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 72h 0m 0s pick one colony from the plate and inoculate 80 ml fresh ^GYCC medium with appropriate antibiotic (if necessary) in 125mL baffled side arm flask.

Incubate at 33°C shaking at 125rpm 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.

Inoculate ^GYCC or MR26 agar plates from a glycerol stock containing appropriate antibiotic if necessary and incubate at 33°C .

After 72h 0m 0ss pick one colony from the plate and inoculate 25 ml fresh medium of ^GYCC or MR26 containing appropriate antibiotic (if necessary) in 50mL flask

Incubate at 33°C with shaking at 125rpm 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

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 . Analyze and visualize the data by any software.