Slide Making with 61°C Wax

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mrsonchus
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Slide Making with 61°C Wax

#1 Post by mrsonchus » Sun Apr 04, 2021 2:19 pm

Hi all, some will have seen my earlier post re making slides from sectioned Lichen wherein I mentioned that there had been a small amount of tissue-folding during sectioning.....

I decided to try a different paraffin embedding wax in an attempt to mitigate and ideally eliminate this phenomenon which I'm pretty sure is caused by the combination of a relatively (to other plant tissues I have experience of sectioning) hard tissue - that of Lichen thallus and apothecia, with a rather 'softer' wax.
This wax as used has a melting-point of 52°C which is below the 'std' (in my tiny lab until I ran out of it!) wax of 56°C melting point. This character makes the wax less effective as a support-matrix for harder tissue as above, although for softer tissue such as for example tiny plant-ovaries and ovules, it is usually better as it matches the tissue hardness and consequently paraffin-wax sectioning performance more closely than a harder wax.

The preferred sectioning condition is to have an embedding-wax (AKA "support matrix") within (from infiltration stages) and around (from embedding stages) a tissue that is of a similar hardness - the idea being to give a hopefully optimal sectioning result through what will be a material of reasonably homogeneity in terms of resistance to the cutting blade, namely the 'wax block' containing the tissue. This tissue is to be sectioned with my Shandon rotary E320 AKA 'The Mighty Shandon' microtome.

Anyway, I still had some tissue in the softer wax left over in the form of what I think of as one of my 'bulk blocks' - large wax blocks that I cast with any spare tissue that I think may be of use or interest at a later date following a working block-casting session. The tissue in question is of some wall or English-ivy ) Hedera.helix) that I made slides of and detailed in an earlier post from last year in fact. The tissue block was re-melted, the ivy tissue removed from the molten wax and placed into some of the new 61°C wax used here for re-infiltration. This was alongside as it were some tissues collected this year including Montrbretia - a colourful monocot related to the irises and young poppy plant of the decorative purple variety grown in gardens from seed.

Apologies for the above rather long-winded preamble, just though a little explanation of the rationale and a link to the earlier adventure may be of interest...
Here are a few images of these tissues, (finally I may hear some scream)

This is the ivy tissue during processing last year, in the tissue cassettes I use,
Image

Here is a nice selection of 'fixed' (i.e. killed and preserved) Montbretia tissue in a bowl of 70% alcohol about to begin full processing to culminate in the production of wax blocks then of course sections and slides....
Image

Some poppy tissue at the same stage as above,
Image

Note [In terms of 'hardness' I would rate the ivy as the hardest, the montbretia as the middle and the poppy as the 'soft' one...]

O.K. moving on a bit,

This is a flower from the montbretia tissue, in molten-wax in a block-mould before the block has it's white plastic lid added and it solidifies. This orientation will when sectioned give longitudinal sections through the whole flower (hopefully).
Image

Here are a few images of the blocks and some test-sections cut a few days ago,

I cast 34 wax-blocks form various tissue pieces as detailed above, and cut, selected and mounted onto slides about 40 test-sections to start me off...
Image

A nice selection - I'm going to be busy!
Image


This is a block with an ivy stem in LS and TS.. a good test for this harder wax.
Image


I sectioned these test slides at 10µ, 6µ, 5µ and 3µ as far as I recall. All seemed to section well.
Image

Here's one such (montbretia flower LS) section floated onto a slide and ready for the draining and drying - usually for between 2 and 24 hrs depending.
Image

This looks to be a pretty promising start as all wax-blocks look good, and all sections appear cleanly-cut when examined under my stereo 'scope for a first-peek...

