Oct 07, 2022
Conditions for growth of Rhodobacter sphaeroides in different media
- Jaya Yakha1,
- Deborah K. Hanson1,
- Rosemarie Wilton1,
- Philip D. Laible1
- 1Argonne National Lab
Protocol Citation: Jaya Yakha, Deborah K. Hanson, Rosemarie Wilton, Philip D. Laible 2022. Conditions for growth of Rhodobacter sphaeroides in different media . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9emog8j/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: September 20, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 70308
Keywords: rhodobacter sphaeroides in different media, culturing rhodobacter sphaeroides wildtype, rhodobacter sphaeroide, rhodobacter sphaeroides wildtype, other flask, shake flask, baffled side arm flask
Abstract
This protocol provides a method for culturing Rhodobacter sphaeroides wildtype 2.4.1 and its KO strains in shake flask and BioTek Epoch 2, 48-well microplates. A baffled side arm flask is used but other flasks can be used with appropriate modifications.
Materials
- Incubator at 330C
- GYCC agar plate and liquid medium
- Centrifuge
- Sterile tooth pick
- Colorimeter/spectrophotometer
Troubleshooting
Conditions for growth of Rhodobacter sphaeroides in GYCC medium in shake flasks
Inoculate a GYCC agar plate from a glycerol stock containing appropriate antibiotic (if necessary) and incubate at 33 °C in an incubator in dark.
After 72:00:00 pick one colony from the plate and inoculate 80 ml fresh GYCC medium with appropriate antibiotic (if necessary) in 125 mL baffled side arm flask.
3d
Incubate at 33 °C shaking at 125 rpm for 72 -96 hours.
Monitor the growth of the culture with regular measurement of turbidity. Side-arm flasks are especially convenient for the determination of culture turbidity with a Klett-Summerson colorimeter. The equivalent OD600 may also be used if side arm flasks are not used.
Conditions for growth of R. sphaeroides in GYCC or MR26 medium in Microplate
Inoculate GYCC or MR26 agar plates from a glycerol stock containing appropriate antibiotic if necessary and incubate at 33 °C .
After 72:00:00 s pick one colony from the plate and inoculate 25 ml fresh medium of GYCC or MR26 containing appropriate antibiotic (if necessary) in 50 mL flask
3d
Incubate at 33 °C with shaking at 125 rpm for 72 -96 hours.
Monitor the growth of the culture with measurement of turbidity. Once the culture reaches to the log phase (OD600~0.7), the starter culture is ready for inoculation of the microplate.
For each well 600, µl of fresh GYCC or MR26 medium was added. It is recommended to use Microplate Greiner BioOne 48 well (Item no. 677180) for compatibility with a BioTek Epoch2 microplate reader
Procedure Details in microplate reader
Plate Type: Greiner BioOne 48-wellv2 (Use plate lid)
Eject plate on completion
Set Temperature: Setpoint 33°C, Gradient 2 °C
Start Kinetic: Runtime 96:00:00 (HH:MM:SS), Interval 0:15:00, 385 Reads
Shake Double Orbital: Continuous
Frequency: 548 cpm (2 mm)
Read:A600
Absorbance Endpoint
Full Plate
Wavelengths:600
Read Speed: Normal, Delay: 200 msec, Measurements/Data Point: 8
End Kinetic
Once the run is complete, export the data in a .txt or .xls file and save in an appropriate location. Analyze and visualize the data by any software.