Culturing the Rapidly Growing Bacterium Vibrio natriegens

Take a tube containing a culture of Vibrio natriegens, a fast-growing bacterium.

Centrifuge the culture to pellet the bacteria. Discard the supernatant containing residual media.

Resuspend the bacterial pellet in fresh media and transfer it to a conical flask containing the same media for bacterial culture preparation.

Incubate the culture with shaking under aerated conditions.

Due to a short generation time, Vibrio natriegens utilizes nutrients and rapidly divides to generate a concentrated bacterial population.

Using a spectrophotometer, regularly monitor the optical density (OD) to determine the bacterial concentration.

Grow the culture until the OD reaches approximately one, indicating an exponential growth phase with high metabolic activity.

Transfer the culture to centrifuge tubes.

Centrifuge at low temperature to separate the bacteria while preserving the cellular integrity.

Place the tube on ice and remove the supernatant containing cellular debris.

Store the bacterial pellet at ultralow temperature for downstream analysis.

To begin, wash one milliliter of the overnight Vibrio natriegens culture by centrifuging it in a benchtop centrifuge at 10,600 times g for one minute.

Aspirate the supernatant without disturbing the pellet, and resuspend in one milliliter of fresh LB-V2 media. Inoculate one milliliter of washed overnight culture into one litre of fresh LB-V2 growth media in an autoclaved 4 litre baffled Erlenmeyer flask with sterile cover to achieve one to one thousand dilution ratio. Grow culture at 30 degrees Celsius while shaking at 225 RPM.

Use a spectrophotometer to monitor the culture's OD 600, and when it reaches 1.0 plus or minus 0.2, harvest the culture by centrifuging it in two 500-milliliter tubes at 3,500 times g for 20 minutes at four degrees Celsius, and then place on ice. Aspirate the supernatant and process the pellet immediately.