Hi Gunnar, I standardly extract pyraloid DNA from the abdomen of the adults, so that the abdomina are readily macerated for genitalia dissection. I, too, observed a certain fading of the material in this process. In cases where maceration from the DNA extraction was not sufficient I macerate the abdomen for 10-15 minutes in 10% KOH, but warm it up on a cooking plate to about 50-60°C, and this has no obvious fading effect on the material. One should keep in mind that KOH solution has a delay in boiling, so that if it gets too hot it will suddenly start boiling and spout out of the bowl or dish. Heating the KOH solution in a waterbath is the safer option.

I hope that helps. Best wishes, Richard

Hi Gunnar,

We sometimes do that with some of our material (putting it in DNA extraction buffer) and usually we do not do anything else with it and in my specimens of Empididae you can see everything clearly, in case that you still do not see it, maybe you can put the material for a short while in lactic acid, I use 90 % lactic acid. Either I put it for 24 h or I warm it to 90 C and then it takes only few minutes. Lactic acid is not so aggressive as KOH. I hope it helps.

Marija

Hi Gunnar,

In our experience, chironomids that have been through lysis overnight are usually completely cleared (much better than with KOH) and can be mounted directly in Euparal after thorough rinsing in 96% EtOH. 

Torbjørn

Marija's suggestion of lactic acid is worth investigating. The real positive thing about lactic acid is that it is far more selective about which tissue is cleared and which is not (it preferentially target hard chitinous tissue leaving soft tissue intact) , and crucially, unlike KOH, the clearing process reaches an end point... even after many hours at 90C it will not clear any more than it has during the first 1-2h. If you did that with KOH the specimen would eventually dissolve completely!. It is probably advisable to give the specimen an aqueous or neutral pH buffer wash   (at low ionic strength to keep solutes to a minimum) before a final 90-100% EtOH wash and eventual mounting in euparal.

My experience with chironomids is also that after lysis specimens can be mounted directly in Euparal.

I just wanted to give a word of caution as I did not have much luck using lactic acid. The tissue within several specimens expanded so much that they broke apart and distorted. Neither did it clear them sufficiently. This happened regardless of leaving them overnight in room temperature or heating the specimens briefly.

I would try leaving them for a short while in KOH if they are not sufficiently cleared during lysis.

Thanks for the answers everybody! The specimens look incompletely cleared for my purposes, so some further maceration will be neccessary. I think changing the maceration agent to lactic acid is the best bet; as Rüdiger Wagner uses it for his psychodid slides.