Karyotyping, Giemsa Staining - (Aug/26/2014 )
I am preparing chromosomes for karyotyping, because I need to know the chromosome number of my study organism (ant).
So I just need to count them.
I used a chromosome preparation protocol suitable for ants (including colchicin treatment) and did a standard Giemsa stain.
But I never see clear metaphase chromosomes, they more like round bubbles and they stick really close together, so that I am not able to count.
Now I read that Giemsa is making chromosomes bulky. Perhaps if my chromosomes are very short, this leads to their round appearance?
I do not think that its a problem with the microscope, but I am not sure, as pictures do not get too clear. (I have 1600x magnification available)
I attached a picture!
Can somebody recommend another method of staining? Or help me anyway?
Thank you so much in advance!
First of all, it seems to me that your photo is out of focus. Are you experienced in using a microscope? Have you added immersion oil on the slide (your lens is oil-immersed, am I right?)? Nevertheless, I would suggest cleaning the objective lens, the stage and the illuminator very well.
Apart from these, you are right, your metaphase is poorly spread and the chromosomes are not in a great state. I could suggest some stuff but everything is based on human cell cultures (don't know if they apply...) To get well-spread metaphases I would suggest experimenting with ΚCL incubation (longer incubations). I use pre-warmed KCL and add it to the cell suspension slowly under gentle vortexing.
Mitogen incubation could also affect chromosome appearance and metaphase number.
Another thing that might affect chromosome appearance (elongated chromosomes) and metaphase spreading are slide spreading conditions (humidity, room temperature, as well as the height from here you drop your cell suspension on the slides). Slides should be cold (stored at -20C in methanol). According to my experience I get well- spread long chromosomes at about 65% humidity, at room temperature less than 22C. Drying time shouldn't take too long. You can use a blow dryer (at the lowest scale) but not to close because you will destroy the chromosomes (you have to experiment on that). By blowing only at a specific angle you can also improve your metaphase spreading. Slides shouldn't also dry too quickly...
You also have to keep in mind that some drugs and medical conditions affect chromosome structure. But I guess this does not apply to your case...
You could also add thymidine in your cell culture so as to get elongated chromosomes.
I don't know if I helped or confused u even more but metaphase spreading is a try and see thing. Spread one slide at a time, take note of all the conditions, check on the microscope...if it doesn't work make some changes and try again. Good luck! :)
Hello cell slave,
thanks so much for your help!
Your totally right, I have to work on using the microscope.
Yes, I have added immersion oil on the slide.
I will try to clean everything and improve the pictures.
Thanks for all your suggestions, I will definitely try out some of them.
The major problem is, that I do not work with cell culture.
For chromosome preparation, I dissect male ants, isolate their testis und pull testis into pieces to release cells (everything is happening on the slide already).
I incubate in colchicine solution to stop meiosis in metaphase.
KCl isn´t included in my protocol, but instead a hypotonic solution of Na3Citrat x 2H2O
Do you think the kind of hypotonic solution makes a difference?
Consequently, I do not drop anything on the slide, as testis are so tiny I would definitely lose them in a tube.
Nevertheless, I will try storing slides in -20°C Methanol, as you suggest.
Humidity is a good point as well, it´s also mentioned in the protocol I use, I will try that as well.
I am not sure about 2 things you mentioned: mitogen and thymidin:
I do not use any mitogen, as cells in male testis are dividing anyway.
Do you think I should add it to the protocol?
As far as I know thymidin is inhibiting DNA synthesis, right?
As I already use colchicine to arrest in metaphase, I do not know if I should try?
After colchicine treatment, I use three different fixatives stepwise: 2 different Ethanol-GAA mixtures and finally GAA purely.
Than I apply a standard Giemsa stain.
So you think it´s not a problem with the stain but with the chromosome preparation, right?
I am very grateful for your help!
I know that want I´m doing is totally different from human cell culture, but I will try out your suggestions!
Yes,based on my experience Giemsa is not the problem.
The type of the hypotonic solution shouldn't hypothetically matter. Just be careful, cause prolonged incubation might also lead to chromosome loss (metaphases might spread too much so will not be able to accurately determine chromosome number/metaphase).
I am sorry, I meant mitotic inhibitors such as colchicine. I have noticed that prolonged incubation, affects the total number of metaphases and chromosome morphology.
Thymidine and methotrexate as well as other factors are used in order to obtain elongated chromosomes (important in karyotyping analysis by G- or R-banding). But I think that this is the last thing to try...As I can understand u just want to count the number of chromosomes per metaphase so you don't really need to get elongated chromosomes but just well-spread complete metaphases.
Another important thing that I forgot to mention is the time of staining. I always allow my slides to dry for 24 hours and then I stain them. I have noticed that I get "prettier" chromosomes. Sometimes I also dry them at 90 C or 65C for 2-3 minutes just before staining (One slide at a time, so as to find what works best..).
I have found some papers on the internet that might help u more than my suggestions, although u might propably already have them because they are specific for chromosome preparations from ants.
Good luck Ant_Ony!!! :)
Hi cell slave!
Thanks again for the help and for the papers!
I partly know the papers, the protocol I am using is actually from the guy of the third paper, so really ant specific.
Yes, my only goal is to count chromosomes, to get to know the chromosome number of the species.
I will keep working and follow your suggestions.
I will get back to you, when I will hopefully succeed...