Aloe-leaf epidermal peel - stomata

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mrsonchus
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Aloe-leaf epidermal peel - stomata

#1 Post by mrsonchus » Sun Jan 10, 2016 1:09 am

Hi all, I've been putting some freshly-cut Aloe-leaf into fixative this evening and thought I'd make a quick & dirty epidermal-peel. Stained briefly with Toluidine-blue (aq) then water-mounted with coverslip, very colourful.
Had a quick try of a cardboard 'Matthias-arrow' I 'made' when reading the great Walter Dioni's articles when I started-out - it's really not very good compared to the simply fantastic oblique-illumination often seen of this forum (to say the least! :oops: ) but gives a little relief at least.

Here are a few pictures, apologies for the poor quality, it was a very quick foray as I was actually fixing tissue rather than meaning to do this, but though it'd be nice to have a peek. I'll definitely go back and do some 'proper' ones I think, maybe mounted in glycerin or perhaps an alcohol-based mountant, anyway here are a few pictures to peruse!

A real 'purple-haze' :)
ws_aloe_epidermal_peel_x4.jpg
ws_aloe_epidermal_peel_x4.jpg (171.98 KiB) Viewed 3528 times
and closer-in,
ws_aloe_epidermal_peel_1.jpg
ws_aloe_epidermal_peel_1.jpg (268.08 KiB) Viewed 3528 times
more,
ws_aloe_epidermal_peel_2.jpg
ws_aloe_epidermal_peel_2.jpg (248.33 KiB) Viewed 3528 times
and a rather tragic attempt at oblique by way of a handy Matthias-arrow..
ws_oblique_1.jpg
ws_oblique_1.jpg (332.31 KiB) Viewed 3528 times
That's all I have, good fun having a go though, must do some more live-tissue when I get a chance.
To sleep for me, back soon.. :D :D
John B

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gekko
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Re: Aloe-leaf epidermal peel - stomata

#2 Post by gekko » Sun Jan 10, 2016 7:51 pm

Very beautiful, John, and absolute image clarity.

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Seb28
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Re: Aloe-leaf epidermal peel - stomata

#3 Post by Seb28 » Sun Jan 10, 2016 7:57 pm

I agree with Gekko,beautiful pictures.
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Re: Aloe-leaf epidermal peel - stomata

#4 Post by rnabholz » Sun Jan 10, 2016 8:28 pm

Hey John,

Not seeing the poor quality you are apologizing for, great images!

This is more the approach that I see myself undertaking, great to see such excellent results are possible.

Any tips or links on staining specimens under this protocol? I am ham-fisted in my efforts with staining.

Great stuff as usual!

Rod

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Re: Aloe-leaf epidermal peel - stomata

#5 Post by mrsonchus » Sun Jan 10, 2016 9:12 pm

rnabholz wrote:Hey John,

Not seeing the poor quality you are apologizing for, great images!

This is more the approach that I see myself undertaking, great to see such excellent results are possible.

Any tips or links on staining specimens under this protocol? I am ham-fisted in my efforts with staining.

Great stuff as usual!

Rod
Thanks Rod! I buy my Toluidine-blue stain, the very best stain for living (e.g. hand-cut) tissue (plant) in my experience, from this e-bayer in the UK for what is frankly a ridiculously low price http://www.ebay.co.uk/itm/Histology-Sta ... jx9U6JCThw
In the USA perhaps this one would be OK? http://www.ebay.co.uk/itm/EK-Industries ... Sw3xJVZ4Hp

I buy the 0.5% aqueous mix and dilute with water (de-ionised is best) to give my working-solution of 0.05% aqueous. This strength is for my tissue fixed to slides and left in the stain, for 'on-the-fly' staining I use 0.5% from a dropper-bottle straight onto the tissue.
ws_toluidine_blue.jpg
ws_toluidine_blue.jpg (62.91 KiB) Viewed 3502 times
It's a superb stain for live tissues and simplicity to use.
Dropper plenty (of the 0.5%) onto your tissue, on a slide or in a dish etc and let the stain act, probably for about 2 minutes, vary as required. When you think it's right, rinse with water to remove excess stain, look at the staining with a 'scope and stain further if necessary or rinse in water or OH to remove some stain (easier just to run a series of tests than to remove over-staining though).

This stain will act metachromatically and usually gives beautiful rich blues, pinks, reds etc as partly seen in my pictures. A brief and gentle rinse in tap-water often adds a little intensity to a stain once at the desired level of staining. I think the alkalinity causes an effect known as 'bluing' - certainly works for me.

Best then to coverslip for a good water-mount, keeps everything flat and stable! :D

Go to it, you'll really enjoy the results. Have a look at some of my posts re hand-sections, the stain is demonstrated in them with some really beautiful results. Hand-sections can be just as rewarding and beautiful as the full-blown permanent sections that I make, especially now we can photograph them to such high standards for permanent record!

