mrichter0 · June 28, 2025 12:57
diff --git a/0grant.txt b/0grant.txt
 Methods
 Bacterial Strains and Growth Conditions
 All experiments will be performed with Bacillus anthracis Sterne (34F2) and Bacillus thuringiensis HD1 as representative spore-forming bacilli. Cultures will be grown in Brain Heart Infusion (BHI) or Tryptic Soy Broth (TSB) at 37°C with shaking at 200 rpm until mid-log phase (OD600 ~1.0) is reached. For sporulation, cells will be transferred to Schaeffer’s sporulation medium and incubated at 37°C for up to 72 hours with agitationprotocols.

 Spore Production and Harvest
 Sporulating cultures will be monitored by phase-contrast microscopy for the appearance of refractile spores. After 48–72 hours, cultures will be harvested by centrifugation at 4,000 × g for 10 minutes at 4°C. Pellets will be washed three times with ice-cold sterile distilled water to remove residual media. Spore preparations will be incubated at 4°C overnight to facilitate separation of spores from vegetative cells and debrisprotocols.

 Spore Purification
 Spores will be further purified by enzymatic digestion and density gradient centrifugation. Briefly, pellets will be resuspended in 1 mg/mL lysozyme solution and incubated at 37°C for 30 minutes to lyse remaining vegetative cells. The suspension will then be treated with 0.1 mg/mL proteinase K at 55°C for 1 hour. Following enzyme digestion, spores will be washed in cold sterile water and purified by centrifugation through a 50% Histodenz gradient at 10,000 × g for 20 minutes. The purified spore pellet will be washed three times with sterile water and stored at 4°C until use. Spore purity will be assessed microscopically and by plating on nutrient agar to confirm the absence of vegetative cellsprotocols.

 Quantification and Characterization
 Purified spores will be enumerated using a hemocytometer and serial dilution plating on nutrient agar. Spore preparations will be characterized by phase-contrast and electron microscopy to verify morphology and purity. Particle size distribution will be measured by dynamic light scattering (DLS), and hydrodynamic diameter as well as polydispersity index (PDI) will be reportedprotocols.

 Aerosolization Procedures
 Spore suspensions will be prepared at a concentration of 10⁸ CFU/mL in sterile phosphate-buffered saline (PBS). Aerosols will be generated using a six-jet Collison nebulizer connected to an Aerodynamic Particle Sizer (APS) for real-time monitoring and calibration of particle size distribution. The aerosol stream will be delivered to a Class III biosafety cabinet or custom-built exposure chamber. Aerosolization efficiency will be validated by sampling air with impingers or filter cassettes placed at various locations within the chamber. Air samples will be plated on nutrient agar and incubated at 37°C for 24–48 hours to quantify viable spore countsprotocols.

 Environmental Stability and Surface Persistence
 To assess environmental persistence, defined quantities of aerosolized spores will be deposited onto stainless steel, plastic, and glass surfaces. Surfaces will be placed in controlled environmental chambers set to 22°C and 40% relative humidity. At defined intervals (0, 1, 7, 30 days), spores will be recovered by swabbing and elution into PBS. The number of recoverable spores will be determined by serial dilution plating. Spore survival kinetics will be modeled to determine decay rates under each environmental conditionprotocols.

 Animal Inhalation Exposure Model (If applicable)
 Animal exposure studies will utilize nose-only inhalation chambers. Mice (C57BL/6, 8–12 weeks) will be anesthetized and exposed to aerosolized Bacillus spores for 15 minutes. The actual inhaled dose will be calculated using real-time aerosol particle counts and total exposure time. Post-exposure, animals will be monitored for clinical signs, and bronchoalveolar lavage fluid (BALF) and lung tissue will be collected for spore enumeration and downstream analysesprotocols.

