15.6. 1975
Specialia
a d e q u a t e m i c r o g r a p h s . E l e c t r o d e s m a y b e viewed u n c o a t e d a t low a c c e l e r a t i n g v o l t a g e s (less t h a n 2 kV) b u t the. r e s o l u t i o n o b t a i n e d is n o t sufficient to e x a m i n e fine e l e c t r o d e tips. T h e p r o b l e m s a s s o c i a t e d w i t h s e c o n d a r y e m i s s i o n Call be e l i m i n a t e d b y u s i n g t r a n s m i s s i o n microscopy. S a m p l e s do n o t n e e d a m e t a l c o a t i n g a n d r e q u i r e m u c h less s p o t c u r r e n t ( a b o u t 1 pA). T h e t r a n s m i s s i o n m o d e h a s t h e a d d e d a d v a n t a g e of g r e a t e r a t t a i n a b l e r e s o l u t i o n t h a n t h e s e c o n d a r y e m i s s i o n mode. T h e s i l h o u e t t e i m a g e o b t a i n e d in t h e t r a n s m i s s i o n m o d e c a n b e i m p r o v e d b y u s i n g d a r k field w h i c h allows d e t e r m i n a t i o n of t i p l u m e n diameter. I n s u m m a r y , glass m i c r o e l e c t r o d e s were e x a m i n e d w i t h d a r k field s c a n n i n g t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y w i t h o u t t h e n e c e s s i t y of h e a v y m e t a l coating. T h e elect r o d e s were n o t d a m a g e d a n d could b e used for p h y s i o logical r e c o r d i n g a f t e r e x a m i n a t i o n ~.
697
Zusammen/assung. M i k r o g l a s e l e k t r o d e n w u r d e n i m rasterelektronenmikroskopischen Dunkelfeld transmissionsoptisch ohne Metallbeschichtung untersucht, wobei die E l e k t r o d e n n i c h t besch~tdigt w e r d e n u n d fiir physiologische A b l e i t u n g e n w i e d e r b e n i i t z t w e r d e n k 6 n n e n . D, M. FRY
Department o/Zoology, University o/Melbourne, Parkville, ( Vicloria 3052, Australia), 10 January 7975.
9 I wish to thank R. D. PURVESfor his advice and for the use of the resistance measuring equipment. This work was supported by the Australian Research Grants Committee and the National Heart Foundation of Australia.
A Simple Method for the Microscopic Examination of Unsquashed, Stained Organs of Insects I n cytologic-microscopic e x a m i n a t i o n of u n s q u a s h e d obj e c t s of insects, w i t h t h e dyes a c e t i c - l a c t i c o r c e i n 1, carbolIuchsin (Fa. Merck), w h i c h are used for t h e s t a i n i n g of tile nucleus, t h e d i f f i c u l t y arises, t h a t e i t h e r t h e o b j e c t c a n o n l y b e exa'mined in t h e u p p e r or lower level b a s e d on tile s t r o n g colouring, or t h e d y e is n o t able to p e r c o l a t e c o m p l e t e l y b y s h o r t e r d u r a t i o n of staining. To m a k e possible a n e q u a l c o n s i d e r a t i o n of t h e nuclei a t all levels, a m e t h o d was e v o l v e d w h i c h p r o v e d useful for t e s t e s follicles of insects, a n d w h i c h is also s u i t a b l e for d i s c o v e r i n g t h e m e t a p h a s e - I - s t a g e s in oocytes. T h e testicle is d i v i d e d in R i n g e r ' s s o l u t i o n in s e p a r a t e follicles. T h e follicles are t r a n s p o r t e d t o a s m a l l dish w i t h a c e t i c - l a c t i c o r c e i n 1 a n d are left c o v e r e d for a t least 2 h a t r o o m t e m p e r a t u r e u n t i l t h e o b j e c t is t o t a l l y s t a i n e d . Sub-
s e q u e n t t y t h e follicle is t r a n s p o r t e d t o a slide, on w h i c h b r o k e n f r a g m e n t s of cover-glass are placed. T h e coverglass f r a g m e n t s m u s t b e h i g h e r t h a n t h e o b j e c t t o a v o i d s q u a s h i n g . A f t e r c o v e r i n g w i t h a cover-glass, 6 0 % acetic acid is a d d e d on one side, a n d on tile o t h e r side tile dye is sucked off w i t h filter paper, u n t i l t h e l i q u i d - s u c k e d off is n e a r l y colourless. T h e cover-glass is sealed w i t h coverglass c e m e n t . A c c o r d i n g t o t h e t h i c k n e s s of t h e object, t h e slide is k e p t on for 15 m i n t o 2 h a f t e r t h e a d d i t i o n of t h e acetic acid. D u r i n g t h i s t i m e t h e dye, w h i c h still r e m a i n e d in t h e c y t o p l a s m s of t h e follicle, b e c o m e s t r a n s p a r e n t a n d shows in all p a r t s a u n i f o r m colouring.
