Guidance with stains.

Guidance with stains.

Postby Richard Scott » Sun May 09, 2010 12:48 pm

Could someone point me in the right direction for determining which stain to use under various circumstances.
Also I find that when I drop stain onto my specimen the colour is so strong that it impedes the image especially at x1000.
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Re: Guidance with stains.

Postby Neil » Sun May 09, 2010 11:23 pm

Hello Richard,

It may be the case of you're leaving the sample to soak in the stain for too long prior to putting it to the microscope, I honestly don't know what you're doing 'wrong.'

The subject is too complex to tell you here, so I suggest you use Google and type in 'Guide to chemicals for fungal microscopy', but I also believe MJ did an article in the Forayer not too long ago.

Personally, I tend to use congo red (in ammonia) for just about everything and Melzers for most discomycetes.
Some get by just by using tap water, but they must struggle.

Neil Mahler.
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Re: Guidance with stains.

Postby Leif Goodwin » Mon May 10, 2010 5:55 pm

Sounds like it is too strong. Dilute it down and see how you go.

I agree with Neil that Congo Red is an excellent general purpose stain. I dissolve the powder in household ammonia bought from a chemists. Unfortunately it does not always show spore ornamentation, so I also use use lactophenol cotton blue. Melzers is excellent - or so I am told - but it contains a controlled substance making it hard to obtain.

I tried a google, but did not find much. Most discussion of microscopy stains relates to non-mycological uses.
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Re: Guidance with stains.

Postby Richard Scott » Tue May 11, 2010 5:50 pm

Neil/Leif, I think following your replies that I have been making a wrong assumption...
When I have cut my specimen, I have placed it on the microscope slide and added a little KOH followed by a drop of stain (I have tried Congo red & cotton blue).
I have then immediately put on the slide cover. From your replies should I be applying a drop of stain to my specimen, leave it for a while, then remove it from the stain and place it on the microscope slide?
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Re: Guidance with stains.

Postby Roy Miller » Tue May 11, 2010 7:26 pm

Personally, I use different strategies, with Congo Red I may use it either in water, ammonia or KOH, but I rarely exceed a staining period of 30 secs - at which point I drop water one side of the stain and absorb with a tissue/kitchen towel from the other side so that the water is drawn through the sample flushing it out. There are of course times when you may need to apply ammonia or KOH for more than 30 secs, in these cases I do the staining separately before flushing it out.

I expect everyone has their own way of doing things, whatever works is good.
Last edited by Roy Miller on Tue May 11, 2010 11:11 pm, edited 1 time in total.
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Re: Guidance with stains.

Postby Leif Goodwin » Tue May 11, 2010 10:15 pm

I used to dissolve Congo Red in water but found that it precipitated out of solution rather quickly, with lots of particles floating about. I think it was okay if used immediately. A jar of prepared CR in ammonia solution keeps for weeks which is good for lazy people like me. I've not tried KOH. I think Roy's flushing technique is widely used. It certainly makes sense if you want to limit the staining.

A point worth making, and hopefully not too obvious, is that you usually want a very thin/small sample of tissue. A widely used technique is to use needle nosed tweezers to pull off tiny pieces. This is then stained and squashed under the cover glass, taking care not to break the glass. Recently I found a more brute force approach of squashing stained tissue between two slides created very thin samples, which could then be studied in the usual manner on a slide with a cover glass. Maybe I'm a bit of a thug, and more skilled workers are wincing, but it can sometimes be hard to isolate cystidia.

Roy and others: could you please explain the purpose of KOH with stain. I know it is used with some pyrenomycetes. Thanks.
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Re: Guidance with stains.

Postby Roy Miller » Tue May 11, 2010 11:07 pm

The KOH/CR combination works particularly well with Calocera (and some of the other obstinate ones who don't wanna soften). My reasoning is why do something in 2 stages if you can do it in one, but of course one has to be mindful of the concentration/ratio of the stain in longer soaks.
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Re: Guidance with stains.

Postby Neil » Wed May 12, 2010 12:12 am

The reason I obtained my bottle of KOH was to soften up the tough tissue of brackets, but I don't find it really helps.

I've often heard about Leif's use of squashing between two slides, I must give this a try, but I normally use the flat face of a scalpel blade (and end up loosing sight of the dammed sample) !

Neil.
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Re: Guidance with stains.

Postby Leif Goodwin » Wed May 12, 2010 1:13 pm

Thanks, it makes sense.
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Re: Guidance with stains.

Postby Andreas Gminder » Mon Jun 07, 2010 8:55 am

Hello,

the usual reciepts for Congo Red always talk about a saturated solution, be it in water, in NH3 or in SDS. The problem is, that when the saturated solution gets colder, the congo red starts falling out. Same is true for the solution in NH3, because with every opening of the bottle some NH3 vanishes, and soon the solution becomes over-saturated and falls out. You can fix that by warming your bottle a little bit, so that the solution is no longer oversaturated and gets clear again. If you mix your solution not saturated, you get worse results, so this is no good idea.

As I feel that there is a need for explanation about use of stains, I will prepare a longer answer in a separate thread.

best regards,
Andreas
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