Butterfly Bush - Buddleja davidii

[Ecki]

The Butterfly Bush is growing in our garden. This is a cut of the shoot axis, stained with fuchsine, chrysoidine, alciangreen (mix of alcianyellow and alcianblue). I hope I spelled the color names correctly.


20 x EC Plan Neofluar, DIC, Zeiss 1.6x T2-T2, Nikon D5000, ISO 200, 1/200 sec, Red 1 & Petridish as dummy objects

The blue is the background - although first it appears as if the blue is on top of the red. If you look or stare for a while longer, this is reversed and you will see the red like a maze on top of the blue. This looks quite spectacular.

best regards
Ecki

[rekuwi]

Dear Ecki,

well done, I like the colours.

Best regards
Regi

[rjlittlefield]

Ecki,

It is very colorful and well photographed.

I confess, I know almost nothing about stains. What structures or compounds are highlighted by the various stains? Are the stains applied all at once as a mixture, or in sequence? If sequenced, does the order matter? How long does the process take?

Sorry for the very basic questions!

--Rik

[Ecki]

Rik,

it is, to my own surprise, not that difficult. I have a tablet made for me. This protects my terrace table from stains and allows me to quickly move everything to my "lab".



The procedures are as follows:

Take a fresh carrot and cut of a neat piece. Use a spine to drill a channel into the carrot piece. Make sure the channel is not to large and push the leafstalk into it. Than put the carrot in a microtome (seen left in the above picture). No chop with a microtome knife small slices (25-60 µm thickness) of the leafstalk and put them into AFE (90 % alkohol, 5% glacial acetic acid, 5% formalin).

The AFE kills everything inside the slices and removes the plasma.

Then transfer in 70% alcohol, 50% alcohol, 30% alcohol and finally distilled water. Change water 3 times to remove any remains of the AFE and the alcohol.

Now the slice can be stained. This slide was made with a very simple stain that can be applied as a mixture (it contains fuchsine, chrysoidine and alciangreen). Leave it in the stain for 10 minutes, remove the stain and use isopropyl to remove the remaining water. Change isopropyl 3 times to make sure there is absolutely no water left. Put on a slide, apply a drop of euparal (a resin) and top it with a coverglas. Bake it for 48 hours in 40° Celsius.

If you have done everything right under the microscope you will see something like this:


20 x EC Plan Neofluar, bright-field, Zeiss 1.6x T2-T2, Nikon D5000, ISO 200, 1/200 sec

With the help of DIC and dummy objects you get something like the first picture.

best regards
Ecki

[NikonUser]

Rik:
It's differential histological staining, stains are designed such that different chemical compounds in plant and animal tissues stain differnntly. Xylem and phloem cell walls in plants are great examples. Lots of info on internet.

[rjlittlefield]

Ecki, thanks for the description of procedure and showing us your equipment. This helps a lot.

differential histological staining ... Lots of info on internet.

True, but sometimes it's difficult to get the desired level of detail.

In this case, I was hoping for an answer along the lines of "tissue XXX is red because stain YYY binds to molecule ZZZ".

Despite several Google searches, I did not find what I was looking for.

There is an example HERE that notes "It is imperative that you learn the typical staining reactions of Toluidine Blue & Phloroglucinol" and has captions like "Vascular Tissues of Coleus Stained with Phloroglucinol: The Fibers and Xylem stained Red due to the presence of Lignin. The Phloem is always unstained!"

But the stains used in that article have different names from the ones used in this thread, so I have no idea how these stains relate to the structures.

Can someone perhaps point to a page that discusses the stains used here?

Thanks,
--Rik

[Ecki]

Rik,

if there is interest, I can (with a little help from my friends) show a couple of different staining techniques and also explain what is what inside a botanical cut. The biggest challenge is finding the correct translations - does anybody know an online dictionary with biological terms?

When you cut a stem and put it under the microscope without further treatment, all you will see is green, grey and muddy. The plant architecture will be difficult to see. Therefore, the purpose of staining is to differentiate the different cell types.

The staining that I used was developed by Dr. Helmut Etzold to provide students with a staining technique that can be applied easily - the ingredients can be mixed and applied in one step to the botanical cut. The mixture is cheap, for 10$ you can stain a couple of hundred cuts. If somebody wants chemicals for staining, please send me a private message.

The Etzold staining technique delivers the following result:

Woody cell-walls: red. Often the colors vary, sclerenchyma purple, xylem more brick-red or yellow red. The chrysoidine reacts with lignin, the protein that makes cell-walls woody.

Cutin cell-walls: yellow to orange, sometimes red

Cell-walls without cutin or lignin: green

Cork: colorless, but middle lamellae red as they are woody

Plasma: light red

I have asked a friend to annotate the butterfly bush. I hope to provide this later this evening.

If there is interest, I can (with a little help from my friends) show a couple of different staining techniques and also explain what is what inside a botanical cut.

Best regards
Ecki

[rjlittlefield]

Ecki, this is great -- thanks very much!

If there is interest, I can (with a little help from my friends) show a couple of different staining techniques and also explain what is what inside a botanical cut.

I don't know about anybody else, but I'm bouncing in my seat with excitement about seeing such material.

Probably the best place to put it would be in Macro and Micro Technique and Technical Discussions. There it will be easier to find in the future, and also you folks can include as much detail and as many images as you want.

--Rik

[lauriek]

I'd also be very interested in the info, it's this kind of stuff that's stopped me getting more into the microscopy side of things!

Lovely shots btw!

[NikonUser]

And some more information on the hand microtome, please. Different from the rotary microtome I used in high school with paraffin-wax embedded specimens.

[Ecki]

I had asked a friend to help me annotate the picture. He has read this thread, sent me this and asked me to post it here.

Dear Rik,

Ecki asked me to help him with your question about the staining of the different tissues of his Butterfly Bush slice.
Here we go ;-).

At first: what are the tissues in the slice?



Looking from the centre to the outside of the slice there are:
- Pi - pith parenchyma
- PXl - protoxylem or primary xylem, also dead tissue but not totally sclerified
- Xl - xylem, wood cells
- Ph - phloem, living cells
- Sc - sclerenchyma, bast fibres
- Ep - epidermis

There is no cuticula to be seen.

Alciangreen stains live tissues, which are not or not totally sclerifyed, that is PXl , Ph and Ep.
Fuchsine, which is normally red, is altered by the chrysoidin to a lively pink, stains the sclerified tissues such as Pi, Xl and Ep.
The cuticula, if there is any, would be stained light yellow by the chrysoidin.

I hope, I was able to help Ecki and answer your questions.
Joerg

[rjlittlefield]

Excellent, thanks very much for the tissue identifications!

I notice there is no "Ph" in the image as it appears right now, and in the list, there is no "Pl".

Also the illustration shows a "Ca", which is not in the list.

So I am still a little confused. Can you clarify?

Thanks again!
--Rik

[NikonUser]

So Ca is the cambium which gives phloem to the outside and xylem to the inside.
Pl is Ph (Phloem)