This article details the process of producing and purifying spores from Bacillus subtilis, a resilient spore-forming bacterium. The methodology includes inoculation, sporulation, centrifugation, and confirmation of spore purity.
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 double strength liquid Schaeffer sporulation medium and cultivate it with vigorous aeration at 37 degrees Celsius for at least 72 hours or longer until greater than 95% of the culture has been sporulated. Harvest spores in 15-milliliter tubes by centrifugation at 3000 g for 15 minutes and purify the samples using sterile distilled H2O in repeated washing steps.
Check for purity and germination status by phase contrast microscopy and ensure that spore suspensions consist of greater than 99% of phase bright spores and are free of vegetative cells, germinated spores, and cell debris, otherwise further microscopy experiments can be disturbed. Determinate the spore titer by plating 50 microliters of ten-fold serial dilutions on LB agar to calculate the CFUs and incubate the plates at 37 degrees Celsius overnight. Following CFU determination, adjust the sample to 10 to the nine spores per milliliter by concentrating or using sterile water to dilute the samples.
Place a sample carrier in the form of sterilized microscopic slides or round 25-millimeter cover slips inside the electrically operated aerosol spraying unit in alignment with the nozzle. Transfer the spore culture to the nozzle fluid inlet and initiate the spraying at 0.15 seconds with a pressure of 1.3 bar. The sprayed spore suspension forms a thin film on the microscopic slide that dries rapidly within seconds to form a uniformly distributed spore monolayer. Store the treated sample carriers in a sterile container at room temperature.
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