Shattered cells in paraffin section

[mrsonchus]

Great progress my friend!

As you may see from the sections, you have a good degree of integrity in your sections, and judging by the look of the vasculature a good orientation - that is to say a nice perpendicularity to the vessel axis.

De-waxing - needs at least 10 minutes twice-over then wash away with pure-as-you-have OH, before you begin to move to water.....

Make sure you allow your nearly-vertically stood and dust-protected sections to air-dry for at least 12hrs. Inadequate drying will lead to loss of cell contents further-on.

The choice of 10µ is perfect for this work, no worries there.

I'm off to pm you now my friend, see you 'on the other side'

John.

[DeeJay]

I have much better integrity but there are still damaged cells. Do you have any idea what I can improve? I am using cheap Chinese razors. Maybe I will get better results with official disposable microtome blades.

..and judging by the look of the vasculature a good orientation - that is to say a nice perpendicularity to the vessel axis.

I lost you after the word vasculature. Can you elaborate?

-DJ

[mrsonchus]

Hi DeeJay - yes, what I meant (apologies for my verbosity... ) was the vessels of the root/stem look like they have been cut nicely across the stem (i.e. not at an angle, which is easily seen in sections as a 'blurriness' - deceptive but a sure sign of an obliquely-cut section).
The parenchyma cells are usually quite shrivelled-looking as mentioned in an earlier post, so no worries there. The edges look pretty good, remember that orchid 'air roots' have a peculiar surface - empty cell walls with extra spirals of lignin that enable the outer surface of the root (velamen) to hold water as it were....

My advice would be to get some proper blades ASAP and above all at this time to practice your technique, as the improvements to these basically sound results will come quite quickly.
Experiment within-reason with cutting angle and with the orientation of the wax-block to the blade - although looking good it is always a great benefit to become familiar with a few factors at a time and to practice - it really is amazingly gratifying to see the improvements begin to appear.

I really must recommend also that you look-up my posts from my own stumbling beginnings back in 2015 I think - you'll find I documented the entire process, pitfalls, investigations into reasons, corrections etc all the way to the sections I can now cut today.

Get those blades and a holder my friend - this is your major hiatus - I've been there myself!

p.s. just search e-bay for "disposable microtome blades" and be sure to get a 'low profile' holder and of course blades - 'Feather S35' or similar are the standard that I use....

Good luck, John B.

[DeeJay]

I bought a displosable blade holder but it turned out that it was too big to fit in the microtome. So I decided to make two indentations where the microtome clamps grab it. It took me two hours of filing but I finally have it in place.

I did some cuts with both 15micron and 10 micron thicknesses. Before I started sectioning I also trimmed the block a little. For the first time (after soma failures) I was able to create some decent ribbons. I don't know if that is due to the new blades or because I am just getting better at it.
I also lowered the temperature of the water a bit but I still had some bubbles appear. I simply removed them with a spatula. There were no new ones formed so that was one problem out of the way.

So the next step is to re-hydrate them and seeing what they look like. I hope that they will be completely in tact this time.

[MicroBob]

Plant cells are quite big. So it is a sign of the right thickness of cut to open part of the cells. Too thick a cut and you have more than one layer of cell which is not good for the clarity of the image, too thin a cut and it looks unnatural and more like a knitted table cloth. A good starting point is 50µ in my opinion.

To quickly test the blade you can try to cut a paper tissue. The blade shouldn't take too much force to cut into it from the side and should cut on cleanly with a slightly angled blade.

Try to make freehand cuts from fresh plants. When you try to start this and cut a wedge, ending with zero thickness, you will be able to identify an area that has just the right thickness of cut. This would be the thickness to try to get with your microtome.

FCA and W3A Sim are nice simultaneous multi stains.

Bob

[billbillt]

Hello ..

I can see one obvious thing you need to do to make things work much smoother, is polish the rust from the ways on this machine.. It can't be expected to perform to it's peak if the sliding part is rusted to the bed of the machine!!..

BillT

[DeeJay]

Good point. The sliding part can still move freely but it is a good idea to clean it before it becomes a problem.

I viewed the sections under the microscope and there still seems to be a lot of damaged cells. I included pictures of a section of an orchid air root with damaged cells. I also included two pictures of a berry I sectioned. The cells in the middle seem to be ok but the ones on the edge are damaged. I think I did something wrong. Do I have to adjust the angle of the blade? Did I make a mistake in de-hydrating or re-hydrating? Are the slices to thin?

I think it is easy to experiment with the thickness of the slices and the angle of the blade.

[mrsonchus]

Hi DeeJay,
and congratulations for the excellent adaptation to the blade-holder - you now have all the essentials in-place and working well!
Your sections and eventually staining and slide-making will only get better from here-on my friend!

