n s of the drug only, by i n - . i e c t i n g i n t r a p e r i t o n e a l l y into guinea pigs i; c.c. of the sa l i n e washings. Prom each p a i r of plat e s s l i d e s were made and stained f o r a c i d f a s t organisms and f i l e d . The t o t a l amount of drugs used per .400 gram u n i t of guinea p i g i n vivo 36b. amount of drugs used per 400 gram u n i t of guinea p i g i n the vivo experiment of Horn was 2.5 c o . This corresponds to .625 gr. per 100 grams of body weight or 625 gr. per 100 grams 0 f weight of c i r c u l a t i n g r e t i c u l o e n d o t h e l i a l substance approx-imated as l / l O of t o t a l body weight. The amounts of drug i n t h i s experiment were kept w i t h i n - t h i s range as f a r as was possible w i t h the l i m i t e d supply of these chemicals. OBSERVATIONS. A l l p l a t e s inoculated with X - I I I ( a t y p i c a l ) showed no , signs of growth a f t e r three months incubation. S l i d e s made from the washings of the blanks did not reveal presence of a c i d f a s t organisms. I n j e c t i o n into guinea p i g from washing of blank plates inoculated with X - I I I proved non-infectious. A l l p l a t e s inoculated with X-II and with an admixture of ether compounds 1, 2 and 3 gave no v i s i b l e growth of the organ-ism. The 24 blank plates of Batch A a f t e r 7 weeks showed small colonies of X-II appearance. These colonies a f t e r 9 weeks did not develop further and d i d not give the lux u r i a n t growth as i s experienced on the ordinary X-II medium without added c r y s t a l - v i o l e t . S l i d e s were made from plate washings of the drug admixture showed no presence of a c i d f a s t organisms. Several s l i d e s made, from lower concentrations [ ,9c/6) showed small areas of acid f a s t s a l i n e but no organism could be detected. S l i d e s from blanks in-every case showed a c i d f a s t organisms present when smear was made from one of the small wrinkled colonies present. The observations were i d e n t i c a l In a l l three series of t e s t s with compounds 1, 2 and 3 r e s p e c t i v e l y . The growth of Mycobacterium t u b e r c u l o s i s s t r a i n X-II had been completely i n -h i b i t e d on a l l plates containing from 0.7^ to b% of any of the drugs under t e s t . I t had not been i n h i b i t e d on the co n t r o l p l a t e s . The growth of Mycobacterium tuberculosis s t r a i n X - I I I had been i n h i b i t e d on a l l p l a t e s , i n c l u d i n g the c o n t r o l s . 38. DISCUSS10H. Many of the problems encountered have been d i s c u s s e d dur-i n g the d e s c r i p t i o n of the experiments, The method used i n t e s t i n g , the drugs j u s t l y draws c r i t i c i s m . , To impart the drugs i n t o a medium, and then incubate f o r months to see whethe'r growth w i l l take p l a c e , w i l l not y i e l d information as to what such drug w i l l do i n a s h o r t e r time of c o n t a c t , to which i t no doubt w i l l be l i m i t e d d u r i n g i t s presence i n the animal body. On the other hand, .when i n v e s t i g a t i n g the g e r m i c i d a l p r o p e r t y i n r e l a t i o n to e x t e r n a l c o n t a c t , a method f o r m u l a t e d a l o n g the l i n e of procedure as used i n d e t e r m i n i n g the phenol c o e f i c -ient would be i n d i c a t e d . The use of the l a t t e r method i n our case had been c o n s i d e r e d and abandoned. The p e c u l i a r eeto-p l a s r s i e s t r u c t u r e of the organism makes i t a most u n r e l i a b l e f a c t o r of comparison when, used i n co n t a c t t e s t s . We know that phenol i n 5fo c o n c e n t r a t i o n s w i l l not k i l l the organism i n 12 or more hours. ( K e l s e r , 1928) I n a d d i t i o n to t h i s the i n s o l -u b i l i t y of the drugs i n water makes i t i m p o s s i b l e to use v a r i e d c o n c e n t r a t i o n s . I t was n o t i c e d that i n d i s p e r s i o n i n the s o l i d media the emulsoid g l o b u l e s were " d i s t r i b u t e d at d i f f e r e n t den-s i t i e s , depending upon the c o n c e n t r a t i o n s . I n a mixture con-t a i n i n g ,7$ d r u g , f o r example, the d i s t a n c e between dispersed, g l o b u l e s v.