Ilex leaf with worm hole - pen ink stain

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MicroBob
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Ilex leaf with worm hole - pen ink stain

#1 Post by MicroBob » Sun Dec 13, 2020 8:07 pm

Hi together,
while preparing our last group meeting for this year I did some ilex leaf cross sections. On the leaf I saw tiny vore holes towards the central vein of the leaf. When doing experiments on staining processes I hit one of these worm holes and got a nice photo here. My idea is that the attack was directed at the phloem to subtract some energy. 50µ section with cylinder microtome and blade holder, stained with safranine, differentiated with 70% Ethanol and a tiny amount of HCl, counter stained with Herlitz "Königsblau" school pen ink.

Bob
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Safranin-Salzsäurealkohol-Herlitz Königsblau Ilex Blatt querschnitt 50µ DSC_1030 1024.jpg
Safranin-Salzsäurealkohol-Herlitz Königsblau Ilex Blatt querschnitt 50µ DSC_1030 1024.jpg (250.87 KiB) Viewed 1232 times

Wes
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Re: Ilex leaf with worm hole - pen ink stain

#2 Post by Wes » Sun Dec 13, 2020 8:22 pm

Very nice section and image Bob. Have you tried to section sick plants such as ones infected by fungi/bacteria?
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/(epi)FL/Jamin-Lebedeff
Youtube channel

MicroBob
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Re: Ilex leaf with worm hole - pen ink stain

#3 Post by MicroBob » Sun Dec 13, 2020 8:30 pm

Wes wrote:
Sun Dec 13, 2020 8:22 pm
Have you tried to section sick plants such as ones infected by fungi/bacteria?
No, this was just by accident. There are special stains to make fungi visible in plant tissue, perhaps for bacteria too. Might be a very attractive image of a destructive condition!

MicroBob
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Re: Ilex leaf with worm hole - pen ink stain

#4 Post by MicroBob » Mon Dec 14, 2020 10:20 am

For Fungi Pianese stainig would work: Fungus pink, plant green.

Wes
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Re: Ilex leaf with worm hole - pen ink stain

#5 Post by Wes » Mon Dec 14, 2020 10:26 am

MicroBob wrote:
Mon Dec 14, 2020 10:20 am
For Fungi Pianese stainig would work: Fungus pink, plant green.
Do you have this stain at your disposal?
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/(epi)FL/Jamin-Lebedeff
Youtube channel

MicroBob
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Re: Ilex leaf with worm hole - pen ink stain

#6 Post by MicroBob » Mon Dec 14, 2020 10:33 am

No, I just happened to read it here on page 18: https://www.waldeck-ms.de/wp-content/do ... eutsch.pdf

They try to sell us a ready mixed stain, but Eisner has the ingredients here: http://www.aeisner.de/methoden/farb118.html
I have one Fuchsine, but there are at least three types, I don't know which one I have right now. Malachit green is not too special but I have never hear anyone uses Martius yellow, might be less common.

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mrsonchus
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Re: Ilex leaf with worm hole - pen ink stain

#7 Post by mrsonchus » Mon Dec 14, 2020 12:14 pm

Hi, I use pre-made lactophenol cotton-blue for live staining fungus - high contrast and pretty good.
Here's an image of some fungus which as far as I can remember came from a rotting plant bud. Water-mounted with coverslip and stained with LP stain....
fungus stain.jpeg
fungus stain.jpeg (91.85 KiB) Viewed 1185 times
John B

MicroBob
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Re: Ilex leaf with worm hole - pen ink stain

#8 Post by MicroBob » Mon Dec 14, 2020 3:25 pm

Hi John,
nice contrasty stain! Was this fungus in air or had it entered ito the tissue?

Bob

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mrsonchus
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Re: Ilex leaf with worm hole - pen ink stain

#9 Post by mrsonchus » Mon Dec 14, 2020 3:51 pm

Hi, this was in the tissue - a typically 'mushy-brown' appearance of an infected closed-vegetative bud.
It's a handy stain and very cheap.

I also have sets of slides that I have made of infected plant tissue, e.g. Sonchus.arvensis stem and leaf tissue. The permanently-mounted slides are great as I am able to try several different stain combinations and protocols, as I always do, each of which tends to emphasise different structures in different proportions giving a nice variety of views on the infected tissue and it's assailant...

A few images stained with permanent combinations - fungi in this condition will stain with just about anything I find, with varying results...

Image
Image

Image
Image
John B

MicroBob
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Re: Ilex leaf with worm hole - pen ink stain

#10 Post by MicroBob » Tue Dec 15, 2020 7:53 pm

Hi John,
your images make me think to have a closer look into fungus infested plants! It shouldn't be too difficult to find suitable plant material, I would perhaps take a closer look at our fruit trees.
What stains did you use for the last 2 images?

Bob

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mrsonchus
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Re: Ilex leaf with worm hole - pen ink stain

#11 Post by mrsonchus » Tue Dec 15, 2020 8:46 pm

Hi Bob - yes, not only do such 'infections' make good images, they're pretty easy to achieve also.
The penultimate image is safranin + alcian-blue (an alternative to safranin + fast-green).
I prefer fast-green for accuracy and subtlety of finest detail but alcian-blue for more 'drama' in vivid colours - which (the alcian-blue that is) for the more technical sections I find a little too bright/glaring.

The last image is with safranin + alcian-blue plus a third stain, orange-g. This has the pleasing effect of making the alcian-blue greener (and is to some extent quite controllable in this respect) whilst adding subtlety and detail of the orange-g. This is much the same principle as I use when combining rhodamine-B, acriflavin and alcian-blue for triple-staining based upon Wacker's stains but with contemporary stains that are, unlike the originals, available still. The effect of the 'greening' of the alcian-blue by the acriflavin (which is yellow rather than orange) may be seen in the 2 images below of a tri-stained root TS...
ws_06_02_19_179.jpg
ws_06_02_19_179.jpg (130.36 KiB) Viewed 1129 times
ws_06_02_19_167_stitch.jpg
ws_06_02_19_167_stitch.jpg (141.03 KiB) Viewed 1129 times
Beautiful colours for certain, but not my go-to combinations for more specialised images rather than the mophological 10-6ish µ sections which enable the uptake of a lot more stain than the thinner say sub-5µ specialised sections.

Here's a link to a whole folder of images from my infected Sonchus.arvensis slides....
John B

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Re: Ilex leaf with worm hole - pen ink stain

#12 Post by MicroBob » Tue Dec 15, 2020 9:09 pm

Hi John,
thank you very much for the detailed explanations! I haven't looked closer at my staining results for many years - it became colourful - good! :oops: While preparing my part of our last group meeting I took the time to do some more experimenting with botanical stains and made a little progress. Staining really takes a lot of practical experience and careful experimenting to improve the results. Also our discussion on the meeting was quite interesting when comparing my "bread slices" of hand cut sections to evenly cut thin microtome cuts from paraffin and the influences on the staining.

Bob

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