fixative for botany vs zoology

[mete]

I see many fixatives are said to be for botany or zoology, some says it is for both. Why is that ? Due to cell wall in plants or different stains employed or only because it is just not tested or not used much with other types of samples ?

For example the list in http://www.aeisner.de under Rezepte.

[MicroBob]

I think the typical FAE botanical fixative is focussed on the cell walls. A good zoological fixative will be more involved. A lot of work has been carried out on this topic so it probably is unlikely that a simple but good method has been overlooked.

[mete]

I am looking into coagulant/non-formalin formulas and new ones are published in recent years because of the issue with formalin but either because of the sources I am looking or maybe because of the main use I dont know, I always see their use with animal tissue in the papers. So I wonder if it is just because the authors interest is not botany or the proposed fixative would have disadvantages when used with plants.

[ldflan]

I think there has simply been vastly more histological work and research on animal tissue than there has been on plant tissues. Why? The money has been in animal tissue research, not plants. Probably as simple as that.

For coagulating botanical fixatives there is really nothing very good that I know of which does not contains formalin, paraformaldehyde, glutaraldehyde, or else some nasty heavy metal like osmium tetroxide or mercuric chloride. Zirkle's type dichromate fixatives might be the one exception, if a basic fixative is acceptable for your purposes.

Couple of good sources for botanical microtechnique:

Berlyn & Miksche, "Botanical Microtechnique and Cytochemistry" (1976)
Ruzin, "Plant Microtechnique and Microscopy" (1999)

For basic botanical histochemical techniques that are reasonably accessible to the amateur scientist:

Jensen, "Botanical Histochemistry" (1962)

If you are not embedding and sectioning, but are making hand sections or whole mounts of leaf or similar tissue, you may want to look at Visikol for Plant Biology (TM). The product contains a precursor to chloral hydrate for tissue clearing, and a fixative mixture consisting of methyl alcohol and trichloroacetic acid, with glycerol as a plasticizer. The best part about Visikol is it's legal where chloral hydrate often is not; the downsides are that it is quite expensive, very very very difficult to make permanent slides from the stuff, and it tends to make staining using standard histological dyes difficult. It is also not a good cytoplasmic fixative for use prior to paraffin embedding, in my experience.

I am not a great expert on the subject, but I have been working on plant and insect tissue for a number of years now. The best and most accessible general purpose coagulant fixatives for botanical samples I have used either employ formalin or (if possible and better) ice cold paraformaldehyde in an appropriate stabilization buffer. (e.g., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625903/)

Bottom line, though, is that anything that works well as a cellular fixative - whether for plant or animal tissue - is by definition going to be toxic to your body's cells and therefore dangerous to handle! That's just an inescapable fact. My approach has been to accept that, install a fume hood, avoid the toxic heavy metal fixatives, and to be really careful!

If you are embedding, the next and equally critical topic for good results is the dehydration process. The best approach I have found involves a graded N-buty or tertiary butyl alcohol series. These are also paraffin solvents, so xylene can be eliminated from the process.

Leonard

[mete]

Many thanks for your detailed reply.

I think there has simply been vastly more histological work and research on animal tissue than there has been on plant tissues. Why? The money has been in animal tissue research, not plants. Probably as simple as that.

I understand, it makes sense. I think maybe it is also due to medical research, that naturally applies mostly human or animals more than plants.

For coagulating botanical fixatives there is really nothing very good that I know of which does not contains formalin, paraformaldehyde, glutaraldehyde, or else some nasty heavy metal like osmium tetroxide or mercuric chloride. Zirkle's type dichromate fixatives might be the one exception, if a basic fixative is acceptable for your purposes.

I am at no point to say if a fixative is good enough, but from the recent (animal histology) literature I see it is argued some are quite good (indistinguishable from traditional ones at least for some purposes) and they do not contain something too toxic (formaldehyde, mercury etc.) (e.g. https://pubmed.ncbi.nlm.nih.gov/27221702/). However, all such things I see is tested on animal tissue, hence my question. Also I think I did not learn yet how to search info on plant research.

Couple of good sources for botanical microtechnique:

Berlyn & Miksche, "Botanical Microtechnique and Cytochemistry" (1976)
Ruzin, "Plant Microtechnique and Microscopy" (1999)

For basic botanical histochemical techniques that are reasonably accessible to the amateur scientist:

Jensen, "Botanical Histochemistry" (1962)

For some weeks, I am actually scanning many books since early 1900s. I had 2 of the books above, I will check Jensen probably this week.

