Hello.....I've been culturing human peripheral blood for karyotype analysis. However, in the past few days when i band my slides using giemsa and observe them under the microscope there is a cytoplasmic background that doesn't allow me to observe the chromosomes nicely and hinders the banding pattern.

 

Can anyone tell me the reason why i am getting it and how do i get rid of this dense cytoplasmic background???

 

Secondly, there's splitting of chromosomes often seen while analysing the cells...any specific reason for splitting??? and how can it be avoided??

 

thanks in advance

I would try giving the cells an extra wash in fixative, maybe two more, but I wouldn't do more than 3-5 total washes. That should alleviate the cytoplasm problem. As for the chromatids splitting, I'm not sure. We sometimes have that happen too, but I'm only a "year old" in this lab and I'm not sure why that happens yet. I know your question is from a while ago so I'm betting you already fixed your problem...?