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

[deBult]

Hello,

Beginners question: how long does it take for Euparal in Isopropanol to cure/settle/dry?

6 weeks ago I received a beginners kit from Klaus H. And some example slides from Wolfgang (thanks again)

Started with botanical stem slices (50u thickness approximately) with WAsim III (one combined solution variant) de-hydration with Isopropyl alcohol (10 seconds-30 seconds-2 minutes). Embedded in Euparal in Isopropanol.

Slides are on a warming plate 40 Degrees Celsius for 6 weeks now: and still the Euparal has not cured/dryed completely: when cleaning the slides for observation it is very easy to move the coverglass and distroy the slide.
I received some example slides from Wolfgang dated September 2019 sing Euparal: the are dry/cured completely.

What am I doing wrong?
Guidance appreciated on how to improve my way of working.

Best,
Maarten

[Klaus Herrmann]

Hi Maarten,

Slides are on a warming plate 40 Degrees Celsius for 6 weeks now: and still the Euparal has not cured/dryed completely:

I put my slides over night into the lab-oven at 50° and then they are done! I can only think, that the thickness is too high. Is it possible, that your cuts are 100 µ or more?
Could you show a picture?

[deBult]

Slide thickness: yes this may be the issue as cutting with the table microtome and the Leitz blades is still a struggle.

Will try and get a picture up tomorrow.

Thanks for feed back.

[Fahrenheit]

Hi Maarten,

Euparal takes about 6 months to cure completely. My slides sit on a hot plate at about 45 to 50 degree for one week, befor I do the labeling. That's enough to put them into a slide box in upright possision but one should be carefull when cleaning the cover glass. So Klaus seems to be somewhat hasty ... 

If You have a hot plate too You might want to weight the slides with a 8 nut on the cover glass to keep the specimen level.

Good luck for the picture! Camera adaption is not always easy ...

Best
Jörg

[deBult]

First try on a photo. (well trying to figure out how to upload (the upload instruction appear to be in the 2009 edition of the forum and I have no access there).

Feed back on cut (thickness OK?), staining and photo quality appreciated.

[Bob]

Hi Maarten,

the picture upload has been changed since 2009. I attatch a picture that shows how to do this now. There are size limits to be considered. Irfan View is a nice program to reduce images in resolution and also to reduce to a given file size. It is ideal to use the forums image host as most external image hosts have a questionable future.

The section looks nicely made. There is a bit of residual red stain in the left side that could have been washed out a bit better. On the right side some torn cell walls seem to be drawn over the section. When have you started to make botanic sections? Not bad!

Bob

[deBult]

Bob,

Thank you.

This is my 2nd try on sections and staining and first photo with my newly acquired Trino head.

Maarten

[Fahrenheit]

Good Morning Maarten,

the photo looks fine so far. When the whole section ist the same it's not to thick and the artefacts Bob mentioned will vanish when You geht used to slicing. Are You using a microtom knife or single use blades? Even if these are expensive, they should be used only once.

After staining and rinsing with Aqua dest. You can try slow differenciation by leaving the stained sections in Aqua dest. for up to 24 h. This will remove the red from the cells where it does not belong.

Best
Jörg   

[deBult]

Thank you all for guidance: new MK2 version: feed back appreciated


- Klaus: practicing on getting thin cuts

- Jorg: New cut with Table microtome, SHK and fresh knife (Yes I was reusing an old knife  ): so less torn cell walls now

- Bob: Check: I think I need to improve on the staining and washing: we have a lot of green residual stain

Protocol:
   * AFE 2 days => 70 % alcohol
   * Table microtome, SHK and fresh knife, Schneidehilfe
   * A drop of 70% alcohol on knife and sample each cut
   * Use a small brush to move the cut
   * 70 % Alcohol => 30 %, toilet paper to remove the fluids
   * 5 min staining on the slide itself: 2 drops WAsimIII combined solution from Klaus
      and a bit of moving/shaking the slide to distribute the stain very 30 seconds
   * 30 sec Alcohol 30 %=> 30 sec 70 % => 30 Sec 90%  (on the slide): I  loose a lot of red stain here
   * Isopropanol 30 seconds 3 times
   * Euparal

Noot: still practicing on taking pictures as well

[Fahrenheit]

Hi Maarten,

then let us take the next step now.

