[NHCOLL-L:1900] transfer of amphibian larvae - ethanol to formalin

Fri Apr 18 07:17:26 EDT 2003

John,

What about transferring amphibian larvae originally preserved and stored in 
ethanol into formalin.  (We generally fix and store amphibian larvae in 10% 
buffered formalin, but occasionally get collections that have larvae that 
were initially fixed in ethanol or originally fixed in formalin and 
transferred to ethanol.) I recently spoke to Ronn Altig about this.  We 
agreed that tadpoles do not do as well in ethanol because they do not fix 
as well initially and are soft and may deteriorate over the long 
term.  Also, they may become dehydrated and shriveled when stored in ethanol.

I have transferred ethanol preserved tadpoles into formalin which 
re-hydrates them and makes them more usable (un-shriveled).  We do not know 
the effects of this.  Ronn said the transfer to formalin will not improve 
fixation of poorly fixed specimens, but we did not know any reason not to 
transfer them.

Do you have any thoughts about this procedure?

Greg

At 02:00 PM 4/16/2003 -0500, you wrote:
>There really isn't a "correct procedure" for doing this, but based on what
>has been done, I recommend staging between fluids to avoid too much stress
>to the specimens.  What steps you use will depend in large part on what the
>concentrations of the ethanol and isopropyl are.  Below is a section from
>"Herpetological Collecting and Collections Management" that addresses this
>issue.
>
>--John
>
>John E. Simmons
>Collection Manager, Natural History Museum
>and
>Coordinator, Museum Studies Program
>University of Kansas
>Dyche Hall
>1345 Jayhawk Boulevard
>Lawrence, Kansas 66045-7561
>Telephone 785-864-4508
>FAX 785-864-5335
>jsimmons at ku.edu
>www.ku.edu/~museumst/
>
>
>
>Selection of a Preservative
>Since the mid-1600s, when the preservative properties of spirit of wine were
>discovered, the overwhelming favorite fluid preservative has been ethyl
>alcohol, usually in a 60-75% concentration.  The second most common
>preservative (though far less common than ethyl alcohol) is isopropyl
>alcohol, which is less expensive and easier to obtain.  Isopropyl alcohol
>was not used as a preservative until sometime after 1920 when commercial
>production of it began (Hatch 1961); thus, we have fewer than 80 years of
>experience with it as a preservative.  The third common preservative is
>formaldehyde, which was not used as a preservative until sometime after 1893
>(Simmons 1995).  Methyl alcohol (wood alcohol) is unsuitable for use as a
>preservative.
>
>The biggest drawbacks to ethyl alcohol are its expense, the necessity of
>obtaining a Federal permit to purchase it tax-free, and the potential fire
>hazard it poses.  The arguments in favor of using isopropyl alcohol are that
>it is less expensive and easier to obtain (a Federal permit is not
>required), and that it leaves the specimens more flexible (Walker et al.
>1995).  However, there are many more arguments against its use.  Isopropyl
>alcohol poses a fire hazard, it is twice as toxic as ethyl alcohol, and many
>people find its odor objectionable.  The greater flexibility of the
>specimens may mask warning signs of deterioration.  Isopropyl alcohol does
>not have the 350+ year record of use in museums that ethyl alcohol does.
>The use of isopropyl alcohol as a preservative makes specimens unsuitable
>for most types of histological preparation (Jones and Owen 1987).  It is
>difficult to measure the density of isopropyl alcohol solutions,
>particularly with a hydrometer, as its density is close to that of water.
>In low concentrations (below 45%), isopropyl may promote a rapid clearing of
>tissues.  Isopropyl alcohol is difficult to mix with water and prone to
>layering in containers (Fink et al. 1979).  In long-term storage, it has
>been shown to soften bone (Steedman 1976).  While perhaps an argument might
>be made to continue using isopropyl alcohol for fish collections already in
>it (Walker et al. 1995), the argument does not justify using a preservative
>other than ethyl alcohol to preserve herpetological collections.
>
>Just because most fluid preserved collections use ethyl alcohol does not
>mean that it is the best preservative to use.  However, no data indicate
>that any other preservative is superior, or even the equal of ethyl alcohol
>(Simmons 1995).  Some alternate preservative formulas have been proposed for
