A Few Colourful Stains Compared

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mrsonchus
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A Few Colourful Stains Compared

#1 Post by mrsonchus » Sat Sep 19, 2015 6:00 am

Hi all, I've been trying to organize some of my material and found a few nice staining-test pictures that you may like a look at. I've been dabbling with stains for some time now, with the added and parallel problem of section-integrity that was, pre-Shandon days, causing something of a hiatus in my progress - recently I've been able to get some nice results with the 2-stain procedure using Fast-Green - a very well-known stain that is great for plant tissue, and the equally well-known and suitable Safranin, also excellent with plant tissue.
Before this I got to grips with TBO - 'Toluidine Blue' - a metachromatic (i.e. it gives a series of colours different to the colour it has when you apply it) stain that gives excellent tissue differentiation to just about everything I use it for! TBO when used on living tissue (plant) is a thing of absolute beauty..
Anyway, here are a few colourful pictures, some I may have posted before, but the Safranin/Fast-Green combination of stain and counterstain is a new one that I'm starting to see some pretty nice results with;

This is Fuchsine,
ws_700x525_x20_basic_fuchsine_stack_carex.jpg
ws_700x525_x20_basic_fuchsine_stack_carex.jpg (135.79 KiB) Viewed 7079 times
Methylene Blue (also metachromatic),
ws_700x525_S03_s_asper_stem-0007.jpg
ws_700x525_S03_s_asper_stem-0007.jpg (171.24 KiB) Viewed 7079 times
Now, the above are certainly colourful - but not very subtle, to say the least! Things started to improve quickly when I started trying TBO, much better definition and of course the metachromasia gives very nice differentiation to the tissues,
(coupled with better technique to be fair, most of my 'test-runs' start out with the aim of simply getting the stain to actually stick to the tissues!) - subtlety doesn't come easily - very often my best results are as much by luck as judgement!
Here's an improvement brought about by the superb TBO stain,
ws_x20_SA20_8mic_1.jpg
ws_x20_SA20_8mic_1.jpg (223.65 KiB) Viewed 7079 times
Here's my favourite so far, the combination of first applying Safranin, which will stain everything pink until it is 'differentiated' with careful OH rinse/s (this removes the stain gradually, but at different rates from different tissue-types within the section, if judged correctly the vascular bundles for example can be left stained whilst the rest is removed, ready for another stain to be applied, in this case Fast-Green). The ability to selectively and gradually remove the stain after effectively 'overstaining' with it makes this stain (Safranin) 'regressive' - i.e. - overstain->remove (differentiate) until desired amount remains... Next a 'progressive' stain is added (in this case Fast-Green - so-called because, well, it's fast! About 10 seconds does the trick) that has to 'be added a bit at a time' up to the correct/desired level of staining, it cannot be selectively removed in the same way as the Safranin was. Together the aim of this 2-part double-staining combination is to stain, in this case, vascular bundles with (pink/red) Safranin and cytoplasm with the Fast-Green - other elements will also be stained by these stains, for example the nuclei will be stained by Safranin, and Fast-Green can vary between green and bluish-tones...
ws_700x525_ws_quick_pic-0008.jpg
ws_700x525_ws_quick_pic-0008.jpg (104.17 KiB) Viewed 7079 times
I thought the TBO was the best until I finally got the hang of the Safranin/Fast-Green combination - a large amount of information, not to mention pleasing colour, has been revealed!

Finally, here's a picture of TBO doing what I think, up to now that is, it does best of all - the staining of live/fresh tissue - this picture is of the papillae of a plant-stigma that has part of (the annulus) a fern-sporangium (Hart's-tongue fern I suspect) attached! The TBO has picked out the detail beautifully and quite delicately...
ws_700x525_qsave-15-07-23-20-36-32.jpg
ws_700x525_qsave-15-07-23-20-36-32.jpg (71.72 KiB) Viewed 7079 times
Anyway, better get to bed, just thought I'd post a few pictures as I'm a bit busy at the moment with various aspects but have little ready to post yet - more soon though when I get a chance to put together a more helpful and organized post or two. Hope you like the pictures. :)
Last edited by mrsonchus on Sun Sep 20, 2015 10:48 pm, edited 1 time in total.
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Re: A Few Colourful Stains Compared

