MicrobeHunter.com Microscopy Forum

Hi together,
here a first try to show mitosis in onion root tips. Stained in carmin -acetic acid.
I tried to freeze the slide to secure the onion cells to the slide, remove the cover slip and add mountant but his needs some more work.

Bob

Looks great to me!
Very nice, and I like the stain too.

Great demo - I'd love to see some more!

mrsonchus wrote: ↑

Sun Sep 22, 2019 8:59 pm

Looks great to me!
Very nice, and I like the stain too.

Great demo - I'd love to see some more!

Likewise, Bob!

Very nice and interesting ! Thank you.

This is a very classic biological experiment. I find it interesting that it can actually make chromosomes visible without much effort. Usually the onion root tip is just squashed with a drop of carmin in 50% acetic acid. The acid fumes are not healthy for the microscope though so I'm looking for an improved protocol that hopefully leads to a nice permanent slide with just one snap of the fingers
Apart from carmin orcein can be used to stain the chromosomes.
We did this once as as a small part of a microscopy meeting but only one member got a halfway acceptable result. So I'm working on an improved process for our November meeting. The picture here was just from my first and only quick try to verify the general method an to test the stain which was found in an unlabeled bottle but smelled and looked just right.

Bob

MicroBob wrote: ↑

Tue Sep 24, 2019 9:01 am

... a drop of carmin in 50% acetic acid. The acid fumes are not healthy for the microscope though so I'm looking for an improved protocol that hopefully leads to a nice permanent slide with just one snap of the fingers

Bob, they are even less healthy for our respiratory system... I would love to see a different fixative, mitosis is an attractive idea.

Hi Doron,
the acetic acid is part of the stain and applied at 80°C. I did this outside while sitting in the sun with my wife, drinking coffee. The amout of vapor is small as very little is needed, but for the microscope the fumes from nearby can be harmful.

Bob

Very interesting Bob. I think a pinch more of de-staining would be make for absolutely great slides. Your method is likely suitable for insect gonads where you can observe the different stages of meiosis (prophase ot meiosis I is separated into 5 stages characterized by different level chromosome compaction, namely leptotene, zygotene, pachytene, diplotene and diakinesis. In the later stages you will find chromosomes engaged in crossing over to facilitate exchange of genetic material.

May I ask where did you source the carmine from? Recently I searched for carmine to feed Peranema with (apparently its loves the stuff and get stuffed with it) but didn't find any particular source that I liked.

Hi Wes,
I've done some further testing today. I had put some root tips into 50% acetic acid at let it sit for two days. My impression was that this made the root tips too hard to flatten and didn't de-stain a lot at the same time. So far I have stained the whole root tips for 20 min at about 80°C. I will do that more precisely the next time to have a more dependable basis for further experiments. Today I squashed a root tip under a cover slip, then iced the slide and popped of the cover slip. I could also do the staining at this stage.

I got the chemicals from a group member, ancient stock, so I have no idea where to get it now. I ordered orcein from ebay.

Bob

Hi Culicoides and John,
thank you for you staining tips in the other thread, the can be applied to my task at hand too.
It was quite easy to get the first acceptable result, but to get a much better result is some work.
Growing onion roots was easy so I have enough supply for further tests.

Bob

Urk, I posted my last post re staining in the wrong thread.....

In response to Culicoides' mention of the use of Crystal-violet for chromosome staineing I wrote,

Hi, for the chromosomes most 'basic' (in pH terms not complexity) stains will do, including of course the venerable and excellent Crystal-violet, suitably mordanted with Lugol's. Another suitable (basic) stain is Safranin - which is far more stable in the presence of alcohol than C-violet, and is able to give a nice metachromatic stain if you're lucky. You may even try to differentially stain the mitotic spindles along which the chromosomes travel as they divide, with an 'acid' stain such as Fast-green.

Be very reserved when using Crystal-violet as it is an extremely potent stain and will overstain details horribly in the blink of an eye...
I'd start with 0.1% Crystral-violet (mayeb start at 10-30 seconds) and a short (maybe 10 seconds) period of Lugol's after a water-rinse of excess C-v.

My area isn't really live staining but I may well have a quick go as you've gone and got me all interested with your excellent posts!

Keep-up the good work!

Apologies for the mix-up...

John B.

mrsonchus wrote: ↑

Wed Sep 25, 2019 7:20 pm

mix-up

And I thought this was just to test the reader!

A senior-moment I'm afraid...

Hi together,
I have made some progress: I can now turn the squashed slide into a permanent slide. without acid fumes around it (picture). I'm now experimenting with the staining/destaining process to get better coulour contrast in the image. I have mixed orcein acetic stain and made some slides but they have to dry for a while. I'm now staining the unsquashed root tip, but think about trying destaining in squashed state. The squashing can be done better with wax paper than with a cover slip as the cover slip too easily rips parts of the specimen with it when it is removed. Befor removing the wax paper I cool with cooling spray.

Bob

Great progress, following with interest.

Hi Bob, this looks pretty good, its as if I can see the two chromatids paired together. Looking forward to more chromosome shots from you.

Hi together,
I have spent some more time on the process and tried this and that. Enclosed is a picture of a slide I made this way:

-Fresh morning-harvested onion root tips
-20 Min in aceto carmine at about 75°C (hot plate 85°, water bath)
-Squashed under waxep paper, iced with cooling spray, waxed paper removed again
- Cooked one time in aceto orcein with cigarette lighter
- Removal of aceto orcein (tissue)
- destained with 50% acetic acid, two times
- Removal of acetic acid (tissue)
- Isopropanol 2x to remove rest of aceritc acid and water
- Removal of Iprop (tissue)
- Drop of Euparal, cover slip

The vegetables try to drive me mad - perfect roots but not always a lot of mitotic stages visible.
I was told to better harves in the morning, but have yet to find a good way to have them ready in the afternoon. I also had the impression that too little water level below the tips is a problem and overcast sky too. Black cats from left to right are a problem in any case.

Bob

Hi Bob,

If you want to increase the mitotic index of your samples you can do so by adding cytokinins which are plant hormones driving cell division in roots and shoots. You can probably buy a powder preparation over the counter but a classical cytokinin called Zeatin is found in coconut milk in active concentrations. Maybe you can see more chromosomes if you let the roots soak in coconut milk overnight. Another classical cytokinin is kinetin which is produced by autoclaving DNA (a good pressure cooker would work and DNA is easily extracted in high amounts).

Hi Wes,
thank you for this good hint!
I always prefer to use widely available stuff for my recipes so they can be useful everywhere and even on restricted budget.
For my own needs I'm happy now with the best of my slides, but I work on stabilizing the technique and would like to make it possible for everybody to get a good result without too much trouble. Lots of dividing cells in the onion root are for sure a good boost for this task! As far as i know the best time for a good amount of dividing cells is the morning, but our meetings are in the afternoon.

One of my sons came upon the idea to make a cup from a cocoa nut.

Bob

Hi together,
here a couple of photos on mitosis stages, bright field, NPL Fluotar 100 1,32, acetic-orceine stain.

Bob

rest

Excellent series Bob. Really good to see the progression of phases.

Nice work old chap, and superbly interesting!

This is very interesting and diligent work. I love the precise inquiry into methods to get better results and your stacks. Great for the forum . Helps take the entire website to a level it won't achieve without such input.

This fine project invokes appetite to try my hands in mytosis as well.