The steps for isolating mitochondria from microorganisms are described below. 

1. Transfer the yeast culture grown overnight into two 15ml tubes in a sterile manner. 
2. Spin the tubes in a centrifuge at 500g for 10 minutes at 4°C. 
3. Carefully take out the supernatant without disturbing the pellet. 
4. Wash the pellet with 1 ml of 0.9% sodium chloride using a micropipette. 
5. Dispose of the sodium chloride liquid from the tube using a micropipette. 
6. Mix the pellet with 1 ml of cold lysis buffer using a pipette
7. For 10 minutes, let it incubate a shaker at 4°C. 
8. Centrifuge the mixture at 1000g for 10 minutes at 4°C then remove the supernatant. 
9. Resuspend the cell pellet in 1.5ml of cold disruption buffer and disrupt the cells using a blunt needle.
10. Centrifuge the lysate at 1000g for 10 minutes at 4°C
11. Transfer the supernatant to a new tube. 
12. Centrifuge this supernatant at 6000g for 10 minutes at 4°C and discard the liquid part.
13. Wash the remaining pellet with a mitochondria storage buffer. 
14. Centrifuge the washed pellet at 6000g for 20 minutes at 4°C. 
15. Resuspend the final pellet in the mitochondria storage buffer and store it at -20°C.