[NHCOLL-L:4596] Re: Use of isopropyl for fixing and storing invertebrates

[John E Simmons]

Lex and Ryan
Isopropyl can be used as a preservative,but be aware that it has problems.
I have inserted a section of text below from the book I am writing on fluid
preservation that summarizes the literature on isopropyl as a preservative.

I would not use isopropyl at 50% as it can clear tissues at around 45% or
less.  If I had to use it, I would use a 70% solution mixed with deionized
or distilled water.

If I may ask, why are you buffering the alcohol, and to what pH?  Most
alcohol is slightly acidic (which makes a better preservative). I would be
concerned, particularly with isopropyl, about making too basic a solution
which could clear tissues.  This could particularly be an issue with
crayfish and mollusk shells, I would think, as they are going to further
buffer the solution, aren't they?  In any case, if you do have a good reason
to buffer, I would use a more stable buffer than CaCO3.

What is the glycerin for?  Glycerin acts as a scavenger that will attract
relative humidity and any contaminants in the air into your solution if your
containers leak.

Here is the text from the draft of the book, with citations:

Isopropyl alcohol was first made in 1855, but it did not go into commercial
production until 1920 in the United States (Hatch 1961).  It was recommended
as an alternative preservative to ethanol, without being tested, as early as
1922 (Griffin 1922).  A notice in *Turtox News* in 1934 complained of the
high taxes on ethanol, the red-tape necessary to buy it tax-free, and the
additives used to produce denatured ethanol.  The recommended solution was
isopropyl alcohol.  The recommendation for its use came despite the fact
that “Sufficient experimentation has not been carried on in connection with
its use in biological work to warrant a dogmatic statement from us that it
can *always* be used as a substitute, but our preliminary work justifies
this *belief*” (Windsor 1934:216).  There was a conflict-of-interest in the
recommendation to change to isopropyl alcohol:  “Where can you buy
isopropanol?  Turtox will gladly accept your orders for it” (Windsor
1934:217).  At that time, anhydrous isopropanol was $3.75 per gallon (for an
88% solution), but “the grade used for general preservation” was just
$2.50/gallon (Windsor 1934:217).


In 1922, Philip Pope preserved one specimen of a newt each in jars of 40%,
50%, 60%, 70% and 80% isopropanol, with one specimen in 70% ethanol for a
control (Pope 1928).  Pope examined the newts after a mere six years in the
preservative, and decided that the 40-50% isopropyl preserved specimens were
fine, even though he could not find the control to compare them to.  The
advantages that Pope listed for isopropyl were that it had no restrictions,
was inexpensive, could be used in low concentrations, was not irritating
like formaldehyde, and did not soften bones or teeth like formaldehyde (Pope
1928).  However, 45-50% isopropanol has been shown to cause considerable
shrinkage of specimens, it can be difficult to mix thoroughly, it is prone
to layering in specimen containers, it may soften bone, and it may cause
clearing of tissues if concentrations fall below 30-40% (Fink et al. 1979).
DiStefano et al. (1994) found 50% isopropyl failed to prevent tissue decay
in fish and crayfish.  The permeability of the epidermis (the solute
absorbed per ml of tissue) for methanol is 0.6, for ethanol 0.9, and for
propanol 1.3, thus isopropyl penetrates epidermis best (Schaefer et al.
1982).  It is important to note that isopropyl is twice as toxic as ethanol
(Monick 1968).


King and Porter (2004) reported observational data that long-term storage in
isopropyl alcohol results in greater fading of ant specimens, probably due
to the greater propensity of isopropyl and other long-chain alcohols to
extract lipids, proteins, and pigments from cellular membranes at high
concentrations (Goates and Knutson 1994).  Some workers, particularly
ichthyologists, have reported that they prefer isopropyl preserved specimens
because they are more flexible than alcohol preserved specimens (Fink et al.
1979, Walker et al. 1995); however, isopropyl preserved specimens are
flexible because the tissue matrix has undergone more breakdown that tissues
do in ethanol (Stoddard 1989); in an accelerating aging study, Von Endt
(2000) found that isopropyl induces more deterioration of collagen than
ethanol.


When isopropyl is mixed with water, the resulting solution undergoes
contraction, so solutions should be mixed on a weight basis rather than a
volume basis (Hatch 1961).



All preserving alcohols cause tissue shrinkage, but the amount of shrinkage
is variable, with less shrinkage occurring in methyl and more in isopropyl
compared to ethyl alcohol (Ciferri 1971).