 Data Analysis
 Spore counts from environmental and biological samples will be log-transformed and analyzed by one-way or two-way ANOVA as appropriate, with Bonferroni post hoc correction for multiple comparisons. Decay curves will be fit using nonlinear regression, and statistical significance will be set at p < 0.05.
	Methods
	Bacterial Strains and Growth Conditions
	All experiments will be performed with Bacillus anthracis Sterne (34F2) and Bacillus thuringiensis HD1 as representative spore-forming bacilli. Cultures will be grown in Brain Heart Infusion (BHI) or Tryptic Soy Broth (TSB) at 37°C with shaking at 200 rpm until mid-log phase (OD600 ~1.0) is reached. For sporulation, cells will be transferred to Schaeffer’s sporulation medium and incubated at 37°C for up to 72 hours with agitationprotocols.

	Spore Production and Harvest
	Sporulating cultures will be monitored by phase-contrast microscopy for the appearance of refractile spores. After 48–72 hours, cultures will be harvested by centrifugation at 4,000 × g for 10 minutes at 4°C. Pellets will be washed three times with ice-cold sterile distilled water to remove residual media. Spore preparations will be incubated at 4°C overnight to facilitate separation of spores from vegetative cells and debrisprotocols.

	Spore Purification
	Spores will be further purified by enzymatic digestion and density gradient centrifugation. Briefly, pellets will be resuspended in 1 mg/mL lysozyme solution and incubated at 37°C for 30 minutes to lyse remaining vegetative cells. The suspension will then be treated with 0.1 mg/mL proteinase K at 55°C for 1 hour. Following enzyme digestion, spores will be washed in cold sterile water and purified by centrifugation through a 50% Histodenz gradient at 10,000 × g for 20 minutes. The purified spore pellet will be washed three times with sterile water and stored at 4°C until use. Spore purity will be assessed microscopically and by plating on nutrient agar to confirm the absence of vegetative cellsprotocols.

	Quantification and Characterization
	Purified spores will be enumerated using a hemocytometer and serial dilution plating on nutrient agar. Spore preparations will be characterized by phase-contrast and electron microscopy to verify morphology and purity. Particle size distribution will be measured by dynamic light scattering (DLS), and hydrodynamic diameter as well as polydispersity index (PDI) will be reportedprotocols.

	Aerosolization Procedures
	Spore suspensions will be prepared at a concentration of 10⁸ CFU/mL in sterile phosphate-buffered saline (PBS). Aerosols will be generated using a six-jet Collison nebulizer connected to an Aerodynamic Particle Sizer (APS) for real-time monitoring and calibration of particle size distribution. The aerosol stream will be delivered to a Class III biosafety cabinet or custom-built exposure chamber. Aerosolization efficiency will be validated by sampling air with impingers or filter cassettes placed at various locations within the chamber. Air samples will be plated on nutrient agar and incubated at 37°C for 24–48 hours to quantify viable spore countsprotocols.

	Environmental Stability and Surface Persistence
	To assess environmental persistence, defined quantities of aerosolized spores will be deposited onto stainless steel, plastic, and glass surfaces. Surfaces will be placed in controlled environmental chambers set to 22°C and 40% relative humidity. At defined intervals (0, 1, 7, 30 days), spores will be recovered by swabbing and elution into PBS. The number of recoverable spores will be determined by serial dilution plating. Spore survival kinetics will be modeled to determine decay rates under each environmental conditionprotocols.

	Animal Inhalation Exposure Model (If applicable)
	Animal exposure studies will utilize nose-only inhalation chambers. Mice (C57BL/6, 8–12 weeks) will be anesthetized and exposed to aerosolized Bacillus spores for 15 minutes. The actual inhaled dose will be calculated using real-time aerosol particle counts and total exposure time. Post-exposure, animals will be monitored for clinical signs, and bronchoalveolar lavage fluid (BALF) and lung tissue will be collected for spore enumeration and downstream analysesprotocols.

	Data Analysis
	Spore counts from environmental and biological samples will be log-transformed and analyzed by one-way or two-way ANOVA as appropriate, with Bonferroni post hoc correction for multiple comparisons. Decay curves will be fit using nonlinear regression, and statistical significance will be set at p < 0.05.