Preparation of a Single-Cell Bacterial Inoculum

Take a metabolically active culture of Mycobacterium marinum. This ensures high bacterial viability and optimal physiological state.

Centrifuge the culture to pellet the bacteria.

Discard the excess supernatant and resuspend the pellet in fresh media containing a dispersing agent.

Transfer the suspension to microcentrifuge tubes and sonicate under controlled conditions.

Sonication disrupts bacterial aggregates and disperses the cells, resulting in a viable single-cell suspension.

Collect the sonicated suspension in a syringe and pass it through a membrane filter to remove any remaining clumps, ensuring a uniform single-cell population.

Using a spectrophotometer, measure the optical density of the bacterial suspension.

Dilute the suspension with fresh cryoprotectant-containing media to reach the desired bacterial concentration while preserving cell viability.

Aliquot the final single-cell suspension and store it at ultra-low temperatures. The cryoprotectant helps maintain bacterial viability during long-term storage.

After inoculation according to the manuscript, centrifuge the culture at 3000 times G for 10 minutes to collect the Mycobacterium marinum as a pellet. Discard all but 300 microliters of the supernatant, and re-suspend the pellet.

Add three milliliters of 7H9 medium with 10% glycerol to further re-suspend the pellet, and then sonicate the suspension in a water bath at 100 watts, with 15 seconds on and 15 seconds off, for a total of two minutes to achieve a single cell homogenate. Transfer the bacterial suspension to a 10 milliliter syringe, and pass through a five micron filter to remove any bacterial clumps. Using a spectrophotometer, measure the optical density of the suspension, and dilute it with 7H9 media containing 10% glycerol to OD 600 at one.

Divide the suspension into 10 microliter aliquots, and store at negative 80 degrees Celsius freezer for further use.