Another Moss Dissection

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mrsonchus
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Another Moss Dissection

#1 Post by mrsonchus » Wed Nov 14, 2018 9:37 pm

Hi again all, I've had another go at moss dissection and thought I's share a few images that you may find interesting.

This is a different moss to the one in my first dissection post, that seems to have some rather interesting features. This time I mounted the moss capsule (sliced crudely lengthways) in PVA and further leaf details, including some hand transverse (cross) sections, in water, again of course with coverslip applied, but no additional pressure as I applied to the more robust PVA mount....

So, here are a few recently gathered mosses in various containers, the bottom-right of which is the one dissected in this post. Also pictured are some of my forceps, the black ones are from one of the various super-cheap sets available for very little. The steel ones are 'superfine' and cost a lot more, but really do have the most amazingly fine tips that actually meet properly too!
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This moss was growing beneath the now dead 'canopy' of a patch of lilies in my garden, within the lawn too. It has a dark glossy green appearance with a prominent mid-line 'vein' or 'nerve' on each leaf. A few immature capsules were also present as a bit of a bonus....
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I removed a single shoot - this moss has no branches, just what look like individual shoots with the capsule (and seta) borne terminally - probably an 'acrocarpous' moss......
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Here's a close-up of one of those prominent teeth along the leaf edges,
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More details on next post, I've run out of images on this one and I haven't uploaded them for linking-to yet.....

Back soon in next post.
John B

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Re: Another Moss Dissection

#2 Post by mrsonchus » Wed Nov 14, 2018 9:55 pm

Soooo, to continue with this lovely-looking moss, a few more details.

Closer-in the 'ribs' along the top of the leaf nerve are seen a little more clearly as well as the teeth and general outline,
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Here the top image is of the cells of the lamina from the top, and the second image is the bottom surface of the ridged (on upper surface) nerve, the cells of which appear rectangular and not ridged. The lamina cells may be papillose but to see better I think I'll need some oblique-lighting.... All my images thus far are simple brightfield of course and so really quite limited when studying live whole tissue, even single-cell thick moss leaves as here...
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Here are images of a transverse-section across the leaf, taken by hand with a single-edged razorblade using a slide across the leaf as a straight-edge. The real detail of those intriguing ridges (actually known correctly as 'lamellae' (pl) I think) begin to emerge, prompting me to consider making some 'proper' permanent and stained microtome-sections of this type of moss.
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Really quite a striking structure when seen in section.....
Interesting to see what may be a hint of papillae on both sides of the leaf's lamina, and what may be adapter water-conducting cells (not however vessels and lignified as in 'higher' vascular plants - mosses are far simpler) running through the nerve?
ws_edit19.jpg
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More details from the PVA-mounted capsule in the next post....
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Re: Another Moss Dissection

#3 Post by mrsonchus » Wed Nov 14, 2018 10:12 pm

Right, to some basic details from the sliced-open capsule, seen here in a PVA mount.

This thick (relatively) capsule is mounted beneath a coverslip that is itself atop a pair of coverslips, one either side of the capsule on a slide. This of course gives the extra depth needed to both avoid squashing the capsule and enable the top coverslip to be mounted nicely parallel to the slide's surface...
ws_4pic1.jpg
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The outer cells of a moss capsule can yield very valuable taxonomic details for distinguishing some very similar mosses, so I thought I'd have a good look at them...

The cells are rectangular, their longest side is parallel to the long-axis of the capsule. Interestingly the longer cell-walls are significantly thicker than the transverse cell-walls appear to be - this may well be a valuable taxonomic character. Many moss capsules also have stomata (unlike anywhere else on a moss gametophyte or sporophyte) on the area at the base of the capsule where it meets the seta - although I haven't any images of this region yet... Very interesting things these tiny plants are!
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Closer-in (apologies for the rather poor image quality...),
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Finally here are what I think are immature spores that have escaped from the sliced-open capsule - again rather poor image but all I could manage today.
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Sorry this is such a quick-tour - It's only really practice - the serious and very long process of producing permanent slides is starting to call me....

Thanks for looking.

