Dec 12, 2022
Preparing_Duckweed-Bacteria_Cultures
- 1Rutgers University
Protocol Citation: Kenneth Acosta 2022. Preparing_Duckweed-Bacteria_Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjd7kvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 11, 2022
Last Modified: December 20, 2022
Protocol Integer ID: 73842
Abstract
Protocol to generate duckweed-bacteria cultures to study duckweed-bacteria interactions.
Guidelines
Some bacteria may grow slowly. In this case, more time is needed for bacterial cultures to grow to sufficient concentrations.
Some bacteria do not pellet easily. Longer centrifugation times and higher centrifugation speeds may be needed for these types of bacteria.
Materials
Note: All reagents/materials should be sterilized.
- H2O
- media
- 30 mL round-bottom centrifuge tubes
- 50 mL centrifuge tubes
- baby jars
Day 1
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
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)
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.