Retrospective Theses and Dissertations
1967
A study of the fine structure of the blue-green alga,
Nostoc muscorum
Judith Ann Reese
Iowa State University
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Recommended Citation
Reese, Judith Ann, "A study of the fine structure of the blue-green alga, Nostoc muscorum " (1967). Retrospective Theses and
Dissertations. Paper 3175.
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REESE, Judith Ann, 1941A STUDY OF THE FINE STRUCTURE OF THE BLUEGREEN ALGA, NOSTOC MUSCORUM.
Iowa State University of Science and Technology,
Ph.D., 1967
Botany
University Microfilms, Inc., Ann Arbor, Michigan
A STUDY OF THE FINE STRUCTURE OF THE
BLUE-GREEN ALGA, NOSTOC MUSCORUM
by
J u d i t h Ann R e e s e
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The R e q u i r e m e n t s f o r t h e D e g r e e o f
DOCTOR OF PHILOSOPHY
Major Subject:
Cell Biology
Approve
Signature was redacted for privacy.
Signature was redacted for privacy.
Chairman A d v i s o r y Committee
Cell Biology Program
Signature was redacted for privacy.
Chairman o f M a j o r D e p a r t m e n t
Signature was redacted for privacy.
College
Iowa S t a t e U n i v e r s i t y
Of Science and Technology
Ames, Iowa
1967
TABLE OF CONTENTS
INTRODUCTION
LITERATURE REVIEW
Light Microscopy
General Electron Microscopy
Gel 1 Wal1
Photosynthetic Apparatus
Nucleoplasm
Inclusions
MATERIALS AND METHODS
Culture Techniques
Fixation Procedures
P r e p a r a t i o n o f C e l l Homogenates
Centrifugation Procedures
Spectrophotometric Measurements
Microscopy
Negative Staining Procedure
OBSERVATIONS
Sectioned Cells
Osmium f i x a t i o n
Glutaraldehyde fixation
Potassium permanganate fixation
Solvent Extraction
Enzyme T r e a t m e n t s
C e l l Homogenates
iii
Sectioned pellets
34
Negatively stained preparations
39
DISCUSSION
42
The C e l l E n v e l o p e
42
Photosynthetic Apparatus
45
Nucleoplasm
49
Inclusions
50
Tubule-like Elements
56
SUMMARY
58
BIBLIOGRAPHY
50
ACKNOWLEDGEMENTS
68
APPENDIX A:
69
FIXATION, DEHYDRATION AND EMBEDDING
Fixation
69
D e h y d r a t i o n a n d Embedding
71
APPENDIX B:
FIGURES
Key t o a l l F i g u r e s
73
73
1
INTRODUCTION
The c e l l u l a r d i f f e r e n t i a t i o n o f t h e C y a n o p h y c e a e i s s i m i l a r i n
many r e s p e c t s t o t h a t o f t h e b a c t e r i a a n d t h e s e two g r o u p s a r e d i s t i n c t
from all higher plants and animals.
The c e l l s l a c k t h e membrane-
bound o r g a n e l l e s s u c h a s n u c l e i , G o l g i , m i t o c h o n d r i a , p l a s t i d s a n d
endoplasmic reticulum found in the cells of higher organisms.
Because
of these differences i t has been proposed that the blue-green
a l g a e a n d b a c t e r i a be c o n s i d e r e d t o g e t h e r a s t h e P r o c a r y o t a a s
opposed to all cells with true nuclei, the Eucaryota (Stanier and
Van N i e l , 1 9 5 2 ) .
As more p r o c a r y o t e s are s t u d i e d a t t h e f i n e s t r u c t u r e l e v e l ,
t h i s s e p a r a t i o n s e e m s t o become more c l e a r l y a f f i r m e d a n d more w i d e l y
accepted.
The c e l l e n v e l o p e o f t h e b l u e - g r e e n s c l o s e l y r e s e m b l e s t h e
cell wall characteristic of gram-negative bacteria.
Both of these
wall types were found to contain a wide range of amino acids and
d i a m i n o p i m e l i c a c i d , b u t t h e l a t t e r compound h a s n o t y e t b e e n f o u n d i n
h i g h e r o r g a n i s m s (Work a n d Dewey, 1 9 5 3 ; F r a n k e t a l . , 1 9 6 2 b ) .
In
g e n e r a l , m o s t compounds a r e s h a r e d by t h e p r o c a r y o t e s a n d e u c a r y o t e s .
However, s t e r o l s have been r e p o r t e d t o be a b s e n t f r o m b o t h b l u e - g r e e n s
and bacteria although they are of general occurrence in the cells of
higher organisms (Levin and Block, 1954).
S t e r o l s a r e commonly f o u n d
i n t h e i n t r a c e l l u l a r membranes o f e u c a r y o t e s ^ a n d i t h a s b e e n s u g g e s t e d
t h a t t h e l a c k o f s t e r o l c o n t e n t may b e s i g n i f i c a n t i n e x p l a i n i n g t h e
l a c k o f membrane-bound o r g a n e l l e s i n t h e p r o c a r y o t e s ( S t a n i e r , 1 9 6 1 ) .
The b l u e - g r e e n s s h a r e t h e b a s i c p h o t o s y n t h e t i c p i g m e n t s .
2
c h l o r o p h y l l a and ^ - c a r o t e n e w i t h h i g h e r p l a n t s a n d i n b o t h c a s e s f r e e
oxygen i s a p r o d u c t o f p h o t o s y n t h e s i s .
Both g r o u p s h a v e & < - l i n o l e n i c
acid-containing galactolipids in the photosynthetic apparatus (Levin
et a l . , 1964).
T h e s e p a r t i c u l a r compounds h a v e been s u g g e s t e d t o
play a role in the process of oxygen evolution and are not found in
bacteria.
In bacteria other substances, such as sulfur, are reduced.
The p h o t o s y n t h e s i s o f b l u e - g r e e n s r e s e m b l e s t h a t o f b a c t e r i a i n i t s
relationship to respiration in that light appears to inhibit oxygen
u p t a k e by b o t h g r o u p s ( S t a n i e r , 1 9 5 1 ) .
R i s a n d C h a n d l e r ( 1 9 5 3 ) compared t w o m a j o r t y p e s o f g e n e t i c
systems which differ significantly in molecular organization.
In
p r o c a r y o t e s a n d v i r u s e s t h e g e n t i c s y s t e m c o n s i s t s o f DNA n o t bound
with hi stone-type proteins.
I n t h e e u c a r y o t e s t h e genome c o n s i s t s
o f chromosomes i n w h i c h t h e DNA m o l e c u l e s a r e combined w i t h b a s i c
proteins.
De ( 1 9 5 5 ) h a s s u g g e s t e d t h a t t h i s f i n d i n g i s u n d e r s t a n d a b l e
i n v i e w o f t h e work on t h e r o l e o f h i s t o n e s i n t h e d i f f e r e n t i a t i o n o f
DNA a n d t h a t t h e a b s e n c e o f h i s t o n e s i n p r o c a r y o t e s may e x p l a i n t h e
diffuse state of their nuclear material.
Although the subcellular structure and organization observed in
th e b l u e - g r e e n s i s q u i t e d i f f e r e n t f r o m t h a t o f h i g h e r o r g a n i s m s , t h e
m e t a b o l i c p a t h w a y s s o f a r i n v e s t i g a t e d i n b l u e - g r e e n s a r e i n many
respects similar to those recognized in higher organisms (Black e t
a l . , 1 9 5 3 ; Holm-Hansen a n d Brown, 1 9 5 3 ; F u j i t a e t a l . , 1 9 5 4 ; S u s o r a n d
Krogman, 1 9 6 4 ; B e r n s e t a l . , 1 9 6 5 ) .
E x a c t l y how t h e p r o c a r y o t e c e l l s p e r f o r m s i m i l a r f u n c t i o n s t o
3
t h o s e o f t h e e u c a r y o t e c e l l s i n w h i c h t h e membrane-bound o r g a n e l l e s
are recognized to spatially segregate certain pathways i s interesting
but l i t t l e understood.
I n f a c t many o f t h e s t r u c t u r e s r e c o g n i z e d i n
b l u e - g r e e n c e l l s remain a t p r e s e n t c h e m i c a l l y u n i d e n t i f i e d and t h e i r
metabolic involvements are even less understood.
The p r e s e n t s t u d y
was u n d e r t a k e n p r i m a r i l y t o s t u d y c y t o p l a s m i c i n c l u s i o n s w h i c h a p p e a r
to be characteristic of blue-green cells.
A better insight into the
morphology of these structures, their structural relationships and
possibly their chemical composition might aid in eventually delineating
their functional relationships and giving a better understanding of
the cellular organization and metabolism of these organisms.
4
LITERATURE REVIEW
Light Microscopy
The s t r u c t u r e o f b l u e - g r e e n a l g a l c e l l s h a s b e e n s t u d i e d f o r
many y e a r s , a n d much o f t h e l i g h t m i c r o s c o p y work h a s b e e n r e v i e w e d
by Fritsch (1945).
According to these early studies, the cell
e n v e l o p e was d i v i d e d i n t o t w o l a y e r s , t h e i n n e r i n v e s t m e n t w h i c h
completely surrounded the protoplast,and the cell sheath which
enveloped single cells in the coccoid forms or formed a cylinder
a r o u n d a n e n t i r e t r i chôme i n t h e f i l a m e n t o u s f o r m s .
The p r o t o p l a s t
was g e n e r a l l y d i v i d e d i n t o two a r e a s , t h e c e n t r o p l a s m a n d t h e c h r o m a t o plasm.
The c e n t r o p l a s m was r e p o r t e d t o c o n t a i n v o l u t i n g r a n u l e s ,
metachromatic granules, and polyphosphate bodies.
Fuhs ( 1 9 5 8 a )
suggested that the polyphosphate bodies were identical to the meta­
chromatic and volutin granules of the older studies.
These granules
h a v e been s u g g e s t e d v a r i o u s l y t o c o n t a i n p o l y p h o s p h a t e s , o r g a n i c
bases or nucleic acid (Fritsch, 1945).
Ebel and Muller (1958)
d e t e r m i n e d t h a t t h e p r e s e n c e o f p o l y p h o s p h a t e s was c h a r a c t e r i z e d b y
a m e t a c h r o m a t i c c o l o r a t i o n w i t h some b a s i c d y e s i n c l u d i n g t o l u i d i n e
blue.
They a l s o s t a i n e d w i t h m e t h y l g r e e n a n d p y r o n i n e w h i c h was
used to stain nucleic acids.
The most specific reagent for inorganic
p h o s p h a t e was f o u n d t o b e l e a d n i t r a t e a t pH 3 . 5 .
A positive reaction
with this reagent in association with a positive metachromatic
reaction permitted positive diagnosis of polyphosphate.
Talpasayi (1963) described polyphosphate bodies as determined
by t h e a b o v e p r o c e d u r e .
They were freed from the cell as distinct
5
g r a n u l e s a n d showed a r e d f l u o r e s e n c e o f RNA when viewed w i t h u l t r a ­
violet light after treatment with buffered acridine orange.
No
m e t a c h r o m a t i c s t a i n i n g was o b s e r v e d a f t e r e x t r a c t i o n w i t h 10% t r i ­
c h l o r o a c e t i c a c i d (TCA).
Fuhs ( 1 9 5 8 a ) r e p o r t e d them t o b e i n t e n s e l y
r e d when t r e a t e d w i t h t h e F e u l g e n r e a g e n t w i t h o u t h y d r o l y s i s b u t f u l l y
removed w i t h HCl a t 5 0 ° C .
The h i g h d e g r e e o f p o l y m e r i z a t i o n g a v e a
c h e m i c a l r e a c t i o n s i m i l a r t o n u c l e i c a c i d s , a n d Fuhs ( 1 9 5 8 a ) c o n s i d e r e d
that methyl green and pyronine could not be used to distinguish
nucleic acid content because the polyphosphates gave an even darker
stain.
The polyphosphate bodies were not disturbed by either RNase or
D N a s e , a n d Fuhs c o n c l u d e d t h a t t h e g r a n u l e s c o n t a i n e d p o l y p h o s p h a t e s
but not nucleic acid.
Cassel and Hutchinson (1954) also reported
negative tests for metachromatin or polyphosphates after digestion
i n IN HCl a t 6 0 ° C .
T h i s t r e a t m e n t a l s o removed t h e c y a n o p h y c i n g r a n u l e s .
T h e c e n t r o p l a s m a l s o c o n t a i n e d w h a t was b e l i e v e d t o be t r u e c h r o m a t i n
i n a l o o s e t h r e e d i m e n s i o n a l n e t w o r k whose a p p e a r a n c e v a r i e d d e p e n d i n g
upon f i x a t i o n a n d s t a i n i n g c o n d i t i o n s .
M i t o t i c f i g u r e s h a v e been
r e p o r t e d , b u t i t i s now g e n e r a l l y a g r e e d t h a t t h e r e i s n o t r u e chromosome
o r membrane-bound n u c l e u s .
The e x a c t mechanism o f c e l l u l a r d i v i s i o n
in these cells remains unsolved.
The b o u n d a r y b e t w e e n c e n t r o p l a s m a n d c h r o m a t o p l a s m was i l l d e f i n e d , b u t i n g e n e r a l t h e c h r o m a t o p l a s m was p e r i p h e r a l a n d , a s t h e
name s u g g e s t e d , c o n t a i n e d t h e p i g m e n t s .
In addition to chlorophyll,
carotenes, and phycocyanin, cells contained varying amounts of
phycoerythrin and xanthophylls.
The p i g m e n t s w e r e c o n s i d e r e d a s
6
diffuse throughout the chromatoplasm or as contained in small vesicles
or granules.
The p h o t o s y n t h e t i c p r o d u c t , a g l y c o g e n - ! i k e s u b s t a n c e ,
was l o c a t e d i n t h e p e r i p h e r a l r e g i o n , b u t p e r h a p s t h e m o s t c o n s p i c u o u s
components of this area were the cyanophycin granules, generally
s u g g e s t e d t o be a f o o d r e s e r v e s u b s t a n c e ( F r i t s c h , 1 9 4 5 ) .
General Electron Microscopy
The very small size of the vegetative c e l l s , approximately 10
m i c r o n s i n d i a m e t e r , made t h e i r s t u d y a t t h e l i g h t m i c r o s c o p e l e v e l
difficult, particularly with respect to subcellular organization.
R e c e n t work a t t h e e l e c t r o n m i c r o s c o p e l e v e l h a s d o n e much t o c l a r i f y
the structure of these cells.
The many s t u d i e s i n c l u d e t h o s e o f
N i k l o w i t z a n d Drews ( 1 9 5 5 , 1 9 5 7 ) , Drews a n d N i k l o w i t z ( 1 9 5 6 , 1 9 5 7 ) ,
Fuhs ( 1 9 5 8 , 1 9 6 3 ) , Hopwood a n d G l a u e r t ( 1 9 6 0 ) , L e f o r t ( 1 9 5 0 a , 1 9 6 0 b ) ,
R i s a n d S i n g h ( 1 9 6 1 ) , P a n k r a t z a n d Bowen ( 1 9 5 3 ) , G i e s y ( 1 9 6 4 ) , a n d
Jost (1965).
A general pattern of organization has developed from
t h e s e s t u d i e s a n d e a c h s u c c e e d i n g work s e e m s t o c o n f i r m a n d e x t e n d
the general description.
T h e same p r o l i f e r a t i o n o f t e r m i n o l o g i e s
which characterized the descriptions given by the light microscopists
i s evident in the electron microscope studies.
C e l l Wall
Ris and Singh (1961) described the cell envelope of Anacystis
nid u l a n s a s c o n s i s t i n g o f 1 ) an e x t e r i o r s h e a t h , 2 ) a 7 mu o u t e r membrane
w h i c h h a d t h e a p p e a r a n c e o f a t y p i c a l u n i t membrane w i t h v a r y i n g
d e g r e e s o f u n d u l a t i o n , 3 ) a n i n t e r m e d i a t e , d e n s e a n d r a t h e r homogeneous
l a y e r o r i n n e r i n v e s t m e n t w h i c h was 1 2 - 2 0 mu t h i c k , a n d 4 ) t h e 7 mu
7
p l a s m a membrane i t s e l f which was a u n i t membrane.
The i n n e r i n v e s t m e n t
extended into the cross walls separating neighboring cells.
The t h r e e
i n n e r l a y e r s w e r e e q u i v a l e n t t o t h e i n n e r i n v e s t m e n t d e s c r i b e d by t h e
light microscopists.
P a n k r a t z a n d Bowen ( 1 9 6 3 ) r e p o r t e d t h a t t h e o u t e r
l a y e r was d i f f u s e a n d , a l t h o u g h i t was m o d e r a t e l y d e n s e a f t e r OsO.
s t a i n i n g , i t d i d n o t s t a i n w e l l w i t h KMnO. a n d was n o t b e l i e v e d t o b e
a u n i t membrane.
J e n s e n ( 1 9 6 5 ) d e s c r i b e d p a p i l l a e 3 0 - 4 5 mu w i d e a n d
3 0 - 1 5 0 mu l o n g p r o j e c t i n g f r o m t h e o u t e r membrane.
They w e r e common
in several of the species examined, and he speculated on a possible
r o l e o f t h e s e p a p i l l a e i n t r i c h ô m e movement w i t h i n t h e s h e a t h .
E c h l i n ( 1 9 6 3 ) a l s o o b s e r v e d a s e r i e s o f 4 0 - 8 0 mu b l e b s o r p r o t r u s i o n s
around the cell periphery.
T h e i r o u t e r b o u n d a r y was f o r m e d by t h e
o u t e r membrane, and t h e i n n e r i n v e s t m e n t p r o j e c t e d i n t o t h e p r o t r u s i o n s .
T h e e l e c t r o n - d e n s e i n n e r membrane o f 1 0 - 1 5 mu d i d n o t t a k e p a r t i n
the formation of the protrusions.
The s t r u c t u r e o f t h e c e l l e n v e l o p e o f t h e b l u e - g r e e n s was
s t r i k i n g l y s i m i l a r t o t h e model o f t h e c e l l w a l l o f g r a m - n e g a t i v e
b a c t e r i a a s p r o p o s e d by G l a u s a n d Roth ( 1 9 6 4 ) .
I n t h i s model t h e 8 mu
p l a s m a membrane a n d t h e 9 mu c e l l w a l l membrane, b o t h o f u n i t n a t u r e ,
w e r e s e p a r a t e d by a 5 - 1 0 mu l e s s d e n s e i n n e r l a y e r s u g g e s t e d t o
c o n t a i n a mucocomplex a n d t o i m p a r t r i g i d i t y t o t h e c e l l w a l l .
Chemical
c o m p a r i s o n s i n w a l l c o m p o s i t i o n s h a v e b e e n made b y F r a n k e t a l . ( 1 9 6 2 b )
a n d Drews a n d Meyer ( 1 9 6 4 ) .
The l a t t e r a u t h o r s compared t h e w a l l s
o f t h e g r a m - n e g a t i v e b a c t e r i u m E s c h e r i c h i a c o l i w i t h Phormidium u n c i n a t u m
and Anacystis nidulans and found muramic acid, diaminopimelic acid.
8
g l u c o s a m i n e and e i g h t amino a c i d s i n common.
The f a c t t h a t d i a m i n o -
p i m e l i c a c i d a n d muramic a c i d h a v e b e e n r e p o r t e d o n l y i n t h e w a l l s o f
blue-greens and bacteria suggests a taxonomic relationship between
t h e s e g r o u p s ( F o g g , 1 9 5 6 ; S t a n i e r a n d Van N i e l , 1 9 5 2 ) .
The b l u e - g r e e n
c e l l w a l l was o b s e r v e d by e l e c t r o n m i c r o s c o p i c s t u d i e s t o h a v e s e v e r a l
layers.
I s o l a t e d c e l l w a l l s o f A n a c y s t i s n i d u l a n s w e r e shown t o c o n t a i n
polysaccharides, proteins, and lipids in addition to mucopolymers.
No
morphological positions were suggested for any of the components
(Drews a n d G o l l w i t z e r , 1 9 6 5 ) .
Protoplasts were successfully produced from several blue-greens
by t h e a c t i o n o f l y s o z y m e ( C r e s p i e t a l . , 1 9 6 2 ) , a n d i t was s u g g e s t e d
t h a t t h e l y s o z y m e - s e n s i t i v e mucopolymers s h a r e d by t h e b l u e - g r e e n a l g a e
a n d b a c t e r i a w e r e l o c a t e d b e t w e e n t h e p l a s m a membrane a n d t h e i n n e r
investment of the blu^-green colls (Frank e t a l . , 1962a).
