Harvest of in vivo spores

  1. Mix black mummies with 5 ml of sterile distilled water
  2. Shake for 5 min
  3. Homogenize the spore solution with a glass tissue grinder for 5 min
  4. Centrifuge to spin-dry at 3500 rpm for 5 min
  5. Discard the supernatant
  6. Add iodine-povidone (50 % in distilled sterile water). Iodine-povidone is added to avoid bacterial contamination
  7. Shake for 10 min
  8. Centrifuge to spin-dry at 3500 rpm for 5 min
  9. Discard the supernatant
  10. Wash three times by adding 5 ml of sterile distilled water
  11. Shake for 5 min
  12. Spin-dry at 3500 rpm during 5 min
  13. Add 1 ml water
  14. Prepare a dilution series to 10-2 or 10-3 for counting and calculating the concentration of the undiluted suspension with a haemocytometer (refer to the BEEBOOK paper on miscellaneous methods (Human et al., 2013).

Spores from mummies can also be harvested using the in vitro harvest method described above. To get a better homogenization, scrape the spore cysts from the outside of the mummy before grinding. In vivo spores must be used the same day they are prepared, due to the presence of other microorganisms on the mummies even though the broad-spectrum bactericide iodine-povidone is used to avoid bacterial contamination in the spore solution preparation. Dry dark mummies with viable spores can be preserved for several years (at least 9 years) when stored dry in a closed bottle (Flores, pers. comm.)