Pursuit of detail - pollen revisited - stain-attack!

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mrsonchus
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Pursuit of detail - pollen revisited - stain-attack!

#1 Post by mrsonchus » Sun Jul 10, 2016 5:27 pm

Hi all, I've been sectioning a new batch of Sonchus.oleraceus flower-heads and florets etc therein with a view to improving on last year's results, given how much I've learned since then both with technique and knowledge of staining etc.
Anyway, you may have seen my earlier post containing pollen-stacked to show the 3D nature of the complex grains of the Sonchus genus.

Here's a 15µ section stained with Safranin and Fast-green to differentiate between the spiny outer 'exine' and the inner-wall known as the 'intine'. Here the intine has been stained by the red Safranin while the exine has been nicely picked-out by the fast-green - a pleasing result which enabled a good impression of the 3D shape of the grains to be approximated.

The staining and the thickness (15µ) are (all being well with technique) the main factors enabling this view - a whole grain is about 32µ across, and a 15µ section, when it 'lands just right' during sectioning, will maximize the chance of a full 'half-grain' section, as seen here. It's just a matter of taking multiple sections and searching for those that are 'just right' - here I had good luck!
ws_pollen_stacked_1.jpg
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Now, when we switch to a far thinner section of 4µ things start to go more-or-less 2D, which is very valuable for fine detail while being completely different from the 'morphological thickness' of the above 15µ section...
I find for this pollen with it's spiny and brittle exine that 4µ is optimal; thinner may not yield significantly more detail but is far harder to make work, as the exine tends to shatter into fragments rather than present as true sections, so a practical limit at this time of 4µ for the spiny Sonchus pollen I think...
This slide has been stained not with 'Fast-green' as commonly seen and indeed preferred by me in combination with Safranin, but with my newly-mixed and first-time used 'Light-green' stain which like Fast-green is an 'acid stain' and targets cytoplasm preferentially rather than nuclei and lignin (the job of Safranin). It seems a little 'bluer' than Fast-green and is clearly capable of great subtlety and therefore accuracy I think,
ws_cleaned_IMG_5088.jpg
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The very pleasing thing about these sections as stained above is the visibility for the first time (I didn't manage to do this last year) of the nuclei within the grains - here it seems that the Sonchus has a tri-nucleate grain. You may remember an earlier post of mine using the softer Lily pollen that also showed nuclei, the Lily in that case had 2 nuclei as I remember. Anyway, the number of nuclei within pollen grains at varying times in their development is a good taxonomic character to study if you're interested like me in the similarities and differences of similar species....
Here's a closer look at those nuclei, the stains have made a great job of differentiating here...
ws_pollen_labelled_1.jpg
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After reading billben74's account of the use of the troublesome 'Orange-G' stain I thought I'd give it a try too, the results aren't too bad really as the stain took pretty well and did give a great subtlety also... It is a voracious stain that very quickly displaces other stains such as in this case Safranin that have already been applied during attempts to multi-stain as with the Safranin and Fast-green combinations above.
Here the Orange-G has fortunately 'left alone' the Safranin staining the big (seen red) 'tube nucleus' (destined to become the pollen-tube), here (it may actually be a nucleolus, must check) allowing differentiation - no sign though of the two smaller curved 'generative nuclei' (destined to become sperm-nuclei) visible using the Light-green with Safranin...
ws_pollen_orange_g.jpg
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Having got a little overexcited I then added Light-green to the staining protocol for an attempted tri-stain - always worth experimenting given the time...
Apologies for the image 'muckyness' - I was using a grubby Meiji projection eyepiece for this one - I just can't seem to get it clean! Anyway, the result has differentiated into 3 stains thankfully - the Light-green has stained the spiky exine 'fins' nicely, the Orange-G has stained the intine and other tissue of the containing anther-tube while the Safranin has manage to pick out the nuclear component! I pleased with this as a first attempt, and obviously intend, once I've cut some more suitable sections, to continue the quest for detail from pollen sections, one of my favourite topics! :D :D
ws_pstack1.jpg
ws_pstack1.jpg (155.42 KiB) Viewed 5717 times
These pictures are from 'wet' slides - the mountant is nowhere-near it's optical and physical best yet - maybe in a week I will retake a new set of pictures...
So far so good! The following post, as I've used my 5 pictures here, has a couple of extra pictures i thought you may like to see of the surrounding tissue and stigmatic structures therein..
Last edited by mrsonchus on Wed Jul 13, 2016 10:46 pm, edited 3 times in total.
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#2 Post by McConkey » Sun Jul 10, 2016 5:37 pm

