1. Using an 18-gage syringe needle, add five drops of reconstituted colcemid (10 pg/mL) to each culture.
2. After mixing well, incubate the cultures for 1-2 h at 37°C.
3. Centrifuge at 250^ for 10 min.
4. Remove the supernatant and break up the pellet by agitation with a vortex mixer.
5. Add 10 mL of hypotonic KCl-Na citrate mixture to each culture. Suspend the cells by gently inverting the centrifuge tubes manually. Allow the tubes to stand at room temperature for 30 min.
6. Gently suspend the cells again by inverting the tubes. Then, add 2 mL of freshly prepared methanol-glacial acetic acid fixative directly to the contents of each tube. Mix the contents by inverting the tubes, and centrifuge the tubes again at 250^ for 10 min.
7. Remove all of the supernatant from each culture. Gently "thump" the tubes to break up the cellular pellets.
8. Add 10 mL of fresh fixative to each culture. Resuspend the cell pellets by inverting the tubes and allow the cultures to stand at room temperature for 10 min, and centrifuge.
9. Repeat step 8.
10. The next steps are performed in a "harvesting room" with a humidifier. For maximal chromosome spreading, adjust the humidity to 55-65%.
11. Set a hot plate to 65°C and check the temperature with a surface thermometer.
12. Centrifuge the cell suspension at 250^for 10 min.
13. Remove the supernatant from the centrifuge tubes and add fixative to the pellet drop by drop until the suspension becomes "semiclear." The amount of fixative required will depend on the size of the pellet. The final cell concentration may have to be adjusted after evaluation of the first slide.
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