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In vitro cultivation of Philonema (Nematoda: Dracunculoidea), a parasite of the body cavity of salmonid… Copping, Barbara Elizabeth 1970

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THE I_N VITRO CULTIVATION OF PHILONEMA (NEMATODA: DRACUNCULOIDEA), A PARASITE OF THE BODY CAVITY OF SALMONID FISHES by BARBARA ELIZABETH COPPING B . S c , U n i v e r s i t y o f B r i t i s h C olumbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF J u l y , BRITISH COLUMBIA 1970 In presenting th i s thes i s in pa r t i a l f u l f i lment o f the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes i s f o r f i nanc ia l gain sha l l not be allowed without my wr i t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada ( i ) ABSTRACT / j The s u r v i v a l i n v i t r o of t h i r d s tage l a r v a e o f Philonema  o h c o r h y n c h i and Philonema ubernacu1um, nematode p a r a s i t e s o f s a l m o n i d f i s h e s , was s t u d i e d . The l a r v a e were c u l t u r e d i n v a r i o u s d e f i n e d media and i n media c o n t a i n i n g a d d i t i v e s . The e f f e c t of a n t i b i o t i c s i n the c u l t u r e media, the e f f e c t of g a s s i n g the c u l t u r e s w i t h 5% carbon d i o x i d e i n a i r and the e f f e c t o f s h a k i n g the c u l t u r e s on the s u r v i v a l o f l a r v a e were s t u d i e d . A s t a t i s t i c a l a n a l y s i s o f the f a c t o r s was performed to see w h i c h c o n d i t i o n s s u p p o r t e d the l o n g e s t s u r v i v a l of the l a r v a e . / G a s s i n g and a n t i b i o t i c s were found to i n c r e a s e the l a r v a l s u r v i v a l , w h i l e s h a k i n g the c u l t u r e s d r a s t i c a l l y d e c r e a s e d the s u r v i v a l . The b e s t medium,, w i t h o u t a d d i t i v e s , was CMRL 1066 w i t h E a g l e ' s s a l t s , and the b e s t a d d i t i v e was c h i c k serum. The optimum medium c o m b i n a t i o n was Medium 199 + 10%. c h i c k serum. The l a r v a e were f o u n d . t o s u r v i v e a maximum of 86 days and an average o f 46.3 days i n Medium 199 + 101 c h i c k serum, w i t h a n t i b i o t i c s and when gassed w i t h 51 carbon d i o x i d e i n a i r . F o u r t e e n l a r v a e m o u l t e d and c o m p l e t e l y e xsheathed i n Medium 199 + 10% d e x t r o s e + 101 horse serum, a l t h o u g h the l a r v a e would s u r v i v e l o n g e r i n o t h e r media c o m b i n a t i o n s . ( i i ) TABLE OF CONTENTS INTRODUCTION 1 MATERIALS AND METHODS 10 E x p e r i m e n t a l d e s i g n 10 Source o f worms f o r c u l turning 10 P r e p a r a t i o n o f l a r v a e f o r c u l t u r e 12 C u l t u r e m a n i p u l a t i o n 12 R e c o r d i n g o f d a t a 14 Measurement o f dead worms 15 P r e p a r a t i o n o f apparatus 15 P r e p a r a t i o n o f media 16 P r e p a r a t i o n o f t r o u t l i v e r and egg e x t r a c t s 19 D e t e r m i n i n g o s m o t i c p r e s s u r e s o f media 21 A n a l y s i s o f r e s u l t s 21 RESULTS 1. Most s u c c e s s f u l medium c o m b i n a t i o n 41 a) C h e m i c a l l y d e f i n e d media 41 b) E f f e c t o f a d d i t i v e s 41 2. A n t i b i o t i c s v s . no a n t i b i o t i c s 43 3. Shaking and g a s s i n g v s . no s h a k i n g and 44 no g a s s i n g 4. M o u l t i n g o f l a r v a e i n c u l t u r e 44 5. C o n d i t i o n o f worms i n c u l t u r e 46 6. Measurement o f dead worms 46 7. Osmotic p r e s s u r e s o f media 47 ( i i i ) TABLE OF CONTENTS (Cont'd) DISCUSSION 49 SUMMARY 65 BIBLIOGRAPHY 66 ( i v ) LIST OF TABLES I Components o f d e f i n e d media 20 I I L i s t o f a l l components and amounts of compounds 71 i n d e f i n e d media I I I Average s u r v i v a l o f l a r v a e c u l t u r e d under a l l 23 c o n d i t i o n s IV Maximum s u r v i v a l (days) o f l a r v a e c u l t u r e d 25 under a l l c o n d i t i o n s V A n a l y s i s o f v a r i a n c e 81 VI M o u l t i n g o f l a r v a e i n Medium 199 + 10% horse 45 serum + 10% d e x t r o s e V I I Average l e n g t h ( m i l l i m e t r e s ) o f dead worms 82 V I I I M o l a l i t i e s o f Media 83 LIST OF FIGURES 1. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media 27 w i t h no a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . 2. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media 28 w i t h no a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . 3. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media 29 w i t h a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . 4. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media 30 w i t h a n t i b i o t i c s , under the c o n d i t i o n s o f no shaking/no g a s s i n g . 5. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + 31 a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f no shaking/no g a s s i n g . 6. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + 32 a d d i t i v e s and w i t h no a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . 7. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + 33 a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f g a s s i n g / n o s h a k i n g . 8. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + 34 a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f ga s s i n g / n o s h a k i n g . 9. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + 35 a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f shaking/no g a s s i n g . 10. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + 36 a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f shaking/no g a s s i n g . 11. The l e n g t h o f s u r v i v a l o f l a r v a e i n E a r l e ' s 37 B a l a n c e d S a l t S o l u t i o n + a d d i t i v e s , w i t h no a n t i b i o t i c s . 12. The l e n g t h o f s u r v i v a l o f l a r v a e i n E a r l e ' s 38 B a l a n c e d S a l t S o l u t i o n + a d d i t i v e s , w i t h no a n t i b i o t i c s . ( v i ) LIST OF FIGURES (Cont'd) 13. The l e n g t h o f s u r v i v a l o f l a r v a e i n c h i c k serum 39 + f o u r d i f f e r e n t b a s i c d e f i n e d media w i t h no a n t i b i o t i c s . 14. The l e n g t h o f s u r v i v a l o f l a r v a e i n c h i c k serum 40 + f o u r d i f f e r e n t b a s i c d e f i n e d media w i t h no a n t i b i o t i c s . 15. C o r r e l a t i o n between s u c c e s s o f media i n s u p p o r t i n g 48 l a r v a l s u r v i v a l and between m o l a l i t i e s o f the media. ( v i i ) ACKNOWLEDGMENT I would s i n c e r e l y l i k e t o thank Dr. J . R. Adams f o r s u g g e s t i n g t h i s s t u d y and f o r h i s c o n t i n u e d h e l p throughout the work, and a l s o d u r i n g the w r i t e - u p o f t h i s paper. The h e l p o f Mr. Steve Borden i n p r o c e s s i n g the s t a t i s t i c a l d a t a was v e r y much a p p r e c i a t e d . The second y e a r o f t h i s work was s u p p o r t e d by a g r a n t from the N a t i o n a l Research C o u n c i l o f Canada. M i s s Norma P i a g g i o v e r y k i n d l y spent many hours t y p i n g t h i s t h e s i s and s i n c e r e thanks i s due t o h e r . INTRODUCTION In v i t r o c u l t u r e g i v e s i n s i g h t i n t o many a s p e c t s o f the ho s t - p a r a s i t e r e l a t i o n s h i p . I_n v i v o a p a r a s i t e i s d i f f i c u l t t o st u d y because i t i s so c l o s e l y a s s o c i a t e d w i t h i t s h o s t and f o r t h i s r e a s o n , o n l y the most e a s i l y r e c o v e r e d s t a g e s r e c e i v e any g r e a t amount of a t t e n t i o n . The s u p p l y and p r o p o r t i o n o f n u t r i e n t s , as w e l l as the p h y s i c o c h e m i c a l c o n d i t i o n s r e q u i r e d f o r growth, development and r e p r o d u c t i o n of p a r a s i t e s can be s t u d i e d by in. v i t r o t e c h n i q u e s . O f t e n each new stage i n a p a r a s i t e ' s l i f e - c y c l e i s d i f f e r e n t from the o t h e r s i n the f a c t o r s t h a t i t r e q u i r e s f o r development. A p a r a s i t e t h r i v e s b e s t i n a h o s t t h a t p r o v i d e s i t s needs, so by u n d e r s t a n d i n g these needs, we u n d e r s t a n d more f u l l y v a r i o u s a s p e c t s o f h o s t - s p e c i f i c i t y . A l s o , w i t h s u c c e s s f u l i n v i t r o c u l t i v a t i o n o f h e l m i n t h s , methods t o combat d i s e a s e s caused by these p a r a s i t e s are e a s i e r t o st u d y and p r o v i d e a good approach t o chemotherapy. In a c h e m i c a l l y d e f i n e d medium, i t i s p o s s i b l e t o a n a l y z e p r o d u c t s formed by the m e t a b o l i z i n g worms. These p r o d u c t s are o f t e n t o x i c and can cause h o s t - r e a c t i o n s such as a l l e r g i e s or even the f o r m a t i o n o f cancerous growths. Growing Philonema i n v i t r o c o u l d prove b e n e f i c i a l t o the f u r t h e r u n d e r s t a n d i n g o f t h i s p a r a s i t e . Philonema i s a member o f the s u p e r f a m i l y D r a c u n c u l o i d e a and as y e t t h e r e have been no r e p o r t s on any h e l m i n t h s i n t h i s group h a v i n g been c u l t u r e d fin v i t r o . - 2 -P l a t z e r (1964) s o l v e d the l i f e - c y c l e o f Philonema  o n c o r h y n c h i . Philonema i s a p a r a s i t e i n the body c a v i t y o f s a l m o n i d f i s h e s . The copepod, C y c l o p s b i c u s p i d a t u s , s e r v e s as the i n t e r m e d i a t e h o s t . A d u l t , g r a v i d , female worms are r e l e a s e d w i t h the roe when the f i s h spawn. The worms b u r s t i n the water and f i r s t s t age l a r v a e are r e l e a s e d . The copepods eat the f i r s t s t age l a r v a e which then burrow i n t o the haemocoele o f the copepod. Here the worms undergo two m o l t s t o become t h i r d s tage l a r v a e , which are i n f e c t i v e t o the f i s h . What s t i m u l u s i n the f i s h a c t s upon Philonema, c a u s i n g the l a r v a e t o a g a i n p r o c e e d w i t h development? Adams (1969) found t h a t once i n s i d e the f i s h the t h i r d s tage l a r v a e m i g r a t e d , m a i n l y through the r e g i o n o f the p y l o r i c c ecae, i n t o the swimbladder. A f t e r some time i n the swimbladder, the worms are found i n the body c a v i t y where they mature. E i t h e r j u s t a f t e r o r j u s t b e f o r e l e a v i n g the swimbladder, the l a r v a e moult to f o u r t h s t a g e . What are the s t i m u l i t h a t p r o v i d e the cues f o r t h i s r o u t e o f m i g r a t i o n o f the l a r v a e i n s i d e the f i s h ? C u l t u r i n g Philonema i n v i t r o makes p o s s i b l e the st u d y o f the n u t r i t i o n a l and p h y s i c o c h e m i c a l c o n d i t i o n s t h a t c o u l d a c t as s t i m u l i , c a u s i n g the worm t o pro c e e d w i t h development and c a u s i n g the l a r v a e t o choose such a r o u t e o f m i g r a t i o n i n s i d e the f i s h . F u r t h e r m o r e , i f the t h i r d s tage l a r v a e c o u l d be in d u c e d t o moult i n v i t r o , a v e r y good s i t u a t i o n would e x i s t - 3 -f o r m o r p h o l o g i c a l s t u d i e s o f the f o u r t h stage l a r v a e . A t p r e s e n t , t h e r e have been no d e t a i l e d m o r p h o l o g i c a l s t u d i e s on any Philonema l a r v a e beyond t h i r d s t a g e . F o u r t h stage l a r v a e are d i f f i c u l t t o o b t a i n i n v i v o and because the e x a c t m o u l t i n g times o f the l a r v a e i n s i d e the f i s h are not known, i t i s o f t e n d i f f i c u l t t o t e l l what stage o f worm has been found. Any m o u l t i n g i_n v i t r o c o u l d , w i t h c a u t i o n , h e l p t o determine m o u l t i n g times o f the worms i n v i v o . The problem o f s t u d y i n g Philonema i n v i v o i s the time i t t a k e s f o r the worms to mature. Philonema o n c o r h y n c h i l i v e s i n anadromous h o s t s w i t h a 4 y e a r spawning c y c l e . The worms do not mature i n the salmon u n t i l the 4th y e a r , when the f i s h r e t u r n t o f r e s h water to spawn. B a s h i r u l l a h (1966) has produced e v i d e n c e t h a t the m a t u r i n g o f the worms i s l i n k e d w i t h the hormonal c y c l e i n the f i s h . Both the worms and the f i s h r e a c h m a t u r i t y at the same tim e . T h e r e f o r e , when the salmon spawn, the f i r s t stage l a r v a e i n s i d e the a d u l t worms are a l s o f u l l y d e v e l o p e d and v i a b l e when r e l e a s e d i n t o the water w i t h the r o e . The e f f e c t o f hormones on the development o f Philonema c o u l d be s t u d i e d by i n v i t r o t e c h n i q u e s . By adding hormones to the c u l t u r e medium, perhaps as w e l l as i n d u c i n g development o f the l a r v a e , the l a r v a l development might even p r o c e e d f a s t e r i_n v i t r o than i n  v i v o . Philonema aqubernaculum i s adapted to r e s i d e n t , f r e s h -w a ter f i s h . There i s s t i l l u n c e r t a i n t y i n the time t h a t - 4 -i t t a k e s f o r t h i s s p e c i e s t o mature. However, B a s h i r u l l a h (1966) r e p o r t e d Philonema agubernaculum t h a t had matured i n l e s s than a y e a r i n t r o u t . P erhaps, the development o f the two worms c o u l d be compared more s u c c e s s f u l l y , by i n v i t r o t e c h n i q u e s . S e v e r a l workers have made s i g n i f i c a n t c o n t r i b u t i o n s to the i n v i t r o c u l t i v a t i o n o f nematodes and have found v a r i o u s f a c t o r s t o be e s s e n t i a l t o the success o f t h e i r c u l t u r e s . G l a s e r (1940) was the f i r s t t o succeed i n c u l t i -v a t i n g , a x e n i c a l l y , a p a r a s i t i c nematode through i t s whole l i f e - c y c l e . He was a b l e to c u l t u r e N e o a p l e c t a n a g l a s e r i , (superfam. R h a b d i a s o i d e a ) , through s u c c e s s i v e g e n e r a t i o n s , w i t h o u t the p a r a s i t e r e t u r n i n g t o i t s normal h o s t . The worms were c u l t u r e d i n an aqueous s u s p e n s i o n o f growing y e a s t o v e r l a i d on s t a n d a r d meat i n f u s i o n . S t o l l (1959) found t h a t p o p u l a t i o n y i e l d s o f N. g l a s e r i were g r e a t e r when the c u l t u r e s were p l a c e d on a s h a k i n g machine. T w e n t y - s i x c u l t u r e tubes t h a t were shaken y i e l d e d 95% more worms than 26 p a r a l l e l c u l t u r e s on a t a b l e - t o p . He a l s o found t h a t t h e r e was a g a i n i n y i e l d (35% i n c r e a s e ) i n c u l t u r e s m a i n t a i n e d i n a dark room, as opposed to those exposed to l i g h t . S t o l l had a l s o attempted to d e f i n e the c u l t u r e medium s t i l l f u r t h e r . He found t h a t the