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Sporulation

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)

Induction of sporulation

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