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Investigations on the effect of colchicine on yeasts McCarter, Alec 1941

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INVESTIGATIONS ON THE EFFECT OF COLCHICINE ON YEASTS by John. Alexander McCarter, B. A. A thesis submitted in p a r t i a l fulfilment of the requirements for the degree of Master of Arts in the Department of Chemistry The University of B r i t i s h Columbia A p r i l 1941 ACKNOWLEDGEMENT The author wishes to express h i s i n d e b t e d  ness to Dr. John Allardyce under whose h e l p f u l direction t h i s work was undertaken. The a u t h o r a l s o wishes t o thank Dr. R. H. Clark, Dr. B. A. Eagles, Dr. D. G. B. Duff, Dr. William .Ure* Dr. F. Dickson, Dr. ¥©rnon Brink and - many others, p a r t i c u l a r l y those interested people i n the Department of Bacteriology of the University for their helpful suggestions, and cooperation. TABLE OF CONTENTS Page E a r l y Work w i t h C o l c h i c i n e and i t s S i g n i f i c a n c e 1 Occurrence of C o l c h i c i n e 1 E x t r a c t i o n o f C o l c h i c i n e 2 S t r u c t u r e and Chemical P r o p e r t i e s of C o l c h i c i n e 2 P h y s i o l o g i c a l P r o p e r t i e s of C o l c h i c i n e 4 The P o s s i b l e R e l a t i o n of C o l c h i c i n e t o the Cancer Problem 7 C y t o l o g y of Y e a s t s 10 E x p e r i m e n t a l / 19 T est Organisms 19 The E f f e c t . o f C o l c h i c i n e on the Budding Y e a s t s 21 The Medium 22 The I n c u b a t o r 23 P r e p a r a t i o n o f the C o l c h i c i n e S o l u t i o n s 24 The Haemoeytometer Method 24 Table 27 D i s c u s s i o n o f R e s u l t s 28 The P h o t o t u r b i d o m e t r i c Method 31 D i s c u s s i o n of R e s u l t s & Method 34 Page Summary of .the Work on Budding of Ye a s t 34 The E f f e c t o f C o l c h i c i n e on S p o r u l a t i o n of t h e Yeast 35 Summary of the Work on S p o r u l a t i o n of the Yeast 40 F u r t h e r E x p e r i m e n t a l w i t h S u g g e s t i o n s f o r Fut u r e Work 41 C o n c l u s i o n s 45 T i t l e Page i Acknowledgements i i Graphs (Haemocytometer Method) i i i Ta b l e s ( P h o t o t u r b i d o m e t r i c Method) i v , v, v i Graphs ( P h o t o t u r b i d o m e t r i c Method) v i i , v i i i P r i n t i x B i b l i o g r a p h y x, x i , x i i " INTRODUCTION Early Work with Colchicine and i t s Significance: Several years ago i t was discovered that by treating the leaves and stems or seeds of some plants with a solution of the alkaloid colchicine, a r t i f i c i a l mutations could be induced. This was a tremendously important discovery from the point of view of the genetecist who could now speed up the processes of evolution more or less at w i l l . It was also important to the plant breeder who could now produce new and improved economically important species. But i t was especially important perhaps from the point of view of the biochemist for i t i s possible that the application of the colchicine technique to a study of c e l l d i v i s i o n might give an insight into the chemistry of that process. It i s with t h i s l a t t e r p o s s i b i l i t y that the present work i s concerned. Occurrence of Colchicine: The alkaloid was f i r s t noted by P e l l e t i e r and Caventou ( 1 ) as occurring in a l l parts of the plant Colchicum autumnale or the autumn crocus. Colchicine also occurs in the followingj Colchicum alpinum, C. arenarium, C. monatum, Gloriosa superba, Androcymbium gramineum, and Mendera bulbo- codium. Farr and Wright ( 2 ) found that the alkaloid content of different samples of seeds (C. autumnale) may vary between •54 and .79$. L. P. Liptak (3 ) reports that colchicine occurs in highest concentrations in the inner layer of the seed coat. E. C. Davies (4 ) reports that the seeds of C. autumnale y i e l d .75$ and the corms .38$ of the a l k a l o i d . P. Chemitus (5 ) claims that the ripe seeds of C. autumnale contain from .4 - •9$ of the al k a l o i d , Androcymbium gramineum Mac Bridge i s a colchicine containing plant of the Southern Sahara. The y i e l d from the seeds i s .37$. The bulbs y i e l d .29$. Mendera bulbo- codium Ram contains 19$ of colchicine in the dry f i n e l y powdered corm. Extraction of Colchicine: The alkaloid may be extracted from the seeds or corms of Colchicum autumnale as the commonest source, by several different methods. Recommended are the procedures employed by E. C. Davies and J. Greir ( 6 ) and B e i l s t e i n ( 7 ). Fortunately the al k a l o i d may be obtained pure from several d i f f e r e n t supply houses. The colchicine used in t h i s investigation was obtained from Mallinckrodt U.S.P. Amorphous alka l o i d , chloroform-free. Structure and Chemical Properties of Colchicine: The al k a l o i d i s a derivative of phenanthrene. The structure as f i r s t suggested by Windaus and Scheele ( 8 ) i s : The s t r u c t u r e was f u r t h e r i n v e s t i g a t e d by K. B u r s i a n ( 9 ). Ac c o r d i n g t o t h i s author the s t r u c t u r e of the "C" r i n g i s s t i l l u n c e r t a i n , and i s p r o b a b l y CHOVne When c o l c h i c i n e i s heated w i t h a c i d i f i e d water i t l o s e s methyl a l c o h o l and y i e l d s c o l c h i c e i n e . = C H o IA E t h e r i f i c a t i o n of c o l c h i c e i n e w i t h methyl a l c o h o l y i e l d s c o l c h i c i n e . , Pure c o l c h i c i n e forms n e a r l y c o l o r l e s s or p a l e y e l l o w inodorous p l a t e s of no sharp m e l t i n g p o i n t . D r i e d oyer con c e n t r a t e d S u l p h u r i c a c i d i t s o f t e n s a t 142°C and m e l t s a t 147°C. I t i s l a e v o r o t a r y . C o l c h i c i n e i s s o l u b l e i n water t o the ex t e n t of 4.54$ a t 20°C and i s v e r y s o l u b l e i n a l c o h o l and c h l o r o f o r m . I t i s f a i r l y s o l u b l e i n benzene and o n l y s l i g h t l y s o l u b l e i n dry e t h e r . When one p a r t of the a l k a l o i d i s d i s s o l v e d i n t h r e e p a r t s of water ( w i t h heat) the s o l u t i o n d e p o s i t s a f t e r some time l a r g e g l i t t e r i n g y e l l o w i s h rhombic c r y s t a l s h a v i n g the co m p o s i t i o n CggHg^NOg*1.5 HgO. Anhydrous c o l c h i c i n e i s amorphous. There are two c r y s t a l l i n e compounds of the a l k a l o i d w i t h c h l o r o f o r m C 2 2 H 2 5 N 0 6 * G H C 1 3 c o l o r l e s s g l i t t e r i n g needles which lose CH013 at 100°C and (C 22Hg 5N0 6 ) 2 » C H C 1 3 , fine needles arranged in rosettes. It i s precipitated by phosphotungstic acid and s i l i c o t u n g s t i c acid in hydrochloric acid solution ( 1 0 ) . B e i l s t e i n states that colchicine gives a bright yellow color i n concentrated HC1, and a v i o l e t color in concentrated HNO3. An alcoholic solution of colchicine gives a garnet color with FeCl3. Boyland and Huntsman (.11) describe a quantitative determination of colchiceine in the presence of colchicine by a colorimetric method based on the green color given by colchiceine and FeClj in chloroform. Under the same conditions colchicine gives no color. Physiological properties of Colchicine: It i s with the physiological properties of colchicine that we are most interested. It was noticed by the Bulgarian botanist Dontcho Rostov ( 12) that sometimes after spraying plants with nicotine sulphate sports or mutants developed. He was led to study the effects of other alkaloids on c e l l d i v i s i o n in the production of mutants, and found that among other substances colchicine was the most e f f e c t i v e . Alpha naphthyl acetic acid i s probably next most e f f e c t i v e . He stated that acenaphthene produces less severe changes in c e l l d i v i s i o n than does colchicine (12,13,14 ). Simonet and Guinochet (15) report that alpha chloro-, and alpha bromo-naphthalene have a colc h i c i n e - l i k e action on the caryo- kinesis of plants. Colchicine has the s p e c i f i c property of i n h i b i t i n g the formation of the spindle in somatic mitosis* Perhaps i t •would not be out of place to review the general phenomena of mitosis. In this regard a diagram i s probably most he l p f u l . Concerning mitosis Sharp says "The outstanding and si g n i f i c a n t feature of somatic mitosis i s t h i s ; each chromo some i s accurately divided into two exactly equal longitudinal halves which are distributed to the two daughter nu c l e i . The two daughter nuclei thus receive exactly similar halves of the chromatin of the mother c e l l . " ( 16 ) Colchicine by i n h i b i t i n g the formation of the mitotic spindle at the metaphase creates conditions for chromosome doubling ( 13,14,17,18,19,20,21) that i s the chromosomes s p l i t longitudinally f i r s t in readiness for ultimate d i v i s i o n of the c e l l , then, when the spindle does not appear they s p l i t once more. When f i n a l l y the process of c e l l d i v i s i o n i s resumed, as when the colchicine i s removed, the daughter c e l l s receive doubled chromosomes. The o r i g i n a l papers outline in d e t a i l the procedures followed with plants and the results obtained. It i s enough to note here that s t e r i l e hybrids may become f e r t i l e 'as a result of chromosome doubling for d i f f e r e n t i a t i o n of the l! ' • 6 if ij c e l l into male and female gametes becomes possible when the fj ij chromosome sets are completed. Increase in size of pollen j . grains i s used as an indication of polyploidy. In general the size of the c e l l s , and correspondingly of the plants i s much increased. Experiments with animals have f a i l e d to produce l i v i n g polyploids. It i s true that the chromosomes of the j f e r t i l i z e d ova of rabbits have been doubled, but the embryos have never developed. Employing chick embryo heart fibroblasts and chick i r i s epithelium, growing in v i t r o Gavrilov, Dina, and Bistram ( 22) showed that even in dilutions of 1:25,000,000 colchicine arrested and distorted development of the c e l l s . The authors emphasized the fact that colchicine even in very weak solutions is a c e l l poison. Verne and V i l t e r (23) corroborated these findings, but report a higher d i l u t i o n of the alkaloid required to check d i v i s i o n of the f i b r o b l a s t s . ''They—reported that colchicine 1:4,000,000 checked the c e l l d i v i s i o n in twenty minutes in v i t r o . Colchicine i s very toxic to the animal organism. E. Maurel (24) found that 0.005 gm/kgm of body weight i s s u f f i c i e n t to k i l l a rabbit whether administered hypodermically or by stomach. Colchicine i s mildly carcinogenic, which i s not sur prising in that i t i s related to phenanthrene. It i s capable of causing the production of tumours in plants and animals (25 ). In addition to being able to cause tumours in plants, colchicine in higher concentrations can check the growth of tumours. This effect i s probably related to the inhibitory action on c e l l 7 d i v i s i o n . By p a i n t i n g tumours such as those eaused by B a c i l l u s tumefaciens on geranium and c a s t o r seed p l a n t s w i t h 0.5% c o l c h i c i n e i n l a n o l i n , the growth of the tumour can be cheeked. (26,27) B r u s h i n g the s u r f a c e s of i n d o l a c e t i c a c i d tumours w i t h 2% c o l c h i c i n e s o l u t i o n s i n h i b i t s f u r t h e r growth i n many i n s t a n c e s . Under c e r t a i n c o n d i t i o n s t h e p l a n t tumour can be c o m p l e t e l y k i l l e d by c o l c h i c i n e p r e p a r a t i o n s . I n a n i m a l s ( r a t s ) the i n j e c t i o n of c o l c h i c i n e s o l u t i o n s i n t o spontaneous mammary tumours r e n d e r s the tumours more s e n s i t i v e to the a c t i o n of X-rays ( 28 ). The P o s s i b l e R e l a t i o n of C o l c h i c i n e t o the Cancer Problem: I t i s i n t e r e s t i n g to note t h a t t h e most h i g h l y c a r c i  nogenic compounds are a l s o i n h i b i t o r s of c e l l d i v i s i o n , but are v e r y much l e s s e f f e c t i v e i n t h i s r e s p e c t t h a n c o l c h i c i n e (29), I t i s q u i t e d e f i n i t e l y a f a c t t h a t the most h i g h l y c a r c i n o g e