T h i s muco-
p o l y m e r was s u g g e s t e d t o be l o c a l i z e d i n t h e i n n e r l a y e r o f g r a m - n e g a t i v e
bacterial walls (Claus and Roth, 1964).
Photosynthetic Apparatus
The e l a b o r a t i o n o f l a m e l l a e t h r o u g h o u t t h e c e l l o r i t s p e r i p h e r a l
r e g i o n s h a s b e e n n o t e d by many w o r k e r s .
The concept of centroplasm
v e r s u s c h r o m a t o p l asm became u n t e n a b l e when t h e e x t e n t o f t h e s e l a m e l l a e
and the lack of a clear demarkation between the lamellae and the
a n a s t o m o s i n g n u c l e o p l a s m was o b s e r v e d .
lamellar units thylakoids.
membrane-bound s a c .
Menke ( 1 9 5 1 a ) t e r m e d t h e s e
Each t h y l a k o i d c o n s i s t e d o f a f l a t t e n e d
I n b l u e - g r e e n s t h e o p p o s i n g u n i t membranes may be
q u i t e c l o s e l y a p p r e s s e d o r b e s e p a r a t e d by an i n t r a t h y l a k o i d a l s p a c e
9
( D r a w e r t a n d M e t z n e r 1 9 5 8 ; Hopwood and G l a u e r t 1 9 5 0 ; L e f o r t 1 9 6 0 b ;
R i s a n d S i n g h 1 9 5 1 ; P a n k r a t z and B o w e n , 1 9 6 3 ) .
P a n k r a t z a n d Bowen
o b s e r v e d f l a t t e n e d t h y l a k o i d s a v e r a g i n g 1 8 mu w i d e a f t e r osmium f i x a ­
t i o n , t h e 6 - 7 mu u n i t membranes s e p a r a t e d b y a 4 - 5 mu l e s s d e n s e s p a c e .
A f t e r KMnO^ f i x a t i o n t h e a p p r e s s e d t h y l a k o i d c o n s i s t e d o f a 3 mu
e l e c t r o n - d e n s e median r e g i o n s e p a r a t e d f r o m l a t e r a l 2 mu d e n s e z o n e s by
3 mu l e s s d e n s e z o n e s ,
dost (1965) studied the fine structure of
Q s c i l l a t o r i a by s e v e r a l t e c h n i q u e s i n c l u d i n g f r e e z e - e t c h i n g a n d r e p o r t e d
t h a t t h e u n i t membranes o f t h e t h y l a k o i d s e x h i b i t e d a c e r t a i n p o l a r i t y .
He o b s e r v e d g l o b u l a r p a r t i c l e s on b o t h s i d e s o f t h e m e m b r a n e s .
T h o s e on
the outside were 50-70 A in diameter and remained visible after negative
staining.
The 1 0 0 - 2 0 0 A p a r t i c l e s o b s e r v e d on t h e i n s i d e o f t h e mem­
branes were suggested to be similar to the quantasomes of Park and Biggins
(1964) and were lost during the process of negative staining.
Jost
concluded that the thylakoids consisted of a support layer with 50-70 A
g l o b u l a r p a r t i c l e s , a homogeneous l a m e l l a r p o r t i o n , a n d t h e q u a n t a s o m e s .
The q u e s t i o n a s t o w h e t h e r t h e q u a n t a s o m e s w e r e i n o r o n t h e membrane
was l e f t o p e n .
Menke ( 1 9 5 5 ) s u g g e s t e d t h a t t h e t h y l a k o i d membrane was
a s y m m e t r i c w i t h an inhomogeneous l a y e r o f 3 5 A g l o b u l e s on t h e o u t e r
s i d e a n d a homogeneous l a y e r o f a l i p i d n a t u r e on t h e i n n e r s i d e .
The
c e n t e r - t o - c e n t e r s e p a r a t i o n o f t h e g l o b u l a r p a r t i c l e s w a s 3 4 - 4 0 A, a n d
t h e y w e r e s u g g e s t e d t o be p r o t e i n .
The o r i g i n o f t h e t h y l a k o i d s i s n o t y e t c l e a r .
P a n k r a t z a n d Bowen
(1963) favored the idea that the thylakoids were subdivided during
c y t o k i n e s i s by t h e a n n u l a r g r o w t h o f t h e c r o s s - w a l l , a n d Chapman a n d
10
S a l t o n ( 1 9 5 2 ) s u g g e s t e d t h e y a r o s e d e novo f r o m t h e c e n t r a l p o r t i o n o f
the cell and migrated to the periphery.
Several workers have suggested
t h a t t h e y may a r i s e f r o m t h e p l a s m a membrane ( P a n k a r a t z a n d Bowen, 1 9 5 3 ;
Echlin and Morris, 1965; Jost, 1965).
Echlin considered that the
t h y l a k o i d s w e r e a complex b r a n c h i n g r e t i c u l u m f o r m e d by e x t e n s i v e
i n v a g i n a t i o n o f t h e p l a s m a membrane.
J o s t p r o p o s e d a model f o r t h e
f o r m a t i o n o f t h e t h y l a k o i d membranes by s u c h an i n v a g i n a t i o n .
Small
v e s i c l e s w e r e formed by t h e i n v a g i n a t i o n , a n d t h e v e s i c l e s t h e n
expanded into the thylakcidal system of the cell.
Pigments including chlorophyll a, xanthophylls, carotenes, and
phycobilins are found within the blue-green cell.
The chlorophyll
and carotene were observed to be associated with particulate elements
p e l l e t e d f r o m t h e c e l l h o m o g e n a t e s ( C a l v i n and L y n c h , 1 9 5 2 ; P e t r a c k ,
1959; Shatkin, 1950).
C a l v i n a n d Lynch n e g a t i v e l y s t a i n e d t h e p a r t i c l e s
and observed rounded 2 u vesicles which they considered to be grana
equivalent to those of higher plant chloroplasts, but Shatkin considered
t h a t t h e v e s i c l e s were a r e s u l t o f membrane d i s r u p t i o n .
Petrack
observed that, in addition to the pigments, the photophosphorolation
a c t i v i t y was a s s o c i a t e d w i t h t h e p a r t i c l e s .
Although the chlorophyll
a n d c a r o t e n o i d s a p p e a r e d t o be a s s o c i a t e d w i t h t h e i n t e r n a l m e m b r a n e s ,
t h e i n t r a c e l l u l a r l o c a l i z a t i o n o f t h e p h y c o b i l i n s was l e s s o b v i o u s ,
particularly since they remained in the supernate after centrifugation.
Bergeron (1963) suggested that the phycocyanins were located between
the lamellae.
P h y c o c y a n i n , a w a t e r s o l u b l e p r o t e i n a c e o u s p i g m e n t was
u s u a l l y r e l e a s e d upon c e l l u l a r d i s r u p t i o n .
R e c e n t l y S u s o r a n d Krogman
11
( 1 9 5 4 ) s t u d i e d p h o t o s y n t h e s i s i n p i g m e n t - c o n t a i n i n g p a r t i c l e s from
Anabaena v a r i a b i l i s .
The p h y c o c y a n i n r e m a i n e d w i t h t h e c h l o r o p h y l l
a n d c a r o t e n o i d s a s s o c i a t e d w i t h t h e membrane f r a g m e n t s i f F i c o l l w e r e
a d d e d t o t h e h o m o g e n i z i n g medium.
O'hEocha (1965) reported that
p h y c o c y a n i n s w e r e more r e a d i l y d i s s o c i a t e d a n d d e n a t u r e d t h a n t h e
phycoerythrins.
Fuhs ( 1 9 6 4 ) r e p o r t e d t h a t t h e l a m e l l a e c o n s i s t e d o f
layers of closely packed 80 A spherical units,
A preparation of
p h y c o c y a n i n g i v e n t o him by O ' h E o c h a showed a 8 0 A m o l e c u l e a f t e r
negative staining.
He c o n c l u d e d t h a t t h e s p h e r i c a l u n i t s w h i c h f o r m e d
the photosynthetic lamellae were partly phycocyanin particles.
I n v e s t i g a t i o n s by G a n t t a n d C o n t i ( 1 9 6 6 ) h a v e r e v e a l e d g r a n u l e s
attached to the chloroplast lamellae of the red alga Porphyridium
cruentum after aldehyde fixation.
T h e s e 3 5 mu g r a n u l e s w e r e a r r a n g e d
i n r e p e a t i n g rows w i t h a c e n t e r - t o - c e n t e r d i s t a n c e o f 4 0 - 5 0 mu.
They
were believed to be the s i t e of phycoerythrin concentration within the
cells.
Such g r a n u l e s w e r e o b s e r v e d i n a marine o s c i l l a t o r i a - l i k e
organism but were not found in organisms which lacked phycobilins.
L e f o r t ( 1 9 6 5 ) s t u d i e d t h e e n d o s y m b i o n t Glaucocystis n o s t o c h i n e a r u m
a n d r e p o r t e d d e n s e 3 0 mu g r a i n s d i s p o s e d r e g u l a r l y i n t h e i n t e r l a m e l l a r
s p a c e s a f t e r OsO^ f i x a t i o n .
She suggested that these grains represented
a cross section of a series of long filamentous structures located on
the external surface of the lamellae.
They were peculiar to symbiotic
b l u e - g r e e n s , a n d no s u g g e s t i o n was o f f e r e d , f o r t h e i r c h e m i c a l c o m p o s i t i o n
or possible function.
The p r o p o s e d f u n c t i o n s o f t h e t h y l a k o i d s i n c l u d e n o t o n l y p h o t o ­
12
s y n t h e s i s b u t a l s o r e s p i r a t i o n , n i t r o g e n fixation, n i t r a t e r e d u c t i o n ,
and protein synthesis.
Cox e t a l . ( 1 9 5 4 ) s t u d i e d Anabaena c y l i n d r i c a
and found nitrogen fixation activity in the pellet sedimenting between
15,000 and 35,000 g for 20 minutes,and suggested that this fixation
a b i l i t y was l o c a t e d i n t h e p h o t o s y n t h e t i c l a m e l l a e w h i c h c o n t a i n e d
c h l o r o p h y l l and c a r o t e n o i d s .
Cox ( 1 9 5 5 ) r e p o r t e d t h a t work w i t h p h o t o -
s y n t h e t i c i n h i b i t o r s s u g g e s t e d t h a t some p a r t o f t h e n i t r o g e n f i x a t i o n
p r o c e s s was a l l i e d t o t h e r e s p i r a t o r y p r o c e s s r a t h e r t h a n t h e p h o t o synthetic pathway.
He f u r t h e r s u g g e s t e d t h a t n i t r a t e r e d u c t i o n may
be as versatile as nitrate fixation, using either light or dark generated
r e d u c i n g power a n d ATP a c c o r d i n g t o p r e v a i l i n g c i r c u m s t a n c e s , u n l e s s
s p a t i a l o r g a n i z a t i o n o f t h e n e c e s s a r y enzymes p r e v e n t e d t h i s .
Jost ( 1 9 6 5 ) o b s e r v e d s m a l l c h a i n s o f 110 A p a r t i c l e s on t h e
thylakoids in his freeze-etched material and suggested that they
were polyribosomes.
J o s t a n d M a t i l e ( 1 9 6 6 ) r e p o r t e d RNA i n a t h y l a k o i d
f r a c t i o n i s o l a t e d by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n .
Nucleoplasm
The p r e s e n c e o f DNA i n t h e c e n t r a l r e g i o n o f t h e c e l l s a s i n d i c a t e d
b y a p o s i t i v e F e u l g e n r e a c t i o n h a s been o b s e r v e d b y many w o r k e r s
i n c l u d i n g C a s s e l and H u t c h i n s o n ( 1 9 5 4 ) , Leak a n d W i l s o n ( 1 9 5 0 ) , Fuhs
( 1 9 6 3 ) , a n d P a n k r a t z a n d Bowen ( 1 9 6 3 ) .
I n a s e r i e s o f s t u d i e s on t h e n a t u r e o f t h e DNA, B i s w a s ( 1 9 5 7 a ,
1 9 5 7 b ) and B i s w a s a n d Meyers ( 1 9 6 0 a , 1960b) d e t e r m i n e d t h a t t h e
n u c l e i c a c i d s o f t h e b l u e - g r e e n s c o n t a i n e d t h e f o u r common n u c l e o t i d e s
and methyl cytidine.
They also found a polysaccharide closely
13
associated with the nucleic acid.
T h e y s u g g e s t e d t h a t t h e c e n t r a l body
c o n t a i n e d h i s t o n e s bound t o t h e n u c l e i c a c i d s b e c a u s e n e i t h e r p e p s i n
nor trypsin digested the central body.
T r e a t m e n t w i t h 5% TCA f o r
15 m i n u t e s a t 90°C l e f t t h e c e n t r a l body i n t a c t a n d s u g g e s t e d t o Biswas
t h a t t h e n u c l e i c a c i d s w e r e bound by s t r u c t u r a l p r o t e i n s .
De ( 1 9 5 5 )
t e s t e d s p e c i e s o f O s c i l l a t o r i a , A p h a n o c a p s a , Anabaena,and P o l y c y s t i s
with 3 histochemical tests specific for histone.
None o f t h e s p e c i e s
t e s t e d showed a p o s i t i v e r e a c t i o n , a n d h e c o n c l u d e d t h a t t h e c e n t r a l
body l a c k e d h i s t o n e .
The l a b i l i t y o f b a c t e r i a l n u c l e o p l a s m t o w a r d f i x a t i o n was f i r s t
d e m o n s t r a t e d by K e l l e n b e r g e r e t a l . ( 1 9 5 8 ) .
Versene treatment after
fixation led to a coarse-structured nucleoplasm but uranyl acetate
treatment led instead to a fine fibrillar organization consisting of
25 A fibrils.
Similar observations held for the fixation of nucleoplasms
of blue-green algae (Ris and Singh, 1961).
They described the central
r e g i o n o f t h e b l u e - g r e e n . c e l l a s composed o f 2 5 A f i b r i l s a n d 1 0 - 1 5 mu
ribosomes.
The r i b o s o m e s a r e p r e s e r v e d w i t h e i t h e r a l d e h y d e o r
osmium b u t n o t w i t h KMnO. f i x a t i o n .
Ris and Chandler (1963) suggested
t h a t DNA f i b r i l s w h i c h w e r e n o t c o m b i n e d w i t h b a s i c p r o t e i n t e n d e d t o
agglutinate during dehydration unless complexed with heavy metal ions
such as the uranyl ion.
De ( 1 9 6 5 ) s u g g e s t e d t h a t t h e a b s e n c e o f h i s t o n e s
in bacteria and blue-green algae could explain the diffuse s t a t e of the
nuclear materi al.
Inclusions
In addition to the thylakoids and nucleoplasm, the blue-green
14
c e l l c o n t a i n s a number o f i n c l u s i o n s , s o m e o f w h i c h a p p e a r t o be common
t o most b l u e - g r e e n s , a n d o t h e r s o f w h i c h a p p e a r t o be s p e c i e s o r
collection-speci fic.
Several workers have noted the appearance of small granules
po s i t i o n e d between t h e t h y l a k o i d s ( N i k l o w i t z a n d D r e w s , 1 9 5 7 ; R i s a n d
S i n g h , 1 9 6 1 ; Menke, 1 9 6 1 b ; P a n k r a t z a n d Bowen, 1 9 6 3 ; J o s t , 1 9 6 5 ) .
Ris and Singh reported that they were heavily stained with lead and had
an a v e r a g e d i a m e t e r o f 2 5 mu.
He t e r m e d t h e s e g r a n u l e s ' g r a n u l a r
inclusions' and postulated that they were photosynthetic products.
P a n k r a t z a n d Bowen ( 1 9 6 3 ) d e s c r i b e d 30 mu m e d i u m - d e n s e g r a n u l e s w h i c h
were frequently elongate, extended between the lamellae, and surrounded
the structured granules.
They t e r m e d t h e m c < - g r a n u l e s a n d s u g g e s t e d t h a t
they were a food reserve or had a specific metabolic function related
to photosynthesis or respiration.
Giesy (1964) studied the inter-
lamellar granules in Oscillatoria and concluded that since their
a p p e a r a n c e r a n g e d f r o m t h e ^ ^ ( - g r a n u l e s ( P a n k r a t z a n d Bowen, 1 9 6 3 ) t h r o u g h
granular inclusions (Ris and Singh, 1961) and crystalline structures
(Menke, 1 9 6 1 b ) t o t h e r o d - s h a p e d i n c l u s i o n s o b s e r v e d i n h i s own s t u d y ,
that these variously described inclusions were the same.
Exposure
t o 2% d i a s t a s e f o r 2 0 m i n u t e s removed b o t h t h e i n t e r l a m e l l a r g r a n u l e s
a n d a P A S - p o s i t i v e s u b s t a n c e o b s e r v e d i n t h e c h r o m a t o p l a s m by l i g h t
microscopy.
Giesy concluded that the granules were polyglucoside in
n a t u r e , a n d on t h e b a s i s o f o b s e r v a t i o n s c o r r e l a t e d w i t h g r o w t h s t u d i e s
suggested that they were food reserves.
Jost (1965) described 'botuli'
w h i c h he c o n s i d e r e d t o b e t h e same a s t h e i n t e r l a m e l l a r g r a n u l e s
15
d e s c r i b e d by G i e s y ( 1 9 6 4 ) .
The ' b o t u l i ' w e r e 35 mu i n d i a m e t e r a n d
a p p r o x i m a t e l y 300 mu l o n g .
They were often observed to be hexagonally
p a c k e d a n d were s u g g e s t e d t o b e a r e s e r v e s u b s t a n c e r e l a t e d t o t h e
developmental cycle of the thylakoids.
Several workers have described osmiophilic granules ranging in
d i a m e t e r f r o w 3 0 - 9 0 mu (Drews a n d N i k l o w i t z , 1 9 5 5 ; F u h s , 1 9 5 8 b ; S h a t k i n ,
1 9 6 0 ; P a n k r a t z and Bowen, 1 9 6 3 ) .
Fuhs s u g g e s t e d t h a t t h e y w e r e p o l y ­
phosphate,and Shatkin suggested that they were storage products.
P a n k r a t z a n d Bowen ( 1 9 6 3 ) r e p o r t e d them t o b e f o u n d m o s t f r e q u e n t l y
near the cross walls and along the lamellae.
On t h e o t h e r h a n d . Leak
and Wilson (1965) reported that they were randomly distributed through­
out the cell.
T h e i r e l e c t r o n d e n s i t y f o l l o w i n g osmium f i x a t i o n a n d p o o r
p r e s e r v a t i o n a f t e r KMnO^ f i x a t i o n s u g g e s t e d t h a t t h e y w e r e l i p o i d a l
in nature.
P a n k r a t z a n d Bowen ( 1 9 6 3 ) t e r m e d t h e s e o s m i o p h i l i c b o d i e s
(^granules and suggested that they were comparable to the osmiophilic
globules observed in chloroplasts.
The o s m i o p h i l i c g l o b u l e s o f h i g h e r p l a n t c h l o r o p l a s t s h a v e b e e n
s t u d i e d b y many w o r k e r s i n c l u d i n g Murakami a n d T a k a m i y a ( 1 9 6 2 ) a n d
Greenwood e t a l .
(1963),
Murakami a n d T a k a m i y a r e p o r t e d t h a t t h e
d i a m e t e r o f g l o b u l e s i n s p i n a c h c h l o r o p l a s t s v a r i e d b e t w e e n 70 a n d
1 2 0 m u , a n d on t h e b a s i s o f s o l v e n t e x t r a c t i o n p r o c e d u r e s s u g g e s t e d
that these globules contained carotenoids and were lipid in nature.
Greenwood e t a l . r e p o r t e d t h a t t h e g l o b u l e s o f t h e V i c i a f a b a c h l o r o p l a s t
w e r e 1 0 - 5 0 0 mu i n d i a m e t e r a n d w e r e l o c a t e d t h r o u g h o u t t h e c h l o r o p l a s t
matrix.
I s o l a t e d g l o b u l e s c o n t a i n e d no m e a s u r a b l e c a r o t e n e a n d t h e s e
16
workers suggested that they functioned in the chloroplasts as a general
d e p o s i t o f i n s o l u b l e l i p i d much a s s t a r c h s e r v e d a s t h e c a r b o h y d r a t e
storage.
J e n s e n and Bowen ( 1 9 5 1 ) s t u d i e d t h e c e n t r o p l a s m o f N o s t o c
p r u niforme.
One inclusion type regularly located in this area was
r o u g h l y i s o d i a m e t r i c a n d e x h i b i t e d a p o l y g o n a l p r o f i l e f r o m 5 0 - 3 6 0 mu
i n d i a m e t e r when o b s e r v e d i n c r o s s s e c t i o n .