- Fixation is fine, You may also try to cut fresh samples and do a slice fixation instead (min. 20 min. AFE, then rinse with Ethanol 70%)

- Important: bring the slices into Aqua dest. prior to staining. This should happen in steps (descending Ethanol row of 70% Ethanol, 50%, 30%, drop some water in, more water when the sections stops dancing ..., water).
- Do the staining for 15 minutes with some heating but not boiling (all steps best done with the slices sitting in a watch glass ....), shake now and then during the staining time
- rinse with Aqua dest.
- leave section in Aqua dest. for 1 to 24 hours to differentiate, change Aqua dest. now and then, You can stop the Process, if the water is clear. There is no danger to overdifferentiation.

- Get the slices in Isopropanol quick (no ascending Ethanol row)
  (3 * fresh Isopropanol 100% for some seconds, 2 * fresh Isopropanol for 1 minute, 2* fresh Isopropanol for 5 minutes each).
- Euparal

Best
Jörg

[deBult]

Retry No 3: updated staining protocol as per Jorg guidance:

Protocol v 3 (UPDATES IN CAPITAL)
   * AFE 2 days => 70 % alcohol
   * Table microtome, SHK and fresh knife, Schneidehilfe
   * A drop of 70% alcohol on knife and sample each cut
   * Use a small brush to move the cut
   * DESCENDING ALCOHOL 70 => 50 => 35 => 0 %
   * STAINING NOW IN WATCH GLASES
      - (no longer on the slide itself) diluted (2 drops + a little remaining water in watch glass) WAsimIII combined solution from Klaus
      -15 minutes on the warming plate approx 50 Degrees Celcius and a bit of moving/shaking the slide to distribute the stain
   * DIFFERENTIATION 1.5 hour in aqua dest (mainly loosing some red)
   * (skipped the ascending Ethanol steps)
   * Isopropanol : twice fast change, twice 1 minute, twice 5 minutes
   * Euparal

Observations:
- I think the cut is still to thick?
- Stain remains in some cells: how to prevent / correct this?

note: working on Photo quality and parfocality: updated version

[Fahrenheit]

Hi Maarten,

looks fine so far. I think that even the red colour in some bark parenchyma cells would still be removed if they were left in distilled water for a longer period of time. On the other hand: you won't get rid of all artefacts even with a verry, very long time of differentiation.

best
Jörg

[Klaus Herrmann]

Hallo Maarten,
da du ja Deutsch verstehst schreibe ich in Deutsch. Die wichtigsten Informationen hat Jörg dir ja schon gegeben.
Die dunklen Flecken sind gefärbte Zellwände, das hat man oft wenn der Schnitt etwas dicker ist als eine Zelle. Die Zellen sind ja 3dimensionale "Behälter" komplett von einer Zellwand umhüllt. In einem dünnen Schnitt sind die oben und unten liegenden Wände abgeschnitten, so bleibt nur das "Häkeldeckchen-Muster" übrig.
Diese störenden Reste kann man mit einer Behandlung der Schnitte mit Natrium-Hypochlorid entfernen. Handelsname in Deutschland ist Clorix. Es wird nicht konzentriert sonder verdünnt eingesetzt. Über Dauer der Einwirkung und Konzentrationen können dir die Spezialisten sicher Auskunft geben. Nach der Behandlung muss sehr gut ausgewaschen werden eventuell sogar noch neutralisiert mit Essigsäure. Sonst wird die anschließende Färbung durch Oxidation zerstört.

[Fahrenheit]

Hi Maarten,

yes, Klaus is right. Sodium dichlorite or potassium hypochlorite bleache the cell walls and dissolve remains of cell content. If used to long, both also dissolve the middle lamella of the tissue and you will end up with single cells at last.

So if You want to use Klorix (the blue bottle, not the green one) You should use 1 part Klorix and 4 parts Aqua dest. You also can use Eau de Javel, which is sold by DM (at least in Germany). To geht used to ist You might also dilute with 2 parts of Aqua dest.

I usually use times around 60 to 90 seconds with lens controll.

Important: rinse until You don't smell chlorine anymore.

Best
Jörg

[deBult]

Jorg und Klaus: danke.

I will practice my cutting to get thinner slides (both fresh and AFE stored) and try the NaClO bleach.

Best
Maarten