Staining techniques

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AntoniScott
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Joined: Tue Dec 24, 2019 3:54 pm

Staining techniques

#1 Post by AntoniScott » Mon Dec 28, 2020 5:59 pm

As a lifelong enthusiast of the microscope I have never tried counter-staining. I have some prepared slides of stem crossections that show two different colors and would like to duplicate this process. I tried to make some permanent slides of simple things like onion skin with just one stain but during the dehydrating (70% to 100% alcohol) and clearing (xylene) procedure most of the color is washed out.

If anyone knows how to make the stains stay put I would love to hear from them how they do it.
Antoni Scott

tgss
Posts: 223
Joined: Wed Jan 25, 2017 3:48 am
Location: Ontario, Canada

Re: Staining techniques

#2 Post by tgss » Mon Dec 28, 2020 11:55 pm

Hi AntoniScott
There are a great many references that could be given in answer to your question, but two that I would recommend are:

Practical Microscopy by J. Eric Marson. Available from Brunel Microscopes in the UK for 12 GBP. This is general in its coverage (including botanical stains), but geared to the hobbyist, and is, as the title suggests, very practical in its approach. (http://www.brunelmicroscopes.co.uk/books.html)

Specifically for botanical work: Plant Microtechnique and Microscopy by Steven E. Ruzin (1999). This is geared more to the professional worker but is written in a very accessible style. I believe it's quite expensive new, but is available used at quite reasonable prices, from the likes of ABE Books, or perhaps even Amazon.
Tom W.

MicroBob
Posts: 3154
Joined: Sun Dec 25, 2016 9:11 am
Location: Northern Germany

Re: Staining techniques

#3 Post by MicroBob » Tue Dec 29, 2020 5:15 am

Hi Antoni,
the probably best source on botanic micro technique for the amateur is: http://www.mikroskopie-bonn.de/biblioth ... index.html
If you can't read german you will have to use google translator or DeepL but this site is worth the hassle.
A very good database on stains is: http://www.aeisner.de/

For a differentiating multiple stain you need stains that attatch to different structures, like lignified / non lignified cell walls. In the end you have to bring your section into the mountant without removing the stain, which can be difficult. A water based stain should not be drawn out too much by water, and often they survive in pure alcohol but water/alcohol mixed remove them quickly. So dehydrating in steps might not be possible with some stains. In Germany we mostly use Euparal for this purpose which can cope with a little water in the section and can be used with isopropanol.

Most quick and still beautiful is the Etzold FCA simultaneous triple stain, Fuchsine,Chryosidine,Astrablau:
- Plant part in 70% Ethanol anfter 24 hours in FAE fixative
- Make section
- Bring into water
- Apply stain on section on the slide, warm a bit without cooking
- Remove stain with water
- Apply 100% isopropanol, replace quickly, another 3x
- Euparal, cover slip

Another very nice triple stain is Wacker W3A, also available as simultaneous stain, nice fluorescence.

What stains do you have available? Perhaps we can find a promising combination to experiment with.

Bob
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AntoniScott
Posts: 108
Joined: Tue Dec 24, 2019 3:54 pm

Re: Staining techniques

#4 Post by AntoniScott » Tue Dec 29, 2020 12:21 pm

Hello MicroBob:
Thank you for the informative response. You have given me a lot of information to research. If I may ask what you mean by "FAE fixative" ? The German documents translated well.
I have several stains available. I'm not at my home right now and can't remember exactly all of them but I have Methylene Blue (two kinds, one is Loeffler's), Eosin, Hematoxylin, Carbol Rose, and a few others, but if I need a specific stain I am sure I can find them.
I was attracted to the idea of section staining from some inexpensive but beautifully stained slides from China. The colors are quite vivid. Based on the price, I'm sure a lot of time was not spent on staining and preparing these slides.
Antoni

AntoniScott
Posts: 108
Joined: Tue Dec 24, 2019 3:54 pm

Re: Staining techniques

#5 Post by AntoniScott » Tue Dec 29, 2020 12:28 pm

Hello TomW:
Thank you for your informative response. I contacted Brunel and will order the book you recommended.
Antoni

MicroBob
Posts: 3154
Joined: Sun Dec 25, 2016 9:11 am
Location: Northern Germany

Re: Staining techniques

#6 Post by MicroBob » Tue Dec 29, 2020 12:28 pm

AntoniScott wrote:
Tue Dec 29, 2020 12:21 pm
If I may ask what you mean by "FAE fixative" ?
http://www.aeisner.de/rezepte/fixant2.html

tgss
Posts: 223
Joined: Wed Jan 25, 2017 3:48 am
Location: Ontario, Canada

Re: Staining techniques

#7 Post by tgss » Wed Dec 30, 2020 3:43 pm

Hi Antoni
Glad to hear you are thinking of ordering the book; I think you'll find it very helpful. I should mention that the author, Eric Marson, operated a business called Northern Biological Supply (NBS) which sold slides and slide making equipment and supplies. So many of the reagents mentioned in the book are referred to by NBS proprietary names, without any information on their composition, presumably as a way of supporting sales. A gentleman by the name of Doug Richardson had many of these reagents analyzed (both Mr. Richardson and Mr. Marson are now, unfortunately, deceased) and the results are published in a document called "Observations", that collects some of his microscopical work over many years. This can be downloaded here: https://www.quekett.org/wp-content/uplo ... ations.pdf.

In fact, however, as far as I can tell, most of Mr. Marson's methods and materials were fairly standard, well known protocols, with the details of use adapted to suit the amateur worker. His Safranin and Fast Green method for botanical sections, for example, is basically that of Johansen, which is covered in fair detail in Ruzin's book mentioned in my previous post, but you can also find the information with an internet search, for example here: http://microscopy.berkeley.edu/Resource ... aining.htm.
I also should mention that, if you are not already familiar with them, mrsonchus's excellent and extensive series of posts on this forum describing his adventures in plant microtechnique, which are informative, entertaining and inspiring! Many others on the forum have also contributed excellent material.

I offer the above not as an expert on the subject by any means, but as someone, like you, finding my way in this field. I'm just mentioning resources that I have found helpful in the hope that they will be as useful to you.
Tom W.

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