3.9.
BUFFERED ALDEHYDES AS PRIMARY FIXATIVES
53
work which has considered or relied upon formalin fixation. This includes the comparative study of different aldehydes performed by Sabatini, Bensch, and Barrnett (1963), discussed below, who were not enthusiastic about the value of formaldehyde as a fixative. In private
conversation with Dr. Barrnett, however, it seems likely they used reagent
grade formalin which probably did contain alcohol.
Methanol-free formalin apparently can be purchased in the United
States. However, formalin is notorious for the decomposition of the aldehyde to form formic acid. Thus, at best, it would be undesirable to use
anything but a relatively freshly prepared solution. The answer to these
difficulties is simple; start with paraformaldehyde and make one's own
solution!
Paraformaldehyde is a simple polymer of formaldehyde which easily
disassociates in the presence of water. To achieve disassociation in a
strong solution, the temperature has to be raised to about 60°C, and the
solution must be slightly alkaline. If the powder is added to warm water,
nothing seems to happen until a small quantity of sodium hydroxide is
introduced. Drops of alkali should be introduced, one at a time, with
stirring in between. Suddenly, the beaker will start to clear, and solution
will take place within seconds. The pH at which this occurs will be just
on the alkaline side of neutrality, about 7.2. If one attempts to make a
40% formaldehyde solution, a slight milkiness may persist, but this will
disappear upon dilution.
Holt and Hicks (1961) experimented with 10% formalin (4% formaldehyde) used with a variety of different buffers including Veronal,
phosphate, collidine, triethanolamine, and acetate. Other parameters
included variations in the molarity of the buffers, and the presence or
absence of sodium and calcium salts. Their general conclusion was that
4% formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, with
7.5% sucrose was most effective. They specified that their stock formalin
solution contained "up to 10% methanol as a stabilizer." Micrographs
illustrating the preservation produced with this fixative indicate that it
did in fact preserve quite well indeed, although a direct comparison with
our experience is not possible since their material was embedded in
methacrylate, and no precautions were taken to prevent subsequent
damage.
Sabatini, Bensch, and Barrnett (1963) used 10% formalin (4% formaldehyde) in 0.1 M phosphate or cacodylate buffers, with small quantities of
sucrose added.
BUFFERED ALDEHYDES AS PRIMARY FIXATIVES
53
work which has considered or relied upon formalin fixation. This includes the comparative study of different aldehydes performed by Sabatini, Bensch, and Barrnett (1963), discussed below, who were not enthusiastic about the value of formaldehyde as a fixative. In private
conversation with Dr. Barrnett, however, it seems likely they used reagent
grade formalin which probably did contain alcohol.
Methanol-free formalin apparently can be purchased in the United
States. However, formalin is notorious for the decomposition of the aldehyde to form formic acid. Thus, at best, it would be undesirable to use
anything but a relatively freshly prepared solution. The answer to these
difficulties is simple; start with paraformaldehyde and make one's own
solution!
Paraformaldehyde is a simple polymer of formaldehyde which easily
disassociates in the presence of water. To achieve disassociation in a
strong solution, the temperature has to be raised to about 60°C, and the
solution must be slightly alkaline. If the powder is added to warm water,
nothing seems to happen until a small quantity of sodium hydroxide is
introduced. Drops of alkali should be introduced, one at a time, with
stirring in between. Suddenly, the beaker will start to clear, and solution
will take place within seconds. The pH at which this occurs will be just
on the alkaline side of neutrality, about 7.2. If one attempts to make a
40% formaldehyde solution, a slight milkiness may persist, but this will
disappear upon dilution.
Holt and Hicks (1961) experimented with 10% formalin (4% formaldehyde) used with a variety of different buffers including Veronal,
phosphate, collidine, triethanolamine, and acetate. Other parameters
included variations in the molarity of the buffers, and the presence or
absence of sodium and calcium salts. Their general conclusion was that
4% formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, with
7.5% sucrose was most effective. They specified that their stock formalin
solution contained "up to 10% methanol as a stabilizer." Micrographs
illustrating the preservation produced with this fixative indicate that it
did in fact preserve quite well indeed, although a direct comparison with
our experience is not possible since their material was embedded in
methacrylate, and no precautions were taken to prevent subsequent
damage.
Sabatini, Bensch, and Barrnett (1963) used 10% formalin (4% formaldehyde) in 0.1 M phosphate or cacodylate buffers, with small quantities of
sucrose added.