Hey Yonara, the software Axiovision can help you with counting (you can download ir for free on the Zeiss website). There is an option to just click onto each chromosome and the program will keep track of the total number for you.

Regarding the metaphase spreading, I found this really thorough answer by an expert on the subject. Take a look!

"The most significant factor in chromosome spreading is the humidity. You need to have around 50% humidity for the best results. Below and above that % gives increasingly poorer results. You can make a small chamber and control the humidity with an inexpensive hydrometer. The size of the drop and the amount of fiixative can have an influence. For this we use P. pipettes with different size openings. Larger drops spread more.

The water bath technique can help but is not sufficiently controlled for consistent results. Dropping distance or slide angles are not important. Fixative running off the end of the slide onto absorbent paper means you are losing metaphases too. I use a dry slide on a flat surface and this provides for a nice circle of metaphases in a consistent postion on the slide which is convient for later in situ hybridization. There is no reason to have poor spreading if humidity is correct and the cells have had correct hypotonic and fixation." Roscoe Stanyon

https://www.researchgate.net/post/Spreading

hello

You can contact Pr Marek Switonski whos is working on canidae chromosomes

Prof. Dr. Marek Switonski,

[email protected]

corresponding member of the Polish Academy of Sciences

Department of Genetics and Animal Breeding

Poznan University of Life Sciences

Wolynska 33

60-637 Poznan

Poland

Hi Yonara;

the software suggested by Guilherme is a good idea. There have been several programs developed over the years but, if the one from the Zeiss web site can be downloaded for free, then I'd just go for it.

Regarding spreading techniques, Roscoe Stanyon suggestions are basically sound. After several decades of experience, I found pre-freezing the slides to be consistently effective. We used to clean the slides in abs EtOH and wiped them dry. Then placed them flat-down on a tray and placed them in a deep-freeze about 20 min before dropping the fixed-cell suspension onto the slides. As you take them out of the freezer, you'll notice a thin, even film of moisture forming on top of the slides. It's important to drop the cell suspension ASAP, before this film dries out or condenses into bigger drops of water. Immediately thereafter, I used to transfer these slides directly onto a hot surface (incubator set at 65-67 deg. C). This caused a quick drying of both, cell suspension and water moisture, and consistently good spreads.

If you want to get much number of chromosome spreads, it will be worth to try PCC use with Calyculin A. I have not experienced PCC for dog cells, but it can be working.

You could use Adobe Photoshop CS6 (extended). This software has also a counting tool that allows clicking onto each chromosome and track the total number of chromosome.

you have already been replayed by Roscoe Stanyon,  The best in this field. 

It is a matter of relative humidity.

best regards

ls

Hi Yonara,

The Metasystems Ikaros software has a counting ability and a karyotype layout that you can use. I've done this once myself...a daunting task at that, but makes it easier.

cheers

Jasen

Which procedures and tissue did you used for the preparation of chromosomes?

Yes I have six years experience in the field of cytogenetics and I can help for preparation of chromosome from any animal. Anil kumar NDRI Karnal

The dog has 78 chromosomes, with all autosomes acrocentric; this is one of the worst organisms for karyotyping. Without perfect metaphase preparation you will be lost. Change conditions (especially hypotonic treatment) until the spreads look perfect. Once you have perfect spreads you can do G-banding and this should help too for identification of chromosomes.

I use Colchicine solution [16ug/mL) for 1 hour, and after centrifugation, the pellet is ressuspended  in 0.075 M KCl hypotonic solution for 20 min.

After hypotonization, we add 5 drops of a freshly prepared ice-cold fixative (3 methanol: 1 acetic acid)  and the cell suspension is stirred gently at room temperature. After 5 min,  the cell suspension is gently stirred with a Pasteur pipette and centrifuged at 1200 rpm for 5 min. Then we ressuspend the pellet in 1 ml of 3:1 methanol:acetic acid, and 3 drops  of the concentrated cell suspension are dropped (from a 30cm distance) onto clean slides and allowed to dry, before Giemsa staining.

Which changes do you recommend in hypotonic treatment that could help? Does the water-bath at 60ºC make difference?

Thanks a lot for all the anwers! 

Yonara, your methodology is certainly adequate.  Are you culturing the blood or using bone marrow aspirate?  That information may help to clarify the missing links.

None, the cells I use are from primary culture.. Mammary cancer cells. 

If hypotonization is a problem, you will often have burst cells with the chromosomal elements dispersed in space.  Temperature facilitates hypotonization but you are in order if you do not have broken cells.  I suggest that you skip colchicine treatment for some trials and see what that does to your chromosome lengths.

you can also try using sodium carbonate in place of potassium chloride as hypotonic and try different concentrations of colchicine for karyotyping

Thank you people!

colchicine treatment time may be decrease