If you are not embedding and sectioning, but are making hand sections or whole mounts of leaf or similar tissue, you may want to look at Visikol for Plant Biology (TM). The product contains a precursor to chloral hydrate for tissue clearing, and a fixative mixture consisting of methyl alcohol and trichloroacetic acid, with glycerol as a plasticizer. The best part about Visikol is it's legal where chloral hydrate often is not; the downsides are that it is quite expensive, very very very difficult to make permanent slides from the stuff, and it tends to make staining using standard histological dyes difficult. It is also not a good cytoplasmic fixative for use prior to paraffin embedding, in my experience.

I am interested in stem and leaf sections. I dont plan to do paraffin embedding for sectioning (at least for now) and I dont plan to make very permanent slides. For permanent slides, it is good for me to have them up to a week or month maybe, so I am looking for either simple or aq. based mounting solutions. I havent heard about Visikol before, I will check. I was going to say it might be OK to be expensive, but looking at its price, it is really expensive, even more than the water based mounts used for fluorescence.

I am not a great expert on the subject, but I have been working on plant and insect tissue for a number of years now. The best and most accessible general purpose coagulant fixatives for botanical samples I have used either employ formalin or (if possible and better) ice cold paraformaldehyde in an appropriate stabilization buffer. (e.g., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625903/)

Thanks for the link, good to know the name of the journal.

Bottom line, though, is that anything that works well as a cellular fixative - whether for plant or animal tissue - is by definition going to be toxic to your body's cells and therefore dangerous to handle! That's just an inescapable fact. My approach has been to accept that, install a fume hood, avoid the toxic heavy metal fixatives, and to be really careful!

I have a simple recirculating desktop fume extractor and I agree with your point but still I feel there is a difference between handling something like alcohol, reasonably diluted acetic acid, glycol etc. and a toxic gas or mutagenic/carcinogenic material, that is why I am at this point (+I work inside my apartment, +I have kids). I am also trying to be very picky on dyes.

[ldflan]

Oh yes, do avoid the nasty stuff like cooking paraformaldehyde in an apartment! I would probably be ok with using FAA or FPA myself, but not heating it. But I can certainly understand avoiding it with kids around, too.

Of the alcohols, I think a methanol would be your best bet. Of course it is extremely volatile and flammable, so there is that consideration... In any case, it penetrates very well and does not harden much. Acetone would be my next choice, but it probably doesn't meet your safety concerns in terms of mutagenic activity, and is similarly a fire hazard.

I've read the article you noted about buffered ethanol. Haven't tried the stuff. As I recall, their big concern was a fixative that does not denature proteins so much that it impairs immunohistochemical tagging. Not so important for your work, I expect.

From your description of what you are doing, I would highly recommend that you do try Visikol for Plant Biology, despite the expense. It only takes 1-2 drops per slide. It can in some measure be reused if you want to immerse larger pieces in it, and can be diluted with methanol to some extent. If you can lay hands on 2,2,2 trichloroethanol and don't mind potentially violating the Visikol patent (if it is valid), you can try to mix up a version of the stuff for yourself. They use tricholoracetic acid and methanol as the fixative components, but I expect acetic acid and methanol would probably work fine instead.

You are right to expect that the usual dyes are also dangerous, as are fixatives in general. Anything that binds to intracellular components and dyes them effectively will bind to the organelles in your cells, too!

If you try the BE70, let us know how it works!

[mete]

Of the alcohols, I think a methanol would be your best bet. Of course it is extremely volatile and flammable, so there is that consideration... In any case, it penetrates very well and does not harden much. Acetone would be my next choice, but it probably doesn't meet your safety concerns in terms of mutagenic activity, and is similarly a fire hazard.

I am using IPA at the moment.

I've read the article you noted about buffered ethanol. Haven't tried the stuff. As I recall, their big concern was a fixative that does not denature proteins so much that it impairs immunohistochemical tagging. Not so important for your work, I expect.

Correct, the problem with recent methods I think they are mainly targeted for advanced applications, not surprisingly. I am interested in the safety side of it.

If you try the BE70, let us know how it works!

I am considering to try it, there is a pH adjustment needed to make it absolutely same as the article, I am not so sure about that. If interested, there is a reply from the author to a question regarding the preparation of the fixative, in the same journal.

[Microscopy_is_fun]

I have a simple recirculating desktop fume extractor and I agree with your point but still I feel there is a difference between handling something like alcohol, reasonably diluted acetic acid, glycol etc. and a toxic gas or mutagenic/carcinogenic material, that is why I am at this point (+I work inside my apartment, +I have kids). I am also trying to be very picky on dyes.

In case you don't know about the SafeLine products of Morphisto, here is a link. These chemicals are supposed to be used in a non-professional environment and are of low or no toxicity.

https://www.morphisto.de/fr/shop/list/f ... dukte/176/

[mete]

In case you don't know about the SafeLine products of Morphisto, here is a link. These chemicals are supposed to be used in a non-professional environment and are of low or no toxicity.

https://www.morphisto.de/fr/shop/list/f ... dukte/176/

I havent seen it, thank you.