Production and Aerosol-Based Deposition of Bacillus subtilis Spores

Begin with a culture of Bacillus subtilis, a highly resistant spore-forming bacterium.

Inoculate the culture into a nutrient-limited medium and incubate with aeration to induce sporulation.

Sporulating cells produce dormant spores with protective layers.

Transfer the culture into a tube and centrifuge to pellet the spores. Remove the supernatant containing the residual cells and debris.

Wash the pellet with sterile distilled water, repeat centrifugation, and resuspend the spores in sterile water.

Using a phase-contrast microscope, confirm spore purity by identifying bright spores and verifying the absence of contaminants.

Perform serial dilutions of the spores, plate on nutrient agar, and incubate to allow germination of viable spores into colonies. Count the colonies to determine spore concentration.

Based on this concentration, adjust the spore amount and load the diluted sample into an aerosol nozzle. Spray it onto a sterile glass surface where the suspension dries rapidly to form a thin, uniform monolayer of spores.

To carry out spore production, transfer a 5 milliliter overnight culture of the respective B.subtilis strain supplemented with appropriate antibiotics to 200 milliliters of