Propagate spores as described in Section 3.1 to achieve the highest sporulation efficiency possible. At the appropriate time point(s), verify spore formation via phase contrast microscopy as detailed above.
Remove plates from the chamber and resuspend cells from one-half of a plate in 1 ml sterile, ice cold dH2O. Repeat for the remaining half.
Centrifuge at 13000 rpm for 2 min at 4°C. Carefully remove supernatant and resuspend in 1 ml sterile, ice cold dH2O. Wash with sterile, ice cold dH2O two more times.
Incubate the spore preparation at −20°C for 48 h to facilitate the lysis of mother cells and subsequent release of mature endospores.
After incubation, centrifuge at 13000 rpm for 2 min at 4°C, decant the supernatant and resuspend the spore preparation with 1 ml sterile, ice cold dH2O. Repeat for a total of five washes in sterile, ice-cold dH2O as described in Step 3.
After the final wash, combine both cell pellets in 3 ml sterile, ice cold dH2O.
Apply the entire 3 ml spore preparation slowly to the top of a 10 ml 50% sucrose gradient in a 15 ml polypropylene conical tube.
Centrifuge in a swinging bucket rotor at 4000 × g for 20 min at 4°C. The vegetative cells and debris will collect at the interface and throughout the gradient, while the spores move through the gradient and form a pellet at the bottom of the centrifuge.
Remove the cell debris at the interface, then remove the rest of the solution, leaving the spore pellet.
Resuspend the spore pellet in 1 ml of sterile dH2O. Centrifuge at 13000 rpm for 2 min at room temperature, decant the supernatant and resuspend the spore preparation in 1 ml sterile dH2O.
Repeat step 10 for a total of five washes in sterile dH2O and three washes in 1X PBS + 1% BSA.
Resuspend the final spore preparation in 1 ml 1X PBS + 1% BSA and quantify as described in Section 3.4.
