many reasons... Are you sure of the cells exploded to reveal chromosomes or if you keep the cells in hypotonic over much, cells may be dispersible before fixing. If you explain your method, we can discuss more.

Yes...there could be many reasons...if culturing is not for sufficient time or harvest is not proper...proper washing and fixation ...and preparation of chromosome spread and staining....also protocol that you follow decides the quality of your chromosome spread...

the reasons may be due to the mitogen (PHA) cocentration and the colchicine concentration.. because the colchicine only help us to arrest at metaphase stage during cell cycle... kindly ensure the colchicine concentration.. 

Dear Dian, it would be interesting to comment as did the cultuing methods, so in this way can figure out which is the reason for the absence of chromosomes

Dear Dian, to comment it, I need to know more about the procedure of culturing. We do this method very often. So I am waiting for more info

Dear Dian,

without exact knowledge of your protocoll(s) it is not possible to make a proper troubleshoot! Most critical steps on your way to make chromosomes are:

1 Blood sample itself -> Heparinized blood NO EDTA-Blood! Direct after taking the blood the vacutainer should mixed head over tail a few times to avoid agglutination of the blood.

2 Culturing (72h-Culture) -> proper Conditions and Medium -> 1ml blood + 9ml culture medium (for example: RPMI 1640) + PhA. 1.5 hours prior to harvesting add Colcemid or Colchicin to the culture! 

If u want to establish synchronized "high resolution" culture you have to add MTX to the culture(s) at 12 to 16 hours before harvesting

3 Harvesting -> After spinning the cultures remove the supernatant, but leave about 1 cm of fluid above the pellet and do NOT remove the upper layer of the pellet coz here are the most metaphase cells!!!! Add about 8 ml prewarmed (37 degrees) hypotonic solution and mix by gently drawing the cells up and down with a Pasteur pipette and incubate for 10 minutes at 37 degrees or 15 minutes at room temperature (20 -22 degrees); mix gently once during incubation. After that spin and remove supernatant cautious, kleaving about 1 cm above the pellet. Resuspend cells carefully and start fixation -> hold vessel in a sloping position and start with 3 drops of freshly prepared ice chilled (-20 degrees) fixative (3:1 methanol:acetic acid) but DONT let them fall into the suspension!!! This step is very critical! Make sure that the first drops of fixative run down the wall of the vessel otherwise u might loose a lot of metaphases because they will burst if first fixation step is to rough! After u have added about 10 drops in that way mix gently with a Pasteur pipette and add another 5ml of fixative, mix gently and leave it for 10 minutes. Spin, remove the supernatant (red) but leav about 0.5 cm of it and add 8 ml fresh cold fixative, resuspend the pellet and spin again. Repeat this 1-2 times and after last soin remove supernatant except for the last 0.5 ml and resuspend cells.

4 Preparation of Slides -> important factors are: humidity, temperature and "condition" of ur slides. Start with 2-3 drops and air dry (or dry on dryplate) and check cell density under phase contrast microscope. If cell density is not sufficient just dilute or concentrate your suspension.

Rading the previous answers I can identify almost all the critical points related to  metaphase' s chromosome yield.  Culture time, proper concentration of phytoaemaglutinin when treating with blood cells. Colcemid concentration and exposure time. The age of a culture, if primary or the confluence at the moment of adding the colcemid. Ther are also several critical steps regarding pellet handling, not only the "fixative related burst" but also the pellet  slide  after hypotonic treatment or during one of the several fixation steps.

If everything ok, means the procedure.

I want to know have you seen the blast cells under microscope?

If not the culture is procedure defective and or infected.

Have to check all steps. Then only can comment.

Dear Colleague: Besides the cases already cited by our colleagues,  the most important reason for not getting chromosomes think it is due to phytohemagglutinin. I advise you to first of all change phytohemagglutinin by a different and new batch to make sure it is 100% active. You must also use phytohemagglutinin as "M". 

Moreover, I make some comments. Culturing peripheral blood is always better in a closed system (without CO2) by adding sodium bicarbonate (200 microliters) to the culture medium (alternatively be used 200 microliters of Hepes buffer solution) and to support the tube always at an angle (no bottle). These tubes have to be removed (gently agitated) once every 24 hours.

Incubation times in thermal bath, hypotonic shock, exposure to fixative are variable according to the protocols, but they seem well you use. Step # 6 must be specified also must be added dropwise. Step # 10 must refer to a refrigerator.

Except for the issue of phytohemagglutinin not understand why the chromosomes do not appear.

Greetings for You.

Dear Dian

What kind of culture are you doing to get chromosomes?

if chromosome not appear you should think first all in antimitotic quality is a very important step. please tell me what kind of culture are you doing?

greetings luis