Molluscan Research: Techniques for collecting, handling, preparing ...

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GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27
throat, even at very low concentrations. It is
carcinogenic and regular handling frequently causes
allergy (short term exposure maximum concentration
1 ppm). For some people allergic reactions may appear
after only a short time, for others it never happens, but
great caution should always be taken to avoid contact
with the fluid or inhaling the vapour. Preserved material
and the solution should always be stored in ventilated
cabinets or a fume hood, never in a closed cabinet. If
stored enclosed, formaldehyde vapour will accumulate
and the concentration may rise to dangerous levels due
to evaporation.
It is fairly cheap and simple to test laboratories
and storage space for presence of formaldehyde in the
air and the test has sensitivity of less than one
thousandth of the maximum allowed concentration. Test
badges (GMD systems 570 series), available at http://
www.scottinstruments.com/products/product_list.cfm
or their local representative are placed at various
locations, a protective cap is removed, the badge is left
in place for 24 hours then resealed and sent to a certified
laboratory for analysis. Quantities down to less than
0.001 ppm are registered. Few (or none) of people in
charge of collecting or collections seem to have
accepted the warnings of Simmons (1991) about the use
of formaldehyde, although many of his statements
about formaldehyde are obviously wrong or
exaggerations.
Addition of a small quantity of ammonia to
formalin fixed specimens has been used to remove the
smell, but the procedure is based on the formation of
formamide (HCONH 2, CAS number 75-12-7) and
hexamethylene tetramine (CAS number 100-97-0)
which are odourless, but the practice is deceptive since
the formalin smell is a good warning to improve
ventilation or do the work in a more suitable place. For
similar reasons, decalcification of large specimens in
formalin with hydrochloric acid must be done in a fume
hood because of the formation of phosgene (carbonyl
chloride, COCl 2 CAS no 75-44-5), also highly
poisonous (threshold limit for allowable air
concentration 0.05 ppm.). Therefore, formalin fixed
specimens must be well rinsed before they are
processed.
Many different qualities of formalin are available
on the market. The cheap qualities are fully functional
for normal fixation if used only for a short time and
adequately buffered. Even so-called acid-free qualities
which are buffered by addition of dolomite (calcium
carbonate) change their pH after one or two years
because the solution cannot dissolve enough dolomite
to buffer for a long time.
Formation of Paraldehyde and
Paraformaldehyde [(CH2O)n (n = 6–50)] takes place
when formalin is stored at temperatures below +5–
10°C; the conversion is faster at lower temperature,
high concentration of formaldehyde and high pH. The
polymer is insoluble in water, ethanol, xylene, acetone,
but can be destroyed by treatment with bleach. If such
deposits have precipitated on shells, it is usually
possible to get rid of them by soaking them in warm
water and brushing off the deposits. It is said that the
polymerised form can be dissolved by autoclaving.
Polymerisation may also occur directly if formalin is
allowed to evaporate, so it is important to rinse formalin
preserved specimens intended to be dried.
One advantage with formaldehyde fixation of
specimens intended for dry collections is that they are
much less likely to be eaten by insects. Specimens
preserved only in alcohol are as likely to be attacked by
insect as those simply dried without any fixation
(common with land snails).
The commercial solution of formaldehyde usually
contains 5–20% methanol, to prevent generation of
formic acid by the so-called ‘Cannizzaro reaction’,
where an aldehyde produces equal amounts of the
corresponding alcohol and acid. This lowers the pH
towards 3 in old, low grade formalin. This can lead to
destruction of sensitive shells in less than a day. To
avoid this the formalin should be buffered, preferably
with sodium tetraborate, which is a nontoxic, cheap and
stable substance. This neutralizes the formic acid and
raises the pH to ca 7.5–8.0 in 40% and to 8.0–9.2 in 4%
formalin.
The solubility of borax in formalin (37% and
3.7%) is slightly more than 50 g/litre at room
temperature (lower at lower temperatures). Addition of
this much borax, however, will increase the risk of
formation of paraldehyde and (1 tablespoon) 10 g / litre
37% formalin is more than enough to ensure a stable pH
above 7.5 for two years.
Prolonged storage of organic tissues in borax
buffered formalin must be avoided because of the onset
of histolysis at this high pH, but this need not be
considered for several months.
To measure pH in formalin is simple and may be
done with indicator paper (preferably special types for a
narrow range, of which several are available, for
example pH 6.0–10.0) or with an electric pH meter.
A general conclusion of this and existing literature
is that it is not advisable to store, only to fix, molluscs or
any animals with a calcareous skeleton in formalin. The
formalin needs buffering, at least to a pH of 7.5–8.0 to
prevent damage of calcium carbonate. At this high pH
there is a risk of polymerisation. For tissue samples a
storage at the isoelectric point of proteins, pH 6–7, is
considered advantageous (Steedman 1976) because at
this level they have the minimum of solubility. At this
low pH, however, the protein chains are believed to
become more brittle and anatomical details, from cilia
to legs, break off more easily (Steedman 1976). More
information on formalin can be found in Carter (1997).
Formic acid. CAS no 64–18-6. Formic acid is often added to
fixatives for combined fixation and decalcification.
Gooding and Stewart's fluid is an aqueous mixture of