Staining Practice

[mrsonchus]

Hi all, I've been experimenting with staining of some Dandelion-root sections I recently made and have a few images some may find interesting.
The sections are cut on my rotary Shandon microtome - AKA 'The Mighty Shandon' of many past posts....
Wax embedded and sectioned at I think 6, 8 or 10µ (I should have made notes but I was really just playing-about a bit ).

Some I pre-treated with immersion in bleach ('Domestos' is 2.5% Sodium hypochlorite and does the job well) for about 4 minutes prior to staining to clear (remove) cell contents, not my usual technique as I prefer to have cell contents included in my slides, but such bleaching does give a cleaner outline of the cell-walls if that's the main feature under scrutiny. Some are cleared this way, some have intact cell contents and consequently nuclei etc which have also been stained within the cell walls....

Anyway, the bi-stain I've been working on for the last few days is a simple one combining Safranin with Alcian blue. These two stains, unlike the classic combination of Safranin and Fast-green, are completely compatible in the sense that they do not intrude into each-other's preferred target areas to stain. This has the very convenient consequence of making differentiation between the two, usually with acetic-acid alcohol, unnecessary!, When rinsed with de-ionised or distilled water in the usual way for such essentially aqueous stains, the two stains are beautifully separated within the tissue...


Here's a root TS with Safranin + Alcian-blue applied.

ws_alcian blue and safranin dandelion root.jpg (102.09 KiB) Viewed 2472 times

I've also been trying to get a tri-stain to work, including three stains, beautiful red Rhodamine-B, Alcian-blue and Acriflavin (orange). They're actually surprisingly tolerant of each other, applied serially not as a combination as with the bi-stain. The main challenge at first attempt seems to be the tendency of the orange stain (Acriflavin) to turn the Alcian-blue green-ish, although the Rhodamine-B red seems pretty independently stable. First attempts were however pretty nice, so I thought I'd post some images from tonight's staining session.....

Here are sections from the same root stained with the tri-stain,

Dandelion-root TS of single tap-root

ws_06_02_19_181_stitch.jpg (111.68 KiB) Viewed 2472 times

The same root in a splitting region as a way perhaps to avoid an obstacle to growth of the root, the same cause of a 'two-pronged' carrot....

Dandelion-root TS across region that is splitting into two

ws_06_02_19_167_stitch.jpg (120.38 KiB) Viewed 2472 times

Also seen are what looks like two lateral-roots emerging from the 'parent' tap-root...

Splitting Dandelion's tap-root with two emergent lateral-roots.

ws_06_02_19_179.jpg (112.18 KiB) Viewed 2472 times

I may get back to these stains tomorrow if I get a chance, but I'd like to make some nice fresh tissue-blocks as I'm starting to dream of Dandelion-roots!

Hope you find these interesting, John B.

[billbillt]

Hi John B.,

These photos are a work of art!... You did your usual perfect job!...

Regards,
BillT

[MicroBob]

Hi John,
interesting sections with the lateral root and the splitting root!
Have you tried Etzold FSA or FCA or Wacker W3A triple stains? They are quite popular here in Germany.

Bob

[mrsonchus]

Thanks Bill, pleased you like them.

[mrsonchus]

Hi Bob, yes there's more than a smattering of W3 inspiration in these tests. I would like to find some ready-made W3 as the ingredients are hard to find. Trouble is I really enjoy the challenge of investigating the interactions of various stains with each other of course and the tissue-features with which they bind.

Staining is an enormously complex and relatively little understood area. It's quite difficult to decide at which level to investigate. At the basic level there's the 'stain it and see what happens' approach, then the consideration of the many, many factors contributing to results. I've really just scratched the surface even after 3 years now of microscopy!

There's nothing I've seen better that the Wacker stains - for either hand or microtome sections. I think I'll see if I can find some ready-made if all else fails.

Thanks for your input Bob, pleased you like the root anatomy. I've a really interesting feature to come of these Dandelion roots that I'm having trouble sectioning cleanly at this time, although I plan to try some more tomorrow or this weekend.

The Dandelion has 'contractile' roots as do many plants that overwinter under or close to the ground, bulbs, corms etc, or in the case of the fascinating and officially-declared ineradicable Dandelion a flat but nevertheless photosynthesizing leaf-rosette to keep it going.... The contractile roots have regions where cells alter their growth-rate in certain planes and effectively collapse, causing the root to 'pull down' on the upper stem and either force it into the ground to an optimal level, or to bring the crown into contact with the substrate - fascinating.
In short, these collapses cause the root to have gnarly ridges on it's outer surface, and internally a 'snaking' central cylinder of vasculature that at first glance when sectioning longitudinally down the root, made me think - what on Earth has happened to my sections....
After a little research I discovered that the Dandelion also has a contractile tap-root! I have sectioned this phenomenon but am unhappy with the quality as the vessels are horribly difficult (actually it's a matter of finger-crossed with each root-section) to get a nice presentation with relatively intact vessels - little chance considering the contorted (in three dimensions) shape that they are forced into.

To follow I hope to have some nice longitudinal sections of a contarcted section of the root to show as it's amazing to discover that plants have a way of effectively 'planting themselves'!!

Back soon with something better hopefully.
I also have about a dozen stained with Safranin & Fast-green that are quite nice, I may add some images of those with some interesting features picked-out.

John B.