n i c agents are the l e a s t i n h i b i t o r y of c e l l d i v i s i o n , and the l e a s t c a r c i n o g e n i c are the most h i g h l y i n h i b i t o r y of c e l l d i v i s i o n . On the b a s i s of the work of o t h e r a u t h o r s , the i n v e s t i g a t o r has been l e d to s p e c u l a t e t h a t tumour g e n e s i s depends on the pro d u c t i o n of a mutant. S i n c e c o l c h i c i n e i s c a r c i n o g e n i c , and s i n c e i t produces mutants i n b o t h p l a n t s and a n i m a l s , i t i s r e a s o n a b l e t o suppose t h a t o t h e r c a r c i n o g e n i c substances a c t i n a s i m i l a r way. V/e are thus l e d t o a s s o c i a t e c a r c i n o g e n i c i t y v e r y c l o s e l y w i t h i n h i b i t i o n of c e l l d i v i s i o n . I t would be expected t h a t substances which are h i g h l y i n h i b i t i v e of c e l l d i v i s i o n would be r e q u i r e d i n v e r y s m a l l c o n c e n t r a t i o n t o produce the temporary i n h i b i t i o n o f c e l l - d i v i s i o n w i t h 8 subsequent resumption of the p r o c e s s r e q u i r e d to produee a mutant. S i m i l a r l y , r e l a t i v e l y l a r g e q u a n t i t i e s of h i g h l y c a r c i n o g e n i c agents (low i n h i b i t o r y power of c e l l d i v i s i o n ) would be r e q u i r e d to produce t h i s e f f e c t . F u r t h e r , we would expect t h a t the s m a l l q u a n t i t y of the f i r s t substance would be e l i m i n a t e d r a t h e r r a p i d l y from the animal organism, c e r t a i n l y more r a p i d l y than the l a r g e q u a n t i t y of the l a t t e r substance. C o n s i d e r the cases of c o l c h i c i n e and m e t h y l c h o l a n t h r e n e as examples. The r e l a t i v e l y s m a l l q u a n t i t y of c o l c h i c i n e r e q u i r e d t o produce an i n h i b i t i o n of c e l l d i v i s i o n would not remain l o c a l i z e d f o r l o n g due t o e l i m i n a t i o n or d e t o x i f i c a t i o n of the a l k a l o i d . The r e l a t i v e l y l a r g e q u a n t i t y of me t h y l - c h o l a n t h r e n e r e q u i r e d t o produce a s i m i l a r e f f e c t on d i v i s i o n of the c e l l would c e r t a i n l y take a l o n g e r time t o be e l i m i n a t e d I t t h e r e f o r e seems v e r y p r o b a b l y t h a t t h e r e i s more chance of pro d u c i n g a mutant i n v i v o u s i n g m e t h y l c h o l a n t h r e n e than t h e r e i s of p r o d u c i n g a mutant w i t h c o l c h i c i n e . That i s t o say, on the b a s i s of the above h y p o t h e s i s m e t h y l c h o l a n t h r e n e would be more c a r c i n o g e n i c than c o l c h i c i n e , which i t i s . There i s ample evidence t o support the view t h a t cancer i s a mutant (30) The h y p o t h e s i s a l s o may be made t o e x p l a i n s u s c e p t i b i l i t y v a r i a t i o n s i n d i f f e r e n t t i s s u e s and s t r a i n s of an i m a l s . D i f  f e r e n t t i s s u e s f o r example are e f f e c t e d d i f f e r e n t l y by a f i x e d c o n c e n t r a t i o n of c o l c h i c i n e . T h i s v a r i a t i o n i n s u s c e p t i b i l i t y m a n i f e s t s i t s e l f even i n the A s p e r g i l l i ( 3 1 ) . We may b r i e f l y r e v iew the f a c t s and the h y p o t h e s i s : (1) C o l e h i c i n e i s h i g h l y i n h i b i t i v e of c e l l d i v i s i o n t hrough p r e v e n t i o n of the f o r m a t i o n of the m i t o t i c s p i n d l e i n 9 somatic m i t o s i s , (2) C o l c h i c i n e i s weakly c a r c i n o g e n i c i n v i v o . (3) M e t h y l c h o l a n t h r e n e i s weakly i n h i b i t i v e of c e l l - d i v i s i o n . (4) M e t h y l c h o l a n t h r e n e i s h i g h l y c a r c i n o g e n i c i n v i v o . ' I t i s suggested t h a t the p r o p e r t y of i n h i b i t i o n of c e l l - d i v i s i o n i s a r e l a t i v e l y c o n s t a n t c h a r a c t e r i s t i c of the compound used f o r any one t i s s u e . I t i s suggested f u r t h e r t h a t the c a r c i n o g e n i c i t y of t h i s substance f o r the same t i s s u e v a r i e s a c c o r d i n g to whether the p r o d u c t i o n of a mutant t a k e s p l a c e i n v i v o or i n v i t r o . I t i s known t h a t methycholanthrene has never been a b l e t o produce a tumour i n v i t r o , but i n a n i m a l s , m e t h y l c h o l a n t h r e n e i s h i g h l y c a r c i n o g e n i c . A f a c t o r of e l i m i n a t i o n p r e s e n t i n v i v o , but not i n v i t r o i s p r o b a b l y i n v o l v e d , as e x p l a i n e d i n more d e t a i l - above, I t would be p r e d i c t e d on the b a s i s of the above h y p o t h e s i s t h a t (1) C o l c h i c i n e s h o u l d be more c a r c i n o g e n i c i n v i t r o than i n v i v o . S i n c e the f a c t o r of e l i m i n a t i o n i s not p r e s e n t , a v e r y s m a l l c o n c e n t r a t i o n (as l i t t l e as 1:25,000,000) may be s u f f i c i e n t to cause the p r o d u c t i o n of a mutant. (2) M e t h y l c h o l a n t h r e n e s h o u l d be l e s s c a r c i n o g e n i c i n v i t r o t han i n v i v o . A r e l a t i v e l y l a r g e q u a n t i t y o f the substance should be r e q u i r e d to produce a mutant. We have, t h e n , the p o s s i b i l i t y t h a t c o l c h i c i n e i n v i t r o i s more c a r c i n o  g e n i c than m e t h y l c h o l a n t h r e n e . The obvious t e s t of t h i s hypo t h e s i s i s t o submit an i n v i t r o c u l t u r e of normal t i s s u e t o 9a t h e a c t i o n of c o l c h i c i n e ; t o wash the t i s s u e f r e e o f the a l k a l o i d u s i n g R i n g e r ' s s o l u t i o n , say, and to t r a n s f e r the t i s s u e i n t o an animal t o see whether or not a tumorous t i s s u e has been produced. L a c k i n g the f a c i l i t i e s t o c a r r y out t i s s u e c u l t u r e methods however, i t was d e c i d e d to employ u n i c e l l u l a r organisms to study t h e e f f e c t of c o l c h i c i n e . Only v e r y r e c e n t l y has any success been secured w i t h the a p p l i c a t i o n of the c o l c h i c i n e t e c h n i q u e t o a study of u n i  c e l l u l a r organisms. S t e i n b e r g and Thorn (31) have r e p o r t e d the p r o d u c t i o n of mutants i n c e r t a i n members of the genus A s p e r g i l l u s employing the a l k a l o i d . They found too t h a t they c o u l d secure a r e v e r s i o n of the mutants to the normal by i n c l u d i n g d - l y s i n e i n the media i n which the organisms were growing. They found i t necessary t o i n c l u d e s o l i d excess C a l c i u m carbonate i n t h e media t o prevent h y d r o l y s i s of the c o l c h i c i n e . They suggest t h a t the p r o d u c t i o n o f a c o l c h i c i n e mutant, a t l e a s t i n t h i s genus i s connected w i t h the d e s t r u c t i o n of f r e e amino groups on the p r o t e i n m o l e c u l e s c o m p r i s i n g the n u c l e a r m a t e r i a l . T h i s c l a i m i s based on t h e i d e a t h a t f r e e amino groups i n p r o t e i n m o l e c u l e s are concerned w i t h the presence i n the m o l e c u l e of l y s i n e . The E f f e c t of C o l c h i c i n e on Y e a s t s I t must be a d m i t t e d t h a t t h e o r i g i n a l i d e a behind the study of the e f f e c t of c o l c h i c i n e on t h e y e a s t s was not as e l a b o r a t e as t h a t put f o r w a r d above. 0. W. R i c h a r d s (32) working w i t h Saccharomyces c e r e v i s i a e ( s t r a i n 4360 A.T.C.) found t h a t i n c l u s i o n of c o l c h i c i n e i n the medium over a range of 1:10,000,000 to 4.5:100 had no i n h i b i t o r y e f f e c t on the 10 f o r m a t i o n of the y e a s t bud. The o r i g i n a l i d e a was t h e n t o t r y t o answer the q u e s t i o n "Why i s one c e l l a f f e c t e d by c o l c h i c i n e w h i l e another i s n o t ? " Why f o r example, i s the budding y e a s t not a f f e c t e d by c o l c h i e i ne even i n 4» &% concen- t r a t i o n when t h e c h i c k embryo f i b r o b l a s t i s a f f e c t e d by 1:25,000,000? The o b v i o u s answer i s of course t h a t i f c o l c h i c i n e i s a b s o l u t e l y s p e c i f i c f o r i n h i b i t i n g m i t o s i s then a l l c e l l s c o u l d be grouped as (1) Those r e s p o n d i n g to c o l c h i c i n e i . e . e x h i b i t i n g m i t o s i s . (2) Those f a i l i n g t o respond t o c o l c h i c i n e , i . e . e x h i b i t i n g a m i t o s i s . The c h i c k embryo f i b r o b l a s t may, t h e r e f o r e , be regarded as b e l o n g i n g t o group one, and t h e y e a s t t o group two. However, t h e r e i s evidence t o show t h a t s p o r u l a t i o n i n the y e a s t i s m i t o t i c , and the p o s s i b i l i t y e x i s t s , t h e r e f o r e , of an organism e x i s t i n g i n b o t h groups. B e f o r e going on t o the next s t e p i n the development of t h e h y p o t h e s i s i t w i l l be necessary t o c o n s i d e r t h e p h y s i o l o g y of t h e y e a s t s i n more d e t a i l . C y t o l o g y of Y e a s t s . The y e a s t s are u n i c e l l u l a r f u n g i c l a s s e d among the Ascomycetes. They are g e n e r a l l y found l i v i n g i n t h e i s o l a t e d s t a t e , but may under r a r e c i r c u m s t a n c e s produce rudimentary m y e e l i a . The u s u a l method of m u l t i p l i c a t i o n i s by budding, but t h e r e are a few s p e c i e s which m u l t i p l y by t r a n s v e r s e f i s s i o n . To be more e x p l i c i t , budding c o n s i s t s o f the appearance of a s m a l l prominence on t h e y e a s t c e l l which i s sep a r a t e d from the mother c e l l by a narrow c o l l a r . The bud appears t o be made up of very dense m a t e r i a l which has emigrated from the mother c e l l When the bud has reached a c e r t a i n s i z e the nucleus of the mother c e l l b e g i n s t o d i v i d e . The nu c l e u s does not change i t s p o s i t i o n even i f s i t u a t e d at the o p p o s i t e end of the c e l l from the bud. I t e l o n g a t e s and assumes the appearanee of a dumbell. One head of the dumbell e n t e r s the bud, the oth e r remains i n the mother c e l l , and the two are then separated by a c o n s t r i c  t i o n of the narrow c o l l a r a t t a c h i n g the bud t o the mother c e l l . G u i l l i e r m o n d ( 3 3 ) s t a t e s , t h a t "The n u c l e a r d i v i s i o n does not o f f e r the c h a r a c t e r i s t i c s of k a r y o k i n e s i s , c o n t r a r y t o the o p i n i o n of oth e r a u t h o r s ( S w e l l e n g r e b e l and Puhrmann). I t seems t o c o n s i s t s i m p l y of d i r e c t d i v i s i o n . " I t sometimes happens when a y e a s t i s growing r a p i d l y , as when under optimum c o n d i t i o n s , t h a t the mother c e l l produces s e v e r a l buds, and the buds themselves may b e g i n t o m u l t i p l y b e f o r e s e p a r a t i n g . The genus Sehizosaccharomyces i s the o n l y genus of y e a s t s which does not d i v i d e by budding. T h i s group of organisms d i v i d e s by t r a n s v e r s e f i s s i o n which c o n s i s t s simply of the f o r m a t i o n of a w a l l i n the middle of t h e c e l l which d i v i d e s the c e l l i n t o two daughter c e l l s . I f the y e a s t i s c u l t i v a t e d under f a v o r a b l e c o n d i t i o n s f o r growth, i t d i v i d e s a c t i v e l y u n t i l the c o n d i t i o n s become u n f a v o u r a b l e e i t h e r through a c c u m u l a t i o n o f waste p r o d u c t s or from l a c k of f o o d . I t then ceases t o d i v i d e and produces forms 12 which a l l o w the organism t o p e r p e t u a t e i t s e l f over u n f a v o r a b l e c o n d i t i o n s . The c e l l s may develop t h i c k w a l l s , and l a r g e s t o r e s of g lycogen and f a t s may appear. These are the durable c e l l s of W i l l and C a s a g r a n d i . The u s u a l method of p e r p e t u a t i o n of the s p e c i e s employed by the t r u e y e a s t s i s t h a t of s p o r u l a t i o n . A c e r t a i n number of i n t e r n a l or endospores are formed