Posted up to here to avoid any 'mishaps' with this post.... More to follow....
John B

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Re: Slide Making with 61°C Wax

#2 Post by mrsonchus » Sun Apr 04, 2021 3:24 pm

Here are some first-look slides from the other day, a selection of different staining and pre-treatments were tried here to see what worked, and of course what I liked.... Some better individual slide-images to follow.
1000px WS_first slide set.jpeg
1000px WS_first slide set.jpeg (129.14 KiB) Viewed 1273 times


Here's a safranin + alcian-blue stained LS of a monbretia flower. The red chunks are I think tannin deposits strongly-stained by the safranin. The tissue looks pretty good at this point. The stem of the flower is below, then the developing ovary containing developing ovules attached to a central 'plcenta' and the stigmas above but only slightly visible above that.. This tissue has a relatively large amount of what I think is tannin at this early stage.
Image


Next a poppy leaf of the soft flimsy (young too) type in TS - stained also with safranin and alcian-blue, dodgy WB - apologies.
Image

Closer-in the very many chloroplasts are seen stained red by safranin in this mixture. There is no real cuticle and as said, this leaf is really quite fragile - no fibrous or collenchymatous cell-wall thickening to help either it seems.
Image


This is another poppy leaf TS but with the addition of a measure of orange-G stain to see what effect it has, hard to tell really from this rather poor image but more to follow with far more detail soon!
Image

These are from a quickly taken set of images very soon after I had a staining session of basically throwing a few different staining protocols at the test sections, not too bad but far better to come I'm certain....


Almost forgot, I had a quick look through polarization although the birefringence is quite weak with this tissue, at least in this section,
the leaf midvein xylem is showing, as are some cell-walls on the lower midrib below the bundle that may well be laying down extra cellolosic layers in this location rather than full-blown lignification.
Image

A (TS) region through the leaf's blade has very many birefringent chloroplasts by virtue of their starch-grain inclusions,
Image

I think this is polarization with the inclusion of a full-lambda retardation-plate, very useful when birefringence is low and making detail hard to see - or I may have simply 'uncrossesd' the polarization somewhat I can't remember or tell as I took these images in mono... I also appear to have overexposed the highlighs I'm afraid and lost some detail. Still, the starch in the chloroplasts is quite visible in a rather blurry way...
Image

Back with better images soon. Also I have what I think will be more interesting blocks to section - I started with the most 'banal-looking' as a test for sectioning of this harder wax as I was unsure of the outcome and didn't want to risk damaging better blocks at this early stage.
John B

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Re: Slide Making with 61°C Wax

#3 Post by MicroBob » Tue Apr 06, 2021 7:23 pm

Hi John,
thank you for this interesting description! For me it is always helpful to read about your plans, decision making, observations and results. I use Leica Paraplast plus with DMSO, not because I chose it, it came to me. It melts at 56°C and shouldn't be heated above 62°C. I have no real comparison and get on with this paraffin but of cause I see that some objects are harder than my paraffin. Right now I'm dehydrating a bunch of objects to embed them with paraffin tomorrow. Some have been swimming in FAA for quite a while and have become fairly hard.

Recently I have been testing a new black stain and compared thicker sections enclosed in carrot with thin ones embedded in paraffin and found it interesting how different these sections took on the stains.


Bob

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Re: Slide Making with 61°C Wax

#4 Post by mrsonchus » Tue Apr 06, 2021 11:25 pm

Hi Bob, pleased to know you find them interesting - I began to wonder if it was worth the effort of posting to be honest, so little interest outside of equipment and pond-water at the moment. Understandable though as the live-mounts are so accessible. Most folk aren't as time-rich as I am, even with the current situation - I have the time to devote to the long and steep learning-curve of microtechnique to be fair.

The realm of Botany simply fascinates me personally, and the more I read/learn the more fascinating it becomes, as the structural details relate ever-more to what I learn of plant development right from the meristems of the shoot-apex and root-apex.....

Microtechnique is as you know yourself very rewarding indeed, as long as the will to analyze and experiment goes beyond a single session as the 'scope.