l a r v a e developed much b e t t e r i f raw l i v e r e x t r a c t was added to the v e a l i n f u s i o n b r o t h . J a c k s o n (1961, 1962) was a b l e to c u l t u r e t h i s - 5 -p a r a s i t e t h r o u g h one complete l i f e - c y c l e i n a f u l l y d e f i n e d medium. The medium was composed of s a l t s , v i t a m i n s , p u r i n e s , p y r i m i d i n e s and v a r i o u s amino a c i d s . N i p p o s t r o n g y l u s m u r i s (superfam. S t r o n g y l o i d e a ) was the f i r s t nematode p a r a s i t e o f a v e r t e b r a t e to be s u c c e s s f u l l y c u l t u r e d in_ v i t r o t h r ough e v e r y s t a g e o f i t s l i f e - c y c l e ( W e i n s t e i n and J o n e s , 1956). However, the c u l t u r e o f s u c c e s s i v e g e n e r a t i o n s has not y e t been a c h i e v e d . Here, W e i n s t e i n and Jones f i r m l y e s t a b l i s h e d c h i c k embryo e x t r a c t as one o f the p r i m a r y components of media f o r i_n v i t r o s t u d i e s p a r a s i t i c nematodes. S i n c e t h a t t i m e , i t has been f r e q u e n t l y used by many wo r k e r s . Many o t h e r members o f the s u p e r f a m i l y S t r o n g y l o i d e a have been c u l t u r e d t h rough p a r t s o f t h e i r l i f e - c y c l e s . W e i n s t e i n (1949, 1953) found t h a t when s m a l l amounts of c h i c k embryo e x t r a c t were added t o Tyrodes S o l u t i o n , a p p r o x i m a t e l y 81% o f the l a r v a e o f Ancylostoma caninum and A. duodenale developed to the i n f e c t i v e s t a g e . Without c h i c k embryo e x t r a c t , l i t t l e development was o b t a i n e d . S i l v e r m a n and h i s c o l l e a g u e s , S i l v e r m a n , 1962), ( S i l v e r -man and Podger, 1962) t r i e d to o b t a i n development o f D i c t y o c a u l u s v i v i p a r u s l a r v a e i n a v a r i e t y o f media. They e v e n t u a l l y had s u c c e s s w i t h a medium c o n s i s t i n g o f c h i c k embryo e x t r a c t , sheep l i v e r e x t r a c t , c a s e i n h y d r o l y s a t e , and sheep serum. - 6 -Diamond and Douvres (1960) have c u l t u r e d Pesophagostomum. They o b t a i n e d f o u r t h s t a g e l a r v a e i n a medium composed o f t r y p t i c a s e , y e a s t e x t r a c t , g l u c o s e , sheep serum and r a b b i t and p i g l i v e r e x t r a c t s . G a s s i n g w i t h 5% CO2 i n a i r was found to be i m p o r t a n t f o r .Pesophagostomum c u l t i v a t i o n . L e l a n d (1961, 1963, 1965) c u l t u r e d Pesophagostomum to f i f t h s t age i n a medium o f c h i c k embryo e x t r a c t , serum, Na c a s e i n a t e , v i t a m i n s and p i g l i v e r e x t r a c t i n b a l a n c e d s a l i n e . A l l the c u l t u r e s were gassed w i t h 51 CP2 i n a i r . S i l v e r m a n a l s o used t h i s gas m i x t u r e i n h i s c u l t u r e s o f Pesophagostomum. He found t h a t the l a r v a e exsheathed i n a b a l a n c e d s a l i n e s a t u r a t e d w i t h CP2 and devel o p e d t o f o u r t h s t a g e i n E a r l e ' s o r Hank's b a l a n c e d s a l t s o l u t i o n s a t u r a t e d w i t h C P 2 . They then developed to f i f t h s t a ge i n a more complex medium o f c h i c k embryo e x t r a c t , beef l i v e r e x t r a c t , serum and Na c a s e i n a t e . The f i r s t s u c c e s s f u l growth i n v i t r o o f T r i c h i n e l l a  s p i r a l i s ( s u p e r f a m . T r i c h u r o i d e a ) was o b t a i n e d by W e l l e r (1943). He grew the l a r v a e i n c u l t u r e s c o n t a i n i n g c h i c k embryo t i s s u e s , w i t h an o v e r l a y medium composed o f Simms s o l u t i o n , CEE, and c h i c k serum. He o b t a i n e d a few s e x u a l l y d i f f e r e n t i a t e d l a r v a e which had i n c o m p l e t e l y moulted t h e i r 2nd and 3 r d s h e a t h s . They then degenerated. B e r n t z e n (1965) found the e f f e c t o f g a s s i n g h i s c u l t u r e s o f T r i c h i n e l l a s p i r a l i s to be d r a s t i c . W ith the c o r r e c t gas m i x t u r e (85% N 2 - 5% CP 2 - 10% 0 2 ) , the i n f e c t i v e muscle stage l a r v a e d e v e l o p e d to s e x u a l l y - 7 -d i f f e r e n t i a t e d a d u l t s . T h i s o c c u r r e d i n a v a r i e t y o f media, but i f the gas phase was changed f o r even a s h o r t t i m e , m u l t i p l e s h e a t h f o r m a t i o n o c c u r r e d . From the s u p e r f a m i l y A s c a r i d o i d e a , A s c a r i s l u m b r i c o i d e s has been the most f r e q u e n t l y c u l t u r e d . P i t t s and B a l l (1955) and P i t t s (1962) o b t a i n e d l i m i t e d growth o f l a r v a e from eggs. In b a l a n c e d s a l i n e and p i g serum, some l a r v a e showed an i n c r e a s e i n s i z e up to 30% i n l e n g t h and up to 25% i n d i a m e t e r . In E a g l e ' s Minimum E s s e n t i a l Medium and 10% r a b b i t serum, some l a r v a e had a 304% i n c r e a s e i n l e n g t h and a 500% i n c r e a s e i n d i a m e t e r . C l e e l a n d (1963) and C l e e l a n d and Lawrence (1962) c u l t u r e d the l a r v a e i n v a r i o u s d e f i n e d media such as Medium 199. An i n c r e a s e i n s u r v i v a l r e s u l t e d i f serum was added to Medium 199. The f i r s t a ttempts at i_n v i t r o c u l t u r e o f members o f the s u p e r f a m i l y F i l a r i o d e a were d i r e c t e d towards maintenance o f m i c r o f i l a r i a e i n whole b l o o d (Hobson, 1948). E a r l (1959), c a r r i e d out e x p e r i m e n t s on the maintenance i_n v i t r o , o f b o t h the a d u l t s and the m i c r o f i l a r i a e o f D i r o f i l a r i a i m m i t i s , i n a v a r i e t y o f media. The f i r s t s i g n i f i c a n t s u c c e s s i n the c u l t i v a t i o n o f m i c r o f i l a r i a e was t h a t o f Sawyer and W e i n s t e i n (1962) , when they o b t a i n e d development o f D i r o f i l a r i a  i m m i t i s m i c r o f i l a r i a e t o a stage comparable to 3 - 4 days d i f f e r e n t i a t i o n i n a mosquito h o s t . T h i s development o c c u r r e d i n c u l t u r e s o f h e p a r i n a t e d whole b l o o d . - 8 -S i l v e r m a n (1963, 1965) c u l t u r e d t h i r d s t age i n f e c t i v e l a r v a e o f D i r o f i l a r i a i m m i t i s i n NCTC 109 alone and w i t h serum supplements. In the serum f r e e medium, the l a r v a e l i v e d 3 days. However, when 10% human or horse serum was added t o NCTC 109 and i f the c u l t u r e s were gassed w i t h 5% C O 2 i n a i r , the l a r v a e s u r v i v e d 12 - 15 days and a p p r o x i -m a t e l y 100% o f the l a r v a e exsheathed w i t h i n 72 ho u r s . The o n l y worm b e l i e v e d t o have been c u l t u r e d from the s u p e r f a m i l y S p i r u r o i d e a i s Gnathostoma s p i n i g e r u m , a p a r a s i t e o f c a t s (Oba, 1959). T h i r d stage l a r v a e were m a i n t a i n e d f o r 69 days i n s l i c e s o f c a t l i v e r i n R i n g e r s s o l u t i o n . When the medium was supplemented w i t h c h i c k embryo e x t r a c t , a d u l t worms were a b l e to s u r v i v e i n c u l t u r e . In view o f p r e v i o u s work, done on nematode c u l t u r i n g , i t was d e c i d e d t o t e s t v a r i o u s p h y s i c o c h e m i c a l and n u t r i t i o n a l f a c t o r s which might cause the i n f e c t i v e t h i r d s tage Philonema l a r v a e t o d e v e l o p . Here an attempt was made to p e r f o r m a complete s t a t i s t i c a l a n a l y s i s o f the f a c t o r s t e s t e d , r a t h e r than randomly adding components to a medium u n t i l the worms moulted. The f i r s t i t e m t o be concerned w i t h seemed t o be the n u t r i t i o n o f the worms i n c u l t u r e . The e f f e c t o f b a l a n c e d s a l t s o l u t i o n s and more complex c h e m i c a l l y d e f i n e d media on the s u r v i v a l o f the l a r v a e was t e s t e d . An attempt was made to use media t h a t were q u i t e d i f f e r e n t from each o t h e r . - 9 -The e f f e c t o f u s i n g a d d i t i v e s such as embryo e x t r a c t s , a m n i o t i c f l u i d and v a r i o u s s e r a to d e f i n e d media was t e s t e d . Two f i s h e x t r a c t s were p r e p a r e d and were compared to a d d i -t i v e s from h e t e r o l o g o u s s o u r c e s . Because v a r i o u s workers had r e p o r t e d t h a t the s h a k i n g o f c u l t u r e s g r e a t l y added t o the success o f t h e i r work, i t was d e c i d e d to t e s t the s u c c e s s o f s h a k i n g c u l t u r e s o f Philonema. From r e a d i n g v a r i o u s works, g a s s i n g a l s o seemed to be i m p o r t a n t f o r the s u c c e s s f u l i n v i t r o c u l t i v a t i o n o f nematodes. F u r t h e r m o r e , S i l v e r m a n (1965) i n h i s r e v i e w on the i_n v i t r o c u l t i v a t i o n o f h e l m i n t h s r e p o r t e d t h a t o f a l l the p h y s i c o c h e m i c a l f a c t o r s t e s t e d , g a s s i n g was the most n e g l e c t e d . T h e r e f o r e , g a s s i n g was another f a c t o r t e s t e d f o r the i n v i t r o c u l t i v a t i o n o f Philonema. The f i n a l f a c t o r chosen to be t e s t e d i n the c u l t u r e s was a n t i b i o t i c s . Most cases o f c u l t i v a t i o n are not t r u l y a x e n i c but r a t h e r are c a r r i e d out w i t h the use o f a n t i -b i o t i c s , which m a i n t a i n a b a c t e r i o s t a t i c environment. A n t i b i o t i c s s e r v e as a c r u t c h u n t i l the time t h a t an a x e n i c c u l t u r e i s developed. However, a n t i b i o t i c s can prove h a r m f u l t o c e r t a i n h e l m i n t h s (Gochnauer et a l , 1954). Even organisms h e l d i n a b a c t e r i o s t a t i c s t a t e may a l t e r the medium and t h e r e f o r e make d e t e r m i n a t i o n o f the v a l u e o f the components o f the medium more d i f f i c u l t . For these reasons the a u t h o r t e s t e d the r o l e p l a y e d by a n t i b i o t i c s i n the i n v i t r o c u l t i v a t i o n o f Philonema. - 10 -MATERIALS AND METHODS A f a c t o r i a l d e s i g n was used f o r t h i s experiment. F a c t o r one was medium and 30 d i f f e r e n t media were t e s t e d . F a c t o r two was the a n t i b i o t i c s and was t e s t e d at two l e v e l s ; one w i t h a n t i b i o t i c s and one w i t h o u t a n t i b i o t i c s . F a c t o r t h r e e was g a s s i n g , s h a k i n g and no g a s s i n g , no s h a k i n g and was t e s t e d a t t h r e e l e v e l s : 1) no shaking/no g a s s i n g 2) g a s s i n g / n o s h a k i n g 3) shaking/no g a s s i n g For each d i f f e r e n t s e t o f c o n d i t i o n s , 20 worms were c u l t u r e d , 5 p e r f l a s k . One hundred e i g h t y d i f f e r e n t con-d i t i o n s e x i s t e d . The experiment i n v o l v e d 720 c u l t u r e s c o n t a i n i n g 3600 l a r v a e . However, some c u l t u r e s had to be r e p e a t e d because o f c o n t a m i n a t i o n and because o f l o s s e s o f worms. The main c r i t e r i o n f o r the success o f a t e s t was the l e n g t h o f s u r v i v a l o f the worms, a l o n g w i t h t h e i r m o t i l i t y and g e n e r a l c o n d i t i o n . Other c r i t e r i a used were s i z e i n c r e a s e changes i n morphology, and m o u l t i n g o f l a r v a e . SOURCE OF WORMS FOR CULTURING F i r s t s t age l a r v a e o f Philonema agubernaculum were o b t a i n e d from g r a v i d female worms r e c o v e r e d from spawning rainbow t r o u t , caught at Pennask Lake, B.C. The f i r s t s t age l a r v a e o f Philonema o n c o r h y n c h i were o b t a i n e d from g r a v i d female worms i n the body c a v i t y o f spawning sockeye salmon, Oncorhynchus n e r k a caught at L i n d e l l Beach, C u l t u s Lake, B.C. - 11 Copepods ( C y c l o p s b i c u s p i d a t u s ) , caught at C u l t u s Lake, B.C. were i n f e c t e d i n the l a b o r a t o r y w i t h the f i r s t s t age l a r v a e . C o l l e c t i o n o f the copepods, i n f e c t i n g o f the copepods, and maintenance o f the i n f e c t e d copepods were a l l p erformed w i t h o n l y a few m o d i f i c a t i o n s , a c c o r d i n g t o Ko (1966). There was about 90% success i n i n f e c t i n g the copepods and about 250,000 were i n f e c t e d . The copepods were f e d weekly on Paramecium. Wheat g r a i n s were b o i l e d i n d e c h l o r i n a t e d water and then the Paramecium were i n o c u l a t e d i n t o t h i s . A few days were a l l o w e d f o r the Paramecium p o p u l a t i o n to r e a c h a c o n s i d e r a b l e s i z e . F r e s h c u l t u r e s were made eve r y two weeks. A l l the i n f e c t e d copepods were m a i n t a i n e d at 10 degrees c e n t i g r a d e i n a c o n t r o l l e d environment room. N e a r l y a l l the Philonema l a r v a e had grown to t h i r d s tage by 5^ t o 6 weeks a f t e r i n i t i a l i n f e c t i o n i n t o the copepods. The t h i r d s tage l a r v a e are i n f e c t i v e to f i s h and i t was t h i s s tage t h a t was c u l t u r e d . No f u r t h e r development of the worms took p l a c e i n the copepod. T h i r d stage l a r v a e were m a i n t a i n e d i n the copepods up to 8 months and no r e d u c t i o n i n v i a b i l i t y seemed e v i d e n t . However, w i t h time i n f e c t e d copepods became v e r y d i f f i c u l t t o f i n d due t o deaths i n the o r i g i n a l i n f e c t e d p o p u l a t i o n and a l s o to the growth of a new u n i n f e c t e d p o p u l a t i o n . 