Because of their
characteristic shape these bodies were termed polyhedral bodies.
In
c e l l s f i x e d w i t h KMnO^, t h e y p o s s e s s e d a f a i r l y u n i f o r m medium e l e c t r o n
density, showed no regular internal structure,and sometimes appeared
t o b e c l o s e l y s u r r o u n d e d by a n u n s t a i n e d s h e a t h 2 0 - 3 0 mu w i d e .
Similar
b o d i e s have been t e r m e d f o r m e d b o d i e s ( H a l l a n d C l a u s , 1 9 5 2 ) , c r y s t a l ­
l i n e g r a n u l e s (Wildon a n d M e r c e r , 1 9 6 3 ) , e l e c t r o n - d e n s e b o d i e s
(Kawamatu, 1 9 6 5 ) , a n d g r a n u l a r b o d i e s ( L e a k a n d W i l s o n , 1 9 6 5 ) .
Ris
a n d S i n g h ( 1 9 6 1 ) c o n s i d e r e d t h a t s u c h b o d i e s w e r e t h e t h i r d component
( i n a d d i t i o n t o DNA a n d r i b o s o m e s ) o f t h e n u c l e o p l a s m w i t h w h i c h t h e y
usually appeared to be closely associated.
P a n k r a t z a n d Bowen ( 1 9 6 3 )
o b s e r v e d t h a t p o l y h e d r a l b o d i e s i n Symploca muscorum w e r e o c c a s i o n a l l y
s u r r o u n d e d by a 5 mu c l e a r s p a c e and a 3 mu d a r k l i n e a f t e r osmium
fixation.
They w e r e p r e s e r v e d by a v a r i e t y o f f i x a t i o n s i n c l u d i n g
osmium, p o t a s s i u m p e r m a n g a n a t e , a n d f o r m a l i n - o s m i u m .
Costerton (1960)
o b s e r v e d 2 5 0 - 5 0 0 mu b o d i e s w h i c h w e r e a s s o c i a t e d w i t h t h e n u c l e o p l a s m
and, b e c a u s e t h e y w e r e removed by a c i d d i g e s t i o n , s u g g e s t e d t h a t t h e y
were protein bodies.
The f u n c t i o n o f t h e p o l y h e d r a l b o d i e s i s l i t t l e u n d e r s t o o d b u t
E c h l i n ( 1 9 6 6 ) l a b e l s them p r o t e i n r e s e r v e s on t h e b a s i s o f C o s t e r t o n ' s
17
work.
Lang ( 1 9 6 5 ) , i n a s t u d y o f h e t e r o c y s t d e v e l o p m e n t i n Anabaena>
r e p o r t e d a d e c r e a s e i n p o l y h e d r a l body s i z e a n d t h e i r g r a d u a l d i s ­
appearance as the heterocysts matured.
L a r g e d e n s e g r a n u l e s h a v e been r e p o r t e d o f t e n n e a r t h e c r o s s w a l l s
(Fuhs, 1958b) and periphery of osmium-fixed c e l l s , and they correspond
c l o s e l y i n s i z e a n d p o s i t i o n t o t h e c y a n o p h y c i n g r a n u l e s d e s c r i b e d by
light microscopists.
Drews a n d N i k l o w i t z ( 1 9 5 5 , 1957) termed t h e s e
g r a n u l e s ' s t r u c t u r e d g r a n u l e s ' , a n d on t h e b a s i s o f t e t r a z o l i u m a n d J a n u s
green reduction suggested that they were mitochondrial equivalents.
Fuhs ( 1 9 5 8 b ) s u g g e s t e d t h a t more work s h o u l d be done b e f o r e t h e y b e
considered mitochondrial equivalents.
Fogg ( 1 9 5 5 ) c o n s i d e r e d t h a t
t h e g r a n u l e s w e r e p r o t e i n a c e o u s a n d r e p o r t e d them t o h a v e a h i g h
arginine content.
T h e y have been s u g g e s t e d t o be p h o s p h o l i p i d i n n a t u r e
(Fuhs, 1958b; Marcenko, 1951/1962).
Marcenko r e p o r t e d t h a t c y a n o p h y c i n
g r a n u l e s w e r e n o t s t a i n e d w i t h Sudan I I I o r w i t h J a n u s g r e e n .
Ris
a n d S i n g h ( 1 9 6 1 ) c o n s i d e r e d t h e s t r u c t u r e d g r a n u l e s o f Drews a n d
N i k l o w i t z t o be a t y p e o f membrane d i f f e r e n t i a t i o n d i s t i n c t f r o m t h a t
of the lamellae.
P a n k a r a t z a n d Bowen ( 1 9 5 3 ) o b s e r v e d s i m i l a r g r a n u l e s
in Symploca muscorum, but failed to find evidence of a membranous
i n t e r n a l o r g a n i z a t i o n o r o f a l i m i t i n g membrane.
The g r a n u l e s d i d
show an i n t e r n a l p a t t e r n o f i r r e g u l a r d e n s e and l e s s - d e n s e r e g i o n s ,
a n d t h e y w e r e p o o r l y p r e s e r v e d by KMnO. f i x a t i o n .
In addition to the inclusions previously described which were
common t o m o s t b l u e - g r e e n s ( ^ - g r a n u l e s , p o l y h e d r a l b o d i e s , c ^ - g r a n u l e s ,
a n d s t r u c t u r e d g r a n u l e s ) , t h e r e h a v e been many i n t e r e s t i n g i n c l u s i o n s
18
r e p o r t e d w h i c h w e r e n o t a s w i d e s p r e a d i n o c c u r r e n c e and, i n some c a s e s ,
a p p e a r e d t o be s p e c i f i c t o a g i v e n c o l l e c t i o n ( U e d a , 1 9 6 5 ; J e n s e n ,
1965).
Several blue-green algae contain highly refractile areas called
gas or pseudovacuoles.
Bowen a n d J e n s e n ( 1 9 5 5 ) d e s c r i b e d h e x a g o n a l l y
close-packed arrays of gas vesicles.
Single vesicles had conical
e n d s a n d r a n g e d from 0 . 2 t o 1 . 0 u i n l e n g t h w i t h an a v e r a g e d i a m e t e r
o f 7 5 mu.
T h e s e v e s i c l e s w e r e l i m i t e d by a s i n g l e e l e c t r o n - d e n s e mem­
b r a n e 2 mu w i d e a n d , u n l i k e t y p i c a l u n i t m e m b r a n e s , t h i s membrane was
n o t p r e s e r v e d by KMnO^ f i x a t i o n .
Jost (1965) described 'hohlspindeln'
which he observed in freeze-etched preparations and which appeared
t o b e s i m i l a r t o t h e g a s v e s i c l e s o b s e r v e d by Bowen a n d J e n s e n ( 1 9 6 5 ) .
The h o h l s p i n d e l n were a b o u t 6 5 mu i n d i a m e t e r , o f v a r i a b l e l e n g t h ,
and had a complex outer surface structure consisting of 40 A ribs.
19
MATERIALS AND METHODS
Culture Techniques
E a r l y s t u d i e s e m p l o y e d N o s t o c muscorum s t r a i n 1 0 3 7 o b t a i n e d a s a
uni a l g a l c u l t u r e from t h e I n d i a n a U n i v e r s i t y c u l t u r e c o l l e c t i o n .
This
c u l t u r e was b a c t e r i a l l y c o n t a m i n a t e d a n d a N o s t o c s p . s t r a i n M - 1 2 . 4 . 1
obtained as a unialgal culture from the Kaiser Research Foundation and
s u n t i l i t was b a c t e r i a f r e e was a d o p t e d
for routine use.
T h i s o r g a n i s m h a s r e c e n t l y b e e n i d e n t i f i e d a s N_.
muscorum by D r . F r a n c i s D r o u e t ,
The strain exhibited very flocullent
g r o w t h i n b o t h m e d i a e m p l o y e d a n d , s i n c e i t f o r m e d no c l u m p s o r s h e e t s ,
i t was c o n s i d e r e d d e s i r a b l e f o r t h e c u r r e n t s t u d i e s .
Two c u l t u r e m e d i a w e r e u s e d r o u t i n e l y .
Most o f t h e c u l t u r e s w e r e
grown i n Chu ( C h u , 1 9 4 2 ) w i t h s o i l e x t r a c t a d d e d a t t h e r a t e o f 4 0
ml p e r l i t e r .
et a l . , 1950).
O t h e r c u l t u r e s w e r e grown i n G e r l o f f ' s medium ( G e r l o f f
One ml o f c e l l s u s p e n s i o n was u s e d a s i n o c u l u m f o r
100 ml o f medium i n 2 5 0 ml E r l e n m e y e r c u l t u r e f l a s k s .
Cultures were
generally maintained on a 12-hour alternating dark and light regime.
T h e y w e r e grown i n h i g h - l i g h t ( 1 0 0 f o o t c a n d l e s ) f o r 1 0 - 2 0 d a y s a n d
then transferred to low-light conditions (20 foot candles).
The c u l t u r e
chamber contained fluorescent light banks and a single 25-watt incan­
descent bulb.
The t e m p e r a t u r e was m a i n t a i n e d a t a p p r o x i m a t e l y 2 0 ° C .
An e f f o r t w a s made t o u s e c u l t u r e s b e t w e e n 2 a n d 5 weeks o l d a n d t o
standardize the time of day at which cultures were harvested.
20
Fixation Procedures
S i n c e t h e c u l t u r e was f i n e l y s u s p e n d e d , w h o l e c e l l s w e r e c o n c e n ­
t r a t e d f r o m t h e c u l t u r e medium b y c e n t r i f u g a t i o n i n a c l i n i c a l c e n t r i ­
f u g e a t a p p r o x i m a t e l y 1 , 0 0 0 rpm f o r 2 m i n u t e s .
Cool 4% a g a r was u s u a l l y
used to solidify the concentrated cells into a block which could be
c u t i n t o s m a l l p i e c e s a n d h a n d l e d more e a s i l y d u r i n g t h e s u b s e q u e n t
fixing, dehydrating,and embedding procedures.
Occasionally the cells
w e r e f i x e d i n t h e s u s p e n d e d s t a t e and t h e n p u t i n t o a g a r j u s t p r i o r t o
dehydration.
All of the pellets of subcellular components prepared
b y t h e v a r i o u s c e n t r i f u g a t i o n p r o c e s s e s r e q u i r e d a g a r embedding p r i o r
to fixation.
Several fixation procedures were employed,and the details of each
a r e o u t l i n e d i n A p p e n d i x A.
Most o f t h e c e l l s w e r e f i x e d i n 1% osmium
t e t r o x i d e i n v e r o n a l a c e t a t e b u f f e r a t pH 6 . 1 a s d e s c r i b e d by
Kellenberger et al.
a n d Bowen ( 1 9 5 3 ) .
current studies.
( 1 9 5 8 ) a n d m o d i f i e d f o r b l u e - g r e e n s by P a n k r a t z
T r y p t o p h a n was o m i t t e d from t h e f i x a t i v e i n t h e
O t h e r f i x a t i v e s e m p l o y e d i n c l u d e d 4% u n b u f f e r e d
p o t a s s i u m p e r m a n g a n a t e , ( L u f t , 1 9 5 5 ; M o l l e n h a u e r , 1 9 5 9 ) 4% f o r m a l d e h y d e
( P e a s e , 1964) o r 3% p h o s p h a t e - b u f f e r e d g l u t a r a l d e h y d e ( S a b a t i n i e t
a l . , 1 9 5 3 ) w i t h o r w i t h o u t p o s t - f i x a t i o n i n osmium.
Dehydration,
i n f i l t r a t i o n , a n d e m b e d d i n g i n Epon 8 1 2 w e r e p e r f o r m e d a s d e s c r i b e d
i n A p p e n d i x A.
S e v e r a l enzyme d i g e s t i o n p r o c e d u r e s w e r e e m p l o y e d
as outlined in text Figure 1.
21
P r e p a r a t i o n o f C e l l Horaogenates
The r o u t i n e h o m o g e n i z i n g p r o c e d u r e e m p l o y e d t h e M i c k l e d i s i n t e g ­
rator.
C e l l s w e r e c o n c e n t r a t e d f r o m t h e c u l t u r e medium b y c r u d e
c e n t r i f u g a t i o n , and t h e c o n c e n t r a t e d s a m p l e was t h e n a d d e d t o a p p r o x ­
i m a t e l y a t e n - f o l d volume o f 0 . 2 mm g l a s s B a l b i n i h o m o g e n i z i n g b e a d s .
T h e b e a d s w e r e p r e s o a k e d i n t h e c h o s e n h o m o g e n i z i n g medium,and t h e
t o t a l volume o f beads a n d s a m p l e f i l l e d t h e M i c k l e s a m p l e c u p .
Two
s i m i l a r s a m p l e s were p r e p a r e d a n d r u n i n t h e c o l d f o r 5 - 2 5 m i n u t e s
w i t h t h e M i c k l e a d j u s t e d f o r maximum d i s p l a c e m e n t .
The r e s u l t i n g b r e i
was u s e d i m m e d i a t e l y f o r n e g a t i v e s t a i n i n g s t u d i e s , d i l u t e d a n d s u b ­
j e c t e d t o d i f f e r e n t i a l c e n t r i f u g a t i o n , o r l a y e r e d d i r e c t l y on a p r e ­
pared gradient for density gradient studies.
If sucrose or Ficoll
(Pharmacia Fine Chemicals) were used during homogenization, however,
t h e b r e i was n o t s u i t a b l e f o r n e g a t i v e s t a i n i n g .
Centrifugation Procedures
In addition to routine harvesting of cells by low-speed centrifugation in a clinical centrifuge, several other centrifugation
procedures were employed.
A S p i n c o Model L p r e p a r a t i v e c e n t r i f u g e
w i t h a #40 h e a d was u s e d f o r t h e d i f f e r e n t i a l c e n t r i f u g a t i o n .
A
SW-50 s w i n g i n g - b u c k e t r o t o r was u s e d on t h e same c e n t r i f u g e f o r t h e
density gradient preparations.
Density gradients were prepared in
5 ml c e l l u l o i d t u b e s by c a r e f u l l y p i p e t t i n g l a y e r s o f t h e d e s i r e d
sucrose or sucrose-Pi coll solutions.
Continuous gradients were pre­
p a r e d by e s t a b l i s h i n g a d i s c o n t i n u o u s g r a d i e n t a n d p e r m i t t i n g i t t o
diffuse overnight at 4°C,
D i s c o n t i n u o u s g r a d i e n t s w e r e u s e d immed­
iately after preparation.
A S o r v a l l S S - 3 w i t h a SM-24 h e a d and 3 ml
t u b e s was o c c a s i o n a l l y u s e d f o r d i f f e r e n t i a l c e n t r i f u g a t i o n .
S p e c t r o p h o t o m e t r i c Measurements
A b s o r p t i o n s p e c t r a w e r e o b t a i n e d u s i n g a Beckman DU s p e c t r o p h o t o ­
meter equiped with quartz cuvetts.
t h e 2 ml c u v e t t e .
The s a m p l e s w e r e d i l u t e d t o f i l l
No q u a n t i t a t i v e d e t e r m i n a t i o n s w e r e made.
The
absorption spectra for the solvent extraction experiments were obtained
u s i n g a Bausch a n d Lomb S p e c t r o n i c 2 0 c o l o r i m e t e r .
Microscopy
S e c t i o n s w e r e c u t w i t h a DuPont diamond k n i f e on a n LKB U l t r o t o m e .
The 4 0 - 8 0 mu s e c t i o n s w e r e r o u t i n e l y p i c k e d up on c l e a n , e t c h e d , 4 0 0 mesh c o p p e r g r i d s , b u t 150-mesh c a r b o n - c o a t e d f o r m v a r - s u p p o r t e d g r i d s
were used for the negative staining procedures.
The s e c t i o n s w e r e
routinely stained 10-15 minutes in methanol-uranyl acetate (Stempak
and Ward, 1954).
O c c a s i o n a l l y l e a d c i t r a t e s t a i n was e m p l o y e d ( V e n a b l e
and Coggeshall, 1955).
S p e c i m e n s w e r e o b s e r v e d u s i n g an RCA EMU-3F e l e c t r o n m i c r o s c o p e
a t 5 0 kV w i t h e i t h e r a 25 u o r a 3 0 - 4 0 u o b j e c t i v e a p e r t u r e .
Some
o f t h e n e g a t i v e l y s t a i n e d s p e c i m e n s w e r e o b s e r v e d a t 1 0 0 kV.
Micro­
graphs were taken at 14,000 to 17,000 machine magnification on Kodak
h i g h c o n t r a s t p l a t e s w h i c h w e r e d e v e l o p e d f o r 2 - 4 m i n u t e s i n Kodak
D-19 d e v e l o p e r , o r C r o n a r f i l m w h i c h was d e v e l o p e d f o r 1 - 2 m i n u t e s .
T h e n e g a t i v e s w e r e r o u t i n e l y e n l a r g e d 4 . 2 t i m e s on Kodak F2-F5
23
Kodabromide paper.
Negative Staining Procedure
The crude brei prepared by homogenizing the cells in the Mickle
d i s i n t e g r a t o r was d e c a n t e d f r o m t h e h o m o g e n i z i n g b e a d s a n d n e g a t i v e l y
stained by the loop-film technique described by Murray (1963).
The
suspension to be examined was mixed in a small staining dish as
follows:
t h r e e d r o p s o f s u s p e n s i o n , t h r e e d r o p s o f 4% p h o s p h o t u n g s t i c
a c i d (PTA) a t pH 7 . 2 , t h r e e d r o p s o f d o u b l e - d i s t i l l e d w a t e r , a n d o n e
d r o p o f b o v i n e serum a l b u m i n s o l u t i o n .
The mixture was s t i r r e d and
permitted to stand 1-3 minutes so that the larger particles settled.
A 5 mm p l a t i n u m t r a n s f e r - l o o p was u s e d t o t r a n s f e r a s a m p l e d r o p t o
a 150-mesh carboned formvar-coated grid.
i m m e d i a t e l y a t e i t h e r 5 0 o r 1 0 0 kV.
The specimen was examined
24
OBSERVATIONS
Sectioned Cells
Osmi urn f i x a t i o n
T h e g e n e r a l p r e s e r v a t i o n o f c e l l u l a r c o m p o n e n t s a f t e r osmium
fixation and uranyl acetate staining appeared excellent (Figure 2).
Unlike other strains of Nostoc muscorum, the strain employed for
t h e m a j o r p a r t o f t h i s s t u d y s e l d o m h a d an o b s e r v a b l e s h e a t h .
The
w a l l c o m p l e x c o n s i s t e d o f two p a r t s , t h e o u t e r member a n d t h e i n n e r
investment.
T h e o u t e r membrane was o f t e n s l i g h t l y u n d u l a t o r y b u t
m e a s u r e d 6 - 8 mu i n w i d t h a n d e x h i b i t e d t h e t r i p a r t a t e p r o f i l e o f a
unit membrane.
T h e i n n e r i n v e s t m e n t w a s u s u a l l y o b s e r v e d a s a medium
d e n s e h o m o g e n e o u s 6 - 9 mu z o n e a p p r o x i m a t e l y e q u i d i s t a n t b e t w e e n t h e
o u t e r membrane a n d t h e p l a s m a m e m b r a n e .
T h e 7 - 8 mu p l a s m a membrane
a p p e a r e d i n p r o f i l e a s a u n i t membrane a n d h a d a s l i g h t l y u n d u l a t o r y
a p p e a r a n c e s i m i l a r t o t h a t o f t h e o u t e r membrane ( F i g u r e s 2 a n d 5 ) .
When s e c t i o n e d i n o b l i q u e p l a n e s , t h e i n n e r i n v e s t m e n t was n o t a l w a y s
e v i d e n t , a n d t h e r e g i o n b e t w e e n t h e p l a s m a membrane a n d t h e o u t e r mem­
brane appeared to contain a uniform less dense material (Figure 2).
The photosynthetic apparatus consisted of an irregular array of
thylakoids extending throughout the cytoplasm but most often observed
in the peripheral region of the cell.
A f t e r osmium f i x a t i o n a n d u r a n y l
acetate staining, the thylakoids characteristically appeared as a 5p a r t e d m y e l i n - l i k e c o m p l e x 1 5 - 1 8 mu w i d e c o n s i s t i n g o f t w o e x t e r i o r
2 - 3 mu d e n s e z o n e s s e p a r a t e d f r o m a c e n t r a l 5 - 6 mu d e n s e z o n e b y t w o
25
2 - 3 mu l e s s d e n s e a r e a s (Figure 2 ) .
