Slide Making with 61°C Wax

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mrsonchus
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Re: Slide Making with 61°C Wax

#31 Post by mrsonchus » Mon Apr 19, 2021 10:46 am

Sm33 wrote:
Mon Apr 19, 2021 7:25 am
Which stain was used for the slide of Root Tip?
The first and last images above of the embryonic (hence the abscence of mitosis) root-tip is a safranin/fast-green one, with the addition of orange-g which as well as acting as a differentiator for safranin, adds to the staining protocol to create a rather nice triple-stain - squential not simultaneous! See 'yellowsolve' methods.....
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Re: Slide Making with 61°C Wax

#32 Post by tgss » Mon Apr 19, 2021 7:08 pm

Thank you for the clarification mrsonchus. I didn't realize you were not using Johansen's formula for the safranin O. I'm wondering if you have found the addition of the formalin, as mordant, changes the response of the stain during differentiation relative to your normal formulation?
Tom W.

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Re: Slide Making with 61°C Wax

#33 Post by mrsonchus » Mon Apr 19, 2021 8:04 pm

Yes I have found this to be the case. The formulation with mordant and intensifier does appear to differentiate more effectively than the 'plain' formulation. By this I mean that the differentiation (essentially controllable and hopefully selective removal of safranin) appears to be faster, greater and more accurate (in the sense of discrimination that is) than usual. This was with the use of (acetic-from glacial) acidified alcohol - 95% (i.e. the 'purest' form available) isopropanol + 1% V/V glacial acetic acid. I also use 5% when necessary or optimal.

This result is in isolation however at the moment as I haven't had a chance to duplicate and confirm let alone isolate it to the presence of either/both the mordant Formalin or the accentuator Sodium acetate...
I do however have a complete set of 10µ sections taken serially through (in LS) a still-closed flower from the slightly immature inflorescence of montbretia from my earlier sectioning.
These are under a dust-cover and await flotation onto slides, being serial I hope to be able to get to a 'sweet-spot' in the tissue for a good view of the structure and arrangement of the ovary and ovules. Being from the same tissue-block and cut at the same thickness under precisely the same conditions these also offer a good source for such tests as the efficacy of the additives to safranin discussed above, as well of course as other comparative examinations of protocol (staining in this case)....

A nice set waiting to be mounted etc... Maybe I should've subbed 100!
Image


Today I also mixed and applied a 'subbing' solution to 50 plain slides to be used with these relatively thick sections, as an aid to their edhesion to the glass. It's often the case with larger tissue at thicker sections (here 10µ) that they have a tendency to fix poorly to the slide or even come away in places - the subbing-solution helps this a lot. The one I use is based on chrome-alum-phosphate (see link). In the past this method has been so effective that I have literally had to scrape repeatedly at a slide to remove the dried tissue/wax section of a part of the mount that I wished to remove from the final result! This is by far the strongest and maybe leasy likely to background-stain subbing method I know and have used, and I've tried a few over the last 5 years....
Last edited by mrsonchus on Mon Apr 19, 2021 11:30 pm, edited 1 time in total.
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Re: Slide Making with 61°C Wax

#34 Post by BramHuntingNematodes » Mon Apr 19, 2021 8:40 pm

even better than egg white and glycerin i bet!

meanwhile my superfrost + arrived all broken and my fast green is actually green lake-- useless! Congrats on getting any slides made. It is a lot of heartache.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

tgss
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Re: Slide Making with 61°C Wax

#35 Post by tgss » Mon Apr 19, 2021 10:50 pm

Thank you for the response re: differentiation of safranin O. That's an impressive array of sections you've produced and the close-ups look really good. There will be a lot of people on the forum interested to see the results after staining I'm sure, and any further information on your experience with the staining protocol would be very welcome. From your comments on subbing I take it you're no longer using positively charged slides (as in Plus sides etc.)?
Tom W.

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Re: Slide Making with 61°C Wax

#36 Post by mrsonchus » Mon Apr 19, 2021 11:29 pm

tgss wrote:
Mon Apr 19, 2021 10:50 pm
Thank you for the response re: differentiation of safranin O. That's an impressive array of sections you've produced and the close-ups look really good. There will be a lot of people on the forum interested to see the results after staining I'm sure, and any further information on your experience with the staining protocol would be very welcome. From your comments on subbing I take it you're no longer using positively charged slides (as in Plus sides etc.)?
Tom W.
I still use charged slides occasionally but routinely use plain uncharged without subbing either. I find that good sections and clean slides very nearly always give excellent results - adhesion is very rarely a problem. An exception as mentioned may be a particularly inflexible tissue cut at a thicknes off above 10µ or more.... I rarely go above about 8µ and routinely find 5µ to be about right - depends upon subject and degree of staining required. Leaf sections - 6-10µ will give a balanced result, thicker will become 'cluttered' as more than one tissue plane is taken. Thinner will often cause paucity of tissue as spongy parenchyma can look almost absent if sectioned too thinly, on the other hand if fine detail of stomata is desired, down as low as perhaps 2µ may be optimal - depending upon section plane and stomate geometry! For tiny cells of say ovules and meristems, 2µ is usually optimum for detail. Conversely, for what I call 'morphological' slides 8-10µ will give a vivid overall tissue structure to a slide.... Stems, between about 5-10µ for detail and anatomical/morphological emphasis respectively... The subject goes on a long, long way!

