My New Microtome

[billbillt]

Hi John,

I am following what you do with great interest!....
BillT

[vasselle]

Bonjour.
Très belle article et bien détaillée du microtome.
Merci pour le partage.

[mrsonchus]

Hi all, I had a go at processing some of those 20-odd slides I 'cooked' yesterday..... They came up pretty well. Bearing in mind that ordinarily they wouldn't have finished drying & bonding to the slides for another 2-3 days to make a proper job of it. I thought I may just as well dewax some and have a proper look at the condition of the tissue therein - if there was anything left to see!
I used them to practice 2-stain (stain 1 and counterstain 2) staining protocols based upon & including the technique of 'Sass's Safranin & Fast Green counterstain method.

Firstly, the factor I was aiming to asses with this normally considered premature dewaxing was the effect, expected to be a bad one... on the tissue of the overheating to the point of partial melting of the wax in which the sections were cut. Well in that respect they fared pretty well, small amounts of tissue were clearly lost, but I rather suspect this to be because of the prematurity of my dewaxing rather than the temperature peak.

The staining tests also went well - I stained four slides,
1) Safranin & Fast green (ala Sass as related in Ruzin's superb book),
2) Safranin & 'Crystal' violet, see below;
Slides mounted but not yet dried so not at their best yet, slides always improve as they harden, flatten and develop their full optical potential... These pictures are of 'raw' slides but show some information..

Quite good differentiation?

ws_slide_2_saf_and_c_violet.jpg (103.96 KiB) Viewed 12227 times

The tissue has held up rather well considering...

3) Safranin and a 'general' (old-fashioned too some say) stain called 'Bismark brown' that is quite a favourite of mine for simple contrast-enhancement for example. see below;

A nice detailed stain - Bismark brown over Safranin

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4) Safranin & 'TBO' (Toluidine blue - a super metachromatic stain when used with live tissue such as hand-cut sections, not very good for tissue fixed in FAA such as in paraffin sectioning)

Here's my mixture for the (aqueous) Safranin at 0.5% w/v being run through filter-paper after being freshly prepared..

Filtering freshly-mixed aqueous Safranin

ws_filtering_stain.jpg (135.08 KiB) Viewed 12227 times

This is the rig I use to take sections from alcohol --> water for aqueous staining, then back through to alcohol again (i.e. dehydration) in preparation for mounting in solvent-compatible mountant (artificial resins replace Canada Balsam and they don't yellow as much) via 'Histoclear' (a xylene and/or toluene replacement) which removes the alcohol to leave the section in a solvent that is compatible with the resinous mountant - I use 'Numount' - it's easy to get, easy to use and cheap to buy!

Staining rig - basic

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Soooo... so far very good, I'm a happy fellow. Surprised me that the 'overheating' of the wax sections didn't totally wreck the slides - also those old tissue-blocks have come in very handy for practice with the new beast.

Soon have some new specimens processed for cutting sections to keep! Making progress, given the ability of the beast to cut sections reliably I can concentrate on specimen preparation (up to & including wax-embedding) and staining techniques - there's a lot to learn before I can target whatever specimens I choose - but I'm gradually getting there!

Oh yes, this evening I cut some more sections from one of the last 2 tissue-blocks I have left! I'd assumed the cooked ones were a loss...
I've cut about 25 sections at each of 5 different thicknesses for practice and evaluation;
I've now got strips of, 12µ, 10µ, 8µ, 5µ and (pointlessly maybe but I thought I'd try) at 3µ!

Should have these stretched and into drying in the next couple of days (must watch the temperature this time!

Back soon.

[billbillt]

Hi John,
You are certainly on a roll now.. You have really made a lot of progress with this.. Your samples look very good to me... Keep us updated!...

BillT

[mrsonchus]

Hi John,
You are certainly on a roll now.. You have really made a lot of progress with this.. Your samples look very good to me... Keep us updated!...

BillT

Thanks Bill, I'm very pleased you're enjoying the adventure.