John, I love reading your posts and seeing your journey unfold!! Your getting some really awesome results and the detailed descriptions of what's going on is a huge help! I'm not even close to the slicing and staining of my own specimens yet but I know when I get there I'll have your posts to look back on and use as guidance!

Keep up the great work!
Karl
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Re: Pursuit of detail - pollen revisited - stain-attack!

#3 Post by mrsonchus » Sun Jul 10, 2016 5:41 pm

Hi again, here are a couple more from the above staining adventures...
This is the result of washing out (i.e. differentiating) the Light-green stain, helped with a little acidified OH - the stain has become very subtle and has very nice clarity and accuracy - I'll use this in the fiture I'm sure - more notes for my 'staining-book'! Such great fun! :D :D
This shows one 'arm' of the bifid stigma of the Sonchus, as it emerges from the 'anther-tube' that forms the fascinating 'pollen-pump' apparatus covered in earlier posts - the 'brushes' that push the pollen grains up and out are on the left surface, the right surface is the papillate pollen-receptive stigmatic surface that is exposed as the stigma emerges and opens ready to receive pollen-grains..
ws_IMG_5112.jpg
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Given the 'Orange-G treatment' wonderful detail revealed itself, much to my delight! :D :D
One stigma arm has been caught in LS and the other (of the pair) is seen as a TS tip section - very lucky! The surfaces described above are clearly defined, as are the pigment grains that give the anther-tube top its dark appearance in the living floret - fascinatin. There's even a hint of vasculature running up the middle of the LS arm - a great result and very encouraging for me to persue Orange-G as a very valuable addition to my ever-growing stain-kit!
ws_stigma_tips_orange-G.jpg
ws_stigma_tips_orange-G.jpg (216.64 KiB) Viewed 5713 times
Having great time in the lab but I'm watching football on TV tonight! I hope you like the adventures! :D :D
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#4 Post by mrsonchus » Sun Jul 10, 2016 6:18 pm

McConkey wrote:John, I love reading your posts and seeing your journey unfold!! Your getting some really awesome results and the detailed descriptions of what's going on is a huge help! I'm not even close to the slicing and staining of my own specimens yet but I know when I get there I'll have your posts to look back on and use as guidance!

Keep up the great work!
Many thanks Karl - it's an amazing pursuit for sure! When you start on the road to your own sections, we'll all be here to keep you company and maybe even give some help if you like! :D :D
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#5 Post by JimT » Sun Jul 10, 2016 7:35 pm

Very well done! Enjoy the game.

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Re: Pursuit of detail - pollen revisited - stain-attack!

#6 Post by zzffnn » Sun Jul 10, 2016 7:44 pm

Impressive work, John B. Indeed, you are squeezing out even more details there.

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Re: Pursuit of detail - pollen revisited - stain-attack!

#7 Post by billbillt » Sun Jul 10, 2016 8:13 pm

Hello John,

I was once again astonished to view this wonderful chapter in the travels you are taking all of us!.. I also like the "orange-g" stain, as I think it gives such pleasing colors.. Am looking forward to the next "chapter"!...

Warmly,
BillT

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Re: Pursuit of detail - pollen revisited - stain-attack!

#8 Post by gekko » Sun Jul 10, 2016 9:22 pm

As always, fantastic work (all steps including photomicrogrpahy).

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Re: Pursuit of detail - pollen revisited - stain-attack!

#9 Post by mrsonchus » Sun Jul 10, 2016 10:58 pm

Hi fellows and thanks for your kind comments, great to know you like the adventure. I'm always working on ways to improve and explore techniques and love every minute of it! :D So many approaches, combinations etc to try - luxury.

I've a whole set of 10 wax-blocks cast from the series of which these slides are part - these are literally 'a chip off the block' and while they are from Sonchus flower-heads bearing many florets each, they are the start of the block - the whole-florets are yet to be reached, sections of which will contain not only the flowers and pollen as here but the ovaries and cypselas upon which they sit!

