Intramitochondrial Fibers With DNA Characteristics

8/2/2019 Intramitochondrial Fibers With DNA Characteristics

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I N T R A M I T O C H O N D R I A L F I B E R SW I T H D N A C H A R A C T E R I S T I C SI . Fixat ion and Elec tron Sta ining React ions

MAR GI T M. K. NASS , Ph.D., and SYL VAN NASS, Ph.D.From the Wenner-Gren Inst itute for Experimental Biology, Stockholm University,Stockholm, Sweden

ABSTRACTChick embryo mitochondria, studied with the electron microscope, show crista-free areasof low electron opacity. These areas are observable after fixation with osmium tetroxide,calcium perm angana te, potassium permanganat e, formaldehyde, acrolein, acrolein followedby osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resultedin an increased density of a fibrous material within these areas. The appearance of thefibrous structures varied with the fixative employed; after fixation with osmium tetroxidethe material was clumped and bar-like (up to 400 A in diameter), whereas after treatmentof osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrousstructures could be visualized as 15 to 30 A fibrils. Treat men t with ethylenedi amin e-tetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. Afterfixation with calcium permanganate or potassium permanganate, or a double fixation byuranyl acetate followed by calcium permanganate, the fibers appeared to have a patternand ultrastructure similar to that observed after the osmium tetroxide-uranyl acetatetechnique, except that some of them had a slightly greater diameter (up to 50 A). Otherfixatives did not preserve the fibers so well. The fibers appeared strongly clumped byformaldehyde fixation, and were difficult to identify after fixation with acrolein or aceticacid-alcohol, The staining of nucleic acid- conta ining structures by uranyl acetate an dlead hydroxide was improved by trea tment of osmium tetroxide-fixed sections with hydrogenperoxide, a nd t he mito chondrial fibers also had an increased density in the electron beamafter this procedure. The staining characteristics suggest the fibrous material of chickembryo mitochondria to be a nucleic acid-co ntain ing structure, and its variable appearanceafter different fixations parallels that previously reported, or described in this paper, for thenucl eoplasm of bacte ria and blue-gr een algae. The results, in ad ditio n to those describedin the accompanying communication, indicate that these mitochondria contain DNA.

I N T R O D U C T I O NRecent progress in preparative techniques hasadvanced our knowledge of nucleic acid structureand has led to some underst andin g of the essentialrequirements for adequate fixation and visualiza-

tion of nucleic acids and nucleic acid-containingstructures in elec tron microscopy (13, 16, 17, 40,42, 43).

Some of these techniques have been a dapted by

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u s in a n a t t e m p t t o c h a r a c t e r iz e c h e m i c a l l y af i b r o u s s t r u c t u r e f i rs t o b s e r v e d w i t h i n c h i c ke m b r y o m i t o c h o n d r i a a f t er t h e u s e o f e l e c t r o ns t a i n i n g r e a c t i o n s ( 2 6 , 28 ) . T h e l o w c o n t r a s t o ft h e s t r u c t u r e s a f t e r f i x a t i o n w i t h b u f f e r e d o s m i u mt e t r o x i d e s o l u t i o n s , t h e i r i n c r e a s e d d e n s i t y a f t e rs t a i n i n g w i t h l e a d s a lt s a n d u r a n y l a c e t a te , a n dt h e i r s t r u c t u r a l s i m i l a r i t y t o s o m e k n o w n D N A -c o n t a i n i n g r e g i o n s i n o t h e r o r g a n i s m s a n d o r -g a n e l l e s l e d u s t o a t t e m p t a m o r e s y s t e m a t i cs u r v e y o f t h e s i m i l a r i t i e s b e t w e e n t h e r e a c t i o n s o ft h e m i t o c h o n d r i a l f ib e r s a n d t h e r e p o r t e d c h a r -a c t e r i s ti c s o f p u r i f i e d D N A a n d b a c t e r i a l n u c l e o -p l a s m .

T h i s f i r s t c o m m u n i c a t i o n d e s c r i b e s t h e a p p e a r -a n c e o f t h e f i b r o u s m a t e r i a l i n q u e s t i o n a f t e r an u m b e r o f " s i n g l e " a n d " d o u b l e " f i x at i on s a n da l s o t h e e f f ec t s o f s o m e e l e c t r o n c o n t r a s t i n g a g e n t s .A t t e m p t s h a v e b e e n m a d e t o d e m o n s t r a t e t h a t( a ) t h e a r e a s o f l o w d e n s i t y o b s e r v e d i n c h i c ke m b r y o m i t o c h o n d r i a a r e a n o rm a l c o m p o n e n to f t h e s e m i t o c h o n d r i a , ( b) t h e r o d - l i k e f i b r o u ss t r u c t u r e s i n t h e s e a r e a s h a v e e l e c t r o n - s t a i n i n gc h a r a c t e r i s t i c s c o n s i s t e n t w i t h t h e v i e w t h a t t h e yc o n t a i n n u c l e i c a c i d , a n d ( c) t h e e f f e ct s of v a r i o u sf i x a ti v e s u p o n t h e m i t o c h o n d r i a l f ib r o u s s t ru c -t u r e s p a r a l l e l t h e r e p o r t e d e f f e ct s o f t h e s e a g e n t su p o n t h e b a c t e r ia l n u c l e o p l a s m a n d p u r i f i e d D N A .T h e r e p o r t t h a t f o l lo w s d ea l s w i t h e n z y m a t i ct r e a t m e n t s o f f ix e d t i s su e s .M A T E R I A L S A N D M E T H O D S

F I X A T I O N : C h i c k e m b r y o s w e r e i n c u b a t e d f r o m0 h o u r s t o 8 d a y s a t 3 7 C . T h e f i x a t io n p r o c e d u r ed e s c r i b e d r e fe r s to t h e 1 9 - h o u r e m b r y o ( d e f i n it i v es t r e a k s t a g e ). T h i s s t a g e h a s b e e n u t i l i z e d f o r m o s t o ft h e i l l u s t r a t i v e m a t e r i a l i n o r d e r t o f a c i l i t a t e c o m -p a r i s o n s b e t w e e n v a r i o u s l y t r e a t e d t i s s u e s . A f t e r t h ee g g w a s o p e n e d a n d t h e o v e r l y i n g e g g w h i t e r e -m o v e d , t h e f i x a t iv e w a s q u i c k l y p i p e t t e d u p o n t h el i v e e m b r y o in situ. A f t e r 5 m i n u t e s ' f i x a t i o n t h ee m b r y o w a s e x c is e d , t r a n s f e r r e d t o a b u f f e r e d s o l u t i o n( v e h i c le o f t h e f i x a t i v e) w h e r e a d h e r e n t y o l k a n d t h ev i te l li n e m e m b r a n e w e r e r e m o v e d w i t h i n a b o u t 3 0s e c o n d s , a n d t h e n f i x a t i o n w a s c o n t i n u e d . F u r t h e rd i s s e c ti o n w a s p e r f o r m e d d u r i n g t h e s u b s e q u e n t3 0 - m i n u t e w a s h i n g p e r i o d .

T h e f i x a t i v e s a n d c o n d i t i o n s o f f i x a t i o n e m p l o y e da r e s u m m a r i z e d i n T a b l e I . T h e s e c o n d f ix a t iv e w a sa p p l i e d e i t h e r d i r e c t l y a f t e r t h e f i r s t f i x a t i o n a n dw a s h i n g p e r i o d o r a f t er s o m e i n t e r v e n i n g c h e m i c a lt r e a t m e n t ( 2 9 ) .

Escherichia coli ce l l s we r e f i x ed wi th f i x a t iv e s 1

( 0 .3 4 M, p H 8 . 0) o r 8 , T a b l e I , a t r o o m t e m p e r a t u r ef o r 1 h o u r , w a s h e d , a n d s u b s e q u e n t l y m i x e d w i t hag a r ( 1 7 ) .

