giant chromosomes of blood worms

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chrisimbee
Posts: 104
Joined: Fri Mar 19, 2021 5:34 pm

giant chromosomes of blood worms

#1 Post by chrisimbee » Thu Jun 10, 2021 8:09 pm

Hello,

I 've found out that there was a protocol to observe the giant chromosomes présent inside the nucleus of the salivary glands of blood worms ( larvae of chironomes).

I wasnt successfull in dyeing them properly yet with methylen blue or toluidin blue so I tried with orange acridine under Uv-blue light.

So here are some essay :) The chromosome appears green with the dark striations or gene band on some kind or ruban more or less coiled inside the nucléus.
chromosome géant essai1 sharp.jpg
chromosome géant essai1 sharp.jpg (37.97 KiB) Viewed 270 times
10 juin 21 chironomes chromosomes essai 3 epi.jpg
10 juin 21 chironomes chromosomes essai 3 epi.jpg (68.49 KiB) Viewed 270 times
10 juin 21 chironomes chromosomes essai 2 epi.jpg
10 juin 21 chironomes chromosomes essai 2 epi.jpg (68.17 KiB) Viewed 270 times
10 juin 21 chironomes chromosomes essai 1 epi.jpg
10 juin 21 chironomes chromosomes essai 1 epi.jpg (87.08 KiB) Viewed 270 times

If some of you know how to dye then properly with toluidine blue in bright field I'd be very happy to learn...

Thanks

Chris
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

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Wes
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Joined: Sat Mar 09, 2019 12:58 pm

Re: giant chromosomes of blood worms

#2 Post by Wes » Thu Jun 10, 2021 8:49 pm

Nice!

If you can get lactic acid and aceto-orcein you can try to squash the cells and explore polytene bands, Balbiani rings, chromocenters etc.
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/FL/Jamin-Lebedeff
Youtube channel

chrisimbee
Posts: 104
Joined: Fri Mar 19, 2021 5:34 pm

Re: giant chromosomes of blood worms

#3 Post by chrisimbee » Fri Jun 11, 2021 6:09 am

In fact I've just found out after a fex trial the missing important information :

None of the protocols mention FIXATION and i'm going to show you the effect of the addition of a few drops of a mix of alcool-acetique acide : its immediat : the DNA precipitate and the chromosomes become visibles even without any coloration .

Ive made a mix 4 parts alcool + 1 part acetic acid (14°)

Here's WITHOUT any fixative :
11 juin 21  glande salivaire SANS fixation.jpg
11 juin 21 glande salivaire SANS fixation.jpg (107.22 KiB) Viewed 235 times
and then just 1 seconde after adding the mix :
11 juin 21 glande salivaire AVEC fixation.jpg
11 juin 21 glande salivaire AVEC fixation.jpg (106.79 KiB) Viewed 235 times
And then I added the acridine orange :


11 juin 21 chromosomes chironomes x10.jpg
11 juin 21 chromosomes chironomes x10.jpg (91.75 KiB) Viewed 235 times
11 juin 21 chromosome chironome epifluo ++++.jpg
11 juin 21 chromosome chironome epifluo ++++.jpg (72.17 KiB) Viewed 235 times
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

chrisimbee
Posts: 104
Joined: Fri Mar 19, 2021 5:34 pm

Re: giant chromosomes of blood worms

#4 Post by chrisimbee » Fri Jun 11, 2021 6:11 am

11 juin 21  chromosomes chiironome x40 epifluo.jpg
11 juin 21 chromosomes chiironome x40 epifluo.jpg (84.66 KiB) Viewed 234 times
11 juin 21  chromosome chironome ++ x16.jpg
11 juin 21 chromosome chironome ++ x16.jpg (76.14 KiB) Viewed 234 times
11 juin 21 chromosome chironome x40  bis.jpg
11 juin 21 chromosome chironome x40 bis.jpg (60.46 KiB) Viewed 234 times
So in conclusion : the fixation - precipitation is essential for good results :

now i'm trying to find out if the alcool alone would work..
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

chrisimbee
Posts: 104
Joined: Fri Mar 19, 2021 5:34 pm

Re: giant chromosomes of blood worms

#5 Post by chrisimbee » Fri Jun 11, 2021 7:25 am

So I tried with acetic acid 100% alone and then I rince and dye with gentian violet (crystal violet).

