Bleaching of black insect exoskeletons.

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AntoniScott
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Bleaching of black insect exoskeletons.

#1 Post by AntoniScott » Thu Aug 19, 2021 9:10 pm

During the potassium hydroxide step where the chitinous exoskeleton of an insect is softened and partially bleached, this works well with most tan or brown coloured insects and less well with black insects with thick chitinous exoskeletons. Further processing, by allowing longer times in the potassium hydroxide solutions, is required. The risk of further processing is the over-softening of limbs, etc which results in loss of these parts.
I experimented with bleaching these parts in a 2% solution of hydrogen peroxide or regular household bleach ( sodium hypochlorite) with less than a successful outcome. Yes, the dark exoskeleton was bleached but it also resulted in the appperance of bubbles that can't be removed or an exoskelton with no detail.

Does anyone know of a method to bleach black exoskeletons without inducing the fomation of bubbles within the limbs of exoskeletons ?

BramHuntingNematodes
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Re: Bleaching of black insect exoskeletons.

#2 Post by BramHuntingNematodes » Thu Aug 19, 2021 9:49 pm

have you tried dilute sodium hypochlorite?
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Re: Bleaching of black insect exoskeletons.

#3 Post by BramHuntingNematodes » Thu Aug 19, 2021 9:57 pm

Gray also mentions oxalic acid, barium chlorate with sulfuric acid, potassium chlorate with acid.
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AntoniScott
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Re: Bleaching of black insect exoskeletons.

#4 Post by AntoniScott » Thu Aug 19, 2021 10:21 pm

yes I tried diluting both the sodium hypochlorite and hydrogen peroxide but they both produce bubbles. I experimented with both diutions and it is extremely time sensitive. A minute too long and the sample almost becomes invisible !!! Leaving the insect for a longer time in the Potassium Hydroxide only further degrades the structure with no bleaching effect.


I haven't tried oxalic acid or barium or potassium chlorate with acid.
Thanks for the information. This Gray manual you referenced - what is the name of the book, perhaps I can get it ?

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Re: Bleaching of black insect exoskeletons.

#5 Post by BramHuntingNematodes » Thu Aug 19, 2021 11:47 pm

It is his famous Microtomist's Formulary and Guide, with more chemical suggestions than you will ever need or perhaps even want. It is an older book, and some of the suggested formulations are extremely dangerous, so take it with a grain of salt.
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MicroBob
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Re: Bleaching of black insect exoskeletons.

#6 Post by MicroBob » Fri Aug 20, 2021 12:21 am

Hoyers mix might solve your problem. The longevity of the slides varies from a few months to many years.

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Re: Bleaching of black insect exoskeletons.

#7 Post by jimur » Fri Aug 20, 2021 10:45 am

For anyone interested, the book mentioned above by BramHuntingNematodes is available in pdf as a free download at https://archive.org/ (registration is free)
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EYE C U
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Re: Bleaching of black insect exoskeletons.

#8 Post by EYE C U » Fri Aug 20, 2021 9:24 pm

bleach works great for getting meat off bones and turtle shells

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Re: Bleaching of black insect exoskeletons.

#9 Post by BramHuntingNematodes » Sat Sep 18, 2021 3:40 am

AntoniScott wrote:
Thu Aug 19, 2021 9:10 pm
During the potassium hydroxide step where the chitinous exoskeleton of an insect is softened and partially bleached, this works well with most tan or brown coloured insects and less well with black insects with thick chitinous exoskeletons. Further processing, by allowing longer times in the potassium hydroxide solutions, is required. The risk of further processing is the over-softening of limbs, etc which results in loss of these parts.
I experimented with bleaching these parts in a 2% solution of hydrogen peroxide or regular household bleach ( sodium hypochlorite) with less than a successful outcome. Yes, the dark exoskeleton was bleached but it also resulted in the appperance of bubbles that can't be removed or an exoskelton with no detail.

