Festuca Grass Hand Sections

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mrsonchus
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Festuca Grass Hand Sections

#1 Post by mrsonchus » Tue Oct 01, 2019 8:43 pm

Hi all, I though I'd share a few images of some hand-sections I took this afternoon. These are from a remnant of some ornamental Festuca genus (unsure of species) grass we have 'surviving' in a pot in the garden. If you've ever seen a cross-section, and they're pretty ubiquitous, of marram grass, you'll see some similarities. The reason I took these sections is as usual to see what slides of this grass may give, i.e. if they will be of interest.
The Festucas ar a 'beach grass' as are the marrams, and have infolded leaves that aid in the minimisation of drying-out in their usual harsh, impoverished environment that is dunes on a coast.

Anyway, these I stained with a tri-stain mixed as follows: (this stain formula is not useful for permanent mounts but good for fresh tissue)
Image

Nice colourful images but tragically thick sections, a rush-job before shopping I'm afraid...
The infolded nature of the leaf-blade, the inner trichomes and the thick pink-stained fibers beneath the epidermis are all immediately apparent and tell of this plant's anatomical features to suit it's growing environment I think.
Image

Those inner trichomes help to stabilise the micro-environment (mainly air-movement) above the epidermis, where there are undoubtedly stomata (air-pores) that sadly, in this thick section, are not visible....
Image

Coser-in view of a vascular-bundle with a surrounding sheath of asymmetrically thickened cells, not certain that the thickening is lignin though, it may be suberin and a barrier rather than a strengthening tissue - haven't had time to check.... Nice colours.
Image

I labelled the above-mentioned thickening here as lignin - this may well be wrong though. Interesting to see the stratified deposition of this layer - this isn't often visible in permanent slides - highlighted in mag-box in image.
Image

The grass itself is an unassuming glaucous, thin almost wiry-leaved plant that is often in association with marram grass and helps to preserve the integrity of such sandy dune banks.

Sorry to be so rushed and with meagre information, but I thought some may find the colours and structure interesting.
John B

MichaelG.
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Re: Festuca Grass Hand Sections

#2 Post by MichaelG. » Tue Oct 01, 2019 9:56 pm

Beautiful work, as ever, John

Oh to be able to do something that good as a 'rush job' !!

MichaelG.
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Re: Festuca Grass Hand Sections

#3 Post by MicroBob » Wed Oct 02, 2019 11:44 am

Hi John,
beautifully stained sections and an interesting object you picked!
This is round about a Wacker W3A-II stain with Acridinred (only available as an expensive solution today) exchanged for Rhodamine-B.
Why do you think it unsuitable for permanent mounts? I think it has been used successfully for this pupose.

These grasses are really a great topic. By eye the folded structure is detectable but it doesn't make much sense. The differentially stained section shows how this plant works and what makes it survive in a harsh environment.
When you look for interesting objects there is a long list of covered topics in the german mikroskopie-forum: http://www.mikroskopie-forum.de/index.php?topic=21220.0

Bob

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mrsonchus
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Re: Festuca Grass Hand Sections

#4 Post by mrsonchus » Wed Oct 02, 2019 2:52 pm

Hi Michael, thanks old chap, pleased you like them. :D
John B

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Re: Festuca Grass Hand Sections

#5 Post by Sauerkraut » Wed Oct 02, 2019 3:07 pm

Your work is so beautiful. Thank you for sharing them as well as the stain solutions.

I could easily see such images in textbooks and wall posters.

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Re: Festuca Grass Hand Sections

#6 Post by mrsonchus » Wed Oct 02, 2019 3:40 pm

Many thanks Sauerkraut, it's easier than you may think!

An exotic mix of multiple stains isn't by any means essential, have you had a look at my simple single-stain (aqueous Safranin in fact) hand sections of Bamboo from one of my earlier posts? Just use a single-edged razorblade and a 1g per 100ml deionised (i.e. battery topup) water mix of Safranin stain powder.

The Safranin powder is dirt-cheap on e-bay,
Image

The results are beautiful,
Image

Here's a link to my bamboo post...
John B

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Re: Festuca Grass Hand Sections

#7 Post by MicroBob » Wed Oct 02, 2019 7:23 pm

Hi together,
I had a look through the garden but found no interesting grasses. This is probably something to keep in mind when going to the sea.
Perhaps they have some at the botanic garden. They have big signs there that warn you about proscution should you take a leaf or whatever so I would have to make an appointment or ask somebody there.

Bob

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Re: Festuca Grass Hand Sections

#8 Post by Wes » Wed Oct 02, 2019 7:23 pm

Very interesting exploration mrsonchus. I find the lignified cell-wall very interesting, given its multilayered structure I do wonder how it would look like under polarized light.