T h e s p e c i m e n s w e re d e h y d r a t e d i n a n e t h y l a l c oh o ls e r ie s ( 3 0 t o 1 0 0 p e r c e n t ) a n d e m b e d d e d i n E p o n8 1 2 ( 2 2 ) .

] ~ L E C T R O N M I C R O S C O P Y : Sec t io n s , ap p r o x i -m a t e l y 6 0 t o 9 0 m / z , w e r e c u t w i t h a P o r t e r - B h i mm i c r o t o m e e q u i p p e d w i t h g l a s s k n i v e s , p i c k e d u p o nu n c o a t e d o r F o r m v a r - c o a t e d g r i d s , a n d e x a m i n e d i na S i e m e n s E l m i s k o p I , o p e r a t e d a t 6 0 k v , w i t h ac o n d e n s e r a p e r a t u r e o f 1 0 0 o r 2 0 0 / z a n d a n o b j e c ti v ea p e r a t u r e o f 2 0 o r 5 0 # . T h e e l e c t r o n o p t i c a l m a g n i f i -c a t i o n s v a r i e d b e t w e e n 1 , 00 0 a n d 3 0 , 00 0 .

T R E A T M E N T O F S E C T I O N S : M o s t s e c t i o n s w e r es t a i n e d w i t h a l k a l i n i z e d l e a d a c e t a t e ( 4 2 ) f o r 1 t o 2m i n u t e s , l e a d h y d r o x i d e ( 1 4) f o r 1 5 m i n u t e s , u r a n y la c e t a t e ( 1 3 ) f o r 3 t o 5 h o u r s , o r l a n t h a n u m n i t r a t e( 4 0 ) fo r 1 5 m i n u t e s , s i n g l y o r i n c o m b i n a t i o n .

S o m e s e c t i o n s f i x e d w i t h o s m i u m t e t r o x i d e o ra c r o l e i n f o l l o w e d b y o s m i u m t e t r o x i d e w e r e o x i d i z e dw i t h 2 p e r c e n t h y d r o g e n p e r o x i d e f o r 3 0 m i n u t e sb e f o r e s t a i n i n g ( 9 4 ).R E S U L T SA. Osmium Tetroxide

F i x a t i v e 1 ( T a b l e I ) , ~ 0 . 3 4 ~ , p H 8 . 0, w a sf o u n d t o b c m o s t s a t i s fa c t o ry f o r o s m i u m t c t r o x i d ef i x a t io n s . T h e p H o f t h c f i x a t i v e w a s a c r i t i c a lv a r i a b l e f o r t h e s e t i s su e s ; a t a n d b e l o w p H 7 . 4t h e r e w a s a p a r t i a l d i s r u p t i o n o f t h e c e ll s , e s p e c i a l l yo f t h e n u c l e a r c o n t e n t s a n d t h e c e ll m e m b r a n e s .

F i g . 1 i s a s u r v e y m i c r o g r a p h w h i c h i l l u s t r a t e st h e e c t o d e r m a l c e ll s o f t h e 1 9 - h o u r c h ic k e m b r y oi n t h e r e g io n o f H e n s e n ' s n o d e . T h e " g e n e r a l "f i x a t i o n q u a l i t y , c x c e p t fo r ce l l m e m b r a n e s , a p -p e a r s t o b e g o o d . I n t h i s d o u b l e - t r e a t e d t i s su c( o s m i u m t e t r o x i d e f ol l ow e d b y u r a n y l a c e t a t e ) ,c o n t r a s t i n g o f t h e s e c t i o n s w i t h l e a d h y d r o x i d ei n c r ea s e s t h e e l c c t r o n o p a c i t y o f m a n y s t r u c tu r e s ,i n c l u d i n g t h e n u c l c ol i . U n l i k e t h e c h r o m a t i n o fm o s t a d u l t n u c l e i , h o w e v e r , th e c h r o m a t i n i ca r e a s o f t h c s c la r g c e m b r y o n i c n u c l e i a r c n o t w e l ld e f i n ed a n d c o n s e q u e n t l y n o t w e l l c o n t r a s t e d b yt h e s e p r o c e d u r e s .

I t i s t o b e o b s e r v e d t h a t a n a r e a ( 0. I t o 0 .3 # ) o fv e r y l o w e l e c t r o n o p a c i t y i s p r e s e n t w i t h i n t h em a t r i x o f m o s t o f t h e l o ng , s l e n d e r m i t o c h o n d r i a( w h i c h a r c a b o u t 0 . 5 # in d i a m e t e r a n d u p t o 2 0# i n le n g t h ) . T h e s e " c l e a r " a r e a s r e m a i n e d u n -a l t e r e d i n s i z e w h e n t h e t o n i c i t y o f t h e f i x a t iv ew a s r a i s e d t o 0 . 4 5 M .

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T A B L E ITime and Time andFixative temperature Second fixative temperature

1 . 2 p e r c e n t o s m i u m t e t r o x i d e 1 h o u r a t 0 t o - - - -i n b u f f e r e d s a l t s o l u t i o n , 2 C0.34 to 0 .45 M (46) , pH 7 .0 to8 .6

3.* " "

4 . 1 p e r c e n t o s m i u m t e t r o x i d ei n b u f f e r e d s u c r o s e s o l u t i o n(4 ) , pH 8 .0

5 . 4 p e r c e n t f o r m a l d e h y d e , 7 .5p e r c e n t s u c r o s e , i n p h o s -p h a t e b u f f e r ( 1 1 ) , p H 8 . 06. ~c t~

7 . 1 0 p e r c e n t a c r o l e i n i n p h o s -p h a t e b u f f e r ( 2 1 ) , p H 8 . 0

8. " "

" " 0 . 5 p e r c e n t u r a n y l a c e - 2 h o u r s a t r o o mt a t e ( 17 ) i n b u f f e r e d t e m p e r a t u r es a l t s o l u t i o n ( 4 6) , p H5 .3

" " 0 . 25 M e t h y l e n e d i a m i n e - " "t e t r a a c e t a t e( E D T A ) ( 17 ) i n b u f f -e r e d s a l t s o l u t i o n ( 4 6)a s i n f i x a t i v e 2

11/~ hou r s a t 0 to - - - -2 C

I h o u r a n d 2 4hou r s a t 0 to 2 (2

3 0 m i n u t e s a tr o o m t e m p e r -a t u r e

9. " " " "

1 0. 3 p e r c e n t p o t a s s i u m p e r m a n - 1 0 m i n u t e s a tg a n a t e ( 20 ) i n b u f f e r e d s a l t r o o m t e m p e r -s o l u t i o n ( 4 6) , p H 8 . 0, o r i n a t u r ew a t e r

1 1. 3 p e r c e n t c a l c i u m p e r m a n g a - 1 0 m i n u t e s a th a t e ( 1) i n b u f f e r e d s a l t s o - r o o m t e m p e r -l u t i o n ( 4 6) , p H 8 . 0 a t u r e

1 2. 2 p e r c e n t u r a n y l a c e t a t e , i n 1 h o u r a t 0 t o 2 Cb u f f e r e d s a l t s o l u t i o n ( 4 6) ,p H 4 . 6

1 3. A c e t i c a c i d - a l c o h o l , 1 : 3 ( 5) 3 0 m i n u t e s a t 0t o 2 C

14 . " " " "

F i x a t i v e 4 1 h o u r a t r o o mt e m p e r a t u r e o r 0t o 2 C

F i x a t i v e 1 ( 0 .3 4 M , p H 2 h o u r s a t r o o m8 . 0 ) t e m p e r a t u r e

U r a n y l a c e t a t e a s i n f i xa - 2 h o u r s a t r o o mt i v e 2 t e m p e r a t u r e

F i x a t i v e l l 1 0 m i n u t e s a t r o o mt e m p e r a t u r e

U r a n y l a c e t a t e a s i n f i x a- 2 h o u r s a t r o o mt i v e 2 t e m p e r a t u r e

* I n t h e s e e x p e r i m e n t s , e a c h e m b r y o w a s b i s e c t e d a c r o s s t h e p r i m i t i v e s t r e a k ; o n e h a l f w a s t r e a t e d w i t hu r a n y l a c e t a t e , a n d t h e o t h e r h a l f w i t h E D T A . I t w a s p r e v i o u s l y a s c e r t a i n e d t h a t t h e r e w a s n o a p p a r e n ts t r u c t u r a l v a r i a t i o n b e t w e e n m i t o c h o n d r i a f r o m d i f f e r e n t a r e a s o f t h e p r i m i t i v e s t r e a k e c t o d e r m .

