2.7. Karyotype analysis

The passaged cells in the exponential phase of growth were treated with 0.5 µg/mL colchicine for 2 h. Cells were then harvested and treated with 8 mL hypotonic solution of potassium chloride at 0.075 mol/L at 37 °C for 30 min. Then, added 8 mL stationary liquid which fixed in solution with a volume ratio of acetic acid to methanol of 1:3. After centrifuging, added 8 mL stationary liquid to precipitate and incubated at 37 °C for 20 min and then kept in ice for 10 min. Repeating the previous step, then collected the precipitate and added 500 µL stationary liquid for resuspending. The samples were stored at 4 °C, and analyzed of karyotype by practical automatic analyzer.