"Clearing" arthropods

[shawngibson]

Hi all,

I've been reading about the processes required to get an arthropod onto a slide, properly.

One of the processes involved is "clearing". Does this actually make the chitin/pigments transparent, so that you could take, say, something like an ant or a small cockroach and transmit light through it?

Another thing is Canada Balsam, which I adore as a painter. But apparently, you need to use some very toxic substance if you choose CB as your mounting medium. How toxic, exactly? is it ok around cats?

There seem to be 4 stages to mount an arthropod. Get rid of tissues, remove water, clear/remove pigment, and mount. Am I even remotely close to correct?

Chime in to fix any part of my stupidity here, please and thank you:)

Shawn

[mrsonchus]

Hi Shawn, interesting question.. I've very little experience of mounting insects or even non-botanical specimens but I do have some, and the general principles for the process are the same....

A while ago I had occasion to perform brain-surgery upon a fly, unfortunately the fly didn't make it , however a surprise occupant - the cause of this fly's persistent headaches, was removed, and the head of this former squatter was, quite easily, mounted permanently by myself for our Grandson to marvel at with his then newly-acquired microscope.
This was by way of a 'family discussion' deemed to be far too ghastly, likely to give nightmares, permanently-damaging.... etc to be presented to him, and so it remains in with my botanical slides to this day, and is still in perfect condition.

The culprit - a maggot found in the fly's head, ironically perhaps; below is a picture of the maggot's own head, removed from that of the fly, and mounted for posterity!

ws_fly_head_maggot_measured.jpg (221.99 KiB) Viewed 4481 times

To the point, apologies, I seem to have blathered-on somewhat;
I placed the curious 'Russian-Doll' fly into a fixative called 'FAA' (a mixture of formalin, alcohol and acetic-acid) for a few hours to literally 'stop the rot' and render the whole stable and ready for it's next stage - dissection - during which the maggot was discovered and removed, and at this time the maggot's head unfortunately fell rather unceremoniously off .

Anyway, "one can't simply throw away a perfectly good head" as I remarked to my darling Wife as she ran screaming from the room!

Then I moved the head into 95% Isopropanol which is cheap and easy to find to remove the more 'active' acid and formalin elements of the FAA and leave the specimen ready to simply mount straight into any 'alcohol-based mountant' ('Euparal' http://www.bioquip.com/search/DispProdu ... mnum=6372A is a branded version I think) - again easy to find for sale and very cheap - I use a generic and it's perfect.

Cover-slip the whole and let it dry for a week or so before running a thin layer of clear nail-varnish around the edges of the cover-slip/slide join to permanently seal the mount. The slide will be perfectly OK to use and view etc as soon as the mountant is dry, say 1hr after cover-slipping.

If you prefer to mount in a resin, forget real CB and use a replacement such as 'Numount' which is even better. Don't worry about toxic solvents for the resinous mountant such as xylene etc - you don't need them these days. I use a product called 'Histoclear' http://www.brunelmicroscopes.co.uk/stains.html which does the same job of preparing a sample for resinous (Numount/balsam) mounting. It's based upon oils from oranges and is the std replacement for xylene that I use for all slides. It isn't used for dehydration however, that's the job of the IPA, for which Histoclear is then used to 'drive-away' ready for resin..

With such small samples I would try the following;
Place into 95% (sold as 99% and definitely near-enough) IPA for about 2 hours
Move to Histoclear for another 2 hours
Place onto slide with a good soaking of Histoclear upon it
Allow to 'settle' for about 1 minute (don't let it dry-up)
Drop a small drop of Numount onto or next to the specimen
Allow to 'settle' for about 10 seconds
Place cover-slip onto specimen and allow to 'find it's level' as the mountant/Histoclear styarts to ooze slightly from the edges - just leave this to happen, it's normal but of course experience will soon anable to to judge quanities to just-right and this excess will almost completely disappear from your mounts as you get better at it.

In brief,

To mount in balsam/Numount,
into OH as above
into Histoclear
mount in balsam

To mount in alcohol-based-mountant/Euparal
into OH (perhaps 2/3 changes would be good)
mount in alcohol-based-mountant
seal with resin or bail-polish after about 1 week to make permanent

I used an additional stage at the start of the 'head-mount' of FAA simply because I had lots in stock that I use to fix all my botanical specimens...

p.s. Nail polish is often mooted as a quick substitute for resin, which it is, but it shrinks and cracks awfully and I wouldn't bother with it except as a good cover-slip sealant if I were you - I tried it many times but it never quite did the job, always spoiling after 1-2 weeks...