The current batch of new blocks I have are a real treat and I'm just about ready to explore them for their content now that I'm happy with my early findings re the new 61°C wax which is performing very well. I think the increase in mp is going to be very advantageous especially as the weather warms-up here in the U.K. The last time I cut sections with the 52°C wax I had the lab windows open to keep temperatures down and was perishingly-cold throughout the session - not nice....

That said the tissue type is paramount in this respect I find - softer tissue may well section better in the lower mp, but section thickness below about 5µ really begins to need a harder wax I've found. The tissue-integrity of the harder-wax cut sections not least due to their exceptionally good attachment to the slides I've found is superb.

A good counter-example may be the fact that this wax sections so beautifully with the soft tissue that is a young poppy stem - which really is a soft plant - very thin cell-walls non-lignified with sub-epidermal collenchyma which has thickened periclinal (non-lignified) cell-walls but very thin anticlinal cell-walls, which is the reason so many slides are seen with torn collenchyma tissue - it's just such a delicate arrangement for the passing of the blade. The harder wax has transformed this area's sectioning to perfect integrity - I think the wax simply stops or at the very least significantly reduces uneven stresses of these two vastly different wall-structures during sectioning. This is usually only achievable by embedding with plastics as used for sub-µ sectioning with an ultra-microtome, one section at a time!

The minute I was able to cut long ribbons of (near) perfectly-cut 3µ sections from the very varied tissue of the montbretia flowers I new I'd made progress - looking forward to exploring the blocks I cast with the new wax, and taking this forward to more targeted slide production. The tissues I used for the current batch were taken from my sample jars, all at least 1 year old, some 4-5 years since collection......

Great to know someone's exploring plant sectioning - please let me know how you get on - I'd be very interested indeed to see your progress, always so much to learn afresh!

Thanks for the encouragement Bob!
John B

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Re: Slide Making with 61°C Wax

#5 Post by PeteM » Wed Apr 07, 2021 1:46 am

Super informative post, John. Thank you! Ideally this will be up for years (decades?) to come and a guide for aspiring microscopists.

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Re: Slide Making with 61°C Wax

#6 Post by deBult » Wed Apr 07, 2021 2:54 am

Same here, keep them stories coming please!

If you are able to read The German language, there is a wealth of information on botanical cuts:

Overview list of contributions per species
https://www.mikroskopie-forum.de/index. ... ic=21220.0

The site of the MKB
http://www.mikroskopie-bonn.de/bibliothek/index.html

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Re: Slide Making with 61°C Wax

#7 Post by MicroBob » Wed Apr 07, 2021 5:58 am

Hi John,
it really is not always obvious whether it was worth the effort to compose a detailed forum thread. But I think much of the effect is invisible and still very much present. Many of the more active microscopists generally post very little in forums like this, many only start to post results when they are really happy with the reached level. And there also is a non specific effect: People see that it is possible for normal people to get into paraffin sectioning and they themselves take the step to actively prepare e.g. diatom slides.
What I like most about your posts is that they helped me to find into a practical paraffin embedding process and that you explanation help me to better understand what to look for an to interpret it. With limited time at hand it is always good to have a path to follow that leads to a result.
Much information about paraffin technique I found was from pathology uses and not 1:1 applicable to small scale botanic sectioning. And in the german forum very few people actively do botanic paraffin sections.
Good to know that you also work from longer stored material!

Here a sample stain of part of a palm tree leaf. Chryosidine, safranine and an iron gall based black stain.

Bob
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Re: Slide Making with 61°C Wax

#8 Post by mrsonchus » Wed Apr 07, 2021 9:01 am

PeteM wrote:
Wed Apr 07, 2021 1:46 am
Super informative post, John. Thank you! Ideally this will be up for years (decades?) to come and a guide for aspiring microscopists.
Thanks PeteM, very kind to say such - much appreciated. It's always great to know folks may have taken a little pleasure from a post, as I do everyday reading through this fine forum.
John B

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Re: Slide Making with 61°C Wax

#9 Post by mrsonchus » Wed Apr 07, 2021 9:09 am

Thanks deBult, very nice links.
That site is jammed with interesting exploits and no trouble to set the Google translator onto it!

