To begin, with a serological pipette dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes. inside a laminar hood. Add 20 microliters of filter sterilized 5%Tween 80 to the test tubes with a micro pipette.
Next, add the cryostock of Mycobacterium smegmatis into a tube with an inoculation loop. Incubate the tubes for 48 hours in a shaker incubator at 300 rotations per minute and 37 degrees Celsius. To prepare the secondary cultures, dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes.
Then use a micro pipette to add 20 microliters of filter sterilized 5%Tween 80 to the test tubes. Next, with a micro pipette, transfer 20 microliters of the primary culture to one of the tubes. Incubate the cultures in a shaking incubator at 300 rotations per minute and 37 degrees Celsius until they reach an OD600 of 0.6 to 0.8.
