Hello Arian Rahimi ,

You can find below a general protocol working well :

Grow cells in rich medium at 37°C with shaking (200 rpm).

Dilute an overnight culture of S. aureus back to about an OD of 0.5 in 50 ml prewarmed TSB.

Re-incubate for 30 min (to allow the bacteria to grow to OD 0.6-0.8).

Place the vessel in an ice-water slurry for 10 min (Keep on ice when on bench).

Harvest the cells at 3,900 x g for 10 minutes at 4 °C.

Discard the supernatant. Add in 50 ml of sterile ice cold milliQ water

Harvest the cells, 3,900 x g for 10 minutes at 4 °C.

Discard the supernatant. Add in 50 ml of sterile ice cold milliQ water.

Harvest the cells, 3,900 x g for 10 minutes at 4 °C.

Discard the supernatant. Resuspend the pellet by pipetting in sterile ice cold milliQ water. Make up to 5 ml.

Harvest the cells in a swinging bucket centrifuge at 3,900 x g for 10 minutes at 4°C. Discard the supernatant, resuspend the pellet by pipetting in sterile ice cold 10% (w/v) glycerol. Make up to 1 ml.

Harvest the cells at 3,900 x g for 10 minutes at 4°C.

Discard the supernatant, resuspend the pellet by pipetting in 210 μl of sterile ice cold 10% (w/v)

glycerol.

Dispense into 50 μl aliquots and freeze at -80°C.

Hope this will help,

Best regards

Hello Yousef Maali

Thank you sir for your efficient and quick answer. I'm very grateful.