1 L. F. LA COUR, Stain Techn. /6, 169 (1941).
Part of a testicle follicle of Oncopeltus ]asciatus (Heteroptera), 5th larval stage, from an unsquashed preparation. Acetic-lacticorcein. Note the nuclei situated ill cysts.
698
Specialia
T h e n t h e follicle c a n be e x a m i n e d w i t h o u t difficulty u n d e r t h e microscope a t all levels. T h e F i g u r e shows a p a r t of a testicle follicle f r o m Oncopeltus /asciatus ( H e t e r o p t e r a ) t r e a t e d in t h e w a y described. T h e nuclei ( s e c o n d a r y s p e r m a t o g o n i a ) are seen to b e a r r a n g e d i n cysts of d i f f e r e n t size. I n t h e nuclei t h e sex c h r o m o s o m e s are r e c o g n i z a b l e as h e t e r o c h r o m a t i c bodies. W i t h t h e help of t h e d e s c r i b e d m e t h o d it was possible t o show t h a t in Oncopeltus ~ t h e t r a n s f o r m a t i o n of t h e second sex c h r o m o s o m e i n t o t h e h e t e r o c h r o m a t i c s t a t e t a k e s place following t h e 6 t h ( = last) m i t o t i c d i v i s i o n of t h e s e c o n d a r y s p e r m a t o g o n i a , i.e. d u r i n g t h e f o r m a t i o n of t h e 64-cell-cyst ( P u b l i c a t i o n in p r e p a r a t i o n ) . A f t e r t h a t , the primary spermatocytes develop from the spermatogonia. This method has the considerable advantage, compared w i t h t h e e x a m i n a t i o n b y aid of series sections 2, t h a t no t i m e - c o n s u m i n g p r e p a r a t i o n m u s t b e d o n e a n d t h a t no s u m m a r y of t h e i n t e r p r e t a t i o n of single sections is n e c e s s a r y t o o b t a i n t h e result. I n a d d i t i o n , if c o m p a r e d w i t h t h e s q u a s h - p r e p a r a t i o n s a, t h i s m e t h o d is s u p e r i o r
EXPERIENTIA 31/6
w h e r e t h e p r o b l e m is t h a t t h e nuclei are t o b e c o u n t e d or t h e succession of t h e single m i t o t i c or meiotic stages in a testicle follicle is to b e e x a m i n e d .
Zusammen/assung. A m Beispiel v o n H o d e n f o l l i k e l n y o n Oncopeltus ]asciatus ( H e t e r o p t e r a ) w i r d eine M e t h o d e b e s c h r i e b e n , die bei gefiirbten, u n g e q u e t s c h t e n O b j e k t e n eine H c h t m i k r o s k o p i s c h e U n t e r s u c h u n g in allen E b e n e n zul~isst. H. MESSTHALER 4
Lehrstuhl [i~r Spezielle Zoologie der Ruhr- Universittit, D-463 Bochum (German Federal Republic, BRD), 77 December 7974. 2 B. ROMEIS, Mikroskopische Technik (R. Oldenburg Verlag, ~finchen 1968). a C. D. DARLI~GTON, L. F. LA COUR, Methoden der Chromosomenuntersuchung (Franekhsehe Verlagshandlung, Stuttgart 1963). 4 Present address: Biologische Station der Fa. Celamerek GmbH, D-6501 Sehwabenheim/Selz, Germany.
