Plant histology -first attempt

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Seb28
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Plant histology -first attempt

#1 Post by Seb28 » Fri Mar 20, 2015 2:05 pm

Hi
This is my first attempt to the histology process of the stem of Kalanchoe plant.
The Kalanchoe stem were cut into 3-4mm pieces and fixed in 4% Formaldehyde.
Then dehydrated through a series of Isopropyl alcohol or aceton,the next step was removing the alcohol by immersed in clove oil.
Embedded in paraffin and cuting on a rocking microtome at arround 6-10microns.
The thin sections were then transfered to water bath at 40*celsius to strech the parffin section and attached to the slide and left overnight to dry at room temperature.
To remove the parrafin from the slide I used cedarwood oil (because I do not have access to xylene).
Not all sections were perfect,some had some cracks in the center.
I need to more experimenting.
congo red
congo red
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Acridine orange
Acridine orange
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AO, inverted image
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Methylen blue
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-Reichert Polyvar
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Seb28
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Re: Plant histology -first attempt

#2 Post by Seb28 » Fri Mar 20, 2015 2:09 pm

polarized light
polarized light
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AO
AO
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Rhodamine B
Rhodamine B
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Rhodamine B,invertet image
Rhodamine B,invertet image
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-Reichert Polyvar
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JimT
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Re: Plant histology -first attempt

#3 Post by JimT » Fri Mar 20, 2015 2:20 pm

Very good work. I would be proud of these sections and the images.

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75RR
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Re: Plant histology -first attempt

#4 Post by 75RR » Fri Mar 20, 2015 6:08 pm

So that is how thin and perfectly even sections are meant to look like.
Night and Day to my efforts.
You have also convinced me of the value of staining.

Second image would make an excellent poster.

Will now keep an eye out for a rocking microtome.
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gekko
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Re: Plant histology -first attempt

#5 Post by gekko » Fri Mar 20, 2015 7:49 pm

I don't think any apology is needed for being your first attempt. Virtually perfect sections, perfect staining, perfect imaging. Very beautiful. The sections appear to be less than one cell thick; any idea of how thick they are?

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Re: Plant histology -first attempt

#6 Post by einman » Fri Mar 20, 2015 8:47 pm

I believe he said 6-10 microns in his initial post. very Nice. I just purchased a hand microtome. Will see what results I get.

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gekko
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Re: Plant histology -first attempt

#7 Post by gekko » Fri Mar 20, 2015 8:58 pm

Thanks einman and apologies to everyone: I must have skipped that line when reading the post :oops:

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mrsonchus
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Re: Plant histology -first attempt

#8 Post by mrsonchus » Fri Mar 20, 2015 9:44 pm

Stunning results - I'm going to hurl my hand-microtome into the cracks of doom and buy a rocking-microtome ASAP! These are simply superb sections - these are what I want to achieve. :mrgreen:
I'm interested in your use of clove & cedarwood oils as clearing agents rather than something such as Histo-clear for example - what form do you purchase them in?
Your truly professional standard results have made my mind up - I've been looking at rocking microtomes for a while, one reason I bought the cheapest hand microtome I could find, and I'm now 100% certain that I want one! I'm going to tell my Wife it's all your fault ;) :D
Well done - very, very well done - truly inspiring and at the same time depressing - my efforts now seem so puny and tragic :cry:

You are a star - you've shown us all what can be achieved!

Many thanks, I'm off to weep over my hand microtome then order a rocking microtome! :D :) :mrgreen: :lol: ;)
John B

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Crater Eddie
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Re: Plant histology -first attempt

#9 Post by Crater Eddie » Fri Mar 20, 2015 10:42 pm

Simply amazing!
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Seb28
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Re: Plant histology -first attempt

#10 Post by Seb28 » Sat Mar 21, 2015 12:45 pm

Thank you very much for your kind comments.

mrsonchus,
I used clove oil,cedarwood oil as an alternative to Xsylene which is not available in Ireland.I buy them in pharmacy as essential oils in small bottles.
The rocking microtome is a piece of good equipment for beginners.I did some improvements in the microtome blade I'll post some photos later.I found also information that one can somehow convert roking microtome by means of which sections 0.02 μ thick can be cut,but there is a charge for the PDF article.
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hkv
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Re: Plant histology -first attempt

#11 Post by hkv » Sun Mar 22, 2015 9:47 pm

Stunning results!

Regarding the 0.02 micrometer cuts. That would equal to 20 nano meters. That is smaller than 1/10th of the wavelength of light. What type of knife edge would you need for these cuts? The edge of the blade would have to be very thin the the edge would not be optically resolvable in light microscopy as it is smaller that the diffraction limit permits. I am no expert, but this sounds to good to be true.

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Re: Plant histology -first attempt

#12 Post by Peter » Mon Mar 23, 2015 2:02 am

Hi HKV,
I believe sections in the order of the thinness you are describing are used for electron microscopy, the knife blade is often a freshly broken piece of glass.
Peter.

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Re: Plant histology -first attempt

#13 Post by mrsonchus » Mon Mar 23, 2015 11:36 am

Seb28 you have made me spend more money with your fabulous pictures - I told my Wife it's all your fault and she replied 'you'd better order the Rocker before they sell out'! Green light is given - order has just been placed - oh Seb28.....what have you done to us all.....

It's ordered! It's on It's way to me! 5µ or bust! I dream of 5-10µ sections because of you Seb28!

Can't wait - I've even ordered Histoclear & 'proper' FAA! :lol:
I'll post a video of the process as soon as I get the hang of it! Probably about a fortnight hence...

Hussar & Hurrah! :D
John B

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75RR
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Re: Plant histology -first attempt

#14 Post by 75RR » Mon Mar 23, 2015 11:46 am

This is a link to an article on a Rocking Microtome that appeared in 2013. (Old Forum)

http://www.microbehunter.com/forum/arti ... me-review/
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mrsonchus
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Re: Plant histology -first attempt

#15 Post by mrsonchus » Mon Mar 23, 2015 2:51 pm

75RR wrote:This is a link to an article on a Rocking Microtome that appeared in 2013. (Old Forum)

http://www.microbehunter.com/forum/arti ... me-review/
Great article - thanks for that!
John B

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Re: Plant histology -first attempt

#16 Post by gekko » Mon Mar 23, 2015 3:54 pm

Seb28: Always first rate images from every technique you try.

mrsonchus: What a wonderful wife you have! And congratulations on your new rocking microtome! I trust and hope that we'll be seeing the beautiful results not too long after you receive it.

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Re: Plant histology -first attempt

#17 Post by 75RR » Thu Mar 26, 2015 4:53 pm

Seb28 wrote:I found also information that one can somehow convert roking microtome by means of which sections 0.02 μ thick can be cut,but there is a charge for the PDF article.
This is the introduction to the Cambridge Rocking Microtome pdf I found on the web. Have not found Leaflet # 293A yet.

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