Although this appressed condition
was most characteristic, occasionally the two unit membranes were sep­
a r a t e d b y an i n t r a t h y l a k o i d a l s p a c e .
S p h e r i c a l 3 0 - 1 2 0 mu e l e c t r o n - d e n s e ^ - g r a n u l e s a p p e a r e d s c a t t e r e d
throughout the thylakoidal region (Figure 2).
T h e 2 5 - 3 0 mu ©«^-granules
were of low electron-density and were usually located between the
t h y l a k o i d s ( F i g u r e 9 ) , b u t t h e y w e r e s e l d o m w e l l p r e s e r v e d b y osmium
fixation.
Many p r e p a r a t i o n s i n w h i c h t h e ^ ( - g r a n u l e s w e r e n o t p r e s e r v e d
showed electron-transparent areas in the interthylakoidal regions.
Thylakoids,when viewed in oblique section,were observed to support a
regular array of rod or disk-like particles (Figure 5).
The array
u s u a l l y a p p e a r e d t o c o n s i s t o f r o d s a p p r o x i m a t e l y 6 - 7 mu w i d e a n d 2 5 4 0 mu i n l e n g t h s p a c e d 6 mu a p a r t .
R e l a t i v e l y d e n s e 9 - 1 2 mu p a r t i c l e s a s s u m e d t o b e r i b o s o m e s
appeared to be concentrated in the nucleoplasm.
The anastomosing
nucleoplasm had areas of less electron-density in which the only
o b s e r v a b l e s t r u c t u r e was t h a t o f a f i n e n e t w o r k c o n s i s t i n g o f DNA
fibers (Figures 2 and 5).
A third component observed within the
n u c l e o p l a s m was t h e p o l y g o n a l p o l y h e d r a l b o d y .
Single moderately
e l e c t r o n - d e n s e _ p o l y h e d r a l b o d i e s w e r e u s u a l l y 2 0 0 - 3 0 0 mu i n d i a m e t e r
and were often observed in clusters (Figures 2 and 3).
A 3 - 5 mu
dense zone appeared to limit these bodies but was seldom seen in section
to completely surround them.
Where t h i s z o n e a p p e a r e d t o b e i n t e r ­
rupted, the polyhedral bodies appeared continuous with the rest of the
25
nucleoplasm (Figures 2 and 5).
One o f t h e l e a s t e l e c t r o n - d e n s e c o m p o n e n t s o f t h e c e l l was t h e
structured granule (Figures 2 and 3).
These granules were roughly
s p h e r i c a l , r a n g e d i n s i z e up t o 5 0 0 m u , a n d w e r e u s u a l l y o b s e r v e d i n
the peripheral portions of the cells.
The granules sometimes contained
i r r e g u l a r , m o r e e l e c t r o n - d e n s e a r e a s b u t n o l i m i t i n g membrane was
ever observed.
F i g u r e 6 s h o w s t h e c e l l u l a r c o m p o n e n t s after OsO^ f i x a t i o n b u t
without a subsequent stain.
In addition to the general lack of contrast,
several other differences were observed.
T h e o u t e r membrane a n d t h e
i n n e r i n v e s t m e n t w e r e n o t r e a d i l y v i s i b l e , b u t t h e 7 - 8 mu p l a s m a
membrane w a s s t a i n e d .
Q u i t e u n l i k e m a t e r i a l f i x e d t h e s a m e way b u t
stained with uranyl acetate (Figure 2), the thylakoids (Figure 6)
a p p e a r e d a s a 1 5 - 1 7 mu c o m p l e x c o n s i s t i n g o f t w o 6 - 8 mu d e n s e l i n e s
s e p a r a t e d 'by a 3 - 4 mu l e s s d e n s e s p a c e .
uniform low electron-density.
The p o l y h e d r a l b o d i e s w e r e o f
A 2 - 3 mu s l i g h t l y m o r e d e n s e z o n e w a s
o b s e r v e d s u r r o u n d i n g some o f t h e b o d i e s , b u t i t a p p e a r e d q u i t e d i f f e r e n t
from, and less dense than,the unit membranes of the thylakoids.
The
structured granules (Figure 6) appeared more electron-dense than a f t e r
staining with uranyl acetate (Figure 3) and contained irregular areas
of even greater electron-density.
The^-granules were osmiophilic but
the nucleoplasm appeared of low electron-density.throughout.
Figure 7 shows the appearance of cells stained with lead c i t r a t e
a f t e r OsO^ f i x a t i o n .
T h e g r e a t l y i n c r e a s e d s t a i n i n g o f t h e 9 - 1 2 mu
r i b o s o m e s w a s t h e m o s t s t r i k i n g d i f f e r e n c e o b s e r v e d when c o m p a r e d w i t h
27
the unstained cell (Figure 6).
T h e o C - g r a n u l e s a n d t h e DNA f i b r i l s
w e r e a l s o s t a i n e d , b u t t h e r e was l i t t l e c h a n g e i n t h e a p p e a r a n c e o f t h e
other cellular components.
Glutaraldehyde fixation
Glutaraldehyde fixation alone provided l i t t l e contrast and the
cells were barely distinguishable from the embedding medium.
Such
c e l l s a f t e r staining with uranyl acetate (Figure 8) had a relatively
high electron-density throughout but exhibited l i t t l e contrast.
Each
t h y l a k o i d ( F i g u r e 3 ) a p p e a r e d a s a 4 - 5 mu c e n t r a l d e n s e l i n e f l a n k e d
o n e i t h e r s i d e by l e s s e l e c t r o n - d e n s e z o n e s 5 mu w i d e .
The nucleo­
plasm had a finely textured appearance, and the well preserved polyhedral
b o d i e s w e r e s u r r o u n d e d b y a 3 mu e l e c t r o n - d e n s e z o n e .
and ^-granules were not stained.
The ^(-granules
Cells fixed in glutaraldehyde,
p o s t f i x e d i n osmium, a n d s t a i n e d w i t h u r a n y l a c e t a t e ( F i g u r e s 9 a n d
10), appeared similar t o those fixed in osmium (Figure 2) but<K-granules
were more frequently observed (Figure 9 ) .
I n t h e e a r l y work e m p l o y i n g N o s t o c m u s c o r u m 1 0 3 7 , s m a l l t u b u l e ­
l i k e s t r u c t u r e s 1 2 - 1 5 mu i n w i d t h a n d o f u n d e t e r m i n e d l e n g t h w e r e o c c a ­
sionally observed after glutaraldehyde-osmium fixation (Figure 4).
The tubules had no apparent association with other cellular structures
and were not observed in Nostoc muscorum M-12.4.1 which was used f o r
the major portion of this study.
P o t a s s i um p e r m a n g a n a t e f i x a t i o n
C e l l s f i x e d w i t h KMnO^ a n d s u b s e q u e n t l y s t a i n e d w i t h u r a n y l a c e t a t e
28
(Figure 11) had a contrast comparable to the osmium-uranyl acetate
preparations (Figure 2).
The nucleoplasm assumed a greater electron-
density in comparison to the peripheral cytoplasm.
The polyhedral
b o d i e s showed a g r e a t l y i n c r e a s e d e l e c t r o n - d e n s i t y b u t n o s u r r o u n d i n g
d e n s e z o n e was o b s e r v e d a f t e r KMnO. f i x a t i o n .
The DNA s t r a n d s w e r e
visible although they often exhibited a clumped appearance.
The
/^-granules were represented by small round electron-transparent
areas (Figure 11).
The a p p e a r a n c e o f c e l l s f i x e d i n KMnO. b u t n o t
s u b s e q u e n t l y s t a i n e d i s shown i n F i g u r e 1 2 .
Each thylakoid appeared
a s a 1 4 - 1 7 mu c o m p l e x c o n s i s t i n g o f two 6 - 7 mu u n i t m e m b r a n e s s e p a r a t e d
by a 3 - 4 mu s p a c e .
The polyhedral bodies appeared s l i g h t l y electron-
dense, and the nucleoplasm appeared finely granular with regions of low
e l e c t r o n d e n s i t y , e s p e c i a l l y when c o m p a r e d t o t h e p o s t - s t a i n e d c e l l
(Figure 11).
The ^ - g r a n u l e s ( F i g u r e 1 2 ) w e r e r e p r e s e n t e d b y s m a l l
electron-dense rings, but the structured granules were not preserved.
Solvent Extraction
Unfixed cells and cells previously fixed in glutaraldehyde were
t r e a t e d w i t h s e v e r a l s o l v e n t s (100% m e t h a n o l , 100% a c e t o n e , o r 20%
acetone-80% methanol) in an attempt to extract pigments and to
observe any correlated morphological change.
U n f i x e d c e l l s w e r e t r e a t e d w i t h 20% a c e t o n e - 8 0 % m e t h a n o l a s
described by Gantt and Conti (1966) and fixed in osmium.
The control
consisted of similarly fixed cells from the same culture which had not
been solvent treated.
The control appeared similar to the osmium-
fixed cells previously described (Figure 2), but the treated cells were
29
quite altered (Figure 13).
T h e y a p p e a r e d q u i t e cremated, a n d t h e o u t e r
membrane a n d i n n e r i n v e s t m e n t w e r e s e p a r a t e d f r o m t h e p l a s m a membrane
and the protoplast.
The o u t e r membrane a p p e a r e d h i g h l y c o n v o l u t e d , a n d
many s m a l l p a p i l l a e w e r e o b s e r v e d .
The cell contents appeared clumped
a n d t h e r e w e r e m y e l i n - l i k e membranous w h o r l s i n t h e c y t o p l a s m .
polyhedral bodies remained recognizable.
The
The thylakoids consisted of
t w o 3 mu e l e c t r o n - t r a n s p a r e n t l a y e r s s e p a r a t e d by a n e l e c t r o n - d e n s e
l a y e r 4 - 5 mu w i d e .
T h e 3 mu d e n s e z o n e s w h i c h f l a n k t h i s c o n f i g u r a t i o n
in the control were hard to distinguish or were absent.
The ^-granules
were also less apparent than those in the control.
B e c a u s e o f t h e a p p a r e n t e x t e n s i v e damage t o the s o l v e n t - t r e a t e d
unfixed cells, cells were prefixed with gl utaraldehyde, then treated
f o r 1 h o u r w i t h e i t h e r 100% m e t h a n o l , 100% a c e t o n e , o r 20% a c e t o n e 80% m e t h a n o l , a n d p o s t - f i x e d i n o s m i u m .
p r e s e r v a t i o n was i m p r o v e d .
In general, the cellular
The controls appeared similar to glutar-
aldehyde-osmium fixed cells previously described (Figure 10).
T h e c e l l s t r e a t e d w i t h 100% a c e t o n e h a d a s l i g h t l y g r a n u l a r
appearance.
Few e l e c t r o n - d e n s e / ^ g r a n u l e s w e r e o b s e r v e d a f t e r t h e
solvent extraction and several less-dense areas were observed from
w h i c h t h e / ^ - g r a n u l e s may h a v e b e e n e x t r a c t e d ( F i g u r e 1 4 ) .
The appear­
ance of the thylakoids and other structures was similar t o t h a t of the
control.
The a b s o r p t i o n s p e c t r u m o f t h e s o l v e n t e x t r a c t i n d i c a t e d
carotenoid extraction but l i t t l e extraction of chlorophyll.
C e l l s t r e a t e d w i t h 100% m e t h a n o l ( F i g u r e 1 5 ) s h o w e d a s l i g h t
derangement of the thylakoids and the electron-dense outer zones of
30
t h e t h y l a k o i d s were d i f f i c u l t t o d i s t i n g u i s h .
Some o f t h e p e r i p h e r a l
^ g r a n u l e s w e r e removed l e a v i n g l e s s e l e c t r o n - d e n s e a r e a s .
Many o f
the more interior/^-granules remained intact but t h i s could be accounted
for i f the solvent gradually penetrated the cell.
A thin electron-
t r a n s p a r e n t z o n e was o b s e r v e d i n t e r i o r t o t h e i n n e r i n v e s t m e n t , a n d t h e
p l a s m a membrane was n o t v i s i b l e .
This zone was not observed in e i t h e r
the control or the acetone-extracted cells.
were not visibly changed.
Other cell components
The absorption spectrum of the extract
indicated both carotenoid and chlorophyll extraction.
Cells extracted
w i t h 20% a c e t o n e - 8 0 % m e t h a n o l a p p e a r e d s i m i l a r t o t h e m e t h a n o l - e x t r a c t e d
cells just described (Figure 15),and the absorption spectra of the
extracts were also similar.
The absorption spectra of the cells after solvent extraction
i n d i c a t e d t h a t some o f t h e c h l o r o p h y l l a n d c a r o t e n o i d s r e m a i n e d
u n e x t r a c t e d , b u t t h e c e l l s e x p o s e d t o 100% m e t h a n o l a n d t o 20% a c e t o n e 80% m e t h a n o l r e t a i n e d r e l a t i v e l y l i t t l e c h l o r o p h y l l .
The phycocyanins
w e r e n o t removed f r o m t h e c e l l s b y t h e s o l v e n t s e m p l o y e d .
Enzyme T r e a t m e n t s
S e v e r a l enzymes i n c l u d i n g p e p s i n , t r y p s i n , c h y m o t r y p s i n , l i p a s e ,
deoxyribonuclease (DNase) and ribonuclease (RNase) were used in an
e f f o r t t o e x t r a c t known compounds f r o m i n t a c t c e l l s ( F i g u r e 1 ) .
An i n i t i a l e x p e r i m e n t w a s d o n e i n w h i c h u n f i x e d c e l l s w e r e e x p o s e d
t o RNase b u t t h e c o n t r o l ( w h i c h h a d b e e n e x p o s e d t o b u f f e r o n l y )
s h o w e d s u c h e x t e n s i v e damage t h a t s u b s e q u e n t d i g e s t i o n s w e r e performed
using pre-fixed cells.
The cells were fixed with e i t h e r glutaraldehyde
31
o r formaldehyde, r i n s e d , incubated i n t h e enzyme o r c o r r e s p o n d i n g c o n ­
trol conditions, post-fixed in OsO., dehydrated, and embedded.
A s t u d y o f F i g u r e 1 s h o w s that, w i t h o n e e x c e p t i o n , n o s p e c i f i c
e x t r a c t i o n a t t r i b u t a b l e t o t h e enzyme e m p l o y e d was a t t a i n e d .
Of
particular interest, however, were the cells prefixed in formaldehyde
a n d e x p o s e d t o RNase a c c o r d i n g t o t h e m e t h o d o f J a c o b s o n e t a l ,
(1963).
A l t h o u g h t h e c e l l u l a r p r e s e r v a t i o n was l e s s t h a n d e s i r a b l y t h e
results were consistent.
The nucleoplasm in the digested material
(Figure 16) consisted of a single, moderately electron-dense mass and
the polyhedral bodies could not be seen as discrete e n t i t i e s .
The
control (Figure 17) also exhibited a clumped appearance, but the poly­
hedral bodies remained structurally intact.
In both instances the outer
membrane o f t h e c e l l w a s q u i t e u n d u l a t o r y a n d s o m e t i m e s formed p a p i l l a e
(Figures 15 and 17).
T h e f o r m a t i o n o f p a p i l l a e w a s a l s o common i n
c e l l s treated a t 60°C (Figure 21) or with Tris buffer (Figure 20).
T h e r e m a i n d e r o f t h e enzyme t r e a t m e n t s f a i l e d t o v i s i b l y a l t e r t h e
cellular components i f the treated cells were carefully compared with
their corresponding controls.
interest.
However, several of the controls were of
Since formaldehyde-osmium fixation had not been employed in
the current work prior to t h i s study, i t was interesting to note
that i t compared favorably with the glutaraldehyde-osmium fixation
often employed (Figures 18 and 19).
In glutaraldehyde-fixed cells incubated at 60°C (Figure 21),
peripheral electron-dense myelin-like whorls were observed.
The
structured granules usually observed in this region were not found.
FIXATION*
COMMENTS
ENZYME TREATMENT
4 hr F
RNase ( 0 . 0 7 % ) i n d i s t . FLO pH 6 . 6
4 h r . 60OC, TCA ( 5 % , 4 ° C , 3 0 m i n ) .
Specific extraction. Figures 16 and 17.
Poor fixation, crenation, papillae.
12 hr G
RNase ( 0 . 0 7 % ) i n d i s t . H^O pH 6 . 6
4 h r , 60OC, TCA ( 5 % , 4 ° C , 3 0 m i n ) .
No s p e c i f i c e x t r a c t i o n . F i g u r e 2 1 . F i x a t i o n
poor. Slight crenation and formation of
papillae. Myelin-like whorls.
2 hr G
RNase ( 0 . 0 8 % ) i n d i s t . hUO, pH 6 . 8
1 0 h r , 37OC.
No s p e c i f i c e x t r a c t i o n .
Fixation poor.
2 hr G
RNase ( 0 . 0 5 % ) i n p h o s p h a t e b u f f e r
pH 7 . 4 , 4 h r , 3 2 0 C .
No s p e c i f i c e x t r a c t i o n .
Fixation poor.
4 hr F
RNase ( 0 . 0 7 % ) i n T r i s - H C l b u f f e r
pH 7.6, 18 hr, 20°C, TCA (5%, 4OC,
30 min).
12 hr G
RNase ( 0 . 0 7 % ) i n T r i s - H C l b u f f e r
pH 7.6, 18 hr, ZO^C, TCA (5%, 4°C,
30 min).
No s p e c i f i c e x t r a c t i o n . F i g u r e 2 0 . F i x a t i o n
poor. Slight crenation and formation of
papillae.
2 hr G
Lipase (0.2%) in phosphate buffer
at pH 7.4, 4 hr, 320C.
No s p e c i f i c e x t r a c t i o n .
•No s p e c i f i c e x t r a c t i o n . F i x a t i o n p o o r .
Walls separated from cell. Cells crenated.
*F=Formaldehyde fixation
G=Glutaraldehyde fixation
C e l l s w e r e p o s t - f i x e d i n OsO, a f t e r t r e a t m e n t .
Figure 1.
Enzyme T r e a t m e n t s Employed
F i x a t i o n fair.
FIXATION
ENZYME TREATMENT
COMMENTS
2 hr G
Trypsin (0.1%) in phosphate buffer
pH 8 . 0 , 4 h r , 3 2 ° C .
No s p e c i f i c e x t r a c t i o n .
Fixation fair.
12 hr G
Chymotrypsin (0.05%) in Tris-HCl
b u f f e r pH 7 . 6 , 1 8 h r , 2 0 ° C
No s p e c i f i c e x t r a c t i o n .
fai r.
Figure 19.
Fixation
4 hr F
Chyniotrypsin (0.05%) in Tris-HCl
b u f f e r pH 7 . 6 , 1 8 h r , 2 0 ° C .
No s p e c i f i c e x t r a c t i o n .
fai r.
Figure 18.
Fixation
2 hr G
P e p s i n ( 0 . 1 5 % ) i n 0 . 0 1 N MCI, 4
hr. 320c.
No s p e c i f i c extraction. F i g u r e s 23, 24,and
25. P o l y h e d r a l b o d i e s r e m o v e d . S l i g h t
formation of papillae.
12 hr G
DNase (1%) i n 0 . 0 0 3 M MgSO, pH
6 . 6 , 4 h r , 2 0 0 C , TCA (5%, 4 0 C ,
30 min.)
No s p e c i f i c e x t r a c t i o n .
Fixation poor.
Cells granular.
4 hr F
DNase (1%) i n 0 . 0 0 3 M MgSOa pH
6 . 6 , 4 h r , 20OC, TCA ( 5 % , 4 0 C ,
30 min.)
No s p e c i f i c e x t r a c t i o n .
Fixation poor.
Figure 1. continued
34
F i g u r e s 23, 2 4 , a n d 2 5 show t h e r a n g e o f v a r i a t i o n o b s e r v e d i n t h e c e l l s
e x p o s e d t o 0 . 0 1 N HCl w h i c h w a s t h e c o n t r o l f o r t h e p e p s i n t r e a t m e n t .
T h e r e was a s l i g h t f o r m a t i o n o f p a p i l l a e f r o m t h e o u t e r membrane
(Figure 24).
The nucleoplasm was very electron-dense and had a clumped
rather than fibrillar appearance.
The appearance of the polyhedral
b o d i e s v a r i e d g r e a t l y a n d w a s p a r a l l e l e d by s i m i l a r c h a n g e s i n t h e i n t e r thylakoidal regions of the cell.
I n some c e l l s t h e y w e r e t o t a l l y
removed (Figure 23), but in others they were observed as electrontransparent regions containing small clumped f i b r i l s (Figure 25).