A Technique for C h r o m o s o m e Analysis of Small Rodents Without Sacrificing the Animal I n m a n y e x p e r i m e n t a l designs it would b e g r e a t l y convenient to have information on the chromosomal c o n s t i t u t i o n of s m a l l r o d e n t s w i t h o u t killing t h e specimen. T h u s far, l e u k o c y t e or skin 1-8 c u l t u r e s h a v e b e e n t h e most common methods employed to study the chromosomes of l i v i n g animals. H o w e v e r , b o t h t y p e of c u l t u r e s are difficult or e v e n i m p o s s i b l e t o o b t a i n in r o d e n t s . L e u k o c y t e or skin c u l t u r e s f r o m v a r i o u s species of vole mice of t h e g e n u s A k o d o n ( R o d e n t i a Cricetidae) cons t a n t l y fail to yield s u i t a b l e a m o u n t s of m i t o s i s for c y t o g e n e t i c analysis. T h u s , we decided t o e m p l o y a m o d i f i c a t i o n of t h e in v i v o c h a m b e r t e c h n i q u e used to s t u d y t h e k i n e t i c s of a n t i b o d y r e s p o n s e or t o grow t u m o u r cells 4, 5. T h e m e t h o d b a s i c a l l y consists in: a) t o ineroduce a m i c r o c h a m b e r c h a r g e d w i t h a n a n t i g e n i n t o t h e abd o m i n a I c a v i t y of t h e a n i m a l t o s t u d y , b) t o e x t r a c t t h e m i e r o c h a m b e r w h e n it is s u r r o u n d e d b y a n a c t i v e l y d i v i d i n g p o p u l a t i o n of i m m u n o c o m p e t e n t cells (the a n i m a l is colchicinized 3 h before e x t r a c t i n g t h e microc h a m b e r ) , c) t o h a r v e s t t h e i m m u n o c o m p e t e n t cells b y m e a n s of t h e e n z y m e p r o n a s e , d) to p r e p a r e m e t a p h a s e s p r e a d s b y h y p o t o n i c s h o c k a n d air d r y i n g procedures. Materials and method. Diffussion c h a m b e r s (0.18 m l c a p a c i t y ) were c o n s t r u c t e d b y c e m e n t i n g a circular S a r t o r i u s filter (0.2 [xm p o r e size) to e a c h side ot a n acrylic r i n g w i t h a n e x t e r n a l a n d i n t e r n a l d i a m e t e r of 14 a n d 12 m m r e s p e c t i v e l y (the c e m e n t e m p l o y e d is M F C e m e n t Millipore Co). C h a m b e r s a r e p l a c e d i n o p e n P e t r i dishes a n d sterilized a t 80~ for 48 h. P e t r i dishes are c o v e r e d a n d t h e s t e r i l i z a t i o n is c o m p l e t e d b y p l a c i n g t h e dishes a t 37~ for 24 h a n d a t 80~ for a n o t h e r 48 h. T h e sterilized c h a m b e r s are filled w i t h t o t a l blood f r o m a n h e t e r o l o g o u s species h a l f d i l u t e d w i t h H a n k s or E a r l e saline (for A k o d o n we usually e m p l o y mice or r a t blood). T h e c h a m b e r filling is p e r f o r m e d w i t h a n e e d l e a n d a syringe t h r o u g h a hole p e r f o r m e d in t h e wall of t h e acrylic ring. T h e n t h e hole is sealed w i t h a d r o p of sterile paraffin. R e c e p t o r a n i m a l s are a n a e s t h e t i z e d w i t h 1 [zg/g b o d y w e i g h t of N a p e n t o b a r b i t a l (the N a p e n t o b a r b i t a l s o l u t i o n is p r e p a r e d b y dissolving 550 m g of t h e d r u g in a m i x t u r e of p r o p y l e n e glycol 20 mI, a b s o l u t e e t h a n o l 10 hal, distilled w a t e r 80 ml). A f t e r w a r d s t h e a b d o m e n is opened, 1 or 2