1. UNSTAINED SECTIONS OF MITOCHONDRIAF i g. 2 r e p r e s e n t s a h i g h e r m a g n i f i c a t i o n o f 2

m i t o c h o n d r i a f r o m a n o s m i u m t e t r o x id e - f i x ede m b r y o ( p r i n t e d fo r m a x i m u m c o n t r a s t ) . W i t h i n

t h e m i t o c h o n d r i a l a r e a s o f l o w d e n s i t y m a y b eo b s e r v e d c l u m p e d f i b er s of l o w c o n t r a s t ( a r r o w s ).T h e r e g i o n s o f l o w e l e c t r o n o p a c i t y a r e n o tb o u n d e d b y m e m b r a n e s .

MAI~GIT M. K. NASS AND SYLVAN NASS I n t r a m i t o e h o n d r i a l D N A F i b e r s . l 59 5



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~. SECTIONS OF MITOCHONDRIA CONTRASTEDWITH HEAVY METAL IONS

Staining with uranyl acetate and /o r lead com-pounds, based on procedures described by Huxleyand Zubay (13) (but after a ~ -h ou r or 1-hourfixation period), or with lanthanum nitrate (40)increases the general contrast of most cell struc-tures, but most dramatically enhances the densityof the clumped mitochondri al fibers. In compari ngFigs. 2 and 3, it becomes apparent that the mito-chondrial fibers after staining with uranyl acetatehave increased in contrast relative to the mito-chondrial membranes and are now of approxi-mately equal density. The fibrous structures arecontrasted to a similar extent by treatment ofsections with uranyl acetate or lead hydroxide,but sequential staining further increased theircontrast.

In any tissue section there is a wide variationin the number of electron-transparent areas permitochondrion and in the number, size, and shapeof the structures st ained by the heav y metal com-pounds. The diameters of the fibers vary between15 and 400 A, and the thicker bar -shape d struc-tures are sometimes resolvable into a numberof parall el strands. In many sections the thinne rfibers appear to extend or attach to the mem-branes of the mito chondria (26). Mitochond rialfibers have been observed at all developmentalstages studied.3. TREATMENT OF SECTIONS WITH HYDROGENPEROXIDE

Treatment of osmium tetroxide-fixed tissuesections with hydrogen peroxide increases thestaining of nucleic acid-containing structures byheavy metal compounds (24). Sections were

treated with 2 per cent hydrogen peroxide for30 minutes (24), rinsed, and then contrasted withuranyl acetate and/o r lead hydroxide (Fig. 4)Under these conditions, membranes have a lowdensity, and the ribosomes and mitochondrialfibers are the only cytoplasmic structures whichare strongly electron-opaque.

The peroxide treatment removed the mito-chondrial dense granules, leaving holes in thesection, and thus this reagent is also of value indistinguishing between mitochondri al fibers whichhave been cut in cross-section and the densegranules.

Sectioned Escherichia coli fixed in os mium tetrox-ide as used for chick embryos, are shown un-stained in Fig. 5 and after the peroxide treatmentand staining in Fig. 6. The nucleoplasm has thetypical clumped appearance seen after osmiumfixation. After the peroxide and staining proce-dures the clumped nucleoplasm and the ribosomesare heavily contrasted (Fig. 6), and previouslyundetected fine strands can be seen radiatingfrom the dense, aggregated portions of the nucleo-plasm. It is apparent that both the mitochondrialfibers, described above for chick embryo, andthe bacterial nucleoplasm have a high contrastafter these procedures, but they are not visiblyaltered in structure.4. URANYL ACETATE AND EDTA TREATMENTSOF TISSUES

Figs. 7 and 8 represent micrographs from ad-jacent ectodermal regions of a single embryoafter osmium tetroxide-fixation followed by im-mersion in uranyl acetate or ED TA solutions (17).After these treatments the mitochondrial fibersno longer appear as bar-like structures (up to 400

Ex p l a n a t i o n o f F i g u r e sArrows, mitochondrial electron-trans- n, nucleus

parent areas and/or mitochondrial nu, nueleolusfibers v, cytoplasmic vesiclecm , cell membrane y, yolk globule, at various stages of di-er, endoplasmic reticulum gestionm, mitochondrionAll micrographs of chick embryos depict the 19-hour Hensen's node or primitive streak.FmvaE 1 Survey micrograph of chick node (ectoderm) fixed with buffered ~ per centosmium tetroxide, with salts added to ~ 0.34 M, at pH 8.0; tissues treated with 0.5 percent uranyl acetate, sections stained with lead hydroxide. Almost all the mitochondriawithin tile field have areas of low density in thei r matrices. X 8,200.

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MARC, T M . K. NA ss AND SYLVAN NASS Intramitochondrial DNA Fibers. I 59 7



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A i n d i a m e t e r ) t y p i c a l o f o s m i u m t e t r o x i d e - f i x e dm i t o c h o n d r i a b u t m a y b e r e c o g n i z e d a s c l u st e rso f f in e f i b ri ls . T o f a c i l i t a te c o m p a r i s o n o f t h ee f fe c ts o f t h e s e tr e a t m e n t s , b o t h m i c r o g r a p h s a r ee n l a r g e d t o 8 0 , 0 0 0 d i a m e t e r s . A t t h i s m a g n i f i c a -t i o n t h e m i t o c h o n d r i a l f i b r i ls ( 1 5 t o 3 0 A ) o f t h eu r a n y l a c e t a t e - t r e a t e d t i s s u e s m a y n o t b e v i s i b l ei n t h e r e p r o d u c t i o n o f t h e p r i n t ( s e e F i g . 9 f o r th ef i n e s t r u c t u r e o f t h e f b r i l s a f t e r t h i s t r e a t m e n t ) ,b u t t h e E D T A - t r e a t e d t i s s u e s c l e a r l y s h o w m i t o -c h o n d r i a l f i b e r s ( 3 0 t o 6 0 A ) . A h i g h e r m a g n i f i -c a t i o n o f a m i t o c h o n d r i o n a f t e r u r a n y l a c e t a t er i n s in g i s p r e s e n t e d i n F i g . 9. T h e b r a n c h e d a n di r r e g u l a r b e a d e d a p p e a r a n c e o f t h e f i b ri ls isc h a r a c t e r is t i c o f t h e i r s t r u c t u r e a f t e r t h is t r e a t m e n to r a f t e r f i x a t i o n w i t h c a l c i u m p e r m a n g a n a t e( se e s e c t i o n B ) .B. Calc ium and Potass ium Permanganate