i n the i n t e r i o r of each c e l l , the c e l l thus b e i n g transformed i n t o a sporangium c a l l e d an a s c . In c e r t a i n s p e c i e s the ascospores are formed by a s e x u a l process which may be e i t h e r i s o - or heterogamic. The asc r e s u l t s from the f u s i o n of two c e l l s . C oncerning the s e x u a l p r o c e s s G u i l l i e r m o n d s t a t e s (33) "Two c e l l s i d e n t i c a l i n c h a r a c t e r i s t i c s and l y i n g a d j a c e n t t o each other i n t h e same c o l o n y are j o i n e d by means of a c o p u l a t i o n c a n a l formed by the f u s i o n of two l i t t l e p r o  j e c t i o n s put out by each c e l l . The middle w a l l which s e p a r a t e s the two c e l l s i s r a t h e r q u i c k l y d i s s o l v e d and the n u c l e i , t r ansformed thus i n t o gametes pass down the c o p u l a t i o n c a n a l where they f u s e t o form an egg or zygospore. Formed i n t h i s manner by isogamic c o n j u g a t i o n the egg soon germinates. I t i n c r e a s e s i n volume w h i l e i t s n u c l e u s undergoes two s u c c e s s i v e d i v i s i o n s , sometimes t h r e e which g i v e s f o u r or e i g h t n u c l e i . Then t h e s e become d i s t r i b u t e d about t h e zygospore, and s u r  r o u n d i n g themselves w i t h a zone of p r o t o p l a s m form f o u r or e i g h t a s c o s p o r e s . The zygospore i s t h e n t r a n s f o r m e d i n t o an as c . " The commonest groups of y e a s t s to form spores i n t h i s way are the Schizosaccharomyces and the Zygosaccharomyces. In the case of heterogamy observed i n the case of Zygosaccharo myces c h e v a l i e r i c o p u l a t i o n i s c a r r i e d out by two c e l l s of d i f f e r e n t dimensions. One i s v e r y s m a l l and r e p r e s e n t s t h e male gamete. I t i s young, w h i l e the o t h e r , the female gamete, i s l a r g e r and much o l d e r . The two c e l l s u n i t e by a c o p u l a t i o n c a n a l and the c o n t e n t s of the male gamete pass i n t o the female gamete i n which the p r o t o p l a s m i c and n u c l e a r f u s i o n takes p l a c e . A f t e r - t h i s has taken p l a c e the female gamete s e p a r a t e s i t s e l f by means of a w a l l and produces from one t o f o u r a s c o s p o r e s . In the g r e a t m a j o r i t y of the y e a s t s one does not f i n d any t r a c e of s e x u a l i t y . In t h i s case ascospores a r i s e by par t h e n o g e n e s i s . The common beer y e a s t , Saccharomyces c e r e v i s i a e i s an example of t h i s t y pe. The s e x u a l p r o c e s s has p r o b a b l y been l o s t t h r o u g h l o n g a d a p t a t i o n to c e r t a i n c o n d i t i o n s , and t h i s y e a s t u s u a l l y forms spores t o the number of f o u r by two s u c c e s s i v e d i v i s i o n s of the n u c l e u s i n the s i n g l e c e l l . G e r m i n a t i o n of the ascospores i n the p r e c e d i n g case i n v o l v e s a s w e l l i n g of the spores w i t h i n the ascus w i t h a sub sequent b u r s t i n g of the s u r r o u n d i n g membrane. The spores are thus r e l e a s e d and begin t o bud as s i n g l e c e l l s i n the normal f a s h i o n . In y e t o t h e r y e a s t s , the asc i s formed by partheno- g e n s i s , but the ascospores conjugate two by two immediately p r i o r to g e r m i n a t i o n . Such a y e a s t i s Saccharomyces L u d w i g i i . C o n c e r n i n g t h e l a t e r work of G u i l l i e r m o n d ( 3 4 ) he d e f i n i t e l y proposes a chromosomal b a s i s f o r s p o r u l a t i o n . Q u i l l i e r m o n d suggests t h a t the f o l l o w i n g types of y e a s t must be r e c o g n i z e d : (1) The h a p l o b i o n t i c y e a s t s : wherein i s o - o r h e t e r o - gamic c o n j u g a t i o n precedes ascus f o r m a t i o n (Schizosaceharomyces, Zygosaccharomyces, Z y g o p i c h i a , Debaromyces, Nadsonia, Nematospora) 14 (2) The d i p l o b i o n t i c y e a s t s : wherein c o n j u g a t i o n i s postponed u n t i l l a t e r i n the c y c l e u l t i m a t e l y t o occur between ascospores about to germinate. (S. L u d w i g i i , Hansenula Saturnus, and numerous o t h e r s p e c i e s o f Saccharomyees) " H a p l o b i o n t i c " i s a term a p p l i e d t o any p l a n t where m e i o s i s , or r e d u c t i o n d i v i s i o n takes p l a c e as soon as the zygote g e r m i n a t e s , the d i p l o i d phase b e i n g r e s t r i c t e d to the zygote. The term " d i p l o b i o n t i c r e f e r s t o any p l a n t where m e i o s i s immediately precedes gamete f o r m a t i o n , the haplophase b e i n g , t h e r e f o r e , r e s t r i c t e d t o the gametes." I l l u s t r a t e d g r a p h i c a l l y : '8 Q e <3 (9 (9y H a p l o b i o n t i c D i p l o b i o n t i c a. haplophase b. d i p l o p h a s e c. m e i o s i s d. m i t o s i s e. g e r m i n a t i o n of ascospores f. .. budding phase G u i l i i e r m o n d s t a t e s f u r t h e r t h a t c e r t a i n a s p e c t s of the f o r m a t i o n o f a s c o s p o r e s seem t o i n d i c a t e t h a t t h e n u c l e u s d i v i d e s by k a r y o k i n e s i s , or i n d i r e c t d i v i s i o n . C e l l s which are p r e p a r i n g t o s p o r u l a t e assume a v e r y complex s t r u c t u r e . Two s o r t s o f a l v e o l i o r foam s t r u c t u r e s appear i n the cytoplasm. Some are f i l l e d w i t h metachromatic g r a n u l e s , o t h e r w i t h glycogen. The c o r p u s c l e s i n t h e former a l v e o l i i n c r e a s e i n 15 number and d i m i n i s h i n volume u n t i l they are q u i t e d i f f u s e throughout the a l v e o l i . D u r i n g t h i s time the nucleus undergoes i t s f i r s t d i v i s i o n s . The a c t u a l mechanism i s d i f f i c u l t t o observe, a c c o r d i n g t o G u i l l i e r m o n d , no one has y e t been a b l e to see chromosomes i n a y e a s t n u c l e u s , but one i s a b l e t o see two small- n u c l e i c l o s e l y r e l a t e d t o each o t h e r . Each s m a l l n u c l e u s emigrates one t o each p o l e of the c e l l . 1 t h i n t h r e a d of plasma u n i t e s them s t i l l , and the t h r e a d has been l i k e n e d to the a c h r o m a t i c s p i n d l e . The t h r e a d soon d i s a p p e a r s and each nucleus undergoes another d i v i s i o n . The condensed cytoplasm or sporoplasm as i t i s termed, surrounds each n u c l e u s w i t h t h e r e s u l t t h a t f o u r spores are formed. In Schizosaccharomyces o c t o s p o r u s the c y t o l o g i e a l phenomena of s p o r u l a t i o n are e s p e c i a l l y easy t o observe. G u i l l i e r m o n d has shown t h a t a k a r y o k i n e s i s s i m i l a r t o t h a t e x i s t i n g i n the Ascomycetes e x i s t s i n t h i s y e a s t a t t h e stage o f s p o r u l a t i o n . To sum up t h e n , and r e l a t e t h i s b r i e f r e view of the c y t o l o g y of the y e a s t s t o the h y p o t h e s i s , i t seems t h a t c e r t a i n a t l e a s t of t h e y e a s t s are organisms which should be i d e a l t o work w i t h i n s t u d y i n g the e f f e c t of c o l c h i c i n e on c e l l d i v i s i o n . I n Schizosaccharomyees o c t o s o p o r u s we have an organism which a p p a r e n t l y e x h i b i t s a m i t o s i s d u r i n g t r a n s v e r s e d i v i s i o n , and m i t o s i s d u r i n g s p o r u l a t i o n . I n the Zygosaccharomyces and i n the o t h e r h a p l o b i o n t i c s p e c i e s , and indeed i n the Saccharomyces t o o , we have s i m i l a r phenomena. I n o t h e r words, we have organisms which a p p a r e n t l y f a l l i n t o b oth groups one and two on the b a s i s of m i t o s i s and a m i t o s i s , and might p o s s i b l y f a l l i n t o b o t h groups on the b a s i s o f r e a c t i o n t o c o l c h i c i n e t r e a t m e n t . 16 We are now i n a p o s i t i o n t o proceed t o t h e next s t e p i n the h y p o t h e s i s . Suppose t h a t c o l c h i c i n e i s w i t h o u t e f f e c t on budding, as we might expect i t t o be, and as R i c h a r d s has a p p a r e n t l y proved, but i n t e r f e r e s i n some way w i t h the phenomena of s p o l  i a t i o n . What may we expect as a r e s u l t of t h i s i n t e r f e r e n c e ? In the case of the h a p l o b i o n t i c s p e c i e s we might expect t o f i n d c o n j u g a t i o n w i t h the p r o d u c t i o n of a zygospore, but s i n c e m e i o s i s and m i t o s i s are i n h i b i t e d , we would not expect t o f i n d ascospores, In the case of d i p l o b i o n t i c s p e c i e s we might expect an i n h i b i  t i o n of m e i o s i s w i t h the r e s u l t t h a t no gametes would be formed, and c o n s e q u e n t l y no spores would be produced. In the case of pa r t h e n o g e n s i s w i t h o u t parthenogamy we might expect i n h i b i t i o n of s p o r u l a t i o n . I t a ppears, t h e r e f o r e , t h a t the h a p l o b i o n t i c s p e c i e s p r o v i d e a good t e s t group as i t should be c o m p a r a t i v e l y easy t o study t h e e f f e c t o f c o l c h i c i n e on the number of spores produced. In the case of t h e d i p l o b i o n t i c s p e c i e s , n o n - s p e c i f i c i n h i b i t i o n of s p o r u l a t i o n , i f i t e x i s t e d would p r o v i d e c o n f u s i n g evidence. F i n a l l y , l e t us suppose t h a t we have found t h a t c o l c h i c i n e does not a f f e c t budding, but p r e v e n t s f o r m a t i o n of ascospores i n the h a p l o b i o n t i c s p e c i e s . Why does c o l c h i c i n e f a i l t o a c t on the one hand and cause an e f f e c t on the o t h e r ? Perhaps t h e r e i s some fundamental c h e m i c a l o r p h y s i c a l b a s i s f o r c e l l d i v i s i o n . Perhaps t h e r e i s some (and t h i s i s h i g h l y s p e c u l a t i v e ) h o r mone-like substance which r e g u l a t e s t h e pheno mena of d i v i s i o n i n such a way t h a t i t s presence o r absence s p e l l s the d i f f e r e n c e between m i t o s i s and a m i t o s i s . S i n c e the phenomena of c e l l d i v i s i o n a re c l o s e l y r e l a t e d t o those of g e l a t i o n and s o l u t i o n , perhaps the e f f e c t of c o l c h i c i n e i s o n l y of a p h y s i c a l n a t u r e , but i t i s p r o b a b l y s a f e t o say t h a t i f the a c t i o n of c o l c h i c i n e i s p h y s i c a l , then we should not expect to f i n d a r r e s t e d development of c h i c k embryo f i b r o b l a s t s i n such a s m a l l c o n c e n t r a t i o n as 1:25,000,000. B h a d u r i suggests t h a t c o l c h i c i n e appears to c a t a l y z e c h e mical r e a c t i o n s which i n c r e a s e the f l u i d i t y of t h e c y t o p l a s m and n u c l e a r m a t e r i a l thus i n h i b i t i n g s p i n d l e f o r m a t i o n . ( 35 ) The advantages of working w i t h the y e a s t s now become obvious. The ease of m a n i p u l a t i o n of these organisms and the v a s t amount of l i t e r a t u r e p e r t a i n i n g t o them are two advantages. But f o r the purposes of t h i s i n v e s t i g a t i o n , and i t i s hoped, f u t u r e investigations„ the most important p r o p e r t y of the y e a s t s i s t h a t they d i v i d e by two methods. I f the c e l l - d i v i s i o n r e g u l a t i n g substance e x i s t s then we might expect t o f i n d i t p r e s e n t at one stage and not a t a n o t h e r , or perhaps p r e s e n t i n s m a l l e r c o n c e n t r a t i o n a t t h i s l a t t e r s t a g e . I f c o l c h i c i n e i n t e r f e r e s w i t h c e l l d i v i s i o n t h r o u g h r e a c t i n g i n some p h y s i c a l or c h e m i c a l way w i t h t h i s s u b s t a n c e , then i t i s very l i k e l y t h a t f r a c t i o n a t i o n of t h e y e a s t a t the two s t a g e s , and comparison of s i m i l a r f r a c t i o n s i n t h e i r r e a c t i o n toward c o l c h i c i n e would show i n what f r a c t i o n t h i s substance e x i s t s . , I t i s suggested t h a t i f t h i s substance e x i s t s i t would pro b a b l y be found i n the y e a s t at the s p o r u l a t i n g s t a g e , o r immed i a t e l y p r i o r to t h i s s t a g e . The work of R i c h a r d s has been mentioned. He found t h a t c o l c h i c i n e when weighed out and added to the medium f o r 18 S. C e r e v i s i a e ( W i l l i a m s medium) over a c o n s i d e r a b l e range of c o n c e n t r a t i o n s was w i t h o u t i n h i b i t o r y e f f e c t on the f o r m a t i o n of the yea s t bud. He d e s c r i b e s the occ u r r e n c e of two c y c l e s i n the growth of t h i s y e a s t i n W i l l i a m s medium, which he e x p l a i n s by s a y i n g t h a t when the c o n c e n t r a t i o n of t h e u n f a v o r  a b l e p r o d u c t s of metabolism r e a c h a c e r t a i n l i m i t , or when the c o n c e n t r a t i o n of the food f a l l s o f f t o a c e r t a i n v a l u e , t h e r e r e s u l t s a s e l e c t i v e k i l l i n g of the l a r g e r buds. The a d d i t i o n of food j u s t b e f o r e the end of the f i r s t c y c l e p r e v e n t s the r e t a r d e d growth, and the p o p u l a t i o n grows d i r e c t l y to a maximum cro p . F u r t h e r , s i n c e he found t h a t the a d d i t i o n of c o l c h i c i n e a l s o e l i m i n a t e s the p e r i o d of r e t a r d e d growth, he s t a t e s t h a t c o l c h i c i n e must a c t e i t h e r as a food o r as a b u f f e r i n l e s s e n i n g the i n c r e a s i n g l y adverse c o n d i t i o n s e x i s t i n g i n the medium. He d i d not i n v e s t i g a t e the a c t i o n of c o l c h i c i n e on the s p o r u l a t i o n of h i s y e a s t as he i s i n t e r e s t e d o n l y i n normal growth.