74
3.
FIXATION
nated. At this point the vacuum was released before the methacrylate was
warm enough to have an appreciable vapor pressure. Sjostrand and
Baker (1958) interpreted their success with this method as indicating
that gaseous films at atmospheric pressure prevent adequate wetting of
the dry specimen by methacrylate monomers.
Grunbaum and Wellings (1960), Hanzon and Hermodsson (1960), and
also Elfvin (1963) have all used vacuum impregnation, although not the
high vacuum and low temperature employed by Sjostrand and Baker.
Most simply, Hanzon and Hermodsson put a layer of the embedding
mixture in the bottom of a large test tube at room temperature, and the
specimens were placed on a shelf hanging from the cork. This was connected to a mechanical pump through a stopcock. The pump was turned
on and the stopcock opened judiciously so that a controlled boiling
occurred. The embedding mixture was allowed to boil for a few minutes
until it was essentially degassed, and the residual atmosphere was the
plastic monomer. At that point the specimens were tipped off from the
platform and allowed to fall into the embedding mixture. The procedure
originally was devised for methacrylate impregnation, but has been used
also with Araldite and Vestopal.
People who have worked with freeze-drying all emphasize that perfection is too much to hope for. There surely will be considerable areas
of tissue that are seriously damaged by artifact. One must therefore be
selective and critical of the state of preservation. One hopes and expects
that small areas will show a structural pattern which can be interpreted
as indicating a good state of ultrastructural preservation. Perhaps the
most extraordinary finding of Sjostrand and Baker (1958), confirmed by
Hanzon and Hermodsson (1960), was that ribosomes are not evident in
the best material, suggesting that their usual appearance after fixation
may represent a condensation artifact.
Since materials which are preserved by freeze-drying without fixation
presumably retain their original solubilities, special difficulties arise
during sectioning. As will become apparent in Chapter 5, sections as
they are cut normally are floated upon aqueous solutions, which undoubtedly would leach water-soluble materials out of frozen-dried sections. Perhaps this is why Sjostrand and Baker (1958) and Hanzon and
Hermodsson (1960) did not observe undenatured ribosomes. In the
future, it will be desirable for people to show ingenuity in collecting
specimens without wetting them with water. One might return to the
techniques of the earliest days of ultrathin sectioning when dry sections
3.
FIXATION
nated. At this point the vacuum was released before the methacrylate was
warm enough to have an appreciable vapor pressure. Sjostrand and
Baker (1958) interpreted their success with this method as indicating
that gaseous films at atmospheric pressure prevent adequate wetting of
the dry specimen by methacrylate monomers.
Grunbaum and Wellings (1960), Hanzon and Hermodsson (1960), and
also Elfvin (1963) have all used vacuum impregnation, although not the
high vacuum and low temperature employed by Sjostrand and Baker.
Most simply, Hanzon and Hermodsson put a layer of the embedding
mixture in the bottom of a large test tube at room temperature, and the
specimens were placed on a shelf hanging from the cork. This was connected to a mechanical pump through a stopcock. The pump was turned
on and the stopcock opened judiciously so that a controlled boiling
occurred. The embedding mixture was allowed to boil for a few minutes
until it was essentially degassed, and the residual atmosphere was the
plastic monomer. At that point the specimens were tipped off from the
platform and allowed to fall into the embedding mixture. The procedure
originally was devised for methacrylate impregnation, but has been used
also with Araldite and Vestopal.
People who have worked with freeze-drying all emphasize that perfection is too much to hope for. There surely will be considerable areas
of tissue that are seriously damaged by artifact. One must therefore be
selective and critical of the state of preservation. One hopes and expects
that small areas will show a structural pattern which can be interpreted
as indicating a good state of ultrastructural preservation. Perhaps the
most extraordinary finding of Sjostrand and Baker (1958), confirmed by
Hanzon and Hermodsson (1960), was that ribosomes are not evident in
the best material, suggesting that their usual appearance after fixation
may represent a condensation artifact.
Since materials which are preserved by freeze-drying without fixation
presumably retain their original solubilities, special difficulties arise
during sectioning. As will become apparent in Chapter 5, sections as
they are cut normally are floated upon aqueous solutions, which undoubtedly would leach water-soluble materials out of frozen-dried sections. Perhaps this is why Sjostrand and Baker (1958) and Hanzon and
Hermodsson (1960) did not observe undenatured ribosomes. In the
future, it will be desirable for people to show ingenuity in collecting
specimens without wetting them with water. One might return to the
techniques of the earliest days of ultrathin sectioning when dry sections