 Ciferri, A.  1971.  Swelling and phase transition of insoluble
collagen.  Pages
101-151 *in* Elden, H. R. (editor). *Biophysical Properties of the Skin*.  John
Wiley and Sons, Inc., New York, vii + 645 pages.

DiStefano, R.J., M.J. Roell, B.A. Wagner, and J.J. Decoske.  1994.  Relative
performances of four preservatives on fish and crayfish.  *Transactions of
the American Fisheries Society* 123:817-823

Fink, W.L., K.E. Hartel, W.G. Saul, E.M. Koon, and E.O. Wiley.  1979.  A
report on current supplies and practices used in curation of ichthyological
collections.  American Society of Ichthyologists and Herpetologists ad hoc
Subcommittee Report, 63 pages.

Goates, C.Y. and K. Knutson.  1994.  Enhanced permeation of polar compounds
through human epidermis.  I.  Permeability and membrane structural changes
in the presence of shorter chain alcohols.  *Biochimica et Biophysica
Acta*1195:169-179.


Griffin, L.F.  1922.  Practicable substitutes for grain alcohol.
*Science*55(1419):262-263.


Hatch, L.F.  1961.  *Isopropyl Alcohol*.  McGraw-Hill Book Company, Inc.,
New York, x + 184 pages.

King, J.R., and S.D. Porter.  2004.  Recommendations on the use of alcohols
for preservation of ant specimens (Hymenoptera, Formicidae).  *Insect
Sociaux* 51(2):197-202.

Monick, J. A.  1968.  *Alcohols:  Their Chemistry, Properties and
Manufacture*.    Reinhold Book Corporation, New York, xiv + 594 pages.

Pope, P.H.  1928.  Isopropyl alcohol as a preservative.
*Science*68(1768):487-488.


Schaefer, H., A. Zesch, and G. Stuttgen.  1982.  *Skin Permeability*.
Springer-Verlag,
Berlin, ix + 541-896 pages.

Stoddard, R. W.  1989.  Fixatives and preservatives:  their effects on
tissue.  Pages 1-25 *in* Horie, C. V. (editor).  *Conservation of Natural
History Specimens:  Spirit Collections*.  The Manchester Museum and
Department of Environmental Biology, The University of Manchester,
Manchester, viii + 115 pages.

Von Endt, D.W.  2000.  Staying in shape: the stability of structural
proteins in natural history museum storage fluids.  *Polymer
Preprints*41(2):1794-1795.

Walker, H.J., G.H. Burgess, A.Y. Suzumoto, C.I. Klepadio, L. VanGuelpen, and
J.M. Humphries.  1995.  Isopropanol revisited.  *Curation Newsletter*11:3-4.


Windsor, A. S.  1934.  Alcohol notes.  *Turtox News* 12(11):215-217.

--John

John E. Simmons
Museologica
128 E. Burnside Street
Bellefonte, Pennsylvania 16823-2010
simmons.johne at gmail.com
303-681-5708
www.museologica.com
and
Adjunct Curator of Collections
Earth and Mineral Science Museum & Art Gallery
Penn State University
19 Deike Building
University Park, Pennsylvania 16802-2709
jes67 at psu.edu


On Thu, Nov 5, 2009 at 4:35 PM, Alexandra M Snyder <amsnyder at unm.edu> wrote:

> Any ideas and/or caveats re: use of isopropyl for invertebrate
> preservation? See letter below.  Thank you in advance for information
> provided.  Lex
>
> Lex,
>
> I just exhausted my last supply of ethanol, so I'll be
> fixing crayfishes and freshwater mollusks in iso and
> storing as well (theoretically).  It is 99% ACS Iso and as
> far as I can ascertain using Google, not denatured.  The
> plan for now is to try and buffer (CaCO3) and add a little
> glycerin as I do with denatured ethanol. My guess was
> diluting to 50% isopropyl.
>
> Ryan
>  Ryan Evans, Aquatic Zoologist
> Kentucky State Nature Preserves Commission
> 801 Schenkel Lane, Frankfort, KY 40601
> 502-573-2886; ryan.evans at ky.gov
>
> The world is made for people who aren't cursed with self
> awareness. - Annie Savoy
>
> **************************************
> Alexandra M Snyder
> Collections Manager-Fishes
> Museum of Southwestern Biology MSC03-2020
> 302 Yale NE
> University of New Mexico
> Albuquerque NM 87131 USA
> PH/FAX 505.277.6005
> http://www.msb.unm.edu/fishes/index.html
>
>


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