-as wider than i n h i g h e r c o n c e n t r a t i o n s . The a c t i o n of the drugs, t h e r e f o r e , must e v i d e n t l y e i t h e r reach beyond the of the i n d i v i d u a l g l o b u l e i n t o the rued ium not v i s i b l y d i f f u s e d by i t , or an i n f i n i t e s m a l amount of i t hi the s o l -39. u t i o n may he able to i n h i b i t the growth and b i l l the organism w i t h i n the time e x p e r i e n c e d . Gome c o n t a m i n a t i o n by molds was • , e x p e r i e n c e d on a s m a l l percentage of p l a t e , Even i n t o the most c a r e f u l t e c l m i c t h i s cannot be e n t i r e l y e l i m i n a t e d . The rubber tape f i t s w e l l over the edges of t h i s n e t r i p l a t e and c o v e r , and stands up w e l l under c o n d i t i o n s of i n c u b a t i o n . I t s . uso was found more convenient than P e t r o f f ' s method of s l i p p i n g on bands made and shaped from inner t u b i n g of t i r e s , (Petroff-, 1930) The organism X - I I I d i d not grow'at a l l on medium R - I I , p l u s dye, as observed by the f a i l u r e . t o g i v e growth on the var-i o u s c o n t r o l plates., I t was n o t i c e d t h a t X - l l was d i s t i n c t l y r e t a r d e d as.compared to growth on the medium R-I I w i t h o u t the a d d i t i o n of dye, bait gave c o l o n i e s on every b l a n k . Both s t r a i n s grow w e l l on R - I I medium wit h o u t the a d d i t i o n of c r y s -t a l v i o l e t , and i t i s safe to assume t h a t the presence(1:30000). o f - t h e dye c o m p l e t e l y i n h i b i t e d the a t y p i c a l s t r a i n , That the c o l o n i e s of X - I I d i d not develop beyond a c e r t a i n stage of growth may be due t o . t h e f a c t t h a t the oxygen t e n s i o n became too low f o r the m i c r o a e r o p h y l i c tendency of the organism i n the p l a t e s s e a l e d almost h e r m e t i c a l l y .by the rubber tape to prev-ent d e s s i c a t i o n . The compounds Meta-Iodo-bemzyl-einnamate, 2 oxy-5 odo-orthopleno'l-phenol and b e n z y l cinnamate must have a s o l u b i l i t y - o f very low c o n c e n t r a t i o n , which seems, however, to be s u f f i c i e n t to a c t g e r m i c i d a l l y on Mycobacterium tuber-c u l o s i s . This, h y p o t h e s i s seems to be j u s t i f i e d s i n c e no p r e s -ence .of organism c o u l d be demonstrated by i n v i v o and i n v i t r o t e s t s of the s a l i n e washings of 3 month o l d p l a t e s which d i d 4 0 . not show a p e r c e p t i b l e degree of d e s s i c a t i o n , and co n t a i n e d the drugs i n a c o m p a r a t i v e l y rough emulsoid s u s p e n s i o n . F u r t h e r t e s t s w i t h the use of t h i s adaptable medium are necessary to f i n d the minimum l e t h a l d i l u t i o n of any of the drugs i n q u e s t i o n , I t i s a d v i s a b l e , however, not to use the a d d i t i o n of dyes such as c r y s t a l v i o l e t . I n s t e a d of p l a t i n g , the use of s l a n t c u l t u r e s .in l a r g e tubes {•§ x- 6 inches) stopp-ered w i t h c o r k s would e l i m i n a t e an absolute s e a l and a f f o r d enough p r o t e c t i o n a g a i n s t i n f I l l a t i o n of contaminants "through" the c o t t o n p l u g and against d e s s i c a t i o n , ' In such tubes the m i e r o - a e r o p h y l i c c o n d i t i o n s seem to be j u s t r i g h t and the o r -ganism grows e x c e l l e n t l y , In p r e p a r i n g the medium R-II the author found i t most convenient to weigh out a l l the i n g r e d -i e n t s f o r s o l u t i o n at the c l o s e of the day and to l e t ' them stand o v e r n i g h t to d i s s o l v e i n a covered beaker. Then g r i n d the p o t a t o e s the next morning, add y e a s t s o l u t i o n and mix to whole, W i t h i n an hour the medium was ready f o r d i s p e n s i n g and the auttyelav^e. With age, p a r t i c u l a r l y when inc u b a t e d f o r very l o n g p e r i o d s , the medium somewhat b r i g h t e n s i n colour w i t h no e f f e c t on i t s' q u a l i t y . The c o m p a r a t i v e l y speedy growth and q u a n t i t a t i v e l y high-y i e l d w i t h a slow growing s t r a i n of Mycobacterium t u b e r c u l o s i s would suggest the medium f o r the use of p r e p a r i n g Tuberculin B . E. from s t r a i n s of s i m i l a r n a t u r e , Hothing can-be s a i d of. the s u i t a b i l i t y of the medium for i s o l a t i o n of Mycobacterium t u b e r e u l o s i s from specimens since the time has been too l i m i t e d to give i t an e x t e n s i v e t r i a l . 