This site is where I started with microscopy back in 2015 - feels like home here, and the amount of help and encouragement I've benefited from over those years from the great folk here is truly amazing, a great place to visit!

Hi Bob, many thanks, very encouraging message indeed! I love the section, especially like the differentiation of the cuticle, which with fully processed sections can be quite a difficult feature to pick out with clarity. Beautiful intense colouring coupled with superb clarity - very, very nice job!
John B

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Re: Slide Making with 61°C Wax

#10 Post by jimur » Wed Apr 07, 2021 10:00 am

mrsonchus wrote:
Tue Apr 06, 2021 11:25 pm
- I began to wonder if it was worth the effort of posting to be honest, so little interest outside of equipment and pond-water at the moment.
Oh John, perish the thought! Count me among the percieved few, but my interest is great. I'm such a clutz with the process I would drive you nuts with my inquiries as well as expose my inability. Know that I appreciate and need your informative posts from which to learn. Thanks.

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Re: Slide Making with 61°C Wax

#11 Post by dtsh » Wed Apr 07, 2021 12:52 pm

mrsonchus wrote:
Tue Apr 06, 2021 11:25 pm
I began to wonder if it was worth the effort of posting to be honest, so little interest outside of equipment and pond-water at the moment. Understandable though as the live-mounts are so accessible.
I certainly enjoy them and find them very infromative. I don't frequently comment unless I think I have something useful to add as I feel a bunch of "looks great" comments only detracts from the info. That is one of the ways that I think "like" buttons are useful.

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Re: Slide Making with 61°C Wax

#12 Post by pippo1234 » Wed Apr 07, 2021 12:59 pm

Indeed we noobies learn a lot from these posts, on top of the sheer pleasure and inspiration we derive from the beauty/quality of the images.

@John: As I wrote in another thread, a post of yours in this thread https://www.microbehunter.com/microscop ... dcc#p53305 provided the only info I could find on staining for aqueous mounts and taught me that you cannot use fast green with water. The social value of all these posts is really high. Even more on a topic, such as specimen preparation, that has a steep learning curve.

@Bob: I love the palm tree leaf image. The combination of colours and fantastic sharpness really does it for me.

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Re: Slide Making with 61°C Wax

#13 Post by PeteM » Wed Apr 07, 2021 3:56 pm

My take is that exceptional skill and a willingness to help others - often at some effort - should be recognized. John and Bob are among those who have my respect and gratitude for their many contributions. I'll often provide links to their comments for kids aspiring to learn more about science starting from the micro world up. Could well be they'll have a greater impact than they might know.

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Re: Slide Making with 61°C Wax

#14 Post by mazo4033 » Wed Apr 07, 2021 6:50 pm

Great and very informative post, John, thanks for sharing! It is always a treat to read these kinds of posts :D Wonderful stain result as well with the safranin + alacian blue, especially on the monbretia flower. Your posts and advice do have a big impact - per your advice on a different post, I went out and got some safranin which produced an excellent stain (I'll post a picture when I get a good section :) ) it is definitely a very useful stain! So thank you, and keep in mind that though you may not see the impact your posts make, they are a really big help to all exploring botanical sectioning, especially to beginners like me.

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Re: Slide Making with 61°C Wax

#15 Post by mrsonchus » Thu Apr 08, 2021 9:41 pm

Thanks Jim, dtsh, pippo1234, Pete and mazo4033, that's really nice to know, it's great to be able to share adventures, but I sometimes let my posts get a little dense I think, I have such an interest and enjoyment of Botany and microscopy that I'm inclined to get a little carried away... Thanks for your kind comments, it's always a great boost for me just to know that others are finding my posts enjoyable.
John B

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Re: Slide Making with 61°C Wax

#16 Post by deBult » Sat Apr 10, 2021 4:21 am

Yes, keep them coming please, and to me the process details in your posts make them into an interesting read.

