Daff-Dismantled

[mrsonchus]

Hi all, found a moth-eaten bunch of Daffodils for sale in the 'bargain-bin' today for £0.40! Snapped them up, took them home, and had a rather awkward conversation with my darling Wife that began something like - (my Wife..) "Hmmmm...for me, you shouldn't have.." and progressed through to (me) "aren't they great - they're for sectioning..", followed by a rather protracted silence....

Anyway, my rather tragic-looking 'bunch' of Daffodils in hand and awaaaayyyy to the lab!!

Yes - it's Histology Time again! Hussar!

Pre-op, leaf-sections, pedicel (flower-stem) sections,

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and the really interesting parts - the reproductive structures

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Should be able to take some sections through the pollen-producing anthers, the receptive stigma
and hopefully the un-fertilized ovule within the immature ovary! Should be very interesting indeed!

While cutting those pieces for the fixative-stage that starts-off the whole process that ultimately ends with a wax-block containing the tissue, ready for microtomy, I took a few quick hand-sections and dropped a little Toluidine-blue ('TBO') stain onto them to bring-out a little detail...
These are I'm afraid very rough-and-ready, no coverslip etc just a big drop of water and a little stain. I had a 'wander' around the tissue and found a few interesting bits & pieces that I thought you'd like to have a peek at;
(I've doubled-up the pictures to fit them into this one post)

Here are a couple of pictures of the outer-'skin' (epidermis) of the flower-stalk (pedicel) atop which is the beautiful trumpet of the Daffodil in life. A living stomate or 'stomatal-pore' has been sectioned quite by fortunate accident and is quite interesting to see,

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The stain has rather nicely emphasized the epidermal cell-walls.

Here are a few views of some water-conducting vessels (xylem) in an 'unwound' state, not cross-sections rather more a 'pulling-apart' of the vessels lengthwise - again the stain has added considerable contrast as an aid to imaging..

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and

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In a few days I should have some nice Daffodil-sections to play with!!

Meanwhile I've been investigating the effect of alcoholic and (or) aqueous 1% acetic-acid (white-vinegar is all I'm using for the acid ) as a post-aqueous-staining differentiator and colour intensifier - it seems to work very well with Methylne-blue and with Safranin with sections of Sonchus-florets used for testing - I'll post a few of the very colourful results in a seperate thread for a quick perusal.

It's been a full month now since I suffered a ghastly complication whereby I had delivered by my usually totally reliable supplier, Brunel Microscopes the wrong-type of Histoclear clearing-agent, which consequently led to my needing to throw away all my histology mixtures both Histoclear and Histoclear/alcohol blends and replace them, together with the loss of some 2-dozen slides - the Histoclear (turned out to be vII - supplied to them also in error they said..) they supplied reacted badly with the mountant I always use - (supplied always by BM!) with disastrous results .
Anyway, in the meantime I ordered a (US) gallon of Histoclear vIII elsewhere (and it wasn't cheap!) which was desribed as "compatible with all synthetic resinous mountants- unlike vII" - the manufacturterer's own description, not mine.. Well, the fun continued, while waiting for Brunel Microscopes to send replacements for their original mistake (and they took their time as they were 'waiting for a delivery') I started to use the newly-purchased and much-celebrated 'Histoclear III' - you guessed it - also incompatible with my favourite mountant (the superbly optically-precise 'Numount' synthetic resin) and another horrendous mess, loss of reagents and yet more sections, only about 4 this time though..
Soooo - the Histoclear III from my other supplier (AGTC) has now been replaced by them with my original Histoclear I and everything's now, finally, running smoothly again!!! . In the meantime the replacement bottles from Brunel Microscopes also finally arrived - phew, I've had a rather trying Christmas & New-Year period as far as lab-work is concerned. Anyway all's finally well and going along nicely again.

Back soon with some more pictures.

[75RR]

Nice images.

... not cross-sections rather more a 'pulling-apart' of the vessels lengthwise ...

messy cut ? :)

[JimT]

Nice. Looking forward to more

[mrsonchus]

Nice images.

... not cross-sections rather more a 'pulling-apart' of the vessels lengthwise ...

messy cut ?

Very! It's always great to 'poke around' the edges and mucky-bits of hand-cut materials, you never know what perspective or surprise parts you may find!

[mrsonchus]

Thanks Jim, more on the way.

[charlie g]

Beautiful 'hand sections of live material', John B. Do you use a simple hand held razor...or an actual hand microtome device for your cuts? I ask as the stomata in epidermis section really kept it's structure.

Will you do something tricky to keep pollen attached to the :ovary/stigma/stamen unit?

