Chromosome spread preparation and staining

CHROMOSOME ANALYSIS

Aim

  • Preparation of chromosome spread from the cultured leucocytes, staining of chromosomes with Giemsa stain and analysis.

Materials required

  • 0.075 M KCl, Fixative solution (Absolute Methanol (3) : (1) Glacial acetic acid), Slides, Coverslips, Alkaline solutions, 70% and 95% ethanol, and Giemsa stain.

Procedure

  • To the lymphocyte cell suspension prepared earlier, add 10 ml of 0.075 M KCl, dropwise at first and with gentle agitation to the centrifuge tube. Gently mix with each drop.
  • Let the cells stand exactly 6 minutes in the hypotonic KCl.
    • Note : The hypotonic solution should not be in contact with the cells in excess of 15 min from the time it is added.
  • Centrifuge the cells at 1000 rpm for 6 minutes. Aspirate the KCl and discard all but 0.5 ml of the supernatant. Gently resuspend the cells in this small volume of fluid.
  • Add 10 ml freshly prepared fixative solution (3 parts Methanol and 1 part glacial acetic acid) dropwise at first and then with gentle agitation. Gentle and continuous agitation is important at this step to prevent clumping of the cells. If the cells were not properly resuspended in step 3, the cells will clump beyond any further use.
  • Allow the cells to stand in fixative at room temperature for 30 minutes.
  • Centrifuge at 1000 rpm for 5 minutes and remove all but 0.5 ml of supernatant. Resuspend the cells in fresh fixative.
  • Wash the cells twice more in 10 ml of volumes of fixative. Add the fixative slowly, recentrifuge and aspirate the fixative as previously directed.
    • Note : The fixed, pelleted cells may be stored for several weeks at 4 ° C.
  • Resuspend the pellet of cells in just enough fixative to give a slight turbid appearance.
  • Use a Pasteur pipette to draw up a few drops of the suspended cells and drop the cells onto the surface of the chilled slides. Spreading of the chromosomes may be enhanced by dropping the cell suspension from a height of at least 12 inches. As soon as the cells strike the slide, blow hard on the slide and rapidly spread the cells.
  • Allow the slides to air dry and then flame the slides in room temperature for 30 seconds.
  • Rinse the slides thrice in saline-citrate solution for 5-10 minutes each.
  • Incubate the slide in saline-citrate solution at 65 ° C for 60-72 hrs.
  • Treat with 3 changes of 70% ethanol and 3 changes of 95% ethanol (3 minutes) each and then air dry the cells.
  • Stain the slides in Giemsa stain for 5 minutes.
  • Rinse the slide in distilled water and air dry the slide.
  • Examine the slide under oil immersion of a microscope.
  • Take the photograph of chromosome spread and then cut out the individual chromosome pairs for alignment (Karyotyping)

Question
Last modified: Wednesday, 20 June 2012, 6:21 AM