Mitosis in onion root tip

[pwnell]

I never had the chance to perform awesome experiments such as staining the DNA in the cells of an onion's roots and seeing mitosis happening as I opted for different subjects than Biology when I was in high school. Now I am playing catchup.

Fredlab's photos inspired me so I tried my hand at this technique. The instructions I found in my "kit" was horribly wrong - I did not manage to get any DNA stained. So I found another resource and it seems like the trick is to incubate the tips with 1N HCl at a temperature of 60C. Without doing that I did not get anything stained. What do you think of these two photos?


20130626-DSLR_IMG_0079-Edit.jpg by pwnell, on Flickr
Onion root tip dna stained - mitosis, 60x, DIC


20130626-DSLR_IMG_0108.jpg by pwnell, on Flickr
Onion root tip dna stained - mitosis, 60x, DIC

[Pau]

They look excellent, a nice combination of stain and DIC.

The instructions I found in my "kit" was horribly wrong - I did not manage to get any DNA stained. So I found another resource and it seems like the trick is to incubate the tips with 1N HCl at a temperature of 60C. Without doing that I did not get anything stained.

You need to attack the cell wall to allow some stains get inside and to dissolve hemicellulose in the lamina media to allow separatate the cells. There are several methods but all I know imply acids, some hot and some at room temperature. Could you describe yours?

[pwnell]

Yep. I basically followed these steps:

1) Wash tips in RO water
2) Incubate at 60C for 12 minutes in 1N HCl
3) Wash tips again
4) Submerge in Leucofuchsin dye for 12 minutes.
5) Wash tips again
6) Place on slide and squish with cover plate to flatten sample

The kit had additional chemicals to bleach the non stained areas - but I have not used those for these photos.

[bromodomain]

Wow, these are awesome, you could sell them to a biology textbook publisher

I did something similar as a fresher in university. I recall using 5N HCl and toluidine blue.

[discomorphella]

Nice staining. The method you're using is called the Feulgen technique. It uses HCl (or other acids for staining biomolecules other than DNA) to hydrolyze the DNA, specifically the deoxyribose backbone, to an aldehyde, that the leucofuschine reacts with with to make a magenta colored dye. The leucofuschine reagent is called Schiff's reagent, since the final colored product is an imine, or Schiff's base. The stain is typically followed by a rinse or two in a weak sulfurous acid bath to help bleach the colored Schiff base from everything other than the DNA that you were trying to stain (a variety of differentiation step). So next time you can try the extra sulfurous acid washes and see if the nuclear staining changes for the better. These look quite good, however the extra acidified bisulfite rinses may crisp the staining up a bit. The hydrolysis time and temperature is, as you've discovered, crucial for getting good staining. In fact, the hydrolysis time / temp schedule depends on both the sample and the fixative that was used. Additionally, since the Schiff reagent reacts with aldehydes, if you use something like formaldehyde or glutaraldehyde as a fixative you have to wash the tissue and then the sections very thoroughly, or you'll get a lot of background staining. Glutaraldehyde is really not a good fixative to use if you're going to employ Schiff's reagent since it induces a lot of background staining. Its as nice technique, but for DNA staining its been more or less supplanted by the more useful DNA staining fluorochromes Sytox, DAPI, TOTO etc).
You might try fixing the root tips with some ethanol/acetic acid and then washing and starting your staining schedule. It should permeablize the cells nicely for your stain. You will have to adjust the hydrolysis time after you fix.

David

edit: a good reference if you're curious is
"Histological and Histochemical Methods", J.A. Kiernan, 4th ed, 2008
Scion

[Marek Mis]

Brillant and very clear images !

Marek

[Jacek]

Very interesting, beautiful pictures, I have to try

[RogelioMoreno]

Beautiful!

Rogelio

[pwnell]

Thanks for the comments, especially for discomorphella's detailed comment. I will certainly try your suggestions and post back.

[yvan_be]

Very nice slide and pictures!

(To be honest: I find most of the pictures here jaw dropping... Very jealous of your skills and your knowledge, guys).

The microtechnique behind this type of slides is not that difficult and it can be done easily at home. I wrote an article in Micscape a few years ago on squash slides. It can be found here: http://www.microscopy-uk.org.uk/mag/ind ... quash.html. It describes only one of the numerous techniques (there are hundreds).

Those interested in this type of work should realy look for a second hand copy of what stil is THE essential manual on the subject: C.D. Darlington & L.F. LaCour: The Handling of Chromosomes (1960's, publisher: Unwin BrothersLimited, London).
It's also availlable (second hand that is) in a German edition, published at the time by KOSMOS/Franckh: Methoden der Chromosomenuntersuchung.

Staining Procedures 4th ed. (ISBN 0-683-01707-1) has some 20 pages on plant cytogenetic methods, from Belling's original iron acetocarmine up to in situ rRNZA/DNA hybridization and autoradiography.

And a very usable manual on plant microtechnique in general, in English is Steven E. Ruzin's Plant Microtechnique and Microscopy. The copy I have is ISBN 0-19-508956-1.

Perhaps of interest for those looking for another color for artistic reasons: once the DNA in the sample is hydrolized using an acid, allmost any stain/dye can be used to stain the DNA/chromosomes/nuclei: any haematoxylin/haemateïn staining solution, including Mayer, Delafield, Gill, Mallory's PTAH, ...
Any staining solution containig carmine, such as aceto-carmine, Grenacher's alcoholic boraxcarmine, Orth's lithiumcarmine, ...
Any of the following "nuclear" dyes, dissolved 1% in water: methylene blue, crystal-/ gentiana violet, safranin, thionin, fuchsin basic, malachite green, nile blue, ...

[carlos.uruguay]

Impressive.
Thank you for sharing it