I am working with wheat chromosomes and trying to stain the chromosomes. However, during staining with Giemsa dye chromosomes appear totally dark. I'm not sure why this might be. Anyone have any ideas?
The Wright or Giemsa-stains can be influenced by the pH of the rinsing solution after the incubation in the dye-solution. The lower the pH the more pink will be the result. The higher the pH the more blue will be the result. Usually Giemsa-stains are differentiated at an approximately neutral pH. (Weise buffer pH 7,2 or acidified water pH 6,5). To lower the pH you can add few drops of acetic acid. This will also enhance the speed of differentiation.
Giemsa-stains are also sensitive for too long rinsing in diluted ethanol. The color turns blue and more faint.
Also it might be the trypsin that can digest some region rich in C or G. It's not condensed so appears light but the other region is very condensed so the trypsin can't enter and digest this region, which appears dark or the opposite.