(32) 19 ' EXPERIMENTAL Test Organisms Three attempts were made t o o b t a i n c u l t u r e s of Schizosaccharomyces o c t o s p o r u s . Twice c u l t u r e s a r r i v e d from., the American Type C u l t u r e C o l l e c t i o n , but both times they were dead on a r r i v a l . An u n s u c c e s s f u l attempt was made t o o b t a i n a c u l t u r e of t h i s organism from Lochead and P a r r e l l of t h e C e n t r a l E x p e r i m e n t a l Farm a t Ottawa, but t h e i r c u l t u r e had d i e d out. They p r o v i d e d two o t h e r y e a s t s however, t h e i r c u l t u r e s 138 and 58, Zygosaccharomyces p r i o r i a n u s , and Zygosaccharomyces b a r k e r ! r e s p e c t i v e l y . (36) Meanwhile a sample o f f e r m e n t i n g honey was o b t a i n e d through Dr. E a g l e s who i n t u r n o b t a i n e d i t t h r o u g h Mr. J . D i c k . Employing a s e p t i c p r e c a u t i o n s t hroughout, d i l u t i o n s of 1:10, 1:100, 1:1000, and 1:10,000 of the honey i n water were made. 1.0 ec of each of t h e above d i l u t i o n s was i n o c u l a t e d i n t o P e t r i p l a t e s by the poured p l a t e c u l t u r e method, t o o b t a i n i s o l a t e d c o l o n i e s of t h e organisms c a u s i n g f e r m e n t a t i o n . The medium used was t h a t employed by Lochead and F a r r e l l ( 36 ). The p l a t e s were i n c u b a t e d at 28°C and were examined a f t e r t h r e e days. Smears were made from i s o l a t e d c o l o n i e s , and were s t a i n e d w i t h c r y s t a l v i o l e t f o r e x a m i n a t i o n under t h e o i l immersion l e n s of the m icroscope. The organisms were r a t h e r l a r g e o v a l budding y e a s t s . 20 A c c o r d i n g t o Lochead and F a r r e l l , of a l l t he y e a s t s which t h e y i s o l a t e d from f e r m e n t i n g honeys, the m a j o r i t y were of the genus Zygosaccharomyces. A y e a s t was i s o l a t e d from the f e r m e n t i n g honey which l a c k i n g a complete c l a s s i f i c a t i o n o f the organism has been c a l l e d Zygosaccharomyces X. The c e l l s are m o s t l y o v a l i n young c u l t u r e . The s i z e i s 2.5 x 3.15 microns. In o l d e r c u l t u r e s t h e c e l l s may be l a r g e r , and i r r e g u l a r l y round. , The a c t i v e l y growing c e l l s m u l t i p l y by budding. The yeast grows r a p i d l y i n 30$ glucose b r o t h w i t h a f i n e even t u r b i d i t y * A s u r f a c e r i n g i s almost never formed. The y e a s t forms a s c o s p o r e s v e r y s l o w l y on P l a s t e r o f P a r i s b l o c k s , and not a t a l l i n Gorodovska* s medium. Ascos pores a r e , however, r e a d i l y formed on 30$ g l u c o s e agar by both isogamy and p a r t h e n o g e n e s i s . The number of spores formed by isogamy v a r i e s from two to f o u r w i t h a t l e a s t one spore i n each e n l a r g e d p o r t i o n o f t h e a s c . P a r t h e n o g e n e t i c a s c i r e g u l a r l y c o n t a i n f o u r a s c o s p o r e s . C o n j u g a t i o n between e x a c t l y s i m i l a r c e l l s makes i t s appearance on the agar i n 36 hours at 30°C and ascospo r e s may u s u a l l y be observed a f t e r 48 hours under these same c o n d i t i o n s . The spores are o v a l and v e r y smooth. They average about 1.6 x 2.4 microns i n s i z e . The g i a n t c o l o n y on 30$ gluc o s e agar i s very charac t e r i s t i c . I t may be observed by p l a c i n g a l o o p f u l of inoculum on t h e s u r f a c e of the agar i n a P e t r i p l a t e and i n c u b a t i n g a t room temperature f o r f o u r or f i v e weeks. The c o l o n y i s at f i r s t smooth white and round, convex r a i s e d w i t h an e n t i r e edge. I t l a t e r becomes roughened about the edge, and f i n a l l y t r e e - l i k e 21 growths appear a l l around the edge of the c o l o n y . The c e n t e r p o r t i o n i s v e r y smooth. The organism ferments the f o l l o w i n g s u gars, d e x t r o s e , g a l a c t o s e , f r u c t o s e , s u c r o s e , m a l t o s e , r a f f i n o s e . I t ferments 1-arabinose and m a n n i t o l o n l y f e e b l y , and does not ferment x y l o s e , d u l c i t o l , l a c t o s e , s a l i c i n , i n u l i n , or d e x t r i n . The organism grows r a p i d l y i n '60% glucose b r o t h w i t h the p r o d u c t i o n of a f i n e even t u r b i d i t y . No a g g l o m e r a t i o n of c e l l s o c c u r s , t h a t i s , the c e l l s remain q u i t e s e p a r a t e and the organism i s i d e a l f o r a study of r a t e s of growth by v i s u a l c o u n t i n g methods u s i n g an haemocytometer, and by t u r b i d o m e t r i c methods u s i n g a mod i f i e d F i s h e r E l e c t r o p h o t o m e t e r . The organism forms spores r a p i d l y on "60% g l u c o s e agar. T h i s y e a s t i s , t h e r e f o r e , a good one to study. The o t h e r Zygosaccharomyces which were o b t a i n e d are not s u i t a b l e f o r study f o r s e v e r a l reasons. #138 forms a s u r f a c e r i n g , and forms spores very s l o w l y on a l l media t r i e d . #58 grows s l o w l y i n b r o t h w i t h a g r a n u l a r growth. I t forms spores more r a p i d l y than #138, however. Another yeast c a l l e d Zygosaccharomyces Y a l s o i s o l a t e d from f e r m e n t i n g honey produces a s u r f a c e r i n g . The E f f e c t of C o l c h i c i n e on the Budding Y e a s t . I f c o l c h i c i n e i n t e r f e r e s i n some manner w i t h the f o r m a t i o n of y e a s t buds, then of c o u r s e , the r a t e of growth of the y e a s t under d e f i n e d c o n d i t i o n s should be a f f e c t e d . I f the p r o c e s s of budding i s m i t o t i c , we would expect t o f i n d a decrease i n the growth r a t e , and an i n c r e a s e i n t h e s i z e of the c e l l . R i c h a r d s has r e p o r t e d t h a t c o l c h i c i n e s t i m u l a t i o n of 22 y e a s t growth f a i l s to r e v e a l m i t o s i s . While t h i s may be t r u e f o r Saccharomyces v e r e v i s i a e , i t i s not n e c e s s a r i l y t r u e f o r a member of the Zygosaccharomyces u n t i l we have proved i t so. The f i r s t s t e p i n the E x p e r i m e n t a l was, t h e r e f o r e , t o see what e f f e c t i f any c o l c h i c i n e had on the budding Zygosaccharornycete. R i c h a r d s employed three d i f f e r e n t methods i n h i s work on r a t e s of growth; t h a t i s , d i r e c t count u s i n g the haemocytometer, c e n t r i f u g a t i o n of the c e l l s i n t o graduated c e n t r i f u g e t u b e s , and a p h o t o t u r b i d o m e t r i e method. Du r i n g t h i s i n v e s t i g a t i o n , o n l y two methods have been used, t h a t i s , the haemocytometer method, and p h o t o t u r b i d o m e t r i c measurements. The Medium. The medium used f o r a l l e x p e r i m e n t a l work was based on a s i m i l a r medium employed by Lochead and F a r r e l l i n t h e i r work on t h e o s m o p h i l i e y e a s t s ( 3 6 ) . They used peptone as the source of n i t r o g e n . In an attempt to approach a s y n t h e t i c medium and t o . e l i m i n a t e the c o l o r which might be due to the peptone, a s p a r a g i n e was used. d e x t r o s e 300. grams B.D.H.AnalaR as p a r a g i n e 0.5 D i f c o KgHP0 4 1.0 B a k e r s ' C P . MgS04-i7Hg0 • 1.0 B a k e r s ' C P . N H ^ t a r t r a t e 0.5 B.D.H. KaCl 0.1 Bakers' C P . C a C l 2 . 2 H 2 0 0.1 B a k e r s ' C P . Yeast e x t r a c t 1.0 S. O r l a - J e n s e n Water 1 l i t r e . d i s t i l l e d F o r s o l i d media 2% of agar (B.D.H.) was i n c l u d e d and the m i x t u r e was s t e r i l i z e d by a u t o c l a v i n g a t 15 l b s . steam p r e s s u r e f o r o n e - h a l f hour. For 1 i q u i d media the above medium (minus the agar of 23 course) was s t e r i l i z e d by S e i t z f i l t r a t i o n ( B . K . f i l t e r ) . The medium was kept i n 250 ce l o t s i n s t e r i l e 500 cc Erlenmeyer f l a s k s , and the f l a s k s were s e a l e d w i t h P a r a f i l m u n t i l used. The v a r i o u s l o t s of media were checked f o r s t e r i l i t y by i n  c u b a t i n g a t 28°C f o r 48 hours p r i o r t o b e i n g s t o r e d . Any f l a s k which showed c o n t a m i n a t i o n was d i s c a r d e d . The medium r e s u l t s i n a pH of 6.8. I t i s almost c o l o r l e s s . I t suppor t s the growth of the Zygosaccharomyces v e r y w e l l , e s p e c i a l l y Zygosaccharomyces X which g i v e s a f i n e even t u r b i d i t y i n 20 hours a t 28°C. The I n c u b a t o r . Two i n c u b a t o r s were used. The f i r s t was an a i r . i n c u b a t o r . The temperature of a l a r g e a sbestos l i n e d box was kept c o n s t a n t by the use of a s e n s i t i v e mercury expansion th e r m o s t a t and r e l a y . H e a t i n g was b y a carbon f i l a m e n t lamp. The o t h e r i n c u b a t o r was a c o n s t a n t temperature water bath. H e a t i n g was by a 125 watt k n i f e h e a t e r . The temperature was c o n t r o l l e d by an e a s i l y c o n s t r u c t e d t h e r m o s t a t , and a r e l a y was used. D e t a i l s of the c o n s t r u c t i o n of the th e r m o s t a t appear below. - T . i - i l b«.- ir . T h i s apparatus c o n t r o l l e d the temperature of the water bath t o w i t h i n 0.05°C of the r e q u i r e d temperature. 