m i c r o c h a m b e r s are p l a c e d i n t o t h e p e r i t o n e a l c a v i t y a n d t h e a b d o m i n a l wall is s u t u r e d . W h i l e t h e a n i m a l is b e i n g o p e r a t e d , t h e b l o o d - c h a r g e d m i c r o c h a m b e r s are m a i n fiained in sterile H a n k s solution. W e h a v e t r i e d v a r i o u s t i m e - l a p s e s t o d r a w microc h a m b e r s . H o w e v e r we h a v e f o u n d t h a t t h e p e a k of m i t o t i c a c t i v i t y in t h e i m m u n o c o m p e t e n t cells w h i c h surround the microchamber appears at about 8 days after t h e i r i n t r o d u c t i o n i n t h e r e c i p i e n t a n i m a l . Accordingly, a t t h i s m o m e n t t h e m i c r o c h a m b e r s are t a k e n f r o m t h e p e r i t o n e a l c a v i t y of a n i m a l s . 3 h before r e m o v i n g ehe m i c r o c h a m b e r s t h e a n i m a l s are i n j e c t e d i.p. w i t h 1 [xg/g b o d y w e i g h t of colchicine (0.04% s o l u t i o n in distilled water). Immediately after being removed the microchambers are p l a c e d in a 0.5% s o l u t i o n of p r o n a s e (Calbiochem) in n o r m a l saline a n d g e n t l y s h a k e d for 10 to 15 m i n to free t h e cell coat w h i c h covers t h e m . T h e p r o n a s e s o l u t i o n is c e n t r i f u g e d a t 600 r p m for 7 m i n a n d t h e cell p e l l e t o b t a i n e d r e s u s p e n d e d ill a 0.7 M s o l u t i o n of KC1 a t 37 ~ for 15 m i n . T h e n ceils are c e n t r i f u g e d a g a i n a n d fixed in 3/1 a b s o l u t e e t h a n o l / g l a c i a l acetic acid. A f t e r one w a s h i n g in fresh fixative, c h r o m o s o m e s p r e a d s a r e p r e p a r e d b y air d r y i n g a n d s t a i n e d w i t h c a r b o l - f u c h s i n , giemsa, acetic orcein or a n y o t h e r c h r o m o s o m a l stain. Results and discussion. W e h a v e s t u d i e d w i t h t h e a b o v e p r o c e d u r e a large n u m b e r of r a t s a n d mices a n d m o r e t h a n 50 s p e c i m e n s of A k o d o n b e l o n g i n g to 3 d i f f e r e n t species. I n all cases we were able to o b t a i n a c o n s i d e r a b l e a m o u n t of good m e t a p h a s e s s u i t a b l e for c h r o m o s o m e a n a l y s i s or b a n d i n g procedures. D u r i n g t h e first t r i a l s 3 0 % of a n i m a l s died a f t e r t h e r e m o v a l of m i c r o c h a m b e r s (no a n i m a l died d u r i n g or a f t e r t h e first o p e r a t i o n ) . H o w e v e r , as we g a i n e d e x p e r i e n c e t h e m o r t a l i t y d e c r e a s e d t o zero
1 M. RAY, Can. J. Genet. Cytol. 12, 87 (1970). I. JOHNSON, P. A. SULLIVAN, P. CI~I. CHAN, J. LoBuE, F. C. MONETTE and A. S. GORDON, Ann. N. Y. Aead. Sci. 25, 807 (1967). a R. L. HYBERTSON and H. D. BRYAN, Life Sei. 6, 1047 (1967). 4 S. A. GOODMAN,M. G. CHE~ and T. MAKINODAN, J. Immun. 108, 1387 (1972). 6 L. RUMI, I. LARRIPA S. B. DE SALUMand C. D. PASQUALINI, Eur. J. Cancer, in press.