F i x a t i o n w i t h e i t h e r 3 p e r c e n t c a l c i u m p e r -m a n g a n a t e ( l ) o r p o t a s si u m p e r m a n g a n a t e ( 20 )i n b u f f e r e d s a l t s o l u t i o n ( 4 6 ) , a t p H 8 . 0 , g e n e r a l l yr e s u l te d i n b e t t e r p r e s e r v a t i o n o f m e m b r a n o u sc o m p o n e n t s o f t h e c e l l t h a n d i d f i x a t i o n w i t h a n yo f t h e o s m i u m f i x a t iv e s e m p l o y e d . T h e s u r v e ym i c r o g r a p h ( F i g . 1 0 ) s h o w s th e j u n c t i o n b e t w e e ne c t o d e r m a n d m e s o d e r m . T h e m i t o c h o n d r i aa p p e a r s w o l l e n ( d i a m e t e r s u p t o 1 / z) r e l a t i v e t ot h e i r a p p e a r a n c e a f t e r o s m i u m t e t r o x i d e - f i x a ti o n .I t is a g a i n a p p a r e n t t h a t m o s t o f t h e m i t o c h o n d r i ai n t h e f i e ld h a v e a n a r e a o f l o w d e n s i ty w i t h i n t h e i rm a t r ix . A f t e r e i th e r p o t as s iu m p e r m a n g a n a t e - o rc a l c iu m p e r m a n g a n a t e - f i x a t i o n t h e m i t o c h o n d r i alf i b e r s a r e f a i n t l y v i s i b l e w i t h i n t h e l o w d e n s i t ya r ea s . U r a n y l a c e t a t e - t r e a t m e n t o f ti ss u es , s ta i n -i n g o f se c ti o n s w i t h u r a n y l a c e t a te , l e a d h y d r o x i d e ,o r d o u b l e s t a in i n g p r o d u c e a n o v e r - a l l i n c r e a s e i nc o n t r a s t o f a l l c e l l s t r u c t u r e s . I n m a n y s e c t io n s o fm i t o c h o n d r i a v e r y f in e fi b ri ls a p p r o x i m a t e l y 1 5t o 3 0 A i n d i a m e t e r a n d s o m e f i b e r s o f s l i g h t l yg r e a t e r d i a m e t e r s ( u p t o 5 0 A ) a r e m o r e r e a d i l yo b s e r v e d a f t e r t h es e c o n t r a s t in g p r o c e d u r e s ( F i g.l 1 ). T h e f i n e f i b r il s a p p e a r b e a d e d a n d p o r o u s

a l o n g t h e i r l e n g t h a n d f r e q u e n t l y s e e m t o m e r g ea n d b r a n c h .C. A erolein

T i s s u e s f ix e d w i t h l 0 p e r c e n t a c r o l e i n ( 2 1 ) i np h o s p h a t e b u f f e r , p H 8 . 0 , a n d p o s t f ix e d w i tho s m i u m t e t r o x i d e s h o w , i n a d d i t i o n t o t h e m i t o -c h o n d r i a l a r e a s o f l o w d e n s i t y , t h e e x c e l l e n t p r e s -e r v a t i o n o f c e ll u l a r m e m b r a n e s , e s p e c i al l y t h o s eo f t h e e n d o p l a s m i c r e t i c u l u m ( F ig . 1 2 ). T h en u c l e u s , a f t e r s t a i n i n g w i t h l e a d s a l t s , c l e a r l ys h o w s a n u c l e o l u s a n d a n a r r o w , i n d i s t i n c t c h r o m a -t i n i c m a t e r i a l b o r d e r i n g t h e in n e r n u c l e a r m e m -b r a n e . A l t h o u g h t h e m i t o c h o n d r i a l f ib e r s a r eo f t e n i d e n t i f i a b l e a s f i n e f i b r i l s a f t e r t h e s t a i n i n go f a c r o l e i n - O s O 4 - f i x e d t i s s ue s , t h e y a r e n o t p r e -s e r v e d i n a r e g u l a r o r d i s t i n c t p a t t e r n ( F i g . 1 2 ).U r a n y l a c e t a t e t r e a t m e n t o f ti ss u es f ix e d i n a c r o -l e i n a ls o d i d n o t p r e s e r v e t h e f i b e r s in a n y d e f i n i t e l yr e c o g n i z a b l e f o r m . S i m i l a r ly , t h e b a c t e r i a l n u c l e o -p l a s m a f t e r d o u b l e f i x a t i o n w i t h a c r o l e i n a n dO s O 4 ( a n d s t a in i n g o f s ec t io n s w i t h l e a d o r u r a n y li o n s ) i s r e l a t i v e l y i n d i s t i n c t (cf. Fi g s . 6 a n d 1 3 ) .N e v e r t h e l e s s , f i b r i l l a r m a t e r i a l ( 2 0 t o 5 0 A i nd i a m e t e r ) n o t u n l i k e t h a t o b s e r v e d i n m i t o c h o n -d r i a a f t e r t h e s e p r o c e d u r e s (of. Fi g s . 1 2 a n d 1 3 )i s m o r e a p p a r e n t i n t h e b a c t e r i a l n u c l e o p l a s mt h a n i n t h e c h i c k m i t o c h o n d r i a b e c a u s e o f t h eh i g h e r c o n c e n t r a t i o n o f t h is m a t e r i a l i n t h e b a c -t e r i u m .D. Formaldehyde

F i x a t i o n w i t h f o r m a l d e h y d e o r w i t h f o r m a l d e -h y d e f o l l o w e d b y o s m i u m t e t r o x i d e c au s e s am a r k e d s h r i n k a g e o f t h e m i t o c h o n d r i a r e l a t i v e t ot h e i r a p p e a r a n c e a f t e r a l l o t h e r f i x a t i o n m e t h o d s .D e s p i t e t h e s h r i n k a g e a n d g e n e r a l i n c r e a s e i ne l e c t r o n o p a c i t y o f t h e m i t o c h o n d r i a l m a t r i x , t h ea r e a s o f l o w d e n s i t y a r e s t i ll a p p a r e n t ( F i g . 1 4 ),i n d i c a t i n g t h a t t h e s e r e g i o n s a r e , a t l e a s t i n t h e i rr e s p o n s e t o f i x a t i o n , r e l a t i v e l y r ig i d s t r u c t u r e s . Af e w d e n s e f i b e r s w i t h i n t h e s e a r e a s o f lo w d e n s i t y

FmUB E ~ Un s t a i n e d se ct io n s o f mi t o c h o n d r i a . T i s su e f ix e d wi t h t h e o smi u m t e t ro x i d ef i x a t i v e d e sc r ib e d i n l e g e n d t o F i g . 1 . Th i s m i c ro g ra p h sh o ws so me fa i n t l y v i s ib l e ma t e r i a lwi t h i n t h e e l e c t ro n - t ra n sp a re n t a re a s o f t h e mi t o c h o n d r i a . )< 5 7 ,0 0 0 .FIGUR E $ Se c t i o n o f mi to c h o n d r i o n o f a 5 5 -h o u r e mb ry o ( t a il b u d ) s t a i n e d wi t h u ra n y la c e t a t e . Al t h o u g h t h e re i s a g e n e ra l i n c rea se in c o n t ra s t , i t i s a p p a re n t t h a t t h e n o w c l e a r lyv i s i b l e c l mn p e d mi t o c h o n d r i a l f i b e r s a re p re fe re n t i a l l y s t a i n e d a s c o mp a re d t o t h e i r a p -pearance in Fig . 2 . X 105 ,000 .

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i n fo r m a l d e h y d e - f i x e d , l e a d - s t a in e d m i t o c h o n d r i am a y a l s o b e s e e n i n F i g . 1 4.E . O t h e r F i x a t i v e s

T w o o t h e r f i x a ti v e s w e r e s t u d i e d, a c e t i c a c i d -a l c o h o l , 1 : 3 , a n d 2 p e r c e n t u r a n y l a c e t a t e f o l-l o w e d b y c a l c i u m p e r m a n g a n a t e . A f t e r s t a i n in gt h e a c e t i c a l c o h o l - f i x e d m a t e r i a l w i t h u r a n y la c e t a t e a n d / o r l e a d h y d r o x id e , o r a f t e r u r a n y la c e t a t e - t r e a t m e n t o f t is s ue b lo c k s, t h e m i t o -c h o n d r i a w e r e b a r e l y v i s i b l e ( n o m e m b r a n e w a ss e e n) , t h e c e n t e r a n d i n n e r m a r g i n s o f t h e n u c l e iw e r e h e a v i l y s t a in e d , a n d t h e r i b o s o m e s s h o w e di n t e n s e c o n t r a s t . T h e m i t o c h o n d r i a , s o m e t i m e so b s e r v e d a s a r e a s o f f a i n t d e n s i t y , h a d t r a c e s o fw h a t a p p e a r e d t o b e t h e f ib r o u s c o m p o n e n t .

