DMSO stock preparation
Akashdutta
Abstract
Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4oC. Long-term storage of bacteria is best at temperatures around -80oC. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol or DMSO (anti-freezing agents) is added to the media to store samples at such temperatures.
Steps
From the streaked plate of the bacteria, pick one colony and inoculate into a 250mL flask containing 100mL culture.
Grow the culture till it reaches OD∼ 0.6-0.8
Take the 100mL culture and transfer 40mL each into two falcon tubes.
Leave the remaining 20mL culture in the flask. To that, add 80mL of micronutrient enriched BG-11 media.
Take the two falcon tubes and centrifuge them at 5000rpm,25°C .
Discard the supernatants of the centrifuged pellets.
Add 2mL of BG-11 into each of the emptied falcon tubes. Use a 1mL micropipette to mix such that the pellet is resuspended into a concentrated culture.
Add 140µL of 7% DMSO.
Take 4 cryovials. Add 1mL of the liquid in each cryovial.
Flash-freeze the vials using liquid nitrogen.
Store the stocks at -80°C