It works but it shows more blurring of the definition...
21 juin 21  chromosome chironome fixation acétique violet x20 2.jpg
21 juin 21 chromosome chironome fixation acétique violet x20 2.jpg (57.8 KiB) Viewed 224 times
21 juin 21  chromosome chironome fixation acétique violet x20 3.jpg
21 juin 21 chromosome chironome fixation acétique violet x20 3.jpg (63.26 KiB) Viewed 224 times
11 juin 21 chromosome chironome fixation acetique violet gentiane x20.jpg
11 juin 21 chromosome chironome fixation acetique violet gentiane x20.jpg (53.86 KiB) Viewed 224 times
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

dtsh
Posts: 378
Joined: Wed May 01, 2019 6:06 pm

Re: giant chromosomes of blood worms

#6 Post by dtsh » Fri Jun 11, 2021 1:27 pm

Quite interesting chrisimbee!
You refer to them as giants, how large are they compared to other chromosomes or a rough size in microns?

DrPhoxinus
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Location: Rochester Hills, MI

Re: giant chromosomes of blood worms

#7 Post by DrPhoxinus » Fri Jun 11, 2021 2:21 pm

Acridine orange is a powerful and dangerous mutagen.
I have used it to stain DNA in agarose gels but it is very dangerous for the hobbyist.

Giant salivary chromosomes can be found in most dipterans including fruit flies and mosquitoes

I have dissected the salivary glands out of a fruit fly maggot
and have gotten good results with aceto-orcein

See if you can find Lochoptera dubia if you live in North America. I have collected these in Michigan.
They are all female, a parthenogenic relic of 2 sexual species that have gone extinct.

Chromosomes show weird loops because of synapsis of 2 different species

Gerard

DonSchaeffer
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Location: Winnipeg, Manitoba, Canada

Re: giant chromosomes of blood worms

#8 Post by DonSchaeffer » Fri Jun 11, 2021 2:26 pm

Wow

DrPhoxinus
Posts: 139
Joined: Mon Jan 28, 2019 5:17 pm
Location: Rochester Hills, MI

Re: giant chromosomes of blood worms

#9 Post by DrPhoxinus » Fri Jun 11, 2021 3:12 pm

Don,

They are found in your province.

They are beautiful under a stereo scope.
They are called spear wing flies because of the usual wing crossveins

You might want to get a sweep net and take some swipes over moist grass. The diversity of fly species is amazing

Best,

Gerard

chrisimbee
Posts: 104
Joined: Fri Mar 19, 2021 5:34 pm

Re: giant chromosomes of blood worms

#10 Post by chrisimbee » Fri Jun 11, 2021 3:29 pm

I'm living in France.. so i dont know about the "Lochoptera dubia" but i'll ask the entomology dept at work.

And about the carcinogeny of acridine orange can you give any data and sources ? And aceto -orcein is hard to buy and also pretty expensive. The amount needed of acridin orange was really really minute : i just had to use a dust speck of it for the slide ! When i use it to dye living paramecia its the same : the amount is so minute i cant weight it with my lab scale ( 1mg 10% precision). And all the dyes are pretty toxic and dangerous. I'm not a newbee. (but thanks anyway). ;)

The toxicoly data sheet doesnt say much and there is no restrictions in its use nor its selling.

These chromosomes are huge compared to the one you can see in garlic root tip mitosis. I was able to see them easily with the x10 objective already to give you an idea.

These chromosomes are called polythene chromosomes and its a classic observation in biology lesson like the oignon root mitosis. Its just a bit tricky to extract the salivary glands when you're not used to disect insects ( I've been doing queen bee dissection for diagnosis in beekeeping for years now :) )

https://en.wikipedia.org/wiki/Polytene_chromosome
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850506/.
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

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Wes
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Re: giant chromosomes of blood worms

#11 Post by Wes » Fri Jun 11, 2021 7:53 pm

DrPhoxinus wrote:
Fri Jun 11, 2021 2:21 pm
I have used it to stain DNA in agarose gels but it is very dangerous for the hobbyist.
I think my morning cigarette is orders of magnitude more dangerous than a wee bit of AO. This reminds me of the ethidium bromide paranoia.
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/FL/Jamin-Lebedeff
Youtube channel

DonSchaeffer
Posts: 1470
Joined: Sun Mar 22, 2020 10:06 am
Location: Winnipeg, Manitoba, Canada

Re: giant chromosomes of blood worms

#12 Post by DonSchaeffer » Fri Jun 11, 2021 7:56 pm

DrPhoxinus wrote:
Fri Jun 11, 2021 3:12 pm
Don,

They are found in your province.

They are beautiful under a stereo scope.
They are called spear wing flies because of the usual wing crossveins

You might want to get a sweep net and take some swipes over moist grass. The diversity of fly species is amazing

Best,

Gerard
I am hung up on smaller things and I haven't got the skills to even aspire to what you dis. Congratualtions.

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