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Re: Festuca Grass Hand Sections

#9 Post by Roldorf » Wed Oct 02, 2019 7:42 pm

Hey John
Nice job 'Multi Colour Staining' I think you are going to start a trend. Lovely. Just got back from a 'working holiday' and to see this first thing is fabulous.
Many thanks
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Re: Festuca Grass Hand Sections

#10 Post by mrsonchus » Wed Oct 02, 2019 7:50 pm

MicroBob wrote:
Wed Oct 02, 2019 7:23 pm
Hi together,
I had a look through the garden but found no interesting grasses. This is probably something to keep in mind when going to the sea.
Perhaps they have some at the botanic garden. They have big signs there that warn you about proscution should you take a leaf or whatever so I would have to make an appointment or ask somebody there.

Bob
Hi Bob, "...no interesting grasses...."! :o
Bob,....Bob...they're all interesting my friend! :D :D

Monocotyledon leaf structure is nice in cross-section. The veins all being parallel will give nice clean and well-oriented views of the vascular-bundles. Primarily make sure you cut straight across the leaf to get the best result. The monocotyledon vascular-bundles are often said to resemble faces!

Go-on Bob, have-at those grasses, they're really rather splendid in section - lots of silica within cells also.

The way I often approach new subjects is to take several different plant types, all cuts from the same tissue-type in each, e.g. leaf, stem.... Then, when they are hand-sectioned (or of course microtome-sectioned) the differences in their anatomy will be revealed. That's where the questions begin to form in the old grey-matter. Why are they different, how are they different, what do the differences imply or mean in terms of function or even response to environment (not all anatomical features are 'endogenous' and present genetically)? An example may be an increase in strengthening-structures such as lignified or thickened cell-walls in response to wind stress...

I'm currently nursing an interest in such anatomical comparisons of grasses, sedges and reeds - the Poaceae, Cyperaceae and Juncaceae Families respectively!

Oh, I'm going-on as my Wife would say..... :oops:

Go-get 'em Bob, and perhaps a simple Safranin stain! Great intrigue, adventure and downright fun awaits the Botanical inquisitor. :D
John B

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Re: Festuca Grass Hand Sections

#11 Post by mrsonchus » Wed Oct 02, 2019 7:56 pm

Wes wrote:
Wed Oct 02, 2019 7:23 pm
Very interesting exploration mrsonchus. I find the lignified cell-wall very interesting, given its multilayered structure I do wonder how it would look like under polarized light.
Arggghhh.....pht......
Wes, I'm currently having nightmares trying to add polarisation to my BX40! :D :oops:

I'll make some better hand-sections maybe tomorrow, perhaps even with removal of chlorophyll prior to staining for increased clarity (and hopefully some nice thin un-rushed sections).
The tatty remains of the Festuca in the plantpot also have a fungal visitor - interesting microtome sections for sure, must also place some samples into fixative to that end...

Thanks for looking Wes, great to see folk interested in the green-stuff! :D :D
John B

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Re: Festuca Grass Hand Sections

#12 Post by mrsonchus » Wed Oct 02, 2019 7:57 pm

Roldorf wrote:
Wed Oct 02, 2019 7:42 pm
Hey John
Nice job 'Multi Colour Staining' I think you are going to start a trend. Lovely. Just got back from a 'working holiday' and to see this first thing is fabulous.
Many thanks
Thanks Roldorf - great to know others like them! :D
John B

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Re: Festuca Grass Hand Sections

#13 Post by mrsonchus » Wed Oct 02, 2019 8:13 pm

MicroBob wrote:
Wed Oct 02, 2019 11:44 am
Hi John,
beautifully stained sections and an interesting object you picked!
This is round about a Wacker W3A-II stain with Acridinred (only available as an expensive solution today) exchanged for Rhodamine-B.
Why do you think it unsuitable for permanent mounts? I think it has been used successfully for this pupose.

These grasses are really a great topic. By eye the folded structure is detectable but it doesn't make much sense. The differentially stained section shows how this plant works and what makes it survive in a harsh environment.
When you look for interesting objects there is a long list of covered topics in the german mikroskopie-forum: http://www.mikroskopie-forum.de/index.php?topic=21220.0

Bob
Hi Bob - I seem to have messed-up my reply made earlier - sorry my friend, It's not here!