S o m e p o r t i o n s o f t h e t i s su e w e r e p r e s e r v e d a f t e rs u c ce s s iv e tr e a t m e n t s w i t h u r a n y l a c e t a t e a n dc a l c i u m p e r m a n g a n a t e . T h e u r a n y l f i x a t iv e ( p H4 . 5 ) , k n o w n t o p r e s e r v e p u r i f i e d D N A f i b e r s (4 0 ) ,k e p t m a n y o f t h e 1 5 t o 3 0 A m i t o c h o n d r i a l f i b r il si n t a c t , d e s p i t e d i s r u p t i n g m o s t o t h e r c e l l c o n -s t i t u en t s (2 8 ) .D I S C U S S I O NT h e r e s u l t s p r e s e n t e d i n d i c a t e t h a t e l e c t r o n -t r a n s p a r e n t a r e a s w i t h in c h i c k e m b r y o m i t o -c h o n d r i a a r e r e p e a t e d l y o b s e r v e d a f te r m a n yd i f f e r e n t f i x a t i o n p r o c e d u r e s . T h e y a l s o s h o w t h a tt h e v a r i o u s t e c h n i q u e s r e p o r t e d t o i n c re a s e t h es p e c i f i c it y o f t h e s t a i n i n g o f n u c l e i c a c i d - c o n -t a i n i n g s t r u c t u r e s b y h e a v y m e t a l c o m p o u n d si n c r e a s e t h e d e n s i t y o f f ib r o u s s t r u c t u r e s f o u n d i nt h e l o w d e n s i ty r e g io n s . F u r t h e r , r e a g e n t s k n o w nt o s ta b i l i z e t h e b a c t e r i a l n u c l e o p l a s m a n d p u r i f i e dD N A f i be r s h a v e t h e s a m e e f f e c t o n t h e m i t o -c h o n d r i a l f i b e rs ( o s m i u m t e t r o x i d e + u r a n y la c e t a t e , u r a n y l a c e t a t e , a n d p o t a s s i u m p e r m a n -g a n a t e ) . A g e n t s , s u ch a s E D T A , w h i c h p a r t i a l l y

a g g r e g a t e t h e b a c t e r i a l n u c l e o p l a s m , o r f i x a t i o ni n o s m i u m t e t r o x i d e o r f o r m a l d e h y d e , w h i c hr e s u lt s in s t r o n g c l u m p i n g o f t h e n u c l e o p l a s m ,o r a c r o l e i n f i x a t i o n , a l l h a v e v e r y s i m i l a r e f f e c t su p o n t h e m i t o c h o n d r i a l f ib e r s .

M I T O C H O N D R I A L A R E A S O F L O W E L E C -T R O N O P A C I T Y : A l t h o u g h m a n y r e p o r t s i n t h el i t e r a t u r e r e l a t e t h e l o ss o f d e n s i t y o f t h e m i t o -c h o n d r i a l m a t r i x t o f i x a t i o n a r t i f a ct s , p o s t m o r t e mc h a n g e s , n e o p l a s t i c t r a n s f o r m a t i o n s , a n d t o th ee f f e c ts o f c e r t a i n c h e m i c a l s a d m i n i s t e r e d t o a n i -ma l s (1 5 , 4 6 , cf . r e f e r e n c e s 3 0 a n d 3 6 ) , t h e r e s u l t so f th e p r e s e n t i n v e s t i g a t i o n d o n o t i n v i t e s u c h i n -t e r p r e t a ti o n . T h e m i t o c h o n d r i a l a r e a s o f l o w e le c -t r o n o p a c i t y a r e c o n s i d e r e d t o b e a s t r u c t u r a l c o m -p o n e n t b e c a u s e t h e y w e r e o b s e r v e d a f t e r f i x a t i o nb y a l l t h e m e t h o d s e m p l o y e d , a n d a t v a r i o u s p Ha n d s a lt c o n c e n tr a t io n s . F u r t h e r m o r e , t h e f a c t t h a te a r l y c h i c k e m b r y o s c a n b e f i x e d d i r e c t l y w i t h o u tp r i o r k i l l i n g a n d d i s s e c t in g o f t h e e m b r y o , a n d t h a tt h e f i x a t i v e s p e n e t r a t e v e r y r a p i d l y i n t o a l l p a r ts o ft h e s e s m a l l o r g a n i s m s s e e m s t o r u l e o u t a c o n -s i d e r a ti o n o f p o s t m o r t e m c h a n g e s o c c u r r i n g b e f o ref i x a t io n . G e n e r a l l y , r a r i f i c a t i o n i s o b s e r v e d t oo c c u r t h r o u g h o u t t h e m a t r i x o f t h e m i t o c h o n d r i aa n d i s f r e q u e n t l y a c c o m p a n i e d b y s w e l l i n g o f t h ee n t i r e o r g a n e l l e , b u t t h e m i c r o g r a p h s p r e s e n t e dh e r e s h o w n o o v e r - a l l lo s s o f d e n s i t y o f t h e m i t o -c h o n d r i a l m a t r i x , a n d e v e n t h e r e l a ti v e l y s h r u n k e nm i t o c h o n d r i a o b s e r v e d a f t e r f i x a t i on w i t h f o r m -a l d e h y d e s h o w c l e a r a r e a s ( F i g . 1 4 ). T h e l a t t e rf i n d i n g a l s o s u g g e s t s t h a t s u c h a r e a s m a y h a v es o m e s t r u c t u r a l r i g i d i t y .

S P E C I F I C I T Y O F S T A I N I N G R E A C T I O N S :T h e e f fe c ts p r o d u c e d b y h e a v y m e t a l c o n t r a s ti n ga g e n t s u p o n c h i c k e m b r y o t i s s u e s f i x e d i n v a r i o u sw a y s i n d i c a t e t h a t u n d e r c e r t a i n c o n d i t i o n s t h ek n o w n n u c l e i c a c l d - c o n t a l n i n g s t r uc t u r es o f th ec e ll a re m o r e h i g h l y e l e c t r o n - o p a q u e t h a n a r e

FIGURE 4 Chick node t i ssue f ixed wi th the osmium te t roxide f ixat ive described in legendto Fig . 1 . Sect ions t reated for 80 minutes wi th ~ per cent hydrogen peroxide prior to s ta in-i n g w i t h u ran y l ac e t a t e fo l lo w ed b y l ead h y d ro x i d e . Th e co n t ra s t o f t h e mi t o ch o n d r i a lf ibers and ribosomes i s great ly increased over that of the rest of the cy toplasmic com-ponents . The e lect ron-opaque granules of mi tochondria are speci fical ly removed by t i lehydroge n peroxide t reatm ent . )< 60 ,000.FIGURES 5 AND 6 Escherichia col i cel l s f ixed wi th the osmium te t roxide f ixat ive describedin legend to Fig . 1 . Comparison between the nucleoplasms in unsta ined sect ion (Fig . 5)and sect ion t reated wi th hydrogen peroxide and sta ined as described in legend to Fig . 4(Fig . 6) . I t i s apparent that the peroxide t reatment and s ta in ing great ly increases cont rastbut does not v is ib ly a l ter the s t ructu ral appea rance. )< 60,000.