Does the vital stain you use...stain your fingers if you have a slip up? all the best, and thanks. Charlie guevara

[Crater Eddie]

Thanks for the "early on" pics! Its always interesting to see the zylem.
CE

[billbillt]

Another very interesting and complete post by the master!...

BillT

[rnabholz]

Popcorn popped, waiting for the show.

Thanks for taking the time. Doing it all is hard enough without remembering to stop and document it. I appreciate the effort!

Rod

[mrsonchus]

Beautiful 'hand sections of live material', John B. Do you use a simple hand held razor...or an actual hand microtome device for your cuts? I ask as the stomata in epidermis section really kept it's structure.

Will you do something tricky to keep pollen attached to the :ovary/stigma/stamen unit?

Does the vital stain you use...stain your fingers if you have a slip up? all the best, and thanks. Charlie guevara

Hi Charlie and thanks for your encouragement.

Now then,
I have a 'proper' bench-hand-microtome (see my posts re hand-sectioning with it - video too) but as I've become better equipped and more experienced I find that for a quick stem or leaf section I don't really need to use it anymore.
I now use 'used' disposable microtome-blades from the Mighty Shandon (there's one in a picture above) which may be sub-optimal for the rotary microtome cutting 5µ, but for hand sectioning is absolutely deadly! Also the fact that I cut them under the stereo 'scope is a huge advantage as I can see exactly what's happening as I make the cuts, and am able to achieve the vital perpendicularity relative to the axis of the tissue that a good section requires - not perfectly of course, but good enough.
A plain single-sided safety razor is perfectly suitable and I use these also - don't scrimp on the blades, use a new one every time you section. Blade sharpness I've found outweighs all other factors by at least an order of magnitude as regards section quality, be it rotary at 5µ or a hand-cut section at 100µ. the clean-cut of the stomate is due to the sharpest blade and a steady and uninterrupted cutting stroke. Always section by hand with 'wet' tissue and blade of course, alcohol or water etc, but liquid.

To keep the pollen in place may but very likely will not be a problem I find. Earlier sections I took of Sonchus florets whole actually have sectioned pollen-grains still within and upon the (longitudinally) sectioned anther-tubes that the Sonchus posseses. I was even able to stain the grain-exine differentially from the interior this way.. Many anthers produce a sticky substance called 'pollenkit' and I think this helps, as the pollen-grains are coated in it (it's the yellow 'waxy' coating of many pollens).
Can't resist a quick picture!

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and

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Apologies for the gratuitous pictures, just couldn't resist another look at them..

Having said that, I'll probably have a go at using the now reliable double-embedding technique used so effectively with my Sonchus cypselas a short while back - straight from FAA (where they are now in a cassette to keep the 'assembly' together) into a nice cosy doubly-embedding agar or agar/gelatin-overcoat!
I should be able to handle and orientate as desired individual anthers with this technique! Can't wait to get at them!

Ah, colourful hands - I don't really get much stain on my hands or even fingers as a rule, but a quick dip and wash with whatever solvent carries the stain (water, alcohol etc) followed by the usual 'dish-washing-up' liquid and warm water usually removes at least 90% of it.

Pleased you like it Charlie, keep your blade sharp and your fingers clean!

[mrsonchus]

Popcorn popped, waiting for the show.

Thanks for taking the time. Doing it all is hard enough without remembering to stop and document it. I appreciate the effort!

Rod

Thanks Rod, I love it! Great to know folk like it!

[mrsonchus]

Another very interesting and complete post by the master!...

BillT

Bill, thank you - very pleased you like them.

[mrsonchus]

Thanks for the "early on" pics! Its always interesting to see the zylem.
CE

Thanks Eddie, we're in for another grand adventure!
Ahhhh....the seductive smell of Histoclear and IPA, the faintly-stained fingers, the 600-ton pile of kitchen-tissues and fragments of wax! Sounds like heaven!

Strap yourselves into the lab-chairs chaps, we off again!

[KurtM]

Doing it all is hard enough without remembering to stop and document it. I appreciate the effort!

^^^ That silver-tongued devil has a way with words, and once again expresses my own thoughts more perfectly than I can myself. Simply wonderful stuff, mrsonchus! After diatoms, pollen particularly fascinates me.

[mrsonchus]

Doing it all is hard enough without remembering to stop and document it. I appreciate the effort!

^^^ That silver-tongued devil has a way with words, and once again expresses my own thoughts more perfectly than I can myself. Simply wonderful stuff, mrsonchus! After diatoms, pollen particularly fascinates me.

Haha Kurt - that's what I started with - pollen! You're on the event-horizon of the botanical-black-hole-of-wonder! Jump in Kurt! You'll never regret it!

It's available, it's easy to examine mounted or not, it's easy to permanently mount without any sectioning etc - great for mounting-practice! Also a collection of pollen slides is a beautiful thing, and the imaging of pollen never fails to please as every now-and-then a particularly fine image pops-out! Great interest to be had in the comparison of pollens from related species too!