Specialia
a d e q u a t e m i c r o g r a p h s . E l e c t r o d e s m a y b e viewed u n c o a t e d a t low a c c e l e r a t i n g v o l t a g e s (less t h a n 2 kV) b u t the. r e s o l u t i o n o b t a i n e d is n o t sufficient to e x a m i n e fine e l e c t r o d e tips. T h e p r o b l e m s a s s o c i a t e d w i t h s e c o n d a r y e m i s s i o n Call be e l i m i n a t e d b y u s i n g t r a n s m i s s i o n microscopy. S a m p l e s do n o t n e e d a m e t a l c o a t i n g a n d r e q u i r e m u c h less s p o t c u r r e n t ( a b o u t 1 pA). T h e t r a n s m i s s i o n m o d e h a s t h e a d d e d a d v a n t a g e of g r e a t e r a t t a i n a b l e r e s o l u t i o n t h a n t h e s e c o n d a r y e m i s s i o n mode. T h e s i l h o u e t t e i m a g e o b t a i n e d in t h e t r a n s m i s s i o n m o d e c a n b e i m p r o v e d b y u s i n g d a r k field w h i c h allows d e t e r m i n a t i o n of t i p l u m e n diameter. I n s u m m a r y , glass m i c r o e l e c t r o d e s were e x a m i n e d w i t h d a r k field s c a n n i n g t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y w i t h o u t t h e n e c e s s i t y of h e a v y m e t a l coating. T h e elect r o d e s were n o t d a m a g e d a n d could b e used for p h y s i o logical r e c o r d i n g a f t e r e x a m i n a t i o n ~.
697
Zusammen/assung. M i k r o g l a s e l e k t r o d e n w u r d e n i m rasterelektronenmikroskopischen Dunkelfeld transmissionsoptisch ohne Metallbeschichtung untersucht, wobei die E l e k t r o d e n n i c h t besch~tdigt w e r d e n u n d fiir physiologische A b l e i t u n g e n w i e d e r b e n i i t z t w e r d e n k 6 n n e n . D, M. FRY
Department o/Zoology, University o/Melbourne, Parkville, ( Vicloria 3052, Australia), 10 January 7975.
9 I wish to thank R. D. PURVESfor his advice and for the use of the resistance measuring equipment. This work was supported by the Australian Research Grants Committee and the National Heart Foundation of Australia.
A Simple Method for the Microscopic Examination of Unsquashed, Stained Organs of Insects I n cytologic-microscopic e x a m i n a t i o n of u n s q u a s h e d obj e c t s of insects, w i t h t h e dyes a c e t i c - l a c t i c o r c e i n 1, carbolIuchsin (Fa. Merck), w h i c h are used for t h e s t a i n i n g of tile nucleus, t h e d i f f i c u l t y arises, t h a t e i t h e r t h e o b j e c t c a n o n l y b e exa'mined in t h e u p p e r or lower level b a s e d on tile s t r o n g colouring, or t h e d y e is n o t able to p e r c o l a t e c o m p l e t e l y b y s h o r t e r d u r a t i o n of staining. To m a k e possible a n e q u a l c o n s i d e r a t i o n of t h e nuclei a t all levels, a m e t h o d was e v o l v e d w h i c h p r o v e d useful for t e s t e s follicles of insects, a n d w h i c h is also s u i t a b l e for d i s c o v e r i n g t h e m e t a p h a s e - I - s t a g e s in oocytes. T h e testicle is d i v i d e d in R i n g e r ' s s o l u t i o n in s e p a r a t e follicles. T h e follicles are t r a n s p o r t e d t o a s m a l l dish w i t h a c e t i c - l a c t i c o r c e i n 1 a n d are left c o v e r e d for a t least 2 h a t r o o m t e m p e r a t u r e u n t i l t h e o b j e c t is t o t a l l y s t a i n e d . Sub-
s e q u e n t t y t h e follicle is t r a n s p o r t e d t o a slide, on w h i c h b r o k e n f r a g m e n t s of cover-glass are placed. T h e coverglass f r a g m e n t s m u s t b e h i g h e r t h a n t h e o b j e c t t o a v o i d s q u a s h i n g . A f t e r c o v e r i n g w i t h a cover-glass, 6 0 % acetic acid is a d d e d on one side, a n d on tile o t h e r side tile dye is sucked off w i t h filter paper, u n t i l t h e l i q u i d - s u c k e d off is n e a r l y colourless. T h e cover-glass is sealed w i t h coverglass c e m e n t . A c c o r d i n g t o t h e t h i c k n e s s of t h e object, t h e slide is k e p t on for 15 m i n t o 2 h a f t e r t h e a d d i t i o n of t h e acetic acid. D u r i n g t h i s t i m e t h e dye, w h i c h still r e m a i n e d in t h e c y t o p l a s m s of t h e follicle, b e c o m e s t r a n s p a r e n t a n d shows in all p a r t s a u n i f o r m colouring.