41 The c o s t of the medium as c a l c u l a t e d a c c o r d i n g to p r i c e s p a i d l o c a l l y f o r s m a l l l o t s of the c o n s t i t u e n t s i s 22 cents r per 100 l a r g e s l a n t s , (.£ x 5 i n c h t u b e ) . There seems to be a heat s t a b l e f a c t o r i n the e x t r a c t s of y e a s t and B i o s from d i f f e r e n t sources which a i d s the growth of Mycobacterium tu b e r c u l o s i s i n v i t r o , T his same f a c t o r may be secured i n water e x t r a c t s from macerated m y c e l i a of the pen-i e i l l i u m genus, • . ' The a c t i o n of added c r y s t a l v i o l e t i n r e l a t i o n to the OR of the medium has been d i s c u s s e d elsewhere i n t h i s paper, (Page 28 ). In f u t u r e i n v e s t i g a t i o n of t h i s and r e l a t e d problems i t would be i n t e r e s t i n g to see whether a m o d i f i c a t i o n of the methods of H e i s t and Long (1922) would permit combined i n v i v o - i n v i t r o t e s t s , I n t h i s method eap^ile/y tubes are being made use of. i n which, the humoral f l u i d of t r e a t e d and c o n t r o l animals are brought i n c o n t a c t w i t h the organisms of i n f e c t i o n . Subsequ-ent 'subculturing of "specimens" continues the t e s t izr< v i t r o . 42. SUMMARY. 1. Growth a c c e l e r a t i o n substances were s t u d i e d w i t h the aim to a c c e l e r a t e the growth of a s low-growing s t r a i n of Myco-bacter ium t u b e r c u l o s i s on a r t i f i c i a l media . 2 . . An inexpens ive new medium i s presented i n which agar as a c o l l o i d base i s r e p l a c e d by the po ly sachar ide s conta ined i n raw p o t a t o . 3 . T h i s medium i s a true s o l u t i o n of n u t r i e n t s a l t s and a suspens ion of p o l y s a c h a r i d e s of low v i s c o s i t y , before h e a t i n g . A f t e r h e a t i n g to 8 0 ° C . f o r 10 minutes a c o l l o i d change takes p lace impar t ing to the medium a very h igh i r r e s e r v i b l e v i s c o s i t y , which Is not changed d u r i n g a u t o c l a v i n g . Upon c o o l i n g , the med-ium becomes s o l i d . 4 . The a d d i t i o n of b o i l e d yeast e x t r a c t , B io s or mold f i l t r a t e had an a c c e l e r a t i n g i n f l u e n c e on the growth of a slow growing s t r a i n of Mycobacterium t u b e r c u l o s i s i n v i t r o when the above medium was used . The a c c e l e r a t i n g a c t i o n was not ex-ceeded by that of egg or B lood or B lood serum, a l l of which had hot been.heated but f o l l o w e d through B e r k e f e l d c a n d l e s . b.. The a d d i t i o n of c r y s t a l v i o l e t (1 : 30,000) to t h i s new- medium was found to i n h i b i t the growth of an a t y p i c a l d i s s -o c i a n t of My^cj^bacJterJ^ t u b e r c u l o s i s 'used i n t h i s experiment. 6. C r y s t a l v i o l e t (1 : 30,000) was found to r e t a r d the growth of the S ( t y p i c a l ) s t r a i n of the organism and to extend a b a c t e r i o s t a t i c a c t i o n i n t o the l a t e r stage of growth. 7 . M e t a - i o d o - b e n z y l cinnamate, 2 oxy-5 ibdo-or thopheny l -43. phenol and benzy l cinnamate added i n concentra t ions from Afo to .7$ complete ly i n h i b i t e d growth of Myoobaoter ium t u b e r c u l o s i s i n media of h i g h and low p r o t e i n c o n c e n t r a t i o n . 8. Washings of p l a t e s used i n above t e s t s (7) were used i n s u b c u l t u r i n g i n v ivo and i n v i t r o . The presence of Mycobac te r ium t u b e r c u l o s i s c o u l d not be demonstrated a f t e r 3 months i n c u b a t i o n on a medium c o n t a i n i n g the drugs i n concent ra t ions 0 .7$ and upwards to 4$ . 44, BIBLIOGRAPHY, , Alexander, Jackson E., 1936. Am. Rev. Tub. Calmette s 1923,, "Tubercle B a c i l l u s i n f e c t i o n s and Tuberculo s i s i n man and animal." B a l t i m o r e , Williams and Wilkins Co. Corperj H.J., 1920. Am. Rev-. Tub. vol.. 3. 8. Corner, H.J. and Cohn M.L.. 1933 Am. Journ* Hyg. s v l i , 1., Corner , H..J. 1933. . Journ. Am. Med. 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