House fly - beheaded and cleared in KOH (process explained)

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daruosha
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Location: Tehran, Iran

House fly - beheaded and cleared in KOH (process explained)

#1 Post by daruosha » Wed Feb 26, 2020 9:50 pm

Look at my eyes :evil:
Image
10x NA 0.25 / Phase Contrast / OnePlus 6 , handheld

The wife found a live fly and managed to put it in a clear plastic bag (I don't know how!) as a surprise for me. When I came home, I immediately anesthetized the insect by injecting some Acetone vapors in the bag and after a few seconds the poor fly was motionless. Then the usual process of fixing in alcohol/formalin and then the surgery comes in. With a pair of dissection scissors and help of my stereo microscope, I carefully removed the body parts (wings, legs, head and mouth parts), and put them in separate watch glass containers.

For clearing the body parts of insects I usually use 24 hours Lactic Acid followed by 12 hours of %35 hydrogen peroxide treatment. But I couldn't help myself and take the shortcut route: Put the head in the %30 (w/v) of Potassium Hydroxide solution and boiled it for 5 minutes and voila. Done. Afterwards I removed the KOH from specimen and dehydrated it with alcohol.

My phase contrast microscope doesn't have a trinocular head (I have ordered it, but it takes 2 months to arrive), so please forgive the low image quality.

I hope you like it.

Update: the proper dilution is %30 weight by volume (w/v). I made a mistake and wrote v/v in the initial post. My apologies.
Last edited by daruosha on Thu Feb 27, 2020 4:01 pm, edited 4 times in total.
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Wes
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Re: House fly - beheaded and cleared in KOH (process explained)

#2 Post by Wes » Wed Feb 26, 2020 11:23 pm

Thanks for the interesting description, I always enjoy reading how people prepare their samples.

Quite nice photo too, I wouldn't have come up with phase contrast for this particular subject but it really makes it stand out. Quite alien really. Nice one!

WhyMe
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Re: House fly - beheaded and cleared in KOH (process explained)

#3 Post by WhyMe » Thu Feb 27, 2020 2:15 am

Thanks for the explanation. I think the photo is very nice!

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daruosha
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Re: House fly - beheaded and cleared in KOH (process explained)

#4 Post by daruosha » Thu Feb 27, 2020 2:34 am

Image

Another shot, with a better focus.

Thank you all for your encouragements and kind comments.
Last edited by daruosha on Thu Feb 27, 2020 10:51 pm, edited 2 times in total.
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MicroBob
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Re: House fly - beheaded and cleared in KOH (process explained)

#5 Post by MicroBob » Thu Feb 27, 2020 10:37 am

Hi Daruosha,
thank you for posting images and preparation process!
Can you add a bright field image? I can imagine that the fine structures of the insect head could profit from the subtle details of plain bright field.

A quick way to make semi-permanent slides from insects is Hoyers medium, if one can obtain the ingredients. It brightens the specimen over a couple of weeks but sometimes tends to crystallize after half a year or so which destroys the slide.

Bob

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daruosha
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Re: House fly - beheaded and cleared in KOH (process explained)

#6 Post by daruosha » Thu Feb 27, 2020 1:19 pm

Thanks Bob for your tips and comments. Your suggestion are always welcomed.

Here in Iran, it's very difficult to obtain any specific microscopy stuff, all i found was Entellan and Canada Balsam solid bars (you have to dissolve them in Xylene) at a very expensive price. I paid nearly 250 USD equivalent for a bottle of 500ml Entellan. Other mounting mediums are next to impossible to find in the local market. And it's even more costly to import them (custom fees, shipping, legal sanctions, blah blah, don't get me started :) ).

For a few years I lived in Stockholm (Sweden) and it was a heaven (taxes were crazy though). Now i have to stick to what I have.

I'll post BF version soon tonight.

Update: I update the estimated price of Entellan (I cannot even get my currency rates correct :D ).
Daruosh.

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daruosha
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Re: House fly - beheaded and cleared in KOH (process explained)

#7 Post by daruosha » Thu Feb 27, 2020 4:03 pm

Update: the proper dilution is %30 weight by volume (w/v). I made a mistake and wrote v/v in the initial post. My apologies.
Daruosh.

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daruosha
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Re: House fly - beheaded and cleared in KOH (process explained)

#8 Post by daruosha » Thu Feb 27, 2020 10:16 pm

For the sake of comparison, I took all shots with my cell phone (I can attach a DSLR to my BF microscope and take better shots if you are interested).

Here we go:

Image
Bright field - 10x NA 0.25 / Condenser aperture: 0.10 / Handheld, OnePlus 6, Leveled in PS


Image
Bright field - 10x NA 0.25 / Condenser aperture: 0.25 / Handheld, OnePlus 6, Leveled in PS


Image
Dark field - 10x NA 0.25 / Condenser aperture: fully open / Patch stop size: 15mm / Handheld, OnePlus 6, Leveled in PS


Image
Oblique illumination - 10x NA 0.25 / Condenser aperture: 0.25 /Handheld, OnePlus 6, Leveled in PS


I think phase contrast did a really good job on this particular subject. Due to the thickness of the specimen, you have to close the aperture to get a better contrast (at the cost of not so desirable artifacts) or do focus stacking(I can do it with 20 or more shots if you like to see a better, more dignified result).
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apochronaut
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Re: House fly - beheaded and cleared in KOH (process explained)

#9 Post by apochronaut » Thu Feb 27, 2020 11:28 pm

Very fine work Daroush. An ounce of commitment and an ounce of skill are worth a ton of high tech.

Your housefly looks familiar.

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