T h e s t r u c t u r e d g r a n u l e s w e r e a l s o r e m o v e d by t h i s a c i d t r e a t m e n t
(Figure 22).
Cell Homogenates
Sectioned pel 1ets
C e l l s h o m o g e n i z e d i n v a r i o u s media, i n c l u d i n g d i s t i l l e d w a t e r a n d
T r i s b u f f e r a t pH 7 . 2 , w e r e s u b j e c t e d t o d i f f e r e n t i a l c e n t r i f u g a t i o n .
In general, whole cells, wall fragments, and large thylakoid fragments
were pelleted f i r s t .
I n c r e a s i n g c e n t r i f u g a l f o r c e s b r o u g h t down
increasingly smaller thylakoidal fragments and finally small f i b r i l s
only.
The blue phycocyanin pigment could not be pelleted from solution
by f o r c e s a s g r e a t a s 1 0 5 , 0 0 0 g f o r 2 h o u r s .
The composition of the
successive pellets overlapped considerably, giving no clear-cut
separation of subcellular particles.
Several subcellular components including polyhedral bodies and
structured granules were not observed in the early work, and since the
35
centn'fugation procedures required considerable time to complete, i t
was thought that fixation of the cells prior to homogenization might
stabilize these structures.
C e l l s f i x e d i n osmium o r g l u t a r a l d e h y d e
prior to homogenization were difficult to rupture.
Whole c e l l s a n d
l a r g e f r a g m e n t s w e r e common, b u t f e w c o m p o n e n t s w e r e r e l e a s e d f r e e i n t o
t h e medium a n d n o s a t i s f a c t o r y s e p a r a t i o n w a s a c h i e v e d .
However, f i x ­
a t i o n a f t e r c e l l h o m o g e n i z a t i o n b u t p r i o r t o c e n t n ' f u g a t i on d i d n o t
a p p r e c i a b l y a l t e r t h e a p p e a r a n c e o r b e h a v i o r o f t h e p a r t i c l e s when
compared with unfixed preparations.
A t t e m p t s t o i s o l a t e p o l y h e d r a l b o d i e s f o l l o w i n g disruption o f
the cells in buffers or distilled water alone failed.
The f i r s t
successful isolation was achieved in a medium of 20% Ficol 1-17% sucrose0 . 0 1 M NaCl a n d s u b s e q u e n t l y i n t h e s a m e medium w i t h o u t F i c o l l .
cell breakage-was decreased in these viscous media.
The
The polyhedral
bodies were found distributed throughout the various pellets after
differential centrifugation, but appeared most numerous in the pellet
obtained between 5,000-8,000 g.
The polyhedral bodies freed from the
cells (Figures 26, 27,and 29) appeared similar to those observed in
intact cells.
They remained very electron-dense and t h e i r characteris­
t i c polygonal profile was observed.
T h e y w e r e s u r r o u n d e d b y 2 - 5 mu
electron-dense 'membranes' but no unit nature was discerned.
In
several of the isolated polyhedral bodies (Figures 25 and 27), there
appeared a clear zone between the body i t s e l f and the associated
'membrane'.
Although most bodies appeared homogeneous (Figures 26
and 27), several appeared partially empty with only granular material
36
r e m a i n i n g ( F i g u r e s 28 a n d 2 9 ) .
Occasionally profiles were observed
of polyhedral body 'membranes' devoid of contents but retaining
their angularity (Figure 28).
In homogenized material, the bodies
often appeared to retain the close association with one another
( F i g u r e s 2 7 a n d 2 8 ) w h i c h was o b s e r v e d i n s e c t i o n e d c e l l s ( F i g u r e s
2, 3, and 10).
The polyhedral bodies usually appeared free from
other cellular components, but occasionally they appeared to be very
closely associated with the thylakoidal fragments (Figure 25).
Structured granules have been observed in several preparations
but relatively pure fractions were not obtained.
Structured granules
observed in glutaraldehyde-osmium fixed cell homogenates (Figure 30)
appeared quite homogeneous with dense wrinkles near the periphery
suggesting a rough surface.
A f t e r osmium f i x a t i o n ( F i g u r e 3 1 ) , t h e
same roughened surface was apparent, and there were small less dense
areas within the granules.
S t r u c t u r e d g r a n u l e s f i x e d i n osmium a n d
exposed to ethanol (Figure 32) appeared t o be partially dissolved
and clumped together in aggregates.
I n an e f f o r t t o i n c r e a s e t h e d e g r e e o f s e p a r a t i o n o f v a r i o u s
components, cell homogenates were subjected to density gradient
centrifugation.
The various layers obtained were harvested from the
b o t t o m o f t h e t u b e by a p u n c t u r e w i t h a s p e c i a l l y d e s i g n e d n e e d l e
device.
A f t e r c e n t r i f u g a t i o n a t 1 0 0 , 0 0 0 g o n a 60-20% s u c r o s e g r a d i e n t
t h e r e was a t h i c k g r e e n p e l l e t a t t h e t u b e b o t t o m a n d b l u e p i g m e n t
in the sample layer at the tube top.
T h e i n t e r m e d i a t e b a n d i n g was
quite variable, but there usually was a yellow band ranging from
37
d i s t i n c t t o r a t h e r d i f f u s e n e a r t h e t u b e bottom.
When t h i s b a n d w a s
h a r v e s t e d , i t was f o u n d t o h a v e a n a b s o r p t i o n maximum n e a r 4 4 0 mu.
A pellet of this material prepared for electron microscopy (Figure
35) was observed to contain thylakoidal vesicles with an average,
d i a m e t e r o f a p p r o x i m a t e l y 2 5 0 mu a n d a h i g h p r o p o r t i o n o f y ^ - g r a n u l e s .
Occasionally the^-granules appeared to be associated with the
thylakoidal vesicles.
the thylakoidal
In lower speed pellets from studies in which
system was less disrupted (Figure 33), the /^-granules
w e r e a s s o c i a t e d w i t h t h e t h y l a k o i d s , a n d a s many a s t h r e e o r f o u r
membranes a p p e a r e d t o i n t e r s e c t a t a ^ - g r a n u l e .
Fractions from the density gradient centrifugations were over­
l a p p i n g a n d t h e s m a l l v o l u m e o f m a t e r i a l o b t a i n e d o n h a r v e s t was e a s y
to lose in the subsequent repelleting and preparation for electron
microscopy.
Occasionally fragments of the cell envelope were observed (Figures
27, 35, and 37).
B o t h t h e o u t e r membrane a n d t h e p l a s m a membrane
r e t a i n t h e a p p e a r a n c e o f u n i t membranes ( F i g u r e 3 5 ) .
The inner invest­
ment i s visible as a comparatively electron-dense zone.
Often the
plasma membrane does not appear with the wall fragments (Figure 27)
b u t i t i s p e r h a p s p u l l e d away a s t h e c e l l c o n t e n t s a r e l o s t ( F i g u r e
37).
I n one o f t h e e a r l y f r a c t i o n a t i o n s t u d i e s , Nostoc muscorum
strain 1037 was employed and a high speed p e l l e t was obtained
( F i g u r e 3 4 ) w h i c h c o n t a i n e d 1 5 mu x 1 0 0 mu r o d o r t u b u l e - l i k e
structures.
T h e s e r o d s a p p e a r e d t o h a v e 4 - 5 mu e l e c t r o n - d e n s e z o n e s
on each side and an electron-transparent interior region giving the
38
appearance of a small tubule.
A l t h o u g h such structures h a v e n o t
been regularly observed in homogenates of strain M-12.4.1, similarappearing rods were occasionally observed (Figure 27).
These rods
w e r e 1 2 - 1 5 mu w i d e a n d 1 0 0 mu i n l e n g t h b u t a p p e a r e d t o b e o f u n i f o r m
electron-density.
Thylakoids separated from cells which were homogenized in water
and subsequently prepared for electron microscopy, had a smooth surface
(Figure 33) and most typically appeared as expanded vesicles.
As t h e
speed required to sediment the fragments was increased, the size of
the vesicles obtained decreased.
Most thylakoid fragments pelleted
f r o m a s u c r o s e h o m o g e n i z i n g medium a l s o h a d a s m o o t h s u r f a c e a n d
r e t a i n e d t h e i r 70 mu u n i t membrane p r o f i l e s ( F i g u r e 4 1 ) .
When F i c o l l
was e m p l o y e d i n t h e m e d i u m , i s o l a t e d t h y l a k o i d f r a g m e n t s had a c o a r s e
or rough surface (Figure 38) and in certain cases were seen to be
studded with 75-90 A particles.
T h e p a r t i c l e s w e r e s e e n on t h e o u t e r
surface of the expanded thylakoidal vesicle.
Sometimes the thylakoids
remained relatively unexpanded and appeared appressed (Figure 40)
similar to those observed in sectioned cells.
Elongate projections were
observed on the extrathylakoidal surfaces but the intrathylakoidal
spaces appeared structureless.
but varied in length.
These projections were 4-7 mu wide
Cells which failed to be disrupted in the Ficoll
medium a p p e a r e d v e r y w e l l p r e s e r v e d ( F i g u r e 4 3 ) .
these cells often appeared to be studded with 5-7
The thylakoids in
mu p r o j e c t i o n s
w h i c h w e r e r e g u l a r l y s p a c e d a n d a b o u t 6 mu a p a r t ( F i g u r e s 4 3 a n d 4 4 ) .
These projections closely resembled those observed in Figure 40 and
39
w e r e f u r t h e r s u g g e s t i v e o f t h e membrane p a r t i c l e s o b s e r v e d i n
Figure 5.
An e f f o r t was made t o s t a b i l i z e c e l l u l a r c o m p o n e n t s b y b r e a k i n g
t h e u n f i x e d c e l l s i n a 1% s o l u t i o n o f g l u t a r a l d e h y d e .
The homogeniza-
t i o n t o o k 2 0 m i n u t e s a n d many o f t h e c e l l s r e m a i n e d i n t a c t o r o n l y
slightly broken.
The c e l l f r a g m e n t s w h i c h w e r e r e l e a s e d h o w e v e r w e r e
quite interesting.
Few o f t h e t h y l a k o i d f r a g m e n t s w e r e f u l l y e x p a n d e d
(Figures 39 and 42) and most retained a partially appressed profile
with only narrow intrathylakoidal spaces.
Some o f t h e m e m b r a n e s
( F i g u r e 4 2 ) a p p e a r e d t o s u p p o r t 2 0 - 3 0 mu g l o b u l a r p a r t i c l e s o n t h e i r
outer surface.
In oblique section these particles appeared to be
a r r a n g e d i n rows a p p r o x i m a t e l y 3 5 mu a p a r t .
In other instances the
thylakoids appeared slightly more expanded (Figure 39), and small
p a r t i c l e s a p p r o x i m a t e l y 6 - 7 mu w i d e a n d 1 0 mu l o n g w e r e o b s e r v e d o n
the outer surface.
These resembled the particles observed in Figure
40 but appeared better preserved.
G r a n u l e s r o u g h l y 1 5 x 4 5 mu a l s o
a p p e a r e d t o b e a t t a c h e d t o some o f t h e s e m e m b r a n e s .
I t is not clear
whether the presence of such granules was due to better preservation
of the components or to a clumping effect not observed in other
preparations.
Negatively stained preparations
S a m p l e s f r o m u n f i x e d h o m o g e n a t e s w h i c h w e r e p r e p a r e d immediately
for electron microscopic observation were far superior to those prepared
from glutaraldehyde or osmium-fixed homogenates.
However, the fresh
40
samples seemed to deteriorate rapidly,and by 30 min?jtes a f t e r cell
r u p t u r e much o f t h e p o t e n t i a l d e t a i l was l o s t from t h e p r e p a r a t i o n .
C e l l h o m o g e n a t e s p r e p a r e d i n t h e Mickle d i s i n t e g r a t o r a n d n e g a t i v e l y
s t a i n e d w i t h 1% u r a n y l f o r m a t e o r 4% PTA w e r e p l a c e d o n c a r b o n e d
formvar-coated grids and observed immediately.
T h e t h y l a k o i d f r a g m e n t s o b s e r v e d i n t h e s e p r e p a r a t i o n s varied
from vesicular or tubular elements to f l a t sheets (Figures 45, 47, 48,
and 49).
O f t e n t h e t u b u l a r r e g i o n s showed a n e x p a a d e d b u l b o u s e n d
or holes at which bulbous or tubular evaginations could have broken
off (Figure 48).
The less disrupted fragments (Figure 45) ware
suggestive of a continuous anastomosing tubular network.
The surface
o f t h e t h y l a k o i d s v a r i e d f r o m a s m o o t h ( F i g u r e s 47 and 4 8 ) t o a
stippled appearance (Figure 45 and 49).
I n t h e most pronounced c a s e
)
( F i g u r e 4 9 ) , t h e membrane p a r t i c l e s h a d a n a v e r a g e d i a m e t e r o f 7 . 5 m u .
S m a l l r o d - s h a p e d p a r t i c l e s ( F i g u r e s 4 7 a n d 4 8 ) a v e r a g i n g 1 4 x 9 3 mu
w e r e o b s e r v e d i n a s s o c i a t i o n w i t h some o f t h e t h y l a k o i d s .
They appeared
s i n g l y ( F i g u r e 4 7 ) o r i n p a c k e t s o f two o r t h r e e ( F i g u r e 4 8 ) .
These
r o d s a p p e a r e d hom ogeneous a n d w e r e s i m i l a r i n d i m e n s i o n s t o t h o s e
o b s e r v e d i n s e c t i o n e d h o m o g e n a t e s o f s t r a i n M-12.4- . 1 ( F i g u r e 2 7 )
and strain 1037 (Figure 34).
E l e c t r o n - t r a n s p a r e n t g l o b u l e s r a n g i n g from 60 - 1 2 0 mu i n d i a m e t e r
were observed in negatively stained preparations ( Figures 45, 48,
and 49) and were considered to be the ^-granules.
They sometimes
a p p e a r e d f r e e f r o m o t h e r c e l l u l a r c o m p o n e n t s b u t i-zere o f t e n o b s e r v e d
in association with the thylakoids (Figure 45).
41
Although o<.-granules were not observed in pellets of cell
honiogenates, they were observed a f t e r negative staining (Figure 45)
and appeared similar to those observed in sectioned cells (Figure 9).
They were composed of two approximately equal parts joined a t a
slight constriction (Figure 45).
T h e i s o l a t e d g r a n u l e s a v e r a g e d 3 0 mu
w i d e and 7 0 mu l o n g a n d w e r e o f u n i f o r m d e n s i t y e x c e p t a t t h e m o r e
electron-dense midline.
T h e s u b s t r u c t u r e was much m o r e e v i d e n t
a f t e r s t a i n i n g w i t h u r a n y l f o r m a t e ( F i g u r e 4 6 ) t h a n w i t h PTA
(Figure 49)i
No s t r u c t u r e s w h i c h correlated w e l l w i t h s t r u c t u r e d g r a n u l e s o r
polyhedral bodies were observed in negatively stained preparations.
42
DISCUSSION
The Cell Envelope
T h e a b s e n c e o f a s h e a t h i n t h e o r g a n i s m s t u d i e d was s u r p r i s i n g
b u t may b e r e l a t e d t o t h e s t r a i n e m p l o y e d , t h e a g e o f t h e c u l t u r e ,
or i t s growth habit.
N o s t o c muscorum s t r a i n 1 0 3 7 w h i c h w a s u s e d i n
t h e e a r l y work w a s o b s e r v e d t o h a v e a s h e a t h a n d t e n d e d t o g r o w i n
thin sheets.
N_. muscorum s t r a i n M - 1 2 . 4 . 1 e x h i b i t e d a v e r y f l o c u l l e n t
g r o w t h a n d no s h e a t h w a s o b s e r v e d .
Cultures of M-12.4.1 were
u s u a l l y 2 - 3 w e e k s o l d when h a r v e s t e d f o r s t u d i e s b u t o l d e r c u l t u r e s
of 1037 were employed.
The structure of the remainder of the cell envelope (Figures 2
and 5) i s in general agreement with the observations by Ris and Singh
(1961) on several related organisms.
The dense inner investment in
t h e N o s t o c s t u d i e d i n t h e c u r r e n t w o r k , h o w e v e r , a v e r a g e s 1 0 mu i n
w i d t h a s c o m p a r e d t o 2 0 mu f o r t h e N o s t o c e m p l o y e d b y R i s a n d S i n g h .
T h i s d i f f e r e n c e i s n o t s u r p r i s i n g s i n c e t h e i n n e r i n v e s t m e n t was
o b s e r v e d t o v a r y i n w i d t h f r o m 1 0 mu i n A n a c y s t i s t o 2 0 0 mu i n
Oscillatoria (Ris and Singh, 1961).
The inner investment was observed
t o b e a p p r o x i m a t e l y e q u i d i s t a n t ( 6 mu) f r o m t h e o u t e r membrane a n d t h e
p l a s m a membrane b u t t h e i n t e r v e n i n g r e g i o n s a p p e a r e d t o b e f i l l e d
with a less electron-dense substance and did not appear to be an
artifact of fixation as suggested by Echlin and Morris (1965).
I n c e l l h o m o g e n a t e s t h e c e l l e n v e l o p e was s o m e t i m e s o b s e r v e d
i n t a c t ( F i g u r e 36% a n d t h e f a c t t h a t t h e l a y e r s w e r e a s s o c i a t e d a n d
t h e o u t e r membrane a n d p l a s m a membrane r e t a i n e d t h e i r r e s p e c t i v e
43
distances from the dense inner investment further suggested that the
i n t e r m e d i a t e r e g i o n s w e r e n o t a r t i f a c t b u t i n d e e d c o n t a i n e d some l e s s
dense substance.
T h e g e n e r a l s t r u c t u r e of t h e N o s t o c c e l l e n v e l o p e w a s s i m i l a r t o
t h a t proposed by Claus and Roth (1964) f o r the gram-negative bacteria
in which the structurally similar wall and plasma membranes were
s e p a r a t e d by a 5 - 1 0 mu l e s s d e n s e ' i n n e r l a y e r ' .
In certain planes of
s e c t i o n t h r o u g h b l u e - g r e e n w a l l s , t h e r e g i o n b e t w e e n t h e o u t e r membrane
a n d t h e p l a s m a membrane a p p e a r e d h o m o g e n e o u s b u t i t w a s a p p r o x i m a t e l y
2 0 mu t h i c k .
The relationship of the dense inner investment usually
observed within this region, to the 'inner layer' of the gram-negative
bacteria is not clear.
Claus and Roth (1964) suggested that the inner
l a y e r c o n t a i n e d t h e mucocomplex w h i c h i m p a r t e d r i g i d i t y t o t h e w a l l .
A l t h o u g h b l u e - g r e e n s a r e known t o c o n t a i n a s i m i l a r m u c o p o l y m e r a n d
protoplasts have been formed in several species by the action of
lysozyme (Crespi e t a l . , 1952) i t i s not yet clear where t h i s muco­
polymer i s located within the wall.
Frank e t al, (1962a) suggested
t h a t i t was l o c a t e d i n t h e r e g i o n b e t w e e n t h e i n n e r i n v e s t m e n t a n d t h e
plasma membrane.
T h e 7 - 8 mu p l a s m a membrane was o b s e r v e d a s d e s c r i b e d
b y L e f o r t ( 1 9 6 0 b ) a n d b e h a v e d s i m i l a r l y t o t h e u n i t membranes o f t h e
thylakoids with respect to fixation and staining procedures (Figures
5 , 6, 7, 11, and 12).
The o u t e r membrane a n d t h e i n n e r i n v e s t m e n t ,
h o w e v e r , w e r e n o t v i s i b l y s t a i n e d a f t e r OsO, ( F i g u r e 6 ) o r KMnO^ f i x a t i o n
(Figure 12) but did increase greatly in electron-density after the
subsequent uranyl acetate staining (Figures 2 and 5 ) .
44
T h i s s u g g e s t e d a c h e m i c a l d i f f e r e n c e b e t w e e n t h e o u t e r membrane a n d
the plasma membrane.
T h i s d i f f e r e n c e w a s a l s o i n d i c a t e d b y some o f t h e
solvent extraction studies.
T h e p l a s m a membrane was removed by m e t h a n o l
(Figure 15) or acetone-methanol treatment, but both the inner invest­
m e n t a n d t h e o u t e r membrane w e r e s t i l l c l e a r l y v i s i b l e .
Although
t h e a p p e a r a n c e o f t h e o u t e r membrane a n d t h e p l a s m a membrane w e r e s i m i l a r
in stained, fixed cells, i t i s suggested that their chemical compositions
a r e q u i t e d i f f e r e n t a n d t h a t , i n p a r t i c u l a r , t h e p l a s m a membrane h a s
a greater lipid content.