Great luck to you!

[vasselle]

Bonjour
Très beau travail.
En plus les photos sont très belles.
Bonne continuation et merci pour le partage.
Cordialement seb

[gekko]

Beautiful images of the pollen!

[mrsonchus]

Beautiful images of the pollen!

Many thanks Gekko - pleased you like them!

[mrsonchus]

Hi all, very quick update - the Daffodil tissue has been in it's first wax-infiltration bath for several hours now (16:00 hrs here). Overnight tonight then 3-4 hrs tomorrow morning and they're ready to cast!
I think the anthers may be a problem, and it now appears that I've sliced the base of the ovary off....
Still, at the very least there's still some cracking leaf and stem tissue to come! I'll return to anthers and use the superb double-embedding technique to try to 'up the quality' very soon.

Exciting, I love the anticipation, 'histology is like a box of chocolates...'

Back soon with all-manner of goings on! Onward-ho!

p.s. still considering which orientation to use for the anthers and stigma, probably easier to go for LS as I've not doubly-embedded them (as I should have.. ).

[mrsonchus]

Bonjour
Très beau travail.
En plus les photos sont très belles.
Bonne continuation et merci pour le partage.
Cordialement seb

Thanks Seb, you're very generous - I'm so pleased folk like them, I love the botany and histology and it's great to have this forum in which we share our many adventures.

[mrsonchus]

There here! Cast 10, yes 10! Shiny-new wax-blocks containing a menu of Daffodil-parts just begging to be sectioned. The protocol-board is finished, the blocks will have 'matured and settled' by tomorrow and sectioning can begin. Tonight I'll 'rough' a few blocks just to prepare the surface.
Here's the complete protocol used, as seen on the fabulous left-hand whiteboard...

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Should have some news tomorrow - watch this space, the adventure is about to pat dividends - I hope!

[mrsonchus]

Hi all, I've been 'roughing' some wax-blocks this evening and as usual have taken a few rough sections to assess the blocks, mainly for their quality in terms of the completeness of their infiltration with wax. They all seem pretty good, looks like a 10/10 for a change!

Anyway, on this menu I've daffodil flower-parts (the reproductive parts specifically) without the inclusion of petals..
The ovary of a Daffodil is as may be clearly seen in any bloom, below the petals, anthers and stigma - it's this ovary, complete with still-attached central-stigma (pollen receptor) and 6 peripherally-attached anther-filaments (the anthers proper being atop the filaments) that are seen in these first few sections. these pictures are obviously of the sections whole - complete with wax that is, that are as they are when floated onto slides before drying and de-waxing etc...

The ovary of a Daffodil contains multiple ovules (to become seeds), each will, when fertilized, have a pollen-tube that has grown right down through the stigma, through it's 'stalk', into the ovary and finally into a gap in the ovule where it's load of 1/2 genome from inside the pollen-grain will fertilize the egg-cell within the ovule, giving rise to a zygote, which of course grows into an embryo within a seed! Amazing journey that one!

A few pictures, still in wax, no cover-slip etc, just as drying on the slide;

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and in closer we see a very complex structure being revealed..

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and

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It looks like this is going to be a very detailed and complex set of sections - plenty of structure to be worked-out! Can't wait to 'get into the block' and start on what should make a fascinating set of slides once stained and mounted! Looking good for the ovary and flower-parts so far..

A long while ago when I first started sectioning, with a rocking-microtome, I sectioned a Daffodil ovary in TS, I still have these slides and they will go nicely with a new set of TS slides.

Here are a couple of pictures of a similar but mature Daffodil-ovary, but this time in TS (cross-section)..
The Daffodil ovary has 3 'chambers' each containing ovules, this is clearly evident in TS, not however in today's LSs - these are from back in May of last year when just starting-out so please excuse the rather 'tatty' condition of them!
This is stained with the 'general stain' 'Bismark-brown', an old stain that I rather like,

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This is (was) a mature and far larger ovary full of nearly mature seeds, unlike the ovary of today that's very small and immature, it's almost impossible, for me at least, to see much correlation between these LS and TS sections, I'll do a 'proper' series on Daffodils a little later in their season to catch them at various and comparable stages of development, but for now, these will be great practice!
Here's an old picture of a 'stain testing' session I had back then, using the Daffodil ovaries as the subjects of several different varieties of stain! Quite colourful these slides...

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So far so good with this batch, the infiltration of wax looks complete and the blocks will I think section very nicely, tomorrow should see some 'proper' sections if I get a chance, the wax will have settled and fully set by then..

Back soon with ovaries, leaves and stems! What an adventure!