Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

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mazo4033
Posts: 107
Joined: Mon Jun 15, 2020 4:52 pm

Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#1 Post by mazo4033 » Mon Aug 02, 2021 8:11 pm

Hi everyone,

I have been drawn to the bright yellow flowers of the yu choy plant so I have spent some time sectioning and staining their stem, using the opportunity to test some crystal-violet stain.

1 - background
Yu choy/choy sum, or Brassica rapa var. parachinensis, is a vegetable very similar to bok choy and is in the same genus as different cabbage varieties. Yu choy, like other members of the Brassica genus, sprouts pretty yellow flowers.

Image
Brassica rapa var. parachinensis (not my image; here is the credit: https://en.wikipedia.org/wiki/Choy_sum# ... stalks.JPG)

The part of the flower which I sectioned is the thicker stem that attaches the flowers to the rest of the plant. Other parts of the flowers like the unopened buds or the thinner stems that the flowers are directly connected to seem like promising subjects to section. It was just too difficult to do so by hand and would require paraffin embedding and microtome sectioning for optimal results.

Image
The yellow flowers

2 - procedure
Sectioning was done by hand with a single-edge razor blade. Temporary mounts were made with a pva glue-based mounting medium (a 1:1 mixture of a pva-based glue like clear Elmer's glue and glycerin combined with some water). Two different stains were used, safranin-o and crystal-violet. Here are the procedures I followed:
Safranin-o:
sections in safranin-o 1% aqueous for about 5 seconds
transfer sections to water to rinse excess stain
transfer sections to 70% ethyl alcohol for ~10-15 minutes. The Brassica rapa var. parachinensis was a very interesting plant since I felt it was much more fragile than other plants I've sectioned. The first set of sections were a failure in fact because the tissues were damaged somewhere in the staining and mounting process. Knowing this, I opted to limit the time they spent in the alcohol. I am not sure if this made a difference, but the second set of sections (pictured in this post) were more successful than the first.
transfer sections back to water and mount
Crystal-violet:
sections in diluted crystal-violet for some time (my experience with crystal-violet is that it is a very potent stain, so I went light on the stain) - 1 drop of stain into quite a bit of water
transfer sections to water to rinse excess stain
transfer sections to 70% ethyl alcohol for ~5-10 min. I found that when I took the section out, too much of the stain had been removed by the alcohol, so I dipped the sections back into the diluted crystal-violet until I felt satisfied with the stain intensity.
transfer sections back to water and mount

I was just experimenting with the stains, especially crystal-violet. I have no idea if I properly applied the stains, but it seemed to work! :lol:

3 - results
Since Brassica rapa var. parachinensis is a dicotyledon, my main goal with these sections was to better visualize the vascular bundles that circle around. Unfortunately I think the sections were a bit too thick to have a great view of the vascular bundles, but oh well...

Safranin-o:

Image
40x

Image
Vascular bundles, 100x

Image
Nuclei, 400x

Crystal-violet:

Image
40x

Image
Vascular bundles, 100x

Image
Xylem in the vascular bundle, 400x

4 - conclusion
Those are the results, I wish the sections were thinner, but I like how the crystal-violet stain turned out since it did not overstain. Now to add to my stain collection... :D

Javier
Posts: 806
Joined: Tue May 09, 2017 11:19 am
Location: Buenos Aires, Argentina.

Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#2 Post by Javier » Mon Aug 02, 2021 8:56 pm

Very nice project. I really like your results using the crystal violet stain. Thanks for sharing!

mazo4033
Posts: 107
Joined: Mon Jun 15, 2020 4:52 pm

Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#3 Post by mazo4033 » Mon Aug 02, 2021 11:47 pm

Thanks Javier!

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mrsonchus
Posts: 4175
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#4 Post by mrsonchus » Tue Aug 03, 2021 8:48 am

Looks like a nice start to me - clearly able to see that the vascular bundles are singles and as you say arranged in a ring. Some plants will have a complete ring, some other arrangements such as having the usual arrangement with 'extra' phloem bundles on the 'inside' of each vascular bundle - seen in milkweed stems I thnk.

Keep up the good work!
John B

mazo4033
Posts: 107
Joined: Mon Jun 15, 2020 4:52 pm

Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#5 Post by mazo4033 » Tue Aug 03, 2021 2:51 pm

Thank you, John!

Interesting arrangement you describe, I’d like to find that sometime!

Kevin_r
Posts: 48
Joined: Tue Aug 04, 2020 1:36 am

Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#6 Post by Kevin_r » Tue Aug 03, 2021 3:47 pm

Superb documentation!
I hope to do this too!
:D

mazo4033
Posts: 107
Joined: Mon Jun 15, 2020 4:52 pm

Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain

#7 Post by mazo4033 » Tue Aug 03, 2021 4:25 pm

Thanks, Kevin! I look forward to seeing your results when you try some sectioning and staining!

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