Charged slides can be almost totally unforgiving also - once the section is picked up by the slide it simply will not be adjustable as it would with a plain slide - the section will not be able to be moved, re-floated etc - if it's not oriented as you intended it's a gonner! They also have a habit of refusing to allow all water to drain from the bottom edge of the newly floated-on section - very often the bottom of the section (bottom as the lowest edge as it's stood on end to dry/drain) seals instantly to the slide and water remains trapped (above the seal) under the section - yes a tiny piece of filter-paper may be touched to it to wick-away the water, but this will then cause the bulging (on account of the water trapped under the section) section to wrinkle if the water doesn't flow out perfectly symmetrically, as it almost never will....

If a section has many unconnected (i.e. non-contiguous) elements such as an anther in the free-pollen stage, a charged slide may be very useful to 'keep the bits on the slide' - although again with a well-cut section and a clean slide even this is rarely a problem.....
John B

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Re: Slide Making with 61°C Wax

#37 Post by mrsonchus » Mon Apr 19, 2021 11:36 pm

BramHuntingNematodes wrote:
Mon Apr 19, 2021 8:40 pm
even better than egg white and glycerin i bet!

meanwhile my superfrost + arrived all broken and my fast green is actually green lake-- useless! Congrats on getting any slides made. It is a lot of heartache.
Urghhhh - that's bad luck. Here in the U.K. over the last 5-6 years I have never had a single damaged box let alone item within, delivered - I've gone and done it now - my next delivery will probably be in 1000 pieces after tweaking the nose of fate!
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Re: Slide Making with 61°C Wax

#38 Post by mrsonchus » Tue Apr 20, 2021 12:14 am

Oh, here are a few images of some slides I mounted unstained, for use in polarization, phase-contrast etc microscopy. This will often reveal or clarify detail that is invisible or uncertain in stained slides. For phase-contrast I find that well mounted unstained sections are far superior to trying to use stained, with almost no halo effect. Great for tiny tissues such as pollen-grains and virtually all investigations of plant reproductive tissue.

These are actually slides of garden poppy ovaries containing as poppies do, very numerous ovules which of course produce the huge number of seeds that ultimately shake out of a mature poppy seed-pod!

The phase used is only basic plan-achro phase objectives with the Olympus phase abbe condenser on my BX50. Great cell-wall clarity and superb nuclear and cytoplasmic information also. Personally I really like these....

A stitch across a poppy ovary with ovules in various orientations, cell-wall arrangement is very clear - this unstained tissue is invisible without phase contrast!
Image

Cells with cytoplasm, some of which appear in active differentiation/mitosis stages - this is after all a developing and immature ovary.
Image

Closer-in the ovules are curving to become 'anatropous' in form in the mature state. On the right cells of one of the ovary's placentae are clear.
Image

Close-in ovule, below the ovule tiny phase-granules are visible, the are not artifacts - they don't extend into the ovule proper but occur in the cells around it's 'stalk' (funiculus) and placenta region.
Image
and
Image


This was an interesting observation under polarization as I remember. The cell walls are more noticeably birefringent at certain angles relative to the polarizers.... Good use for rotatable stage in a basic way at least.
Image

Apologies for throwing in so many mixed images. I like these and just thought I'd share them - I should really have made a proper more organized post for them.
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Re: Slide Making with 61°C Wax

#39 Post by tgss » Tue Apr 20, 2021 2:55 pm

mrsonchus wrote: I still use charged slides occasionally but routinely use plain uncharged without subbing either. I find that good sections and clean slides very nearly always give excellent results - adhesion is very rarely a problem. An exception as mentioned may be a particularly inflexible tissue cut at a thicknes off above 10µ or more.... I rarely go above about 8µ and routinely find 5µ to be about right - depends upon subject and degree of staining required. Leaf sections - 6-10µ will give a balanced result, thicker will become 'cluttered' as more than one tissue plane is taken. Thinner will often cause paucity of tissue as spongy parenchyma can look almost absent if sectioned too thinly, on the other hand if fine detail of stomata is desired, down as low as perhaps 2µ may be optimal - depending upon section plane and stomate geometry! For tiny cells of say ovules and meristems, 2µ is usually optimum for detail. Conversely, for what I call 'morphological' slides 8-10µ will give a vivid overall tissue structure to a slide.... Stems, between about 5-10µ for detail and anatomical/morphological emphasis respectively... The subject goes on a long, long way!

etc.

John B
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Very good information on your experiences with different subbing strategies John. Thank you for that.
The phase and pol images are very interesting and give quite a different perspective from the more usual stained sections. Thanks for sharing. Looking forward to seeing some of those "more usual" images when you get some of these excellent sections stained. Keep then coming!
Tom W.

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