Beginner questions: Part 11: 3rd session with Bob's printed knife holder

[deBult]

Morgen,

New cut and stain (WA-sim III) Ginkgo biloba, leave and leave-stem, collected and stored for 2 months in 70 % of Alcohol (no AFE available at that moment in time).
No klorix used yet.

Still struggling in getting thin cuts (Table Microtome, SHK, Schneidehilfe, lots of Alcohol, new knife for each series of cuts).

The leaves cut relatively easy and relatively thin but still having some torn cell walls.
The stem cuts are thicker and "snap" and jump away when cutting: so probably are to "hard" caused by 2 months in Alcohol?
(I will try to soak the sample for 1-2 days in a mix of Alcohol and Glycerol and see whether this improves the cutting).

Feed Back and tips on moving forward appreciated.

[deBult]

and the stem cut

[Klaus Herrmann]

Guten Morgen Maarten,

Blätter sind schwierig zu schneiden, man muss sie gut einklemmen, damit sie sich nicht bewegen können beim schneiden. Der Schnitt durch den Blattstiel ist doch sehr gut gelungen. So wie es aussieht noch etwas keilförmig. Das liegt sicher auch an ungenügender Klemmung. Ich nehme dafür immer Karotte oder Styrodur.

so probably are to "hard" caused by 2 months in Alcohol?

Das ist sicher richtig. Alkohol härtet das Gewebe. Deshalb ist das AFE besser, weil die Essigsäure den Härtevorgang ausgleicht. Aber das AFE hast du ja jetzt.

[deBult]

Klaus,

Danke.

When I sampled the Ginkgo I was on holiday: only 70 % of Alcohol available.

Somehow my idea was to start with AFE (2 days-1 week) to stop al the chemical processes in the sample and afterwards move the sample to 70 % Ethanol.

Question: so is it better to STORE the samples in AFE ? 

Best,
Maarten

[Klaus Herrmann]

Question: so is it better to STORE the samples in AFE ?

In Ethanol werden sie halt stärker gehärtet. Es ist immer abhängig vom Material, nicht alle Gewebe verhalten sich gleich.

[deBult]

Hello,

As per Klaus H. guidance I now tried to support the Ginkgo leave-stem with Styropor (the green pellets they put in mail-boxes the prevent damage) while cutting.
They are way less damaging to the knife sharpness compared to the Styrodur from the DIY shop.

Still no clean cut and in my impression the thickness is still an issue?

Feed Back appreciated.

Best,Maarten

[Bob]

Hi Maarten,
that is a too thick section for sure. One useful point to remeber is that a section is always the result of not one but two cuts. So if in a cut the knife just scratches over the object instead of cutting it the material is still there and will add to the thickness of the next cut. You might install a magnifying glass on a flexible arm at your cutting place to monitor the cutting process more closely. I try to move the blade along the object with little pressure until I see that it actually enters. Then I finish the cut with a slicing movement. This works quite well for me.

Bob

[deBult]

Bob,

Thank you: I have been practicing cuts for approx. 4 weeks now, Table microtome, SHK, new Leitz knives every time, Schneidehilfe , yes/no carrot or styropor support, lots of alcohol and not getting a cut that is OK.

I really enjoy the exercise including the staining but to be honest having no succes is not vey encouraging.

I took a 1 afternoon course on staining and cutting they were using a "Rasierklingenmikrotom": and the cuts were a bit more thin but damaged: so this does not look like a useful alternative.

I bumped into a rotary microtome (clone from India) but need a to find a knife holder for that one.
Not sure it will be much help as wax embedding the sample is one more step complicating the process.


Next steps, continue practicing with the table microtome:
- Will try your approach (and find a loupe add on for my spectacles)
- As per Jorg's guidance will try to cut some fresh material and apply the AFE etc AFTER the cut

Best,
Maarten

[Fahrenheit]

Hi Maarten,

at first: staining seems to be fine now!

Yes, the last section is not thin enough but Ginkgo is not easy to slice.
To get used to Your microtome equipment start with easy things like the petiole of ivy leaves (Hedera helix). They are quite easy to slice and 8 out of ten should be fine. If so, You can be sure to have mastered Your tools and if sections of other specimens are not so good I think You will be able to figure out what to change.

