פברואר 28, 2024

Mounting arthropods on microscopic slides

My protocol for microscopic slide mounting of Coccoidea (scale insects) and many other insects (inspired by various sources but mostly Kondo & Watson). Total time ~ 1 hour (but you can prepare several specimens in parallel)

  • Clearing: KOH 10%. Pierce the back with a needle (not necessary for small Diaspididae). Duration: a few minutes (if heated) to a few hours, then crush the scale insect with a spatula to evacuate the body contents. If you wait long enough, the contents will become liquid and will drain easily.
  • Rinsing: 80% alcohol 4-5 drops + glacial acetic acid 1-2 drops → aim = to rinse and neutralise KOH to facilitate the next staining step. Squeeze the insect with a spatula to remove any bubbles or remaining body content
  • Staining: acid Fuchsin in lactic acid (10mg powder in 10ml lactic acid). A few minutes (until the specimens are sufficiently colored).
  • Rinsing off excess stain: return to the 80% alcohol + acetic acid bath for a few minutes: aim = to remove excess dye + dehydration. NB : If maceration lasts too long, the dye tends to fade.
  • Dehydration: 99% alcohol (+ 1 drop of glacial acetic acid?) for a few minutes. The acetic acid helps fix the stain. NB : some people use pure glacial acetic acid at this stage.
  • Final clearing: clove oil for a few minutes. In theory, not necessary if you are using Euparal but I usually do it anyway. Purpose = to evacuate alcohol but also the rest of the greasy contents, oils, waxes, etc. You can also conserve the specimens for longer in clove oil
  • Mounting: Euparal : less sensitive to incomplete dehydration, and solvents maybe less toxic (?) than Canada balsam

Misc :

  • Spatulas are essential for moving specimens from one bath to another and for squeezing out specimens. Made from entomological needles, flattened with a hammer, bent and cut to the desired shape with scissors, then attached to a mandrel. Alternative : nylon wire flattened with pliers : advantage = transparency, disadvantage : less thin and less rigid.
  • The staining step varies greatly between protocols, especially in terms of duration (from a few seconds to several hours). I think this is due to the fact that acid fuschin can be prepared in different ways (e.g. in lactic acid, in water) and at different concentrations. If the stain is too concentrated, I tend to lose the sample in it... 10mg powder in 10ml lactic acid 80% (= 0.1% solution) seems to work well for me.
  • Leucaspidini (Diaspidinae) need an extra dissection step (e.g. at the KOH step) to extract the female body from its external protection (skin of the last larval stage).
  • For very waxy species you may need a bath with a detergent such as Decon 90, Histoclear,... Some people do this at the beginning, others at the end...

הועלה ב-פברואר 28, 2024 03:13 לפנה"צ על ידי gillessanmartin gillessanmartin | 0 תגובות | הוספת תגובה

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