Chromsome counts

Today a recent graduate student and I were doing some root tip squashes. This is what we learned:

Carefree Spirit 3x

Spring Fever 2x

Red Drift 2x

All the Rage 3x

Wild Thing 3x


David thanks so much for doing all these chromsome counts. You are really helping a lot of us. By the way I tried germinating some op carefree spirit seeds last Winter and not a single one germinated. Don’t know about its pollen.


Thanks Patrick,

It is really fun to do and interesting when surprises show up. It seems like some of the triploids can set a lot of hips like ‘Carefree Spirit’. I was expecting it would be 4x from all the hips. Maybe with triploids a higher rate of the embryos abort or are aneuploid and don’t develop normally?? Thanks for sharing your experience with germination of C.S.

I’m really excited Andrea started as a graduate student carrying on Vance Whitaker’s black spot research. Andrea has a true passion for plants and really caught on fast and seems to enjoy chromosome counts!! She really loves roses too. I look forward to all she will accomplish.


To do a chromosome count do you just obtain a root tip, that is undergoing rapid mitosis like from a newly struck cutting, and then stain with somethng like methylene blue and do a squash to count the chromosomes when you find a cell at the right mitotic phase?

Hi Simon,

Generally. The key is to get actively growing root tips so there will be a lot of cells in metaphase. I like to collect the root tips and put them in vials with water and put them on ice for up to 24 hours. The cold slows down spindle fiber activity. One can arrest more cells in metaphase this way. People sometimes use colchicine or 8-Hydroxyquinaline citrate to do the same. Ice is easier and I like the fact we are not left with toxic waste to dispose of. The cells are then fixed to kill them. I like to use Farmers solution (3 parts 95% Ethanol to 1 part Glacial Acetic Acid). I then refrigerate the samples until I look at them. Before looking at them it is helpful to soften the cells so the squash and spread will be flatter. I like to use 6N HCl to do this and leave the root tips in that for 90 or so minutes. I replace the acid then with water and am ready to go. I typically stain with acetocarmine. I take the root tips and carefully cut them longitudinally down the middle and gently knead out the very young meristematic cells and get rid of the rest of the tip. The older cells can interfere with their more developed cell walls from getting as flat a spread. I then put significant pressure by tapping on the slide to squash or spread the cells. I like to take the slide with cover slip and put it upside down within a paper towel over and below it before I tap hard. This allows good pressure to be given to the sample without breaking the coverslip.

I like to look at multiple cells to be convinced and cells from multiple tips. Usually everything is consistent. Sometimes I may rarely get a cell in the process of having the sister chromatids separating to the poles for the new daughter cells and the cell looking like it has twice as many chromosomes as it really does. It takes patience and practice looking for those small chromosomes and getting cells where the spread is nice enough to separate them and count them.

Sometimes if I am in a pinch and there is a rose that does not root well from cuttings, I do this technique with a shoot tip. It is more difficult to isolate the meristematic cells and there are a lot more starch and other particles that make it more difficult to clearly see the chromosomes.



Thanks David. I don’t know if we have acetocarmine at work (because it is a school lab and many things have been banned… so the ice thing is a good strategy). Is acetocarmine known by a common name? I seem to remember seeing a stain labelled ‘Carmine Red’ on the stains shelf… I am also trying to find this information but if you know off the top of your head I would love to know (in case I can’t find it rolls eyes). Everything else is easy to source. We have just won a 2 million dollar grant to build a biotech lab at work and we’ve applied for a new digital microscope… I’d love to take some decent photos of things like this :slight_smile: In the absence of acetocarmine how do you think methylene blue would do as a substitute? I use methylene blue with the junior kids to stain proteins and DNA because it is safe to use (but messy in the hands of a year 8 student)… I also get the kids to make root tip squashes of onion roots and these worked quite well but I’ve never sat down and physically counted the chromosome before… looking forward to it. Will start with a multiflora I have that I know is diploid so I know what to expect I think. Thanks again :slight_smile:

Hi Simon,

Wow, it sounds like you have a lot of resources!!! That is great!!

The truth is you really don’t need stain, it just helps. I would go ahead and try your Methylene blue. Acetocarmine is just carmine boiled and dissolved to saturation in acetic acid and a 30ml or so bottle is just a few dollars from some lab supply companies.

It’ll be great to have more of us counting and sharing!!


I finally got time to look up acetocarmine in my Conn’s stains book. It is based on cochineal, which ahs a rather interesting history in Australia according to the WIKI. But important point is it isa food stain for a “natural” red so that part is OK even from middle school. Our usual school suppliers (Wards, Carolina Bio) sell it.

The glacial acetic (50 %) may be a bit rough on the skin. I got a lot of it on my hands as a grad student decades ago and it can cause some dryness or peeling in high doses. But a lot less dangerous than the HCl. Carmine is not very soluble in anything neutral, it needs acid or base to dissolve. So I doubt you could get by with just vinegar, thoguht you might take the stock 50 % acetic and dilute it 10 x for student use. I’ve never tried so can’t say how the solubility would hold up.

To stain chromosomes you want certain properties which methylene blue doesn’t have.

Stay away from acridines. Great stains but carcinogens.

You might also consider Giemsa staining as the corn geneticists describe on an old newsletter posted on the web for seeing pachytene banded chromosomes. But the acetocarmine is probably simplest.

Supposedly aceto-orcein stain is better for staining genetic material. It’s available at the usual places for about the same prices.