Preparing_Duckweed-Bacteria_Cultures
Kenneth Acosta
Abstract
Protocol to generate duckweed-bacteria cultures to study duckweed-bacteria interactions.
Steps
Day 1
Inoculate 5 mL liquid cultures from bacterial glycerol stocks.
Grow 5 mL liquid bacterial cultures overnight at 28C at 250 rpm.
Day 2
Use 500 uL of liquid bacterial culture from Day 1 to inoculate 50 mL liquid cultures.
Grow 50 mL liquid bacterial cultures overnight at 28C at 250 rpm.
Day 3 (t = 0 days, start of experiment)
Transfer 25 mL of bacterial cultures to sterile 30 mL centrifuge tubes.
Spin at 8000 rpm for 5 min at 4C.
Decant SN.
Add 25 mL of sterile H2O to pellets. Resuspend pellets.
Spin at 8000 rpm for 5 min at 4C.
Decant SN.
Add 25 mL of sterile H2O to pellets. Resuspend pellets.
Spin at 8000 rpm for 5 min at 4C.
Decant SN.
Suspend in 25 mL of media.
Read OD600. In a volume of 50 mL dilute to OD600 = 0.2.
Transfer 50 mL cultures to baby jars.
Inoculate cultures with duckweed to cover the surface of the culture.
