Fwd: Re: sectioning insects
From: | "Dr. Ian Montgomery" |
Chloral
hydrate, no problem, you can buy it from Merck (17.90 P Sterling) Use it
regularly for Mayer's (1903) haematoxylin.
Ian.
Date: Wed, 27 Feb 2002 19:24:54
-0600
From: Tom Clarke <tec8435@ksu.edu>
Subject: Re: sectioning insects
To: Barry Rittman <brittman@admin4.hsc.uth.tmc.edu>
Cc: histology <histonet@pathology.swmed.edu>
Hi Barry,
Thanks for the reply. I can probably get my hands on chloral
hydrate as we
have a vet school on campus and I think that drug is still used as an
animal
anesthetic, though I suspect it will be a red-tape hassle. Do you
know if
the chloral hydrate is necessary, (ie., can treating with phenol
have the
same effect?) or if another chemical can substitute it? The
specimens I'm
working on are late instar caterpillars, so the cuticle isn't overly
tough
but still sufficently strong to cause problems in sectioning.
-Tom Clarke-
Barry Rittman wrote:
> Tom
> Most of the experience I had sectioning insects was looking at
> dragonflies and dragonfly larvae almost 40 years ago but I did spend
a
> lot of time researching of methods to produce sections of the
head
> regions.
> The most effective method I found was to concentrate on the
processing
> itself. I mixed melt equal parts of chloral hydrate and phenol
together
> by gentle warming and used as the chitin softening agent (I cannot
claim
> credit for this and chloral hydrate is probably difficult to get
chloral
> hydrate unless in business of shanghaiing sailors!)). This solution
was
> used after dehydration and specimens were left for up to a week in
this
> solution. Usually a day was adequate but did not hurt leaving
much
> longer. Then chloroform as an intermediary agent.
> Most of the other methods that I tried such as softening with
hydroxides
> did not seem to be effective.
> I think that effectiveness may depend largely on the insects and
probably
> will not work with thick shelled coleoptera.
> Some of the older methods suggested snipping off a piece of the
outer
> chitin to allow penetration of agents through the rest of the body
of the
> insects.
> An alternate is to use double embedding with celloidin-wax. Found
this to
> work but not as well as first method.
> Yet another method was to use isopropyl alcohol dehydration in place
of
> ethanol dehydration to minimize hardening. Worked reasonably well
for
> larvae but was not as effective for adult dragonflies.
> If the insects have already been embedded I would recommend soaking
the
> block in water or using Mollifex.
> If the chitin tends to separate you might try to use the technique
of
> wiping a block of 45 degree wax over the surface before cutting
each
> section. Failing this (and probably by this time after several
> Guinesses)- try painting a thin layer of celloidin on the
surface, allow
> to dry and then cut the sections. This is time consuming but
sometimes
> works.
> Effects on DNA - no idea?
> Barry
>
> Tom Clarke wrote:
>
> > Does anyone have any experience sectioning paraffin embedded
insects?
> > Are there any methods that can be used to soften the chitin
without
> > significantly damaging the other tissues (in particular, the
DNA)?
> >
> > -Tom Clarke-
> > Division of Biology
> > Kansas State University
Dr. Ian Montgomery,
Microscopy Service Unit,
Graham Kerr Building,
Institute of Biomedical & Life Sciences,
University of Glasgow,
Glasgow,
G12 8QQ.
Tel: 0141 332 8855
Office: 4652
Lab: 6644.
e-mail: ian.montgomery@bio.gla.ac.uk
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