Dear @Askandar Omer

Getting the metaphase chromosome spreads onto the slides is the most difficult part of chromosome harvesting, and there is plenty of debate on which step is the most influential on the final spread.

Once you are ready to make your metaphase spreads, resuspend your cell pellet in fresh fixative a few times. This will help get rid of any cell clumps before you drop them onto your slides.

The trick is to let gravity do the work for you, but you may need to spend some time optimizing the distance. If the distance between your slide and your pipette is too large, your chromosomes will spread too far apart. When chromosomes are too far apart, it may be difficult to determine if they were from one cell or two cells. This could be a huge problem if you are trying to assess the ploidy of your cells.

If the distance between your slide and your pipette is too small, your chromosomes will overlap and be squished together. When your chromosomes are overlapping, you won’t be able to tell them apart. This will make any staining you might do difficult to evaluate. Use a disposable transfer pipette to drop your cells onto clean slides, but keep in mind that the diameter of the pipette can also influence chromosome spread on the slide.

Humidity is a critical component of the metaphase spread procedure, and it seems to be the most important factor affecting how the chromosomes spread on the slides. The fixative pulls moisture from the air, which results in rehydration of the now broken cells. Controlling this rehydration process is the key to beautiful metaphase spreads!

The two most common methods of maintaining slide humidity are:

1. keeping the slides slightly damp with water (either by dipping the slides in water or pre-freezing them) before dropping the cells onto them, then allowing them to dry on a heat block.

2. using dry slides, dropping the cells, and then immediately placing the slides on a tray in a covered water bath.

Choosing the method is up to the individual, but the overall message is to keep it humid!

When drying the slides, the temperature can vary from 20°C to 75°C depending on the protocol. [6,7,13,18,19] A higher temperature can cause the fixative to dry too quickly. A temperature that is too cold could cause an increase in moisture on your slides, as cold air can’t hold as much water as warm air. This could cause your fixative to dry too slowly. As drying time influences chromosome spreading, it is important to find the right combination of temperature and humidity to optimize chromosome spreading for your cells.

Dear Askander Omar,

Do you use any hypotonic treatment before fixing your slides? Mild hypotony of a cell suspension could help: change exposure time and different solutions (KCl or sodium citrate) for better results.

Dear Eugene Daev

Thank you for your response, We use hypotonic treatment but we constantly have overlapping chromosomes, I don't know if we need another protocol or we should just make some changes in the spreading technic.

if the problem is overlapping chromosomes, you could increase the concentration of the hypotonic solution (e.g. on insects, I use sodium citrate in 0.5% water). I hope you managed to get a good protocol

Hi,

I think that this problem has several workplaces. We also try to arrange the metaphases on the slide as beautifully as possible. Several steps are important - from the cleanliness or degreasing of the glasses, the correct temperature of the frosted glass (depending on whether super-frost glasses or ordinary glass slides are used), but mainly the technique of dropping the suspension on the glass - sufficient height, number of drops on the glass and above all, the quick drying of the dripped suspension with warm air proved useful, but a warm plate is also sufficient. All this will ensure that the metaphase spreads out nicely on the slide. and of course, it also depends on the methodo used, culture media and chemistry used.

I wish you the best of luck in making it happen.