[mrsonchus]

Thank you for inviting all of us to your lab, Mr.Sonchus! charlie guevara

Thanks Charlie, I'm having a lot of fun! To finally be able to concentrate on the Botany is very liberating, gradually each stage, right from the wildflower specimen spotted growing, to it's eventual study and preservation as a permanent section-set, is becoming more stable for me. The discovery of this fantastic microtome for a price I could actually afford was a major leap forward for me - sometimes life treats us poorly, sometimes we get lucky - very lucky. Having a great time.
p.s you're welcome to my 'lab' anytime!

[charlie g]

Thanks for the mention of: Histoclear, and Numount...I like that you say this mountant is not pricey.

Anything to be gained by doing the steps, especially the wax infiltration, with reduced atmospheric pressure, John B.? Or too much footprint cluttering your work areas? Not high vacum ( must be careful with all those glass vessels)...but rduced pressure in a dehydration storage vessel ( a dessicator with a valve on it's cover) with a hand pump say. I guess if all is turning out on schedule..'don't fix it'!

It's like we are at your lab, John B....splendid! I like your minds eye mention of native plants to compile an atlas of.

I wonder if the stomates on say Noding Trilliums differ from stomates in dryer dog days of August foliage...guess I'd need a plant with leaves from spring to fall...? Salidago/Golden Rod? stomates on initial leases...latter leaves...humm.

Thank you for this botanical microscopy. charlie guevara

[mrsonchus]

Hi Charlie, yes the chemicals I use are all very affordable, Histoclear is about £8 per 250ml (goes a long way), Numount I think is about £6 for a 'small bottle' (see pictures below) as far as I can remember, as it's ages since I bought it - I'm still only about 20% through the bottle - it lasts a long time! (also see picture below)...
Here are the fixative (FAA) and Histoclear that I use;

Histoclear & FAA

DSCN1722.JPG (113.26 KiB) Viewed 12210 times

Here is the Numount, a PVAOH-glue I use and an alcohol-compatible mountant for mounting straight from alcohol;

PVAOH, Numount & 'Alcohol-mountant' I use

DSCN1728.JPG (160.47 KiB) Viewed 12210 times

I use a slight (no idea of it's quantification I'm afraid..) vacuum in the early stages of fixation to (theoretically at least) release gases still within tissue, I've a video of one of my very early vacuum-chamber 'improvisations' (I called it the 'Mexican-Hat system' as the rubber jar-lid grip I used as the chamber lid is this shape - and luminous orange!)
Here's a bit of video showing the effect of this slight vacuum (drawn with a syringe through the 'Mexican Hat') as gasses may be seen rising from the specimens and probably the fixative too; The specimens are in a glass bottle (McCartney 30ml) with it's base chopped off and it's metal cap perforated to make a 'tissue-transporter' - saves keep grabbing tissue with forceps during transfers)



This was my first version, I now use a more flexible version that I came up with that uses individual rubber-bungs with tubing through them (fish-tank air line) and a valve (fish-tank again ) on-top to control the vacuum in an individual bottle, jar etc (different-sized bungs for different-sized containers of course). The vacuum can be drawn with a syringe, the valve closed and the syringe removed - I use another valve on the syring to allow it to be emptied of drawn-off air and used repeatedly to build-up a stronger vacuum if more than one syringe-full of air is to be drawn off...

Here's a picture - it's a very flexible, cheap and portable system for use by an amateur at home such as me...

Here is a pair of McCartney bottles under vacuum

ws_vacuum_fixing.jpg (72.39 KiB) Viewed 12210 times

I don't as yet use vacuum for infiltration as I only have a very small 'incubator' - it's actually a tiny oven that I control with a digital thermo-switch and temperature-probe that I bought online for £10...

I think I've used all my media-space for this post - back soon with more sectioning reports.

Oh, maybe I've 1 picture left.... Here's a book that I use constantly, have almost worn the words out on the pages! It's Ruzin's superb tome - my histological bible.

Fantastic resource

ws_ruzin_book.jpg (123.33 KiB) Viewed 12200 times

I've a series of sections through 5 thicknesses to stretch onto slides - I'll photograph them 'still in the wax' (they'll need at least 3 days drying and sticking time before further dewaxing & processing) and we can see if any differences in tissue characteristics are evident at this early stage in their life-cycle... Meanwhile I've a couple of boxes of permanently positively-charged Leica 'X-tra Adhesive' pre-cleaned slides on their way - should help with tissue-to-slide adhesion and prevent tissue loss during processing. 144 slides in total for £4.99 delivered - pretty good.