P a p i l l a e a s s o c i a t e d w i t h t h e o u t e r membrane a n d r e s e m b l i n g those
d e s c r i b e d by s e v e r a l w o r k e r s ( E c h l i n 1 9 6 3 a , 1 9 5 3 b ; J e n s e n 1 9 6 5 ) w e r e
observed.
T h e y w e r e p o s t u l a t e d by J e n s e n t o p l a y a r o l e i n f i l a m e n t
movements.
Most w e l l p r e s e r v e d c e l l s ( F i g u r e s 2 , 5 , a n d 1 0 ) d i d n o t
show these papillae but under several experimental conditions they were
quite evident.
T h e f o r m a t i o n o f p a p i l l a e w a s o b s e r v e d i n some o f t h e
p r e p a r a t i o n s a s s o c i a t e d w i t h enzyme s t u d i e s , b u t t h e b l e b b i n g s e e m e d
t o b e most common i n c e l l s i n c u b a t e d a t 5 0 ° C ( F i g u r e s 2 0 a n d 2 1 ) o r
in acid conditions (Figure 24) and not correlated t o the enzyme
employed.
C e l l s t r e a t e d w i t h 20% a c e t o n e - 8 0 % m e t h a n o l p r i o r t o
fixation showed the most prominent blebbing (Figure 13).
The wall was
s e p a r a t e d f r o m t h e u n d e r l y i n g p l a s m a membrane a n d p r o t o p l a s t , a n d
a l t h o u g h t h e u n d u l a t o r y o u t e r membrane a p p e a r e d t o b e i n c o n t a c t w i t h
t h e i n n e r i n v e s t m e n t , t h e l a t t e r l a y e r was n o t o b s e r v e d t o e x t e n d i n t o
the projections as reported by Echlin and Morris (1955).
The fact
t h a t w e l l p r e s e r v e d c e l l s f a i l e d t o show t h e p r e s e n c e o f p a p i l l a e .
45
c o u p l e d w i t h t h e o b s e r v a t i o n t h a t when p r e s e n t t h e y w e r e a c c o m p a n i e d
by rather poor fixation of the entire cell suggests that the appear­
a n c e o f s u c h p a p i l l a e i n N o s t o c muscorum i s a f i x a t i o n a r t i f a c t
rather than a normal condition of the c e l l .
Photosynthetic Apparatus
The photosynthetic units of Nostoc muscorum, the thylakoids, were
distributed throughout the cytoplasm with the majority positioned near
the cell periphery.
T h e i r d i s t r i b u t i o n a p p e a r e d t o b e m o r e random
than that observed by Pankratz and Bowen (1963) for Symploca muscorum.
The thylakoid (Menke, 1961a) consists of a flattened membrane-bound
v e s i c l e , a n d t h e o p p o s i n g membranes may a p p e a r c l o s e l y a p p r e s s e d
or separated by an intrathylakoidal space varying in width.
The
general appearance and dimensions of the thylakoids observed in
sectioned material agreed with the observations of previous workers
( M e n k e , 1 9 6 1 a ; P a n k r a t z a n d Bowen, 1 9 6 3 ; J e n s e n , 1 9 5 5 ) .
Rod o r d i s c - l i k e p a r t i c l e s 6 mu w i d e s e e n on t h e o u t e r s u r f a c e s
of obliquely sectioned membranes appear to be a previously undescribed
thylakoid component.
They were preserved by osmium, glutaraldehyde-
osmium a n d f o r m a l d e h y d e - o s m i u m f i x a t i o n s b u t h a v e n o t b e e n o b s e r v e d
a f t e r KMnO^ f i x a t i o n .
Similar particles were particularly evident
in sectioned cells and thylakoid fragments isolated from cell homogena t e s p r e p a r e d i n a F i c o l l - s u c r o s e medium ( F i g u r e 4 0 ) .
Although this
medium w a s e m p l o y e d b y S u s o r a n d Krogman ( 1 9 6 6 ) i n o r d e r t o r e t a i n t h e
p h y c o c y a n i n on t h e l a m e l l a e , i t i s n o t c l e a r w h e t h e r o r n o t t h e r o d ­
l i k e e l e m e n t s p r e s e r v e d b y t h i s medium a r e t h e s i t e o f p h y c o c y a n i n
45
association.
T h e f a c t t h a t t h e s e workers c o u l d p r e p a r e membrane
fragments which retained the phycocyanin argues for at least a loose
structural association between these pigments and the photosynthetic
lamellae, although Costerton (1960) suggested the phycocyanins were
confined to the chroniatoplasm but not bound to membranes.
Fuhs ( 1 9 6 4 ) r e p o r t e d t h a t a protein l a y e r o f t h e t h y l a k o i d
c o n s i s t e d o f 8 0 A p a r t i c l e s some o f w h i c h w e r e p h y c o c y a n i n .
In the
p r e s e n t s t u d y i s o l a t e d membrane f r a g m e n t s n o t p o s s e s s i n g p h y c o c y a n i n
a p p e a r e d a s u n i t membranes a n d i t s e e m s u n l i k e l y t h a t a m a j o r membrane
component could have been removed.
Since purified phycocyanin particles were reported t o be 80 A
in diameter (Fuhs, 1964), i t is possible the 75-90 A particles observed
o n n e g a t i v e l y s t a i n e d membrane s u r f a c e s ( F i g u r e s 4 5 a n d 4 9 ) o r t h e 6 0 70 A rod-like particles observed in sectioned cells (Figure 5) are
the phycocyanins.
P h y c o e r y t h r i n , an a c c e s s o r y p i g m e n t o f r e d a l g a e w a s r e p o r t e d t o
b e l o c a l i z e d i n 3 0 mu p a r t i c l e s o n t h e s u r f a c e o f t h e c h l o r o p l a s t
lamellae of Porphyridium cruentum (Gantt and Conti, 1966).
Similar
3 0 mu p a r t i c l e s h a v e b e e n o b s e r v e d o n t h e o u t e r s u r f a c e o f t h e
thylakoids in the blue-green endosymbiont Glaucocystis nostochinearum
(Lefort, 1965) but no function was suggested.
Similar particles have
not been observed in organisms which lack phycobilins as accessory
pigments.
The N o s t o c muscorum e m p l o y e d i n t h e p r e s e n t s t u d y d o e s n o t
e x h i b i t an a b s o r p t i o n a t 5 6 5 mu i n d i c a t i v e o f p h y c o e r y t h r i n s , a n d 3 0 mu
granules have not been observed in sectioned c e l l s .
Gantt and Conti
47
( 1 9 6 6 ) i n d i c a t e d t h a t g l u t a r a l d e h y d e f i x a t i o n was n e c e s s a r y t o
preserve the granules.
I n t h e c u r r e n t s t u d y some o f t h e t h y l a k o i d s
released into glutaraldehyde at the time of disruption exhibited an
a r r a y o f 3 0 mu s p h e r i c a l p a r t i c l e s ( F i g u r e 4 2 ) .
Since the organism
employed does not exhibit a phycoerythrin absorption and since such
3 0 mu p a r t i c l e s h a v e n o t b e e n o b s e r v e d i n s e c t i o n e d c e l l s , i t i s n o t
suggested that the currently observed particles contain phycoerythrins.
However, i t i s possible that phycocyanins and phycoerythrins are
associated within the cell in similar aggregates, and that because the
p h y c o c y a n i n s a r e more e a s i l y . d i s s o c i a t e d a n d d e n a t u r e d ( O ' h E o c h a ,
1965) they have not been adequately preserved in the blue-green algae.
This is particularly possible since the choice fixation for blueg r e e n s i s o s m i u m , a n d osmium i s p a r t i c u l a r l y d e s t r u c t i v e t o t h e
phycobilins (Gantt and Conti, 1966).
A l t h o u g h J o s t ( 1 9 6 5 ) r e p o r t e d 1 0 - 2 0 mu c l o s e l y p a c k e d s p h e r o i d s
w h i c h he c o n s i d e r e d t o b e a n a l o g o u s t o q u a n t a s o m e s on t h e i n n e r s i d e
of the thylakoid after freeze-etching, no similar particles were ob­
served in the current study.
The intrathylakoidal spaces were observed
to be empty a f t e r the treatments employed (Figures 38, 39, and 40).
Chains of 110 A particles similar to those considered by Jost to
b e p o l y r i b o s o m e s w e r e n o t o b s e r v e d b u t t h i s may h a v e b e e n d u e t o t h e
different -preparative techniques.
In negative staining preparations
75-90 A particles were observed on thylakoidal surfaces (Figures 45
a n d 4 9 ) b u t t h e s e p a r t i c l e s w e r e s m a l l c o m p a r e d t o t h e 9 - 1 2 mu
ribosomes observed in sectioned cells (Figure 7).
Since ribosomes
48
were preserved by the methods employed in the current study, i t i s
curious that they were not observed to be associated with the thylakoids
i f indeed this were a regular occurrence.
Jost and Matile (1955)
supported Cost's earlier conjecture of ribosome association with the
t h y l a k o i d s b y o b s e r v i n g RNA i n a t h y l a k o i d p r e p a r a t i o n .
t i o n may, h o w e v e r , b e o p e n t o o t h e r i n t e r p r e t a t i o n s .
This observa­
Although wall
fragments, gas vacuoles and phycobilins were separated by centrifugation
from the thylakoids, the authors did not suggest where other cell
components incl uding ^-granules, cC-granules, free ribosomes, structured
granules, and polyhedral bodies were located.
The d i f f i c u l t i e s
encountered in the current study in separating components from the
t h y l a k o i d s s u g g e s t t h a t t h e p r e p a r a t i o n by J o s t a n d M a t i l e may n o t
be pure thylakoids.
One t h y l a k o i d f r a c t i o n i s o l a t e d b y d e n s i t y g r a d i e n t c e n t r i f u g a t i o n
was o b s e r v e d t o h a v e a b s o r p t i o n maxima a t 4 4 0 a n d 6 7 5 mu i n d i c a t i v e o f
carotenoids and chlorophyll.
The c a r o t e n o i d a b s o r p t i o n r e l a t i v e t o t h a t
o f c h l o r o p h y l l was g r e a t l y i n c r e a s e d c o m p a r e d t o t h e s p e c t r u m o b t a i n e d
from whole c e l l s , and the pellet obtained from this fraction contained
a h i g h p r o p o r t i o n o f y £ ? - g r a n u l e s ( F i g u r e 3 5 ) when e x a m i n e d b y e l e c t r o n
microscopy.
Thylakoids have previously been observed t o contain
both carotenoids and chlorophyll (Calvin and Lynch, 1952; Shatkin,
1960; Susor and Krogman, 1964).
The y^-granules could be removed
from the cell by e i t h e r acetone o r methanol (Figures 14 and 15),
and carotenoid absorption appeared in the extracts.
From t h e p r e s e n t
study i t is concluded that although the thylakoids contain
49
both carotenoids and chlorophyll, the ^S-granules also contain
carotenoids.
T h e ^ - g r a n u l e s h a v e n o t been i s o l a t e d f r e e f r o m t h e t h y l a k o i d
fragments.
The a p p a r e n t l y c l o s e a s s o c i a t i o n o f t h e s e g r a n u l e s w i t h t h e
thylakoids, particularly in cases in which the thylakoids remained
r e l a t i v e l y i n t a c t ( F i g u r e 33), s u g g e s t s t h a t t h e s e two e l e m e n t s c o u l d
be structurally associated.
Although the ^granules never appeared to
a d s o r b t o e a c h o t h e r t h e p o s s i b i l i t y t h a t t h e y a d s o r b t o t h e membrane
after homogenization remains.
The / ^ g r a n u l e s o f b l u e - g r e e n s h a v e b e e n p r e v i o u s l y c o m p a r e d t o t h e
osmiophilic globules observed in the chloroplasts of higher plants
( P a n k r a t z a n d Bowen, 1 9 6 3 ) .
G l o b u l e s i s o l a t e d f r o m s p i n a c h by Murakami
a n d Takamiya ( 1 9 5 2 ) w e r e n o t membrane a s s o c i a t e d b u t d i d e x h i b i t a
high carotenoid content.
Other workers, however, have failed to
d e t e c t c a r o t e n e i n i s o l a t e d c h l o r o p l a s t g l o b u l e s (Greenwood e t a l . ,
1963).
Both ^-granules and the osmiophilic droplets appeared to be
l i p i d i n n a t u r e on t h e b a s i s o f f i x a t i o n and s t a i n i n g p r o c e d u r e s
and are at least spatially associated with the photosynthetic apparatus.
T h e c e l l u l a r f u n c t i o n o f n e i t h e r component i s known.
Nucleoplasm
The n u c l e o p l a s m o f N o s t o c muscorum a p p e a r e d much l i k e t h a t d e s ­
c r i b e d by R i s a n d S i n g h ( 1 9 5 1 ) f o r s e v e r a l b l u e - g r e e n s a n d c o n s i s t e d
o f an a n a s t o m o s i n g n e t w o r k w i t h 3 - 5 A DNA f i b r i l s i n t h e l e s s d e n s e
r e g i o n s a n d many . r i b o s o m e - 1 i k e p a r t i c l e s i n t h e more d e n s e a r e a s
( F i g u r e s 2 and 5 ) .
Fuhs ( 1 9 5 8 a ) i d e n t i f i e d RNA i n t h e n u c l e o p l a s m i c
50
r e g i o n by s t a i n i n g w i t h p y r o n i n e , a n d no s t a i n i n g was o b s e r v e d a f t e r
ribonuclease digestion.
Identification of these particles at the
e l e c t r o n m i c r o s c o p e l e v e l h a s n o t y e t been p e r f o r m e d b u t t h e 9 - 1 2
mu r i b o s o m e s ( F i g u r e 7 ) a g r e e c l o s e l y i n s i z e t o o t h e r p l a n t r i b o somes ( S z a r k o w s k i e t a l . , 1 9 5 0 ) .
L u f t ( 1 9 5 6 ) r e p o r t e d t h a t KMnO^
fixation failed to fix ribosomes.
In the current work, ribosome
particles were not visible after such fixation (Figure 12), but i f
the fixed cells were subsequently stained with uranyl acetate,
t h e n u c l e o p l a s m y i e l d e d a marked i n c r e a s e i n d e n s i t y ( F i g u r e 1 1 ) .
T h e DNA f i b r i l s w e r e s t a i n e d , a n d a g e n e r a l i n c r e a s e i n g r a n u l a r i t y
was o b s e r v e d .
T h i s g r a n u l a t i o n was q u i t e f i n e a n d d i d n o t a p p e a r t o
b e i n t a c t 9 - 1 2 mu r i b o s o m e s .
Ribosomes w e r e m o s t a p p a r e n t a f t e r
osmium f i x a t i o n and l e a d c i t r a t e s t a i n i n g ( F i g u r e 7 ) , b u t e v e n i n t h i s
p r e p a r a t i o n t h e y were n o t o b s e r v e d t o be a s s o c i a t e d w i t h t h e t h y l a k o i d
membranes a s s u g g e s t e d by J o s t ( 1 9 5 5 ) .
Inclusions
T h e 30 mu i n t e r l a m e l l a r g r a n u l e s t e r m e d o<.-granules ( P a n k r a t z a n d
Bowen, 1963) and shown to be polyglucoside in nature (Fuhs, 1963;
Giesy, 1964), are difficult to fix and stain in a consistent manner.
G1 u t a r a l d e h y d e f i x a t i o n g e n e r a l l y a i d s i n t h e i r p r e s e r v a t i o n ( F i g u r e 9 ) ,
b u t i n osmium f i x e d p r e p a r a t i o n s e l e c t r o n - t r a n s p a r e n t r e g i o n s a r e
observed between the lamellae where the drgranules are usually located
(Figure 2).
The ' b o t u l i ' d e s c r i b e d by J o s t ( 1 9 6 5 ) a n d c o n s i d e r e d by
him t o b e e q u i v a l e n t t o A - g r a n u l e s w e r e 35 mu w i d e a n d 300 mu l o n g
with a globular surface appearance in negatively stained
51
preparations.
Jost and Matile (1956) reported the presence of the
'botuli' after centrifugation procedures.
I n t h e p r e s e n t work no
^-granules were recovered after the centrifugation treatments.
They
were probably dissolved before the pellets were prepared and fixed.
They w e r e , h o w e v e r , r e l e a s e d i n t a c t f r o m t h e c e l l s , a n d n e g a t i v e l y
s t a i n e d p r e p a r a t i o n s r e v e a l e d them t o be 3 0 mu by 70 mu b i p a r t a t e
structures similar to those observed in sectioned cells (Figure 45).
No s t r u c t u r e s s i m i l a r t o ' b o t u l i ' w e r e o b s e r v e d .
If indeed the
' b o t u l i ' a r e e q u i v a l e n t t o t h e ^ - g r a n u l e s a s o b s e r v e d by P a n k r a t z
a n d Bowen ( 1 9 6 3 ) , t h e y may m a n i f e s t a d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n .
The s t r u c t u r e d g r a n u l e s o b s e r v e d i n N o s t o c muscorum a p p e a r e d
s i m i l a r t o t h o s e p r e v i o u s l y d e s c r i b e d by many w o r k e r s i n c l u d i n g P a n k r a t z
a n d Bowen ( 1 9 6 3 ) a n d J e n s e n ( 1 9 6 5 ) , b u t t h e y w e r e n e v e r o b s e r v e d t o
b e membrane b o u n d .
Isolated structured granules were observed,
some o f which appeared t o h a v e h o l e s a s i f t h e y h a d b e e n p a r t i a l l y
dissolved (Figure 31).
This observation, coupled with their staining
p r o p e r t i e s a f t e r OsO. f i x a t i o n i n t o more a n d l e s s d e n s e a r e a s
( F i g u r e 6 ) , s u g g e s t e d t h a t t h e y w e r e n o t c h e m i c a l l y homogeneous.
I s o l a t e d s t r u c t u r e d g r a n u l e s f i x e d i n osmium w e r e much b e t t e r p r e s e r v e d
t h a n t h o s e f i x e d i n osmium a n d s u b s e q u e n t l y e x p o s e d t o e t h a n o l
(Figure 32).
The l a t t e r g r a n u l e s a p p e a r e d t o be much l e s s d e n s e .
S t r u c t u r e d g r a n u l e s o b s e r v e d i n i n t a c t c e l l s a f t e r OsO. f i x a t i o n ( F i g ­
u r e 6 ) were much more e l e c t r o n - d e n s e t h a n t h o s e s u b s e q u e n t l y e x p o s e d t o
methanol-uranyl acetate stain (Figures 2 and 3).
The o b s e r v a t i o n t h a t
t h e s t r u c t u r e d g r a n u l e s w e r e m o d e r a t e l y e l e c t r o n - d e n s e a f t e r OsO^
52
f i x a t i o n b u t w e r e n o t w e l l p r e s e r v e d w i t h KMnO
fixation coupled with
t h e , a d m i t t e d l y c i r c u m s t a n t i a l , o b s e r v a t i o n o f d e c r e a s e d s t a i n i n g upon
exposure to ethanol and methanol suggested that the structured
granules were at least partially lipid in nature.
Myelin-like areas
w e r e o b s e r v e d i n some c e l l s where o n e would e x p e c t t o o b s e r v e s t r u c ­
tured granules.
Fuhs ( 1 9 5 8 b ) s u g g e s t e d t h a t t h e s t r u c t u r e d g r a n u l e s
were phospholipid.
Jensen (1965) observed the structured granules
t o b e p a r t i a l l y d i g e s t e d by p e p s i n and s u g g e s t e d t h a t t h e y w e r e p a r t i a l l y
proteinaceous.
In the present work, attempts to repeat this obser­
v a t i o n u s i n g c e l l s w h i c h h a d n o t p r e v i o u s l y been embedded i n Epon
failed.
The g r a n u l e s d i d i n d e e d a p p e a r t o be p a r t i a l l y d i g e s t e d
after exposure to the pepsin, but control cells exposed to 0.01 M
HCl w e r e e q u a l l y a f f e c t e d ( F i g u r e 2 2 ) .
I t i s s u g g e s t e d t h a t t h e low
pH r a t h e r t h a n t h e p e p s i n was r e s p o n s i b l e f o r t h e removal o f t h e
structured granules.
The p o s s i b i l i t y o f a p r o t e i n a c e o u s component h a s
neither been affirmed nor rejected on the basis of the current work.
