Permanent slide preparation of whole mount insects Part 2.

[AntoniScott]

When the technician has decided after a visual examination that the bleaching and softening process has been reached, the insect is transferred to a 1" deep water dish. Squeezing out the abdominal contents is necessary so that the insect can be flattened sufficiently well for microscopic viewing.
I discovered, through trial and error, that the flattening process can be made more easily and safely without the loss of limbs, etc, by squeezing thrrough two glass slides. Depending on the insect, I prevent crushing of the eoskeleton by using a paper shim. Shims can be easily made by using a 3x5 index card cut into thin strips, one at each end of the slide and fixed in place using small paper clips. Larger insects can be flattened by stacking the paper shims. My personal experience has shown that I never need more than two stacked shims on each end.

After the insect has been successfully mounted between the two glass slides, it can be dehydrated from 70% to 90 %, then100% alcohol. Finally the slides are transferred to several changes (at least five changes) of 100% Xylene for final clearing. I usually allow several hours, or even overnight, for each alcohol concentration, and at least 24 hours in each of the xylene clearing steps.

The xylene step is to be done with caution because the vapors are carcinogenic and liquid contact is toxic. I use acrylo-nitirile gloves and an activated carbon facemask. I have used the finger cots because they are so tactile but you have to check them frequently for holes.

When the slides are removed from the xylene I allow the liquid to drain from between the slides before removing the paper clips from each end. To separate the two slides that are very closely bound, I use a 30g syring needle to push between the two glass slides to help separate them. They are held together very tightly due to hydrostatic pressure. Carefully prying apart the slides will make sure the entire insect stays on one slide and not separate.

I visually inspect the insect to make sure there are no bubbles . If bubbles are seen I use a drop of xylene and some manipulating with a blunt object to help move them. The last step is to apply the mounting medium. My preferred mounting medium is Fisher Permount. I prefer to use more Permount than may be necessary rather than less because as the cover slip is slowly lowered I want to make sure that no air bubbles are trapped in low points across the exoskelton. Once you get air bubbles trapped, they are very difficult to remove after the cover slip has been applied. If you think that you have applied too much Permount, they you can "wick" the excess by using a Qtip.

The slides should be stored flat for at least two weeks before storing in slide boxes.