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Re: Slide Making with 61°C Wax

#17 Post by BramHuntingNematodes » Sat Apr 10, 2021 7:04 am

I have noticed quite a bit of compression using paraplast with some FAA-preserved trametes. It's a soft wax to be sure, and my cutting room is closer to 75 F where I think this paraplast really prefers to be cut around 65 F. Trametes are pretty woody in my case, though, so maybe I will try some different, softer mushrooms.
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Re: Slide Making with 61°C Wax

#18 Post by mrsonchus » Sat Apr 10, 2021 8:57 am

BramHuntingNematodes wrote:
Sat Apr 10, 2021 7:04 am
I have noticed quite a bit of compression using paraplast with some FAA-preserved trametes. It's a soft wax to be sure, and my cutting room is closer to 75 F where I think this paraplast really prefers to be cut around 65 F. Trametes are pretty woody in my case, though, so maybe I will try some different, softer mushrooms.
Hmm, if you have any images of the fresh-cut sections straight from the blade I can have a quick look if you like...

I now have a selection of 52°. 57° and 61° infiltration/embedding waxes ehich should give me a good span.
If you also have details of your blade's profile and angle these would help as they're significant factors also.
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Re: Slide Making with 61°C Wax

#19 Post by BramHuntingNematodes » Sun Apr 11, 2021 2:45 am

I don't have any pictures handy right now but the ribbon has a little of that accordion crinkling going on. Going to try to shrink the presentation angle a little bit see if that helps. I rigged up a Sir all ralph knife adapter for the paraffin sectioning kit to hold Feather single edge razors, which are sharper than disposable microtome blades I can get ahold of according to microscopic examination. They are a bit thin and can flex, although there is quite a bit of support in my jig. Nevertheless I think the angle might be causing some scraping where cutting should occur and this soft warm wax is not helping.
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Re: Slide Making with 61°C Wax

#20 Post by mrsonchus » Sun Apr 11, 2021 3:44 pm

BramHuntingNematodes wrote:
Sun Apr 11, 2021 2:45 am
I don't have any pictures handy right now but the ribbon has a little of that accordion crinkling going on. Going to try to shrink the presentation angle a little bit see if that helps. I rigged up a Sir all ralph knife adapter for the paraffin sectioning kit to hold Feather single edge razors, which are sharper than disposable microtome blades I can get ahold of according to microscopic examination. They are a bit thin and can flex, although there is quite a bit of support in my jig. Nevertheless I think the angle might be causing some scraping where cutting should occur and this soft warm wax is not helping.
Hi, you may find this a little counter-intuitive but, increasing the knife's angle of attack (which intuitively may suggest an increase in "scraping") will far more often improve the section. The "crinkling" will according to the books, be caused by 'something loose' such as the blade's clamp or indeed the block's clamp or holder's clamp etc.... This however I have never found to be the cause when, as you can imagine, I've had the same symptom of parallel-to-the-blade 'accordion' type ripples in sections. Although a little dosconcerting I find them to be of little trouble in floating-out as they almost always instantly disappear when hitting the water's surface, with no sign of effect in the section as on the slide when examined.

I very often find that a decrease in the angle of attack of the blade does nothing to help at best.

The connections are between the hardness of the tissue at the time of sectioning,
the temperature of the wax-block (the blade/knife temperature is essentially uncontrollable so forget that as a factor - it will at least be constant for a session),
the angle of attack of the blade,
the condition of the blade's cutting-edge in terms of any nicks or deposits which are able to be seen with a stereo 'scope at 40-50x mag easily enough,
the speed of sectioning,
the condition of the edges (those top and bottom parallel to the blade must be parallel and 'cleanly cut') of the wax block,
the purity in terms of even microscopic 'bits' in the wax,
the hardness of the wax at time of sectioning,

A dull blade, not detectable by any examination - anything that is actually detectable really means the blade's finished for sure - will increase resistance during sectioning and may give the symptom of crinkling or tight-curling (loose curling's no real problem as the floating out will remove this instantly) - very common in fact. This is the time to use a new blade - the old one as it may now be considered to be, will be fine for roughing if it's not causing drag-lines or scores during sectioning...