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s t r u c t u r e s c o n t a i n i n g l i t t l e o r n o n u c l e i c a c i d s(F i g s . 2 t o 4 , an d 1 4 ). A l l o f t h e re su l t s a re co n -s i s t en t w i t h t h o s e r e p o r t e d i n t h e l i t e r a t u r e ( 1 3, 1 7,1 9, 2 4 , 4 1 , 4 2 ) . T h u s , a f t e r s t a n d a r d o s m i u mt e t r o x i d e - f i x a t i o n a n d s e c t i o n s t a i n i n g t h e r e o c c u r sa g e n e r a l i n c r e a s e i n c o n t r a s t o f m o s t o r a l l s t ru c -t u r e s , b u t t h e r e l a t i v e i n c r e a s e i n d e n s i t y o f r i b o -s o m e s , n u c l e o l i, a n d m i t o c h o n d r i a l f i b e r s a f t e rs t a in i n g a p p e a r s t o b e s o m e w h a t g r e a t e r t h a nt h a t o f m e m b r a n o u s s t r u ct u r e s ( e . g . , Fi g s . 2 an d 3 ) ,s u g g e s t i n g t h a t t h e r e i s a h i g h e r d e g r e e o f b i n d i n gb e t w e e n h e a v y m e t a l i o n s a n d n u c l e i c a c i d -c o n t a i n i n g s t r u c t u r e s t h a n b e t w e e n t h e s e i o n sa n d t h e m e m b r a n e s . T h e g e n e r a l i z e d st a i n in ge f f e c t o b t a i n e d b y i m m e r s i o n o f o s m i u m t e t r o x i d e -f i x e d t is s u e b l o c k s i n u r a n y l a c e t a t e s o l u t io n s i sa l s o i n a g r e e m e n t w i t h t h e r e s u l t s o b t a i n e d b yK e l l e n b e r g e r e t a l . ( 1 7 ) w i t h b a c t e r i a . S t a i n i n ga f t e r p o t a s s i u m o r c a l c iu m p e r m a n g a n a t e f i x a ti o ns i m i l a r l y r e s u l te d i n a g e n e r a l i n c r e a s e i n c o n t r a s to f m o s t c e l l s t r u c tu r e s .T h e c o n t r a s t in g b y h e a v y m e t a l i o n s a f t e rf i x a ti o n w i t h n o n - m e t a l l i c r e a g e n ts ( f o r m a l d e h y d e ,a c r o le i n , a c e t ic a c i d - a l c o h o l ) a p p e a r s t o b e m o r es p e c i f ic f o r r i b o s o m e s , c h r o m a t i n i c a r e a s , a n dn u c l e o l i t h a n a f t e r m e t a l l i c f i x a t i o n ( e . g . , r e f e r -e n c e s 1 9 a n d 4 1 ) . F o r m a l d e h y d e - f i x e d a n d s t a i n e dm i t o c h o n d r i a s h o w t h e m i t o c h o n d r i a l f i b e r s t ob e d en s e l y s t a in e d . T h e o x i d a t i o n a n d p e r h a p sr e m o v a l o f t h e r e d u c e d o s m i u m t e t r o x i d e c o m -p o u n d s b y h y d r o g e n p e r o x i d e m a r k e d l y i n c r e a s est h e s t a i n i n g s p e c i f i ci t y o f k n o w n n u c l e i c a c i d -c o n t a i n i n g c e l l c o m p o n e n t s b y h e a v y m e t a l c o m -p o u n d s ( 2 4) . A l l o f t h e s e r e su l t s a r e c o m p a t i b l ew i t h t h e v i e w ( 6 ) t h a t t h e r e l a t i v e n o n - s p e c i f i c i t yo f s t a i n i n g o b s e r v e d a f t e r t h e u s e o f m e t a l l i cf i x at i ve s m a y b e t h e r e s u l t o f a c o m p l e x - i o n f o r m a -t i o n b e t w e e n t h e f i x a ti v e a n d t h e c o n t r a s t i n ga g e n t. T h e r e m o v a l o f r e d u c e d o s m i u m t e t r o x i d ec o m p o u n d s b y h y d r o g e n p e r o x i d e c o u l d t h u sa c c o u n t f o r t h e f a c t t h a t a f t e r t r e a t m e n t w i t h t h i s

r e a g e n t t h e h e a v y m e t a l c o n t r a s t i n g a g e n t s d on o t p r o d u c e a g e n e r a l o v e r - a l l i n c r e a s e i n s t a i n i n gb u t a r e l a t i v e l y s p e c i fi c o n e .

S T R U C T U R E O F T H E M I T O G H O N D R I A L F I -B E R S : T h e a p p e a r a n c e o f t h e m i t o c h o n d r i a lf i b e r s v a r i e s w i t h t h e f i x a t i v e e m p l o y e d , a n d t h ee l e c t r o n o p a c i t y v a r i e s w i t h t h e s t a i n i n g r e a c t i o n su s e d. A f t e r o s m i u m t e t r o x i d e - f i x a t io n t h e m i t o -c h o n d r i a l f i b e r s h a v e a l o w d e n s i t y ( F i g . 2 ) ; t h isi s a l s o t r u e o f th e o s m i u m t e t r o x i d e - f i x e d b a c t e r i a ln u c l e o p l a s m i n a r e a s o f c o m p a r a b l e f i b e r c o n -c e n t r a t i o n ( F i g . 5 ) , a n d m a y p e r h a p s r e l a t e tot h e f a c t t h a t p u r i f i e d p r e p a r a t i o n s o f n u c l e i ca c i d s d o n o t p r o d u c e a v i s i b le r e d u c t i o n p r o d u c tw i t h o s m i u m t e t r o x i d e ( 2 ) . T h e d i f f e r e n c e o b -s e r v e d i n t h e d e n s i ty o f t h e o s m i u m t e t r o x i d e -f i xe d , u n s t a i n ed , c l u m p e d b a c t e r i a l n u c l e o p l a s ma n d s i m i l a r l y f ix e d m i t o c h o n d r i a l f i b e r s i s u n -d o u b t e d l y r e l a t e d t o t h e m u c h g r e a t e r c o n c e n t r a -t i o n of t h e b a c t e r i al D N A p e r u n i t a r e a a n d n o t t oi ts r e a c t io n s w i t h o s m i u m t e t r ox i d e . T h e c l u m p e db a c t e r ia l n u c l e o p l a s m a f t e r f i x a t io n w i t h f o r m -a l d e h y d e h a s a d e n s i t y e q u i v a l e n t t o t h a t o b -s e r v e d a f t e r o s m i u m t e t r o x i d e - f i x a t i o n , a l t h o u g ht h e f o r m e r f i x a ti v e c o u ld h a r d l y i n c r ea s e t h ew e i g h t d e n s i t y o f t h e s t r u c t u r e s s i g n i f i c a n t l y ( 2 3) .T h e m i t o c h o n d r i a l f i b er s a r e a l so c l u m p e d b yf o r m a l d e h y d e , b u t , a s w i t h o s m i u m t e t ro x i d e ,t h e y w e r e n o t r e a d i l y v i s u a l i z e d u n t i l s t a i n i n gw i t h h e a v y m e t a l i o n s w a s c a r r i e d o u t .

A f t e r a l l o f t h e s t a i n i n g p r o c e d u r e s e m p l o y e df o l l o w i n g o s m i u m t e t r o x i d e - f i x a t i o n ( F i g s . 3 , 4 ) ,t h e f ib r o u s c o m p o n e n t o f t h e m i t o c h o n d r i a i sr e c o g n i z a b l e a s a b a r - l i k e s t r u c t u r e ( u p t o 4 0 0 Ai n d i a m e t e r ) , u s u a l l y w i t h s o m e f in e f il a m e n t s( 15 t o 5 0 A ) e x t e n d i n g t o w a r d t h e p e r i p h e r y o ft h e m i t o c h o n d r i a ( 2 6) . T h e b a c t e r i a l n u c le o p l a s ma l s o s h o w s f i b r o u s s t r a n d s e x t e n d i n g f r o m t h el a r g e r c l u m p e d p o r t i o n s ( F i g . 6 ) . I t m a y b e n o t e dt h a t i n u n s t a i n e d s e c t io n s o f b a c t e r i a t h e s e f in ef i l a m e n t s a r e n o t d e t e c t a b l e ; i . e . , t h e y h a v e a l o w

FIGURE 7 Por t ion of primi t ive s t reak fixed wi th osmium te t roxide, t rea ted wi th 0 .5p e r cen t u ran y l ace t a t e fo r ~ h o u rs ; s ec t i o n s s t a i n ed w i t h l e ad h y d ro x i d e . Th e a rea s o flow densi ty in the mi toehondria conta in f ine f ibri l s (between arrows), which are barelyvisib le a t th is mag nificat ion (see Fig . 9 for a magnified image of the f ibers after th is t reat -ment). )< 80,000.FIGU RE 8 Po r t i o n o f p r i mi t i v e s t reak ( f ro m t h e same em b ry o a s i n F ig . 7 ) t rea t ed w i t h0 .25 M ED T A (instead of uranyl aceta te) for ~ hours; sect ions s ta ined wi th lead hydroxide.Mitochondria l f ibers are coarsened. The fibers have a d iameter approximately two toseveral t imes that of f ibers observed after uranyl aceta te t reatment . X 80 ,000.