Anyway, better go to sleep, back soon.

[billbillt]

Hi John,
I am blown away by your work here!... This is fantastic and very interesting... Looks like it would make a good paper for "Microbehunter" magazine to me!...
BillT

[mrsonchus]

Hi John,
I am blown away by your work here!... This is fantastic and very interesting... Looks like it would make a good paper for "Microbehunter" magazine to me!...
BillT

Thanks Bill, I've thought a lot about articles, it's just having the time to compile them....
Thanks for your kind words - I'm glad you're enjoying them, I'm certainly having a great time too!

[mrsonchus]

Hi all, I received my positively-charged super-sticky slides today, together with a set of mixed (used) Shandon embedding cassete (Tissue-Tek) system stainless-steel moulds. I've some cassettes coming from China - cheap and I'm in no rush! Meanwhile, I've cut some wooden-blocks the right size to put onto the moulds instead of a cassette.
This will enable me to use the excellent mould-system that has many differently-sized wax-block options (I'll be embedding some very small specimens as well as larger stem and leaf types..) and at the same time enable me to save the expense of replacing my microtome's all-purpose chuck (block-holder) with a quick-release cassette holding chuck which is very expensive and not as flexible as the all-purpose chuck.
The cassettes fit my all-purpose chuck as well as they fit the quick-release version - phew! So now I have the option of embedding into the stainless-steel cassette moulds (using a wooden block with them or a 'proper' disposable cassette) or my current method of casting with ice-cube moulds or smaller waffle-moulds - all options will work because I'm keeping the all-purpose chuck.

Here's the mixed set of moulds I bought (used) for half the normal price of a set (which I think has to be all the same-sized moulds) online for £19 delivered..

Moulds & positively-charged slides

ws_cassette_mould_set.jpg (178.19 KiB) Viewed 12191 times

Here is a picture (not mine - from an advert I think) of the mould-cassette system

ws_t_tek_picture - Copy.jpg (5.09 KiB) Viewed 12191 times

I cut wooden blocks to the size of the cassettes so they can be used with the moulds in the same way as a cassette if preferred...

ws_cassette_apternative_blo.jpg (147.79 KiB) Viewed 12191 times

They fit like this..

ws_block_on_mould.jpg (132.48 KiB) Viewed 12191 times

The whole thing goes onto the microtome's chuck in the same way as a cassette would;

ws_mould_block_in_chuck_2.jpg (102.91 KiB) Viewed 12191 times

I'll be able to try this system out when I have some specimens ready for embedding - can't wait! Meanwhile I'll cast some empty practice-blocks using these moulds and wooden blocks to test the principle....

More soon..

[charlie g]

Thank you so much for this lab journal complete with source text you highly aclaim! I need to be vigilant...with you doing all this technical but clearly understandible process...there is temptation to tag along at your lab...and not get my own fingers wet! thanks, charlie guevara

[mrsonchus]

Thank you so much for this lab journal complete with source text you highly aclaim! I need to be vigilant...with you doing all this technical but clearly understandible process...there is temptation to tag along at your lab...and not get my own fingers wet! thanks, charlie guevara

Hi Charlie, great to have your company - guess what I've been up to this evening whilst trying to watch some football.....

I've cast a couple of blocks using my new moulds as speculated in my previous post, just to spice it up I sprinkled some long-dried (but completely un-processed) crocus pollen into the moulds as per a regular sample. The moulds performed as hoped, the wood-blocks on, I ran to the mighty Shandon.... It's a goal of mine to produce pollen grain sections.

I have taken pictures and video of my attempts to section the blocks complete with pollen-samples embedded (sort-of ).

I took a whole series of sections from 15µ, 12µ, 10µ, 8µ, 6µ, 5µ ----> yes I kept on turning down the thickness dial, with fingers-crossed and breath-held to 3µ and (gulp) 2µ - the 2µ started to compress a little, but should be OK when stretched on water..