12 -PREPARATION OF THE LARVAE FOR CULTURE A bowl o f i n f e c t e d copepods was p l a c e d on a b l a c k c l o t h . A t a b l e lamp was used to s h i n e l i g h t i n t o the bowl. Each copepod was caught i n d i v i d u a l l y by means o f a f i n e p i p e t t e . A f t e r b e i n g caught the copepods were p l a c e d i n a w e l l -s l i d e and any excess water was sucked up by a p i p e t t e . T h i s p r e v e n t e d too much movement o f the copepods. The copepods were d i s s e c t e d under a d i s s e c t i n g m i c r o s c o p e u s i n g 32 times m a g n i f i c a t i o n by means o f two No. 000 i n s e c t p i n s a t t a c h e d t o two a p p l i c a t o r s t i c k s . Care was taken t o i n s e r t the p i n s at the extreme ends o f the copepods and p u l l v e r y g e n t l y so as not to damage the nematode l a r v a e . The e x t r a c t e d l a r v a e were p i c k e d up by an e y e l a s h a t t a c h e d t o an a p p l i c a t o r s t i c k . The l a r v a e were washed t w i c e i n s t e r i l e g l a s s - d i s t i l l e d water b e f o r e the media were i n o c u l a t e d . CULTURE MANIPULATION S t e r i l e , p l a s t i c , d i s p o s a b l e , 30 ml., screw-cap F a l c o n t i s s u e c u l t u r e f l a s k s were used f o r a l l the c u l t u r e s . A l l c u l t u r e s were m a i n t a i n e d a t 10° C. i n a r e f r i g e r a t e d i n c u b a t o r and a l l media were a d j u s t e d to a pH o f 7.0 to 7.3. The s h a k i n g machine was p l a c e d i n the i n c u b a t o r and the c u l t u r e s were shaken at the lo w e s t speed, 40 s t r o k e s / m i n u t e . T h i s speed gave a p e r s i s t e n t r o c k i n g m o t i o n to the media, but no s p l a s h i n g o r b r e a k i n g o f the s u r f a c e f i l m o c c u r r e d . - 13 -Each worm was i n d i v i d u a l l y i n o c u l a t e d i n t o the medium by b e i n g p i c k e d up on an e y e l a s h a t t a c h e d to an a p p l i c a t o r s t i c k . Care was t a k e n not to t o u c h the s t i c k onto the s i d e s o f the f l a s k and not to put i t i n the medium. F i v e worms were i n o c u l a t e d i n t o each f l a s k . As soon as the l a r v a was put i n t o the medium and the cap screwed onto the f l a s k , the e y e l a s h was obser v e d i n water under the d i s s e c t i n g m i c r o s c o p e t o make c e r t a i n t h a t the l a r v a had been t r a n s f e r r e d s u c c e s s f u l l y and was not s t i c k i n g t o the e y e l a s h . Any worm t h a t was not s u c c e s s f u l l y i n o c u l a t e d i n t o the medium on the f i r s t t r y was not used. As soon as i t was determined t h a t the l a r v a was i n the c u l t u r e f l a s k , the worm was examined i n the medium t o be sure t h a t no i n j u r y had t a k e n p l a c e . A c t i v i t y a f t e r i n o c u l a t i o n had t o be e x a c t l y the same as a c t i v i t y b e f o r e . T h i s was o f utmost importance b e f o r e the i n o c u l a t i o n c o u l d be c o n s i d e r e d s u c c e s s f u l . Any g a s s i n g o f the c u l t u r e s took p l a c e under the u l t r a -v i o l e t hood. The gas m i x t u r e used was 5% carbon d i o x i d e i n a i r . Sodium b i c a r b o n a t e was used as a b u f f e r . Each f l a s k was gassed f o r a p p r o x i m a t e l y 5 seconds. The f i n a l pH was determined by means o f the p h e n o l r e d i n d i c a t o r i n the medium and by means of pH paper. The c u l t u r e s were observed at l e a s t 4 times a week by u s i n g e i t h e r a d i s s e c t i n g or i n v e r t e d m i c r o s c o p e . The worms were t r a n s f e r r e d t w i c e weekly. The o l d medium was poured i n t o a s t e r i l e s y r a c r u s e watch g l a s s . Then the 14 -worms were p i c k e d up by a s t e r i l e p i p e t t e i n as s m a l l an amount o f medium as p o s s i b l e . They were then dropped i n t o f r e s h medium i n a new f l a s k . A g a i n the worms' a c t i v i t i e s were checked b e f o r e and a f t e r t r a n s f e r . I n i t i a l l y , i n t h i s s t u d y the t r a n s f e r s were done by p i c k i n g the l a r v a e up on e y e l a s h e s . However, the l o n g e r a worm was i n c u l t u r e , the more s u s c e p t i b l e i t became to i n j u r y by t h i s method o f t r a n s f e r and too many c u l t u r e s had to be s t a r t e d a g a i n . No i n j u r y was observed by t r a n s f e r r i n g w i t h a p i p e t t e . C o n t a m i n a t i o n became e v i d e n t by means o f a change i n the pheno l r e d i n d i c a t o r . The medium was a l s o observed on the i n v e r t e d m i c r o s c o p e f o r the presence o f m i c r o - o r g a n i s m s . I f any c u l t u r e was found t o be c o n t a m i n a t e d , i t was d e s t r o y e d . RECORDING OF DATA Each f l a s k was numbered and a s e p a r a t e sheet o f paper was used t o c o n t a i n i n f o r m a t i o n about the f a c t o r s t e s t e d i n the c u l t u r e , about the c o n d i t i o n o f the medium and about the c o n d i t i o n o f the l a r v a e . Each time a c u l t u r e was checked or was t r a n s f e r r e d , the date was r e c o r d e d and the c o n d i t i o n o f each worm i n the c u l t u r e was r e c o r d e d . Four c l a s s i f i c a t i o n s o f a c t i v i t y were used, r a n g i n g from (++++) to (+). In the (++++) c o n d i t i o n , the l a r v a e moved so r a p i d l y t h a t t hey were d i f f i c u l t t o see, i n (+++) mot i o n they moved v e r y f a s t , i n (++) a c t i v i t y they moved v e r y s l o w l y and i n (+) c o n d i t i o n , t h e i r body was r i g i d w i t h o n l y the head or - 15 -t a i l moving s l i g h t l y . The day t h a t each worm d i e d was r e c o r d e d and then a few worms were f i x e d , mounted and measured. MEASUREMENT OF DEAD WORMS A few dead l a r v a e from each of the c u l t u r e c o n d i t i o n s were kept f o r measurement. The worms were c l e a r e d i n g l y c e r i n e - a l c o h o l (10 p a r t s g l y c e r i n e , 90 p a r t s 70% a l c o h o l ) f o r a p p r o x i m a t e l y one week o r u n t i l the a l c o h o l had e v a p o r a t e d . The l a r v a e were mounted i n a drop o f g l y c e r i n e on a g l a s s s l i d e . A No. 0, 18 mm. sq. c o v e r s l i p was used and was e i t h e r s u p p o r t e d by 2 p i e c e s o f n y l o n t h r e a d o r 2 p i e c e s o f g l a s s -w o o l , so as not t o c r u s h the worms. The c o v e r s l i p s were s e a l e d w i t h g l y c e e l . Measurements o f the l a r v a e were made on a compound mi c r o s c o p e by means of an o c u l a r micrometer. PREPARATION OF APPARATUS Only n o n - t o x i c and p r o p e r l y c l e a n e d m a t e r i a l s were used. Immediately a f t e r use a l l g l a s s w a r e and o t h e r washable m a t e r i a l s were soaked i n d e c h l o r i n a t e d water and then r i n s e d s e v e r a l t i m e s . A c i d - w a s h i n g was never used and g e n e r a l l y the use o f d e t e r g e n t s was a v o i d e d (Dougherty e t . a l . 1959), (Douvres, 1968). However i f water alone d i d not c l e a n the m a t e r i a l s a d e q u a t e l y , a m i l d d e t e r g e n t was used a f t e r which t h e r e was e x t e n s i v e r i n s i n g i n water. Glassware and o t h e r apparatus were e i t h e r s t e r i l i z e d by dry heat f o r one hour at 180° C. or were s t e r i l i z e d i n an a u t o c l a v e at 15 pounds p r e s s u r e f o r 15 t o 20 minu t e s . 16 -A membrane f i l t e r (pore s i z e , .45 m i c r o n s ) i n a p r e s s u r e f i l t r a t i o n f u n n e l was used to s t e r i l i z e any e x t r a c t s t h a t were made. The f u n n e l , complete w i t h the f i l t e r , was s t e r i l i z e d by a u t o c l a v i n g . A s t e r i l e atmosphere i n which to make the media and a p p l y gas to the c u l t u r e s was o b t a i n e d b y . u s i n g an u l t r a v i o l e t hood. A s m a l l Bunsen b u r n e r was p l a c e d i n the hood so t h a t a l l equipment used t o make the media c o u l d be flamed. Many of the s t e r i l i z e d m a t e r i a l s were put under the hood u n t i l t h ey were used. The g a s s i n g apparatus was a l s o kept under the hood. PREPARATION OF THE MEDIA A s e p t i c t e c h n i q u e s were used d u r i n g the p r e p a r a t i o n and h a n d l i n g o f the media. A l l media were p r e p a r e d under the u l t r a v i o l e t hood. The g l a s s at the f r o n t o f the hood was r a i s e d j u s t enough to a l l o w the hands to f i t under c o m f o r t a b l y . Each c o n s t i t u e n t o f the media was measured i n e i t h e r a s e p a r a t e s t e r i l e s y r i n g e or s e p a r a t e g r a d u a t e d c y l i n d e r . No p i e c e o f apparatus was used t w i c e . A l l media were p r e p a r e d a c c o r d i n g t o the s u p p l i e r s d i r e c t i o n s . A p p r o p r i a t e amounts o f sodium b i c a r b o n a t e , water and o f t e n o t h e r components such as 5% g l u t a m i n e had t o be added. Only s t e r i l e g l a s s - d i s t i l l e d water was used. The a n t i b i o t i c m i x t u r e was put i n the media f o l l o w i n g c o m b i n a t i o n o f the. v a r i o u s components. The pH was then a d j u s t e d by s t e r i l e Na b i c a r b o n a t e t o 7.1 to 7.3. A l l pH 17 -d e t e r m i n a t i o n s were made by the use o f pH paper. When completed the media was poured i n 4 ml. amounts i n t o the screw-capped c u l t u r e f l a s k s . The tubed media was then f r o z e n u n t i l u se, i f the s u p p l i e r s s t a t e d t h a t f r e e z i n g was a d v i s a b l e . T h i r t y d i f f e r e n t media were t e s t e d . Each medium was d i v i d e d i n t o two b a t c h e s , A and B. Batc h A c o n t a i n e d no a n t i b i o t i c s and b a t c h B c o n t a i n e d 50 u n i t s s t r e p t o m y c i n per ml. (6.5 mg.) and 50 u n i t s p e n i c i l l i n p e r ml. (3.15 mg.). The media t e s t e d c o n s i s t e d o f 3 b a l a n c e d s a l t s o l u t i o n s ; 10 c o m m e r c i a l l y a v a i l a b l e , c h e m i c a l l y d e f i n e d t i s s u e c u l t u r e media; Medium 199 and 10 a d d i t i v e s ; E a r l e ' s b a l a n c e d s a l t s o l u t i o n and 4 a d d i t i v e s , and the b e s t a d d i t i v e w i t h the 3 b e s t b a s i c media t e s t e d . The f o l l o w i n g media were used: B a l a n c e d S a l t S o l u t i o n s 1. G.K.N. 2 . E a r l e ' s B.S.S. 3. Hank's B.S.S. Commercial C h e m i c a l l y D e f i n e d Media 4. S c h e r e r ' s Maintenance Medium 5. N.C.T.C. 109 6. R.P.M.I. 1640 7. R.P.M.I. 1603 8. Medium 199 9. Waymouths M.B. 752/1 10. CM.R.L. w i t h E a g l e ' s s a l t s - 18 -11. CM.R.L. w i t h Hank's s a l t s 12. Ham's F-10 13. E a g l e ' s M i n i m a l E s s e n t i a l Medium Medium 199 and: 14. 10% C h i c k Embryo E x t r a c t 15. 10% Beef Embryo E x t r a c t 16. 10% d e x t r o s e 17. 10% d e x t r o s e , 10% Horse Serum 18. 10% Horse Serum 19. 10% C h i c k Serum 20. 10% C h i c k Serum, 10% d e x t r o s e 21. 10% Bovine A m n i o t i c F l u i d 22. 10% T r o u t Egg E x t r a c t 23. 10% T r o u t L i v e r E x t r a c t E a r l e ' s B.S.S. and: 24. 10% Beef Embryo E x t r a c t 25. 10% Horse Serum 26. 10% C h i c k Serum 27. 2% C a l f Serum, 0.5% L a c t a l b u m i n h y d r o l y s a t e ( M e l n i c k Monkey Kidney Medium B) 10% C h i c k Serum and: 28. Waymouths M.B. 752/1 29. Ham's F-10 30. CM.R.L. w i t h E a g l e ' s S a l t s T a ble 1 shows a l i s t o f the components i n the c h e m i c a l l y d e f i n e d media. A complete l i s t i n g o f a l l components and amounts i s i n the appendix i n Ta b l e 2. 19 -PREPARATION OF TROUT LIVER AND EGG EXTRACTS The eggs and l i v e r s were o b t a i n e d from rainbow t r o u t , Salmo g a i r d n e r i , w hich were caught at Pennask Lake, B. C. The e x t r a c t s were p r e p a r e d s i m i l a r t o the method used by W e i n s t e i n and Jones (1956) f o r t h e i r c h i c k embryo e x t r a c t , but w i t h some m o d i f i c a t i o n s . 1) The eggs or p i e c e s o f raw l i v e r were weighed and then p l a c e d i n a b l e n d e r . 2) G l a s s - d i s t i l l e d water was added i n a r a t i o o f 1:1 (1 c c . water to 1 g. m a t e r i a l ) . 3) A f t e r b l e n d i n g , the l i q u i d was c e n t r i f u g e d f o r h hour by o r d i n a r y c e n t r i f u g e . 4) The l i q u i d was decanted o f f and kept and the sediment was d i s c a r d e d . 5) The decanted l i q u i d was c e n t r i f u g e d at 15,000 r.p.m. (1000 g.) i n a h i g h - s p e e d c e n t r i f u g e . 6) A f t e r c e n t r i f u g a t i o n , the l i q u i d was decanted o f f and then s t e r i l i z e d by p r e s s u r e f i l t r a t i o n t h r o u g h a membrane f i l t e r w i t h a pore s i z e o f 0.45 m i c r o n s . 7) The e x t r a c t was then s t o r e d f r o z e n u n t i l ready f o r use. TABLE 1 COMPONENTS OF DEFINED MEDIA Medium No. of S a l t s No. o f Amino A c i d s No. o f V i t a m i n s M i s c e l l a n e o u s G.K.N. 2 - - 1 E a r l e ' s B.S.S. 6 - - 1 Hank's B.S.S. 7 - - 1 S c h e r e r ' s 7 4 10 13 N.C.T.C. 109 6 24 18 19 R.P.M.I. 1640 6 21 11 1 R.P.M.I. 1603 10 18 13 1 Medium 199 7 21 18 13 Waymouth's 8 18 11 2 CM.R.L. w i t h E a g l e ' s 6 21 13 18 CM.R.L. w i t h Hank's 7 21 13 18 Ham's F-10 10 20 11 3 E a g l e ' s M i n. E s s . 8 13 8 1 - 21 -DETERMINING OSMOTIC PRESSURES OF THE MEDIA Osmotic p r e s s u r e s were measured by d e t e r m i n i n g the f r e e z i n g p o i n t d e p r e s s i o n s o f the media. A s m a l l amount o f the medium was ta k e n up i n a m i c r o -h e m a t o c r i t tube and a bubble o f the medium was then dropped i n p a r a f f i n - o i l . P a r a f f i n - o i l a l t e r n a t i n g w i t h medium from the bubble was sucked i n t o a lambda p i p e t t e by means o f a mouth tube. The ends o f the p i p e t t e were then s e a l e d w i t h p l a s t i c c l a y . The medium w i t h i n the p i p e t t e was f r o z e n by d i p p i n g the p i p e t t e i n t o 90% a l c o h o l c o n t a i n i n g p i e c e s o f dry i c e . The m e l t i n g p o i n t was measured and was taken to be the moment t h a t the l a s t c r y s t a l d i s a p p e a r e d . ANALYSIS OF RESULTS A three-way a n a l y s i s o f v a r i a n c e was used on the d a t a t o determine i f any o f the c o n d i t i o n s s i g n i f i c a n t l y i n c r e a s e d the s u r v i v a l o f the worms. The a n a l y s i s was performed on b o t h the a c t u a l numbers and the l o g s o f the numbers. - 22 -RESULTS T a b l e 3 shows a comparison o f the mean s u r v i v a l of the l a r v a e c u l t u r e d under a l l the v a r i o u s c o n d i t i o n s . T a b l e 4 shows a comparison o f the maximum s u r v i v a l . The a n a l y s i s o f v a r i a n c e showed t h a t t h e r e were s i g n i f i c a n t d i f f e r e n c e s between a l l the f a c t o r s t e s t e d . The media were d i f f e r e n t from each o t h e r w i t h r e g a r d s to s u p p o r t i n g l a r v a l s u r v i v a l . The l a r v a e s u r v i v e d l o n g e r i n media c o n t a i n i n g a n t i b i o t i c s , i n media t h a t were gassed and i n media t h a t weren't shaken. A l l the v a l u e s from the a n a l y s i s are l i s t e d i n T a b l e 5. F i g u r e s 1 to 14 show g r a p h i c a l l y the s u r v i v a l p a t t e r n s of the l a r v a e c u l t u r e d under the v a r i o u s c o n d i t i o n s and i n the v a r i o u s media. TABLE III AVERAGE SURVIVAL OF LARVAE CULTURED UNDER ALL CONDITIONS Medium No Shaking/No Gassing No Antibiotics Antibiotics Average Survival (Days) S.E. Average Survival (Days) S.E. No Gassing/Shaking No Antibiotics Antibiotics Average Survival (Days) S.E. Average Survival (Days) S.E. No Shaking / Gas s ing No Antibiotics Antibiotics Average Survival (Days) S.E. Average Survival (Days) S.E. GKN 5 .3 0, .4068 5 .5 0. 2832 3.6 0. 1575 4 .2 0. 1347 4 .9 0 .3149 4, .8 0, .2909 Earles 4 .4 0, .1561 7 .9 0. 7379 3.8 0. 1952 4 .2 0. 