602 TIIE JOURNAL OF CELL BIOLOGY VOLUME 19, 1963



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i n t r i n s i c d e n s i t y c o m p a r a b l e t o th a t o f t h e m i t o -c h o n d r i a l f i b e rs . S i m i l a r f ib r o u s s t r u c t u r e s w i t hs i m i l a r p r o p e r t i e s h a v e b e e n s h o w n t o c o r r e s p o n dw i t h t h e D N A - c o n t a i n i n g p o r t i o n s o f th e k i n e t o -p l a s t - a s s o c i a t e d m i t o c h o n d r i o n o f T r ypanos or na(3 9 ) an d B o d o ( 3 2 ), a n d k a p p a p a r t i c l e s o f P a r a -m e c i u m ( 7 , I 0 ) . S o m e c h e m i c a l l y u n d e f i n e d f i b r i l la rs t r uc t u r es w i t h i n t h e m i t o c h o n d r i a o f g i a n t a m e b a e( 3 1 ) a n d a m a r i n e c i l i a t e ( 9 ) h a v e a l s o b e e n r e -p o r t e d .

T h e u l t r a s tr u c t u r e o f t h e m i t o c h o n d r i a l f i b e r sh a s b e e n f o u n d t o b e s t a b i l i z e d a s b r a n c h i n g 1 5t o 3 0 A f i b r il s ( F i gs . 7 a n d 8 ) a f t e r t r e a t m e n t o ft is s u es b y t h e t e c h n i q u e o f K e l l e n b e r g e r e t a l . (1 7 ) ,w h i c h i s th e g e n e r a l l y a c c e p t e d m e t h o d f o r th ep r e s e r v a t i o n o t t h e b a c t e r i a l n u c l e o p l a s m . T h ef i b r il s a p p e a r m a r k e d l y s i m i l a r i n s u b s t r u c t u r e t op u r i f i e d D N A , a s s h o w n b y S t o e c k e n i u s ( 4 0 ) .U r a n y l a c e t a t e - t re a t m e n t o f o s m i u m t e t r o x id e -f i x ed t i s su es h a s a l so b een sh o w n t o s t ab i l i z e t h eD N A - c o n t a i n i n g r e g i on s o f b l u e - g r e e n a l g a e ( 1 2 ) ,c h l o r o p l a s t s ( 3 5) , a n d k a p p a p a r t i c l e s o f P a r a -m e c i u m ( 3 3 ), a s w e l l a s c h e m i c a l l y u n i d e n t i f i e df i b r il s i n t h e m i t o c h o n d r i o n o f M i c r o m o n a s (34).I t i s o f i n t e re s t t h a t u r a n y l a c e t a t e t r e a t m e n t o fu n f i x e d D N A p r o t e i n f i lm s s t a b il i z e s t h e f i b e r sa g a i n s t a l c o h o l i c d e h y d r a t i o n , a n d i n t h e a b -s e n c e o f u r a n y l a c e t a t e t h e f i b er s a r e c l u m p e d b ya l c o h o l ( 4 0 ) . I t s e e m s p l a u s i b l e t h a t t h e c l u m p e da p p e a r a n c e a s w e l l a s t h e l o w d e n s i t y o f t h em i t o c h o n d r i a l f i b er s a f t e r o s m i u m t e t r o x i d e - f i x a -t i o n m a y b e r e l a t e d t o t h e l a ck o r l o w d e g r e e o ff i x a t i o n o f n u c l e i c a c i d s b y o s m i u m t e t r o x i d e ,w h i c h a l l o w s c l u m p i n g t o t a k e p l a c e d u r i n ga l c o h o l ic d e h y d r a t i o n p r o c e d u re s .F i x a t i o n w i t h e it h e r o f t h e p e r m a n g a n a t e r e -a g e n t s a l s o s t a b i l i z e d t h e m i t o c h o n d r i a l f i b e r s a s ab r a n c h i n g n e t w o r k o f f i b r il s , a n d p o t a s s i u m p e r -m a n g a n a t e h a s b e e n s h o w n t o s t a b i l i z e t h e b a c -t e r i a l n u c l e o p l a s m i n a m a n n e r c o m p a r a b l e t o t h eK e l l e n b e r g e r e t a I . ( 1 7 ) p r o c e d u r e ( 2 5 , cf . r e f e r e n c e1 6) . F u r t h e r s u g g e s t i v e e v i d e n c e t h a t t h e m i t o -c h o n d r i a l f i b e r s c o n t a i n D N A i s t h e c y t o c h e m i c a ld e m o n s t r a ti o n t h a t R N A , b u t n o t D N A , c o r n -

p o u n d s a r e r e m o v e d f r o m c e ll s f i xe d w i t h p o t a s -s i u m p e r m a n g a n a t e ( 3 ).

T h e t r e a t m e n t o f t is s ue s w i t h E D T A i n p l a c e o fu r a n y l a c e t a t e p a r t i a l l y a g g r e g a t e s t h e m i t o -c h o n d r i a l f i b e r s , a g a i n m i m i c k i n g t h e a c t i o n o ft h is m e t a l c h e l a t o r u p o n t h e b a c t e r i a l n u c l e o -p l a s m . T h a t t h e a g g r e g a t i o n i n b o t h b a c t e r i a a n dm i t o c h o n d r i a i s n o t s o g r e a t a s a f t er o s m i u mt e t r o x i d e - f i x a t i o n a l o n e i n d i c a t e s t h a t t h e E D T At r e a t m e n t e f f e c ts s o m e d e g r e e o f s t a b i l i z a t i o n o ft h e f i b e rs .

T h e s i m i l a r a p p e a r a n c e o f t h e b a c t e r i a l n u c l e o -p l a s m a n d t h e m i t o c h o n d r i a l f i b e r s a f t e r a c r o l e i n -o s m i u m t e t r o x i d e d o u b l e f i x a t i o n a n d s t a i n i n ga g a i n a p p e a r s t o r e f l e c t t h e s i m i l a r i t i e s i n t h ec o m p o s i t i o n o f t h e s e s t r u c t u r e s .

I t i s a p p a r e n t t h a t a l l t h e s i m i l a r it i e s b e t w e e nt h e f i b r o u s c o r e s o f t h e c h i c k m i t o c h o n d r i a , a sw e l l a s o f m i t o c h o n d r i a f r o m o t h e r o r g a n i s m s( 2 7 ) , a n d t h e n u c l e o p l a s m o f b a c t e r i a a r e n o ts i m p l y m o r p h o l o g i c a l . T h e s i m i l a r i t ie s in s t r u c -t u r e a f t e r m a n y d i f f e r e n t f i x a t i o n p r o c e d u r e s ,r a t h e r , a r e i n d i c a t i v e o f a s e t o f d i s t i n c t c h e m i c a lr e a c t i o n s w h i c h t h e s e " p l a s m s " s h a r e i n c o m m o n .T h e r e a c t i o n s i n b a c t e r i a a r e c o n s i d e r e d t o b es p e ci f ic f o r h i g h ly h y d r a t e d D N A ( 1 6) n o t c o a t e db y h i s t o n e s ( 44 , 4 7 ) . M a n y o f t h e s e r e a c t i o n sh a v e b e e n d e m o n s t r a t e d t o o c c u r i n o t h e r c y t o -p l a s m i c s t r u c t u r e s c o n t a i n i n g D N A , w h i c h a r ec o n s i d e r e d t o b e " s e l f - r e p l i c a t i n g " (c f . r e f e r e n c e s8 , 1 8 , 3 7 , 3 8 ) . T h e f a c t t h a t m i t o c h o n d r i a , l o n gt h o u g h t t o b e " s e l f - r e p l i c a t i n g " s t r u c t u r e s (c f .r e f e r e n c e s 8 , 1 8 , 3 7 , 4 5 ) , s h o u l d h a v e a c o m p o n e n tw i t h s i m i l a r p r o p e r t i e s i s p e r h a p s n o t s u r p r i s i n g .I t s e e m s t o b e m o s t u n l i k e l y t h a t t h e s t a i n i n g r e a c -t i o n s, w h i c h a r e h i g h l y s u g g e s t i v e o f a n u c l e i c a c i dc o n t e n t i n t h e m i t o c h o n d r i a , a n d t h e p a r a l l e l r e -s u l ts o b t a i n e d i n t h e f i x a t i o n o f m i t o c h o n d r i a l f i b e r sa n d b a c t e r i a l n u c l e o p l a s m a r e s i m p l y f o r t u i t o u s .W h e n w e c o n si d e r t h a t n o o t h e r natur a l l y occur r ings t r u c t u r e t h u s f a r s t u d i e d h a s a ll t h e p r o p e r t i e s d i s -c u s s e d , w i t h r e s p e c t t o f i x a t i o n , s t a b i l i z a t i o n a n ds t a i n i n g , th e u n a v o i d a b l e c o n c l u s i o n a p p e a r s t ob e t h a t t h e m i t o c h o n d r i a l f i b er s c o n t a i n D N A .