24 P r e p a r a t i o n of the C o l c h i c i n e S o l u t i o n s . C o l c h i c i n e was d i s s o l v e d i n water or s a l i n e as the case might be. The s o l u t i o n s were s t e r i l i z e d by f i l t r a t i o n t hrough a S e i t z E. K. f i l t e r . The f i l t e r s were always washed w i t h ' a l a r g e volume of t h e s o l v e n t b e f o r e s t e r i l i z a t i o n . The Haemocytometer Method. I t was e a r l y r e a l i z e d t h a t one f a c t o r which would have t o be e l i m i n a t e d was t h a t of t h e h y d r o l y s i s of c o l c h i c i n e . I f c o l c h i c i n e i s i n c l u d e d i n the medium i n which the y e a s t s a r e growing, t h e r e i s c e r t a i n to be some h y d r o l y s i s of the a l k a l o i d . Boyland and Huntsman ( 11 ) s t a t e t h a t c o l c h i c i n e i s 20% h y d r o l y z e d i n 24 hours a t 37 °C i n 1/50 N HC1. We might expect t h a t R i c h a r d s ' r e s u l t s were a f f e c t e d t o some degree a t l e a s t by the above f a c t , s i n c e the pH of the medium f e l l o f f t o about 3.5 or t h e r e a b o u t s . The e f f e c t i v e c o n c e n t r a t i o n of c o l c h i c i n e was thus reduced. However, he should c e r t a i n l y have found an e f f e c t over the range employed, had such an e f f e c t e x i s t e d . In an attempt t o get around the d i f f i c u l t y o u t l i n e d above, i t was d e c i d e d t o determine a normal r a t e of growth i n the medium, then remove t h e c e l l s a f t e r f i n i s h i n g a run and soak them i n 1% c o l c h i c i n e i n an i s o t o n i c s a l i n e s o l u t i o n f o r about one g e n e r a t i o n time of the organism. A c o n t r o l c u l t u r e would be t r e a t e d s i m i l a r l y except t h a t the c e l l s would be suspended i n s a l i n e o n l y . The r a t e s of growth would then be compared. I t would be expected t h a t i f the y e a s t bud forms by m i t o s i s t h e n t h e i n h i b i t i o n of budding d u r i n g treatment 25 w i t h c o l c h i c i n e w i t h subsequent r e i n o c u l a t i o n i n t o the medium should produce a mutant, and the r a t e of growth should be d i f f e r e n t . The d e t e r m i n a t i o n of normal r a t e of growth would serve as an a d d i t i o n a l c o n t r o l . D e t a i l s of the Method. S i n c e t h e e r r o r s o f the haemocytometer method are many, i t s use must be made the s u b j e c t of c a r e f u l c o n t r o l and s t a n d a r d i z a t i o n o f procedure. To minimize e r r o r s i n sampling 20 ec of medium was used i n 125 cc Erlenmeyer f l a s k s . T h i s p r o v i d e d f o r easy and thorough s h a k i n g t o suspend a l l the growth e v e n l y throughout the c u l t u r e , and to break up any s m a l l c o l o n i e s of c e l l s . I t i s v e r y n e c e s s a r y to have a u n i f o r m s u s p e n s i o n of s i n g l e organisms as i t i s e x t r e m e l y d i f f i c u l t t o count a c o l o n y of c e l l s . The medium was i n o c u l a t e d w i t h 0.2 cc of a 36 hour b r o t h c u l t u r e of Zygosaccharomyces X. The organism had p r e  v i o u s l y been p l a t e d out so as t o secure a pure c u l t u r e . S e v e r a l t e s t runs had shown t h a t the best time t o i n o c u l a t e was on the a f t e r n o o n of t h e day p r e c e d i n g t h a t on which t h e counting was t o be done. N i n e t e e n t o t w e n t y - t h r e e hours was c o n s i d e r e d most s a t i s f a c t o r y . The o r i g i n a l p o p u l a t i o n of the c u l t u r e a f t e r i n o c u l a t i o n was u s u a l l y about 100,000 organisms per c c , as shown by d i r e c t count. The next day the counts were s t a r t e d . Every hour over a p e r i o d of 6 hours t h e f l a s k was removed from the i n c u b a t o r , and the c o n t e n t s were w e l l shaken, care b e i n g taken not t o wet the c o t t o n p l u g . A s m a l l sample was withdrawn w i t h 26 a P a s t e u r p i p e t t e and the c e l l s were counted a t 600X u s i n g the haemocytometer under the microscope. The f l a s k was p l a c e d a t 28°C a g a i n immediately a f t e r sampling. The time and count were recorded each t i m e , and curves were drawn w i t h p o p u l a t i o n s as o r d i n a t e s , and times as a b s c i s s a e . The c o n t r o l and t r e a t e d r a t e s o f growth were ob t a i n e d as f o l l o w s : 10 c c of a 36 hour b r o t h c u l t u r e of the ye a s t were thrown down by c e n t r i f u g a t i o n . The supernatant was removed and the c e l l s were washed t h r e e times by suspending i n i s o t o n i c s a l i n e and r e c e n t r i f u g i n g . The c e l l s were then suspended f o r l£ hours i n 1% c o l c h i c i n e ( R i c h a r d s found t h a t 1% c o l c h i c i n e gave the most s t i m u l a t o r y e f f e c t - when i n c l u d e d i n the medium w i t h S. c e r e v i s i a e ) . F o r the c o n t r o l the c e l l s were merely soaked i n i s o t o n i c s a l i n e f o r the same l e n g t h of time. The c e l l s were then f r e e d of c o l c h i c i n e by t h r e e washings i n s a l i n e as b e f o r e . The c e l l s were f i n a l l y suspended i n s a l i n e and were i n o c u l a t e d i n t o 20 cc of medium as i n the d e t e r m i n a t i o n of normal r a t e s of growth. A l l t i m e s were c a r e f u l l y r e c o r d e d and an attempt was made to c a r r y out the c o n t r o l s i n as c l o s e l y s i m i l a r a way as p o s s i b l e t o the t e s t s . The r e s u l t s o f a t y p i c a l s e r i e s appear below. 1. Normal Growth Rate: Time : Count ( c e l l s per cc.) I n o c u l a t e d 0 19 h r s . 6 min 1 h r . 8 min 375.000 7,880,000 12.470,000 1 h r . 35 min: 21^047,000 1 h r . 16 min: 28,525,000 27 2. Times taken f o r treatment of Test and C o n t r o l . Procedure ( S a l i n e F i r s t ( C e n t r i f u g e s t a r t e d W a s h - ( " stopped ( S a l i n e removed Second Wash T r e a t  ment ( S a l i n e added ( C e n t r i f u g e s t a r t e d ( stopped ( S a l i n e removed ( S a l i n e p l u s c o l c h i c i n e ( added ( T e s t ) ( S a l i n e added ( C o n t r o l ) ( C e n t r i f u g e s t a r t e d stopped S a l i n e p l u s c o l c h i c i n e removed ( Te s t ) S a l i n e removed ( C o n t r o l ) ( S a l i n e added T h i r d ( C e n t r i f u g e s t a r t e d Wash ( stopped ( S a l i n e removed ( S a l i n e added F o u r t h ( C e n t r i f u g e s t a r t e d Wash ( stopped ( S a l i n e removed T o t a l T est C o n t r o l I n t e r v a l s i n Minutes, 0.0 : 0.0 2.0 : 2.0 6.0 : 6.0 1.0 : 1.0 2.0 : 2.0 1.0 : 1.0 5.0 : 5.0 1.0 : 1.0 2.0 58.0 5.0 2.0 •1.0 1.0 6.0 1.0 1.0 2.0 6.0 6.0 1.0 60.0 4.0 2.0 1.0 1.0 6.0 1.0 1.0 2.0 6.0 6.0 109.0 109.0 3. T r e a t e d Growth Rate Time Count ( C e l l s per cc) 0 22 min 38 min 25 min 35 min 42 min 66 min 12.48 12.89 10.1 11.1 19.9 22.59 27.64 1 0 6 ?0 ,<*-° Time i n Minutes 28 4. C o n t r o l Growth Rate T i m e Count ( c e l l s per cc) 0 6.0 x 1 0 6 42 min. 6.75 40 min. 7. 75 60 min. 10.66 53 min. 11.45 62 min. 16.14 The g e n e r a t i o n time of Zygosaccharomyces X i n t h e medium was determined from t r i a l r u n s . T h i s time i s t h a t taken f o r any g i v e n count to be d o u b l e d , and should remain r e l a t i v e l y con s t a n t over the phase o f • l o g a r i t h m i c growth. F o r Zygo X i n the 30$ g l u c o s e medium at 28°C under t h e d e f i n e d c o n d i t i o n s out l i n e d above, i t i s about two hours. I t w i l l be observed t h a t the time of treatment i n each,of the t e s t and t h e c o n t r o l , was about o n e - h a l f the g e n e r a t i o n time of the y e a s t . I t seemed ne c e s s a r y t h a t the s a l i n e used s h o u l d have the same osmotic p r e s s u r e as the medium t o e l i m i n a t e any e f f e c t s which might be due t o changes i n osmotic p r e s s u r e . By c a l c u l a t i o n the c o n c e n t r a t i o n of sodium c h l o r i d e r e q u i r e d was 4.0$. That t h i s c o n c e n t r a t i o n of sodium c h l o r i d e i s i s o t o n i c was proven by washing the c e l l s i n the s a l i n e s o l u t i o n , and l e a v i n g them suspended f o r t w e n t y - f o u r hours. M i c r o s c o p i c e x a m i n a t i o n showed no exploded c e l l s . D i s c u s s i o n of R e s u l t s The r e s u l t s would i n d i c a t e t h a t c o l c h i c i n e treatment 29 of t h e c e l l s i n t h i s f a s h i o n was w i t h o u t e f f e c t . I t i s very- l i k e l y t h a t m u l t i p l i c a t i o n ceases as soon as the c e l l s are removed from the medium. I t might be expected, t h e r e f o r e , t h a t c o l c h i c i n e would be w i t h o u t e f f e c t . I t i s d i f f i c u l t t o suggest an a l t e r n a t i v e method, however, f o r t r e a t i n g the ye a s t at the budding stage i n a n e u t r a l medium. I t was thought t h a t by adding excess s o l i d c a l c i u m carbonate t o the medium con t a i n i n g c o l c h i c i n e t h a t the pH might be kept near t h e n e u t r a l p o i n t . T h i s technique was found t o be w o r t h l e s s i n t h i s case, however, as the p a r t i c l e s of c a l c i u m carbonate made a v i s u a l count of t h e y e a s t p o p u l a t i o n v e r y d i f f i c u l t . I t might now be p r o f i t a b l e t o c o n s i d e r the d i f f i c u l  t i e s of the haemocytometer count method, and r a t e s of growth measurements i n g e n e r a l . I t i s obvious t h a t growth r a t e s are a f f e c t e d by many f a c t o r s . I t might be surmised t h a t the r a t e of growth of a d e f i n e d organism i n a d e f i n e d medium a t a d e f i n e d temperature over a d e f i n i t e p o p u l a t i o n range would be c o n s t a n t . In the case of f a c u l t a t i v e anaerobes such as the y e a s t s , however, a d d i t i o n a l f a c t o r s come i n t o p l a y . The q u a n t i t y of medium employed, the type of medium, and the s i z e and shape of t h e c o n t a i n e r a l l a f f e c t the growth r a t e . The y e a s t s grow f a s t e r i n the presence of oxygen than they do i n i t s absence, t h a t i s , the growth r a t e of y e a s t i s a f f e c t e d by the oxygen t e n s i o n i n the medium. The oxygen t e n s i o n i n t u r n v a r i e s d i r e c t l y as the s u r f a c e a r e a exposed, and i n d i r e c t l y as the depth of the medium. Here l i e s the e x p l a n a t i o n of the importance of f i x i n g the q u a n t i t y of medium t o be employed, and the shape and s i z e 30 of the c o n t a i n e r . I t i s w e l l known t h a t growth r a t e s of mic r o  organisms i n c r e a s e w i t h i n c r e a s e d amounts of inoculum i n a s y n t h e t i c medium. T h i s e f f e c t i s p r o b a b l y due t o the f a c t t h a t organisms such as the y e a s t s r e q u i r e c e r t a i n a c c e s s o r y f a c t o r s f o r growth, f o r example, the s o - c a l l e d B i o s f r a c t i o n s . The y e a s t s are a b l e t o manufacture these substances s i n c e the v a r i o u s B i o s f r a c t i o n s may be e x t r a c t e d from t h e i r c e l l s , but a p p a r e n t l y they do not s y n t h e s i z e enough t o meet t h e i r requirements. I f no b i o - a c t i v a t o r f r a c t i o n s are i n c l u d e d i n the medium, t h e r e f o r e , the growth of the c e l l s i s slow. As soon as some of the o l d e r c e l l s d i e and a u t o l y z e , t h e i r c o n t e n t s are r e l e a s e d to the medium and the growth r a t e becomes f a s t e r . T h i s e x p l a i n s why the growth r a t e i s f a s t e r when a l a r g e inoculum i s used. We might suppose, t h e r e f o r e , t h a t a more c o n s t a n t r a t e o f growth co u l d be o b t a i n e d i f the a c t i v a t o r f r a c t i o n s c o u l d be i n c l u d e d i n the medium. T h i s i s the reason f o r the a d d i t i o n of y e a s t e x t r a c t t o the medium. The y e a s t e x t r a c t was prepared some y e a r s ago by S. O r l a - J e n s e n and was o b t a i n e d from Dr. E a g l e s . The n e c e s s i t y of i n c l u d i n g the p r e p a r a t i o n i n the medium was proven as f o l l o w s . A sample of medium c o n t a i n i n g no y e a s t e x t r a c t , and another sample w i t h the a d d i t i o n of y e a s t e x t r a c t (as i n the complete medium on page (22) were i n o c u l a t e d w i t h Zygosaccharo myces X. V i s i b l e growth o c c u r r e d i n n i n e t e e n hours i n the medium p l u s y e a s t e x t r a c t . No growth appeared i n the medium minus y e a s t e x t r a c t i n two weeks. I d e n t i c a l amounts of inoculum were used. A complete s e r i e s , i . e . one normal r a t e of growth 31 one t r e a t e d r a t e of growth, and one c o n t r o l r a t e of growth r e q u i r e d s i x days to complete. The method i s l a b o r i o u s , but the counts are q u i t e a c c u r a t e as compared to one another. One square was counted t w i c e w i t h the f o l l o w i n g r e s u l t s : Number of c e l l s Number of c e l l s 65 64 67 68 90 92 92 89 89 83 61 • 58 77 77 84 86 Average 78.1 Average 77.1 Count | x 7 8 . 1 x l 0 6 Count | x 7 7 . 