3.13.
RAPID FREEZING
67
frozen-dried specimens, excepting in the latter the lipids are extracted
and negative images of the cytomembrane systems are seen. Also, when
cells are fixed with osmium tetroxide while being continuously viewed
by phase contrast microscopy, the alteration in their structure seems
minimal.
Fixatives which combine osmium tetroxide and permanganate salts
are possible according to Tahmisian (1964). He presents both buffered
and unbuffered variants which are said to have ionic strengths equivalent
to 0.9% NaCl. Their formulations follow:
Buffered osmium-permanganate fixative:
Buffered saline solution
8
ml
(equal parts of 9% NaCl, 0.1 M Na 2 HP0 4 ,
and 0.1 M KH 2 P0 4 )
KMn0 4
1.3 gm
2% Os0 4 solution
50
ml
Distilled water to make
100
ml
(pH should be 6.8)
Unbuffered osmium-permanganate fixative:
KMn0 4
1.3 gm
2% Os0 4 solution
50
ml
9% NaCl solution
2.7 ml
Distilled water to make
100
ml
The present writer cannot speak from personal experience about either
the virtues or disadvantages of these osmium-permanganate mixtures.
Tahmisian's illustration indicates, however, that much more cell structure is preserved than with permanganate alone.
3.13. Rapid Freezing
Thoughtful electron microscopists often have worried about chemical
fixation. They have wondered what might be missed by this method, or
what specific artifacts might be introduced. The situation was particularly
critical so long as they were virtually dependent upon osmium tetroxide
fixation. The recent successful utilization of aldehydes as alternate
chemical fixatives has given us more confidence, but one would nevertheless like to be able to avoid chemical reactions altogether. Thus the
possibility of preserving tissue by purely physical means has been a goal
RAPID FREEZING
67
frozen-dried specimens, excepting in the latter the lipids are extracted
and negative images of the cytomembrane systems are seen. Also, when
cells are fixed with osmium tetroxide while being continuously viewed
by phase contrast microscopy, the alteration in their structure seems
minimal.
Fixatives which combine osmium tetroxide and permanganate salts
are possible according to Tahmisian (1964). He presents both buffered
and unbuffered variants which are said to have ionic strengths equivalent
to 0.9% NaCl. Their formulations follow:
Buffered osmium-permanganate fixative:
Buffered saline solution
8
ml
(equal parts of 9% NaCl, 0.1 M Na 2 HP0 4 ,
and 0.1 M KH 2 P0 4 )
KMn0 4
1.3 gm
2% Os0 4 solution
50
ml
Distilled water to make
100
ml
(pH should be 6.8)
Unbuffered osmium-permanganate fixative:
KMn0 4
1.3 gm
2% Os0 4 solution
50
ml
9% NaCl solution
2.7 ml
Distilled water to make
100
ml
The present writer cannot speak from personal experience about either
the virtues or disadvantages of these osmium-permanganate mixtures.
Tahmisian's illustration indicates, however, that much more cell structure is preserved than with permanganate alone.
3.13. Rapid Freezing
Thoughtful electron microscopists often have worried about chemical
fixation. They have wondered what might be missed by this method, or
what specific artifacts might be introduced. The situation was particularly
critical so long as they were virtually dependent upon osmium tetroxide
fixation. The recent successful utilization of aldehydes as alternate
chemical fixatives has given us more confidence, but one would nevertheless like to be able to avoid chemical reactions altogether. Thus the
possibility of preserving tissue by purely physical means has been a goal