I usually use carrot to embed my samples for sectioning. If You use any plastic it should be Styrodur.

Best
Jörg

p.s.
A rotational microtom  or sliding microtome will not help from learning the section technique needed for each of them and they do differ. If You get one, You'll have to start from scratch.
Especially a rotanional microtom is best used for zoomolgical samples embeded with paraffin. On the other hand: in the time I'll get my sliding microtom ready I'll have the sections ready in AFE using my cylinder microtome ... so I normaly don't use it.
There are exceptions: blossoms for example or cones, that would disintegrate without a paraffin embeding ...

[deBult]

Jorg,

Thanks again for feedback.

Next steps, continue practicing with the table microtome:
- Will try Bobs' approach (and find a loupe add on for my spectacles)
- As per your guidance will try to cut some fresh Hedera helix material and apply the AFE etc AFTER the cut.

Will keep you updated,

Best
Maarten

[Fahrenheit]

Maarten,

good luck! At the end You won't need a loupe any longer if Your eyes are still strong: if You get no slice or a very thin one, You simply drop the next section and go on.

For example: when doing a new specimen I usually try to get 6 to 8 fine slides. Therefor I need about 12 to 16 sections to be on the good side. When präparitions are ready (Mircotom on the taple, watch glas with AFE at hand, knife holder with a fresh blade, Ethanol and a fine brush ready and specimen fixated in the cylinder) it will take about 5 minutes to get the 16 slices needed in good quality.
So it's usually 10 minutes to build the setup, 10 minutes to clean everything after sectioning and 5 minutes sectioning.

Best
Jörg

Don't get frustraded: when specimens are hard to slice I often get only two out of ten. And I think I know my tools

[Bob]

Hi Maarten,
the cylinder microtome / blade holder method really works but there is some room for errors left. So a bit of experience is needed to get the process straight. I'm sure you will succeed soon.

Some further ideas:
- the object has to be held firmly: The thickness of the section varies a lot when the cylinder moves within the microtome or the object is pushed to the side. I use carrot as it is firm and friendly to the knives. On Jörgs MKB-website there is a description how to prepare the carrot - very useful.
- Does your cylinder microtome have slack between outer and inner cylinder - many newer ones have.
- The blade must never touch anything hard like the table surface. A good check of the blade's sharpness is cutting a firmly held paper tissue.

The rotary microtome is only for paraffine sections. Since the cylinder microtome / blade holder method works so well for botanic objects that hold together well I would suggest to concentrate on this now. Some people nevertheless don't get along well with this method - here an intermediate alternative would be cutting fresh, carrot embedded material with a sliding microtome with angled blade. With the rotary this work work.

Bob

[deBult]

Hallo Bob,

Your observation on my microtome may be spot on, it is 30 years old.

In the market section a tischmikrotom from Swift is offered: would this be a better option?

Best
Maarten

[Bob]

Hi Maarten,
the 30 years won't have done any harm - but was it a good fit from new? Can you wiggle the inner cylinder back and forth? I can't comment on the Swift microtome.

A sloppy fit might be curable: Since this is not a crank pin of a race engine two longitudinal stips might be enough to lead to a closer fit. Typical material for this could be beer can sheet metal. You would look for a three line fit, on one side cylinder angainst cylinder, 120° to both sides a strip that closes the gap.

In my old Sartorius hand microtome the cylinder fits very well - until the screw that closes the jaw is tightened that is. 

Bob

[deBult]

Well,


Total failure again in cutting slices of Hedra helix: slices are way to thick and uneven.

Tried the carrot: no succes:
- no there is hardly any play in the inner and outer tube of the microtome
- the underside of the SHK  holder bumps into the top of the carrot halfway the cut
   (see picture  of the underside knife with blue circle)
   ( the part the knife rests on! So I have the knife sticking out 5 mm extra to remedy this)
- slices are so thick I do not even consider staining them and / or view them under the scope

I'm a bit out of options now.