Interesting times ahead as I'm able this year to apply so much more knowledge and experience to the work, can't wait to apply a range of section thicknesses, stains and stain-combinations and hopefully some very high-res cellular-level investigations also. If I get the chance I'll have a go at growing some pollen-tubes also, slides showing the actual sperm nuclei (the curved ones) on their way down a pollen-tube would be great to see! So many things to do! :D :D

Oh yes, I fully intend to pursue the sectioning of those fascinating thallose liverworts (Marchantia) from my other thread also - I had a quick peek at the undersides of the archegonia today and found some nice green fully-formed sporopytes, little 'bags' full of the spores and elaters also seen in my earlier post - fascinating indeed! :D

Thanks again chaps for your company and input. :)
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#10 Post by McConkey » Mon Jul 11, 2016 1:48 am

mrsonchus wrote: I've a whole set of 10 wax-blocks cast from the series of which these slides are part...
Do you have a post which describes the process you took to make these blocks? I took a quick look but couldn't find anything. Thanks!

Looking forward to seeing your posts for when you reach the treasure trove of the block!
Karl
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Re: Pursuit of detail - pollen revisited - stain-attack!

#11 Post by c-krebs » Mon Jul 11, 2016 2:19 am

John,

I'm new here but it sounds like you are having way too much fun at this. (And to think I thought all this subject preparation would be burdensome ;) )

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Re: Pursuit of detail - pollen revisited - stain-attack!

#12 Post by mrsonchus » Mon Jul 11, 2016 11:21 am

c-krebs wrote:John,

I'm new here but it sounds like you are having way too much fun at this. (And to think I thought all this subject preparation would be burdensome ;) )
Hi Charles, you're spot-on there old chap. The 'process' is every bit as stimulating and enjoyable as the end results in my personal experience.
It's a case of the more you put in the more you get out in this field - I absolutely love the pursuit of Botany and am not limited to the microscopic either. I collect herbarium specimens and also a 'wet collection' not to mention the photographic! :D :D
I'm simply fascinated by the variety and in particular the oft-quoted 'form vs function' relationship.

Very recently for example I simply 'pulled up' a clump of 'green-stuff' spotted while out and upon bringing it home and examining it, together with a little time with my extensive collection of tomes, discovered it was a 'thallose liverwort' and further that it is of the species 'Marchantia' - fascinating. Another direction in which to take the preparation, sectioning and mounting of this specimen awaits!

Having a fantastic time, learning every day and all in the company of this friendly forum full of superbly helpful and supportive folk - many of whom were with me from my first day with my old Swift Collegiate 400 'scope and my first tentative steps with the late great Walter Dioni (see his fantastic legacy articles), and continue to be so to this day.
It's all good as you will surely find the longer you spend with this fine forum and it's similarly fine folk. :)
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#13 Post by mrsonchus » Mon Jul 11, 2016 7:47 pm

McConkey wrote:
mrsonchus wrote: I've a whole set of 10 wax-blocks cast from the series of which these slides are part...
Do you have a post which describes the process you took to make these blocks? I took a quick look but couldn't find anything. Thanks!

Looking forward to seeing your posts for when you reach the treasure trove of the block!
Hi Karl, you really must have missed something old chap - I've dozens of posts here that cover all aspects of the process, which is long and multi-stage - I haven't the time now to relate the whole thing again I'm afraid my friend - try another search - it's all in this forum's post records to find... :)

p.s. - after searching for the keyword 'batch' in the section 'specimens, samples & slides', with myself as the author I found all the posts you will need - here's one to go to - viewtopic.php?f=10&t=1981&hilit=batch
I hope this helps. :)
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#14 Post by McConkey » Mon Jul 11, 2016 9:18 pm

Searching by Author! Why i didn't think of that is something we will never know! Really do appreciate you throwing me that link! I'll dive into all your older posts later this evening!

I'll admit i'm a little bit disappointed you didn't re-type every single step you've taken over the last year :lol: :lol: but thats ok!

Thanks again!
Karl
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Re: Pursuit of detail - pollen revisited - stain-attack!

