Botanical Microtechnique: Staining Fresh Material

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Ecki
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Botanical Microtechnique: Staining Fresh Material

Post by Ecki »

Probably the most beautiful results can be obtained by cutting and staining fresh material. In this post I will explain how to fix and stain cuts.

We will need some chemicals and some equipment:

Isopropyl pure
Ethanol 70%
Ethanol 50%
Ethanol 30%
Aqua dest.
Etzold Staining Solution (Astra Blue, Fuchsine, Chrysoidine)
Fixation Solution AFE (90% Ethanol, 5% Formalin, 5% Glacial Acid)
Euparal

Pipette: 4x
Watch Glas

The general procedure will be to leave the cuts in the watch glas and remove the chemicals with a pipette. Use 1 pipette for AFE, 1 for Ethanol, 1 for Aqua dest and stains, 1 for Isopropyl. It is very important that the Isopropyl is as dry as possible. Water in the Isopropyl will remove the Chrysoidine. It is a good idea to shake the watch glas a little bit after a new chemical is applied.


Fill the watch glas (or another small container with a similar form) with AFE. Now cut fresh material either by hand or a small microtome. Each section that is cut will be put into the watch glas. After the last cut is safely in the watch glas, simply follow this procedure:

15 min AFE, then remove with pipette 1
5 min Ethanol 70%, then remove with pipette 2
3 min Ethanol 50%, then remove with pipette 2
3 min Ethanol 30%, then remove with pipette 2
3 min Aqua dest, change 3 times with pipette 3
10 min Etzold Staining Solution, apply heat (50° C), then remove with pipette 3. Use 10 drops of the staining solution so the sections are covered with the staining solution.
Use Aqua dest. to rinse of excess staining solutions, remove with pipette 3
10 sec Isopropyl pure, remove with pipette 4
2 min Isopropyl pure, remove with pipette 4
5 min Isopropyl pure, remove with pipette 4
Isopropyl pure

Now use a Stereo Microscope or something similar to find the best samples and put them with a fine brush onto a freshly cleaned slide. Put a drop of Euparal on it and a cover glas. Apply some pressure (careful!).

Now bake at 40° C for 48 hours. Voila, we are done. If you did everything correct the result will look like this:

Image
English Yew, Taxus baccata

Give it a try and show your results. It is nowhere as difficult as it sounds (even I can do it) and a lot of fun once you mastered it.

Questions are always welcome and if you need help obtaining the Etzold Staining Solution, please drop me a Message and I will help.

Good luck!

ChrisR
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Post by ChrisR »

This is beautiful, Ekhard.
As far as I know though, chemicals like ethanol, isopropyl alcohol, glacial acetic acid, would not be available to individuals in the UK.

rjlittlefield
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Post by rjlittlefield »

A simple question: is "aqua dest." simply high quality distilled water (as suggested by Google search), or is it something else?

--Rik

rjlittlefield
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Post by rjlittlefield »

And a not-so-simple question...

You mention that "Water in the Isopropyl will remove the Chrysoidine."

In the procedure, it looks to me that the staining step occurs just before rinsing with water, after which we switch to several changes of pure isopropyl.

So I am confused. I understand that if the isopropyl has any water in it, that will fog the Euparal. But I don't understand why water in the isopropyl would affect the chrysoidine, since staining is followed by a water rinse.

Can you explain in more detail what happens with the chrysoidine?

Many thanks for writing this description! It is very clear. I am only confused on this one small point.

--Rik

NikonUser
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Post by NikonUser »

Ecki: Thanks, very nice explanation of the methodology.

ChrisR: In Canada Isopropyl Alcohol is readily available as "Rubbing Alcohol"

Euparal is one of several mounting media. I have used it in the past only once or twice. Its odour gives me a terrible migrane headache almost immediately on opening the bottle. I simply cannot use it. I guess there is no way you can predict how anyone will react to Euparal.

One advantage of Euparal is that it is miscible with alcohol. Most of the other mounting media will not mix with alcohol and so you have to go through a Xylene wash before mounting.
Things may have changed as it has been many years since I made permanent mounts.

Rik: Standard practice is (or was) to overstain the subject and then control the destaining under a scope to decide how much intensity you want. So perhaps you overstain with the Etzold soln and then control the final intensity with water washes. You stop the destaining with pure alcohol; but if there is water in the alcohol then some destaining will continue.

But let's wait and see what Ecki has to say.
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

Ecki
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Post by Ecki »

Yes Rik, Aqua dest. is high quality distilled water.

The pigments for this staining are solved in water. Rinsing with water will remove any excess staining solution that is not bound to the cells. The rinsing takes 10 seconds.

If you have some water in the Isopropyl you will see red color rising from the cuts. Quickly remove the Isopropyl and apply fresh Isopropyl. Euparal is indeed miscible with alcohol. While the Euparal "bakes" the Isopropyl gases out. If there was some water in the Isopropyl the Red will get pale in a couple of weeks.

In Germany it is difficult to obtain the chemicals, too. There are many solutions, from finding a pharmacy that understands the need of a microscopist to joining a microscopic society.

Best regards,
Ecki

Harold Gough
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Post by Harold Gough »

NikonUser wrote: In Canada Isopropyl Alcohol is readily available as "Rubbing Alcohol"
The equivalent in the UK is surgical spirit which may contain some oil of wintergreen.

Harold
My images are a medium for sharing some of my experiences: they are not me.

NikonUser
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Post by NikonUser »

ChrisR wrote:This is beautiful, Ekhard.
As far as I know though, chemicals like ethanol, isopropyl alcohol, glacial acetic acid, would not be available to individuals in the UK.
Brunel sells several chemicals:
HERE
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

rjlittlefield
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Post by rjlittlefield »

Ecki wrote:Rinsing with water will remove any excess staining solution that is not bound to the cells. The rinsing takes 10 seconds.

If you have some water in the Isopropyl you will see red color rising from the cuts. Quickly remove the Isopropyl and apply fresh Isopropyl.
Thanks, Ecki -- I understand now.

--Rik

Ecki
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Post by Ecki »

This staining technique delivers the following result:

Woody cell-walls: red. Often the colors vary, sclerenchyma purple, xylem more brick-red or yellow red. The chrysoidine reacts with lignin, the protein that makes cell-walls woody.
Cutin cell-walls: yellow to orange, sometimes red
Cell-walls without cutin or lignin: green
Cork: colorless, but middle lamellae red as they are woody
Plasma: light red

This staining was developed by Dr. Helmut Etzold. It differentiates the different cell types well and can be used as a mixture - thus making the staining process pretty easy.

To produce the staining solution solve in 1 liter distilled water:

Glacial Acid = 20 ml
Astra Blue = 1,25g
Fuchsine = 0,1g
Chrysoidine = 0,143g

Harold Gough
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Post by Harold Gough »

This is slightly off topic but might be of interest:

http://www.microscopy-uk.org.uk/mag/ind ... mount.html

As might this:

http://www.nhm.ac.uk/hosted_sites/queke ... links.html

Harold
My images are a medium for sharing some of my experiences: they are not me.

Graham Stabler
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Location: Swindon, UK

Post by Graham Stabler »

Isopropyl Alcohol can be bought in maplins as a spray can or as a litre bottle.

Graham

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