Re: sectioning insects

From:Geoff McAuliffe

    You might have a look at a paper by Beckel in Nature 184:1584-1585, Nov. 14, 1959. He used tetrahydrofuran extensivly, Mollifex and a tape tranfer system. Also DeGiusti and Ezman Trans. American Micros. Soc. 74:197-201, 1955. Finally, Bolles-Lee in the Microtomist's Vade-mecum, recommends "diaphanol" (a proprietary substance from Leitz) as a chitin softening agent. He points out than ethanols harden chitin more than other alcohols do.

Tom Clarke wrote:

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

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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