I've stretched most of them via water-bath (or 'baking-tray' as it's known at Asda) onto the new Leica charged-slides I received today with the metal moulds. I've taken video of the whole process, from pouring the wax (and pollen) into the moulds, cooling and removing the now mounted blocks - Tissue-Tek shaped, sectioning through the series mentioned, stretching on water, floating onto the slides, and finally I've just started inspecting them with the 'scope (while they're still in wax - if there's anything there that is..). Well, surprise, I think I have sections through pollen-grains at several different thicknesses right down to 3µ - the 3µ ones seem by far to be the best - if what I'm seeing are indeed sectioned pollen-grains and not my hopeful imagination!
They definitely look like pollen-grain sections to me though, and I've studied a lot of pollen (I love Palynology in particular) from first getting a microscope.
I'll post pictures and video-clips of the whole story but not tonight! I'll get a few pictures posted later though when I've looked at the sections and perhaps measured them to see maybe if they could be pollen sections...

What fun, what an adventure! The mighty Shandon just keeps performing brilliantly - the thought making fully prepared and stained pollen-sections is making my mouth water - you never know - It may be possible.

Must go and eat, back later with a few early pictures - should be able to post the whole process tomorrow.

Watch this space, it's going to be an exciting time I hope!
p.s. Thanks for you kind praise Charlie - made me smile.

[charlie g]

Your quest for 2 micron sections somehow feels like 'free diving' to me as I am an excited spectator of your quest, John B.!

With the volume of your nifty wax moulds, and the average surface area of your pollen specimens...2 micron pollen sections sounds like statistics on collisions of molecules in a given volume of a gas...the 'mean free path' between colllisions adhered somewhat to an equation of state for the specific gas molecules. I keep thinking of this as that microtome blade periodically advances through the wax block to a distribution of embedded pollen grains.

I guess the trick is to layer wax+pollen grains, then wax alone, then wax+pollen grains , then gentle temp increase...and touch this mould to a vibrating plate to send waves through that suspenion of pollen.

If you start the sectioning with a block comprized of four layers of pollen...after the brief vibration induced scatter...you might have more chance of midsections of pollen grains?

Heh...it's fun to watch a wonderful quest from this close forum vantage point! all the best, charlie guevara

[mrsonchus]

Your quest for 2 micron sections somehow feels like 'free diving' to me as I am an excited spectator of your quest, John B.!

With the volume of your nifty wax moulds, and the average surface area of your pollen specimens...2 micron pollen sections sounds like statistics on collisions of molecules in a given volume of a gas...the 'mean free path' between colllisions adhered somewhat to an equation of state for the specific gas molecules. I keep thinking of this as that microtome blade periodically advances through the wax block to a distribution of embedded pollen grains.

I guess the trick is to layer wax+pollen grains, then wax alone, then wax+pollen grains , then gentle temp increase...and touch this mould to a vibrating plate to send waves through that suspenion of pollen.

If you start the sectioning with a block comprized of four layers of pollen...after the brief vibration induced scatter...you might have more chance of midsections of pollen grains?

Heh...it's fun to watch a wonderful quest from this close forum vantage point! all the best, charlie guevara

Hi Charlie, glad you came, yes, realistically a 2µ section just isn't feasible with a std rigid blade - there are too many other factors in play at those distances/proximities, the wax itself I think needs to be very hard and micro-crystalline to afford even a remote chance!

But, it's great to 'crank down the dial' even though it's pretty pointless below perhaps a limit of 5 or 6µ... I think most of my sections will be made within a range of say 5 - 12µ.

I've a few quick stills to post here to be going on with, back soon with them

[JimT]

Oh well, "S"tuff happens. Looking forward to next posts.

[mrsonchus]

Some early pictures of Tissue-Tek metal moulds on wooden mounts - with some pollen on the side.

Here's the 'kit' ready for wax;

Moulds keeping warm, block-mounts ready to go

ws_moulds_ready_to_fill.jpg (117.47 KiB) Viewed 12173 times

The principle of the wooden-mounted (rather than cassette) Tissue-Tek style wax-blocks seems to have worked well;

Ready for the microtome's chuck..

ws_blocks_cast_and_trimmed.jpg (100.87 KiB) Viewed 12173 times

The mounted block in the microtome's chuck, ready to go..

Mounted and ready to section

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After sectioning, the cut-face of the wax-block is beautifully even & smooth, the rotary with retraction is working well, the blade used has cut around 600 sections to this point but still has about 60% of it's use-able cutting edge untouched...