0840 4 .3 0 .1465 5. ,6 0, ,5124 Hanks 5 .4 0, ,3003 8 .0 0. 4881 3.3 0. 1983 4 .2 0. 1412 6 .1 0 .3830 5. .9 0. .5565 Scherers 6 .2 0, .3033 6 .5 0. 3201 3.4 0. 2025 2 .9 0. 1955 10 .4 0 .4104 10, .5 0, ,3078 NCTC 109 7 .4 0, .6751 8 .8 0. 5889 5.8 0. 6087 4 .9 0. 2008 10 .4 0 .4104 10. ,7 0, .3078 RPMI 1640 10 .6 0, .5737 13 .5 0!. 8645 5.4 0. 3677 6 .1 0. 3860 14 .5 0 .5019 19. ,1 1, .6083 RPMI 1603 11 .2 0. .5731 11 .2 0. 4665 5.9 0. 3788 4 .9 0. 3111 13 .3 0 .4534 10, .4 0, .7421 Med. 199 10 .5 0. .4497 11 .8 1. 1254 5.0 0. 2769 5 .8 0. 7072 12 .5 0 .5740 8, .0 0, ,6903 Waymouths 7 .4 0, .8095 14 .6 1. 1943 6.1 0. 3606 5 .1 0. 3060 9 .9 0 .6069 14. ,8 0, .7968 CMRL 1066 15 .0 1. .3241 18 .2 1. 0647 6.3 0. 4817 7 .1 0. 4204 19 .9 2 .2860 20, .9 1. .7247 (Eagle) CMRL 1066 6 .2 0. .3513 9 .6 0. 3278 4.3 0. 1465 3 .8 0. 1643 9 .4 0 .6543 17, .2 1, .0140 (Hank) Hams F-10 18 .9 1, .8740 20 .8 1. 3783 8.1 0. 5565 6 .8 0. 3910 13 .5 1 .0747 14, .1 1, .1766 Eagles Min 9 .9 0, .5151 12 .4 0. 8061 4.4 0. 1123 4 .6 0. 1730 12 .4 0 .6757 12, .6 0, .8338 Ess. Med. 199 + C.E.E. 8.3 0.3058 9.9 0.5903 5.3 0.2480 5.2 0.3182 7.2 0.2183 9.6 0.5883 + B.E.E. 10.0 0.4211 9.7 0.4534 4.3 0.1804 4.8 0.1804 11.4 0.4229 10.0 0.3722 + B.A.F. 13.8 1.2490 11.3 0.7978 5.0 0.3021 6.6 0.4104 13.6 0.6201 10.5 0.4373 + Dextrose 10.1 0.5129 10.7 0.3352 4.2 0.2721 4.8 0.1804 7.7 0.6407 10.7 0.5373 + horse ser 6.9 0.2909 12.7 0.8055 7.7 0.4015 6.6 0.2928 20.5 2.0255 14.9 1.7343 + Dextrose Medium No Shaking/No Gassing No Antibiotics Antibiotics Average Average Survival Survival (Days) S.E. (Days) S.E. Med. 199 + horse 16.3 1.5579 12.1 0.9956 serum + chick 20.3 0.7476 20.4 1.2812 serum + Dextrose chick 39.9 1.8291 42.4 1.4419 serum + trout 3.6 0.2025 3.9 0.1808 egg extract + trout 12.8 0.9021 13.1 0.7349 liver ext Earles + B.E.E. 6.5 0.3449 8.5 0.2409 Horse 10.2 1.0131 9.9 0.8909 serum chick 19.7 3.7217 30.7 3.3363 serum calf serum 10.9 1.2876 11.6 0.7996 + lact. hyd. Chick serum + Waymouths 11.1 1.0164 19.3 2.9123 Hams F-10 18.0 0.6509 23.9 1.9734 CMRL 1066 24.3 1.9308 32.9 3.0592 (Eagles) TABLE III (Cont'd) No Gassing/Shaking No Antibiotics Antibiotics Average Survival (Days) S.E. Average Survival (Days) S.E. No Shaking/Gassing No Antibiotics Antibiotics Average Survival (Days) S.E. Average Survival (Days) S.E. 7.0 0.8188 7.0 0.3368 16.6 2.4123 6.8 0.2642 8.7 0.4713 17.0 1.3315 8.6 0.1730 13.7 0.6627 19.8 2.2828 3.2 0.1539 7.1 0.4041 26.1 2.0940 3.3 0.1465 6.9 0.3320 36.2 3.9453 4.0 0.000 9.9 0.3967 46.3 4.7991 4.0 0.0513 8.5 0.6677 5.9 0.3860 7.9 0.4875 9.8 0.7163 5.3 0.4136 5.7 8.4 10.0 6.5 0.2909 0.6833 0.5470 0.5643 9.8 10.9 20.2 9.4 0.3692 0.9394 1.0085 1.1224 8.1 12.8 12.2 38.1 0.1894 0.8183 1.1180 3.7457 4.4 0.1703 9.9 0.7852 8.5 0.8437 4.5 8.9 8.4 0.2832 0.6710 0.4665 23.7 30.3 35.5 1.9051 1.6441 2.7805 17.4 27.1 43.3 2.5376 1.8607 4.2712 TABLE IV MAXIMUM SURVIVAL (DAYS) OF LARVAE CULTURED UNDER ALL CONDITIONS No Shaking/No G a s s i n g No G a s s i n g / S h a k i n g No S h a k i n g / G a s s i n g No No No Medium A n t i b i o t i c s A n t i b i o t i c s A n t i b i o t i c s A n t i b i o t i c s A n t i b i o t i c s A n t i b i o t i c s GKN 7 8 4 5 7 7 E a r l e ' s BSS 5 14 5 5 5 10 Hank's BSS 8 12 5 5 8 11 S c h e r e r s 8 8 4 4 8 8 NCTC 109 13 12 11 7 13 12 RPMI 1640 14 36 8 9 18 41 RPMI 160 3 15 14 8 7 16 16 Med. 199 14 22 8 13 15 13 Waymouths 15 22 8 8 12 19 CMRL 1066 w i t h E a g l e 26 23 11 11 33 35 CMRL 1066 w i t h Hank 8 11 6 5 16 26 Hams F-10 34 30 12 9 20 20 E a g l e M i n. E s s . 14 19 5 6 20 19 Med. 199 + C.E.E. 12 14 7 8 9 12 B.E.E. 12 12 6 6 14 12 B.A.F. 33 17 8 9 17 12 Dex t r o s e 13 14 7 6 12 13 Horse Ser § 9 19 12 9 42 23 D e x t r o s e C h i c k Ser + 26 29 9 14 29 17 De x t r o s e C h i c k Serum 52 48 36 36 66 86 T r o u t Egg 5 5 4 4 4 4 E x t r a c t T r o u t L i v e r 17 17 11 8 13 15 E x t r a c t Horse Serum 33 20 16 9 36 10 TABLE IV (Cont'd) Medium E a r l e ' s + B . E . E . Horse Serum C h i c k Serum C a l f Ser + Lac. Hyd. C h i c k Serum + Waymouths Hams F-10 CMRL 1066 w i t h E a g l e s No Shaking/No Gassing No A n t i b i o t i c s A n t i b i o t i c s No G a s s i n g / S h a k i n g 9 19 54 22 2 i 22 37 10 16 59 17 42 41 48 No S h a k i n g / G a s s i n g No No A n t i b i o t i c s A n t i b i o t i c s A n t i b i o t i c s A n t i b i o t i c s 11 13 6 16 15 7 12 14 11 7 14 14 12 16 24 21 33 41 63 9 16 22 71 35 43 69 - 27 -F i g u r e 1. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media w i t h no a n t i b i o t i c s under the con-d i t i o n s o f no shaking/no g a s s i n g . NUMBER OF " WORMS. : ALIVE D D G K N DAYS OF SURVIVAL © © EARLES — _ HANKS B B SCHERERS NUMBER OF WORMS ALIVE •Q NCTC 109 -© RPM11640, -A RPMI 1603 -§3 MEDIUM 199 10 20" DAYS OF SURVIVAL "30- 40 - 28 -F i g u r e 2. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media w i t h no a n t i b i o t i c s under the con-d i t i o n s o f no shaking/no g a s s i n g . NUMBER OF WORMS ALIVE DAYS OF SURVIVAL a — @ WAYMOUTHS A A CMRL 1066- (EAGLE) © © CMRL 1066 (HAWKS) O — O HAMS F-10 £s & EAGLES mm, ESS. - 29 -F i g u r e 3. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media w i t h a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . 2. N U M B E R OF W O R M S A L I V E -© G K N -@ EARLES H A N K S - 0 SCHERERS 20F N U M B E R OF W O R M S A L I V E 10 0 © © N C T C 109 0 o R P M I 1640 & A R P M I 1603 O- O MEDIUM 199 10 20" 30 D A Y S OF S U R V I V A L f - 30 -F i g u r e 4. The l e n g t h o f s u r v i v a l o f l a r v a e i n d e f i n e d media w i t h a n t i b i o t i c s , under the c o n d i t i o n s o f no shaking/no g a s s i n g . N U M B E R OF W O R M S A L I V E D A Y S OF S U R V I V A L o — — o W A Y M O U T H S D 0 C M R L 1066 ( E A G L E S ) A — & C M R L 1066 ( H A N K S ) — © H A M S F-10 0 — s EAGLES M I N . E S S . - 31 -F i g u r e 5. The l e n g t h o f s u r v i v a l of l a r v a e i n Medium 199 + a d d i t i v e s and w i t h no a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . NUMBER OF WORMS ALIVE O -o MEDIUM |^°\ © © MED.199 & CHICK EMBRYO EXTRACT 0 0 MED.199 & BEEF EMBRYO EXTRACT 2 0 3 0 DAYS OF SURVIVAL 4 0 5 0 "5b NUMBER OF WORMS ALIVE 0 ~ JO -O MED.199 & HORSE SERUM 2 0 3 0 DAYS OF SURVIVAL 5 0 6 0 -S MED.199 & BOVINE AMNIOTIC FLUID -0 MED.199 & DEXTROSE -O MED.199 & HORSE SERUM & DEXTROSE - 32 -F i g u r e 6. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + a d d i t i v e s and w i t h no a n t i b i o t i c s under the c o n d i t i o n s o f no shaking/no g a s s i n g . 2 0 NUMBER OF WORMS 1 0 ALIVE DAYS OF SURVIVAL O O MED, 199 & CHICK SERUM © © MED. 199 & CHICK SERUM & DEXTROSE 0 0 MED. 199 & TROUT LIVER EXTRACT 0 0 MED.199 & TROUT EGG EXTRACT - 33 F i g u r e 7. The l e n g t h of s u r v i v a l o f l a r v a e i n Medium 199 + a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f g a s s i n g / n o s h a k i n g . D A Y S O F S U R V I V A L O O MED.199 & H O R S E S E R U M S- i M E D . 1 9 9 & B O V I N E A M N I O T I C F L U I D © © M E D . 1 9 9 & D E X T K O S E D —a M E D . 1 9 9 & H O R S E S E R U M & D E X T R O S E - 34 -F i g u r e 8. The l e n g t h o f s u r v i v a l o f l a r v a e i n Medium 199 + a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f g a s s i n g / n o s h a k i n g . NUMBER OF WORMS ALIVE 10 -O MED 199 & CHICK SERUM 20 30 DAYS OF SURVIVAL 40 50 60 70 ©- -© MED 199 & CHICK SERUM & DEXTROSE -0 MED 199 & TROUT EGG EXTRACT -O MED 199 & TROUT LIVER EXTRACT - 35 -F i g u r e 9. The l e n g t h of s u r v i v a l o f l a r v a e i n Medium 199 + a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f shaking/no g a s s i n g . NUMBER OF WORMS ALIVE 20 10 o-10 20 DAYS OF SURVIVAL 30 "30 -Q, MEDIUM 109 MED 199 & CHICK EMBRYO EXTRACT -0 MED 199 & BEEF EMBRYO EXTRACT 20R NUMBER OF WORMS ALIVE O-0-©-10 20 DAYS OF SURVIVAL - ° MED 199 & HORSE SERUM -Q MED 199 & BOVINE AMNIOTIC FLUID ~® MED 199 & ©EXTROSE -® MED 199 & HORSE SERUM 30 "4*0 36 -F i g u r e 10. The l e n g t h o f s u r v i v a l of l a r v a e i n Medium 199 + a d d i t i v e s and w i t h no a n t i b i o t i c s , under the c o n d i t i o n s o f shaking/no g a s s i n g . NUMBER Of WORMS ALIVE DAYS OF SURVIVAL © © MED 199+ CHICK SERUM 0 • MED 1994 CHICK SERUM+ DEXTROSE O- o MED 199+ TROUT EGG EXTRACT B a MED 199+ TROUT LIVER EXTRACT 37 -F i g u r e 11. The l e n g t h o f s u r v i v a l of l a r v a e i n E a r l e ' s B a l a n c e d S a l t S o l u t i o n + a d d i t i v e s , w i t h no a n t i b i o t i c s . WO SHAKING; MO GASSSMG NUMBER OF WOK MS AL'VE 1& © EARLS *S •. C O EAGLE'S -r BEEF EMB51YO. EXTRACT.- • A r - A P A T i L E *S - ! - M O R S E S S R U J V S A AEAKLE'S+CHICK SERUM -0 0 SARLE -5 + CALF SERUM +.LACTVHYD. 20 30 DAYS OF SURVIVAL 60 S H A K I N G • WO GASSING NUMBER OF WORMS ALIVE 20 30 DAYS OF SURVIVAL-. 40 50 60 - 38 -F i g u r e 12. The l e n g t h o f s u r v i v a l of l a r v a e i n E a r l e B a l a n c e d S a l t S o l u t i o n + a d d i t i v e s , w i t h a n t i b i o t i c s . G A S S I N G ; N O S H A K I N G N U M B E R O F W O R M S A L I V E D A Y S O F S U R V I V A L © © E A R LE ' S . O O E A K L E ' S + B E E F E M B R Y O E X T R A C T A & E A R L E * S + H O R S E S E R U M & & E A R L E »S + C H I C K S E R U M • 0 E A R L E ' S + C A L F S E R U M + L A C To H Y D . 39 -F i g u r e 13. The l e n g t h o f s u r v i v a l o f l a r v a e i n c h i c k serum + f o u r d i f f e r e n t b a s i c d e f i n e d media w i t h no a n t i b i o t i c s . - 40 -F i g u r e 14. The l e n g t h o f s u r v i v a l o f l a r v a e i n c h i c k serum + f o u r d i f f e r e n t b a s i c d e f i n e d media w i t h no a n t i b i o t i c s . N O G A S S I N G ; S H A K I N G N U M B E R O F W O R M S A L I V E D A Y S O F S U R V I V A L © — © M E D I U M 199 +CH1CK S E R U M o O H A M S F - 1 0 + C H I C K S E R U M A & C M R L 1 0 6 6 CEAGLES) + C H I C K S E R U M & A W A Y M O U T H S + M E D I U M 1 9 9 - 41 -1. Most S u c c e s s f u l Medium Combination The a n a l y s i s o f v a r i a n c e showed a s i g n i f i c a n t d i f f e r e n c e between the media i n s u p p o r t i n g d i f f e r e n t l e n g t h s o f s u r v i v a l o f the- l a r v a e . a) C h e m i c a l l y D e f i n e d Media The d e f i n e d media s u p p o r t e d an average s u r v i v a l o f l a r v a e from 2.9 days f o r S c h e r e r ' s Maintenance Medium to 20.9 days f o r Medium CMRL 1066 w i t h E a g l e ' s S a l t s . The t h r e e b a l a n c e d s a l t s o l u t i o n s s u p p o r t e d s u r v i v a l f o r the fewest days. F i g u r e s 1 to 4 compare the s u r v i v a l of the l a r v a e i n d e f i n e d media. b) E f f e c t o f A d d i t i v e s F i g u r e s 5 to 10 compare the s u r v i v a l o f the l a r v a e i n Medium 199 + a d d i t i v e s . The s u r v i v a l of the l a r v a e i n Medium 199 + a d d i t i v e s ranged from an average o f 3.2 days i n Medium 199 + t r o u t egg e x t r a c t t o 46.3 days i n Medium 199 + c h i c k serum. Medium 199 + c h i c k serum prove d t o be the most s u c c e s s f u l c o m b i n a t i o n o f a l l the media t e s t e d . - 42 -C h i c k serum was by f a r the most b e n e f i c i a l a d d i t i v e used whether f o r s h a k i n g , g a s s i n g , o r no s h a k i n g , no g a s s i n g . The a d d i t i o n o f c h i c k embryo e x t r a c t , b o v i n e embryo e x t r a c t , d e x t r o s e and t r o u t egg e x t r a c t to Medium 199 d i d not i n c r e a s e time o f s u r v i v a l o f the l a r v a e beyond t h a t when they were c u l t u r e d i n Medium 199 a l o n e . Medium 199 + t r o u t egg e x t r a c t s u p p o r t e d the l e a s t s u r v i v a l o f any medium c o m b i n a t i o n t e s t e d . The egg e x t r a c t must have had a t o x i c e f f e c t on the l a r v a e s i n c e t h e i r s u r v i v a l i n i t was even l e s s than i n the t h r e e b a l a n c e d s a l t s o l u t i o n s . E a r l e ' s BSS + a d d i t i v e s Here the average s u r v i v a l o f the l a r v a e ranged from 5.3 days i n E a r l e s + c a l f serum + l a c t a l b u m i n h y d r o l y s a t e to 38.1 days i n the same medium. How-e v e r , c h i c k serum was the most s u c c e s s f u l a d d i t i v e f o r a l l c o m b i n a t i o n s except w i t h a n t i b i o t i c s , c u l t u r e d under g a s s i n g / n o s h a k i n g . F i g u r e s 11 and 12 compare s u r v i v a l o f the l a r v a e i n E a r l e s B.S.S. + a d d i t i v e s . - 43 -C h i c k serum + d e f i n e d media F i g u r e s 13 and 14 compare the s u r v i v a l o f the l a r v a e i n c h i c k serum + d e f i n e d media. Even though Medium CMRL 1066 w i t h E a g l e ' s s a l t s was the b e s t d e f i n e d medium f o r s u r v i v a l when used a l o n e , i t wasn't the most s u c c e s s f u l medium i n c o m b i n a t i o n w i t h c h i c k serum. The b e s t medium w i t h c h i c k serum was Medium 199. The most u n s u c c e s s f u l medium f o r s u p p o r t i n g s u r v i v a l . w i t h the c h i c k serum was Waymouth's. The l a r v a e even l i v e d l o n g e r i n E a r l e ' s BSS + c h i c k serum than they d i d i n Waymouth's + c h i c k serum. 2. A n t i b i o t i c s v s . No A n t i b i o t i c s The a n a l y s i s o f v a r i a n c e showed a s i g n i f i c a n t d i f f e r e n c e between the l e n g t h of s u r v i v a l o f the l a r v a e c u l t u r e d i n media w i t h a n t i b i o t i c s and media w i t h o u t a n t i b i o t i c s . F i g u r e s 1 t o 4 compare s u r v i v a l o f l a r v a e c u l t u r e d w i t h and w i t h o u t a n t i b i o t i c s . As seen from Table 4, the maximum s u r v i v a l o f the l a r v a e which was 86 days o c c u r r e d i n Medium 199 + c h i c k serum c o n t a i n i n g a n t i b i o t i c s . The medium which s u p p o r t e d the next maximum s u r v i v a l was E a r l e ' s BSS + c a l f serum + l a c t a l b u m i n h y d r o l y s a t e . T h i s m i x t u r e s u p p o r t e d a maximum s u r v i v a l o f 71 days. I t a l s o c o n t a i n e d a n t i b i o t i c s . - 44 -3. Shaking and G a s s i n g v s . No Shak i n g and No G a s s i n g The a n a l y s i s o f v a r i a n c e showed a s i g n i f i c a n t d i f f e r e n c e i n the s u r v i v a l o f the l a r v a e c u l t u r e d under the c o n d i t i o n s o f no shaking/no g a s s i n g , g a s s i n g / n o s h a k i n g and shaking/no g a s s i n g . F i g u r e s 5 to 10 compare s u r v i v a l o f the worms c u l t u r e d under these c o n d i t i o n s . G a s s i n g i n most cases i n c r e a s e d the l a r v a l s u r v i v a l beyond t h a t when they weren't gassed. However, under the c o n d i t i o n s o f no gas s i n g / n o s h a k i n g and gas s i n g / n o s h a k i n g a l l 30 media, both w i t h and w i t h o u t a n t i b i o t i c s , s u p p o r t e d l o n g e r worm s u r v i v a l than i f the c u l t u r e s were shaken. 