Does anyone know of a method to bleach black exoskeletons without inducing the fomation of bubbles within the limbs of exoskeletons ?
I have here the antennal socket of an alate queen camponotus sp. (I'll pin it down give me a little bit), perhaps the thickest, blackest part of the large queen after 24 hours in hydrogen peroxide. This specimen should have stayed longer in the sodium hydroxide as it did soften but still cracked a bit under compression but here was the sequence where "to" denotes graduated series

to 99% alcohol
water and sodium hydroxide, 24 hours
squeezed between slides ala mr. sonchus
to 99% alcohol 48 hours
histoclear 2, decided was too dark so
3% hydrogen peroxide 24 hours
to 99% alcohol 24 hours
three changes of histoclear 2 over the course of about a week or two
mount in omnimount

There are not more bubbles than usual than with a specimen this size-- slow lowering of the coverslip is warranted. Mr S uses seven or so changes of xylene to remove all the alcohol and water I think you could take less time than a week this way I let that slide sandwich soak for a long period of time because I forget about it. The bubbles also disappear over this time frame. To do it again I would maybe double or even triple the time in sodium hydroxide as it is pretty gentle. I have never made a slide of an exoskeleton this size and it was fun but the queens may have been ambitious they are large and have delicate parts situated awkwardly for flattening.
Last edited by BramHuntingNematodes on Thu Oct 21, 2021 3:23 am, edited 1 time in total.
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BramHuntingNematodes
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Re: Bleaching of black insect exoskeletons.

#10 Post by BramHuntingNematodes » Sun Sep 19, 2021 5:38 pm

Image
Image
Image
Image
Here we can see a bubble in the head that likely will not be removed. One thing I like about omnimount is in a few days if you decide you want to redo the slide you just pry off the cover slip, put a couple drops of histoclear 2 on it and start new.
Image
Image

So you can see what I mean y some cracking. The skeleton is not perfectly preserved. The bleaching was effected readily, however, and produced a transparent specimen.
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BramHuntingNematodes
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Re: Bleaching of black insect exoskeletons.

#11 Post by BramHuntingNematodes » Thu Sep 30, 2021 2:37 am

Hey wait I am confused that wasn't mr s that used all those changes of xylene it was you! So, when you use hydrogen peroxide, the timing here couldn't be so critical? I had my queen in it for near 36 hours. All the bubbles eventually escaped in the ensuing week. How long can you wait for the bubbles to escape? I assume agitation such as vibration would help. I also wonder about a vacuum chamber. Could this potentially help?
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ldflan
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Re: Bleaching of black insect exoskeletons.

#12 Post by ldflan » Wed Oct 20, 2021 6:04 am

This is a hard problem to solve. You might try Visikol ("for Plant Biology"), or your normal sodium/postassium hydroxide clearing routine followed by a version of Heir's BB-4 1/2 solution. (Viz - 1 part benzyl benzoate plus 9 parts of a solution consisting of 2:2:2:2:1 lactic acid, phenol, clove oil, chloral hydrate, and xylene). Both were "designed" for clearing plant specimens in wholemounts, but can often work pretty well with small insects. You can substitute 2,2,2 trichloroethanol for the chloral hydrate in BB-4 1/2, at the risk of possibly violating Visikol's (in my opinion BS) patent on the stuff.

Getting any mounting medium to set after either of these is a trick; both 2,2,2 trichloroethanol and Visikol (which contains the same stuff) will prevent most mountants from setting up, and will dissolve most ringing cements - even epoxy (I have had some success using PVP for ringing). One kind of fiddly approach that can work is to move from Visikol to BB-4 1/2, then to a rinse solution consisting of 2:2:1 phenol, clove oil,and turpentine; carefully blot off what you can with a Kimwipe after the rinse and mount in gum damar in turpentine- try not to disturb the specimen and mix the clove oil/phenol rinse remaining on the specimen with the gum damar, otherwise it may never set up. The clove oil will at best cause the gum damar to take a loooong time to harden. Also you may get a haze at first, but don't toss the slide right away - gradually the hazy stuff often seems to migrate to the edges leaving the sample clear.

Oh and be really careful. BB 4-1/2 and the phenol/clove oil rinse described above are nasty stuff - the phenol will gradually cause nasty burns if you get it on yourself, and since it's saturated in oil it's very hard to wash it off. Be careful, wear gloves, and dispose of waste Kimwipes, slides, gloves, etc. with care - a face mask is a good idea, not so much because of splashes as to prevent yourself from absent-mindedly touching your face with a contaminated glove.

Leonard

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