Hope this helps a bit, ask away if you need more, there are a lots of very helpful and knowledgable folk on this forum.

Good luck

[Peter]

Hi Shawn,
Clearing is a term which appears to have two related meanings; when tissue is moved from a relatively low refractive index medium e.g. alcohol to a medium of a similar refractive index to that of the tissue itself e.g. benzene then the tissue's optical characteristics change from cloudy to clear, alternatively when moving from dehydrating agent to embedding agent the alcohol must be cleared from the specimen for this purpose a solvent e.g. benzene is used.
As for toxicity all substances are toxic it is only the amount of dosage which varies e.g. 1 tablespoon of table salt will kill you if taken all at once. Cats are extremely sensitive to and highly repulsed by organic solvents, however this should not prove a problem as the micro laboratory is no place for cats (unless they happen to be the specimen at the time).
Are your specimens coming out to dark, if so I believe it is possible to bleach them. If you can give us more information on the processes you are putting your specimens through and the results you're achieving then we may be able to help you further.
Hope this helps.
Peter.

[shawngibson]

Thanks very much for the responses; very, very helpful.

As of yet, I've never done any of this (chemicals with specimens). I've been into microscopy for less than a month, and am coming from an artistic background with a tremendous curiosity largely revolving around: Protostome and Deuterostome differences (I'd really like to create definitive cleavage images from both taxa); the incredibly fascinating reality of how complex tiny Arthropods are; the relationship between Arthropods and older Ecdysozoans (Tardigrades and Velvet Worms...both of which are very beautiful); basic notochord evolution; pigment evolution; various eyes; evolutionary convergence under a microscope; Bacterial and Archaeal endosymbiosis into Eukaryotes (HGT, mitochondria, etc.) ...I could go on and on and babble with the passion of a true idiot lol.

Unfortunately, as an artist, with very little science training, this could all get quite messy, quite quickly.

I'm a little concerned that I have 2 cats and am doing this in an apartment, not a lab. The cats are separated from the room, always; door shut, windows open...is that enough? I received a bunch of stains today, but now I'm afraid to even read the directions lol...

I really have no interest in working with anything alive; just purchased specimens. And what I find day-to-day. For example, last week, a hummingbird clearly hit the tower where I work; it was dead, and while I was a bit sad, I also wanted to throw it into a jar with alcohol from the pharmacy, since I knew it was already lifeless. It could merely rot, per nature's way, or I could maybe in my own small way make images that might add to our knowledge (unlikely) or inspire young people to study biology.

As you can see, I've gone whole hog into an endeavour which will take me a long time to learn.

However, back to the original question, if I aim only to turn dead arthropods into semi-transparent specimens for viewing under the microscope...forgetting about preserving...is that more do-able in a ventilated home environment (no kids or humans but me, and cats not allowed in the room)? So basically, make the specimen ready to photograph, and then remove it, not preserve it...

Shawn

[shawngibson]

Random photo...from tonight. I think it's a roach head, but I'm not sure. It was in a trap. What interested me was the apparent huge hole, as if it had been scavenged in the trap. Or shot by a very tiny American.

[mrsonchus]

Hi and welcome to a fascinating and timeless world - you're in for a treat!
I wouldn't worry too much about the cat - just keep to the basic cautions as we all do working at home; my top priority is always to make sure I don't carry any chemicals around with me - I have a dog and whilst not as sensitive as cats, still need careful consideration. Just make sure you wash your hands or even use gloves when handling the solvent/s such as Histoclear, the OH really shouldn't be a problem at all.
Relatively speaking these chemicals are really very safe, although of course cats require a certain elevated level of caution around the solvent elements. Perhaps a quick consultation with your veterinarian would be a good idea to enable you to receive some specialist advice?
If you are intending to study the topics mentioned you will need to have such materials to hand but good protocol/s will keep things safe when practiced well. Crack-on, you should be fine, as should your cat with a little care.
Good luck, start simple with IPA and de-ionised water mixes and take it from there.


p.s. I suspect your specimen may have had a squatter at some point post-mortem....

102_3219.JPG (157.37 KiB) Viewed 4452 times

Yuketh!