MicrobeHunter.com Microscopy Forum

Hello,

I wanted to try the classic experiment of onion root mitosis as I have a good microscope

The first photos are made with the stain gentian violet ; I took the photos with a green filter as my color camera has difficulties to work with purple colour...

And then I found a similar protocol involving acridine orange and epifluorescence : these are the first trials : I find it pretty intersting as it gives a better view of the DNA and chromosomes..


Chris

Great results chrisimbee.
I would be very interested on the preparation details.

Agreed. Stunning photos, and would also be interesting to learn more about the process involved.

thank you very much it's very encouraging,

here is what i did :

1) first I bought a leek at the market and put it in a glass of water next to my window sill for a few days. But before putting it in the glass of water I cut off the roots and leave 1cm . In doing so i make sure that new fresh roots will grow and that is better material for the experiment. Its also important to change the water every day as i may easily rot.

2) after a few days i selected a root tip and cut 5mm from the tip : the active part (meristem) is at 1-2mm from the very tip.

staining protocol I used :

a) plonge the cut tip into HCl at approx 1 mol/L (the HCl I bought at the market is said to be 27% Hcl so after some calculation I just had to make 1 part acid with 6 parts water. I leave to root for about 5-12min. The HCl breakes the pectin between the cells and make it easier to separate the cells later. If the HCl solution is too strong it prevents the cell from staining properly.

b) after the HCl bath I take the root and rinse it in water to remove the acid ( otherwize the gentian violet will turn green and wont stain the nuclei properly.)

c) I put a drop of water on a slide with the root in it and then with a needle a put a few tiny (tiny weeny minute grains) grains of gentian violet ( i have my stain in dry powder form) in the drop of water to get a violet color of the water (=solution of gentian violet). And then I wait for a few seconds to some minutes depending of how strong the coloration gets.

d) Sometimes i check the staining by putting the slide without the cover glass under the microscope and i check for the nucleus to stain : i use the x10 or even better the x4 as the working distance is further away. And when I think its ok i rince the root with water.


e) Then I put the cover glass and gently crush the root (wich should be tender because of the HCl action. The crushing is a must in order to separate the cells).

I noticed that unlike toluidine blue or methylène blue it works very well and faster with gentian violet and the solution doesnt need to be strong either.

And the fact that i just make a drop of staining solution on the slide allows me to spare a lot of my powder gentian violet as its not easy to buy apart maybe on ebay.

If I want to make the epifluo i just use acridine orange instead . Then the nucleus will become orange and the cytoplasm will stain in green.

I will post photos of the protocol.


For the epifluo I ju

Hi Chris,
thank you for posting this interesting protocol! I used orceine in 45% acetic acid and made permanent mounts from my root tips, but your results are probably better. There is an interesting book with a lot of recipes: https://www.microbehunter.com/microscop ... mes#p72362

Bob

Excellent results, Chris !!

... and thanks for sharing your protocol.

Looking forward to seeing the supplementary photos

MichaelG.

Very nice and interesting.

Do the chromatic issues you mentioned arise from using a Zeiss objective and an Olympus photo eyepiece\projection eyepiece simultaneously ?

no, it only comes from the eyepiece i'm using as a photo eyepiece and the astro camera itself...

When i use my D3100 nikon its ok but theres still abberation produced by the eyepice : i expecting a proper photo eyepiece bought on ebay...

When i observe through the microscope the image is sharp and much much better than the photos.

The zeiss is a neofluar and the eyepiece i look through is an olympus but the x10 i use as photo eyepeice is a standard x10 Bresser

i'm going to try again and also try the giant chromosomes of drosophiles and chironomes larvae..

Very impressive and beautiful images.

thank you very much guys