#2 Post by mrsonchus » Sat Sep 19, 2015 6:20 am

Here are a couple more pictures of the Safranin-Fast-Green combination showing quite a lot of detail/information;
ws_wed_2.jpg
ws_wed_2.jpg (235.48 KiB) Viewed 7077 times
ws_700x525_carex_saf_fgreen-0028.jpg
ws_700x525_carex_saf_fgreen-0028.jpg (94.71 KiB) Viewed 7077 times
ws_700x525_ws_wed_4.jpg
ws_700x525_ws_wed_4.jpg (67.05 KiB) Viewed 7077 times
ws_700x525_ws_x60_carex.jpg
ws_700x525_ws_x60_carex.jpg (112.13 KiB) Viewed 7077 times
Finally, the stigma seen earlier, with tetrads of pollen attached (Epilobium), stained live with TBO.
ws_700x525_qsave-15-07-23-20-50-48.jpg
ws_700x525_qsave-15-07-23-20-50-48.jpg (75.06 KiB) Viewed 7077 times
Back soon, hope you like these. :)
John B

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Re: A Few Colourful Stains Compared

#3 Post by JimT » Sat Sep 19, 2015 3:42 pm

What is the orange and magenta coloured arc in the fifth image? Interesting "Thing".

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Re: A Few Colourful Stains Compared

#4 Post by mrsonchus » Sat Sep 19, 2015 6:35 pm

JimT wrote:What is the orange and magenta coloured arc in the fifth image? Interesting "Thing".
Hi Jim, It looks like part of a spore-case from a fern, and I've a lot of the common 'Hart's Tongue' fern around my garden - I suspect it's from those.... The tiny chambers seen on the undersides of fern-leaves when they are producing spores contain these pods, each of which 'pings open' when the ribbed annulus contracts, and spores are flung everywhere! Here's a few pictures taken from mine a while back;
ws_spore_case-(2).jpg
ws_spore_case-(2).jpg (67.66 KiB) Viewed 7064 times
ws_empty-spore-case.jpg
ws_empty-spore-case.jpg (53.96 KiB) Viewed 7064 times
ws_spore-case-1.jpg
ws_spore-case-1.jpg (47.69 KiB) Viewed 7064 times
I'm pretty sure it's a stray one of those...
John B

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Re: A Few Colourful Stains Compared

#5 Post by gekko » Sat Sep 19, 2015 8:56 pm

Very interesting! And, as usual, beautiful staining and photos that does it full justice.

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Re: A Few Colourful Stains Compared

#6 Post by Dennis » Sun Sep 20, 2015 2:21 am

Geez a few of those you could be selling prints!

-Dennis

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Re: A Few Colourful Stains Compared

#7 Post by mrsonchus » Sun Sep 20, 2015 2:39 am

Thanks Gekko & Dennis, glad you like them! :)
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Re: A Few Colourful Stains Compared

#8 Post by vasselle » Sun Sep 20, 2015 5:01 am

Bonjour
Très bien documenté
Merci pour le partage
Cordialement seb
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Re: A Few Colourful Stains Compared

#9 Post by mrsonchus » Sun Sep 20, 2015 2:21 pm

Thanks Seb, it's really nice to know folk like them. :D
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Re: A Few Colourful Stains Compared

#10 Post by JimT » Sun Sep 20, 2015 3:04 pm

Thanks for the feedback and additional images. Interesting stuff.

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Re: A Few Colourful Stains Compared

#11 Post by charlie g » Sun Sep 20, 2015 3:07 pm

Beautiful work, one can study your sections for their beauty, Mr.sonchas. The structures are fascinating, thanks for shareing your lab activities, JohnB! charlie guevara.