Typical rounded-square of TT system's moulds shows on cut face

ws_block_face_smooth.jpg (65.38 KiB) Viewed 12173 times

A series of raw sections at all different thicknesses (nominal) - lowest useful thickness I think will be 5µ, below this I think is no more than an interesting workout for the microtome... These sections are just placed onto slides for safe-keeping, they've yet to be stretched onto their intended slides for drying & processing..

ws_sections_straight_from_m.jpg (65.63 KiB) Viewed 12173 times

So, the metal moulds are very nice indeed and give perfectly straight, even and aligned blocks easily - they'll probably need a 'mould releasing agent' sprayed on them before casting, I flashed a little heat across their base to release them, but that could be damaging I think. A tin of release spray would be easier and faster I think - faster cooling may also help, this test was all a bit Heath Robinson, but has worked well I think.

[billbillt]

YES!!... This is as fascinating as a "page turner" detective story.. I can't wait to see what happens next!...

BillT

[mrsonchus]

YES!!... This is as fascinating as a "page turner" detective story.. I can't wait to see what happens next!...

BillT

Haha - thanks Bill - I'm excited too!
I still can't believe how lucky I am to have this beautiful microtome - it's a dream to use.
I'm down to 1 maybe 2 tissue blocks, 1 is a leaf (XS orientation) from an enormous ornamental grass that's been trying to take over our garden for the last 10 years or so! It's a nice section, very interesting being a grass and a monocot' too. But, it's a very dry & 'gritty' tissue to section, and I really need to be careful attempting to section it, lots of scores and a few tears always seem to originate as the blade encounter's the vascular bundles - my processing for this tissue has been too harsh as well, still it's all I have left at this time!
I did do a quick comparison between 8µ and 5µ sections of it this evening, only as 'raw sections' stretched rapidly onto slides, the tissue clarity is a little better in the 5µ range I think (very hard to judge with tissue still entombed in wax).
However, this block is a very poor one and the sections are a mess, but I'll show a couple here and you can see what I mean about the vascular tissues and their 'tracking' artifacts....

Here are 2 8µ sections;
All at x20, stretched onto sildes, in air, partially dried only.

8µ through vascular bundles

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Here are 3 5µ sections for comparison

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There's maybe a slight difference but in wax it's pretty hard to see anything meaningful - it'll be interesting though when I;ve got some fully stained & mounted tissues of varying thicknesses for comparison - it'll be a while yet though - lots to do!

I've got to get some more tissue into dehydration, I a few batches already fixed, I may start some off in dehydration tomorrow if I get a chance..
Sorry these images aren't much good, but it's quite interesting to see sectioned tissue as it arrived from the microtome, still within the wax-matrix.

[billbillt]

Hi John,
I think your photos are VERY good!... It does seem that you were destined to have this precision microtome to help you make wonderful sections... You are somewhat pulling me towards the botanical studies away from the "wee critters"!...

[mrsonchus]

Hi John,
I think your photos are VERY good!... It does seem that you were destined to have this precision microtome to help you make wonderful sections... You are somewhat pulling me towards the botanical studies away from the "wee critters"!...

Bill, you really are very generous, thanks for your encouraging words.

I started reading articles by the late great Walter Dioni (in the magazine) and really got the bug (or should I say leaf!).
Sections can be cut well by hand with a razor-blade or £30 hand-microtome, stained and permanently-mounted easily enough for virtually no cost..

Here's a couple of hand-cut sections (using hand microtome and a cut-throat razor), the first completely untreated, the second stained with good old Methylene blue;

Cross section of Aloe plant leaf

ws_enhanced_aloe_leaf_stitch.jpg (179.95 KiB) Viewed 12159 times

Chrysanthemum flower-stalk from a bunch of flowers..

ws_stem_section.jpg (302 KiB) Viewed 12159 times

If you work through Walter's superb examples you'll never look back...

Go on, make an onion-peel, put it on a slide, dribble then rinse a bit of stain through it (Iodine would do - or the blue or green fish treatments) and you'll be amazed at what you've created - dehydrate in alcohol and put a drop of nail-polish onto it, add cover-slip and you have your permanently-mounted slide!