The p o l y h e d r a l b o d i e s o f N o s t o c muscorum a p p e a r e d a s p r e v i o u s l y
d e s c r i b e d f o r s i m i l a r o r g a n i s m s by J e n s e n a n d Bowen ( 1 9 5 1 ) a n d
P a n k r a t z a n d Bowen ( 1 9 6 3 ) ,
I n d i v i d u a l l y t h e y w e r e u s u a l l y b e t w e e n 100
a n d 400 mu i n d i a m e t e r b u t t h e y o f t e n appeared i n c l u s t e r s up t o 1 u
in size.
H a l i c k i ( 1 9 6 4 ) s t a t e d t h a t t h e p o l y g o n a l p r o f i l e was ' d e f i n i t e l y
a result of compression', but from several of the present observations
i t i s concluded that they have an angular shape independent of
compression effects.
Single angular polyhedral bodies (Figures 5 and
53
1 0 ) a n d a n g u l a r bodies on t h e e d g e s o f c l u s t e r s ( F i g u r e s 5 a n d 1 2 )
a r e commonly o b s e r v e d .
The c h a r a c t e r i s t i c p o l y g o n a l s h a p e was a l s o
r e t a i n e d upon i s o l a t i o n from t h e c e l l ( F i g u r e s 2 6 , 2 7 a n d 2 9 ) .
The 3 - 5 mu l i m i t i n g 'membrane' was seldom o b s e r v e d i n s e c t i o n e d
c e l l s ( F i g u r e s 2 , 5 , a n d 1 0 ) t o c o m p l e t e l y s u r r o u n d t h e b o d y , b u t i t was
observed to be continuous in the isolated polyhedral bodies suggest­
i n g t h e p l a n e o f s e c t i o n was r e s p o n s i b l e f o r t h e former o b s e r v a t i o n .
T h i s 'membrane' a p p e a r e d n o t t o b e a mere i n t e r f a c e b e t w e e n t h e b o d y
a n d t h e c y t o p l a s m s i n c e i t was o b s e r v e d s u r r o u n d i n g i s o l a t e d p o l y ­
hedral bodies and also retained i t s polygonal profile when empty
(Figure 28).
The r e l a t i o n s h i p o f t h e p o l y h e d r a l b o d i e s t o e a c h o t h e r a n d t o
t h e r e s t o f t h e c e l l u l a r components i s n o t c l e a r .
Occasionally an
i s o l a t e d p o l y h e d r a l body was s e e n i n c l o s e a s s o c i a t i o n w i t h a
t h y l a k o i d membrane b u t i t i s p o s s i b l e t h a t t h e s e w e r e c a s e s o f
adsorption rather than of structural relationship (Figure 26),
A
c l o s e s p a t i a l r e l a t i o n s h i p was f r e q u e n t l y o b s e r v e d i n s e c t i o n e d
c e l l s b e t w e e n a p o l y h e d r a l body a n d a t h y l a k o i d ( F i g u r e 3 ) , b u t no
d e f i n i t e s t r u c t u r a l r e l a t i o n s h i p was o b s e r v e d .
I n some s e c t i o n s
t h e r e a p p e a r e d t o be a s t r u c t u r a l a s s o c i a t i o n v i a t h e p o l y h e d r a l
body ' m e m b r a n e s ' b e t w e e n members o f t h e same c l u s t e r , b o t h i n w h o l e
cells (Figures 2, 3,and 10), and in homogenates (Figures 27 and 28).
The n a t u r e o f t h e 'membrane' s u r r o u n d i n g t h e s e b o d i e s i s o p e n t o
a certain degree of speculation but i t does not exhibit dimensions or
s t a i n i n g c h a r a c t e r i s t i c s s i m i l a r t o a t y p i c a l u n i t membrane.
I t does
54
n o t h a v e a t r i p a r t a t e a p p e a r a n c e n o r i s i t p r e s e r v e d by KMnO, f i x a t i o n
(Figures 11 and 12).
I t i s p r e s e r v e d by OsO. f i x a t i o n ( F i g u r e 6 ) b u t
becomes much more e l e c t r o n - d e n s e a f t e r s t a i n i n g w i t h uranyl a c e t a t e
(Figure 2).
T h e r e i s a s t r i k i n g s i m i l a r i t y between t h e s e ' m e m b r a n e s '
a n d t h e 2 - 4 mu membrane s u r r o u n d i n g t h e g a s v e s i c l e s o b s e r v e d i n some
b l u e - g r e e n s a n d r e c e n t l y d e s c r i b e d by Bowen and J e n s e n ( 1 9 6 5 ) .
These
membranes e x h i b i t e d f i x a t i o n p r o p e r t i e s s i m i l a r t o t h e p o l y h e d r a l
body ' m e m b r a n e s ' j u s t d e s c r i b e d .
They a p p e a r e d t o m a i n t a i n t h e
c h a r a c t e r i s t i c c y l i n d r i c a l s h a p e o f t h e v e s i c l e s w h i c h had c o n i c a l e n d s .
The ' m e m b r a n e s ' o f t h e p o l y h e d r a l b o d i e s may a l s o b e r e s p o n s i b l e f o r
the characteristic shape of this cell component.
The p o l y h e d r a l b o d i e s
were not easily isolated from the cells unless the homogenization took
p l a c e i n a s u c r o s e medium, s u g g e s t i n g t h a t t h e ' m e m b r a n e ' may be q u i t e
osmotically labile.
Cost and Matile (1956) reported the isolation of
gas vesicles from Osci11atoria.
The i s o l a t e d v e s i c l e s a p p a r e n t l y l o s t
their rigidity and appeared after negative staining as flattened
bladders of various sizes.
The c h a r a c t e r i s t i c a s s o c i a t i o n o f t h e p o l y h e d r a l b o d i e s w i t h t h e
nucleoplasm and the notable lack of other structures in this area,
s u g g e s t e d t h a t t h e y c o u l d be t h e p o l y p h o s p h a t e b o d i e s commonly
o b s e r v e d i n t h i s r e g i o n by t h e l i g h t m i c r o s c o p i s t s .
Although
i n d i v i d u a l p o l y h e d r a l b o d i e s would be d i f f i c u l t t o d e t e c t b e c a u s e o f
t h e i r s m a l l s i z e , t h e c u l t e r s w h i c h a r e o f t e n o b s e r v e d r a n g e up t o
1 u i n s i z e a n d w o u l d , t h e r e f o r e , be r e l a t i v e l y e a s y t o o b s e r v e .
The p o l y p h o s p h a t e b o d i e s , m e t a c h r o m a t i c g r a n u l e s a n d v o l u t i n
55
g r a n u l e s h a v e b e e n s u g g e s t e d t o be I d e n t i c a l ( F u h s , 1 9 5 8 a ) .
The
p o l y p h o s p h a t e b o d i e s h a v e been r e p o r t e d t o b e removed by e x p o s u r e t o
1 N HCl ( C a s s e l a n d H u t c h i n s o n , 1 9 5 4 ; F u h s , 1 9 5 8 a ) .
The f a c t t h a t
t h e p o l y h e d r a l b o d i e s w e r e removed by HCl e x p o s u r e i n t h e c u r r e n t
study further suggested that they were equivalent to polyphosphate bodies.
Talpasayi (1963) and others observed that polyphosphate bodies were
not present in mature akinetes or heterocysts.
I n an e l e c t r o n
m i c r o s c o p i c s t u d y o f h e t e r o c y s t d e v e l o p m e n t i n A n a b a e n a , Lang ( 1 9 5 5 )
reported a gradual loss of polyhedral bodies as the heterocysts matured.
Although the above observations strongly suggest that the poly­
hedral bodies of the electron microscopists and the polyphosphate
b o d i e s o f t h e l i g h t m i c r o s c o p i s t s a r e t h e same c e l l u l a r s t r u c t u r e , t h e
chemical composition of these structures i s less clear.
Although they
a r e c o n s i d e r e d t o be h i g h l y p o l y m e r i z e d p o l y p h o s p h a t e ( T a l p a s a y i ,
1963), they also exhibit several reactions characteristic of nucleic
a c i d s : s t a i n i n g by m e t h y l g r e e n a n d p y r o n i n e ( E b e l a n d M u l l e r ,
1 9 5 8 ) , d i g e s t i o n w i t h c o l d 10% TCA ( F u h s , 1 9 5 8 a ) , a n d r e d f l u o r e s c e n c e
when t r e a t e d w i t h a c r i d i n e o r a n g e a n d v i e w e d w i t h u l t r a v i o l e t l i g h t
(Talpasayi, 1963).
P o l y p h o s p h a t e b o d i e s c o u l d n o t b e removed f r o m
t h e c e l l s w i t h e i t h e r RNase o r DNase, h o w e v e r , a n d o n t h i s b a s i s Fuhs
(1958a) concluded that the bodies did not contain nucleic acids, but
r a t h e r t h a t t h e r e a c t i o n s s h a r e d by p o l y p h o s p h a t e s a n d n u c l e i c a c i d s
w e r e c a u s e d by t h e h i g h d e g r e e o f p h o s p h a t e p o l y m e r i z a t i o n .
I n t h e p r e s e n t s t u d y , e x p o s u r e t o RNase according t o t h e method
d e s c r i b e d by J a c o b s o n e t a l . ( 1 9 5 3 ) l e f t t h e n u c l e o p l a s m u n i f o r m l y
56
e l e c t r o n - d e n s e b u t no p o l y h e d r a l b o d i e s w e r e o b s e r v e d ( F i g u r e 1 6 ) .
S i n c e t h e controls d i d r e t a i n p o l y h e d r a l b o d i e s ( F i g u r e 1 7 ) , t h e
e x p e r i m e n t s u g g e s t s t h a t t h e b o d i e s c o n t a i n RNA.
Several other
d i g e s t i o n c o n d i t i o n s w e r e e m p l o y e d i n w h i c h no a l t e r a t i o n o f t h e .
p o l y h e d r a l b o d i e s was o b s e r v e d ( F i g u r e 1 ) .
Costerton (1960) reported structures, recognizable from his
e l e c t r o n m i c r o g r a p h s a s p o l y h e d r a l b o d i e s , t o b e ' p r o t e i n b o d i e s ' on
the basis of light microscope studies.
He o b s e r v e d g r a n u l e s i n t h e
c e n t r o p l a s m w h i c h w e r e s t a i n e d w i t h m e r c u r i c bromphenol b l u e b u t n o t
with alkaline fast green and subsequently concluded that the bodies
consisted of a non-basic protein.
The m e r c u r i c bromphenol b l u e r e a c t i o n
i s considered specific for proteins provided proper controls such as
pepsin extraction are carried out, but no such controls were reported.
The p o l y h e d r a l b o d i e s a r e c h a r a c t e r i s t i c o f a l l t h e b l u e - g r e e n
algae so far examined at the electron microscope level, and i t i s
tempting to think that they play a prominent role in the cellular
activities.
They p o s e s e v e r a l i n t e r e s t i n g q u e s t i o n s w i t h r e g a r d t o
the nature, role, and origin of their unusual 'membrane', the origin
and maintenance of their polygonal shape, and of course their chemical
composition and metabolic involvements.
On t h e b a s i s o f t h e c u r r e n t
work, i t i s concluded that the polyhedral bodies are probably the
polyphosphate bodies of the light microscopists and furthermore,
t h a t t h e y may h a v e an RNA c o m p o n e n t .
Tubule-like Elements
The 1 2 - 1 5 mu t u b u l e - l i k e e l e m e n t s o b s e r v e d i n N o s t o c muscorum a f t e r
57
g l u t a r a l d e h y d e - o s n i i u m f i x a t i o n w e r e s i m i l a r t o t h e 1 4 mu tubules
o b s e r v e d i n N o s t o c p r u n i f o r m e by J e n s e n ( 1 9 5 5 ) .
T h e y a p p e a r t o be
s m a l l e r t h a n t h e 2 3 - 2 7 mu t u b u l e s r e p o r t e d i n o t h e r p l a n t s ( L e d b e t t e r
a n d P o r t e r , 1953) b u t compare w i t h t h e 1 5 mu s p i n d l e t u b u l e s o r w i t h
bacterial flagella.
A l t h o u g h s i m i l a r t u b u l e s a r e common i n e u c a r y o t e s , t h e y h a v e o n l y
rarely been reported in procaryotic cells; their function in these
c e l l i s , a s y e t , unknown.
The f a c t t h a t t h e y w e r e o b s e r v e d i n
sectioned cells, cell homogenates, and negatively stained preparations,
argues against the possibility that they could be fixation artifacts.
W h e t h e r t h e s e t u b u l e s a r e a normal component o f b l u e - g r e e n a l g a l c e l l s
or the result of a viral infection, i s not yet clear.
58
SUMMARY
1.
The f i n e s t r u c t u r e o f t h e b l u e - g r e e n a l g a , N o s t o c m u s c o r u m ,
was s t u d i e d u s i n g s e v e r a l f i x a t i o n m e t h o d s , enzyme d i g e s t i o n s ,
s o l v e n t e x t r a c t i o n s , and c e l l f r a c t i o n a t i o n p r o c e d u r e s .
2.
The c e l l e n v e l o p e was s i m i l a r t o t h a t o b s e r v e d i n g r a m -
n e g a t i v e b a c t e r i a a n d c o n s i s t e d o f an o u t e r m e m b r a n e , an i n n e r i n v e s t ­
m e n t , a n d a p l a s m a membrane.
Papillae were seen associated with the
o u t e r membrane a s d e s c r i b e d by e a r l i e r w o r k e r s , b u t v a r i e d w i t h
e x p e r i m e n t a l t r e a t m e n t , and w e r e t h e r e f o r e c o n s i d e r e d t o be f i x a t i o n
artifacts.
The p l a s m a membranes a n d t h e o u t e r membrane a p p e a r e d
structurally similar after fixation and staining; however only the
p l a s m a membrane was v i s i b l e p r i o r t o u r a n y l a c e t a t e s t a i n i n g .
On
t h e b a s i s o f s o l v e n t e x t r a c t i o n s t u d i e s , t h e p l a s m a membrane h a s a
greater lipid content.
Therefore i t is concluded that the plasma
membrane and t h e o u t e r membrane a r e c h e m i c a l l y d i f f e r e n t .
3.
The i n c l u s i o n s commonly d e s c r i b e d f o r b l u e - g r e e n a l g a e w e r e
o b s e r v e d ; t h y l a k o i d s , DNA f i b r i l s , r i b o s o m e s , p o l y h e d r a l b o d i e s ,
structured granules, «^-granules, and y^-granules.
4.
P r e v i o u s l y u n r e p o r t e d r o d o r d i s c - s h a p e d p a r t i c l e s 6 - 7 mu
wide were observed on obliquely sectioned thylakoid membranes.
As a
working hypothesis, i t i s proposed that these particles are the
phycocyanins.
5.
T u b u l e - l i k e e l e m e n t s 1 2 - 1 5 mu w i d e w e r e o b s e r v e d i n s e c t i o n e d
c e l l s from s t r a i n 1 0 3 7 , a n d i n h o m o g e n a t e s o f s t r a i n s 1 0 3 7 a n d M - 1 2 . 4 . 1 .
W h e t h e r t h e t u b u l e s a r e normal c e l l u l a r c o m p o n e n t s o r a r e s u l t o f
viral infection has not been determined.
Although similar tubules are
59
common i n e u c a r y o t e s , t h e y h a v e o n l y r a r e l y been r e p o r t e d i n p r o caryotic cells.
6.
A f r a c t i o n c o n t a i n i n g i n t a c t p o l y h e d r a l b o d i e s was i s o l a t e d .
The polyhedral bodies retained their characteristic polygonal pro­
files.
The 2 - 5 mu s u r r o u n d i n g 'membrane' was o b s e r v e d t o b e d i f f e r e n t
f r o m m o s t b i o l o g i c a l membranes, b o t h i n d i m e n s i o n s a n d i n f i x i n g a n d
staining properties.
The p o l y h e d r a l 'membrane' d i d , h o w e v e r , a p p e a r
s i m i l a r t o t h e membranes o f e x p a n d e d g a s v e s i c l e s o b s e r v e d i n some
blue-green algae.
The polyhedral bodies are characteristically
a s s o c i a t e d w i t h t h e n u c l e o p l a s m and a r e d i s s o l v e d by d i l u t e HCl.
On
t h e b a s i s o f t h i s work i t i s c o n c l u d e d t h a t t h e p o l y h e d r a l b o d i e s a r e
t h e p o l y p h o s p h a t e b o d i e s d e s c r i b e d by t h e l i g h t m i c r o s c o p i s t s .
Further­
m o r e , on t h e b a s i s o f s t a i n i n g p r o p e r t i e s a n d enzyme d i g e s t i o n s t u d i e s ,
i t i s c o n c l u d e d t h a t t h e y p r o b a b l y h a v e an RNA c o m p o n e n t .
7.
The s t r u c t u r e d g r a n u l e a p p e a r e d t o b e c h e m i c a l l y h e t e r o g e n e o u s
and to have a lipid component.
8.
Negative staining reveals that the polyglucosidic
-granules
a r e 3 0 by 7 0 mu e l l i p s o i d s w i t h a m e d i a n t r a n s v e r s e c o n s t r i c t i o n .
9.
The ^ - g r a n u l e s w e r e removed f r o m t h e c e l l s by l i p i d s o l v e n t s .
On the basis of absorption spectra obtained from the solvent extracts
and from isolated ^-granule fractions, i t i s concluded that the
^-granules contain carotenoids.
60
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68
ACKNOWLEDGEMENTS
The author wishes to express her sincere appreciation to Dr.
C. C. Bowen f o r h i s e n c o u r a g e m e n t a n d s u p p o r t t h r o u g h o u t t h e c o u r s e
o f t h i s s t u d y , a n d t o D r . H. T . H o r n e r f o r s e v e r a l s t i m u l a t i n g
discussions.
T h i s s t u d y was s u p p o r t e d i n p a r t by an Iowa S t a t e
R e s e a r c h F o u n d a t i o n F e l l o w s h i p ( 1 9 6 3 - 1 9 6 4 ) a n d a NASA T r a i n e e s h i p
( 1 9 6 4 - 1 9 6 5 ) t o t h e a u t h o r a n d by t h e N a t i o n a l I n s t i t u t e o f H e a l t h
g r a n t H DO 1 2 4 8 t o D r . C. C. Bowen.
69
APPENDIX A:
FIXATION, DEHYDRATION AND EMBEDDING
Fixation
1.
Osmium t e t r o x i d e f i x a t i o n
T h e p r o c e d u r e e m p l o y e d was m o d i f i e d f o r b l u e - g r e e n s by P a n k r a t z
a n d Bowen ( 1 9 6 3 ) f r o m t h a t d e s c r i b e d f o r b a c t e r i a by K e l l e n b e r g e r e t
a l . (1958),
I n t h e c u r r e n t work t h e t r y p t o p h a n w a s o m i t t e d ,
a . Preparation of Michael i s buffer;
s o d i u m a c e t a t e (NaC2H302.3H20)
1.94 g
sodium veronal (Barbital)
2.94 g
sodium chloride
3.40 g
distilled water to
ICQ
ml
b. Preparation of fixing buffer:
Michael i s buffer (above)
5 ml
distilled water
1 3 ml
0 . 1 N HCl
7 ml
1 M CaClg s o l u t i o n
F i n a l pH s h o u l d b e 6 . 1
0 . 2 5 ml
to 6.2.
c. Preparation of Fixative;
D i l u t e f i x i n g b u f f e r ( a b o v e ) 1 : 1 w i t h a q u e o u s 2% oxmium
tetroxide.
d . F i x s p e c i m e n s 1 - 3 h o u r s a t room t e m p e r a t u r e .
2. Potassium permanganganate fixation
U n b u f f e r e d 4% a q u e o u s p o t a s s i u m p e r m a n g a n a t e ( L u f t , 1 9 5 6 ;
M o l l e n h a u e r , 1 9 5 9 ) was e m p l o y e d .
a t room t e m p e r a t u r e .
Specimens were fixed 3-5 minutes
70
3.
Glutaraldehyde fixation
P h o s p h a t e b u f f e r e d 3% g l u t a r a l d e h y d e ( S a b a t i n i e t a l . , 1 9 6 3 )
was u s u a l l y employed w i t h p o s t - f i x a t i o n i n osmium.
a.
Preparation of buffer:
0 . 1 M KHgPO
1 3 ml
0 . 1 M Na HPO^
3 7 ml
F i n a l pH s h o u l d b e 7 . 2 t o 7 . 4
b.
Preparation of fixative:
Add 50% g l u t a r a l d e h y d e t o a b o v e b u f f e r i n p r o p e r p o r t i o n t o
o b t a i n a f i x i n g s o l u t i o n o f 3% g l u t a r a l d e h y d e . (1ml g l u t a r ­
a l d e h y d e : 1 5 ml b u f f e r ) .
c.
Fix 1-12 hours at 4°C.
d.
