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

[Bob]

Hi Maarten,
I can recommend to cut the carrot as described on the MKB website (inside out) it really clamps better this way. Remove the peel completely as it can contain sand!
I don't have an SHK holder but I'm sure that it is not intended that there is a gap between blade and holder. The Leica blades are fairly thin and need all the support they can get. The blade should rest against the back rest. This ensures that the edge is at a constant height over it's length. The cuts are made in a slicing action and this doesn't work well when the blade is not parallel to the base. So there are still a few options!

Bob

[deBult]

Bob,

Thanks again, also for the heads up  , next steps:
- The SHK has adjustment screws: wil try to get the blade flush to the support
- cut WITHOUT the carrot support only the "schneidehilfe"

I have not yet been able to locate the recommended blades for your printed knife-holder: as I have 2 of those and 2 initial delivered blades  will try and switch to using that one instead and see whether the cuts improve.

Best,
Maarten

[deBult]

Hello,

Hedera helix cut: still no success:
* fresh cut  => AFE 20 min  => Ethanol
* SHK: now with Leitz 818 flush to the bottom (so no gap at te back side between knife and holder: as consequence the cutting angle has changed)
* Table microtome WITH "Schneidehilfe" plus "back-2-back"carrot support as per Bob's/Jorg's guidance: the slices are way too thick
* Table Microtome "Schniedehilfe" only: slices are somewhat thinner now but:
   - I think the cuts are still way to thick?
   - Also the staining is to intense (15 min WA sim III => wash => 24 Hrs of differentiation in aqua dest => 99.9 Propanol => Euparal).

Your feed back and guidance on next steps is appreciated.

I will start using the  Bob's "Printed" knife holder for next cuts to see whether this helps.

Best,
Maarten

[Fahrenheit]

Maarten,

yes, You'r right, the section shown on the photo is way to thick.
But you startet with a piece of Ivy sprout, not petiolus. This is much harder to section, especially when embedded with carrot.

If You choose a petiolus that is about 3 mm in diameter You won't need the carrot, the "Scheidehilfe" will do. Sprouts I usually cut without embedding (= freistehend).

Try to move the SHK holder with only a slight pressure: I managed to deformate mine (about half a mm) because I pressed it far to much down on the glas plate of the microtome. No other person was able to get a good cut with it and it had to be mendet at Darmstadt (thanks Detlef!).

Best
Jörg

p.s.
Perhaps You can manage to visit Würzburg in spring 2020, as I will show the cutting technique there.

[deBult]

Ha Jorg,

Thanks for feedback.

This time I switched from SHK to Bob's printed knifeholder with Olfa LBB blades (baumarkt) and Schneidehilfe only (so no carrot support) plus an easier sample to cut: the leafstalk (petiole) of a Pelargonium.

Ik think we are getting somewhere now: as always please feed back on this result.

Best,
Maarten

[Klaus Herrmann]

Hallo Maarten,

und jetzt wäre schon der exakte Vergleich mit einem Schnitt mit dem SHK durch das Pelargonium interessant. Ich habe mit Efeu auch keine guten Erfahrungen gemacht.

[Bob]

Hi Maarten,
this looks great now! And you have selected a beautiful object for you first success - is this Pelargonium or Pelarfonium?
You should be able to get the same result with the SHK holder, I think there is still something wrong with the assembly of it. Can you post a picture of it, especially from the side? One image assembled as used for the last cut and one with the knurled nuts and the top removed and the blade positioned?
My blade holder is cheaper on consumables but won't cut significantly better.

Bob

[Fahrenheit]

Hi Maarten,

nothing to complain about. Sectioning and staining fine.

But You should be able to come to the same good results with the SHK assembly.

Best
Jörg

[deBult]

Thnx for all guidance and feed back, next step: the SHK again

Bob: Pelargonium graveolens (corrected: tnx).

Best,
Maarten

[deBult]

Hello,

As per Klaus request I retried the Pelargonium cut with SHK, new Knife, plenty of Ethanol.
(Note: I'm asked questions at the pharmacy on my drinking habits: again 4 new 100 ml bottles of Ethanol).

Wel: no succes: fragmented cuts (zerissen): I must be doing something wrong using the SHK.

Note: I tried an overview picture using a different Photo setup: not very successful as you can see uneven lighting.

[deBult]

Additional SHK cut: twice the thickness

[deBult]

As per Bobs guidance: the SHK knife setup.

Anything here that might cause the unsuccessful cuts?

[Fahrenheit]

Hi Maarten,

I can't see any error with  the SHK setup. The petiolus You've cut with it seems to be smaller than the one You prepared befor with the printed knife holder, which might be a reason for the different results.

On the other Hand I think we are at a point now, where I must actually see, how You do the sectioning to get some new clue or another. Or You might take your self the time and do some 100 sections of the same specimen with both methods to get a hint what might go wrong ...

Best
Jörg

[deBult]

Jorg: tnx for feed back.

Yes it was a fresh petiolus part, so size may differ a bit.

As for the last 2 weeks I did approx over 750 cuts and nearly 80 slides: so I will give it a few days before starting a 100 cut comparison run.  .

Next ambitious project: try and do a CLA (Clean, Lubricate, Adjust) of the BH2 Trinocular  .

Best,
Maarten

[Bob]

Hi Maarten,
the last sections with the SHK holder look quite good, I wouldn't see the torn cell walls as a safe indicator for a problem with blade or holder, they sometimes simply tear.
In my view the assembly of blade and holder should be ok. The maximum clamping force is needed right behind the edge, the blade should have no play there. The clamping force comes from the knurled nuts in the middle of the holder. The other support should be in the back of the flat area. In the first image of your last post it looks as if there were a gap - true? If there is a gap you might close this by inserting a strip of cardboard. The blade should idealy be securely clamped before the knurled nuts are fully tightenend so the cap of the holder exerts some spring action in pressing on the blade.

750 sections x 0,05mm = 37,5mm! 

Bob