Pleased you like the adventure!

[billbillt]

Hi John,
You are sure tempting me... Both of your latest pics are superb...
BillT

[mrsonchus]

Hi John,
You are sure tempting me... Both of your latest pics are superb...
BillT

Go on Bill - just one tiny leaf.... they're so green and lovely...... my tip;
Go get an onion, any onion will do, load up Walter D's onion examples and you're off!

Cell, walls, nuclei, cytoplasmic-strands, plasmodesmata, chloroplasts, stomata and their guard-cells, oxalic-acid crystals.... all there waiting to be spotted in the humble onion-peel - no sectioning required!

Back in February I worked through Walter's articles, here's a few snippets;

When my 'lab' was born

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PVA-OH glue is a good mountant...

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You know you want to......

[gekko]

The onion skin images are excellent. But your aloe leaf and crysanthemum section images are exceptionally beautiful.

[zzffnn]

Beautiful work, John!
Do you plan to section some small insects?

[mrsonchus]

The onion skin images are excellent. But your aloe leaf and crysanthemum section images are exceptionally beautiful.

Hi Gekko and thanks,
Yes I agree completely, the onion slides came up really well, with beautifully clear nuclear images (thanks to Walter's articles) and are what I would think of as a 'technical' preparation, whereas live tissue, the far thicker hand-sectioned versions, give images that are simply beautiful!
The extra thickness, inherent three-dimensionality and absence of chemical treatments (basically mummification...) of the live tissue definitely augments it's beauty I think.
Although of course the pedicel-section has been lightly stained with Methylene blue, which when matured before use will give a lovely (and subtle) metachromasia. A much underrated stain I think (probably because we often have experience of it from early school years and familiarity can sometimes foster contempt..), especially when it's allowed to stand and 'mature' for a few days before use, during which time it forms 3 closely-related compounds (isomers I think?) that together give such a lovely and informative image to the tissue.
The aloe was a pleasant surprise as it's a very succulent and delicate tissue to section, especially with a relatively blunt razor, but sometimes you just get lucky with a lovely section from dozens of so-so ones.

You can't beat the beautifully rich and vivid greens of live tissue.

Green glory!

websize_vasc_bundle_stack_4.jpg (163.21 KiB) Viewed 12150 times

A 'slice of beauty' - nature never fails to delight me....

[mrsonchus]

Beautiful work, John!
Do you plan to section some small insects?

Hi zzffnn, not really, I wouldn't have a clue how to go about it, I suppose the preservation may or may not involve dissolving the soft tissues, depending on whether you want details of the exoskeleton's arrangement (as when the relative positions of plant cells is desired, and the cell contents are deliberately removed or left unstained in favour of the cell-walls) or actual soft-tissue details (similar to imaging nuclei, cytplasm etc within plant cells) in which case a more subtle and non-destructive fixation protocol would be appropriate? ('Bouin's fluid'?)
I'm guessing here as I know nothing about insect preparation beyond straightforward dehydration (wet or dry I suppose?). I think of the 2 approaches as being a little like imaging with X-rays or an MRI...
Sectioning the exoskeleton may require a softening (to avoid shattering and enable clean cutting) together with support from thorough embedding and infiltration, without which the whole thing may well compress upon sectioning and simply collapse..
It sounds like a complex operation but with replaceable blades at just £2 it's possible maybe to just 'have a go'?
I wonder if polymer infiltration and embedding would give better results with insects? That exoskeleton seems to throw up many questions, together with the absence of rigid cell-walls in animal tissue...

But, Botany is my passion I'm afraid, but I suppose I could have a try sometime, just to see what happens.

In brief though, I doubt it, too many fascinating plants to get to grips with, I'm quite interested in the diversity and functional morphology of trichomes at the moment, that and the viscin-threads found with some pollen - so many voyages of discovery! What boundless fun!

[gekko]

Another beautiful section. I also would like to complement you on your excellent photography of your work table and the like.

[mrsonchus]

Another beautiful section. I also would like to complement you on your excellent photography of your work table and the like.

Thanks gekko, pleased you like them.

[billbillt]

Hi John,
I never get tired of viewing your photos... They are very pleasing to the eye with crystal clear sharpness!..
BillT