4. M o u l t i n g o f Larvae i n C u l t u r e The o n l y medium which i n d u c e d m o u l t i n g o f the l a r v a e was Medium 199 + 10% horse serum + 10% d e x t r o s e . Worms moulted and c o m p l e t e l y exsheathed i n t h i s medium, w i t h e i t h e r o f the a n t i b i o t i c m i x t u r e s . A l l moults o c c u r r e d i n the c o n d i t i o n o f no s h a k i n g and no g a s s i n g . At f i r s t the m o u l t i n g worms were t r a n s f e r r e d w i t h an e y e l a s h . Many o f the l a r v a e d i e d d u r i n g t h i s method o f t r a n s f e r so a p i p e t t e was then used f o r the t r a n s f e r s . Only the s u r v i v a l times o f the m o u l t i n g worms t r a n s f e r r e d by p i p e t t e s h o u l d be c o n s i d e r e d , as no damage to the l a r v a e took p l a c e by t h i s method o f t r a n s f e r . Any o f the worms t h a t d i d not moult seemed to be as s u s c e p t i b l e to damage i n t h i s medium c o m b i n a t i o n as the m o u l t i n g worms. - 45 -TABLE VI MOULTING OF LARVAE IN MEDIUM 199 + 10% HORSE SERUM + 10% DEXTROSE Medium Without A n t i b i o t i c s Medium W i t h A n t i b i o t i c s E y e l a s h T r a n s f . P i p e t t e T r a n s f . E y e l a s h T r a n s f . P i p e t t e T r a n s f , Day Day Mo u l t e d D i e d Day Day Mo u l t e d D i e d Day Day M o u l t e d D i e d Day Day Mo u l t e d D i e d 2 2 3 3 5 7 5 5 8 9 12 4 8 15 15 2 3 6 4 17 17 16 15 - 46 -5. C o n d i t i o n o f Worms i n C u l t u r e The a c t i v i t y o f a l l the worms seemed the same i n a l l the v a r i o u s media and c o n d i t i o n s , except f o r one worm c u l t u r e d i n Medium RPMI 1640. G e n e r a l l y , the l a r v a e were i n a s t a t e o f c o n s t a n t r a p i d w r i g g l i n g , u n t i l about 2 - 3 days b e f o r e they d i e d , a t which time they moved v e r y s l o w l y . One day b e f o r e they d i e d , they would j u s t move t h e i r head or t a i l s l i g h t l y , w i t h the r e s t o f t h e i r body r e m a i n i n g r i g i d . The o n l y e x c e p t i o n was one worm c u l t u r e d i n RPMI 1640, w i t h a n t i b i o t i c s , under the c o n d i t i o n s of no g a s s i n g and no s h a k i n g . The l a r v a remained i n a r i g i d s t a t e , w i t h o n l y the t a i l moving, f o r 15 days b e f o r e i t f i n a l l y d i e d on the 36th day. A l l the worms appeared q u i t e g r a n u l a r , towards the end o f t h e i r l i f e i n c u l t u r e . No medium or s e t o f c o n d i t i o n s was s u p e r i o r t o any o t h e r , i n the k i n d of a c t i v i t y the l a r v a e e x h i b i t e d . 6. Measurement o f Dead Worms Tab l e 7 shows the average l e n g t h s o f the c u l t u r e d l a r v a e . The l a r v a l l e n g t h o f the dead c u l t u r e d worms measured from 0.6 mm. to 1.1 mm., w i t h the m a j o r i t y o f the worms measuring i n the 0.8 and 0.9 mm. range. A c c o r d i n g t o Ko (1966), these v a l u e s f a l l w i t h i n the normal range o f measurements f o r 3rd stage l a r v a e , t h e r e f o r e no growth o f the l a r v a e took p l a c e . - 47 -7. M o l a l i t i e s T a b l e 8 shows a l i s t o f the m o l a l i t i e s o f the media. F i g u r e 15 shows the c o r r e l a t i o n between the media su c c e s s i n s u p p o r t i n g l a r v a l s u r v i v a l and the m o l a l c o n c e n t r a t i o n o f the media. - 48 F i g u r e 15. C o r r e l a t i o n between s u c c e s s of media i n s u p p o r t i n g l a r v a l s u r v i v a l and between m o l a l i t i e s o f the media. 40 30 d a y s of s u r v i v a l of l a r v a e 20 10 © ® "7)50 .060 lOTO XSO 1090 .100, TnC .12.0 • . j l O " J < 0 • .150. .160 .170 J 8 0 . molal c o n c e n t r a t i o n of m e d i a - 49 -DISCUSSION I t was hoped i n t h i s work t h a t i n growing Philonema l a r v a e i n v i t r o under d i f f e r i n g n u t r i t i o n a l and e n v i r o n m e n t a l c o n d i t i o n s , t h a t one c o m b i n a t i o n would prove more e f f i c i e n t to the worms w i t h r e g a r d s to t h e i r s u r v i v a l and development. T h i s c e r t a i n l y was the case. In Medium 199 p l u s 101 c h i c k serum, w i t h a n t i b i o t i c s and w i t h g a s s i n g / n o s h a k i n g , the l a r v a e l i v e d an average of 46.3 days and a maximum of 86 days. F u r t h e r m o r e , t h i s medium a l s o s u p p o r t e d the b e s t average and maximum l a r v a l s u r v i v a l under b o t h a n t i b i o t i c c o m b i n a t i o n s and f o r a l l c o n d i t i o n s o f s h a k i n g and g a s s i n g . In the s e l e c t i n g of media t o be t e s t e d , t h e r e was l i t t l e t o go by. T h i s i s o f t e n j u s t a case of guesswork w i t h many wo r k e r s . Many t i s s u e c u l t u r e media or b a l a n c e d s a l t s o l u t i o n s w i t h a d d i t i v e s are t r i e d u n t i l one medium i s found to be s u c c e s s f u l f o r s u p p o r t i n g development o f the h e l m i n t h i n q u e s t i o n . E n t o m o l o g i s t s have seemed the most concerned about the d e t e r m i n a t i o n o f d e f i n e d media f o r the i n v i t r o c u l t u r e o f metazoan organisms. House, Gordon, Sang and o t h e r s have p r o v i d e d d a t a on the growth f a c t o r s r e q u i r e d by a r t h r o p o d s (Dougherty, 1959).. Dougherty developed a c h e m i c a l l y d e f i n e d medium f o r the nematode C a e n o r h a b d i t i s b r i g g s a e , based on the knowledge o f c o c k r o a c h n u t r i t i o n and the concept t h a t a l l metazoa have the same b a s i c n u t r i t i o n a l r e q u i r e m e n t s . D e f i n e d media h e l p us t o u n d e r s t a n d the e s s e n t i a l and n o n - e s s e n t i a l r e q u i r e m e n t s o f metazoan organisms. - 50 -Some o f the d e f i n e d media t e s t e d i n t h i s work have been used f o r the in_ v i t r o c u l t i v a t i o n o f o t h e r h e l m i n t h s . Medium 199 and NCTC 109 seem to have been used the most f r e q u e n t l y . These media have m a i n t a i n e d N e o a p l e c t a n a g l a s e r i ( J a c k s o n , 1961, 1962); A s c a r i s ( C l e e l a n d and Lawrence, 1965); A n g i o s t r o n g y l u s c a n t o n e n s i s ( W e i n s t e i n et. a l . 1962) , N l p p o s t r o n g y l u s muris ( W e i n s t e i n and J o n e s , 1959); and D i r o f i l a r i a i m m i t i s ( E a r l , 1959),(Sawyer and W e i n s t e i n 1961, 1963), ( T a y l o r , 1960). E a g l e ' s Minimum E s s e n t i a l Medium has been used by P i t t s (.1962, 1963) f o r c u l t u r i n g h a t c h e d A s c a r i s l a r v a e . W e i n s t e i n and Jones (1959) t e s t e d E a g l e ' s Medium f o r the c u l t i v a t i o n o f N i p p o s t r o n g y l u s m u r i s , but found t h a t i t d i d not support growth or development. B a l a n c e d s a l t s o l u t i o n s are o f t e n employed w i t h a d d i t i v e s i n c u l t u r i n g h e l m i n t h s . S i l v e r m a n et. a l . (1962) used E a r l e ' s b a l a n c e d s a l t s o l u t i o n and a d d i t i v e s f o r c u l t u r i n g t h i r d stage l a r v a e of D i c t y o c a u l u s , a lungworm of c a l v e s and sheep. T h i r d s t a g e l a r v a e o f Oesophagostomum r a d i a t u m d e v e l o p e d t o f o u r t h stage i n Hanks and E a r l e s S o l u t i o n s ( S i l v e r m a n , 1963). Other d e f i n e d media used i n t h i s work, have been used o n l y o c c a s i o n a l l y i n h e l m i n t h c u l t u r e . CMRL 1066 was used by Y o e l i et. a l . (1964) f o r c u l t u r i n g t h i r d s t a g e D i r o f i l a r i a i m m i t i s l a r v a e . Hansen et. a l . (1966) c u l t u r e d C a e n o r h a b d i t i s b r i g g s a e i n Waymouth's Medium but the Waymouths had t o be d i l u t e d t o one-seventh the o r i g i n a l s t r e n g t h and v i t a m i n s had t o be added. - 51 -The a u t h o r has seen no r e f e r e n c e t o the o t h e r d e f i n e d media which were used i n t h i s paper (GKN, RPMI 1640, RPMI 1603, S c h e r e r s and Ham's F-10), h a v i n g been used b e f o r e i n the i n  v i t r o c u l t u r e o f nematodes. Of the d e f i n e d media t e s t e d i t i s r e a s o n a b l e t h a t the t h r e e b a l a n c e d s a l t s o l u t i o n s s u p p o r t e d the l a r v a e f o r the l e a s t amount o f t i m e . The worms s u r v i v e d an average of 4-5 days i n these s o l u t i o n s . Most i n f e c t i v e l a r v a l s t a g e s possess s u f f i c i e n t n u t r i t i o n a l r e s e r v e s t o enable them t o l i v e f o r s h o r t p e r i o d s o f time i n i s o t o n i c s o l u t i o n s . When these r e s e r v e s run o u t , the worms w i l l d i e u n l e s s the medium s u p p l i e s a d d i t i o n a l m a t e r i a l s , such as amino a c i d s or v i t a m i n s . S c h e r e r ' s Maintenance Medium s u p p o r t e d worm s u r v i v a l l i t t l e beyond t h a t of the b a l a n c e d s a l t s o l u t i o n s . The l a r v a e had an average s u r v i v a l of 6.7 days i n S c h e r e r ' s Medium. T h i s medium d i d c o n t a i n the lo w e s t number of amino a c i d s . I t i s i n t e r e s t i n g t o note the d i f f e r e n c e i n success o f l a r v a e grown i n the same medium, but w i t h the s a l t s d i f f e r i n g . Larvae were c u l t u r e d i n Medium CMRL 1066 w i t h E a g l e s s a l t s and l i v e d an average of 14.5 days. T h i s medium s u p p o r t e d the l o n g e s t average s u r v i v a l of the worms of a l l the t h i r t e e n d e f i n e d media t e s t e d . However, i n Medium CMRL 1066 w i t h Hanks s a l t s , the l a r v a e o n l y l i v e d an average of 8.4 days. Perhaps, the d i f f e r e n c e i n success was due t o the osmotic p r e s s u r e o f the media. CMRL 1066 w i t h E a g l e s s a l t s had a lower osmotic p r e s s u r e than CMRL 1066 w i t h Hanks s a l t s . - 52 -Media RPMI 1640 and 1603 were v e r y s i m i l a r i n c o m p o s i t i o n but the l a r v a e l i v e d l o n g e r i n RPMI 1640 (11.5 d a y s ) , than i n RPMI 1603 (9.5 d a y s ) . RPMI 1603 had 10 i n o r g a n i c s a l t s as opposed t o 6 i n RPMI 1640. RPMI 1640 had 2 more amino a c i d s , but had fewer v i t a m i n s than RPMI 1603. A l s o , the amounts o f the components i n the media v a r i e d . I t i s v e r y d i f f i c u l t t o p i n p o i n t any one t h i n g i n these complex media t h a t c o u l d be c o n t r i b u t i n g t o the s u r v i v a l of the l a r v a e . Medium NCTC 109 c o n t a i n e d the h i g h e s t number o f amino a c i d s ( 2 4 ) , o f v i t a m i n s ( 1 8 ) , and o f m i s c e l l a n e o u s components (.24). However, the l a r v a e o n l y l i v e d an average of 8.0 days i n t h i s medium. A l l the a d d i t i v e s t e s t e d i n t h i s work have been used b e f o r e f o r c u l t u r i n g h e l m i n t h s . C h i c k embryo e x t r a c t has been used the most and has been c o n s i d e r e d the most i m p o r t a n t a d d i -t i v e . W e i n s t e i n and Jones (1956) f i r m l y e s t a b l i s h e d c h i c k embryo e x t r a c t as one o f the p r i m a r y components o f media, f o r p a r a s i t i c nematodes i n v i t r o . I t i s i n t e r e s t i n g t h a t c h i c k embryo e x t r a c t d i d not have a b e n e f i c i a l e f f e c t on the s u r v i v a l o f Philonema. However, i t was o n l y t e s t e d i n c o m b i n a t i o n w i t h Medium 199 and o n l y at a 101 c o n c e n t r a t i o n . Of the a d d i t i v e s t e s t e d , serum c e r t a i n l y was the most s u c c e s s f u l f o r c u l t u r i n g Philonema. G r a f f and McCarty (1958) s t a t e d t h a t metazoan c u l t u r e as opposed t o m i c r o b i o l o g i c a l c u l t u r e r e q u i r e s the i n c l u s i o n o f serum p r o t e i n s . Morgan (1958) s t a t e d t h a t i t i s g e n e r a l l y t h o u g h t , however, t h a t serum - 53 -c o n t r i b u t e s t o s u r v i v a l r a t h e r than the a c t i v e s t i m u l a t i o n of growth. C h i c k serum was most s u c c e s s f u l i n c o m b i n a t i o n w i t h Medium 199, which was not the b e s t d e f i n e d medium. In Medium 199 the l a r v a e l i v e d an average of 8.9 days. In Medium 199 + c h i c k serum, the l a r v a e l i v e d an average of 35.1 days. T h i s medium c o m b i n a t i o n s u p p o r t e d the b e s t average s u r v i v a l and maximum s u r v i v a l under b o t h a n t i b i o t i c c o n d i t i o n s and f o r a l l the c o n d i t i o n s o f s h a k i n g and g a s s i n g . When the a d d i t i v e s were t e s t e d w i t h E a r l e ' s b a l a n c e d s a l t s o l u t i o n , a g a i n c h i c k serum was the most s u c c e s s f u l . The l a r v a e l i v e d an average of 5 days i n E a r l e s and an average of 17.1 days i n E a r l e s + c h i c k serum. Other workers have had the use o f an a d d i t i v e d r a m a t i c a l l y i n c r e a s e the s u c c e s s of t h e i r work. Hatched A s c a r i s l a r v a e l i v e d i n Medium 199 f o r 9-11 days. In Medium 199 + 20% b o v i n e serum, the l a r v a e l i v e d 27 days ( C l e e l a n d and Lawrence, 1962). E a r l (1959) r e p o r t e d t h a t D i r o f i l a r i a i m m i t i s l a r v a e l i v e d 4 days i n Medium 199. They l i v e d 43 days i n Medium 199 + 10% dog serum and l i v e d 61 days i n Medium 199 + 30% dog serum. Only one c o n c e n t r a t i o n o f c h i c k serum (10%) was used i n c o m b i n a t i o n w i t h Medium 199. A f u r t h e r t e s t s h o u l d be done to see w h i c h c o n c e n t r a t i o n i s b e s t . Perhaps 20% or 30% c h i c k serum w i l l even s u p p o r t the l a r v a e l o n g e r than the 10% c o n c e n t r a t i o n . Beyond a c e r t a i n p e r c e n t a g e , the s u r v i v a l u s u a l l y s t a r t s to decrease a g a i n . - 54 -I t has o f t e n been shown t h a t h e t e r o l o g o u s e x t r a c t s are o f t e n more b e n e f i c i a l i n i n v i t r o c u l t u r e than homologous e x t r a c t s . W e i n s t e i n and Jones (1959) c u l t u r e d N i p p o s t r o n g y l u s  m u r i s , which i s a nematode p a r a s i t e o f r a t s . They used a medium composed of c h i c k embryo e x t r a c t and serum. However, they found t h a t human serum gave much b e t t e r r e s u l t s than homologous r a t serum. Perhaps i n homologous serum, a n t i b o d i e s have a l r e a d y been formed a g a i n s t the p a r a s i t e . Sawyer (1963, 1965) c u l t u r e d the t h i r d stage l a r v a e o f D i r o f i l a r i a i m m i t i s , the dog heartworm. In NCTC 109 + 10% human or horse serum, the l a r v a e s u r v i v e d 12-15 days and had e xsheathed by 72 h o u r s . Dog serum was found to be i n h i b i t o r y and s u p p o r t e d l a r v a l s u r v i