#15 Post by mrsonchus » Mon Jul 11, 2016 10:19 pm

Go get 'em Karl - it's a fantastic journey if you stay with it my friend - always ready to pass on what I've learned here in this fine forum old chap - anytime. :)
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#16 Post by mrsonchus » Mon Jul 11, 2016 10:38 pm

McConkey wrote:Searching by Author! Why i didn't think of that is something we will never know! Really do appreciate you throwing me that link! I'll dive into all your older posts later this evening!

I'll admit i'm a little bit disappointed you didn't re-type every single step you've taken over the last year :lol: :lol: but thats ok!

Thanks again!
Oh I've just had a thought that may help,
I've been using a little free program for ages now (at least a couple of years) called 'BullZip PDF printer' - it is a virtual printer that instead of printing to a real (i.e. hardware) printer when selected as a printer to 'print to' this will produce a PDF file of what you selected to print - it even has a 'simplify page' option that cuts off the 'trimmings' all around the 'meat' of a lot of internet content.
If for example you were to go to say my thread pointed to earlier you could, with a RH 'click' and 'print' then select the 'BullZip PDF printer' as the target printer, produce a fine PDF (save this on your desktop perhaps) of the entire thread, posts, pictures and all! It works (on my Windows 7 Home Ed machine) perfectly. I now use it constantly for all manner of things, even batch printing of pictures into one PDF file of all of them - so very handy.

In fact - I've just done exactly as I've just written and uploaded a PDF of the thread mentioned onto a shared place on my G-Drive - here's the link to the whole thread as a PDF...
https://drive.google.com/open?id=0B-LIc ... DJUdWpIcmc

I hope it helps a bit. :)
John B

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Re: Pursuit of detail - pollen revisited - stain-attack!

#17 Post by vasselle » Tue Jul 12, 2016 3:58 pm

Bonjour
Très jolies séries et comme toujours très bien commenter
Merci pour le partage
Cordialement seb
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Re: Pursuit of detail - pollen revisited - stain-attack!

#18 Post by billben74 » Tue Jul 12, 2016 4:18 pm

Lovely work John.
Really liked the triple nuclei on the pollen -> more of your excellent results of really getting down to the biology.

And I can only second mrsonchus enjoyment of all things histological. Its such fun taking a sample through all the various steps to slide.

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Re: Pursuit of detail - pollen revisited - stain-attack!

#19 Post by Crater Eddie » Wed Jul 13, 2016 12:15 pm

Thanks for the mention of that PDF printer program, I have needed something like that many times. I'm off to go download it.
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Re: Pursuit of detail - pollen revisited - stain-attack!

#20 Post by McConkey » Wed Jul 13, 2016 2:08 pm

I agree with Crate Eddie, that PDF program is an excellent resource!
Karl
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Re: Pursuit of detail - pollen revisited - stain-attack!

#21 Post by billbillt » Wed Jul 13, 2016 3:40 pm

Thanks John for the .pdf... Downloading now..

BillT

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Re: Pursuit of detail - pollen revisited - stain-attack!

#22 Post by mrsonchus » Wed Jul 13, 2016 10:20 pm

You're welcome chaps - it's a ridiculously useful little utility as it's usable for anything that may be 'printed'.

p.s. I've been staining and of course mounting some comparisons again of pollen grains, I've Lily pollen sectioned at 3µ and bi-stained with basic-Fuchsine for the intine and Light-green for the exine, and tonight I've mounted some 10µ and some 3µ Sonchus pollen this time stained with the similar (in terms of colour that is) combination of Safranin and Fast-green - an interesting set for comparison!

At first glance the results look very nice, with clearly differentiated nuclei in both pollens at 3µ, and superb exine structure in the more 3D version afforded by the 10µ sections of the Sonchus grains - also fascinating as the 3µ are demonstrably 1 cell thick (the 'surrounding' floret-parts that is, that contain the pollen grains) while the 10µ are very clearly (as the two layers focus very clearly and separately - almost like having '2 sections for the price of one') exactly two layers thick - quite amazing to see! :D :)
Amazing to see the different views afforded by different section thicknesses with various tissue-types. If I get a chance tomorrow I'll post a few pictures of the comparison.
:)
John B

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