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Re: A Few Colourful Stains Compared

#12 Post by apochronaut » Sun Sep 20, 2015 7:32 pm

This really exciting work. John , practical as well as beautiful.
I have a bacteriology textbook from 1912, that I picked up at a junk shop a number of years back. It is almost unbelievable how much knowledge had been gained about the nature and nurture of bacteria up to that time, much of which had been gained through the practice of staining. There already had been exhaustive work done in species differentiation and extensive data had been kept and shared, logging the visual aspects of various stains and stain complexes for that purpose.

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Re: A Few Colourful Stains Compared

#13 Post by mrsonchus » Sun Sep 20, 2015 10:43 pm

Thank you Charlie and apo' - you're very generous.
The old tomes are still very relevant I find, I've several, one's about 120 years old, and is still relevant and useful - simply put, the cutting-edge of those pioneering days are about the same level of facility I for example have at home in my 'lab' come dining-room. No automatic tissue processors, embedding station, incubator etc - just me, the basics, sometimes very Heath-Robinson but equally effective, and a lot of effort! The huge advantage we have today is that even as pure amateurs we're able to purchase just about anything needed for a reasonably affordable price...
This superb forum, full of very friendly and knowledgeable folk is a huge help to me for certain, and more or less everyone else I'm certain.

The spirit of exploration lives on with the amateur in his/her dining-room-cum-laboratory! :D Hussar! :D

Thanks again both, for your kind praise and encouragement. :)
John B

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Re: A Few Colourful Stains Compared

#14 Post by mrsonchus » Mon Sep 21, 2015 1:26 am

Hi all, found a few more colourful pictures, here's a better view of the earlier seen Epilobium-pollen-tetrads in TBO live - the grain shapes are a lot clearer in this one I think...
ws_700x525_qsave-15-07-23-20-40-30.jpg
ws_700x525_qsave-15-07-23-20-40-30.jpg (49.57 KiB) Viewed 7032 times
Here's the 'neck' of the stigma that the pollen was all over, nice colours and very visible vascular elements going to each of the 4 lobes of the stigma;
ws_700x525_epilobium_stigma.jpg
ws_700x525_epilobium_stigma.jpg (96.65 KiB) Viewed 7032 times
This one shows a side-view of the xylem-pits seen in the root pictures, although this is of the vessels within a (LS) stem if I remember correctly.
ws_700x525_quick_pic-0007.jpg
ws_700x525_quick_pic-0007.jpg (87.48 KiB) Viewed 7032 times
This is the 2-part aqueous Safranin and Fast-Green in cellosolve (stains in the presence of OH not water) kit used;
ws_700x525_DSCN2198.jpg
ws_700x525_DSCN2198.jpg (79.5 KiB) Viewed 7032 times
Finally here's a reminder of just how amazingly beautiful the natural (unstained) colours of a section cut with a hand-microtome
can be when the section is simply put into some water/glycerin and straight onto the 'scope... This is a chrysanthemum-stem XS from a bunch of flowers in our front-room, from back in March...
websize_vasc_bundle_stack_4.jpg
websize_vasc_bundle_stack_4.jpg (144.69 KiB) Viewed 7032 times
Anyway, to bed and colourful dreams for me!

Back soon. :D
John B

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Re: A Few Colourful Stains Compared

#15 Post by mrsonchus » Thu Sep 24, 2015 12:35 am

Here are a few more recent slides of various stains;
Pits in the xylem cell-walls in LS;
ws_700x525_edited_S3_308_2.jpg
ws_700x525_edited_S3_308_2.jpg (137.59 KiB) Viewed 7023 times
Safranin is able to stain metachromatically if applied for long enough then differentiated a little;
ws_700x525_s_asper_stitch (2).jpg
ws_700x525_s_asper_stitch (2).jpg (140.36 KiB) Viewed 7023 times
Some nice blue-stained nuclei in this one;
ws_700x525_x60_LS_xylem.jpg
ws_700x525_x60_LS_xylem.jpg (103.39 KiB) Viewed 7023 times
Here are a couple I forgot to post, the phloem sieve-plates are visible here, they lie across the ends of the phloem elements (cells) between cells comprising the phloem (food transport tube as opposed to the xylem which carries water and has no walls between cells but pits on the sides of the cells as seen in earlier pictures stained blue). The phloem in the pictures are the long cells stained purple, the xylem cells (blue) are dead when mature and the phloem cells are alive (and have supporting or 'companion cells' to supply them with their needs)...
ws_700x525_edited_S3_308_3.jpg
ws_700x525_edited_S3_308_3.jpg (133.25 KiB) Viewed 7023 times
ws_700x525_edited_S3_317_7.jpg
ws_700x525_edited_S3_317_7.jpg (99.33 KiB) Viewed 7023 times
Fascinating to see what the stains reveal! :D