v a l f o r o n l y 6-9 days. E x t r a c t s from h e t e r o l o g o u s h o s t s were more b e n e f i c i a l to the Philonema l a r v a e than the two t r o u t e x t r a c t s t e s t e d . T r o u t egg e x t r a c t e x h i b i t e d a t o x i c e f f e c t on the l a r v a e , f o r they l i v e d l e s s i n i t (3.6 days) than they d i d i n the b a l a n c e d s a l t s o l u t i o n s . In Medium 199 + the t r o u t l i v e r e x t r a c t , the l a r v a e l i v e d o n l y 9.6 days. However f i s h serum would have to be t e s t e d b e f o r e drawing any f i n a l c o n c l u s i o n . The o n l y medium i n which the worms moulted c o n t a i n e d a serum (Medium 199 + 10% horse serum + 10% d e x t r o s e ) . F o u r t e e n l a r v a e moulted i n t h i s medium c o m b i n a t i o n and even the non-m o u l t i n g worms were v e r y f r a g i l e and d i f f i c u l t t o h a n d l e . No m oults o c c u r r e d i n any o t h e r media. The l a r v a e l i v e d f o r as l o n g as 15 days a f t e r m o u l t i n g . A l l the l a r v a e t h a t moulted c o m p l e t e l y exsheathed. Many l a r v a e moult i n u n f a v o r a b l e - 55 -c o n d i t i o n s , but produce m u l t i p l e sheaths and f a i l t o shed any ( B e r n t z e n , 1965). However, when the worms were a g a i n t r i e d i n t h i s medium c o m b i n a t i o n , no m o u l t i n g o c c u r r e d . Perhaps t h e r e i s a time p e r i o d , d u r i n g which the i n f e c t i v e l a r v a e must come i n c o n t a c t w i t h the c o r r e c t c o n d i t i o n s , i n o r d e r f o r f u r t h e r development to o c c u r . I t has o f t e n been found t h a t t i s s u e c u l t u r e media are t o x i c t o h e l m i n t h s and are much l e s s s u c c e s s f u l at s u p p o r t i n g p a r a s i t e s i n v i t r o , than a s i m p l e b a l a n c e d s a l t s o l u t i o n , such as E a r l e s or Hanks ( S i l v e r m a n , 1963). For t h i s r e a s o n E a r l e ' s s o l u t i o n , w i t h a few a d d i t i v e s , was t e s t e d . In each i n s t a n c e , the a d d i t i v e s s u p p o r t e d l o n g e r s u r v i v a l o f Philonema when added to Medium 199, than when added to E a r l e ' s b a l a n c e d s a l t s o l u t i o n . G a s s i n g w i t h 5% C O 2 i n a i r was a l s o one of the components i n the c o m b i n a t i o n t h a t s u p p o r t e d the l o n g e s t s u r v i v a l of the Philonema l a r v a e . F u r t h e r m o r e , the a n a l y s i s o f v a r i a n c e showed t h a t g a s s i n g i n c r e a s e d the o v e r a l l average s u r v i v a l of the l a r v a e , to a s m a l l degree. S i n c e the t h i r d stage Philonema l a r v a e m i g r a t e t o the swimbladder a f t e r e n t e r i n g the f i s h , i t was thought t h a t the gas m i x t u r e used s h o u l d be s i m i l a r t o the gases i n the swim-b l a d d e r . However, i n most physostomous f i s h , the swimbladder gases are the same as a i r d i s s o l v e d i n water (Fange, 1966). As the gases pass through the membranes o f the swimbladder at d i f f e r e n t r a t e s , p e r c e n t a g e c o m p o s i t i o n may be h i g h l y v a r i a b l e . - 56 -O f t e n e x t r e m e l y h i g h oxygen t e n s i o n s r e s u l t , e s p e c i a l l y i n r a p i d l y s e c r e t i n g p h y s o c l i s t s , w i t h w e l l - d e v e l o p e d gas g l a n d s . However, i n t r o u t and most s a l m o n i d s , the swimbladder i s a s i m p l e sac w i t h p o o r l y developed s e c r e t o r y s t r u c t u r e s and so gas s e c r e t i o n i s v e r y slow and the oxygen c o n c e n t r a t i o n seldom reaches h i g h v a l u e s . Carbon d i o x i d e has been found t o be a v e r y i m p o r t a n t component o f the gaseous phase, f o r the i n v i t r o c u l t i v a t i o n o f h e l m i n t h s ( S i l v e r m a n 1963, 1965). Indeed, the most f r e q u e n t c h o i c e o f gas m i x t u r e s f o r nematode c u l t u r e has been the same as i n t i s s u e c u l t u r e , 5% carbon d i o x i d e i n a i r . Sawyer and W e i n s t e i n (1963) used t h i s m i x t u r e f o r c u l t u r i n g D i r o f i l a r i a  i m m i t i s , i n v i t r o . T h i s m i x t u r e was a l s o used f o r the i n v i t r o c u l t i v a t i o n of N i p p o s t r o n g y l u s m u r i s ( W e i n s t e i n and J o n e s , 1959). Many o t h e r workers have chosen 51 carbon d i o x i d e i n a i r ( L e l a n d , 1963), ( C l e e l a n d and Lawrence, 1962), (Sawyer, 1963, 1965). I t i s i n d i c a t e d t h a t carbon d i o x i d e i s not j u s t an i n e r t end p r o d u c t i n c u l t u r e ( H a r r i s , 1954). Werkman (1951) p o s t u l a t e d t h a t carbon d i o x i d e i s a g e n e r a l r e q u i r e m e n t of a l l l i v i n g c e l l s . He s a i d t h a t i n a l l forms of l i f e , c arbon d i o x i d e i s an e s s e n t i a l p h y s i o l o g i c a l f u n c t i o n , p r o v i d i n g f o r the s y n t h e s i s o f i n d i s p e n s a b l e m e t a b o l i c i n t e r m e d i a t e s . G r a f f and McCarty (1958), i n p o i n t i n g out the enigma o f carbon d i o x i d e , s t a t e d t h a t i t i s d i f f i c u l t t o t e l l i f the gas i s i n t r o d u c e d i n the c u l t u r e system as a reagent or o n l y as a b u f f e r . However, i t has been shown t h a t carbon d i o x i d e i s i n c o r p o r a t e d i n t o a n i m a l c e l l s . - 57 -Because 5% C O 2 i n a i r has been found to be s u c c e s s f u l f o r the c u l t i v a t i o n o f many h e l m i n t h s and because i t i s n e c e s s a r y f o r c e l l s i n t i s s u e c u l t u r e , t h i s gas m i x t u r e was chosen f o r t h i s work. A n t i b i o t i c s were the f i n a l component p r e s e n t i n the b e s t c o m b i n a t i o n o f f a c t o r s f o r growing Philonema l a r v a e , i n v i t r o . A l s o , the a n a l y s i s showed t h a t the a n t i b i o t i c s s u p p o r t e d an o v e r a l l i n c r e a s e i n s u r v i v a l of the l a r v a e . There i s no need to emphasize the importance of s t e r i l i t y i n i n v i t r o c u l t u r e . For the s u c c e s s f u l c u l t i v a t i o n o f worms, the medium must be f r e e from c o n t a m i n a t i o n . Because of the f a v o u r a b l e n u t r i e n t s i n c u l t u r e , i f c o n d i t i o n s are not s t e r i l e , m i c r o o r g a n i s m s can r a p i d l y m u l t i p l y and a l t e r the environment t o the e x t e n t t h a t the worms might d i e . I t i s e v i d e n t t h a t the aim o f i n v i t r o c u l t i v a t i o n must be to a c q u i r e a t r u l y a x e n i c c u l t u r e . T h i s has been d e f i n e d as the maintenance of a s t e r i l e o rganism, f r e e from the presence of any o t h e r o r g anism, (Dougherty, 1959). Only i n an a x e n i c c u l t u r e can c o n d i t i o n s be t r u l y s t a n d a r d i z e d . However, a t r u l y a x e n i c c u l t u r e i s d i f f i c u l t t o a c h i e v e and a n t i b i o t i c s are u s u a l l y employed to h o l d any m i c r o o r g a n i s m s p r e s e n t i n a b a c t e r i o s t a t i c c o n d i t i o n . A v e r y low c o n c e n t r a t i o n o f a n t i b i o t i c s was chosen f o r t h i s work (50 u n i t s p e n i c i l l i n / m l . and 50 u n i t s s t r e p t o -mycin/ml.) and were t e s t e d a g a i n s t the use of no a n t i b i o t i c s . Townsley et. a l . (1963) used t h i s c o n c e n t r a t i o n o f a n t i b i o t i c s f o r the i n v i t r o m a t u r a t i o n of T e r r a n o v a d e c i p i e n s , a p a r a s i t e i n cod. - 58 -T h i s low c o n c e n t r a t i o n was chosen because i t was thought t h a t such a s m a l l amount of a n t i b i o t i c s would not harm "the l a r v a e . However, i t i s now thought t h a t i f a much l a r g e r amount had been chosen, the t e s t would have been more m e a n i n g f u l . The a n t i b i o t i c s as used p r o v e d s l i g h t l y more b e n e f i c i a l t o the worms than not u s i n g any at a l l . However, most workers have used c o n c e n t r a t i o n s i n the range o f 200 to 400 u n i t s / m l . L e l a n d (1963) used 400 u n i t s p e n i c i l l i n / m l . and 100 u n i t s m y c o s t a t i n / m l . i n h i s c u l t u r e s of nematodes p a r a s i t i c i n sheep and c a t t l e . Sawyer and W e i n s t e i n (1963) used 200 u n i t s p e n i c i l l i n / m l . and 100 ug. o f s t r e p t o m y c i n / m l . i n t h e i r c u l t u r e s o f D i r o f i l a r i a i m m i t i s l a r v a e . S o m m e r v i l l e (1966) used as much as 500 u n i t s p e n i c i l l i n / m l . and 500 mg./ml. s t r e p t o m y c i n i n h i s medium f o r the c u l t u r e o f Haemonchus c o n t o r t u s l a r v a e . Because the s m a l l amount of a n t i b i o t i c s s l i g h t l y improved the s u r v i v a l o f Philonema l a r v a e i n c u l t u r e , the a u t h o r wonders i f g r e a t e r c o n c e n t r a t i o n s would have improved s u r v i v a l t o the same e x t e n t or would l a r g e r amounts of a n t i b i o t i c s have been d e l e t e r i o u s to the l a r v a e . G e n e r a l l y , i t was f e l t t h a t a good degree of s t e r i l i t y was m a i n t a i n e d i n t h i s work. In the i n i t i a l s t e p s of c u l t u r i n g the Philonema l a r v a e , the a u t h o r had many co n t a m i n a t e d c u l t u r e s . However, w i t h p r a c t i c e at s t e r i l e t e c h n i q u e and once the l a r v a e were washed b e f o r e b e i n g i n o c u l a t e d i n t o c u l t u r e , c o n t a m i n a t i o n was g r e a t l y reduced. Of the seven hundred, twenty c u l t u r e s i n - 59 -t h i s e x p e r i m e n t , o n l y seven became contam i n a t e d and had to be r e p e a t e d . Some workers have s t e r i l i z e d the s u r f a c e of h e l m i n t h s w i t h c h e m i c a l s t e r i l i z i n g a g e n t s . N i c h o l a s and McEntegart (1957) s u r f a c e s t e r i l i z e d r h a b d i t i d nematodes w i t h m e t h i o l a t e and hydrogen p e r o x i d e . T h i s can be dangerous because such agents prove h a r m f u l to the worms. As S i l v e r m a n (1963) s t a t e d , one o f the most e f f e c t i v e methods to s t e r i l i z e nematodes i s by r e p e a t e d washing i n s t e r i l e s a l i n e . T h i s was c e r t a i n l y adequate f o r the Philonema l a r v a e . The r e s u l t s from the c u l t u r e s t h a t were shaken were unexpected. The a c c u m u l a t i o n of m e t a b o l i t e s i n c u l t u r e has a d e t r i m e n t a l e f f e c t on the development o f h e l m i n t h s . A g i t a t i o n of c u l t u r e v e s s e l s by a s h a k i n g machine ensures the d i s p e r s i o n and d i l u t i o n o f such t o x i c p r o d u c t s . The problem i s even g r e a t e r f o r s l u g g i s h worms such as tapeworms, which o f t e n r e q u i r e s p e c i a l t ypes of a p p a r a t u s , which p r o v i d e a c o n t i n u o u s f l o w of media and a r a p i d removal o f the waste s u b s t a n c e s . I t i s t h e r e f o r e i n t e r e s t i n g t h a t s h a k i n g d r a s t i c a l l y r educed the s u r v i v a l o f Philonema i n c u l t u r e . I t i s perhaps r e a s o n a b l e t h a t s h a k i n g was not n e c e s s a r y f o r Philonema because the t h i r d s t age l a r v a e are e x t r e m e l y a c t i v e . T h e i r own c o n s t a n t w r i g g l i n g m o t i o n p r o b a b l y d i s p e r s e s any b y - p r o d u c t s t h a t have formed. A l s o , o n l y f i v e l a r v a e , each measuring a p p r o x i m a t e l y 900 m i c r o n s i n l e n g t h , were c u l t u r e d i n f o u r ml. of medium. S i n c e t h e r e was no crowding and such a l a r g e r a t i o - 60 -of medium t o worms, problems caused by the a c c u m u l a t i o n of waste p r o d u c t s p r o b a b l y d i d not r e s u l t . Perhaps t h e r e was a c o n s t a n t s t r e s s produced by the s h a k i n g machine t h a t d e c r e a s e d the s u r v i v a l o f Philonema. S t o l l (1959) r e p o r t e d t h a t f a s t speeds (300 s t r o k e s / m i n u t e ) produced s m a l l e r p o p u l a t i o n o f worms and some had an abnormal morphology. A l l the c u l t u r e s were m a i n t a i n e d a t a c o n s t a n t temperature (10°C). W i t h some p a r a s i t e s , temperature o f t e n a c t s as a t r i g g e r i n g mechanism. The g e n i t a l i a i n p l e r o c e r c o i d s of S c h i s t o c e p h a l u s s o l i d u s become i n d u c e d to mature by a r i s e i n the temperature to 40°C. when they are eaten by a b i r d (Smyth, 1959). However, i n t h i s work temperature was not c o n s i d e r e d to be a major f a c t o r i n t r i g g e r i n g development. The worms are always exposed to a f a i r l y wide range of temperatures w h i l e i n t h e i r p o i k i l o t h e r m i c h o s t s . A good mean temperature of water (10°C.) was t h e r e f o r e chosen f o r the l a r v a e to be c u l t u r e d i n . Some worms show an a b i l i t y to develop i n a wide pH range w h i l e o t h e r s are v e r y l i m i t e d . The Philonema l a r v a e were c u l t u r e d at a pH of 7.0 to 7.5 because t h a t i s the approximate pH o f the f i s h t i s s u e s . A l s o , Ko (unpubl. data) found t h a t the l a r v a e s u r v i v e d b e s t i n a s l i g h t l y a l k a l i n e pH. Ko (1966), found t h a t t h i r d s t a g e Philonema l a r v a e i n the copepod v a r i e d from 0.79 mm i n l e n g t h t o 1.04 mm i n l e n g t h . E a r l y t h i r d s t age l a r v a e measured from 0.79 mm t o 0.96 mm, w i t h an average l e n g t h o f 0.88 mm. O l d e r t h i r d s t age l a r v a e - 61 -ranged from 0.92 mm to 1.04 mm, w i t h an average l e n g t h o f 0.99 mm. These l e n g t h ranges came from measuring twenty e a r l y t h i r d s t a g e l a r v a e and twenty o l d e r t h i r d s t age l a r v a e . In t h i s work, the l a r v a l l e n g t h o f the dead c u l t u r e d worms ranged from 0.61 mm to 1.13 mm, w i t h the m a j o r i t y o f the worms measuring i n the 0.80 mm and 0.90 mm. F i f t y - s i x o f the l a r v a e were s h o r t e r than Ko's minimum measurements f o r t h i r d s t a ge l a r v a e and two were g r e a t e r than the maximum l e n g t h f o r t h i r d s t age l a r v a e . However, f i f t y - f o u r o f the f i f t y - s i x were i n the 0.70 mm range. S i n c e a p p r o x i m a t e l y 440 worms were measured i n t h i s p a p e r , i t i s to be ex p e c t e d t h a t some w i l l f a l l o u t s i d e the l i m i t s found by Ko. T h e r e f o r e , no s u b s t a n t i a l i n c r e a s e o r de c r e a s e i n the l e n g t h of the worms took p l a c e due to b e i n g c u l t u r e d . No growth or development took p l a c e i f an i n c r e a s e i n l e n g t