Rinse in several changes of phosphate buffer.
e.
I f p o s t - f i x a t i o n i s d e s i r e d , f i x 1 - 3 h o u r s a t room t e m p e r a t u r e
i n osmium ( p r e v i o u s l y d e s c r i b e d ) .
4.
Formaldehyde f i x a t i o n
P h o s p h a t e - b u f f e r e d 4% f o r m a l d e h y d e ( P e a s e , 1 9 5 4 ) w i t h osmium
p o s t - f i x a t i o n was e m p l o y e d .
a.
Preparation of stock solutions:
S t o c k - b u f f e r s a l t s o l u t i o n : M o n o b a s i c s o d i u m p h o s p h a t e 2.26%
Stock alkali solution:
b.
s o d i u m h y d r o x i d e 2.52%
P r e p a r a t i o n o f f i x a t i v e (4% f o r m a l d e h y d e ) :
Stock buffer salt solution
8 3 ml
Stock alkali solution
1 7 ml
Paraformaldehyde (powder)
A d j u s t pH t o 7 . 2 - 7 . 4
4 gm
71
c.
F i x 1 - 1 2 h o u r s a t 4°C
d.
P o s t - f i x 1 - 3 h o u r s i n 1% osmium ( p r e v i o u s l y d e s c r i b e d ) a t
room t e m p e r a t u r e .
D e h y d r a t i o n a n d Embedding
D e h y d r a t i o n a n d embedding w e r e p e r f o r m e d a s m o d i f i e d f r o m L u f t
(1951) as follows:
1.
P r e p a r a t i o n o f Epon m i x t u r e s ;
a.
Mixture A
6 2 c c Epon 8 1 2
100 cc DDSA (Dodecenyl s u c c i n i c a n h y d r i d e )
b.
Mixture B
8 9 c c NMA ( N a d i c m e t h y l a n h y d r i d e )
100 c c Epon 812
c.
Add 3 p a r t s m i x t u r e A t o 2 p a r t s m i x t u r e B a n d s t i r t h o r o u g h l y .
Add DMP-30 ( c a t a l y s t ) a t t h e r a t e o f 0 . 2 ml p e r 1 0 ml Epon
mixture.
2.
Mix t h o r o u g h l y .
D e h y d r a t i o n o f s p e c i m e n s a t room t e m p e r a t u r e i m m e d i a t e l y f o l l o w e d
fixation.
3.
a.
5 m i n u t e s e a c h i n 50%, 70%, a n d 95% e t h a n o l
b.
3 c h a n g e s o f 2 - 5 m i n u t e s e a c h i n 100% e t h a n o l
c.
3 changes of 2-5 minutes each in propylene oxide
I n f i l t r a t i o n was a c c o m p l i s h e d i n 4 s t e p s .
a.
5-10 minutes in a mixture of 1 part Epon: 3 parts propylene
oxi de
b.
10-20 minutes in 1 part Epon: 1 part propylene oxide
72
c.
2 0 - 4 0 m i n u t e s i n 3 p a r t s E p o n : 1 part p r o p y l e n e o x i d e
d.
8 - 1 8 h o u r s i n 100% E p o n .
The s p e c i m e n v i a l s w e r e s l o w l y
rotated during this period.
4.
S p e c i m e n s w e r e embedded i n s h a l l o w o p e n aluminum f o i l b o a t s .
5.
T h e p o l y m e r i z a t i o n was p e r f o r m e d i n 3 s t e p s .
a.
8 - 1 2 h o u r s a t 35°C
b.
12 hours at 45°C
c.
3-6 days a t 60°C
73
APPENDIX B;
FIGURES
Key t o a l l F i g u r e s
A
-
-granules
B
-
-granules
D
-
DNA f i b r i l s
G
-
Granules
IV -
Inner investment
IT -
Intrathylakoidal spaces
W
Myelin-like whorls
-
NP -
Nucleoplasm
OM -
O u t e r membrane
P
-
Papillae
Z
-
Peripheral clear zone
PM -
P l a s m a membrane
PB -
Polyhedral bodies
R
Ribosomes
-
SG -
Structured granules
TH -
Thylakoids
T
Tubule-like elements
-
Figure 2.
C e l l o f N o s t o c muscorum f i x e d i n osmium a n d s t a i n e d i n
u r a n y l a c e t a t e . The c e l l e n v e l o p e c o n s i s t s o f t h e o u t e r
membrane (OM), i n n e r i n v e s t m e n t ( I V ) , a n d p l a s m a membrane
(PM). The n u c l e o p l a s m (NP) c o n t a i n s r i b o s o m e s ( R ) , DNA
f i b r i l s (D) a n d p o l y h e d r a l b o d i e s (PB) w h i c h a r e s o m e t i m e s
s u r r o u n d e d by a f i n e d e n s e z o n e . T h y l a k o i d s ( T H ) , / S - g r a n u l e s
( B ) , and a s t r u c t u r e d g r a n u l e (SG) a r e a l s o v i s i b l e .
7 0 , 0 0 0 X.
Figure 3.
P o r t i o n o f a c e l l showing s t r u c t u r e d g r a n u l e s ( S G ) , and a
c l u s t e r o f p o l y h e d r a l b o d i e s ( P B ) . Arrow i n d i c a t e s a c l o s e
s p a t i a l r e l a t i o n s h i p between p o l y h e d r a l body and a
t h y l a k o i d . Osmium f i x a t i o n and u r a n y l a c e t a t e s t a i n i n g .
7 0 , 0 0 0 X.
Figure 4.
P o r t i o n o f a c e l l o f N o s t o c muscorum s t r a i n 1037 showing
t u b u l e - l i k e e l e m e n t s ( a r r o w s ) ^ G1 u t a r a l d e h y d e - o s m i u r n
f i x a t i o n and u r a n y l a c e t a t e s t a i n i n g . 7 0 , 0 0 0 X.
Figure 5.
P o r t i o n o f a c e l l showing r o d - l i k e s t r u c t u r e s on o b l i q u e l y
s e c t i o n e d t h y l a k o i d s ( a r r o w s ) . The c e l l e n v e l o p e c o n s i s t ­
i n g o f t h e o u t e r membrane (OM), i n n e r i n v e s t m e n t ( I V ) and
p l a s m a membrane (PM) i s c l e a r l y v i s i b l e . 1 4 0 , 0 0 0 X.
Figure 6.
P o r t i o n o f an osmium f i x e d c e l l o t h e r w i s e u n s t a i n e d . The
s t r u c t u r e d g r a n u l e s (SG) a n d / S - g r a n u l e s (B) a r e o s m i o p h i l i c .
The p o l y h e d r a l b o d i e s a r e s u r r o u n d e d by a f i n e d e n s e z o n e
( a r r o w ) and t h e c e l l e n v e l o p e i s u n s t a i n e d e x c e p t f o r t h e
plasma membrane (PM). 7 0 , 0 0 0 X.
Figure 7.
P o r t i o n o f an osmium f i x e d c e l l s t a i n e d w i t h l e a d c i t r a t e .
T h e , x - g r a n u l e s ( A ) , DNA f i b r i l s (D) and r i b o s o m e s (R) show
an i n c r e a s e d e l e c t r o n - d e n s i t y . The e d g e s o f s e v e r a l
t h y l a k o i d s ( a r r o w s ) a r e v i s i b l e . 7 0 , 0 0 0 X.
Figure 8.
C e l l f i x e d i n g 1 u t a r a l d e h y d e and s t a i n e d w i t h u r a n y l
a c e t a t e . The l e s s d e n s e a r e a s ( a r r o w s ) a r e s u g g e s t e d t o
have c o n t a i n e d ^ - g r a n u l e s . 7 0 , 0 0 0 X.
Figure 9.
P o r t i o n o f a g l u t a r a l dehyde-ostni um f i x e d c e l l showing good
p r e s e r v a t i o n o f o t - g r a n u l e s ( A ) . Uranyl a c e t a t e s t a i n i n g .
7 0 , 0 0 0 X.
Figure 10.
G l u t a r a l d e h y d e - o s m i u m f i x e d c e l l showing p r e s e r v a t i o n
s i m i l a r t o t h a t o b s e r v e d i n osmium f i x e d c e l l s ( F i g u r e 2 ) .
Uranyl a c e t a t e s t a i n i n g . 7 0 , 0 0 0 X.
Figure 11.
C e l l f i x e d i n KMnO, and s t a i n e d w i t h u r a n y l a c e t a t e showing
i n c r e a s e d e l e c t r o n - d e n s i t y o f n u c l e o p l a s m (NP) and p o l y ­
h e d r a l b o d i e s ( P B ) . The / ^ g r a n u l e s a r e n o t p r e s e r v e d
( a r r o w s ) . The i n t r a - t h y l a k o i d a l s p a c e s a p p e a r s l i g h t l y
e x p a n d e d ( I T ) . 7 0 , 0 0 0 X.
Figure 12.
C e l l f i x e d i n KMnO^ b u t o t h e r w i s e u n s t a i n e d showing plasma
membrane (PM), p o o r l y p r e s e r v e d / ^ - g r a n u l e s ( B ) , p o l y h e d r a l
b o d i e s ( P B ) , and l e s s d e n s e n u c l e o p l a s m ( N P ) . 7 0 , 0 0 0 X.
83
Figure 13.
C e l l t r e a t e d w i t h 20% acetone-80% methanol and t h e n f i x e d
i n osmium showing f o r m a t i o n o f p a p i l l a e ( P ) from t h e o u t e r
membrane. Note t h e m y e l i n - l i k e membranous whorl (W), a n d
p o l y h e d r a l b o d i e s ( P B ) . 7 0 , 0 0 0 X.
Figure 14.
G 1 u t a r a l d e h y d e - f i x e d c e l l , t r e a t e d w i t h 100% a c e t o n e and
p o s t - f i x e d i n osmium showing l e s s d e n s e a r e a s from which
^ - g r a n u l e s have been removed ( a r r o w s ) . 7 0 , 0 0 0 X.
Figure 15.
G 1 u t a r a l d e h y d e - f i x e d c e l l t r e a t e d w i t h 100% methanol and
p o s t - f i x e d i n osmium showing s m a l l l e s s d e n s e a r e a s from
w h i c h / 5 - g r a n u l e s have been removed ( a r r o w s ) , and a
p e r i p h e r a l c l e a r z o n e ( Z ) . No p l a s m a membrane i s v i s i b l e .
7 0 , 0 0 0 X.
Figure 15.
Formaldehyde-fixeci c e l l i n c u b a t e d 4 h o u r s a t 60°C i n
RNase, e x t r a c t e d w i t h TCA, and p o s t - f i x e d i n osmium.
Note t h e c e n t r a l a r e a o f u n i f o r m e l e c t r o n - d e n s i t y . The
o u t e r membrane (OM) i s s l i g h t l y u n d u l a l o r y . 7 0 , 0 0 0 X.
Figure 17.
C e l l t r e a t e d a s i n d i c a t e d above e x c e p t t h e RNase was a b s e n t
from t h e i n c u b a t i o n medium. P o l y h e d r a l b o d i e s (PB) remain
and a few p a p i l l a e ( P ) a r e o b s e r v e d . 7 0 , 0 0 0 X.
Figure 18.
C e l l f i x e d i n f o r m a l d e h y d e and p o s t - f i x e d i n osmium.
7 0 , 0 0 0 X.
Figure 19.
C e l l f i x e d i n g l u t a r a l d e h y d e and p o s t - f i x e d i n osmium,
7 0 , 0 0 0 X.
Figure 20.
Note p a p i l l a e ( P ) on o u t e r membrane o f c e l l i n c u b a t e d a t
60OC ( s e e t e x t F i g u r e 1 f o r t r e a t m e n t d e t a i l s ) . 7 0 , 0 0 0 X.
Figure 21.
P o r t i o n o f g l u t a r a l d e h y d e - f i x e d c e l l i n c u b a t e d a t 60°C
showing m y e î i n - l i k e w h o r l s (W), and a s l i g h t l y p a p i l l a t e
o u t e r membrane. 7 0 , 0 0 0 X.
Figure 22.
Portion of gl utaraldehyde-fixed cell exposed to 0.01
N HCl showing removal o f t h e s t r u c t u r e d g r a n u l e ( S G ) .
7 0 , 0 0 0 X.
89
Figures 23 to 25.
Portions of cells fixed in glutaralceny
e x p o s e d t o 0 . 0 1 N HCl. The p o l y h e o r a l
(PB) have been entirely or partially re
P o s t - f i x e d i n osmium and s t a i n e d witr, u
a c e t a t e . 7 0 , 0 0 0 X.
Figures 23 to 25.
P o r t i o n s o f c e l l s f i x e d i n g l u t a r a l d e h y d e and
exposed t o 0 . 0 1 N HCl. The p o l y h e d r a l b o d i e s
(PB) have been e n t i r e l y o r p a r t i a l l y removed.
P o s t - f i x e d i n osmium and s t a i n e d w i t h uranyl
a c e t a t e . 70,000 X.
m
Figure 26.
P o l y h e d r a l body f r e e d from c e l l i n s u c r o s e medium. The
l i m i t i n g 'membrane' i s p r e s e r v e d b u t t h e c o n t e n t s a p p e a r
p u l l e d from edge l e a v i n g a c l e a r s p a c e . Arrow i n d i c a t e s
a s s o c i a t i o n o f p o l y h e d r a l body w i t h t h y l a k o i d . Osmium
f i x a t i o n and uranyl a c e t a t e s t a i n i n g . 70,000 X.
Figure 27.
C l u s t e r o f i s o l a t e d p o l y h e d r a l b o d i e s . Note small t u b u l e s
( T ) , and p o r t i o n o f wall c o n t a i n i n g o u t e r membrane (CM) and
i n n e r i n v e s t m e n t ( I V ) . Osmium f i x a t i o n and uranyl a c e t a t e
s t a i n i n g . 70,000 X.
Figure 28.
P o l y h e d r a l body 'membranes' d e v o i d o f c o n t e n t s and r e t a i n i n g
a n g u l a r i t y . Osmium f i x a t i o n and uranyl a c e t a t e s t a i n i n g .
70,000 X.
Figure 29.
I s o l a t e d p o l y h e d r a l b o d i e s may a p p e a r e l e c t r o n - d e n s e t h r o u g h ­
o u t o r w i t h varying d e g r e e s o f g r a n u l a r i t y . Osmium f i x a t i o n
and uranyl a c e t a t e s t a i n i n g . 70,000 X.
Figure 30.
Isolated structured granules after glutaraldehyde-osmium
f i x a t i o n showing s l i g h t s u r f a c e p r o j e c t i o n s ( a r r o w ) .
Uranyl a c e t a t e s t a i n i n g . 70,000 X.
Figure 31.
I s o l a t e d s t r u c t u r e d g r a n u l e s a f t e r osmium f i x a t i o n showing
o c c a s i o n a l l e s s dense a r e a s ( a r r o w s ) . Uranyl a c e t a t e
s t a i n i n g . 70,000 X.
Figure 32.
I s o l a t e d s t r u c t u r e d g r a n u l e s a f t e r osmium f i x a t i o n and
e x p o s u r e t o e t h a n o l showing d e c r e a s e d e l e c t r o n - d e n s i t y ,
d e c r e a s e d s i z e and homogeneous a p p e a r a n c e . Uranyl a c e t a t e
s t a i n i n g . 70,000 X.
Figure 33.
Thylakoid v e s i c l e s i s o l a t e d i n d i s t i l l e d w a t e r . Arrow
indicates possible /î-granule association with the
t h y l a k o i d s . Low s p e e d p e l l e t f i x e d i n osmium and s t a i n e d
w i t h u r a n y l a c e t a t e . 70,000 X.
Figure 34.
P o r t i o n o f high s p e e d p e l l e t from crude homogenate o f Nostoc
mus CO rum s t r a i n 1037 showing t u b u l e - l i k e e l e m e n t s ( T ) .
Osmium f i x a t i o n and uranyl a c e t a t e s t a i n i n g . 70,000 X.
Figure 35.
Portion of pellet obtained after sucrose gradient centrifug a t i o n c o n t a i n i n g t h y l a k o i d v e s i c l e s and a high p r o p o r t i o n
o f ^ - g r a n u l e s . Sample a l s o showed high c a r o t e n o i d a b s o r p ­
t i o n . Osmium f i x a t i o n and uranyl a c e t a t e s t a i n i n g .
7 0 , 0 0 0 X.
Figure 36.
C e l l homogenate c o n t a i n i n g a p o r t i o n o f c e l l e n v e l o p e
showing t h e e l e c t r o n - d e n s e i n n e r i n v e s t m e n t (IV) and t h e
u n i t n a t u r e of both t h e o u t e r membrane (OM) and t h e plasma
membrane (PM). Osmium f i x a t i o n and uranyl a c e t a t e s t a i n i n g .
1 4 0 , 0 0 0 X.
Figure 37.
P o r t i o n o f c e l l e n v e l o p e showing plasma membrane (PM)
p u l l e d away from t h e w a l l complex. Osmium f i x a t i o n and
u r a n y l a c e t a t e s t a i n i n g . 140,000 X.
Figure 38.
Thylakoid v e s i c l e showing 75 A p a r t i c l e s ( a r r o w s ) on t h e
outer surface. Isolated in Ficoll-sucrose, fixed in
osmium and s t a i n e d i n uranyl a c e t a t e . 70,000 X.
Figure 39.
T h y l a k o i d s homogenized i n 1% g l u t a r a l d e h y d e showing small
particles associated with the outer surfaces (arrow).
Occasionally, larger elongate granules (6) are observed.
I n d i v i d u a l t h y l a k o i d s o f t e n remain c l o s e l y a p p r e s s e d .
Osmium f i x a t i o n and uranyl a c e t a t e s t a i n i n g . 70,000 X.
Figure 40.
T h y l a k o i d s i s o l a t e d i n F i c o l l medium showing small p r o j e c t ­
i o n s from o u t e r s u r f a c e ( a r r o w ) . I n d i v i d u a l t h y l a k o i d s
remain c l o s e l y a p p r e s s e d . Osmium f i x a t i o n and u r a n y l
a c e t a t e s t a i n i n g . 70,000 X.
Figure 41.
T h y l a k o i d s i s o l a t e d i n s u c r o s e medium a r e u s u a l l y expanded
b u t t h e u n i t n a t u r e o f t h e membranes i s e v i d e n t . Osmium
f i x a t i o n and uranyl a c e t a t e s t a i n i n g . 70,000 X.
Figure 42.
T h y l a k o i d s from c e l l s homogenized i n 1% g l u t a r a l d e h y d e show­
i n g 30 mu g r a n u l e s a s s o c i a t e d w i t h t h e o u t e r s u r f a c e o f t h e
membrane. Osmium f i x a t i o n and uranyl a c e t a t e s t a i n i n g .
70,000 X.
Figure 43.
P o r t i o n o f a whole c e l l from F i c o l l homogenate f i x e d i n
osmium and s t a i n e d w i t h uranyl a c e t a t e . P r o j e c t i o n s 6 - 7
mu wide a r e o b s e r v e d on t h e o u t e r s u r f a c e s o f t h e t h y l a k o i d s
( a r r o w s ) . 70,000 X.
Figure 44.
Enlargement from F i g u r e 4 3 o f membrane p r o j e c t i o n s ( a r r o w s ) .
140,000 X.
Figure 45.
T h y l a k o i d from c r u d e homogenate s h o w i n g / ^ - g r a n u l e s (B) and
knobby s u r f a c e . T u b u l a r system a p p e a r s t o anastomose
( a r r o w s ) . N e g a t i v e l y s t a i n e d w i t h PTA. 70,000 X.
Figure 46.
I s o l a t e d (K-granules showing b i p a r t a t e s t r u c t u r e and e l e c t r o n dense c o n s t r u c t i o n ( a r r o w s ) . N e g a t i v e l y s t a i n e d i n uranyl
f o r m a t e . 70,000 X.
Figure 47.
Thylakoid fragment showing a s s o c i a t e d small rods o r t u b u l e ­
l i k e e l e m e n t s ( T ) . N e g a t i v e l y s t a i n e d w i t h PTA. 70,000 X.
Figure 48.
T u b u l a r t h y l a k o i d fragment showing a p a i r o f small r o d s o r
t u b u l e - l i k e e l e m e n t s ( T ) . N e g a t i v e l y s t a i n e d w i t h PTA.
70,000 X.
Figure 49.
P r e p a r a t i o n n e g a t i v e l y s t a i n e d w i t h PTA showing / > - g r a n u l e s
( B ) , ( X - g r a n u l e s (A) and t h y l a k o i d f r a g m e n t w i t h 75-90 A
s u r f a c e p a r t i c l e s . 70,000 X.
101