John B. :D
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Re: Another Moss Dissection

#4 Post by Hobbyst46 » Wed Nov 14, 2018 10:18 pm

Stunning work! from what I scantily read about mosses, I thought that they do not possess conducting tubes...
As for the images, they very nicely and clearly convey the botanical information!
I notice that the leaves of your moss sample are quite large - 5mm long or so?
BTW: Your "expensive" forceps are what we used to call "watchmaker forceps".
Last edited by Hobbyst46 on Wed Nov 14, 2018 10:25 pm, edited 2 times in total.
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Re: Another Moss Dissection

#5 Post by charlie g » Wed Nov 14, 2018 10:22 pm

Thanks for this study, Mr.Sonchus. I find your brightfield images a great transition from the macro images in this series. And always good for me to see your active bench. I have a collection of fine tweezers like your and use them often, splinter removals, gulp, tick removals, and a dedicated set for microscopy.

You really bring those native mosses to forum in a full dimensioned and clearly alive presentation for us, thank you,thank you. Charlie guevara

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Re: Another Moss Dissection

#6 Post by billbillt » Wed Nov 14, 2018 10:53 pm

Hi John B.,

What a wonderful collection of images!... thanks for sharing.. It is your usual tour-de-force complete study that should be in a text book....

The Best,
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Re: Another Moss Dissection

#7 Post by MichaelG. » Wed Nov 14, 2018 11:57 pm

Great work, John B

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Re: Another Moss Dissection

#8 Post by Roland » Thu Nov 15, 2018 10:49 am

Hi mrsonchus,
Nice images! I look forward to see other pictures of this moss in a "slow-tour" of yours. I think this is Atrichum undulatum, an acrocarp. See my article about this moss some time ago in the Microbehunter Magazine, April 2012 "A moss under the microscope"
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Re: Another Moss Dissection

#9 Post by mrsonchus » Thu Nov 15, 2018 11:47 am

Roland wrote:Hi mrsonchus,
Nice images! I look forward to see other pictures of this moss in a "slow-tour" of yours. I think this is Atrichum undulatum, an acrocarp. See my article about this moss some time ago in the Microbehunter Magazine, April 2012 "A moss under the microscope"
Roland


Hi Roland, thanks for the ID! You seem to be entirely correct olf chap, I've just had a quick look in a lovely old copy of one of my very favourite books, Watson's "British Mosses and Liverworts" and have found it on page 126, Bryidae, Polytrichales, Atrichum.undulatum.....

Coincidentally I was only 1hr ago stacking 2 images of the very distinctive leaf-edge teeth, which are 'doubles' and immediately thought that this character would be very useful taxonomically. Watson has, on page 126, a few very nice drawings, one of which is indeed these double teeth.
Here's a 2-image stack of these fascinating features,
ws_double teeth along leaf edge.jpg
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Also, here's an image to add of those nerve-lamellae, which as well as being 'in fours' in TS may also appear 'in threes' as in this image,
ws_leaf nerve lamellae.jpg
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Thanks for the ID and interest Roland, pleased you like them!

John.
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Re: Another Moss Dissection

#10 Post by mnmyco » Fri Nov 16, 2018 2:31 pm

Hobbyst46 wrote:Stunning work! from what I scantily read about mosses, I thought that they do not possess conducting tubes...
As for the images, they very nicely and clearly convey the botanical information!
I notice that the leaves of your moss sample are quite large - 5mm long or so?
BTW: Your "expensive" forceps are what we used to call "watchmaker forceps".
The vascular tissues in bryophytes are not considered true vascular tissue as they lack the specialization, phloem and xylem, in the true vascular plants.

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Re: Another Moss Dissection

#11 Post by Hobbyst46 » Sat Nov 17, 2018 4:30 pm

mrsonchus wrote:...
I just revealed this downloadable file:

CHAPTER 2-2 LABORATORY TECHNIQUES: SLIDE PREPARATION AND STAINS
Janice M. Glime and David H. Wagner
Chapt. 2-2. In: Glime, J. M. Bryophyte 2-2-1
Ecology. Volume 3. Methods. Ebook sponsored by Michigan Technological University and the International Association of Bryologists.
Ebook last updated 3 August 2017 and available at <http://digitalcommons.mtu.edu/bryophyte-ecology/>.

Might be of interest.
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Re: Another Moss Dissection

#12 Post by mrsonchus » Sat Nov 17, 2018 4:47 pm

Hobbyst46 wrote:
mrsonchus wrote:...
I just revealed this downloadable file:

CHAPTER 2-2 LABORATORY TECHNIQUES: SLIDE PREPARATION AND STAINS
Janice M. Glime and David H. Wagner
Chapt. 2-2. In: Glime, J. M. Bryophyte 2-2-1
Ecology. Volume 3. Methods. Ebook sponsored by Michigan Technological University and the International Association of Bryologists.
Ebook last updated 3 August 2017 and available at <http://digitalcommons.mtu.edu/bryophyte-ecology/>.

Might be of interest.
Very handy link, thanks Hobby'!

John.
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Re: Another Moss Dissection

#13 Post by billbillt » Sat Nov 17, 2018 8:28 pm

Thanks Hobby for the links....

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Re: Another Moss Dissection

#14 Post by PNWmossnerd » Fri Nov 23, 2018 7:49 am

Wonderful preparations of both the leaves and capsules! I agree that it is certainly a member of the genus Atrichum, and probably A. undulatum. This moss is a member of the family Polytrichaceae, which possess the most complex anatomy and morphology of any mosses. The lamellae are actually a way to increase photosynthetic efficiency in high-light environments, and are typical of the family. The Polytrichaceae is also the only moss family which possesses sugar and water conducting tissues in its gametophytes. The large circular cells in the nerve are actually the sugar conducting cells known as "deuters".

The double teeth are diagnostic of Atrichum.

The family Polytrichaceae is my favorite group of mosses. Earlier this year I completed a phylogenetic study of the group which you can read here: https://www.researchgate.net/publicatio ... trichaceae
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Re: Another Moss Dissection

#15 Post by PNWmossnerd » Fri Nov 23, 2018 7:58 am

Also, Mr. Sonchus,
I applaud your desire to make permanent mounts of mosses. Very few mosses have ever been subjected to rigorous anatomical study. Almost certainly if you imbed most moss species in paraffin and make microtome sections, you will be the first person in history to do so.

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Re: Another Moss Dissection

#16 Post by Hobbyst46 » Fri Nov 23, 2018 8:26 am

[quote="PNWmossnerd"]Also, Mr. Sonchus,
I applaud your desire to make permanent mounts of mosses. [quote]
May I pose some questions: for how long can the green color of the moss (the chlorophyll) be preserved in a semi-permanent or permanent mount? I have two 1-year old slides of moss in 80-90% fructose and glycerin, respectively. The green color looks better in fructose. But, a few weeks ago I mounted (the same moss type) in distilled water, and the green color faded within days.
As mrsonchus suggested, I think that PVA should be better than fructose. How well is the green color preserved in PVA? and what concentration of PVA should I use, if I buy solid powder or grains of PVA and dissolve it in water?
I would also like to try mounting moss in Norland 61 adhesive. Any experience with that?
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Re: Another Moss Dissection

#17 Post by SutherlandDesmids » Fri Nov 23, 2018 10:05 pm

This really is a wonderful series. It might be beneficial for beginners to state, if my memory serves me, that acrocarpous mosses bear the sporophyte on the end of the axis of growth, parallel to a vertical growth habit, whereas the pleurocarpous mosses bear the sporophytes on the sides of the axis of growth, appearing perpendicular to a horizontal growth habit.

I particularly like the sequence of photographs increasing in magnification, too often in my opinion section views are disassociated from what the entire plant (gametophyte in this case) looks like.

An two-per-cent solution of potassium acetate, just coloured blue with copper acetate, preserves the pigmentation of the green algae really very well, although it is necessary to seal such a liquid mount under a round cover-slip with gold-size or a proprietary ringing-cement and a rotary ringing table. The method, in a paper from as far back as 1909 (I got it from the ''British Desmidiaceae'') is said to produce good results with the protonemata of mosses and the prothallia of ferns. It's surely worth a try.

https://www.jstor.org/stable/2993242?se ... b_contents
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Re: Another Moss Dissection

#18 Post by Hobbyst46 » Fri Nov 23, 2018 10:43 pm

SutherlandDesmids wrote:An two-per-cent solution of potassium acetate, just coloured blue with copper acetate, preserves the pigmentation of the green algae really very well, although it is necessary to seal such a liquid mount under a round cover-slip with gold-size or a proprietary ringing-cement and a rotary ringing table. The method, in a paper from as far back as 1909 (I got it from the ''British Desmidiaceae'') is said to produce good results with the protonemata of mosses and the prothallia of ferns. It's surely worth a try.

https://www.jstor.org/stable/2993242?se ... b_contents
Thanks for this link and info. Perhaps worth to combine PVA and potassium acetate. The copper, I suspect, has been added to inhibit fungi and bacteria growth; I would add just a trace of copper sulfate. And seal the slide with gel nail polish, since ringing is not within my arsenal.
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Re: Another Moss Dissection

#19 Post by PNWmossnerd » Sun Nov 25, 2018 4:49 am

There is some wonderful discussion of mounting media here!

To specifically address your questions Hobbyist46,
In an appropriate permanent medium, I think the green color may be preserved for years. Unfortunately Eukitt (https://www.sigmaaldrich.com/catalog/pr ... &region=US) is the only permanent mounting medium I have used much. It has an excellent refractive index, dries quickly, and does not yellow with age. However, this medium is hydrophobic, so samples must be dehydrated before mounting and this process often removes the chlorophyll from samples. Additionally, the solvent in Eukitt is xylene which can be nasty stuff if you are regularly exposed to it. Eukitt is also seriously expensive. I only use it because many of my permanent mounts end up in museum collections and may be examined many years in the future so I need to use a mounting medium that doesn't yellow with age.

I do not know how long the green color is preserved in pure PVA, since I have only used a 1:1 mixture of PVA and glycerine. In my experience, the green color begins to fade after 6 months to a year, much as with pure glycerine. I have never used fructose. This thread has inspired me to try a mount using only PVA.

I actually use a Elmer's Clear School Glue which uses PVA as its active ingredient instead of mixing up my own PVA since the glue is cheap, readily available and works as well as home-brewed PVA. I use this PVA-based glue to seal microscope slides instead of nail polish since it is cheaper, water soluble, and doesn't smell.

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Re: Another Moss Dissection

#20 Post by MichaelG. » Sun Nov 25, 2018 7:49 am

SutherlandDesmids wrote: The method, in a paper from as far back as 1909 (I got it from the ''British Desmidiaceae'') is said to produce good results with the protonemata of mosses and the prothallia of ferns. It's surely worth a try.

https://www.jstor.org/stable/2993242?se ... b_contents
Thanks for that link
... I am not familiar with G.S. West's work, but he probably merits futher reading:
https://plants.jstor.org/stable/10.5555 ... m000154585

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Re: Another Moss Dissection

#21 Post by mrsonchus » Sun Nov 25, 2018 11:27 am

SutherlandDesmids wrote:......... The method, in a paper from as far back as 1909 (I got it from the ''British Desmidiaceae'') is said to produce good results with the protonemata of mosses and the prothallia of ferns. It's surely worth a try.

https://www.jstor.org/stable/2993242?se ... b_contents
Thanks for your kind comment and interest, and indeed for the link - downloaded and read!
Very handy and as it turns out very relevant for me right now. Only yesterday I was out roaming the garden and pulled up a piece of mossy-mystery from the lawn, quickly into my tiny lab for a quick look. I love just selecting some Botanical (moss of course in this case) material and identifying or just plain exploring the booty, with the aid always of a coupe of 'scopes and a few books....

The sample had a piece of soil and a tiny piece of what looks like decaying plant stem, possibly Sunflower I think. Anyway, to the relevance....
Within the treasure was a small amount of what I believe to be moss protonema/ta!

I've posted details and images in a separate post to aid clarity.

John B.
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Re: Another Moss Dissection

#22 Post by Hobbyst46 » Thu Nov 29, 2018 4:08 pm

PNWmossnerd wrote:I do not know how long the green color is preserved in pure PVA, since I have only used a 1:1 mixture of PVA and glycerine. In my experience, the green color begins to fade after 6 months to a year, much as with pure glycerine. I have never used fructose. This thread has inspired me to try a mount using only PVA. I actually use a Elmer's Clear School Glue which uses PVA as its active ingredient instead of mixing up my own PVA since the glue is cheap, readily available and works as well as home-brewed PVA. I use this PVA-based glue to seal microscope slides instead of nail polish since it is cheaper, water soluble, and doesn't smell.
I prepared PVA syrup in DW with potassium acetate and copper sulfate. Used vacuum to degas the syrup. The product is a viscous clear stuff, as thick as Elmer's glue or thicker. Preliminary mounting of moss "stems" is promising - preserves the natural green color, no obvious osmotic effects like shrinkage or deformation of the leaves. And the PVA dries at room temperature within 2-3 hours. And excess PVA is easily peeled off with a razor blade or scalpel. However, since PVA is hydrophilic (very!), I think that nail polish or the gel nail polish might be better long term sealants in this case. A year or two will tell how the green color is preserved...

@mrsonchus - thanks again, John B., for reminding about the usefulness of PVA.
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Re: Another Moss Dissection

#23 Post by mrsonchus » Fri Nov 30, 2018 8:21 am

Hobbyst46 wrote:
PNWmossnerd wrote:I do not know how long the green color is preserved in pure PVA, since I have only used a 1:1 mixture of PVA and glycerine. In my experience, the green color begins to fade after 6 months to a year, much as with pure glycerine. I have never used fructose. This thread has inspired me to try a mount using only PVA. I actually use a Elmer's Clear School Glue which uses PVA as its active ingredient instead of mixing up my own PVA since the glue is cheap, readily available and works as well as home-brewed PVA. I use this PVA-based glue to seal microscope slides instead of nail polish since it is cheaper, water soluble, and doesn't smell.
I prepared PVA syrup in DW with potassium acetate and copper sulfate. Used vacuum to degas the syrup. The product is a viscous clear stuff, as thick as Elmer's glue or thicker. Preliminary mounting of moss "stems" is promising - preserves the natural green color, no obvious osmotic effects like shrinkage or deformation of the leaves. And the PVA dries at room temperature within 2-3 hours. And excess PVA is easily peeled off with a razor blade or scalpel. However, since PVA is hydrophilic (very!), I think that nail polish or the gel nail polish might be better long term sealants in this case. A year or two will tell how the green color is preserved...

@mrsonchus - thanks again, John B., for reminding about the usefulness of PVA.
Nicely done my friend!
Can you give a little detail re the amount/s of potassium-acetate and copper-sulphate, also 'how it's combined' into the mounting mix?

I'd love to give it a try also as I'm looking at some ubiquitous Rhytidiadelphus.squarrosus and possibly R.loreus and I'd like to make a few mounts of the leaves, both for differences and the often difficult to see double short costae

John B.

URK - just remembered Sutherland's post above re the formula! Thanks Sutherland'.
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Re: Another Moss Dissection

#24 Post by Hobbyst46 » Fri Nov 30, 2018 11:24 am

mrsonchus wrote:Can you give a little detail re the amount/s of potassium-acetate and copper-sulphate, also 'how it's combined' into the mounting mix?
My starting materials were chemical powders, residues of old chemical supplies. PVA and the acetate are very water-soluble so easy to prepare. Pure PVA white powder, MW around 50,000. To 4g of the powder I added 8g DW in a 20ml capped glass vial. After some swirling and intensive shaking (by hand; I do not have a stirrer or bottle roller), and 15min immersion of the vial in a small pot of boiling water, it dissolved to form a fairly clear syrup. In a separate vial, I added about 8g potassium acetate to 5g DW. Shaking and swirling gave a clear solution within minutes. To this solution I added 10-20 tiny grains of anhydrous copper sulfate. Left overnight, it dissolved completely and tinted the solution very pale blue. 3 drops of the solution was added to 10ml of the syrup, swirled until complete mixing by the eye.

THE CORRECT protocol, that I recommend is somewhat different, as follows:
1. Prepare a 50% solution of potassium acetate in DW. No need to heat for dissolution.
2. Add a few grains (per 10ml, say) of copper sulfate, mix. Ubiquitous copper sulfate (not anhydrous) is in the form of blue crystals, these will dissolve rapidly.
Note: by "grain" I mean a tiny particle, not the "Grain (unit), a unit of mass equal to 64.79891 milligrams, ​1⁄7,000 of an avoirdupois pound" (Wikipedia).
3. Place 5ml (say) of the solution in a capped vial (or beaker, depending on your mixing facilities). Gradually, add the PVA while heating at 80-90C and stirring; or repeat my shortcut, add the full amount, cap the vial, place in a boiling water bath, swirl occasionally, until the syrup emerges.

The best mixer to make syrups AFAIK is an array of two cylindrical rollers in parallel, nearby each other. The rollers are rotated (electric motor) in opposite directions - one CW, the other CCW - such that a bottle or vial that lies between them (along the space between them) slowly rotates around its long axis. IF I had this device, could lay the vial on top , turn rotation on and go away, the mixing would be complete within hours.

P.S I cannot guarantee that the above syrups are totally free from minute particles. They look colloid-like. Can be filtered, not very easy with a syringe and a syringe filter, under mechanical pressure.

Edit: Just discovered a stupid mistake in the "CORRECT" protocol above - the aim is 2-3% acetate in the final syrup, not 25%. So After step 2, dilute the acetate-copper mixture ten-fold, and in the dilute solution dissolve the PVA. Also, less PVA can be taken - say 3g PVA per 10ml of dilute acetate-copper solution, to get a final syrup that is more fluid so easier to apply for mounting.
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