h was used as the c r i t e r i o n . I n a t r u l y s u c c e s s f u l c u l t u r e , the worms grow, d i f f e r e n t i -a t e , become s e x u a l l y mature and l a y f e r t i l e eggs. A p a r t from the few l a r v a e t h a t m o u l t e d , none e x h i b i t e d development. With t h i s c r i t e r i o n perhaps none of the c u l t u r e s c o u l d be con-s i d e r e d s u c c e s s f u l . However, i n s t a r t i n g i n v i t r o s t u d i e s on an a n i m a l , s u r v i v a l i s the f i r s t i n d i c a t i o n o f any su c c e s s at a l l . The a u t h o r f e e l s i t i s f o r t u n a t e t o f i n d a medium f o r Philonema, i n which the l a r v a e do s u r v i v e f o r a l o n g time (Medium 199 + 101 c h i c k serum). They s u r v i v e d up to 86 days i n t h i s medium. I f a worm l i v e s such a l o n g t i m e , i n c u l t u r e , t h e r e i s s u f f i c i e n t time to t r y t h i n g s on i t , t h a t might induce development. - 62 -D i f f e r e n t p r e c u l t u r e t r e a t m e n t s c o u l d be t r i e d on the l a r v a e . T h i s c o u l d i n c l u d e such t h i n g s as immersing the l a r v a e i n a c i d s o l u t i o n s b e f o r e p u t t i n g them i n c u l t u r e . The i d e a l s i t u a t i o n i s to t r y t o s i m u l a t e a s t i m u l u s , t h a t the worms might r e c e i v e in_ v i v o . Philonema l a r v a e might r e c e i v e a s t i m u l u s when they m i g r a t e t h r o u g h the p y l o r i c cecae r e g i o n , i n the f i s h . Such a s t i m u l u s might s e t o f f the " b i o l o g i c a l c l o c k " and l a r v a l development would s t a r t a g a i n (Rogers, 1962). Rogers t h e o r i z e d t h a t the i n f e c t i v e s tage o f p a r a s i t i c a n i m a l s i s a r e s t i n g stage i n which development c e a s e s . I n f e c t i o n t a k e s p l a c e when a h o s t p r o v i d e s the s t i m u l u s needed t o r e s t a r t the development o f the p a r a s i t e . T h i s s t i m u l u s c o u l d a c t on the eggs or the l a r v a e of the p a r a s i t e , c a u s i n g the eggs t o h a t c h or the l a r v a e t o moult. A s t i m u l u s from the h o s t appears t o cause A s c a r i s eggs to s e c r e t e a " h a t c h i n g f l u i d " c a u s i n g them to h a t c h . The exsheathment o f T r i c h o s t r o n g y l u s c o l u b r i f o r m i s l a r v a e r e q u i r e s a h i g h c o n c e n t r a t i o n of u n d i s s o c i a t e d c a r b o n i c a c i d p l u s d i s s o l v e d gaseous carbon d i o x i d e and a pH of 1.5 to 2.5. These c o n d i t i o n s are found i n the stomachs of most v e r t e b r a t e s and i n d e e d , the p a r a s i t e does occur i n many mammals (Rogers (1962). The heads of the bladderworm C y s t i c e r c u s p i s i f o r m i s are k i l l e d by sodium g l y c o c h o l a t e . Hosts whose b i l e c o n t a i n s t h i s s a l t do not get i n f e c t e d . Dogs' b i l e does not c o n t a i n t h i s s a l t so they get i n f e c t e d (Lapage, 1958). Rogers' t h e o r y has been g i v e n s u pport by the i n v i t r o c u l t i v a t i o n of many p a r a s i t e s . L e l a n d (1963) c u l t u r e d C o o p e r i a 63 -p u n c t a t a and found the n e c e s s i t y of two d i f f e r e n t media t o complete the l i f e - c y c l e of the worm. The f i r s t and second s t a g e l a r v a e d e v e l o p e d at 24°C. i n a medium composed o f c h i c k embryo e x t r a c t , v i t a m i n s , sodium c a s e i n a t e , hog l i v e r e x t r a c t and a b a l a n c e d s a l t s o l u t i o n . For the t h i r d stage l a r v a e t o develop to a d u l t s , they had to be c u l t u r e d a t 38.5°C. and c a l f serum had to be added to the medium. W e i n s t e i n and Jones (1956) o b t a i n e d development o f the n o n - p a r a s i t i c s t a g e s of N i p p o s t r o n g y l u s m u r i s i n c h i c k embryo e x t r a c t , but no growth beyond t h i r d stage o c c u r r e d . S e x u a l l y mature a d u l t worms developed i n a medium c o n t a i n i n g c a s e i n a t e , l i v e r f i l t r a t e , c h i c k embryo e x t r a c t and r a t serum. Now t h a t a medium has been found, i n which the Philonema l a r v a e can l i v e a f a i r l y l o n g t i m e , hormones s h o u l d c e r t a i n l y be t e s t e d i n the medium. T h i s c o u l d perhaps be one o f the keys t h a t would s t a r t development o f the l a r v a e . A n other i m p o r t a n t s t e p t o be t r i e d i n the c u l t u r e o f Philonema i s t o grow the worms i n media o f v a r i o u s v i s c o s i t i e s . The v i s c o s i t y and p h y s i c a l n a t u r e o f the medium have been v e r y i m p o r t a n t f o r c u l t u r i n g some h e l m i n t h s . Such t h i n g s as f i l t e r p a p e r , egg y o l k s and agar have been used to p r o v i d e a s u i t a b l e m a t r i x f o r c u l t u r i n g h e l m i n t h s . I_n v i v o , Philonema i s found among the t i s s u e s o f the swimbladder. I f the l a r v a e are p u l l e d out o f these t i s s u e s , they tend to c r a w l back i n . T h e r e f o r e , perhaps a more v i s c o u s medium would be b e t t e r f o r the l a r v a e , as they seem t o p r e f e r c o n t a c t w i t h a s u b s t i a t e . - 64 -Perhaps b e f o r e s t a r t i n g t o c u l t u r e h e l m i n t h s , one s h o u l d v e r y c a r e f u l l y s t u d y the h o s t . Study a l l the areas i n the h o s t t h a t the worm v i s i t s and then c u l t u r e the worm i n c o n d i t i o n s s i m i l a r t o the n a t u r a l environment. N a t u r a l l y , t h i s i s the i d e a i n most workers minds, when they are c u l t u r i n g . However, u s u a l l y l i t t l e i s known about the h o s t , so c u l t u r i n g remains guesswork, t o a l a r g e degree. The d i f f i c u l t i e s o f i n v i t r o c u l t u r e demonstrate what e x a c t and c r i t i c a l c o n d i t i o n s are n e c e s s a r y f o r the development o f a s e x u a l l y mature and normal a d u l t worm. In v i t r o c u l t u r e seems v e r y crude i n comparison w i t h the a s s o c i a t i o n o f a p a r a s i t e and i t s h o s t . One becomes aware of the c o n s i d e r a b l e p h y s i o l o g i c a l and m o r p h o l o g i c a l a d a p t a t i o n s of the p a r a s i t e to be a b l e t o l i v e and m u l t i p l y i n i t , u s u a l l y w i t h o u t harming i t . One becomes aware o f the f i n e l y complex p h y s i o l o g i c a l i n t e r -a c t i o n s , which r e s u l t i n h o s t and h a b i t a t s e l e c t i o n f o r the p a r a s i t e . W i t h f u r t h e r work on i n v i t r o c u l t i v a t i o n o f p a r a s i t e s , the a s s o c i a t i o n between a p a r a s i t e and i t s h o s t w i l l be b e t t e r u n d e r s t o o d . - 65 -SUMMARY 1. The most s u c c e s s f u l s e t o f c o n d i t i o n s f o r s u p p o r t i n g the l o n g e s t s u r v i v a l o f Philonema was Medium 199 + 10% c h i c k serum, w i t h a n t i b i o t i c s , under the c o n d i t i o n o f g a s s i n g and no s h a k i n g . In t h i s s i t u a t i o n , the l a r v a e l i v e d an average o f 46.3 days and a maximum of 86 days. 2. A n t i b i o t i c s were found to s i g n i f i c a n t l y i n c r e a s e the s u r v i v a l o f Philonema i n c u l t u r e . 3. G a s s i n g , w i t h 5% carbon d i o x i d e i n a i r , i n c r e a s e d the s u r v i v a l o f Philonema. 4. Shaking was v e r y d e l e t e r i o u s to the c u l t u r e s and d e c r e a s e d the s u r v i v a l o f the l a r v a e . 5. The d e f i n e d medium t h a t s u p p o r t e d the l o n g e s t s u r v i v a l o f the l a r v a e was Medium CMRL 1066 w i t h E a g l e ' s s a l t s . 6. The most s u c c e s s f u l a d d i t i v e f o r l a r v a l s u r v i v a l was c h i c k serum. 7. The medium c o m b i n a t i o n f o r s u p p o r t i n g the l o n g e s t s u r v i v a l o f Philonema was Medium 199 + 10% c h i c k serum. 8. F o u r t e e n l a r v a e moulted and c o m p l e t e l y exsheathed i n Medium 199 + 10% h orse serum + 10% d e x t r o s e . - 66 -BIBLIOGRAPHY Adams, J.R. 1969. M i g r a t i o n r o u t e of i n v a s i v e j u v e n i l e Philonema o n c o r h y n c h i (Nematoda:Philometridae) i n young salmon. J . F i s h . Res. Bd. Canada 26: 941-946. B a s h i r u l l a h , A.K.M. 1966. The development and m a t u r a t i o n o f Philonema s p e c i e s (Nematoda:Philometridae) i n s a l m o n i d h o s t s w i t h d i f f e r e n t l i f e h i s t o r i e s . Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h C olumbia, Vancouver, B.C. B e r n t z e n , A.K. 1962. 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Energy c o s t s of growth i n c o n t i n u o u s metazoan c u l t u r e s . Cancer Res. 18: 741-746. Hansen, E.L.; S i l v e r m a n , P.H. and Beucher, E . J . 1966. Development o f Haemonchus c o n t o r t u s i n media d e s i g n e d f o r s t u d i e s on C a e n o r h a b d i t i s b r i g g s a e 5 2 / 137-140. H a r r i s , M. 1954. The r o l e o f b i c a r b o n a t e f o r outgrowth o f c h i c k h e a r t f i b r o b l a s t s , i n v i t r o . J . Exp. Z o o l . 125: 85-98. Hobson, A.D. 1948. The p h y s i o l o g y and c u l t i v a t i o n i n a r t i f i c i a l media o f nematodes p a r a s i t i c i n the a l i m e n t a r y t r a c t o f a n i m a l s . P a r a s i t o l 3_8: 183-227. J a c k s o n , G.J. 1961. The p a r a s i t i c nematode N e o a p l e c t a n a g l a s e r i i n a x e n i c c u l t u r e . I . The e f f e c t s o f a n t i b i o t i c s and a n t h e l m i n t h i c s . Exp. P a r a s i t o l 11: 241-247. 1962. 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Sawyer, T.K. and W e i n s t e i n , P.P. 1961. S u r v i v a l o f D i r o f i l a r i a  i m m i t i s m i c r o f i l a r i a e i n m o d i f i e d p h y s i o l o g i c a l s a l i n e s o l u t i o n s . J . P a r a s i t o l 4_7 (No. 5, S u p p l . ) : 24. - 69 -Sawyer, T.K. and W e i n s t e i n , P.P. 1962. Development i n v i t r o t o the sausage stage o f m i c r o f i l a r i a e o f the dog heartworm D i r o f i l a r i a i m m i t i s . J . P a r a s i t o l 48: 35-36 . 1963. S t u d i e s on the m i c r o f i l a r i a e of the dog heartworm D i r o f i l a r i a e i m m i t i s : s e p a r a t i o n of p a r a s i t e s from whole b l o o d . J . P a r a s i t o l 49: 39-45 . ' 1963. The in. v i t r o development o f m i c r o f i l a r i a e o f the dog heartworm D i r o f i l a r i a i m m i t i s t o the "sausage-form". J . P a r a s i t o l 4_9: 218-224. S i l v e r m a n , P.H. 1962. Improvements i n or r e l a t i n g t o v a c c i n e s . Br. 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Werkman, C H . 1951. A s s i m i l a t i o n o f carbon d i o x i d e by h e t e r o -t r o p h i c b a c t e r i a . Ch. 12 i n B a c t e r i a l P h y s i o l o g y (ed. C H . Werkman and P.W. W i l s o n ) . Academic P r e s s , I n c . , New York. Y o e l i , M.; Upmanis, R.S.; Most, H. 1964. S t u d i e s on f i l a r i a s i s . I I I . P a r t i a l growth o f the mammalian stag e s o f D i r o f i l a r i a i m m i t i s i n v i t r o . Exp. P a r a s i t o l 15: 325-334. TABLE I I LIST OF ALL COMPONENTS AND AMOUNTS OF COMPONENTS IN DEFINED MEDIA GKN SOLUTION Chemical Compound NaCl KCl G l ucose 1 L i t e r o f GKN Co n t a i n s 8.00 gm 0.40 gm 1.00 gm EARLE'S SOLUTION Chemical Compound NaCl KCl MgS04-7H20 NaH 2P04-H20 CaCl2(anhyd.) NaHC0 3 Glucose Phenol Red 1 L i t e r of EBSS C o n t a i n s 6.80 gm 0.40 gm 0.20 gm 0.14 gm 0.20 gm 2.20 gm 1.00 gm 0.01 gm HANKS' SOLUTION Chemical Compound NaCl KCl MgS04-7H20 KH2PO4 Na2HP04-7H 20 CaCl2 (anhyd.) NaHC0 3 Glucose Phenol Red 1 L i t e r o f HBSS C o n t a i n s 8.00 gm 0.40 gm 0.20 gm 0.06 gm 0.09 gm 0.14 gm 0.35 gm 1.00 gm 0.02 gm TABLE I I (Cont'd) SCHERER'S MAINTENANCE MEDIUM 1 L i t e r o f 1 L i t e r o f Chemical S c h e r e r ' s Chemical S c h e r e r ' s Compound C o n t a i n s Compound C o n t a i n s Adenine S u l f a t e 6. . 80 mg NaCl 8, . 00 gm p-Amino B e n z o i c A c i d 0. . 01 mg KCl 0. .40 gm Casamino A c i d s 45. . 00 mg MgS04-7H20 0, . 20 gm L - C y s t i n e 15. . 00 mg Na2HP04 •7H 20 0. ,08 gm C y t o s i n e 0. .40 mg K H 2 P 0 4 0, .06 gm G l y c e r o l 500, .00 mg C a C l 2 0. , 14 gm G l y c i n e 20. . 00 mg NaHC0 3 1. , 00 gm D L - H i s t i d i n e ( f r e e base) 20, . 00 mg Glucose 2. , 00 gm L - M a l i c A c i d 5. , 00 mg Phenol Red 0. , 02 gm Guanine HC1 1. .60 mg Sodium A c e t a t e 500. .00 mg Sodium P y r u v a t e 500. , 00 mg S u c c i n i c A c i d 10. . 00 mg Thymine 0. .4 mg DL-Tryptophan 20 , .00 mg U r a c i l 1. . 00 mg X a n t h i n e 1, . 00 mg B i o t i n 0. . 01 mg Ca l c i u m Pantothenate 0. .40 mg C h o l i n e C h l o r i d e 1. .40 mg F o l i c A c i d 0, .01 mg I n o s i t o l 1, .40 mg N i c o t i n a m i d e 0, .40 mg P y r i d o x a l HC1 0, , 40 mg Pyr i d o x a m i n e HC1 0, .40 mg R i b o f l a v i n 0, . 50 mg D-Ribose 0, .40 mg Thiamine HC1 1, .00 mg TABLE I I (Cont'd) NCTC-135 1 L i t e r o f Chemical NCTC-135 Compound C o n t a i n s L - A l a n i n e 31. 48 mg L - A m i n o b u t y r i c A c i d 5. 51 mg L - A r g i n i n e HC1 25. 76 mg L-Asparagine 8. 09 mg L - A s p a r t i c A c i d 9. 91 mg L - C y s t i n e 10. 49 mg D-Glucosamine 3. 20 mg L-G l u t a m i c A c i d 8. 26 mg L-Glutamine 135. 73 mg G l y c i n e 13. 51 mg L - H i s t i d i n e 19. 73 mg H y d r o x y - L - p r o l i n e 4. 09 mg L - I s o l e u c i n e 18. 04 mg L-Leucine 20. 44 mg L - L y c i n e 30. 75 mg L-M e t h i o n i n e 4. 44 mg L - O r n i t h i n e 7 . 38 mg L - P h e n y l a l a n i n e 16. 53 mg L - P r o l i n e 6. 13 mg L - S e r i n e 10. 75 mg L-Ta u r i n e 4. 18 mg L-Threonine 18. 93 mg L-Tryptophan 17. 50 mg L - T y r o s i n e 16. 44 mg L - V a l i n e 25. 00 mg p-Aminobenzoic 0. 125 mg A s c o r b i c A c i d 49. 90 mg B i o t i n 0. 025 mg C a l c i f e r o l 0. 25 mg D-Calcium P a n t o t h e n a t e 0. 025 mg C h o l i n e C h l o r i d e 1. 25 mg F o l i c A c i d 0. 025 mg 1 L i t e r o f Chemical NCTC-135 Compound C o n t a i n s G l u c u r o n o l a c t o n e 1. 80 mg G l u t a t h i o n e 10. 10 mg i - I n o s i t o l 0. 125 mg Menadione 0. 025 mg N i a c i n 0. 0625 mg N i a c i n a m i d e 0. 0625 mg P y r i d o x a l HC1 0. 0625 mg P y r i d o x i n e HC1 0. 0625 mg R i b o f l a v i n 0. 025 mg Sodium A c e t a t e 50. 00 mg Sodium G l u c u r o n a t e 1. 80 mg Thiamine HC1 0. 025 mg Thymidine 10. 00 mg V i t a m i n A 0. 25 mg V i t a m i n B_2 1. 00 mg C o c a r b o x y l a s e 1. 00 mg Coenzyme A 2. 50 mg Deoxyadenos i n e 10. 00 mg D e o x y c y t i d i n e HC1 10. 00 mg Deoxyguanos i n e 10. 00 mg D i p h o s p h o p y r i d i n e N u c l e o t i d e 7. 00 mg F l a v i n Adenine D i n u c l e o t i d e 1. 00 mg 5 - M e t h y l c y t o s i n e 0. 