Incidentally resist the temptation and logic of albeit gently and carefully running a cloth along the blade to 'remove obvious debris' - while this occasionaly does no harm, it nearly always makes the blade worse or ruins it's edge. The best way to 'clear a messy edge' on a blade is to take a few thick sections of perhaps 12-15µ to 'wipe the blade' which is what I've seen it do very well. The wax-block used can be a new one or a currently in use one if you've either not reached the tissue therein yet or are in an area that is not part of your target view.

I always stand my working tissue-blocks face-down on a freezer-pack on my bench with another on the top to make a sandwich as it were during a session. Keeping a blade on such a block is a waste of time - I've tried it - but it returns to room temperature almost the instant it's mounted for use.....
John B

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Re: Slide Making with 61°C Wax

#21 Post by BramHuntingNematodes » Fri Apr 16, 2021 3:30 am

Thanks John. I managed to get a few better cuts but after a few days in safranin and washed with water, all the specimens peel off this cheap slide the second it touches the first alcohol grade. I haven't coated any of these slides with albumen! I got some superfrost + on order and I guess I'll start over.
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Re: Slide Making with 61°C Wax

#22 Post by mrsonchus » Fri Apr 16, 2021 7:35 pm

BramHuntingNematodes wrote:
Fri Apr 16, 2021 3:30 am
Thanks John. I managed to get a few better cuts but after a few days in safranin and washed with water, all the specimens peel off this cheap slide the second it touches the first alcohol grade. I haven't coated any of these slides with albumen! I got some superfrost + on order and I guess I'll start over.
Hi bram' - a few days in safranin is way, way too long! I know all the books seem to say safranin staining of between say 2 - 48hrs is best, and followed by carefull stain-removal as is usual with a 'regressive' stain such as safranin - but I have always, always found in practice that safranin staining is best at about 1min - perhaps 10mins at most with most tissue (botanical).... A huge difference I know, and I can't explain why the tomes give such high durations for safranin, to my mind and over 5 years of experience it makes no sense at all....

There's a simple and useful principle I find, the longer a slide with section attached is left in an aqueous medium (including water alone or an aqueous stain such as safranin) the more tendency, increasing to almost certainty above a couple of hours, there is for the section to become at best partly and at worst completely detached from the slide - in which terminal-case it falls off!

Try 1% safranin for a minute, into deionised water (always DI) to remove excess stain - that is the stain that will simply come away in clouds/whisps without any agitation beyond slow dipping in/out of water for say 1 minute, then if you consider the safranin too strong for your purpose - perhaps better detail or preparation for a counter-stain, you may move the section to alcohol - which doesn't try to peel the section from the slide... Use a graded series of alcohol/water to alcohol, during which more stain will be removed 'regressed' backwards as it were to the stain-condition that you require - with isopropanol this can be harder than you'd think - often the safranin simply refuses to leave, and the addition of an acid such as acetic (glacial) to the alcohol is needed to encourage it's removal - the atomic-bomb for safranin removal is HCl to acidify the alcohol. Once a section is safely into alcohol the risk of detachment is virtually absent.

I may already have asked, but are these wax-infiltrated and microtome-cut sections, floated onto slides in a water-bath prior to draining at near-vertical for about 30mins before leaving to dry completely either flat or at the same angle, with or without any heat applied to hasten drying?
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Re: Slide Making with 61°C Wax

#23 Post by BramHuntingNematodes » Fri Apr 16, 2021 8:39 pm

mrsonchus wrote:
Fri Apr 16, 2021 7:35 pm

I may already have asked, but are these wax-infiltrated and microtome-cut sections, floated onto slides in a water-bath prior to draining at near-vertical for about 30mins before leaving to dry completely either flat or at the same angle, with or without any heat applied to hasten drying?
Yes. They had dried overnight. 4 micron sections in paramat. Only a few looked very good, and now they are gone. I admit that I am following Gray's protocols and seems to ask for long soaking times. Perhaps safranin was weaker in those days. Gray also used adhesives to get his sections to stick, and preferred to artfully position his ribbons rather than use the float method.

