Production and Aerosol-Based Deposition of Bacillus subtilis Spores

Abstract

Source: Fuchs, F. M., et al. Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy. J. Vis. Exp. (2017)

This video demonstrates the production, purification, and aerosol-based deposition of Bacillus subtilis spores to create a uniform monolayer, enabling downstream applications such as microscopy-based analysis of spore germination, viability, or stress response.

Protocol

1. Bacillus subtilis Spore Production and Purification

1. For spore production, transfer a 5 mL overnight culture of the respective B. subtilis strain, supplemented with appropriate antibiotics, to 200 mL double-strength liquid Schaeffer sporulation medium (per liter 16 g nutrient broth, KCl (potassium chloride) 2 g, 0.5 g MgSO₄*7 H₂O (magnesium sulfate heptahydrate), 2 mL 1 M Ca(NO₃)₂ (calcium nitrate), 2 mL 0.1 M MnCl₂ *• 4 H₂O (manganese chloride tetrahydrate), 2 mL 1 mM FeSO₄ (ferrous sulfate), 2 mL 50% (w/v) glucose18) and cultivate it with vigorous aeration at 37 °C for 72 h or until > 95% of the culture has sporulated. The spores of the following strains are used: B. subtilis PY79 (wild type) B. subtilis PY79ΔrecA::neo (deficient of DNA repair protein RecA) B. subtilis PY79 recA-yfp::cat (RecA fused with yellow fluorescent protein [YFP]).
2. Harvest spores by centrifugation for 15 min at 3,000 x g in 50 mL tubes and purify the samples by repeated washing steps (up to 15 times) using sterile distilled H₂O and check for purity and germination status by phase-contrast microscopy. Ensure that spore suspensions consist of phase-bright spores (> 99%) and are free of vegetative cells (rods), germinated spores (black/ grey appearance), and cell debris; otherwise, further microscopy experiments can be disturbed. Wash the sample until the desired purity is reached.
3. Determinate the spore titer by plating out 50 µL of 10-fold serial dilutions on LB (luria-bertani)-agar (i.e.: Use 30 µL sample + 270 µL sterile water for a 1:10 dilution. Take 30 µL from the particular dilution to 270 µL H₂O for a 1:100 dilution and so forth) to calculate the CFU (colony forming units) and incubate the plates at 37 °C overnight. After CFU determination, adjust the sample to 10⁹ spores per mL by concentrating or diluting with sterile water.

2. Sample Preparation of Aerosol-Deposited Bacillus subtilis Spores

NOTE: Accumulation and overlapping of spores might lead to shadowing effects during the treatment, ultimately resulting in falsified inactivation kinetics. To minimize this problem, prepare spore samples by an aerosol-deposition technique. Briefly, control the high-precision two-substance nozzle with an electric timer that regulates the liquid throughput in concert with the flow of pressurized carrier gas (here N₂). Disperse the injected liquid sample through the nozzle outlet using the nitrogen gas flow.

1. Place a sample carrier in the form of sterilized microscopic slides (for survival kinetics) or round 25 mm coverslips (for fluorescent tracking of DNA repair processes/cLSM; confocal laser scanning microscopy) inside the electrically operated aerosol spraying unit in alignment with the nozzle. The used spore concentration needs to correspond to a hundredfold of the desired final concentration.
2. Transfer 1 mL of the spore culture to the nozzle fluid inlet and initiate the spraying process of 0.1 s at a pressure of 1.3 bar. The sprayed spore suspension (1 x 10⁷) 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Two substance nozzle (model 970-8)	Schlick	14,404	230 V, 50 Hz, D 4.484/8, 0.8 mm bore diameter
Luria Bertani Medium	Sigma Aldrich	70122-100G
Tube connectors	Festo	n/a	G 1/8
Glass slides	VWR	48300-026
Attofluor cell chamber	Menzel, Fisher Ref.	3406816	d=25 mm, round
Magnesium sulfate heptahydrate	Sigma Aldrich	13152
Calcium nitrate	Sigma Aldrich	202967
Manganese chloride tetrahydrate	Sigma Aldrich	244589
Ferrous sulfate	AppliChem	13446-34-9
Glucose	Merck	215422
Potassium chloride	Sigma Aldrich	P9541-500G
Nutrient Broth (NB)	Merck	105443
Luria-Bertani (LB)	Merck	110283
96-wellplate	ThermoFisher	243656