1 x l 0 6 - 19.5 x 1 0 6 = : 19.3 x 1 0 6 I n the hands of a p r a c t i c e d person the method g i v e s r e p r o d u c i b l e r e s u l t s , but the p e r s o n a l e r r o r i s n e v e r t h e l e s s l a r g e . No two persons would be l i k e l y t o a r r i v e a t the same count on the same sample. The range of p o p u l a t i o n covered w i t h o u t making d i l u t i o n s i s s m a l l . I f d i l u t i o n s are made, new e r r o r s a re i n t r o d u c e d . The method has so many di s a d v a n t a g e s t h a t i t was decid e d to d i s c a r d i t . The P h o t o t u r b i d o m e t r i c Method. A F i s h e r E l e c t r o p h o t o m e t e r No. 7-089 was m o d i f i e d f o r use i n p h o t o t u r b i d o m e t r i c measurements of growth r a t e s . The i n s t r u m e n t c o n t a i n s a lamp and p h o t o c e l l . The e.m.f. developed by the p h o t o c e l l v a r i e s d i r e c t l y as the i n t e n s i t y of the l i g h t f a l l i n g on i t . The i n s t r u m e n t employs the p o t e n t i o - m e t r i c p r i n c i p l e , t h a t i s , t h e e.m.f. developed by the photo c e l l i s bal a n c e d by a graduated s l i d e - w i r e u s i n g a v e r y 32 s e n s i t i v e galvanometer. The instrument i s made f o r use w i t h g l a s s c e l l s as r e c e p t a c l e s f o r the standards and unknowns. In the p i t f o r the g l a s s boxes a space 5|; em by 5-| cm was a v a i l a b l e so a b l o c k of wood was cut 5g cm on an edge. A 5/8" h o l e was d r i l l e d through i t so t h a t the l i g h t beam from the lamp" would pass through the h o l e on t o the c e n t e r of the photo c e l l . Another 5/8" h o l e was d r i l l e d p e r p e n d i c u l a r t o the f i r s t i n another f a c e of the b l o c k , but not e x t e n d i n g through the b l o c k . T h i s h o l e was t o h o l d the c u l t u r e tubes. The b l o c k was p a i n t e d b l a c k . The instrument was s t a n d a r d i z e d by i n s e r t i n g a tube of d i s t i l l e d water i n the p e r p e n d i c u l a r hole i n the wood-block. A b l a c k cardboard cover .with a h o l e to c o i n c i d e w i t h t h a t i n the wooden b l o c k was p l a c e d over the p i t . S i n c e the r e a d i n g s w i t h the instrument were t o be r e l a t i v e v a l u e s o n l y , i t was easy t o e l i m i n a t e e r r o r s which might' a r i s e from, u s i n g o r d i n a r y c u l t u r e t u b e s . A mark was put on the tube and another was p l a c e d on the cardboard cover. When the two marks were p l a c e d to c o i n c i d e , t h e n f o r c o n s e c u t i v e r e a d i n g s on the same tube the e r r o r s due t o i r r e g u l a r i t i e s and d e f e c t s i n the g l a s s c a n c e l l e d each o t h e r . I t was o n l y necessary to p l o t time a g a i n s t s c a l e r e a d i n g s to o b t a i n growth c u r v e s . T r i a l runs had shown t h a t the b e s t q u a n t i t y of medium t o use i n the tubes (each of which was c a r e f u l l y s e l e c t e d f o r s i z e and freedom from f l a w s ) was 5 c c . The b e s t time f o r i n  o c u l a t i o n was about t h i r t e e n hours b e f o r e c o u n t i n g . The c u l t u r e s were i n c u b a t e d a t 28°C i n the water bat h . I t was d e c i d e d t o r u n a s e r i e s of growth r a t e 33 d e t e r m i n a t i o n s of Zygosaccharomyces X i n the medium p l u s c o l c h i c i n e . A run of 30 tubes was s e t up as f o l l o w s . T e st 1 - 10 5 cc medium p l u s 0.52 ee 1% c o l c h i c i n e , i . e . a d i l u t i o n of 1:1000 Test 11 - 20 5 cc medium p l u s 1.04 cc 1% c o l c h i c i n e , i . e . a d i l u t i o n of 1:500 C o n t r o l 1 - 5 5 cc medium p l u s 0.52 cc 4% s a l i n e C o n t r o l 6 - 1 0 5 cc medium p l u s 1.04 cc 4% s a l i n e The above tubes were i n o c u l a t e d w i t h two l o o p f u l s of a two day o l d pure c u l t u r e of Zygosaccharomyces a t 7:45 P.M. of the day p r e c e d i n g t h a t on which the r e a d i n g s were t o be taken. The c u l t u r e s were i n c u b a t e d a t 28°C i n the water b a t h . To a i d i n i d e n t i f y i n g the tubes each was numbered w i t h a wax p e n c i l , and the tubes were arranged i n th r e e rows of ten each. The p l u g s o f the tubes i n the f i r s t row were p a i n t e d r e d , those i n the second row, b l u e , and the t h i r d were l e f t w h i t e . The next morning the measurements were s t a r t e d and were rep e a t e d on a l l t h i r t y tubes every two hours f o r twenty-four, hours. The in s t r u m e n t was c a l i b r a t e d a g a i n s t the tube of d i s t i l l e d water by a d j u s t i n g the s c a l e and galvanometer r e a d i n g s to z e r o . The c u l t u r e tube was then removed from the i n c u b a t o r , the o u t s i d e of the tube was wiped c l e a n and dry u s i n g a c l e a n s o f t c l o t h , and the c o n t e n t s of the tube were shaken v i g o r o u s l y , care being t a k e n not t o wet the p l u g . The tube was then i n s e r t e d i n the inst r u m e n t and the new s c a l e r e a d i n g was determined. The c a l i b r a t i o n was th e n checked a g a i n b e f o r e r e t u r n i n g t h e c u l t u r e to the i n c u b a t o r . C o l c h i c i n e T e s t s , see page 33 c l > c 2 . e t c r e f e r t o Test 1, Test 2, e t c . :'T" r e f e r s t o Time i n hours as i n s h i p s ' time. "R" r e f e r s t o Instrument r e a d i n g . C l C g C 3 T R T R T R- 915 9.0 916 9.5 917 8.9 1107 9.3 1107* 9.6 1108 10.0 1312 11.1 1313| 10,0 X 3 X 5 1548 16.1 1551 14. 5 X 5 52"g 14.9 1824 19.6 1826 15.4 1827| 18.8 2024 2 4«4 2026 20.1 2026| 24.1 2211 29 * 2 2211| 24.0 2212 2406 34.5 240 71 28. 9 ' 2410 33 o 2 15 XgT 40.2 152^ 33.0 153 38. 7 330 45. 7 332 38.0 333 43.5 500 5 X 9 2 501 44.1 502| 49.1 833 57.6 834 52.5 835 58.0 918 1108* 1315* 15541 1828 2027| 2 2 X 2 *p 2410# 154 334 503* 835§ R T R X X e 2 919 8.6 12.7 1109§ 10.1 X 2»3 1316 10. 7 14.8 X 5 55 g~ 14.8 17.0 1829 17.0 23 © 4 2028 20.9 26.3 2213 26. 7 ;29© *7 2411 32.1 34»2 154| 38. 7 39. 7 334g 43.0 45. 5 504 49.6 56.0 836| 58.6 C 6 C Y c 8 c 9 cio T R ' T , R T R T R T R 921 8.1 92 1 | 7.0 9 25 g 8. 5 1110 8.5 1112 8.0 X X X 3g" 9.0 1317 8.9 1319 8.5 1324 9.6 1556| 14.1 1557 11.4 1600 12. 7 1831 17.0 1833 16.8 1834 14.3 2029 2 X $ 0 2030 22 9 7 2033 19.5 2 2 X 3gr 24 l l | 26.9 2216^ 24.2 2217 23.4 3 2 • 5 2415 28.1 2416f 26.5 1550 37. 7 158* 33.5 159 30. 7 335 44.3 338 , 38.4 338g 35.9 505 49.5 507* 840{ 44. 9 508 41.0 838 58.0 54.8 841 i3 £J ® i3 926 1114| X 325 1601 1834-g 2034 22 X T g 2417 200.0 339f 509 842 8.2 928 6.1 9.0 1115 6.1 9.5 1326 6.4 12.5 1602 8.0 13. 9 1835 9 « 9 17 .5 2037 16.6 2 X 9 2 2218 X 5«X 24.5 2418 19.0 29 • 4 200j 22.8 34.7 341 27.5 39.9 509* 32*3 53.8 842| 44.3 '11 '12 '13 '14 '15 R R R R 934 S s6 934| 7.0 1123 8,6 1123! 7.7 1334 9.6 1334! 8.6 1611 12.2 161l|- 11.2 1840 14.6 1840 l 12.0 2043 - 15.4 2044 14.1 2225 21.2 2226! 16.9 2424 26.9 2425 21.6 206! 31.1 207 25.9 346^ 36.5 347 31.1 514 42.3 515 36.5 847 52.9 848 47.5 C 1 6 C 1 7 °18 C 1 9 °20 T R T . R T R T R T R 935 1124 1336 1612 1841! 2045 2227 2426 208 348 515! 849 8.5 9«5 10. 7 12.6 14.8 17 65 21.2 26.3 31.1 35.7 41.2 54.0 936 1125 1337! 1613 1842! 2048 2228 2426! 209 348! 516 850! 9.6 11.3 14.0 20.3 27.6 34.9 38.4 45.5 49.9 54.1 57.5 65.0 938! 1126 1338 1614 1843 2049! 2229 2427 210 349 517 851 946 1130 1343! 1618 1847 2054 2232! 2431 213 352 520 855! 8.7 9.6 12.7 17.1 23.5 28.0 32.5 40.0 45.3 49.9 53.2 61.9 C o n t r o l s see page 33 K l > K2> e t c * r e f e r to C o n t r o l 1, C o n t r o l 2, e t c . R 947 8.0 1131 6.1 1347 9. 7 1620 12.5 1848 16.0 2055 20.5 2233 24.0 2432 29.6 214 35.2 353 41.5 521 45. 7 856* 55.4 R 12.7 12. 7 14.6 18.1 22.4 27.6 31.3 38.0 43.1 49.2 52.6 62.5 Kg 949 . 1132 1348J 1621 1850| 2056 2235 2433! 216 354 522 858 R R 9.5 10.3 11.5 15.2 19.0 24.5 27.5 33.4 39.6 45.5 49.5 58.4 950 1133 1349 1622 1852 2057 2236 2434 216! 355 522! 859 7.6 9.1 11.4 14.4 18. 5 23.5 27.5 34.0 38.6 47.5 49.3 59.3 950! 1134! 1353 1623 1856 2100 2238 2437 219 35 7 525 903 R 8.3 9.0 11.3 15.1 20.3 26.1 31.9 37.9 42.5 49.8 52.3 60.6 L8 K 10 R R R R R 951! 8.5 1136 8.3 1356 10.3 1625 13. 3 1858 17.0' 2101 22.0 2239 25. 7 2437! 31. 7 220! 35.1 358 41.6 525! 46.6 904 54.9 952 4.7 1137 4. 7 1358 6.1 1626 8.0 1859 10.6 2102 12.8 2240 .16.0 2438! 21.0 221 28.1 359 34.0 526 36.1 904! 47.8 954 5. 5 1139 6.0 1400 6.7 1627 9.0 1900 12.1 2103 15.2 2241 18.5 2439 24.0 222 25.9 359! 30. 7 526! 38.4 905 51.1 957 8. 5 1140 9.0 1402 10.9 1628 14.0 1901 18.6 2104 22. 5 2241! 27.1 2440 32.4 223 38.1 400 43.1 527 46.9 906 56.8 959 4.2 1141 4.5 1404! 6.1 1629 7.8 1901! 12.4 2105 16.5 2242 19*5 2440! 24.3 223! 29.5 400| 35. 7 528 41.1 906! 51.2 rH Z CO EH « t> 3 fx! JS EH CC; &q <cj Q H  S  C5 M 23 CO  E-t Cr4 CD S fx! 25 H K W  <j Q H 2 c5 34 D i s c u s s i o n o f the R e s u l t s and t h e Method I t appears t h a t c o l c h i c i n e i n d i l u t i o n s of 1:1000 and 1:500 i s w i t h o u t e f f e c t on t h e budding of y e a s t . The obvious procedure was t o f o l l o w up t h i s work w i t h i n c r e a s e d c o n c e n t r a t i o n s of c o l c h i c i n e . I t was d e c i d e d , however, not t o continue a l o n g t h i s l i n e as i t i s very d o u b t f u l whether the r e s u l t s o b t a i n e d are of any r e a l s i g n i f i c a n c e i n view of the f a c t of h y d r o l y s i s of the a l k a l o i d i n the medium. That t h i s method of measuring r a t e s of growth i s an e x c e l l e n t one i s e v i d e n t f r o m an e x a m i n a t i o n of the curves o b t a i n e d . The method i s easy to c a r r y o u t , i s q u i t e a c c u r a t e and i s v e r y r a p i d . To c a r r y out a s i m i l a r s e r i e s by the haemocytometer method would have taken s i x months. The instrument i s very s e n s i t i v e , and p e r s o n a l e r r o r i s almost c o m p l e t e l y e l i m i n a t e d . As w i t h o t h e r methods of course a r i g i d c o n t r o l of a l l procedures must be e x e r c i s e d . The method of s h a k i n g f o r example must be r e p e a t e d i n t h e same way f o r each tube. The method i s a p p l i c a b l e over a wide range of p o p u l a t i o n s . That range used i n the above experiments i s most s a t i s f a c t o r y , however, s i n c e i t i n c l u d e s o n l y t h a t p o r t i o n of the curve from the end of the l a g phase and b e g i n n i n g of the l o g a r i t h m i c to the p o i n t o f s a t u r a t i o n of the medium w i t h carbon d i o x i d e . The bubbles of carbon d i o x i d e which appear above t h i s p o i n t i n t e r f e r e w i t h the measurements. Summary of t h e work on the Budding of Y e a s t . I t appears t h a t c o l c h i c i n e does not i n t e r f e r e w i t h the f o r m a t i o n of the y e a s t bud, but t h i s statement i s made w i t h 35 r e s e r v a t i o n s . In the f i r s t method, i n an attempt t o e l i m i n a t e the h y d r o l y s i s of c o l c h i c i n e , perhaps no r e a l t e s t of the e f f e c t of t h e a l k a l o i d was made. I t i s c e r t a i n l y a f a c t t h a t no method t r i e d has e l i m i n a t e d the f a c t o r of h y d r o l y s i s , and i t i s almost i m p o s s i b l e t o c o n c e i v e a q u a n t i t a t i v e method which i s l i k e l y t o get around t h i s d i f f i c u l t y . . Every q u a l i t a t i v e procedure employed i n these i n v e s t i g a t i o n s has, however, i n d i c a t e d t h a t c o l c h i c i n e i s w i t h o u t i n h i b i t o r y e f f e c t on the type of c e l l d i v i s i o n c h a r a c t e r i z e d by budding. T h i s w i l l become e v i d e n t as f u r t h e r e x p e r i m e n t a l work i s o u t l i n e d . I t i s suggested t h a t any s t i m u l a t o r y e f f e c t on c e l l - d i v i s i o n as r e c o r d e d by R i c h a r d s i s connected w i t h a secondary e f f e c t on c e l l metabolism. R i c h a r d s (32 ) s t a t e s t h a t the a l c o h o l pro d u c t i o n i n c u l t u r e s of Saccharomyces c e r e v i s i a e i n c r e a s e s t o a maximum of s i x times the c o n t r o l c u l t u r e c o n c e n t r a t i o n , i n 1% c o l c h i c i n e . T h i s f a c t cannot be e x p l a i n e d on the b a s i s of h y d r o l y s i s o f c o l c h i c i n e a l o n e ; i . e . p r o d u c t i o n of methyl a l c o h o l , even assuming