I was able to mount an unstained specimen, although it was not cut from the block ideally. Fungus has a more hazy, vague structure in my experience than plant sections, and I think a competent staining might have helped. I have an endless supply of sample material at least.
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Re: Slide Making with 61°C Wax

#24 Post by BramHuntingNematodes » Fri Apr 16, 2021 8:44 pm

Also I have been using histoclear 2 and omnimount. I must say I am very pleased with those two.
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Re: Slide Making with 61°C Wax

#25 Post by Roldorf » Sat Apr 17, 2021 4:06 pm

Hi John, glad to see you are still having fun and working hard over there in Cumbria. Thanks for the post.
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Re: Slide Making with 61°C Wax

#26 Post by mrsonchus » Sat Apr 17, 2021 11:40 pm

BramHuntingNematodes wrote:
Fri Apr 16, 2021 8:44 pm
Also I have been using histoclear 2 and omnimount. I must say I am very pleased with those two.
A well-matched combination. I've tried this combo in the past, in fact I still have both in the lab. I reverted back to Histoclear-original a few years back however, combined with 'Histomount' as a replacement for my first permanent mountant which was 'Numount'.

All are pretty good I think.
John B

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mrsonchus
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Re: Slide Making with 61°C Wax

#27 Post by mrsonchus » Sat Apr 17, 2021 11:57 pm

Roldorf wrote:
Sat Apr 17, 2021 4:06 pm
Hi John, glad to see you are still having fun and working hard over there in Cumbria. Thanks for the post.
Hi Roldorf, yes still going strong! Today I mounted a few test-slides of 'broad-bean' (vicia.faba) edible seeds in LS to get a view of the embryonic shoot and root structures. This went quite well considering the sections were 10µ. What did amaze me with these was the absolutely jam-packed starch grains of the cotyledons, quite a sight with crossed polarizers....

Here's a partial section taken before the midline of the seed was reached, good image though of the shoot & root embryonics...
Image

Starch grains within cotyledons as seen through crossed polarizers,
Image

With a full-lambda retarder inserted,
Image

No polarization, just safranin (enhanced) formula, which I have been testing against my 'plain' unenhanced safranin version...
The starch grains appear white in this image...
Image

Here's a version stained with my 'plain' safranin - starch grains here are very slightly pink-stained it seems.....
Image

Root-tip (embryonic within seed),
Image

Great fun exploring the 34 new wax blocks I cast the other day, newly-mixed and filtered stains too!
John B

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Re: Slide Making with 61°C Wax

#28 Post by tgss » Sun Apr 18, 2021 12:16 pm

More terrific pictures John, thank you! But you've done it now... "enhanced" safranin formula??? Did I miss something? Please elaborate.
Tom W.

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Re: Slide Making with 61°C Wax

#29 Post by Sm33 » Mon Apr 19, 2021 7:25 am

Which stain was used for the slide of Root Tip?

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mrsonchus
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Re: Slide Making with 61°C Wax

#30 Post by mrsonchus » Mon Apr 19, 2021 10:43 am

tgss wrote:
Sun Apr 18, 2021 12:16 pm
More terrific pictures John, thank you! But you've done it now... "enhanced" safranin formula??? Did I miss something? Please elaborate.
Tom W.
Hi, the additives are borrowed fromJohansen's protocol - Sodium-acetate at 1% (W/V) as an 'intensifier' and Formalin at 2% (V/V) as a mordant - they seem to work but will almost certainly need to be optimised as this formula is purely an aqueous one, unlike Johansen's.... Both are easy and cheap to buy so worth a try.
John B

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