It is indeed an altered-version of a modified-version of a Wacker stain formula. I substituted rhodamine(B) for acridine-red as I just couldn't find any when I had a look at Wacker's fabulous stains a while-back now. The alcohol dehydration destroys the rhodamine which isn't a problem of course with the hand-sectioned water-mounts. I read a little further after reading your reply and have ordered some triethyl phosphate which was suggested as a good dehydrant that preserves the acridine-red (and I'm hoping it will act the same way for the rhodamine of course... fingers-crossed). I also occasionally use clove-oil as an anhydrous step before Histoclear (limones) pre mount as Histoclear is quite happy to usurp the oil without problems, apart from the relatively large amount needed to be thorough and the hideous cost of Histoclear (about £130 per U.S. gallon!).

I haven't really pursued the Wacker stains yet, but I really should try them as the results are so fantastic- far better than my staining in many respects.
Thanks for the reply Bob, and the handy link - I'll tootle-off and have a look later.
John B

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Re: Festuca Grass Hand Sections

#14 Post by MicroBob » Wed Oct 02, 2019 8:48 pm

Hi John,
as far as I know Robin Wacker held his stain formulations secret for 25 years before he made it public.
Some other people have continued work on his formulations, especially Rolf-Dieter Müller, Klaus Herrmann and Jörg Weiß.
The website of the Mikroskopie-Kolleg-Bonn is my favourite website to learn more about botanic sections as there is a vast amount of proven information and the authors will answer questions if they come up. Here is some intersting information on the staining process. They usually start with a section that was cut enclosed in carrott and have permanent mounts in Euparal in mind.

http://www.mikroskopie-bonn.de/biblioth ... k/296.html
http://www.mikroskopie-bonn.de/biblioth ... k/157.html
http://www.mikroskopie-bonn.de/biblioth ... k/162.html

Some Wacker mixes were not too stable in the bottle so were sold in two bottles.

Bob

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Re: Festuca Grass Hand Sections

#15 Post by mrsonchus » Wed Oct 02, 2019 9:16 pm

Hi Bob - great links - I'll have a proper look over those!

Interesting to immediately see that a proposed replacement for the acridine-red is Rhodamine(B)!

Of course, reading further down it was now I remember the W-ASim II stain that I read of in an article - which I have somewhere. The stains was tested with variations in temperature as I remember with interesting results...

Thanks for the links my friend.

I have a root TS slide (Dandelion I think) somewhere which has a beautifully vivid colour and is stable as a permanent-slide, I'll see if I can find the images I have of it - in fact I have a series of about 2-dozen such slides with variation which I'll get out tomorrow if I've a chance.
John B

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Re: Festuca Grass Hand Sections

#16 Post by MicroBob » Thu Oct 03, 2019 10:07 am

Hi John,
here is a link to the original text from Robin Wacker in the MIKROKOSMOS magazine: https://www.zobodat.at/pdf/Mikrokosmos_95_4_0001.pdf
He describes his staining method as a multi step method. This was the basis for the development of combination stain W3A Sim which is easier to use but of cause cuts some corners.
He writes that Rhodamin can be used instead of Acridin-red. As far as I know the colour of Rhodamin is a bit less striking. Rhodamin-B is a fluorescence stain: https://www.mikroskopie-forum.de/index. ... ic=22654.0 that is excited with blue light.
The whole MIKROKOSMOS magazines from 1907 to 2014 can be downloadad for free. Ideal for everybody who has just got a bigger prison sentence. :lol:

Bob

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Re: Festuca Grass Hand Sections

#17 Post by Hobbyst46 » Thu Oct 03, 2019 11:43 am

MicroBob wrote:
Thu Oct 03, 2019 10:07 am
...He writes that Rhodamin can be used instead of Acridin-red. As far as I know the colour of Rhodamin is a bit less striking. Rhodamin-B is a fluorescence stain: https://www.mikroskopie-forum.de/index. ... ic=22654.0 that is excited with blue light.
Bob, that is a nice link to manny interesting pictures. From just paging through it I have the following impression:
1. 3W monochromatic LEDs produce great fluorescence results at low magnifications (I suppose those are 10X at most ? did not find the optics details).
2. I suspect that most of the colors in the fluorescence images are pseudo-color, not the true colors. Rhodamin fluoresces dark red for example.
3. AFAIK, Rhodamine can be excited with orange light (~590nm), and there is the excitation peak, perhaps 436nm is still exciting; the author of that thread apparently used this blue light because it was targeted at mixed stains, where some were excited by the blue 436nm.