Have fun! :D
Last edited by mrsonchus on Mon Jan 11, 2016 9:58 pm, edited 1 time in total.
John B

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mrsonchus
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Re: Aloe-leaf epidermal peel - stomata

#6 Post by mrsonchus » Sun Jan 10, 2016 9:13 pm

Thanks Seb & Gekko, you always bring a smile to my face with your kind comments. :) Pleased you like them. :D
John B

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Re: Aloe-leaf epidermal peel - stomata

#7 Post by JimT » Sun Jan 10, 2016 9:22 pm

Stain definitely brings out the cell contents. Beautiful images and detail. I like live cell sections the best.

JimT

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Re: Aloe-leaf epidermal peel - stomata

#8 Post by mrsonchus » Sun Jan 10, 2016 10:39 pm

JimT wrote:Stain definitely brings out the cell contents. Beautiful images and detail. I like live cell sections the best.

JimT
You're right there Jim, there's an inherent beauty in the live tissue that I never really see in the processed sections. For sheer detail and information I think the 'full-process' sections are superior, but for anadulterated beauty and a real sense of the plant whence came the tissue, the live sections always come out on top for me.

Thanks chaps for your compliments, I'm very pleased you like these. :D
John B

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Re: Aloe-leaf epidermal peel - stomata

#9 Post by rnabholz » Sun Jan 10, 2016 11:38 pm

Thanks for the run down on staining John.

I need to quit my job to have enough time to do all of this cool stuff.

Maybe the Powerball Lottery is the answer, $1billion this week... That would buy a lot of stain.

Thanks

Rod

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Re: Aloe-leaf epidermal peel - stomata

#10 Post by JimT » Sun Jan 10, 2016 11:54 pm

You just convinced me to order some. You had recommended it before but I went for methylene blue instead. I like it but will give this a try.

JimT

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Re: Aloe-leaf epidermal peel - stomata

#11 Post by mrsonchus » Mon Jan 11, 2016 12:09 am

JimT wrote:You just convinced me to order some. You had recommended it before but I went for methylene blue instead. I like it but will give this a try.

JimT
Go get it Jim - it's definitely superior to Methylene blue, although for processed tissue Methylene blue is capable of very fine detail, it can for some reason cause quite a brutal 'tattering' of some tissue - no idea why though..
You'll see in the pictures that the Toluidine blue will give a very nice (blue) nuclear image too, and doesn't tend to simply 'black-out' the details as a lot of stains often do with nuclei.
Toluidine blue is superb for live tissue, nothing I've tried comes close, although second place is undoubtedly occupied by Safranin I find.
[
If you find your aqueous stains are having trouble penetrating some tissue or areas within, a little OH can often remove certain hydrophobic molecules and help with staining.. Rehydrate the tissue after the OH wash and before staining for best results.
]

Good luck! :)
John B

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Re: Aloe-leaf epidermal peel - stomata

#12 Post by rnabholz » Mon Jan 11, 2016 12:26 am

My Toluidine-blue is on the way too.

Thanks for the direction John.

Rod

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Re: Aloe-leaf epidermal peel - stomata

#13 Post by mrsonchus » Mon Jan 11, 2016 1:04 am

You'll be pleased with it for certain chaps, fun is coming your way! :)

You've probably seen these from my earlier posts re hand-sectioning etc but I thought I'd show what Methylene-blue and Safranin can do with live tissue (used separately that is, not combined)..

Here's a hand-sectioned, Methylene-blue stained vascular bundle, epidermis, cortex and parenchyma-cells;
ws_stem_section.jpg
ws_stem_section.jpg (273.81 KiB) Viewed 3480 times
Here's the hand-sectioned central vascular-cylinder from a grass-stem stained with Safranin - shows several different kinds of vessel-wall structures..
ws_x40_grass_vascular_stem_.jpg
ws_x40_grass_vascular_stem_.jpg (125.25 KiB) Viewed 3480 times
This is a hand-sectioned (chrysanthemum-stem I think...) vascular bundle tri-stained with Crystal-violet, Safranin and I think Fast-green (although I'm not certain of that one.. :oops: )
ws_x40_hand_sec_daf_ped_VB_.jpg
ws_x40_hand_sec_daf_ped_VB_.jpg (287.15 KiB) Viewed 3480 times
Finally here's a hand-sectioned stem rather brutally overstained by me with Toluidine-blue, when I was somewhat 'less experienced... :oops: ' than I (hopefully) am now - subtlety seems to have eluded me here! Some metachromasia though! :D
ws_hand_TBO_stained.jpg
ws_hand_TBO_stained.jpg (252.01 KiB) Viewed 3480 times
Apologies if you've seen these before, just though I'd put them up in case they're new to you.

All were done during my early-days and were just a case of the simple protocol mentioned earlier - a little practice and you're off and running! :D
Staining is a black-art - and superbly rewarding when it goes well - as it always does after a little practice! :D
Good luck chaps! :)
John B

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Re: Aloe-leaf epidermal peel - stomata

#14 Post by vasselle » Mon Jan 11, 2016 3:35 pm

Bonjour
Les photos sont très belles et très bien expliquer.
Merci pour le partage
Cordialement seb
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