3.4.
SPECIAL PURPOSE OSMIUM TETROXIDE FIXATIVES
43
Stock buffer solution:
Sodium Veronal (barbital)
14.7 gm
Sodium acetate
9.7 gm
Sodium chloride
17.0 gm
Distilled water to make
500
ml
Fixative:
Buffer
10
ml
0.1 N HC1
14
ml
M Calcium chloride
0.5 ml
Distilled water
26
ml
Osmium tetroxide
0.5 gm
In designing this as a bacteriological method, the authors were particularly interested in retaining nucleic acids with a minimum of fixation artifact. This fixative, used at pH 6.1, has proven effective in this
connection, but would not be desirable under other circumstances.
Similarly, the post-fixation with uranyl acetate would not be a necessary
concomitant if the general technique was used for other purposes.
about 4000 rpm (1800 G). This is the prefixation phase, which forms a
concentrated pellet. The latter is resuspended in 1 ml fixative and 0.1 ml
of tryptone medium, and left overnight at room temperature. This completes the osmium tetroxide fixation. (If organisms are grown on the surface of a solid medium, the prefixation is omitted and small blocks
of medium and growth are cut out and placed directly in the
fixative
with its tryptone). After about 16 hours, when fixation is regarded as
complete, the suspension is diluted with 8 ml of the buffer alone, and
recentrifuged. This pellet is resuspended in about 0.03 ml of warm 2%
agar, which is mixed in well, and put as a drop on a microscope slide.
These manipulations with the agar must be made at about 45°C. After
cooling and gelation, the drop is cut into little cubes which are treated
for 2 hours with the uranyl acetate solution. This consists of a 0.5% dilution in the acetate-Veronal buffer, the pH of which will be about 5. The
uranyl salt serves as an electron stain, and in the view of Kellenberger
et al. (1958) perhaps also has some fixative properties in so far as nucleoproteins are concerned. It is only after these various treatments that the
specimens are dehydrated and embedded for sectioning.
The fixative employed is made up as follows:
SPECIAL PURPOSE OSMIUM TETROXIDE FIXATIVES
43
Stock buffer solution:
Sodium Veronal (barbital)
14.7 gm
Sodium acetate
9.7 gm
Sodium chloride
17.0 gm
Distilled water to make
500
ml
Fixative:
Buffer
10
ml
0.1 N HC1
14
ml
M Calcium chloride
0.5 ml
Distilled water
26
ml
Osmium tetroxide
0.5 gm
In designing this as a bacteriological method, the authors were particularly interested in retaining nucleic acids with a minimum of fixation artifact. This fixative, used at pH 6.1, has proven effective in this
connection, but would not be desirable under other circumstances.
Similarly, the post-fixation with uranyl acetate would not be a necessary
concomitant if the general technique was used for other purposes.
about 4000 rpm (1800 G). This is the prefixation phase, which forms a
concentrated pellet. The latter is resuspended in 1 ml fixative and 0.1 ml
of tryptone medium, and left overnight at room temperature. This completes the osmium tetroxide fixation. (If organisms are grown on the surface of a solid medium, the prefixation is omitted and small blocks
of medium and growth are cut out and placed directly in the
fixative
with its tryptone). After about 16 hours, when fixation is regarded as
complete, the suspension is diluted with 8 ml of the buffer alone, and
recentrifuged. This pellet is resuspended in about 0.03 ml of warm 2%
agar, which is mixed in well, and put as a drop on a microscope slide.
These manipulations with the agar must be made at about 45°C. After
cooling and gelation, the drop is cut into little cubes which are treated
for 2 hours with the uranyl acetate solution. This consists of a 0.5% dilution in the acetate-Veronal buffer, the pH of which will be about 5. The
uranyl salt serves as an electron stain, and in the view of Kellenberger
et al. (1958) perhaps also has some fixative properties in so far as nucleoproteins are concerned. It is only after these various treatments that the
specimens are dehydrated and embedded for sectioning.
The fixative employed is made up as follows: