Moss Capsule sectioning for slide making

Here you can discuss sample and specimen preparation issues.
Message
Author
User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Moss Capsule sectioning for slide making

#1 Post by mrsonchus » Wed Jan 16, 2019 1:23 am

Hi all, I'm just about to begin an attempt to section some nice capsules of moss collected and placed into fixative a while back. I think the moss is 'Funaria.hygrometrica' but I'm not sure, I'm a total beginner with mosses at the moment.

The plan is to produce permanently-mounted and differentially-stained slides of the capsules of Funaria.hygrometrica in both longitudinal (LS) and transverse (TS) sections, maybe both orientations together on the same slide/s if possible. This may well end in a horrible aborted mess, but then again I'll have to have a go in order to find out! There's an awful lot that could go awry folks; I've never sectioned moss capsules before, so please bear with if things get messy.... :D

I'm going to use the 'double embedding' technique I posted about a year or two ago now, as I think it will help with the orientation of the tiny and irregularly-shaped capsules in order to get some good longitudinal and transverse sections - hopefully anyway! I'll also embed some without the double technique, for a comparison of the logistics/practicalities and indeed results of the two methods.

This thread won't be totally comprehensive in terms of the stages as I've already posted the complete method as some will know, when I sectioned Sonchus seeds and again some Daffodil anthers - a quick search for 'double embedding' and 'mrsonchus' as the subject and author here will lead you to those posts if you would like to read a lot more about this technique.

Here's a link to my first go at double embedding back in 2015...
Here's my next attempt

I've amassed dozens of jars of fixed plant specimens over the last few years, and at least a dozen jars of mosses, here are a few in their little jars of fixative. They may be kept like this for many years and still be fine for slide-making!
Image


Here's a jar containing completely fixed F.hygrometrica moss (if that is indeed the correct ID).
Image

Here's enough agar-agar, the double-embedding medium I use with wax embedding for this technique, to give me four petri-dish 'slabs' of agar to store ready in the refrigerator for use. 1% w/v in de-ionised water, brought to the boil for about 3-4 minutes will give a lovely clear set.
Image

Image

After cooling to about 50-60 deg C (it will begin to set at about 47) the agar is poured into the dishes then left to set, under a dust cover of course, before being stored in the 4 deg C refrigerator until needed (a day or two should see me ready).
Image

Ready for cooler,
Image

Back soon with images of processing.....


John B.
p.s. The double-embedding technique involves first embedding the specimen (in this case the moss capsules) in agar and then embedding the agar-encased block containing the specimen in wax in the usual manner for sectioning with, in my case, a Shandon rotary microtome. The set agar envelopes are able to be trimmed to give a block that holds the capsules in a position optimal for the sections that I wish to make. Without the agar double-embedded technique it's far harder to orientate tiny tissue pieces at the right angle for optimum sectioning planes to be achieved.....
See my earlier posts re double embedding for LOTS of details.
John B

MichaelG.
Posts: 2383
Joined: Mon Apr 10, 2017 8:24 am
Location: NorthWest England

Re: Moss Capsule sectioning for slide making

#2 Post by MichaelG. » Wed Jan 16, 2019 8:37 am

Thanks John ... this should be a great Masterclass.

MichaelG.
Too many 'projects'

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#3 Post by mrsonchus » Wed Jan 16, 2019 10:32 am

Hi Michael, this should be an interesting adventure! I'm really looking forward to this one I've been studying moss for a couple of months now and find them absolutely fascinating. It would be great to have some slides made-up. I'm not sure how this one will go but I'm reasonably confident that something useful will come of it.
Today I'm going to select some nice capsules and start them off into dehydration etc....

Back with a few images later.

Thanks for your company Michael.

John B. :)
John B

Hobbyst46
Posts: 2891
Joined: Mon Aug 21, 2017 9:02 pm

Re: Moss Capsule sectioning for slide making

#4 Post by Hobbyst46 » Wed Jan 16, 2019 11:17 am

Hi John B.
Yes, they are fascinating! Waiting to see your preparations!

Here a cheekily primitive, rough estimation of moss capsule development: Within 20-30 days, "my mosses" developed 2-3cm long stalks, topped with 1-2mm diameter spheroid capsules, soon to bend down. So, a growth rate of ~1mm/day, or ~40 micrometer/hour. Leaf cell length is of the order of 50 micrometers so, very roughly, an order of magnitude of one cell division/hour.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#5 Post by mrsonchus » Wed Jan 16, 2019 3:24 pm

Hi all, well, I've been pretty busy this afternoon preparing the capsules for the next stages, the very next of which is embedding in agar for those capsules I intend to 'doubly embed', some I'll be embedding in the usual way of what I suppose in this context may be termed 'single' (i.e. wax-only, no agar) embedding.

So, the first job was to have a good look over said capsules and to trim off most of the 'stalk' or seta, of the capsules, leaving just a little 'stump' as an aid to handling maybe....

Here they are before trimming from the rest of the plant,
ws_DSCN9899.jpg
ws_DSCN9899.jpg (49.41 KiB) Viewed 6550 times
Into a shallow dish for the trimming, using small scissors,
ws_DSCN9900.jpg
ws_DSCN9900.jpg (42.05 KiB) Viewed 6550 times
After trimming we are left with stalks,
ws_capsule stalks.jpg
ws_capsule stalks.jpg (41.62 KiB) Viewed 6550 times
and a nice set of capsules!
ws_DSCN9904.jpg
ws_DSCN9904.jpg (36.51 KiB) Viewed 6550 times
Those long setae will now need to be trimmed closer to the capsules,
ws_DSCN9910.jpg
ws_DSCN9910.jpg (26.07 KiB) Viewed 6550 times
That's better!

Now, the next stage will be to place some of these capsules into agar and a few left as-is not to be doubly-embedded....
But, there's an important factor to be considered with this 'type' of tissue - by that I mean, unlike say leaf pieces or stem pieces, capsules, ovaries and other 'enclosed' or indeed encapsulated tissue will need in some way to be pierced in order to make the penetration of processing fluids and ultimately wax infiltration more efficient, or in some cases possible at all.

Next job, to piece these tiny capsules in such a way that will not damage areas of the tissue that will be wanted for the finished sections. Some thought therefore must be given to where and how they are to be pierced....
John B

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#6 Post by mrsonchus » Wed Jan 16, 2019 4:07 pm

Here we are then, next job is to pierce the capsules in order to let any air escape (or be usurped perhaps) and to allow processing fluids and ultimately infiltrating-wax to enter every tiny piece and cell of the tissue, as is absolutely necessary for the cutting of quality sections and subsequent slide-making.

I intend to make transverse sections, starting at the very top of the capsules lid, and longitudinal sections taken with the capsule 'on it's side' as presented to the microtome knife, in order to have sections showing maximum structural details throughout the capsule, hopefully!

To that end I am able to trim off the 'stalk end' of those intended for transvers sectioning, as this part of the capsule will not be sectioned, and I will make a tiny hole of slit in one side of a capsule for those intended to be longitudinally sectioned, with the slitted-side opposite the side along which the microtome will make sections, which should allow efficient tissue penetration and be practical in terms of orientation. A steady hand, fine forceps and a sharp scalpel are now needed.....

Here are a number of capsules with their 'tail-end' removed, which are to be transversely sectioned from the other (top) end,
ws_trimmed capsule 2.jpg
ws_trimmed capsule 2.jpg (23.18 KiB) Viewed 6545 times
These have had tiny slits/holes made in one of their sides, all the same side so I know which way up to place them when I embed them of course!
This is a close-up, I think the slit I made in the outer layer is just visible running roughly horizontally in the picture across the side of the capsule....
ws_capsule trimming-0002.jpg
ws_capsule trimming-0002.jpg (24.15 KiB) Viewed 6545 times
Here's a very short and rather poor quality video I took while I cut a slit in the base of a capsule - apologies for the appalling video and reflections of the ringlight of the stereo 'scope used - the cut is at about 26 sec, full screen is perhaps easier to see....


Here's a capsule that I made a slit in without the capsule being covered with liquid, air immediately entered the capsule as can be seen here. I switched to making the trimmings and piercings with the capsules submerged in fixative!
ws_capsule detail-0001(2).jpg
ws_capsule detail-0001(2).jpg (35.87 KiB) Viewed 6545 times
I may get a chance to embed the capsules in agar later, if I do I'll post some more images/details....

John B.
Last edited by mrsonchus on Wed Jan 16, 2019 6:52 pm, edited 1 time in total.
John B

Roland
Posts: 10
Joined: Wed Jul 15, 2015 10:53 am

Re: Moss Capsule sectioning for slide making

#7 Post by Roland » Wed Jan 16, 2019 5:22 pm

Hi John B.,

Nice experiment. Hope that you will succeed in making good sections! I am looking forward to see the next steps you are planning to do.
Capsules will still be full of spores I think.
The moss is Funaria hygrometrica as you already thought it would be. Almost always has asymmetrical capsules (peristome).

Roland

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#8 Post by mrsonchus » Wed Jan 16, 2019 6:07 pm

Hi Roland, yes they have spores, I know because one capsule ruptured as I attempted to make a slit in it and spores issued-forth like I'd hit an artery during an operation!
Another real interest I have is the stomata pattern of the capsule's lower (i.e. more proximal to the seta) epidermis. Apparently they are taxonomic value but rarely used. As I was taking images through the stereo 'scope I thought I'd try a few of the region where they are to be found and did actually find them!

Here's an image I took of their pattern on this Funaria.hygrometrica, wish I'd used the Canon instead of the 5mp USB camera....
ws_capsule stomata 1.jpg
ws_capsule stomata 1.jpg (50.5 KiB) Viewed 6532 times
Fascinating stuff!

I'm going to put these capsules into the agar in a while.....

John B.
John B

mnmyco
Posts: 142
Joined: Tue Aug 28, 2018 11:03 pm

Re: Moss Capsule sectioning for slide making

#9 Post by mnmyco » Wed Jan 16, 2019 7:30 pm

If you want something clearer than agar try gellan gum. I like it myself. Use about 1/2 the amount of agar normally used, and most of the time it needs a small bit of calcium in it to polymerize. Also, it can take higher temps than agar. It is absolutely clear unless you use highly concentrated amounts.

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#10 Post by mrsonchus » Wed Jan 16, 2019 8:49 pm

Thanks for the tip 'myco - I'll give it a look-see.
The agar goes completely transparent - invisible even during the clearing process, and is also clear when infiltrated with wax. It doesn't infiltrate the specimen, only encases it to give the block that is easily orientated.

Here's the agar remelted and allowed to cool to about 50 deg before pouring over the capsules, which I have taken from their fixative and placed ontothe bottom of 2 petri dishes, 1 set for traverse sections the other for longitudinal.
ws_remelted agar.jpg
ws_remelted agar.jpg (42.47 KiB) Viewed 6511 times
Here is one of the dishes with the capsules now encased within setting agar. Once the agar has set overnight at 4 deg C I'll cut -out the individual capsules in their own agar blocks, ready to go on to the dehydration stages....
ws_capsules in setting agar.jpg
ws_capsules in setting agar.jpg (31.93 KiB) Viewed 6511 times
A close-up of an agar-embedded capsule,
ws_cropped capsules in setting agar.jpg
ws_cropped capsules in setting agar.jpg (33.46 KiB) Viewed 6511 times
Tomorrow the doubly-embedded capsules will go through an alcohol series from 50% to 95% for dehydration, then into a couple of clearing stages in the wax-solvent 'Histoclear' (I don't use xylene at all) to remove the alcohol, and on to wax-infiltration.
They should infiltrate with wax tomorrow evening and overnight, and be ready for block-casting (AKA embedding) tomorrow!

This is where the exciting bit begins, sectioning grows nearer! :o
Urk - I hope this goes well, I haven't tried moss capsules before..... :D

Back tomorrow with an update and a few pics of my set-up.

John B.
John B

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#11 Post by mrsonchus » Wed Jan 16, 2019 8:55 pm

mnmyco wrote:If you want something clearer than agar try gellan gum. I like it myself. Use about 1/2 the amount of agar normally used, and most of the time it needs a small bit of calcium in it to polymerize. Also, it can take higher temps than agar. It is absolutely clear unless you use highly concentrated amounts.
Having had a quick look it seems interesting.
Do you use this for double-embedding in the role for which I'm using agar here?
How does this react to dehydration, clearing and wax infiltration?
Agar passes unscathed through dehydration and clearing, and infiltrates fully, along with the tissue it is encasing, just as though it too were part of the tissue.
How does it section between about 12µ and say 4µ, my usual range for plant tissue?
How about staining post sectioning, does the gum interfere with staining?

An interesting idea, thanks for the suggestion. :)
John B

User avatar
Bufo Bill
Posts: 44
Joined: Sat Mar 26, 2016 10:52 pm
Location: Wolverhampton, UK

Re: Moss Capsule sectioning for slide making

#12 Post by Bufo Bill » Wed Jan 16, 2019 10:48 pm

As always old bean, agape and agog to see your results! A masterclass indeed.
Let the Mighty Shandon roar!
Your friend Bill.
My 'scope: Seben SBX-5 Stereo Microscope.

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#13 Post by mrsonchus » Wed Jan 16, 2019 10:59 pm

Hi Bill, great to have you on board my friend, this should be an interesting adventure!

John B. :)
John B

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#14 Post by mrsonchus » Thu Jan 17, 2019 4:59 pm

Update, capsules have been cut out of the agar slabs and are now in their individual small agar blocks, ready to go into the start of dehydration, starting with 50% alcohol to match the 50% alcohol content of the fixative they were in,

Here they are being cut-out,
Image

A single capsule in it's agar jacket, that will be sectioned orientated on it's side for a longitudinal section....
Image

The capsules are placed into a plastic histology-cassette, one for the LS one for the TS capsules.
Image

Oh yes, while I was 'at it' I also placed some extra tissue from Sunflower and from the root of a Dandelion, into cassettes, to be processed and sectioned in the same bath as the capsules - if the capsules go horribly wrong they'll be these to fall back on hopefully!

Here are some Sunflower stem and leaf pieces I've had stored in fixative for a year pr more,
Image

Here are some pieces of Dandelion root for both LS and TS that I've had for nearly 3 years in fixative, some of my first ever tissue....
Image

In their own cassette,
Image

For these larger pieces I've used these red double-sized tissue cassettes, which are very handy for the bigger specimens.

So, the dehydration stages are to come, through 75% and 3 or 4 95% stages before moving on to the start of 'clearing'. Clearing removes the alcohol and replaces it with a solvent of wax, ready for wax infiltration.....

Back soon....

John B.
John B

charlie g
Posts: 1461
Joined: Mon Oct 20, 2014 7:54 pm

Re: Moss Capsule sectioning for slide making

#15 Post by charlie g » Thu Jan 17, 2019 7:30 pm

Thanks, Mr Sonchus for again hosting us well in your microtechnique lab. It's so doable , and the source ingredients so available, these tutorials temp me again and again to aquire a microtome.

I.ve lately been rooting out water bears and protozoa in my areas moss patches. thank you! Charlie guevara

ChrisR
Posts: 138
Joined: Tue Jun 14, 2016 11:01 pm
Location: Surrey, UK

Re: Moss Capsule sectioning for slide making

#16 Post by ChrisR » Thu Jan 17, 2019 11:06 pm

aquire a microtome
That's the easy bit. I have, um, some. I haven't used them any more that the previous fellah.

Went to an interesting talk by Jeff Duckett, on cryo electron microscopy of moss peristomes. As you probably know they're covered in a waxy substance so they're hydrophobic, and the whole "magic" bit is moisture-operated. In normal electron microscopy, the preparation process wrecks the moisture-sensitive cells.
And "Normal" staining is likely to dissolve the wax.

If you just freeze them at minus bejeezus, those doesn't happen.

There are are at least two UK bryo profs on the photomacrography forum. Might be worth another go at a posting.

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#17 Post by mrsonchus » Sat Jan 19, 2019 6:15 pm

Now then, processing finished with overnight and one more change of wax infiltration. I've cast 19 wax-blocks altogether, including of course the capsules which turned out to be dreadfully difficult to orientate even in their agar capsules....
Also included as a little extra I made some Sunflower stem blocks as I'm interested in trying (again) to make some good longitudinal sections of the so-spectacular Sunflower vessels.
Also I embedded some 3yr-in fixative Dandelion root pieces for both transverse and longitudinal sections. Roots have a very interesting anatomy quite different to that of stems.

A few pics of the blocks, may make a few rough sections tonight after the football if I get a chance!

Here are 19 lovely new blocks to section......
Image

Sunflower stem at the node where a leaf/branch is emerging from the stem and the vasculature is diverging into 'traces', which is why the stem is 'streched' to one side as it appears....
Image

Some of the tiny capsules - however their orientation is far from ideal and they may contain air also - drat!
Image

Image

Image

Some promising Dandelion-root pieces that look like they may section well - the hardest part (literally in fact) of root sectioning is their tough outer bark-like epidermis and cork-like cambium.
This block has been 'roughed' and prepared therefore for some 'proper' sectioning with a nice new blade, hopefully later.
With the tissue exposed by the roughing process more detail is able to be seen,
Image

More root pieces, some are orientated for longitudinal sectioning to show the inner core of vasculature that root, unlike stems, have.
Image

Hopefully back tomorrow with some sections to show!

John B. :)
John B

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#18 Post by mrsonchus » Sat Jan 19, 2019 11:57 pm

Hi all, well I had a chance this evening to cut a few quick roughing sections and float them onto slides for dewaxing tomorrow. I thought you may like to see a few images of the sections, which look quite promising even in the wax, which of course obscures a lot of detail...

All together I embedded 19 wax-blocks for sectioning, containing the moss capsules, the root of Dandelion and the stem of Sunflower - the last two are included just to make sure I had something to fall back on if the capsules were a disaster, which happily they were not!

Here are a number of sections cut tonight on the Mighty-Shandon rotary microtome that sits on my desk in my teeny lab at home. Stood nearly-vertical and under a dust cover (actually it's a plastic seed-propagator tay and lid but they are perfect for this job I find)....
ws_DSCN9966.jpg
ws_DSCN9966.jpg (67.62 KiB) Viewed 6361 times
I cut some 10µ and 5µ moss capsule sections, 10µ and 7µ Sunflower stem and 10µ and 5µ Dandelion root sections, floated onto slides and drying.

Here's a Dandelion root wax-block and a section, still in wax, made from it,
ws_dandelion root section twin pic_001.jpg
ws_dandelion root section twin pic_001.jpg (57.91 KiB) Viewed 6361 times
This is a Sunflower stem sectioned at a node,
ws_DSCN9967.jpg
ws_DSCN9967.jpg (37.52 KiB) Viewed 6361 times
Here's a moss (Funaria.hygrometrica) capsule sectioned at 5µ, the peristome and I think the spores are just coming into view. I think this is a side-on view but not certain, the orientation of these tiny blighters was a nightmare! Once some good sections are stained and mounted all will be much cleare. These images are only of the sections still in the wax as cut, before dewaxing, staining and mounting...
Around the tissue is the wax-infiltrated agar, within the tissue is just wax, the agar encapsulates the tissue but doesn't infiltrate into it.
ws_19_01_19_920_stitch.jpg
ws_19_01_19_920_stitch.jpg (62.58 KiB) Viewed 6361 times
The processing and indeed double-embedding with agar seems to have gone very well indeed. Penetration of dehydrant-alcohol and subsequently clearing-agent Histoclear-3 followed by infiltration then embedding with wax all seem to have been good. So far so good anyway, but many blocks and sections have yet to be seen!

If I have time I may even get a few slides stained and mounted tomorrow, although the sections may need a little more drying time.

Back soon.
John B

ChrisR
Posts: 138
Joined: Tue Jun 14, 2016 11:01 pm
Location: Surrey, UK

Re: Moss Capsule sectioning for slide making

#19 Post by ChrisR » Sun Jan 20, 2019 10:09 am

What was hre fixative your dandelion sat in for 3 years, John?

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#20 Post by mrsonchus » Sun Jan 20, 2019 11:08 am

ChrisR wrote:What was hre fixative your dandelion sat in for 3 years, John?
Hi Chris, the fixative, the one I always use, is FAA - Formal Acid Alcohol.

It's very easy to get the ingredients and mix for yourself,

Formalin (which as you'll probably know) is 37% Formaldehyde) and very easy to buy.
The acid is Acetic acid - 'glacial' acetic acid similarly is cheap and easy to buy.
The alcohol is simply 95% Isopropanol (IPA), cheap to buy also.

The formula is, all v/v,
IPA 50%
water (distilled or de-ionised 'car battery water') 35%
formalin (i.e. 37% formaldehyde) 5%,
glacial acetic acid 5%

Simply cut the tissue you wish to preserve and place into a jar of FAA - note, metal lids will over time be damaged by the acid - I use plastic lidded jars. Plastic or glass jars are perfectly fine for FAA.

I dump clumps of all-sorts into it then go back to it for dissection if need-be. If handling fixed material from FAA (e.g. further dissection/grossing) it's best to place it into 50% IPA first as the fumes from FAA are really quite horrible and indeed harmful I sure.
A period of at least 48hrs for complete fixation is usually fine for plant material, of course it will then be fine almost indefinitely in the FAA.

Hope this helps, John B.
John B

ChrisR
Posts: 138
Joined: Tue Jun 14, 2016 11:01 pm
Location: Surrey, UK

Re: Moss Capsule sectioning for slide making

#21 Post by ChrisR » Sun Jan 20, 2019 1:56 pm

Thanks John.
I don't want to derail the thread - if mods would like to split this off?
I can find 40% formaldehyde -ebay etc
Acetic acid I'm finding usually is in water so not "glacial" - I assume that can be used if some of the water is allocated, eg 10% of 50% Acetic Acid?

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#22 Post by mrsonchus » Sun Jan 20, 2019 1:58 pm

A quick slide, from the Dandelion-root.
The rest of the sections cut yesterday still need time to dry fully - I just thought I'd chance making and mounting a quick one to get an idea of the condition of my stains - they may need to be mixed fresh, maybe.

Slides in air-dryer (actually a fruit-piece dryer) for 4hrs at 45 deg C.
ws_DSCN9980.jpg
ws_DSCN9980.jpg (73.14 KiB) Viewed 6326 times
Here are a few images of the first quickly-mounted Dandelion-root slide, stained with Safranin and Alcian-blue.
The section is a transverse section across a particularly large and scaly old Dandelion's main tap root, sections of which I took 3 years ago and have had in fixative for storage since then.

The one slide so far,
ws_DSCN9979.jpg
ws_DSCN9979.jpg (47.77 KiB) Viewed 6326 times
Here's a 24-image stitch of images taken with a 2.5x objective,
ws_dandelion root slide.jpg
ws_dandelion root slide.jpg (112.22 KiB) Viewed 6330 times
A couple stitched of a section of the epidermis and cambium areas,
ws_dandelion root slide 2.jpg
ws_dandelion root slide 2.jpg (76.54 KiB) Viewed 6330 times
It's an interesting and seemingly very complex piece of root that I'll need time to interpret. There are many areas in this section where discreet vascular bundles appear, perhaps branch traces, not sure (yet).

John B.
Last edited by mrsonchus on Sun Jan 20, 2019 2:36 pm, edited 1 time in total.
John B

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#23 Post by mrsonchus » Sun Jan 20, 2019 2:30 pm

ChrisR wrote:Thanks John.
I don't want to derail the thread - if mods would like to split this off?
I can find 40% formaldehyde -ebay etc
Acetic acid I'm finding usually is in water so not "glacial" - I assume that can be used if some of the water is allocated, eg 10% of 50% Acetic Acid?
Hi Chris, don't worry - this thread is for whatever comes along my friend! :D

This is my own mix,
ws_DSCN9975.jpg
ws_DSCN9975.jpg (39.46 KiB) Viewed 6328 times
The Alcohol,
ws_DSCN9978.jpg
ws_DSCN9978.jpg (60.78 KiB) Viewed 6328 times
The Formalin,
ws_DSCN9977.jpg
ws_DSCN9977.jpg (44.47 KiB) Viewed 6328 times
The glacial acetic acid
ws_DSCN9976.jpg
ws_DSCN9976.jpg (61.48 KiB) Viewed 6328 times
I buy the alcohol from 'Shiny Hardware Limited' AKA 'SHL' from Amazon,
Acetic acid and formalin I buy from e-bay......
I think these two, maybe the alcohol too, are available from the ACP brand on e-bay.

It's all pretty cheap too.

John B.
John B

Hobbyst46
Posts: 2891
Joined: Mon Aug 21, 2017 9:02 pm

Re: Moss Capsule sectioning for slide making

#24 Post by Hobbyst46 » Sun Jan 20, 2019 2:57 pm

Since the FAA fixative contains 35% water, the acetic acid need not be glacial (all the better since GAA is corrosive). Just add less water, to balance the amount included as diluent of the acid. BTW formalin is considered a human carcinogen, in spite of its widespread usage.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

ChrisR
Posts: 138
Joined: Tue Jun 14, 2016 11:01 pm
Location: Surrey, UK

Re: Moss Capsule sectioning for slide making

#25 Post by ChrisR » Sun Jan 20, 2019 4:37 pm

Fair enough, thanks.
I spotted this on ebay. For a special friend?? :o 263572761555
lamp.png
lamp.png (160.2 KiB) Viewed 6309 times

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#26 Post by mrsonchus » Sun Jan 20, 2019 9:17 pm

Chris! You're a mad-maniac! :D :D :D :lol:

A few first-images of a quickly-made slide of the Dandelion root TS, the capsules need more drying time....
Stained with Safranin & Alcian blue, a pretty complex-looking tissue that I'll need to study more to identify the various elements within!

22 image stitch through 2.5x objective...
ws_4mp ver pse layer mask edited20_01_19_929_stitch.jpg
ws_4mp ver pse layer mask edited20_01_19_929_stitch.jpg (87.94 KiB) Viewed 6290 times
Closer-in (also 2.5x objective, 2-image stitch) to epidermal/cambial area,
ws_dandelion root slide 2.jpg
ws_dandelion root slide 2.jpg (76.54 KiB) Viewed 6290 times
Looking good this far!

John B.
John B

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#27 Post by mrsonchus » Tue Jan 22, 2019 12:41 am

Well, managed to make a few slides from the sectioned capsules today. Not bad so far, details of peristome outer layer and teeth have been sectioned quite well. The double-embedding has worked very well and sectioned beautifully.

Here's a quick image.
ws_capsule section slide 1.jpg
ws_capsule section slide 1.jpg (47.09 KiB) Viewed 6248 times
John B.
John B

einman
Posts: 1288
Joined: Tue Feb 24, 2015 1:03 am

Re: Moss Capsule sectioning for slide making

#28 Post by einman » Thu Jan 24, 2019 2:07 am

Fascinating, simply fascinating. You should write a book for enthusiasts. Your photos, annotations etc are first rate as always!

User avatar
mrsonchus
Posts: 3782
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Moss Capsule sectioning for slide making

#29 Post by mrsonchus » Thu Jan 24, 2019 3:24 pm

Thanks einman, pleased you like the adventure. I really must agree wholeheartedly with you my friend, the idea of sectioning these tiny capsules is to me simply irresistible! :D

I'm pleased enough with these roughs but am really looking forward to sectioning some of the other blocks also, to see just what views may 'come out' as it were.
I think I'll go for a thicker section at this stage, perhaps increase to 10 or even 12µ, as sometimes, as for those slides I refer to as 'morphological', thinner is not actually better as detail and even tissue may be lost. Not all pertinent detail is best viewed with a x100 objective!
I'm trying to develop a technique (already done of course by others) to enable me to classify capsule/peristome structure along the generic but more likely familial grouping of taxa.

The adventure must start as ever at the beginning, and with a good structured and disciplined approach, perhaps edge towards my goal, albeit a somewhat ambiguous one. My first goal will be the achievement of consistency and reproducibility before I start to go 'off-piste'...
I'd also be very pleased just to produce sections/slides clearly identifiable for what they are :D .

Also have several blocks of Sunflower stem-node to go-at, and similarly several of a tough old Dandelion-root, which I have started with a few slides in different stains already - always good to practice with something relatively straightforward I think.

Having as usual a whole heap of fun with the Mighty-Shandon (et al)!

John B. :D :D
John B

Hobbyst46
Posts: 2891
Joined: Mon Aug 21, 2017 9:02 pm

Re: Moss Capsule sectioning for slide making

#30 Post by Hobbyst46 » Thu Jan 24, 2019 4:29 pm

mrsonchus wrote:The adventure must start as ever at the beginning, and with a good structured and disciplined approach, perhaps edge towards my goal, albeit a somewhat ambiguous one. My first goal will be the achievement of consistency and reproducibility before I start to go 'off-piste'...
This IMHO is the most sternous challenge. May the power ("Schwartz" :lol: ) be with you.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Post Reply