Checklist:

• Use the right Medium.
• Vortex cuvettes if you make a dilution
• Set Nanodrop to cuvettes

1. Overnight culture

• inoculate your culture in 5ml LB-Medium
• let them grow over night at 37°C, 200rpm

2. Exponential growth

• measure the OD600 of your overnight culture
• inoculate 2ml + Xml to measure OD (personal preference) LB-Medium to OD600=0,1 from overnight culture
• let the cells grow to an OD600=0,8 (37°C, 200rpm)

3. Sporulation

• centrifuge the 2 ml of cells at 13.000 rpm for 1 minute
• wash the pellet with PBS
• resuspend the pellet in 1 ml DSM (Difco Sporulation Medium)Culture medium
• let the cells grow (24h,37°C 200rpm)

4. Lysozym treatment

• dilute the cells 6:1 in lysozym solution (15mg/μl)
• incubate for 1 hour at room temperature
• wash 6 times with 1x PBS

5. Count the spores (Neubauer-counting chamber, usually dilute in BPS 1 to 100)

6. Make aliquots (around 100million spores per aliquot)

https://www.researchgate.net/post/How_to_induce_sporulation_of_Bacillus_spp_by_heating_or_any_other_simple_technique
https://www.researchgate.net/post/How_to_sporulate_the_bacteria_or_how_to_induce_the_sporulation_in_bacteria_especially_for_Bacillus_spp