1 L. F. LA COUR, Stain Techn. /6, 169 (1941).
Part of a testicle follicle of Oncopeltus ]asciatus (Heteroptera), 5th larval stage, from an unsquashed preparation. Acetic-lacticorcein. Note the nuclei situated ill cysts.
698
Specialia
T h e n t h e follicle c a n be e x a m i n e d w i t h o u t difficulty u n d e r t h e microscope a t all levels. T h e F i g u r e shows a p a r t of a testicle follicle f r o m Oncopeltus /asciatus ( H e t e r o p t e r a ) t r e a t e d in t h e w a y described. T h e nuclei ( s e c o n d a r y s p e r m a t o g o n i a ) are seen to b e a r r a n g e d i n cysts of d i f f e r e n t size. I n t h e nuclei t h e sex c h r o m o s o m e s are r e c o g n i z a b l e as h e t e r o c h r o m a t i c bodies. W i t h t h e help of t h e d e s c r i b e d m e t h o d it was possible t o show t h a t in Oncopeltus ~ t h e t r a n s f o r m a t i o n of t h e second sex c h r o m o s o m e i n t o t h e h e t e r o c h r o m a t i c s t a t e t a k e s place following t h e 6 t h ( = last) m i t o t i c d i v i s i o n of t h e s e c o n d a r y s p e r m a t o g o n i a , i.e. d u r i n g t h e f o r m a t i o n of t h e 64-cell-cyst ( P u b l i c a t i o n in p r e p a r a t i o n ) . A f t e r t h a t , the primary spermatocytes develop from the spermatogonia. This method has the considerable advantage, compared w i t h t h e e x a m i n a t i o n b y aid of series sections 2, t h a t no t i m e - c o n s u m i n g p r e p a r a t i o n m u s t b e d o n e a n d t h a t no s u m m a r y of t h e i n t e r p r e t a t i o n of single sections is n e c e s s a r y t o o b t a i n t h e result. I n a d d i t i o n , if c o m p a r e d w i t h t h e s q u a s h - p r e p a r a t i o n s a, t h i s m e t h o d is s u p e r i o r
EXPERIENTIA 31/6
w h e r e t h e p r o b l e m is t h a t t h e nuclei are t o b e c o u n t e d or t h e succession of t h e single m i t o t i c or meiotic stages in a testicle follicle is to b e e x a m i n e d .
Zusammen/assung. A m Beispiel v o n H o d e n f o l l i k e l n y o n Oncopeltus ]asciatus ( H e t e r o p t e r a ) w i r d eine M e t h o d e b e s c h r i e b e n , die bei gefiirbten, u n g e q u e t s c h t e n O b j e k t e n eine H c h t m i k r o s k o p i s c h e U n t e r s u c h u n g in allen E b e n e n zul~isst. H. MESSTHALER 4
Lehrstuhl [i~r Spezielle Zoologie der Ruhr- Universittit, D-463 Bochum (German Federal Republic, BRD), 77 December 7974. 2 B. ROMEIS, Mikroskopische Technik (R. Oldenburg Verlag, ~finchen 1968). a C. D. DARLI~GTON, L. F. LA COUR, Methoden der Chromosomenuntersuchung (Franekhsehe Verlagshandlung, Stuttgart 1963). 4 Present address: Biologische Station der Fa. Celamerek GmbH, D-6501 Sehwabenheim/Selz, Germany.