10 mg To c o p h e r o l Phosphate 0. 025 mg T r i p h o s p h o p y r i d i n e N u c l e o t i d e 1. 00 mg Tween 80 12 . 50 mg U r i d i e n T r i p h o s p h a t e 1. 00 mg NCTC-109 (Cont'd) Chemical Compound NaCl KCl CaCl2(anhyd.) MgS04-7H20 NaH2P04-H20 NaHC0 3 G l u c o s e Phenol Red 1 L i t e r o f NCTC-135 Co n t a i n s 6.80 0.40 0. 20 0. 20 0.14 2.20 1.00 20. 00 gm gm gm gm gm gm gm mg RPMI 1640 Chemical Compound 1 L i t e r o f RPMI 1640 Co n t a i n s L - A r g i n i n e ( f r e e base) 200. 0 mg L-Asparagine 50. 0 mg L - A s p a r t i c A c i d 20. 0 mg L - C y s t i n e 50. 0 mg L- G l u t a m i c A c i d 20. 0 mg L-Glutamine 300. 0 mg G l u t a t h i o n e (reduced) 1. 0 mg G l y c i n e 10. 0 mg L - H i s t i d i n e ( f r e e base) 15. 0 mg L - H y d r o x y p r o l i n e 20. 0 mg L - I s o l e u c i n e ( a l i o f r e e ) 50. 0 mg L-Le u c i n e ( M e t h i o n i n e f r e e ) 50. 0 mg L - L y s i n e HC1 40. 0 mg L- M e t h i o n i n e 15. 0 mg L - P h e n y l a l a n i n e 15. 0 mg TABLE I I (Cont'd) Chemical Compound L - P r o l i n e (Hydroxy L - P r o l i n e Free) L - S e r i n e L-Threonine ( a l i o f r e e ) L-Tryptophane L - T y r o s i n e L - V a l i n e B i o t i n C a l c i u m P a n t o t h e n a t e C h o l i n e C h l o r i d e F o l i c A c i d i - I n o s i t o l N i c o t i n a m i d e p-Aminobenzoic A c i d P y r i d o x i n e HC1 R i b o f l a v i n e Thiamine HC1 V i t a m i n B 1 2 NaCl KCl Na2HP04-7H20 MgS04-7H 20 Ca(N0 3)2•4H20 Glucose NaHC0 3 Phenol Red 1 L i t e r o f RPMI 1640 C o n t a i n s 20. 0 mg 30. 0 mg 20. 0 mg 5 . 0 mg 20. 0 mg 20. 0 mg 0. 2 mg 0. 25 mg 3. 0 .g 1. 0 mg 35 . 0 mg 1. 0 mg 1. 0 mg 1. 0 mg 0. 2 mg 1. 0 mg 0. 005 mg 6000. 0 mg 400. 0 mg 1512. 0 mg 100. 0 mg 100. 0 mg 2500. 0 mg 2000. 0 mg 5 . 0 mg RPMI 1603 1 L i t e r o f Chemical RPMI 1603 Compound C o n t a i n s L - A r g i n i n e ( f r e e base) • 200.0 mg L-As p a r a g i n e 50.0 mg L - C y s t i n e 50.0 mg L- G l u t a m i c A c i d 15.0 mg L-Glutamine 500.0 mg G l y c i n e 15.0 mg L - H i s t i d i n e ( f r e e base) 20.0 mg L - I s o l e u c i n e ( a l i o f r e e ) 80.0 mg L-Le u c i n e ( M e t h i o n i n e f r e e ) 80.0 mg L - L y s i n e HC1 25.0 mg L- M e t h i o n i n e 30.0 mg L - P h e n y l a l a n i n e 20.0 mg L - P r o l i n e (Hydroxy L - P r o l i n e Free) 10.0 mg L - S e r i n e 100.0 mg L-Threonine ( a l i o f r e e ) 35.0 mg L-Tryptophane 20.0 mg L - T y r o s i n e 20.0 mg L - V a l i n e 10.0 mg B i o t i n 0.05 mg C a l c i u m P a n t o t h e n a t e 0.25 mg C h o l i n e C h l o r i d e 2.0 mg F o l i c A c i d 0.01 mg F o l i n i c A c i d 0.01 mg i - I n o s i t o l 5.0 mg N i c o t i n a m i d e 0.2 mg N i c o t i n i c A c i d 0.01 mg p-Aminobenzoic A c i d 0.5 mg P y r i d o x i n e H C 1 1.0 mg R i b o f l a v i n e 0.1 mg TABLE I I (Cont'd) Chemical Compound Thiamine HC1 V i t a m i n B12 NaCl KC1 NaH2P04-H20 Na2HP04-7H20 MgS04-7H20 MnS04-H20 Ca(N03)2-4H20 Ca2(S03)4-7H20 ZnS04-7H20 Glucose NaHC0 3 Phenol Red 1 L i t e r o f RPMI 1603 C o n t a i n s 0. 25 mg 0. 05 mg 6000. 0 mg 400. 0 mg 230 . 0 mg 1512. 0 mg 200. 0 mg 1. 0 mg 200. 0 mg 1. 0 mg 2. 0 mg 2500. 0 mg 1500 . 0 mg 5 . 0 mg MEDIUM 199 Chemical Compound Adenine D L - A l a n i n e L - A r g i n i n e H C 1 D L - A s p a r t i c A c i d L - C y s t i n e L - C y s t e i n e H C 1 D i s o d i u m - t o c o p h e r o l Phosphate DL-Glutamic A c i d Monohydrate L-Glutamine G l y c i n e (Aminoa-c e t i c A c i d ) Guanine H C 1 L - H i s t i d i n e H C 1 Hypoxanthine H y d r o x y p r o l i n e D L - I s o l e u c i n e DL-Leucine L - L y s i n e H C 1 DL-Methionine N i a c i n D L - P h e n y l a l a n i n e L - P r o l i n e P y r i d o x a l H C 1 P y r i d o x i n e H C 1 R i b o f l a v i n D L - S e r i n e Sodium A c e t a t e •3 H 2 O 1 L i t e r o f Medium 199 Co n t a i n s 10. 00 mg 50. 00 mg 70. 00 mg 60. 00 mg 20. 00 mg 0. 10 mg 0. 01 mg 150. 00 mg 100. 00 mg 50. 00 mg 0. 30 mg 20. 00 mg 0. 30 mg 10. 00 mg 40. 00 mg 120 . 00 mg 70 . 00 mg 30. 00 mg 0. 025 mg 50. 00 mg 40 . 00 mg 0. 025 mg 0. 025 mg 0 . 01 mg 50 . 0 mg 50. 0 mg TABLE I I (Cont'd) Chemical Compound Thiamine HC1 DL-Threonine Thymine (5-methyl u r a c i l ) L - T y r o s i n e DL-Tryptophan U r a c i l D L - V a l i n e X a n t h i n e Adenosine T r i p h o s p h a t e ( d i s o d i u m s a l t ) A d e n y l i c A c i d p-Aminobenzoic A c i d A s c o r b i c A c i d B i o t i n C a l c i f e r o l C a l c i u m P a n t h o t h e n a t e C h o l e s t e r o l C h o l i n e C h l o r i d e D e s o x y r i b o s e F e r r i c N i t r a t e F o l i c A c i d G l u t a t h i o n e i - I n o s i t o l Menadione N i a c i n a m i d e D-Ribose Tween 80 V i t a m i n A 1 L i t e r o f Medium 199 C o n t a i n s 0. 01 mg 60. 00 mg 0. 30 mg 40. 00 mg 20. 00 mg 0. 30 mg 50. 00 mg 0. 30 mg 10. 00 /mg 0. 2 mg 0. 05 mg 0. 05 mg 0. 01 mg 0. 10 mg 0. 01 mg 0. 20 mg 0. 50 mg 0. 50 mg 0. 10 mg 0. 01 mg 0. 05 mg 0. 05 mg 0. 01 mg 0. 025 mg 0. 50 mg 5 . 00 mg 0. 10 mg TABLE I I (Cont'd) MEDIUM 199 (Cont'd) Chemical Compound NaCl KC1 MgS0 4-7H 20 KH2PO4 Na 2HP0 4.7H 20 C a C l 2 ( A n h y d . ) G l u c o s e NaHC03 Phenol Red 1 L i t e r of Medium 199 Co n t a i n s 8.00 0.40 0. 20 0. 06 0.09 0.14 1.00 0.35 0. 02 gm gm gm gm gm gm gm gm gm WAYMOUTH'S MEDIUM MB 75 2/1 Chemical Compound 1 L i t e r o f Waymouth's Medium C o n t a i n s A s c o r b i c A c i d 17 . , 5 mg C h o l i n e HC1 250 , .0 mg C y s t e i n e HC1 90 , . 0 mg *Glutamine 350. .0 mg Gluco s e 5 . 0 mg G l u t a t h i o n e 15. .0 mg Hypoxanthine 25 . .0 mg NaCl 6, . 00 gm KC1 0. . 15 gm C a C l 2 - 2 H 2 0 0, . 12 gm Chemical Compound 1 L i t e r o f Waymouth's Medium C o n t a i n s M gCl 2-6H 20 0. , 24 gm MgS0 4-7H 20 0. , 20 mg Na 2HP04 0. 30 gm KH 2P04 0. 08 gm NaHC0 3 2. . 24 gm B i o t i n 0. 02 mg Ca l c i u m P a n t o t h e n a t e 1. ,00 mg F o l i c A c i d 0. 40 mg i - I n o s i t o l 1. . 00 mg N i c o t i n a m i d e 1. , 00 mg P y r i d o x i n e HC1 1. . 00 mg R i b o f l a v i n 1. . 00 mg Thiamine HC1 10. , 00 mg V i t a m i n B i 2 0. , 20 mg L - A r g i n i n e HC1 75. , 0 mg L - A s p a r t i c A c i d 60, mg L - C y s t i n e 15. ,0 mg L - g l u t a m i c A c i d 150. , 0 mg G l y c i n e 50. ,0 mg L - H i s t i d i n e HC1 150. .0 mg L - 1 s o l e u c i n e 25. .0 mg L -Leucine 50. . 0 mg L - L y s i n e HC1 240. .0 mg L- M e t h i o n i n e 50, .0 mg L - P h e n y l a l a n i n e 50. .0 mg L - P r o l i n e 50, , 0 mg L-Threonine 75, .0 mg L - T y r o s i n e 40, .0 mg ^Because of i n s t a b i l i t y i n s o l u t i o n , L-glutamine i s o m i t t e d from our l i q u i d media and added at time of use (7 ml o f 5% L-glu t a m i n e p e r l i t e r of Waymouth's Medium 752/1). WAYMOUTH'S MEDIUM MB 752/1 (Cont'd) Chemical Compound L-Tryptophan L - V a l i n e 1 L i t e r o f Waymouth's Medium Co n t a i n s 40.0 mg 65.0 mg CMRL 1066 1 L i t e r o f Chemical CMRL 1066 Compound C o n t a i n s L - A l a n i n e 25. 0 mg L - A r g i n i n e HC1 70. 0 mg L - A s p a r t i c A c i d 30. 0 mg L - C y s t e i n e HC1 260. 0 mg L - C y s t i n e 20. 0 mg L- G l u t a m i c A c i d 75 . 0 mg L-Glutamine 100. 0 mg G l y c i n e 50. 0 mg L - H i s t i d i n e HC1 20. 0 mg H y d r o x y - L - p r o l i n e 10. 0 mg L - 1 s o l e u c i n e 20. 0 mg L- L e u c i n e 60. 0 mg L - L y s i n e HC1 70. 0 mg L- M e t h i o n i n e 15. 0 mg L - P h e n y l a l a n i n e 25. 0 mg L - P r o l i n e 40. 0 mg L - S e r i n e 25. 0 mg L-Threonine 30. 0 mg TABLE I I (Cont'd) Chemical Compound L-Tryptophan L - T y r o s i n e L - V a l i n e p-Aminobenzoic A c i d A s c o r b i c A c i d B i o t i n C a l c i u m P a n t o t h e n a t e C h o l e s t e r o l C h o l i n e C h l o r i d e C o c a r b o x y g l a s e Coenzyme A Deoxyadenosine D e o x y c y t i d i n e Deoxyguanosine D i p h o s p h o p y r i d i n e N u c l e o t i d e E t h a n o l F l a v i n Adenine D i n u c l e o t i d e F o l i c A c i d G l u t a t h i o n e I n o s i t o l 5 - M e t h y l d e o x y c y t i d i n e N i a c i n N i a c i n a m i d e P y r i d o x a l H C 1 P y r i d o x i n e H C 1 R i b o f l a v i n Sodium A c e t a t e 3H2O 1 L i t e r o f CMRL 1066 C o n t a i n s 10. 0 mg 40. 0 mg 25. 0 mg 0. 05 mg 50. 00 mg 0. 01 mg 0. 01 mg 0. 20 mg 0. 50 mg 1. 00 mg 2. 50 mg 10. 00 mg 10. 00 mg 10. 00 mg 7. 00 mg 16. 00 mg 1. 00 mg 0. 01 mg 10 . 00 mg 0 . 05 mg 0. 10 mg 0 . 025 mg 0. 025 mg 0. 025 mg 0. 025 mg 0 . 01 mg 83. 00 mg TABLE I I (Cont'd) CMRL 1066 (Cont'd) 1 L i t e r o f 1 L i t e r o f Chemical CMRL 1066 Chemical F-10 Compound C o n t a i n s Compound C o n t a i n s Sodium G l u c u r o n a t e 4. 20 mg L - H i s t i d i n e HC1 21.00 mg Thiamine HC1 0.01 mg L- I s o l e u c i n e 2.60 mg Thymidine 10.00 mg L-Le u c i n e 13.10 mg T r i p h o s p h o p y r i d i n e L - L y s i n e HC1 29. 30 mg N u c l e o t i d e 1. 00 mg L-M e t h i o n i n e 4.48 mg Tween 80 5. 00 mg L - P h e n y l a l a n i n e 4. 96 mg U r i d i n e t r i p h o s p h a t e 1.00 mg L - P r o l i n e 11.50 mg NaCl 6.80 gm L - S e r i n e 10. 50 mg KC1 0.40 gm L-Threonine 3.57 mg CaCl2(anhyd.) 0. 20 gm L-Tryptophan 0.60 mg MgS04-7H 20 0.20 gm L-Tyros i n e 1. 81 mg NaH 2P04-H20 0 .14 gm L - V a l i n e 3. 50 mg NaHC0 3 2.20 gm B i o t i n 0.024 mg Glucose 1. 00 gm D-Calcium P a n t o t h e n a t e 0.715 mg Phenol Red 0.02 gm C h o l i n e C h l o r i d e 0.698 mg F o l i c A c i d 1. 320 mg Hypoxanthine 4. 080 mg i - I n o s i t o l 0.541 mg HAM'S MEDIUM F-10 L i p o i c A c i d 0. 200 mg Ni a c i n a m i d e 0.615 mg 1 L i t e r of P y r i d o x i n e HC1 0. 206 mg Chemical F-10 R i b o f l a v i n 0. 376 mg Compound C o n t a i n s Sodium P y r u v a t e 110.00 mg Thiamine HC1 1.012 mg L - A l a n i n e 8 . 91 mg Thymidine 0.727 mg L - A r g i n i n e HC1 211.00 mg V i t a m i n B_2 1. 360 mg L - A s p a r t i c A c i d 13.30 mg NaCl 7.400 gm L-Asparagine H2O 15.00 mg KC1 0. 285 gm L - C y s t e i n e HC1 31. 50 mg MgS04-7H20 1.52 gm L-G l u t a m i c A c i d 14.70 mg Na2HP04-7H20 0. 209 gm L-Glutamine 146.20 mg KH2PO4 0 . 083 gm G l y c i n e 7.51 mg CaCl2-2H 20 0.029 gm HAM'S MEDIUM F-10 (Cont'd) Chemical Compound CuS0 4-5H 20 FeS0 4-7H 20 ZnS04-7H 20 NaHC0 3 Glucose Phenol Red 1 L i t e r o f F-10 C o n t a i n s 0.0025 mg 0.834 mg 0.0288 mg 1.200 gm 1.100 gm 0.0012 gm EAGLE'S MINIMUM ESSENTIAL MEDIUM FOR MONOLAYER CULTURE Chemical Compound L - A r g i n i n e ' HC1 L - C y s t i n e *L-Glutamine L - H i s t i d i n e L - 1 s o l e u c i n e L - L e u c i n e L - L y s i n e HC1 L- M e t h i o n i n e L - P h e n y l a l a n i n e L-Threonine 1 L i t e r o f MEM Co n t a i n s 105. 0 mg 24. 0 mg 292 . 0 mg 31. 0 mg 52 . 0 mg 52. 0 mg 58. 0 mg 15 . 0 mg 32. 0 mg 48. 0 mg *Because o f i n s t a b i l i t y i n s o l u t i o n , added at time o f use (6 ml o f 5% L-TABLE I I (Cont'd) Chemical Compound L-Tryptophan L - T y r o s i n e L - V a l i n e C h o l i n e C h l o r i d e F o l i c A c i d I n o s i t o l N i c o t i n a m i d e D-Calcium P a n t o t h e n a t e P y r i d o x a l HC1 R i b o f l a v i n Thiamine HC1 NaCl KCl M gCl 2-6H 20 C a C l 2 ( a n h y d . ) NaH 2P04-2H 20 NaHC0 3 Glucose Phenol Red 1 L i t e r o f F-10 C o n t a i n s 10. 0 mg 36. 0 mg 46. 0 mg 1. 0 mg 1. 0 mg 2. 0 mg 1. 0 mg 1. 0 mg 1. 0 mg 0. 1 mg 1. 0 mg 6. 80 gm 0. 40 gm 0 . 20 gm 0. 20 gm 0. 15 gm 2. 20 gm 1. 00 gm 0. 01 gm lut a m i n e i s o m i t t e d from our l i q u i d media and amine per l i t e r o f s i n g l e - s t r e n g t h E a g l e medium). TABLE V ANALYSIS OF VARIANCE Source o f V a r i a t i o n df SS MS F ( c a l c u l a t e d ) F ( t a b u l a t e d ) 0.01 l e v e l A 29 1. 4317 X 1 0 5 4. 9368 X 1 0 3 187.54 1.6964 B 1 1. 2473 X 10-3 1. 2473 X 1 0 3 47. 38 6.6349 C 29 6. 0193 X 1 0 3 2. 0756 X 1 0 2 7 .88 1.6964 AB 2 4. 6129 X 10* 2. 3065 X 104 876.20 4.6052 AC 58 3. 5510 X 1 0 4 6. 1224 X 1 0 2 23.26 1.4730 : BC 2 6. 7354 X 1 0 2 3. 3677 X 1 0 2 12 . 79 4.6052 ABC 58 1. 1248 X 104 1. 9393 X 1 0 2 7.37 1.4730 E r r o r 3420 9. 0027 X 104 2. 6324 X 1 0 l T o t a l 3599 3. 3402 X 1 0 5 A B C = media = a n t i b i o t i c s = s h a k i n g , g a s s i n g , no s h a k i n g , no g a s s i n g - 82 -TABLE V I I AVERAGE LENGTH (MILLIMETRES) OF DEAD WORMS Shaking G a s s i n g Medium A B A B A B GKN 0. 84 1. 11 1. 02 0. 79 0. 91 E a r l e ' s BSS 0. 78 0. 89 0. 80 0. 79 Hank's BSS 0. 91 0. 96 - 0. 83 0. 86 -S c h e r e r s 0. 88 0. 86 0. 98 0. 84 0. 92 0. 87 NCTC 109 0. 92 0. 89 - 0. 85 0. 91 0. 88 RPMI 1640 0. 8 0. 9 - 0. 9 0. 9 0. 9 RPMI 1603 - 0. 92 0. 92 - 0. 97 0. 87 Med. 199 0. 75 0. 83 0. 82 0. 88 0. 85 0. 81 Waymouths 0. 83 - 0. 97 0. 91 - 0. 82 CMRL 1066 w i t h E a g l e - 0. 88 0. 99 0. 80 - 0. 93 CMRL 1066 w i t h Hanks 0. 89 0. 95 0. 93 0. 87 0 . 94 0. 87 Hams F-10 0. 81 0. 87 - - 0. 86 0. 88 Ea g l e Min E s s . 0. 93 - - 0. 88 0. 99 0. 88 Med. 199 + 0 • £ • £ • 0. 79 0. 80 0. 86 0. 85 0. 92 0. 97 B.E.E. 0. 83 0. 79 0. 86 0. 89 0. 93 1. 0 B.A.F. 0. 79 0. 80 0. 93 0. 80 0. 90 0. 84 De x t r o s e 0. 78 0. 79 0. 88 0. 91 0. 82 0. 96 Horse Serum 0. 81 0. 87 0. 78 0. 92 0. 98 0. 93 Horse Ser + De x t r 0. 80 0. 77 0. 85 0. 78 0. 85 0. 73 C h i c k Ser + De x t r 0. 84 0. 89 0. 88 0. 86 - 0. 93 C h i c k Serum 0. 83 0. 89 0. 93 - 0. 88 0. 88 Tro u t Egg E x t r a c t 0. 86 - 0. 96 0. 85 0. 83 0. 92 T r o u t L i v e r E x t r - 0. 87 0. 96 0. 75 0. 98 0. 98 E a r l e ' s + B.E.E. 0. 84 0. 89 0. 90 0. 94 0. 89 0. 95 Horse Serum 0. 79 0. 76 0. 82 0. 86 0. 83 0. 84 C h i c k Serum 0 . 95 0. 88 0. 84 0. 89 0. 92 0. 92 C a l f Ser. + L a c t . Hyd. - 0. 96 0. 89 0. 91 - 1. 00 C h i c k Serum + Waymouths 0. 84 - 0. 89 0. 77 0. 90 0. 84 Hams F-10 0. 86 1. 06 0. 89 0. 93 0. 91 0. 85 CMR1 1066 w i t h 0. 97 0. 91 - 0. 86 - 0. 87 E a g l e s - 83 -TABLE V I I I MOLALITIES OF MEDIA Medium M e l t i n g P o i n t Ct) Kf OsM GKN 3.58 0.23 0.123 E a r l e ' s BSS 3.65 0.16 0.086 Hank's BSS 3.61 0.20 0.107 S c h e r e r s 3.56 0.25 0.134 NCTC 109 3.62 0.19 0.102 RPMI 1640 3.54 0.27 0.145 RPMI 1603 3.66 0.15 0.081 Med. 199 3.64 0.17 0.091 Waymouths 3.51 0.30 0.161 CMRL 1066 w i t h E a g l e ' s 3.61 0.20 0.107 CMRL 1066 w i t h Hank's 3.54 0.27 0.145 Hams F-10 3.60 0.21 0.113 E a g l e ' s Min E s s . 3.54 0.27 0.145 Med. 199 + C.E.E. 3.61 0.20 0.107 B.E.E. 3.61 0.20 0.170 B.A.F. 3.55 0.26 0.140 D e x t r o s e 3.56 0.25 0.134 Horse Serum 3.69 0.12 0.064 Horse Ser. + D e s t r . 3.63 0.18 0.097 C h i c k Ser. + D e x t r . 3.52 0.29 0.156 C h i c k Serum 3.66 0.15 0.081 Tro u t Egg E x t r a c t - - -T r o u t L i v e r E x t r a c t 3.63 0.18 0.097 E a r l e ' s + B.E.E. 3.69 0.12 0.064 Horse Serum 3.69 0.12 0.064 C h i c k Serum 3.69 0.12 0.064 C a l f Serum + L a c t . Hyd. 3.54 0.27 0.145 Waymouths 3.55 0.26 0.140 Hams F-10 3.64 0.17 0.091 CMRL 1066 w i t h E a g l e ' s 3.61 0.20 0.107 Kf = M e l t i n g p t . d i s t i l l e d H 20 (3.81) - M e l t i n g p t . o f Medium OsM = mKf 1. 86 

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