A general piece of advice I will give you is to concentrate on one or reluctantly two closely-related, factor/s at a time, preferably one.

For your blade's angle for example, set this to the 'zero position' (that doesn't imply or mean zero-degree angle) which should be marked on the holder somewhere with some markings either-side for each-way alterations to blade-angle. In other words, start with the blade at the 'std' angle with no alteration either way. You'll find for 95% of your sectioning that this will actually be the optimum. So, set blade angle to the std position and forget that as a factor.

The rust on the platform is of no consequence at this time as once set the blade-holding apparatus isn't required to move during sectioning.

The condition of the blade may be taken as perfect, these disposable blades are superb. If you want to clean any bits of wax from the blade a very soft cloth and a dip (of the blade) into either your wax solvent (I use 'Histoclear' and I think you're using Xylene) or your strongest alcohol.

Now, the sections;
Your Orchid-root is not at all bad, the 'velamen' epidermis is nicely cut, with the bands of lignin that form a 'mesh' that traps water, nicely defined - the cells are empty so form sort of 'strand-filled boxes' that will hold water - handy for an air-root! No problems there.
The center of the root is nearly all parenchyma - a thin-walled 'general-purpose' tissue with cell-walls that contain in life a high percentage of water. This makes parenchyma far harder to section in a condition that simply 'looks nice'..... Parenchyma very often wrinkles and looks as it does in your sections. This varies enormously through different plants and different species, even different areas of the same plant.
The great challenge in fully-processed plant-tissue sectioning at the ultra-accurate level of the microtome, cutting sections down to 1µ (a whole different World to that of hand-sectioning) is to optimise each section and tissue in terms of so many factors it can make one's head fall-off at the beginning!

I'd take the Orchid sections as very good at this stage and simply practice cutting more - no large changes are needed in your processing or technique at this stage, you're already off to a very sound start.

The berry sections are really very informative also - the wax and the absence of scoring or compression are unmistakable indications that your blades, sectioning-angle, wax-handling, infiltration, embedding and microtome-technique are all in very good shape and need no major tweaks.
The inner tissue has sectioned better than the outer cortex/exocarp maybe (below the epidermis) simply because it is a stronger tissue, fully surrounded by similar tissue, unlike the outer tissue (here it looks like cortex to me) which looks again like parenchyma, with the thin-walled cells prone to the 'shrunken look' as a result of processing - this can be mitigated with protocol changes on a tissue-specific level, but at this stage again I'd say the sections are good enough to concentrate on your technique not only of microtomy but of floating sections onto slides, draining and drying them, de-waxing, hydrating, staining, dehydrating, clearing, mounting etc.....

I hope it's OK to use your image here. From the outside (epidermis) in, the epidermis (outer layer light-grey) has sectioned fine, just the usual nicks etc that are eliminated with practice. Inner to this is the also light-grey parenchyma showing the common 'shrivelled' look - ignore for now. Then we have the band of harder cells that have sectioned quite well, the inner ones of this brownish-band are showing compression - a 'squeezed-up' look to them, very common in collenchyma - a tissue-type comprising cells reinforced with cellulose rather than the more permanent, stiffer, lignin of vessels and fibers. Collenchyma very nearly always compresses like this, unlike the more robust lignin-reinforced cells... A few vasular-bundles are visible within the parenchyma, and being strengthened with lignin, they have, as often is the case, sectioned well by the looks of the image.
The inner cells clearly have thick walls and have sectioned well.

berry center.jpg (270.88 KiB) Viewed 3525 times

So, your basics are all looking good from these sections my friend, a very fine start indeed! You are on your way to excellence, but now is not the time to concentrate on the finer more esoteric details but to make-secure your basic technique, including processing.

Tissue-loss or tearing, dragging etc has many causes, each of which is easy enough to remedy, but very difficult to definitively identify and isolate!

Section thickness, nearly all sectioning will lie in the range of about 15µ at the thickest, to maybe even 1µ but far more likely about 4µ at the thinnest. The two I use for almost everything are 10µ - the very best starting thickness as it's very likely to be optimal, especially for general morphological sectioning such as these, and 6µ - for fine tissue that will give in some cases better detail depending almost entirely upon tissue-type.

Thicker than 15µ is not really in the realms of the high-precision microtomy of the paraffin-sectioning technique - and above about 5µ is too thick for the even-finer plastic-block sectioning of 'ultra' microtomy carried-out under stereo-microscope!

A fantastic start and some honestly very, very good sections at this stage. Keep this up and you'll soon be making beautiful sections, but there is an awful lot of work ahead! Just keep the factors that you work on separate and in a step-by-step organised methodology and you'll find this subject incredibly interesting and rewarding!

Good luck, John B.