complete h y d r o l y s i s of the a l k a l o i d . Experiments suggest t h a t c o l c h i c i n e a c t i v a t e s some enzyme i n the c h a i n of f e r m e n t a t i v e r e a c t i o n s r e s p o n s i b l e f o r Carbohydrate metabolism. I t i s obvious t h a t an i n c r e a s e d r a t e of metabolism of c a r b o h y d r a t e would r e s u l t i n an i n c r e a s e d r a t e of l i b e r a t i o n of energy. P a s t e u r produced evidence t o show t h a t t h e r a t e of growth of y e a s t s was i n c r e a s e d under c o n d i t i o n s of i n c r e a s e d l i b e r a t i o n of energy. (33) The E f f e c t o f C o l c h i c i n e on S p o r u l a t i o n of the Y e a s t I t has been s t a t e d t h a t Zygosaccharomyces does not 36 form spores i n l i q u i d media, forms them only v e r y s l o w l y on P l a s t e r of P a r i s b l o c k s , and forms them q u i c k l y and e a s i l y on agar p l a t e s . The e f f e c t of c o l c h i c i n e on the s p o r u l a t i o n of Zygosaccharomyces p r i o r i a n u s , Zygosaccharomyces b a r k e r ! , and Zygosaccharomyces X was i n v e s t i g a t e d . B r o t h c u l t u r e s of t h e above y e a s t s were made. A f t e r a s u f f i c i e n t time had e l a p s e d t o a l l o w the c e l l s t o b u i l d up glycogen and f a t s t o r e s (determined by m i c r o s c o p i c examination) the c e l l s were removed from the c u l t u r e s by c e n t r i f u g a t i o n , were washed w i t h s a l i n e , and were thence t r a n s f e r r e d t o P l a s t e r of P a r i s b l o c k s . A s e p t i c p r e c a u t i o n s were used throughout. Test b l o c k s were soaked i n 1% c o l c h i c i n e i n 4% s a l i n e . C o n t r o l b l o c k s were soaked i n 4% s a l i n e . The p l a t e s were i n c u b a t e d a t 28°C f o r t h r e e weeks or more. Frequent m i c r o s c o p i c e x a m i n a t i o n s , and f r e q u e n t s u b c u l t u r e s were made i n t o l i q u i d media. Evidence was o b t a i n e d t h a t s p o r u l a t i o n was i n h i b i t e d i n the case of Zygosaccharomyces b a r k e r i f o r a f t e r t h r e e days the c o l c h i c i n e b l o c k s c o n t a i n i n g t h i s organism were s t e r i l e whereas the c o n t r o l b l o c k s were not. In more d e t a i l , the r e s u l t s were t h e s e ; two s u b c u l t u r e s were taken from the c o n t r o l b l o c k and two from the t e s t b l o c k s i n t o b r o t h and then a f t e r s e v e r a l days spread p l a t e s were made from the b r o t h . The two t e s t b r o t h s showed no growth. The c o n t r o l showed heavy growth. The spread p l a t e s from the t e s t b l o c k s were s t e r i l e - the p l a t e f rom the c o n t r o l was not. T h i s o b s e r v a t i o n may be e x p l a i n e d as f o l l o w s . The y e a s t , b e i n g d e p r i v e d of food must form spores to p e r p e t u a t e i t s e l f or d i e . S i n c e c o l c h i c i n e i n h i b i t s m i t o s i s and the f o r m a t i o n of 3? ascos p o r e s seems t o be m i t o t i c , then we s h o u l d expect sporu l a t i o n t o be i n h i b i t e d and the ye a s t would d i e . Some evidence was a l s o o b t a i n e d f o r Zygosaccharomyces p r i o r i a n u s , but the r e s u l t s were not c o n c l u s i v e . They may be t a b u l a t e d as f o l l o w s : Time of s u b c u l t u r e C o n t r o l Test i n b r o t h i n b r o t h 1 week growth growth 2 weeks growth no growth 3 weeks 1 growth 1. growth 2 no growth 2. s i . growth A f t e r t h i s time the c o n t r o l b l o c k was found t o be contaminated w i t h a mold. I t seems l i k e l y , t h e r e f o r e , t h a t chance might have been a f a c t o r i n o b t a i n i n g an inoculum from the b l o c k s . Because i t was d e s i r e d t o work w i t h Zygosaccharomyces X i t was thought n e c e s s a r y t o study the e f f e c t of c o l c h i c i n e on the s p o r u l a t i o n of this y e a s t . More or l e s s by chance i t was d i s c o v e r e d t h a t Zygosaccharomyces X forms spores i n two to th r e e days on the 30$ gluco s e agar a t 28°C. I t was decid e d to i n c l u d e c o l c h i c i n e i n the medium over a c o n s i d e r a b l e range of c o n c e n t r a t i o n s . Granted t h a t h y d r o l y s i s of the c o l c h i c i n e was not e l i m i n a t e d , i t should have been p o s s i b l e by v a r y i n g the c o n c e n t r a t i o n from 1:2000 to s a t u r a t i o n t o cover the range of p o s s i b l e e f f e c t of the a l k a l o i d i f s p o r u l a t i o n c o u l d be induced t o o c cur q u i c k l y and a t a f a i r l y low temperature. A t e s t s e r i e s was run employing a range of 1:2000 to 1:200 u s i n g the t h r e e Zygosaccharomyces on the agar. Sporu l a t i o n was not i n h i b i t e d . C o l c h i c i n e was made up i n water s o l u t i o n i n 2% c o n c e n t r a t i o n and s t e r i l i z e d by f i l t r a t i o n 38 through a S e i t z EK f i l t e r . The next s e v e r a l s e r i e s were run on the same agar t o which t h r e e grams of s o l i d c a l c i u m carbonate had been added i n an attempt t o minimize the h y d r o l y s i s of the a l k a l o i d . A t y p i c a l s e r i e s was se t up as f o l l o w s C°llhitinet 2 % A g a r S u i t i n g d U u t i o n of e o l c h i c i n e 1:2000 0.25 cc 9.75 cc 0.5 cc 9.5 cc 1:1000 1*0 cc 9.0 cc 1:500 „, . 2 - ° c 5 n 8.0 cc 1:250 Water c o n t r o l 2.0 cc 8.0 cc Each p l a t e was d i v i d e d i n t o t h r e e segments (see p r i n t ) and was i n o c u l a t e d w i t h Zygosaccharomyces X, 138, and 58. Growth of the c e l l s was not i n h i b i t e d which i s an added ind i c a t i o n t h a t c o l c h i c i n e i s without i n h i b i t o r y e f f e c t on the budding of y e a s t s . The c a l c i u m carbonate which c o n t r i b u t e d a c e r t a i n o p a c i t y t o the medium was found t o have been d i s s o l v e d around each c o l o n y (see p r i n t ) . The zone around X was l a r g e r than t h a t around 138 and 58 because X grows f a s t e r than 138 or 58. I t was p o s t u l a t e d t h a t t he c a l c i u m carbonate had been d i s s o l v e d by an a c i d d i f f u s i n g from the c o l o n y as a product of metabolism. T h i s was proven by growing the organisms on agar c o n t a i n i n g (a) 1itmus, and (b) bromthymolblue. The presence of a red zone around the col o n y on the l i t m u s agar p l a t e , and a broad y e l l o w zone on the bromthymol blue agar p l a t e i n d i c a t e d the p r o d u c t i o n of an a c i d . S p o r u l a t i o n was not i n h i b i t e d i n any ease above. I n p o u r i n g the p l a t e s the f o l l o w i n g procedure was employed: The r e q u i r e d amount of c o l c h i c i n e s o l u t i o n was put i n 39 the p l a t e and the melted agar ( a t about 40°G)was p i p e t t e d i n . The c o l c h i c i n e s o l u t i o n and the melted agar were w e l l mixed by r o t a t i n g the p l a t e , and the m i x t u r e was c o o l e d q u i c k l y . S ince the pH of the agar i s v e r y c l o s e t o seven when c a l c i u m carbonate i s added, and s i n c e the temperature of the agar f a l l s r a p i d l y when ad m i t t e d t o the c o l d p l a t e , h y d r o l y s i s of the c o l c h i c i n e s h o u l d be almost n i l . I t was now d e c i d e d t o i n v e s t i g a t e the e f f e c t of a c o n c e n t r a t e d s o l u t i o n of t h e a l k a l o i d on s p o r u l a t i o n . 0.5 gm of s o l i d c o l c h i c i n e was added t o 10 cc of the melted CaC03 - 30$ g l u c o s e agar a t 40°G i n a p l a t e and the c o l c h i c i n e was d i s s o l v e d a t l e a s t to the s a t u r a t i o n p o i n t by r o t a t i n g the p l a t e i n the u s u a l f a s h i o n . Some of the a l k a l o i d remained u n d i s s o l v e d a f t e r the agar had s o l i d i f i e d . The p l a t e was i n o c u l a t e d w i t h Zygo saccharomyces X and was i n c u b a t e d a t 28°C. Budding was not i n h i b i t e d , but s p o r u l a t i o n was almost c o m p l e t e l y stopped. P a r t h e n o g e n e t i c a s c i d i d not make t h e i r appearance i n f o u r days whereas i n the c o n t r o l c u l t u r e s spores appeared formed by p a r t h e n o g e n e s i s i n two days. Only a few spores formed by isogamy made t h e i r appearance, and the number of spores was such as t o suggest zygotes and not a s c i were formed. I t i s t r u e t h a t the c o l c h i c i n e added i n t h i s t e s t was not s t e r i l e . Four molds made t h e i r appearance a f t e r f i v e days but the y e a s t had by t h i s time shown the d e s i r e d a f f e c t . A method of p r e p a r i n g a s t e r i l e s a t u r a t e d s o l u t i o n of c o l c h i c i n e was d e v i s e d . I t was d e s i r e d to e l i m i n a t e S e i t z f i l t r a t i o n i f p o s s i b l e as f i l t r a t i o n always i n t r o d u c e s the p o s s i b i l i t y of change i n c o n c e n t r a t i o n due t o a b s o r p t i o n by the f i l t e r pad. 40 C o l c h i c i n e was d i s s o l v e d i n 70$ e t h y l a l c o h o l i n a s t e r i l e f l a s k . The a l c o h o l s t e r i l i z e d t h e c o l c h i c i n e and was evaporated o f f i n vacuo. Experiments are s t i l l p r o c e e d i n g employing t h i s t e c h n i q u e t o prepare c o n c e n t r a t e d s t e r i l e s o l u t i o n s of the a l k a l o i d and t h e i r subsequent use t o d e t e r  mine t h e s m a l l e s t c o n c e n t r a t i o n of c o l c h i c i n e j u s t s u f f i c i e n t to i n h i b i t s p o r u l a t i o n . Two o t h e r methods of a p p l i c a t i o n of t h e a l k a l o i d t o a s t u d y of s p o r u l a t i o n were i n v e s t i g a t e d . The f i r s t of these procedures was c a r r i e d out as f o l l o w s : Zygosaccharomyces X was i n o c u l a t e d on to 30$ gluc o s e agar and mass i n o c u l a were removed from the c o l o n i e s at 24 and 48 hours t o be t r a n s f e r r e d t o water agar (2$ agar i n w a t e r ) , and t o 1:250 c o l c h i c i n e i n water agar. The p l a t e s were i n c u b a t e d a t 28°C. S p o r u l a t i o n d i d not appear on e i t h e r p l a t e i n 26 days. The second method i n v o l v e d the f o l l o w i n g : a l a r g e inoculum of a two day o l d agar c u l t u r e of Zygosaccharomyces X was removed from the p l a t e as above and was washed t w i c e i n 4$ s a l i n e . The c e l l s were f i n a l l y suspended i n 4% s a l i n e i n the c o n t r o l s , and 4% s a l i n e p l u s 1% c o l c h i c i n e i n the t e s t s . The suspensions were i n c u b a t e d a t 28°C. S p o r u l a t i o n d i d not occur i n a month. Summary of the Work on S p o r u l a t i o n of Zygosaccharomyces X. I t seems t h a t c o l c h i c i n e i s w i t h o u t e f f e c t on s p o r u l  a t i o n of Zygosaccharomyces X over a range of c o n c e n t r a t i o n of the a l k a l o i d from 1:2000 t o 1:250. The evidence i s however f o r an i n h i b i t i o n of s p o r u l a t i o n i n c o n c e n t r a t i o n s of 1:100 to 4.5$ 41 and t h i s would seem to i n d i c a t e t h a t the phenomenon of f o r m a t i o n of ascospores i n t h i s y e a s t i s m i t o t i c . I t i s noted t h a t a v e r y g r e a t d i f f e r e n c e e x i s t s between the c o n c e n t r a t i o n s of the a l k a l o i d r e q u i r e d to a r r e s t the development of c h i c k embryo f i b r o b l a s t s (1:25,000,000) and t h a t r e q u i r e d t o check the f o r m a t i o n of spores i n t h e y e a s t s (1:100 t o 4.5$). I t i s a l s o obvious t h a t the y e a s t c e l l i s a r e l a t i v e l y u n d i f f e r e n t i a t e d p r o t o p l a s m compared to the c h i c k embryo c e l l . The y e a s t seems t o be i n t e r m e d i a t e between those c e l l s which d i v i d e by t r u e a m i t o s i s , and those which d i v i d e by m i t o s i s . Perhaps the Zygosaccharomyces are not t h e b e s t organisms t o study i n r e s p e c t to the a p p l i c a t i o n of c o l c h i c i n e t o a study of c e l l u l a r c h e m i s t r y . F u r t h e r E x p e r i m e n t a l w i t h S u g g e s t i o n s f o