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Production of Bacillus subtilis endospores by nutrient exhaustion

Sporulation is induced by limitation of carbon, nitrogen or occasionally phosphorus in the environment. Most methods induce sporulation by either nutrient exhaustion in modified growth media or by washing exponentially-growing cells and introducing them into media lacking one of the above substrates. The former is usually easier and generates larger amounts of spores, the latter results in more synchronised rates of sporulation. Additionally, sporulation on solid media results in higher densities of spores, but liquid medium is more easily scalable and is generally faster.

This protocol originates in Molecular Methods in Bacillus (Harwood and Cutting, 1990), however the method for spore washing is from Anne Moir's group at the University of Sheffield, UK.

1) Inoculate 10-50 ml of LB medium with cells from a fresh colony of B. subtilis.

2) Grow for 6-8 hours at 37°C with shaking at 200 rpm.

3) Dilute 1/200 into 50-10000 ml 2xSG medium in an appropriate vessel and grow at 37°C with shaking at 200 rpm.

4) Check samples for spores daily using light microscopy. Phase contrast microscopy is ideal but if the appropriate filter is not available a Gram stain will distinguish between vegetative (purple) cells and spores (transparent).

5) After 2-3 days >90% of the population should have sporulated.

6) Pellet cells at 9000 rpm for 20 minutes in a JLA 10.500 rotor if possible, keeping temperatures low, otherwise for small quantities a benchtop centrifuge will suffice.

7) Wash spores with ice-cold water 8-10 times to remove residual nutrients and lyse remaining vegetative cells.

8) Store spores at 4°C, with weekly changes of water, or at -20°C for long-term storage.

2xSG medium Difco nutrient broth, 16.0 g; KCl, 2.0 g; MgSO4.7H2O, 0.5 g.

Adjust the pH to 7.0, autoclave.

Add the following sterile component solutions to one litre of the cooled medium prior to use: l M Ca(NO3)2, 1.0 ml; 0.1 M MnCl2.4H2O, 1.0 ml; 1mM FeSO4, 1.0 ml; 50% (w/v) glucose, 2.0 ml.