When John B (mrsonchus) sees those images, I believe that he will switch his resources from phase contrast to fluorescence... 8-)
Last edited by Hobbyst46 on Thu Oct 03, 2019 1:40 pm, edited 1 time in total.
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Re: Festuca Grass Hand Sections

#18 Post by Dave S » Thu Oct 03, 2019 12:46 pm

Nice work John!!
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Re: Festuca Grass Hand Sections

#19 Post by MicroBob » Thu Oct 03, 2019 2:21 pm

Hi Doron,
the big microscope manufacturers have databases to show the use of their filter cubes and to make sure that you buy plenty of this expensive eqipment.
Here is the Zeiss database and they state fpr Rhodamine-B exciting 543 nm and emission 565 nm: https://www.micro-shop.zeiss.com/en/us/ ... t-0000-540
In Hans-Jürgens images I see green and yellow colour where green matched the emission wave length but yellow has to come from something else.
I'm interested in fluorescence microscopy but have so far not spent much time on reading about it. I'm not too keen on using UV light so I will have to see what is possible with exiting light in the visible range.

And yes - printing an article about polychromatic stains with b/w images is quite special. Robin Wacker probably was cross with the publisher for ever!

Bob

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Re: Festuca Grass Hand Sections

#20 Post by Hobbyst46 » Thu Oct 03, 2019 3:04 pm

MicroBob wrote:
Thu Oct 03, 2019 2:21 pm
...Here is the Zeiss database and they state fpr Rhodamine-B exciting 543 nm and emission 565 nm: https://www.micro-shop.zeiss.com/en/us/ ... t-0000-540
I stand corrected about those wavelength... :shock: apparently mixed it up with another stain... :(A long time since I touched Rhodamine...
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Re: Festuca Grass Hand Sections

#21 Post by mrsonchus » Thu Oct 03, 2019 3:10 pm

Hi all, I ran a few - in fact I'm very low now on slides in wax ready to mount, slides of TS of the monocotyledonous garden-plant 'Solomon's seals' through the Wacker-inspired stains that I had mixed back in February I think it was. I quickly tried a few variations and in particular like the way the addition of a little of the acriflavin (which is yellow) to the alcian-blue (I don't have any astra-blue) that I use gives the green-tones.
It's quite difficult to get the yellow to show well.

Here are my Wacker-type test-stains mixed a while back, next to some of the root slides mentioned earlier,
Image
Image

A few images from these mixes. I should really mix fresh and run some tests with some new sections that I have in the pipeline.
At 4x not much visible, this slide is 6µ -at this thickness it needed far longer staining times, although my personal preference is light & accurate staining.
These slides are from this Summer and vary between 6µ and 8µ, taken to give fine tissue detail whilst virtually ignoring cytoplasm, in fact I may even have bleached these - I'm not sure, to remove most of the cytoplasm.
Anyway, Here are a few inages literally still wet from mounting, so optically they're nowhere near ready (and are in fact now in my air-drier for a 4hour 50deg stint) but you can see the idea.


The 4x,
Image

Some idea of colour visible, with good differentiation. Nice to see the yellow of the acriflavin...
Image

Here the green result of adding a little acriflavin to the alcian-blue is evident, I like it.
Image

The Rhodamine(B) red stain needs a minimum of about 15 minutes! It really is easily rinsed-away with dehydration alcohol...

These are just a few quick images from the mixed-protocol 6 slides that I played-around making this afternoon - still wet when photographed! :D :D :D

Washing-up time! So colourful - my Wife said it looks like a mad-Wizzard's cave in the lab! :D
Image

I really must get back to Wacker's amazing stains properly, and soon! :D :D :D :D
John B

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Re: Festuca Grass Hand Sections

#22 Post by MicroBob » Thu Oct 03, 2019 5:38 pm

Hi John,
great sections and a nice stain, just right in my view. Compared with thicker sections the impact of the stain will always be less with thinner sections, but once accustomed to the look it is just right.
Your workplace looks perfectly neat - do you have a background in acounting? :lol:

Bob

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Re: Festuca Grass Hand Sections

#23 Post by MicroBob » Fri Oct 04, 2019 12:22 pm

Hi John,
as far as I know Histoclear is based upon citrus oils. These are also sold for general cleaning purposes. I once bought a bottle of such a natural solvent in a shop for biological building materials for removing stickers in a maintenance workshop, but the maintenance people couldn't stand the smell. :roll: But this might be a cheaper alternative to Histoclear.

Bob

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Re: Festuca Grass Hand Sections

#24 Post by mrsonchus » Sat Oct 05, 2019 12:20 am

Hi Bob - an interesting source for the limones!
Yes Histoclear in the lab does smell a bit of oranges, but not as much as the poor cleaner that has to spread the stuff all-over labels etc!
I can imagine that would be a bit of a horror!

Thanks for the idea, I'll see what I can find-out.
John B

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