>specific functions, such as preserving colors of specimens (see Color
>Preservation), or making specimens useful for specific histological
>techniques.  A few substances, such as phenoxetol, were in use for a decade
>or more before preservation problems were detected (Crimmen 1989).  In the
>absence of carefully controlled, long-term or accelerated aging studies of
>other preservative solutions to demonstrate their superiority, fluid
>preserved specimens should continue to be kept in ethyl alcohol.
>
>Transfer of Specimens from Fixative to Preservative
>It has long been customary to remove specimens from the field fixative,
>rinse or soak them in water in open containers (sometimes for several days),
>and then place them directly in storage-strength alcohol preservative
>(Simmons 1995).  There are several problems with this procedure.  Damage may
>result from soaking specimens too long in water.  Once formaldehyde is
>replaced with water, enzymatic activity in the tissues may resume (Taylor
>1981a) and the water may reverse the chemical crosslinks, permitting
>autolysis to occur.  Specimens will swell as they absorb water during
>rinsing or soaking and shrink once they are placed in an alcohol
>preservative solution.  Some workers have suggested transferring specimens
>directly from the formalin fixative solution to the alcohol preservative
>(Fink et al. 1979).  This procedure has several drawbacks, including severe
>osmotic shock in going from nearly 100% water to 30% water, and the quantity
>of formaldehyde that is released into the alcohol preservative.
>Formaldehyde in the preservative will drive the acidification of the alcohol
>solution, which will decalcify specimens and cause more severe color
>changes, as well as pose serious health risks for people working with the
>specimens.
>
>Based on the studies available on shrinkage, swelling, and other effects on
>specimens during transfer (e.g., Jones and Owen 1987, Lafromboise et al.
>1993; see discussion in Simmons 1995), and considering the effects of
>osmotic pressure changes on tissues (Steedman 1976), a more gradual transfer
>of specimens from a solution of near 100% water (e.g., "10% formalin") to
>30% water (70% ETOH) is recommended.   Because osmotic pressure rises
>steadily with ethanol concentrations below about 75%, it has been suggested
>that approximately equal concentration increments are the most appropriate
>for stepping specimens up to higher ethanol concentrations (Waller and
>Strang 1996).  Following the protocol of Laframboise et al. (1993), I
>recommend using steps of approximately 20% to stage specimens from 10%
>formalin solutions to 70% ethyl alcohol (Figure 9).
>
>Transfer of Specimens between Alcohols
>Specimens should not be changed from one type of alcohol to another,
>especially specimens on loan from other institutions.  Changing a specimen
>from one type of alcohol to another, particularly from isopropanol to
>ethanol, may cause significant shrinkage and other damage (Jones and Owen
>1987) and significantly alter the body proportions of the specimens (see
>review in Simmons 1993).  If it is necessary to change specimens from one
>alcohol preservative to another, follow the protocol of Laframboise et al.
>(1993).
>
>Laframboise, S., R.M. Rankin, and M.M.L. Steigerwald.  1993.  Managing
>change: alcohol transfer at the Canadian Museum of Nature.  Pp. 28-33 in
>Snyder, A.M. (ed.).  The 1992 American Society of Icthyologists and
>Herpetologists Workshop on Collections Care and Management Issues.  ASIH.
>52 pp.
>
>-----Original Message-----
>From: Vusi Mthombeni [mailto:V.Mthombeni at ru.ac.za]
>Sent: Tuesday, April 15, 2003 1:50 AM
>To: NHCOLL-L at LISTS.YALE.EDU
>Subject: [NHCOLL-L:1886] Propyl- and Ethyl alcohol preservatives
>
>Dear NHCOLL list members,
>
>I would like to know what is the correct procedure to transfer specimens
>which
>were originally preserved in propyl alcohol into ethyl alcohol or vice
>versa.
>
>Thanking you in advance.
>
>Vusi Mthombeni
>Collections Officer
>South African Institute for Aquatic Biodiversity
>Private Bag 1015
>GRAHAMSTOWN
>6140
>SOUTH AFRICA
>
>Tel: +27 (0)46 6035800
>Fax: +27 (0)46 6222403

Greg Schneider
Division of Reptiles & Amphibians
Museum of Zoology
University of Michigan
Ann Arbor, Michigan 48109-1079

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