Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
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.
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.
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!
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:
40x
Vascular bundles, 100x
Nuclei, 400x
Crystal-violet:
40x
Vascular bundles, 100x
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...
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.
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.
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!
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:
40x
Vascular bundles, 100x
Nuclei, 400x
Crystal-violet:
40x
Vascular bundles, 100x
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...
Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
Very nice project. I really like your results using the crystal violet stain. Thanks for sharing!
Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
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!
Keep up the good work!
John B
Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
Thank you, John!
Interesting arrangement you describe, I’d like to find that sometime!
Interesting arrangement you describe, I’d like to find that sometime!
Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
Superb documentation!
I hope to do this too!
I hope to do this too!
Re: Brassica rapa var. parachinensis TS safranin-o and crystal-violet stain
Thanks, Kevin! I look forward to seeing your results when you try some sectioning and staining!