r F u t u r e Work. . In the l i g h t of t h e o b s e r v a t i o n s of S t e i n b e r g and Thorn on the A s p e r g i l l i ( 31 ) and those o f the above i n v e s t i g a t i o n on t h e Zygosaccharomyces, i t seems l i k e l y t h a t o t h e r f u n g i might have been b e t t e r s t u d i e d . In f a c t y e a s t s o t h e r than Zygosac charomyces X might have been s t u d i e d to b e t t e r advantage s i n c e i t i s p o s s i b l e t h a t the y e a s t s e x h i b i t t h e v a r i a t i o n i n sus c e p t i b i l i t y t o c o l c h i c i n e treatment as d i d the A s p e r g i l l i s t u d i e d by S t e i n b e r g and Thorn. A y e a s t which s h o u l d p r o v i d e an i n t e r e s t i n g s tudy would be Schizosaccharomyces o c t o s p o r u s . The p h y s i o l o g i c a l c o n d i t i o n s f o r s p o r u l a t i o n of Zygo saccharomyces X s h o u l d be s t u d i e d i n more d e t a i l i n an e f f o r t t o f i n d c o n d i t i o n where h y d r o l y s i s of c o l c h i c i n e would be m i n i m a l . Other compounds such as a l p h a n a p h t h y l - a c e t i c a c i d 42 should be s t u d i e d and t h e i r e f f e c t compared t o t h o s e of c o l c h i c i n e . R i c h a r d s (32) has r e p o r t e d t h a t the a l c o h o l con c e n t r a t i o n s i n W i l l i a m s medium p l u s 1% c o l c h i c i n e r o s e t o a maximum s i x times the c o n t r o l v a l u e . I t i s t r u e t h a t h i s • a l c o h o l d e t e r m i n a t i o n s d i d not a l l o w f o r h y d r o l y s i s of the a l k a l o i d , b u t , as s t a t e d b e f o r e , t h i s h i g h c o n c e n t r a t i o n of c o l c h i c i n e a l o n e . The p r o d u c t i o n of a l c o h o l a c c o r d i n g t o h i s graphs s t a r t s b e f o r e much sugar i s used up, i n d i c a t i n g the h y d r o l y s i s of c o l c h i c i n e w i t h the appearance of methyl a l c o h o l i n t h e medium. I t seems v e r y l i k e l y t h a t c o l c h i c i n e a c t i v a t e s one of t h e enzymes of the zymase complex, and s i n c e a c h a i n of r e a c t i o n s can o n l y go as f a s t as the s l o w e s t l i n k , c o l c h i c i n e p r o b a b l y a c t i v a t e s one of the sl o w e s t enzyme r e a c t i o n s . A t l e a s t i t i s l o g i c a l t o suppose t h a t an i n v e s t i g a t i o n of these systems would be most p r o f i t a b l e . Evidence was o b t a i n e d from a s e r i e s o f experiments t h a t c o l e h i c i n e has an e f f e c t on the enzyme systems of y e a s t . Old c u l t u r e s of y e a s t s which had l o n g s i n c e stopped f e r m e n t i n g may be brought back t o a c t i v e f e r  m e n t a t i o n as e v i d e n c e d by gas p r o d u c t i o n t h r o u g h the i n t r o d u c t i o n of s m a l l q u a n t i t i e s of c o l c h i c i n e . I t i s obvious t h a t excess c a r b o h y d r a t e p e r s i s t s i n t h e medium used (30$ g l u c o s e b r o t h ) . That t h e e f f e c t of c o l c h i c i n e i s not merely due t o a p o s s i b l e f u n c t i o n as a N i t r o g e n source was proved by i n t r o d u c i n g known e x c e l l e n t N i t r o g e n s o u r c e s i n t o o l d c u l t u r e s of t h e y e a s t s . The N-sources used were ammonium s u l p h a t e , and ammonium t a r t r a t e . No e f f e c t was observed i n e i t h e r case. A s e r i e s of experiments 43 was s e t up t o t r y t o l e a r n more of the e f f e c t of c o l c h i c i n e on f e r m e n t a t i o n . I t was proved t h a t c o l c h i c i n e had no e f f e c t on a s t e r i l e f i l t r a t e of an o l d c u l t u r e of Zygosaccharomyces X. L i k e w i s e i t had no e f f e c t on a s t e r i l e f i l t r a t e p l u s o l d washed dead c e l l s ( c h l o r o f o r m k i l l e d ) but gas was produced i n cases where s t e r i l e f i l t r a t e and washed l i v e c e l l s were used. I t appears t h a t the method of k i l l i n g the y e a s t s had something t o do w i t h the observed a f f e c t . C h l o r o f o r m treatment a l t e r e d t h e p e r m e a b i l i t y of the c e l l w a l l . S i n c e a y e a s t growing i n a s h a l l o w l i q u i d medium i s o b t a i n i n g oxygen both by r e s p i r a t i o n ( m o l e c u l a r oxygen) and f e r m e n t a t i o n ( a n a e r o b i c metabolism of car b o h y d r a t e ) i t was d e c i d e d to t r y to t r a c e the e f f e c t of t h e a l k a l o i d s t i l l f u r t h e r . Time d i d not a l l o w a c o n t i n u a t i o n of t h i s work beyond o u t l i n i n g a method of a t t a c k and c a r r y i n g out a few p r e l i m i n a r y experiments. I t i s suggested however t h a t these experiments might w e l l be c o n t i n u e d . I t was d e c i d e d t o i n h i b i t f e r m e n t a t i o n w i t h i o d o a c e t i c a c i d and t o study the e f f e c t of c o l c h i c i n e on r e s p i r a t i o n i n the f i r s t i n s t a n c e , and t o i n h i b i t r e s p i r a t i o n by growing the y e a s t s a n a e r o b i e a l l y t o study f e r m e n t a t i o n alone i n the second i n s t a n c e . An apparatus was d e v i s e d f o r a study of a l c o h o l p r o d u c t i o n i n the presence of c o l c h i c i n e under both r e s p i r a t i o n and f e r m e n t a t i o n . P r o v i s i o n was made f o r p a s s i n g s t e r i l e a i r through the c u l t u r e s i n the presence of i o d o a c e t i c a c i d . Any a l c o h o l produced was r e f l u x e d by a s m a l l condenser run a t 0°C t o r e t u r n t o the c u l t u r e . A f t e r i n c u b a t i o n f o r s e v e r a l days the' m i x t u r e was d i s t i l l e d u s i n g a s m a l l condenser. One cc o f the d i s t i l l a t e was c o l l e c t e d and two cc of 44 c o n c e n t r a t e d s u l p h u r i c a c i d were added. Then 0.1 N potassium dichromate was added u n t i l a permanent c o l o r was o b t a i n e d . A green c o l o r due t o r e d u c t i o n of the dichromate showed the presence of r e d u c i n g substances. T r i a l experiments showed t h a t the t e s t i s s e n s i t i v e to 1:5000 of e t h y l a l c o h o l i n water. I t was found t h a t 1:200,000 of i o d o a e e t i c a c i d was s u f f i c i e n t to i n h i b i t f o r m a t i o n of v o l a t i l e r e d u c i n g substances under c o n d i t i o n s of a e r a t i o n and i n the apparatus mentioned above. T h i s meant t h a t l e s s t h a n 1:5000 o f e t h y l a l c o h o l was produced i n the c u l t u r e . The c u l t u r e showed very heavy growth under these c o n d i t i o n s . The method of t e s t i n g f o r r e d u c i n g substances i s a m o d i f i c a t i o n of the q u a n t i t a t i v e procedure o u t l i n e d by M a t i u Popesco and Popa ( 37 ) f o r a micro d e t e r m i n a t i o n of e t h y l and methyl a l c o h o l s i n m i x t u r e s of the two. The q u a l i t a t i v e t e s t was d e v i s e d as a measure of the a p p l i c a b i l i t y of the q u a n t i t a t i v e method t o any p a r t i c u l a r d e t e r m i n a t i o n . I t i s suggested t h a t i n a m i x t u r e c o n t a i n i n g c o l c h i c i n e where the e f f e c t of the a l k a l o i d on the p r o d u c t i o n of a l c o h o l by r e s  p i r a t i o n or by f e r m e n t a t i o n i s b e i n g measured,that the b e s t procedure would be to p r e c i p i t a t e the c o l c h i c i n e w i t h phos- p h o t u n g s t i c or phosphomolybdic a c i d s . A f t e r f i l t r a t i o n the f i l t r a t e would be d i s t i l l e d and an a l i q u o t p a r t of the d i s t i l  l a t e would be taken f o r a n a l y s i s by the above procedure. In t h i s way the amount of e t h y l a l c o h o l produced by the d e s i r e d r e a c t i o n c o u l d be determined s e p a r a t e from the methyl a l c o h o l r e s u l t i n g from h y d r o l y s i s of t h e c o l c h i c i n e . 45 CONCLUSIONS The work of R i c h a r d s c o n c e r n i n g the e f f e c t of c o l c h i c i n e on the f o r m a t i o n of the y e a s t bud had" been c o r r o  b o r a t e d at l e a s t i n p a r t . Budding seems t o be a m i t o t i c . Some doubt i s t o be g i v e n to r e s u l t s o b t a i n e d by the a p p l i c a t i o n of the c o l c h i c i n e technique to the f u n g i i n the l i g h t of t h e f a c t of p a r t i a l h y d r o l y s i s of t h e a l k a l o i d . An a c c u r a t e method of o b t a i n i n g r a t e s of growth of u n i c e l l u l a r organisms by p h o t o t u r b i d o m e t r i c methods has been d e v i s e d . Evidence has been produced t h a t c o l c h i c i n e i n one to 4.5% c o n c e n t r a t i o n has an i n h i b i t o r y e f f e c t on s p o r u l a t i o n of c e r t a i n members of the genus Zygosaccharomyces. A p o s s i b l e c o n n e c t i o n of the problem w i t h t h e Cancer Problem has been suggested and a mechanism of tumour g e n e s i s has been p o s t u l a t e d . I t i s suggested t h a t the p r o d u c t i o n of a tumour i n s i t u by c a r c i n o g e n i c agents depends on the p r o d u c t i o n of a mutant i n an e x a c t l y s i m i l a r manner t o t h e p r o d u c t i o n of a mutant by c o l c h i c i n e . V a r i o u s l i n e s of approach t o the problem are o u t l i n e d . X BIBLIOGRAPHY 1. P e l l e t i e r and Caventou Ann. Ghim. Phys. 1820 ( i i ) 14, 82. 2. A l l e n ' s Commercial Organic A n a l y s i s 5 t h ed. V o l . V I I pages 147-157. 3. L i p t a k , L.P. Pharm. Monasch. 8, 125-6, (1927). 4. D a v i e s , E.G. Pharm J . 106, 480-1 (1921). 5. Chemitus, F. J . p r a k t . Chem. 118, 29-32 (1918). 6. D a v i e s , E.G. and G r e i r , J . Pharm. J . 109, 210-1 (1922). 7. B e i l s t e i n , "Organische Chemie", I I I A u f l a g e p 873. 8. Windaus, A. and Seheele, H. Ann. 439, 59-75 (1924). 9. B u r s i a n , K, Ber. 71B, 245-57 (1938). 10. K l e i n , G. and P o l l a u f , G. 6 ' s t e r r e i c h B o t . Z e i t s c h r . 78 (3) 250-6 (1929) 11. B o y l a n d , E. and Huntsman. Biochem J . 1204-6 (1938). 12. K o s t o v , D. C u r r e n t S c i . 7, 108-10 (1938). 13. K o s t o v , D„ Nature 142, No. 3599, 753 (1938). 14, Kostov, D. Compt. rend. acad. s c i . U.S.S.K. 19, 197-9 (1938). 15. Simonet, M. and Guinoehet, M. Compt. rend. 208, 1427-8 (1939). 16. Sharp, L.W. " I n t r o d u c t i o n to C y t o l o g y " (1921) page 144. 17. Gavaudin, P. P d m r i a n s k y - K o b o z i e f f , N. Compt. rend. soc. b i o l . 125, 705-8 (1937). 18. K e b e l , B.R. and R u t t l e , M. L. J r . H e r e d i t y 29, 2-9 (1938). 19. E i g s t i , 0. P r o c . Nat. Acad. S c i . 24, 56-63 (1938). 20. B l a k e s l e e and Avery. J r . H e r e d i t y 28 (12) 393-411 (1937). 21. G a r r i g u e s , R. Compt. rend. 208, 461-3 (1939). 22. G a v r i l o v , W. B i s t r a m , Dina v. B u l l assoe f r a n c etude r a i s o n 32, 319-35 (1939). 23. Verne and V i l t e r Compt. rend. soc. b i o l . 133, 618-21 (1940). 24. M a u r e l , E. Compt. rend. soc. b i o l . 67, 687-8 (1910). x i i 25. Annual Review of B i o c h e m i s t r y 1940, pages 440-443. 26. S o l a c o l u , T. and G o n s t a n t i n e s c o , M. and C o n s t a n t i n e s c o , D. Compt. rend. soc. b i o l . 130, 1148-50 (1939). 27. Brown, N e l l i e A. Phytopathology 29, 221-31 (1939). 28. Guyer, M.F. and C l a u s , P.E. Proc. Soc. E x p t l . B i o l . Med. 42, 565-8 (1939). 29. Haddow, A. and Robinson, A.M. P r o c Roy. Soc. (London) B.127, 277 (1939). 30. "Some Fundamental Aspects of the Cancer Problem" Symposium - American Assen. Adv. S c i . (1937) 31. S t e i n b e r g , R.A. and Thorn. P r o c . Nat. Acad. S c i . 26, 363-366 (1940). 32. R i c h a r d s , 0. W. J r . B a c t . 36 (2) 187-195 (1938). 33. G u i l l i e r m o n d , A. "The Y e a s t s " (book) t r a n s l a t e d by Tanner. 34. G u i l l i e r m o n d , A. B o t a n i c a l Review January 1940. 35. B h a d u r i , P. N. J . Roy. Microscop Soc. 59, 245-86 (1939). 36. Lochead and F a r r e l l Can. J . R s r c h . 5. 665-72 (1931). x i i i 37. l o n e s c u - M a t i u , A l . Popesco, E. and Popa, I . B u l l . Acad. Med.. Roumanie 3, 511-24 ef C.A. 25:3932 

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