To study the structure of somatic chromosomes in the bone marrow of rats.
- 0.05% colchicine
- 0.56% KCl
- Centrifuge tubes
- Glass slides
Inject 0.05% colchinine interperitonially into the rat 1½ hours before the
experiment starts. The volume to be injected varies with the weight of the rat.
If the rat is around 40 gms or more, 1 mL of 0.05% colchinine should be injected. Leave the rat for 1½ hours and then kill it by cervical dislocation.
Then, remove the femur bone. Flush the bone marrow in a clear petri dish using
0.56% KCl, which serves as a hypotonic solution. Remove all large particles by
straining the solution in a clear cheesecloth or muslin. Take the filtrate in a
centrifuge tube and spin it at 1000 rpm for 10 minutes. Obtain a cell button after
pouring the supernatant. The fixative (1:3 acetic alcohol), which is
made by mixing 1 part of acetic acid with 3 parts of acetone-free methanol or absolute
alcohol, is added to the cell button and gently mixed. Centrifuge again to get
a fresh cell button and add fresh fixative to get a cell suspension. This method is repeated twice. Finally, drop the cell suspension onto a clear slide, preferably
kept in cold alcohol. Allow the drops on the surface of the slide to dry. This
technique is known as the air drop technique or air dry preparation. Then the
air-dried slides are taken for the staining. Freshly prepared air-dried slides
produce better results for the Gimsa stain. For nonbanded chromosomes, the
standard Gimsa stain is diluted with 6.8 pH phosphate buffer solution. 5 mL
of phosphate buffer solution is taken and added to matured 1 mL of stock
Gimsa stain in a coupling jar, and mixed well. This is the active working Gimsa
stain. Dip the slides in the stain for 15–20 minutes. Wash it under slow
running water until the excess stain is removed. Then rinse it with distilled
water. Allow it to dry. Then observe the slides under the microscope.
Somatic chromosomes of rat bone marrow were observed deep violet under the