Back soon
Last edited by mrsonchus on Tue Nov 29, 2016 11:56 pm, edited 2 times in total.
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Re: A Few Colourful Stains Compared

#16 Post by Raul » Thu Sep 24, 2015 1:37 pm

An almost unbelievable beauty. Thank you for showing these images. Keep up the good work.

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Re: A Few Colourful Stains Compared

#17 Post by billbillt » Thu Sep 24, 2015 5:31 pm

WONDERFUL!!...

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Re: A Few Colourful Stains Compared

#18 Post by Yvan » Mon Oct 24, 2016 9:42 pm

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Re: A Few Colourful Stains Compared

#19 Post by mrsonchus » Mon Oct 24, 2016 10:01 pm

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Last edited by mrsonchus on Mon Nov 28, 2016 12:14 am, edited 1 time in total.
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Re: A Few Colourful Stains Compared

#20 Post by billben74 » Tue Oct 25, 2016 8:58 pm

Fantastic, we have a histologist in the house.
Top tips. I've been wanting a picric acid substitute for some time. I almost tried to get some, but then saw reason...
Zinc chloride will be obtained shortly.
Along with astra blue. I use fast green, but I'm happy to try.
I get that the solution you mention acts to diff as well as stain
I assume you like the colour of astra blue more than fast green? Or is there any other advantage (other than the diff usage).

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Re: A Few Colourful Stains Compared

#21 Post by kit1980 » Wed Oct 26, 2016 7:44 am

Very interesting and informative, thanks for posting!
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Re: A Few Colourful Stains Compared

#22 Post by Yvan » Sat Oct 29, 2016 6:12 pm

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Last edited by Yvan on Sun Dec 04, 2016 3:52 pm, edited 1 time in total.

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Re: A Few Colourful Stains Compared

#23 Post by billben74 » Sat Oct 29, 2016 8:05 pm

First of all - I've never seen phloem sieve elements - more outstanding work from the botanical section master mrsonchus.
And your photographic skill with brightfield is amazing.


Yvan fantastic to hear from someone who has years of professional experience.
I have some phosphotungustic acid so I will try to get hold of some astra blue and give it a whirl.
Can you use dichromate (Cr207) instead of chromate CrO3? (carefully ;)

I have some interest in doing some zoological sections / staining (I had a failed attempt at mallory's trichrome) so I may pm you about this in the future if thats ok?

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Re: A Few Colourful Stains Compared

#24 Post by Yvan » Mon Oct 31, 2016 9:05 am

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Re: A Few Colourful Stains Compared

#25 Post by zzffnn » Sun Nov 27, 2016 2:44 pm

Yvan wrote:I carefully reviewed the pictures, old chap.
Nice slides from a beginning microtomist, but you're not there yet.
All pictures show sectioning artefacts, but it's especially noticeable in the pictures "ws_x20_SA20_8mic_1.jpg" where the outer cell layer has dissapeared (left side) and the collenchym is strongly distorted.
It's also very noticeable in the higher power view "ws_700x525_quick_pic-0007.jpg" where it's hard to find cell wals that aren't distorted. The same goes for "ws_700x525_edited_S3_308_3.jpg".
On staining technique you're not there yet either. The staining techniques you performed lacks intensity and selectivity.
My concludion: very promising first results from a no doubt gifted upcomming microtechnician, but not up to par yet, dear friend.
Yvan,

We respectfully invite you to post some slide images that are of much better quality, from your beginning years. Note those images of JB were posted in more than a year ago, when JB was still not quite experienced, yet. He has since done many more experiments and improvements, that others have seen. At the time, he was at less than 2 months of receiving his new rotary microtome.