FIGURE 9 High er magnificat ion of a mi tocho ndrion from an osmium te t roxide -fixed ,u ran y l ace t a t e - t rea t ed emb ry o , a s i n F i g . 7 ; s ec t i o n s ta i n ed w i t h l e ad h y d ro x i d e . Th e 1 5 t o30 A fibri l s have bulbous and porous enlargements a long thei r length and often appear tob ran ch . (Th e ce l l mem b ran e an d memb ran es o f t h e mi t o ch o n d r i o n a re c l ea r ly v i si b l e a st r i l ami n a t e s t ru c t u re s , ex cep t w h e re t h e memb ran es a re i n i n t i ma t e a s so c i a t i o n w i t h t h ei rregular vesicular s t ructures observable in these cell s .) X ~5,0 00.

604 THE JOUnNALOF CE LL BIOLOGY VOLUME 19, 1963



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T h e f o ll o w i n g p a p e r w i l l p r e s e n t m o r e d i r e c te v i d e n c e t o s h o w t h a t t h i s c o n c l u s i o n is w a r r a n t e d .

I t i s a p l e a s u r e t o a c k n o w l e d g e t h e h o s p i t a l i t y o fP r o f e ss o r O . L i n d b e r g , D i r e c t o r o f t h e W e n n e r - G r e nI n s t i t u t e . T h e a u t h o r s a r e m o s t g r a t e f u l t o D r . B . A .A f z e l i u s f o r i n s t r u c t i n g t h e m i n t h e t e c h n i q u e s o fe l e c t r o n m i c r o s c o p y a n d f o r h i s h e l p f u l a d v i c e a n dc o m m e n t s o n t h e m a n u s c r i p t s . D i s c u s s i o n s a l s o w i t hP r o f e s s o r T . G u s t a f s o n h a v e b e e n m o s t e n c o u r a g i n ga n d s t i m u l a ti n g .

A p r e l i m i n a r y r e p o r t o f t h e s e fi n d i n g s w a s p r e -

s e n t e d a t t h e S y m p o s i u m o n C y t o c h e m i c a l P r o g r e s si n E l e c t r o n M i c r o s c o p y , h e l d i n O x f o r d , E n g l a n d ,Ju ly , 1 9 6 2 ( 2 8 ) .

S o m e o f th e w o r k w a s c a r r i e d o u t w h i l e M a r g i tN a s s w a s a P o s t d o c t o r a l F e l l o w o f th e N a t i o n a lS c i e n c e F o u n d a t i o n . S h e w a s l a t e r a i d e d b y a g r a n tf o r a P o s t d o c t o r a l F e l l o w s h i p f r o m t h e A m e r i c a nC a n c e r S o c i e t y.T h i s w o r k w a s a l s o s u p p o r t e d i n p a r t b y a P u b l i cH e a l t h S e r v i c e F e l l o w s h i p ( C A - 8 5 7 3 ) t o S y l v a n N a s sf r o m t h e N a t i o n a l C a n c e r I n s t i tu t e , U n i t e d S t a t esP u b l i c H e a l t h S e r v i c e .Received for publication, D ecember 13, 1962.

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V o l . 2 , X X - I .

F I GUR E 1 0 Su r v ey m ic r o g r ap h o f ch i ck n o d e ( a t c c to d e r m - m eso d e r m ju n c t io n ) f i xedw i t h 3 p e r c e n t c a l c i u m p e r m a n g a n a t e , w i t h s a l ts a d d e d , a n d b u f fe r e d a t p H 8 .0 ; t i ss u e st r e a t e d w i t h u r a n y l a c e t a t e a n d s e c t i o n s s t a i n e d w i t h l e a d h y d r o x i d e . T h e m i t o c h o n d r i aco n ta in a r ea s o f v e r y l o w d en s i ty i n t h e i r c r i s t a - f r ee r eg io n s . X 9 ,~ 0 0.

6 0 6 T H E J O U R N A L O F C E L L B I O L O G Y V O L U M E 1 9 , 196S



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MARGIT M. K. NASS AND SYLVAN NAss Intramitochondrial D NA Fibers. I 607



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3 5 . R IS , H. , a n d PLAUT, W., J. Cel l Biol. , 1 9 6 2 , 1 3 ,383 .36 . ROUILLER, CH., Int. Rev. Cytol., 1960, 9 , 227 .37 . SALSER, W ., Perspect. Biol. and Med., 1 9 6 1 , 4 ,

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12 , 134 .

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FIGURE 11 Chick node f ixed wi th 8 per cen t ca lc ium perma nga nate in buffered sa l t so lu-t i o n , a t p H 8 .0 ; s e c t i o n s s t a i n e d wi t h l e a d h y d ro x i d e . M i t o c h o n d r i a l f i b ro u s s t ru c t u re sa p p e a r a s 1 5 t o 5 0 A f i b r i l s wi t h p o ro u s a n d b u l b o u s e n l a rg e me n t s a l o n g t h e i r l e n g t h .3< 1~0,000.

6 0 8 T H E J OUIt NAL OF CE L L B IOL OGY VOL UME 1 9 , 1 9 6 3



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MAaGIT M. K. NASS AND SYLVAN NASS lntramitochondrial DN A Fibers. 1 609



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FIOURE 1~ Chick node fixed wUih 10 per cent acro lein buffered at pH 8,0, postfixed with osmium tet-roxide, stained wi th lead hydroxide. Cell membranes and especially the p arfiele-studd ed membranes oftile endoplasmic reticulum are very well defined. The mitochondrial areas of low density are apparent.The mitochondrial fibers are irregularly distrib uted and not easily reeoguizable in a definite and repro-ducible form. X 40,000.

FIGURE 13 Escherichia coli fixed and stained asdescribed in legend to Fig. 1~. The nucleoplasmforms an irregular fibrous network, which is notstrongly contrasted by uranyl or lead staining. X40,000.

610 TH E JOURNAL OF CELL BIOLOGY VOLUM E 19, 1 9 6 8



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FmURE 14 Chick node fixed with 4 per cent forma ldehyde in sucrose, buffered at pH 8.0, for 24 hours;sections stained with lead hydroxide. The mitochondria appear shrunken. The areas of low densitywithin the mitoehondrial matrix are nevertheless apparent. In some of these areas clumped mitochondrialfibers may be seen afte r stainin g (arrows). No memb ranes are visible. At lower left is part of a denselypacked cell characteristically seen after formaldehy de and acrolein fixation. X 4~,000.

MARGIT M. K. NAss AND SYLVAN NASS Intramitochondrial DN A Fibers. I 611


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