A Technique for C h r o m o s o m e Analysis of Small Rodents Without Sacrificing the Animal I n m a n y e x p e r i m e n t a l designs it would b e g r e a t l y convenient to have information on the chromosomal c o n s t i t u t i o n of s m a l l r o d e n t s w i t h o u t killing t h e specimen. T h u s far, l e u k o c y t e or skin 1-8 c u l t u r e s h a v e b e e n t h e most common methods employed to study the chromosomes of l i v i n g animals. H o w e v e r , b o t h t y p e of c u l t u r e s are difficult or e v e n i m p o s s i b l e t o o b t a i n in r o d e n t s . L e u k o c y t e or skin c u l t u r e s f r o m v a r i o u s species of vole mice of t h e g e n u s A k o d o n ( R o d e n t i a Cricetidae) cons t a n t l y fail to yield s u i t a b l e a m o u n t s of m i t o s i s for c y t o g e n e t i c analysis. T h u s , we decided t o e m p l o y a m o d i f i c a t i o n of t h e in v i v o c h a m b e r t e c h n i q u e used to s t u d y t h e k i n e t i c s of a n t i b o d y r e s p o n s e or t o grow t u m o u r cells 4, 5. T h e m e t h o d b a s i c a l l y consists in: a) t o ineroduce a m i c r o c h a m b e r c h a r g e d w i t h a n a n t i g e n i n t o t h e abd o m i n a I c a v i t y of t h e a n i m a l t o s t u d y , b) t o e x t r a c t t h e m i e r o c h a m b e r w h e n it is s u r r o u n d e d b y a n a c t i v e l y d i v i d i n g p o p u l a t i o n of i m m u n o c o m p e t e n t cells (the a n i m a l is colchicinized 3 h before e x t r a c t i n g t h e microc h a m b e r ) , c) t o h a r v e s t t h e i m m u n o c o m p e t e n t cells b y m e a n s of t h e e n z y m e p r o n a s e , d) to p r e p a r e m e t a p h a s e s p r e a d s b y h y p o t o n i c s h o c k a n d air d r y i n g procedures. Materials and method. Diffussion c h a m b e r s (0.18 m l c a p a c i t y ) were c o n s t r u c t e d b y c e m e n t i n g a circular S a r t o r i u s filter (0.2 [xm p o r e size) to e a c h side ot a n acrylic r i n g w i t h a n e x t e r n a l a n d i n t e r n a l d i a m e t e r of 14 a n d 12 m m r e s p e c t i v e l y (the c e m e n t e m p l o y e d is M F C e m e n t Millipore Co). C h a m b e r s a r e p l a c e d i n o p e n P e t r i dishes a n d sterilized a t 80~ for 48 h. P e t r i dishes are c o v e r e d a n d t h e s t e r i l i z a t i o n is c o m p l e t e d b y p l a c i n g t h e dishes a t 37~ for 24 h a n d a t 80~ for a n o t h e r 48 h. T h e sterilized c h a m b e r s are filled w i t h t o t a l blood f r o m a n h e t e r o l o g o u s species h a l f d i l u t e d w i t h H a n k s or E a r l e saline (for A k o d o n we usually e m p l o y mice or r a t blood). T h e c h a m b e r filling is p e r f o r m e d w i t h a n e e d l e a n d a syringe t h r o u g h a hole p e r f o r m e d in t h e wall of t h e acrylic ring. T h e n t h e hole is sealed w i t h a d r o p of sterile paraffin. R e c e p t o r a n i m a l s are a n a e s t h e t i z e d w i t h 1 [zg/g b o d y w e i g h t of N a p e n t o b a r b i t a l (the N a p e n t o b a r b i t a l s o l u t i o n is p r e p a r e d b y dissolving 550 m g of t h e d r u g in a m i x t u r e of p r o p y l e n e glycol 20 mI, a b s o l u t e e t h a n o l 10 hal, distilled w a t e r 80 ml). A f t e r w a r d s t h e a b d o m e n is opened, 1 or 2