You said before that you made thousands of slides yourself and only like less than 100 of those - maybe you can show us one of those 100 best, and compare that with a comparable slide from your early years? I doubt you were always "there", from the beginning.

Also, depending on the microtomist's primary goal, not all structures may be present and not all stains may be intense. For example, if you want to slide a tissue thin enough for 100x NA 1.4 objective to look THROUGH, then not all curved tissues would be intact/present and color inensity may need to be tuned down. I have asked JB to make such ultra high NA slides for me and ignore some integrity / intensity.

Further, some people, such as JB, enjoy sharing/learning and do not mind posting less - than - perfect images. Such people, having a high ego, would rather not post anything that is not perfect.

It is not quite polite to call on people and say " you are not there yet". If one always compare to the next level, very few would be "there" yet. A less experienced microtomist would be less "there" than an experienced one. Your "there" may not be my "there". If I am perfectly " there" in my own mind, I would personally rather help others improve and provide very specific how-to step-by-step, instead of picking on what is not perfect and label "not up to par yet".

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Re: A Few Colourful Stains Compared

#26 Post by Yvan » Mon Nov 28, 2016 5:37 pm

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Last edited by Yvan on Sun Dec 04, 2016 3:54 pm, edited 1 time in total.

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Re: A Few Colourful Stains Compared

#27 Post by zzffnn » Mon Nov 28, 2016 6:18 pm

^ Your previous post was deleted because it was considered as personal attack by forum administrator and many forum members. Remember, when you point one finger towards someone else, three fingers are pointing back to yourself. Your rude wording only reflects poorly on yourself.

This would be my last post to respond to you - after which your post would be ignored - as other forum members have been doing.

In some cases, a microscopist may want to sacrifice some integrity of peripheral tissues to obtain see-through images of tissues such as pollen or chromosome strands, while keeping some (but not all) peripheral context.

It is a personal choice and depends on what you want to study/examine. When I purchased my pollen / flower head slides, I specifically told JB to ignore peripheral integrity, for example.

JB can easily make thicker morphology slides that preserve tissue integrity. Different horses for different courses.

JB's slides have been enjoyed by many forum members - in fact I have not heard one that does not like them, except for you. I consider his pricing very reasonable, considering his personalized service. Do you really think JB can get rich, or recover his time/equipment cost, by selling his slides at current pricing?

I "should be a fool" indeed, if I buy from you. After all, you have not even bothered to reply my polite request of buying slides from you, sent a month ago. How much time does it take you to type "I cannot sell those" or "I don't have them anymore", as reply? Do you know how much time and effort JB uses to communicate with his forum friends and buyers?

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Re: A Few Colourful Stains Compared

#28 Post by Dale » Mon Nov 28, 2016 7:23 pm

Wow, and I thought my unregulated lab heater got hot! I really hope neither of you
decide to leave the forum, your contributions are priceless.
Dale
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Re: A Few Colourful Stains Compared

#29 Post by Yvan » Mon Nov 28, 2016 7:25 pm

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Re: A Few Colourful Stains Compared

#30 Post by zzffnn » Tue Nov 29, 2016 1:19 am

I forgot to mention this, as some forum members already knew.

I was one of the forum members who asked JB to start selling his slides (JB was first hesitant to with the idea, but we pushed him into it). I know at least 5 US microscopists who wanted his slides and are willing to pay. So there was/is a demand. On the other hand, JB needs so more microscopy equipments. Our purchase could help him to pursue microscopy, which would also in turn generate more forum posts on botany for us to enjoy/learn.

That was why JB started his "business". I doubt such a small "business" even earns enough to cover equipment/reagent cost though. It is more of a way to share/pursue his passion, without going broke.

That is it from me. Let us move on.

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