Spirit Collections

Darrel E. Snyder desnyder at PICEA.CFNR.COLOSTATE.EDU
Fri Aug 25 15:15:26 CDT 1995

Many biologists and students are mislead to believe formaldehyde is good for
fixation but not for subsequent preservation because this typical museum
practice for many wet collections.  I believe the preserving of
formaldehyde-fixed specimens (e.g., fish) in alcohol solutions is a
practice imposed primarily to avoid unnecessary exposure of museum personnel
to formaldehyde fumes and solutions (I'm not very fond of alcohol fumes
either!).  However, formaldehyde is a perfectly fine preservative as well
as fixative for many (most?) specimens and tissues.  Once specimens are
fixed they can be transferred to formaldehyde solutions buffered to
near neutral if decalcification or erosion of bony structures is a concern.
Approximately 4% formaldehyde in water (10% formalin) is generally
recommended for fixation.  Once fixed, the specimens can be preserved in
the same solution as long as desired (decades, probably centuries);
however, only enough formaldehyde in solution is required to prevent
reversal fixation (uncross-link proteins?), as would happen in water without
sufficient formaldehyde.  For well fixed fish larvae, we find dilute, 1-2%
formaldehyde solutions (2-5% formalin) are quite adequate.

For large fish, subsequent preservation in alcohol (50% isopropanol or 70%
ethanol are commonly used) is fine expect perhaps when the tissues are to
be studied in a more-or-less natural state. However for delicate,
soft-bodied organisms such as fish larvae and even small juvenile fish,
alcohol dehydrates, deforms, and shrinks specimens and decreases their
flexibility.  These effects are significantly reduced if specimens are
stepped through a graded series of alcohol concentrations, but this is a
very time consuming procedure.  In very high concentrations (e.g., >90%),
specimens also become quite opaque and rigid to brittle and can be very
difficult to work with and may be easily damaged.  I strongly prefer to
retain the specimens for long-term (permanent) storage in formaldehyde
solutions (either the fixative concentration or a more dilute
concentration--3% formalin, phosphate buffered if future skeletal study is
a likely consideration).

The only notable exceptions are fish larvae for which we intend to do
otolith analysis (e.g., to determine age in days) or DNA analysis for
identity verification.  These we "fix?" or preserved directly to straight
(95-100% ethanol).

Recently formed otoliths are very sensitive to erosion in acid solutions
such as unbuffered formaldehyde which is initially required to properly
"fix" tissues.  Fish larvae can be removed to a dilute buffered
formaldehyde after just a few hours but that may be enough to significantly
damage or erode recently formed otoliths.  I suspect that if fixative
solutions of formaldehyde are prepared with very hard water (e.g., water
with a very high calcium content) we may be able to initially fix specimens
for otolith study in formaldehyde them transfer the specimens to dilute,
buffered formaldehyde solutions for long-term storage (preservation).
Except for some promising preliminary experiments, I haven't been able to
pursue this question yet.

There are variations of techiques for DNA study suitable for formalin
preserved specimens (tissues) but our specialist on DNA analysis has
yet to experiment with them and for the present we know we can get good
results with specimens initially and subsequently stored in straight
alcohol.  I expect that we soon will not need alcohol to assure our ability
to determine identity by DNA analysis.

For persons interested in reading up on fixation and preservation
(especially in regard to fish larvae and other zooplanton), I suggest the
book edited by H. F. Steedman (1976.  Zooplankton fixation and preservation.
Monographs on Oceanographic Methodology 4, The Unesco Press, Paris) and two
papers by M. F. DeLeon, R. O. Reese, and W. J. Conley (1991.  Effects of
fixation and dehydration on shrinkage and morphology in common snook
yolk-sac larvae.  NOAA Technical Report NMFS 95:121-128) (Reese et. al.
1991.  Histological effects from long-term storage of common snook yolK-sac
larvae in fixatives and alcohol.  NOAA Technical Report NMFS 95:129-137).

Darrel E. Snyder            Telephone:  (970)491-5295
Larval Fish Laboratory      Fax:        (970)491-5091
33J Wagar Building          E-mail: desnyder at picea.cnr.Colostate.edu
Colorado State University
Fort Collins, Colorado 80523

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