m i c r o c h a m b e r s are p l a c e d i n t o t h e p e r i t o n e a l c a v i t y a n d t h e a b d o m i n a l wall is s u t u r e d . W h i l e t h e a n i m a l is b e i n g o p e r a t e d , t h e b l o o d - c h a r g e d m i c r o c h a m b e r s are m a i n fiained in sterile H a n k s solution. W e h a v e t r i e d v a r i o u s t i m e - l a p s e s t o d r a w microc h a m b e r s . H o w e v e r we h a v e f o u n d t h a t t h e p e a k of m i t o t i c a c t i v i t y in t h e i m m u n o c o m p e t e n t cells w h i c h surround the microchamber appears at about 8 days after t h e i r i n t r o d u c t i o n i n t h e r e c i p i e n t a n i m a l . Accordingly, a t t h i s m o m e n t t h e m i c r o c h a m b e r s are t a k e n f r o m t h e p e r i t o n e a l c a v i t y of a n i m a l s . 3 h before r e m o v i n g ehe m i c r o c h a m b e r s t h e a n i m a l s are i n j e c t e d i.p. w i t h 1 [xg/g b o d y w e i g h t of colchicine (0.04% s o l u t i o n in distilled water). Immediately after being removed the microchambers are p l a c e d in a 0.5% s o l u t i o n of p r o n a s e (Calbiochem) in n o r m a l saline a n d g e n t l y s h a k e d for 10 to 15 m i n to free t h e cell coat w h i c h covers t h e m . T h e p r o n a s e s o l u t i o n is c e n t r i f u g e d a t 600 r p m for 7 m i n a n d t h e cell p e l l e t o b t a i n e d r e s u s p e n d e d ill a 0.7 M s o l u t i o n of KC1 a t 37 ~ for 15 m i n . T h e n ceils are c e n t r i f u g e d a g a i n a n d fixed in 3/1 a b s o l u t e e t h a n o l / g l a c i a l acetic acid. A f t e r one w a s h i n g in fresh fixative, c h r o m o s o m e s p r e a d s a r e p r e p a r e d b y air d r y i n g a n d s t a i n e d w i t h c a r b o l - f u c h s i n , giemsa, acetic orcein or a n y o t h e r c h r o m o s o m a l stain. Results and discussion. W e h a v e s t u d i e d w i t h t h e a b o v e p r o c e d u r e a large n u m b e r of r a t s a n d mices a n d m o r e t h a n 50 s p e c i m e n s of A k o d o n b e l o n g i n g to 3 d i f f e r e n t species. I n all cases we were able to o b t a i n a c o n s i d e r a b l e a m o u n t of good m e t a p h a s e s s u i t a b l e for c h r o m o s o m e a n a l y s i s or b a n d i n g procedures. D u r i n g t h e first t r i a l s 3 0 % of a n i m a l s died a f t e r t h e r e m o v a l of m i c r o c h a m b e r s (no a n i m a l died d u r i n g or a f t e r t h e first o p e r a t i o n ) . H o w e v e r , as we g a i n e d e x p e r i e n c e t h e m o r t a l i t y d e c r e a s e d t o zero
1 M. RAY, Can. J. Genet. Cytol. 12, 87 (1970). I. JOHNSON, P. A. SULLIVAN, P. CI~I. CHAN, J. LoBuE, F. C. MONETTE and A. S. GORDON, Ann. N. Y. Aead. Sci. 25, 807 (1967). a R. L. HYBERTSON and H. D. BRYAN, Life Sei. 6, 1047 (1967). 4 S. A. GOODMAN,M. G. CHE~ and T. MAKINODAN, J. Immun. 108, 1387 (1972). 6 L. RUMI, I. LARRIPA S. B. DE SALUMand C. D. PASQUALINI, Eur. J. Cancer, in press.
