2.2.5.1. Colouration test for spore viability

  1. Add 50 ml of spores in H2O at a concentration of 5 x 105 spores / ml to 1 mM of Sytox green (Molecular Probes, Inc.)
  2. Incubate for 20 minutes at room temperature
  3. Centrifuge for 3 minutes at 1600 x g and discard the supernatant
  4. Homogenize the pellet in H2O and centrifuge for 3 minutes at 1600 x g
  5. Discard the supernatant
  6. Add 100 ml 4’,6-diamidino-2-phenylindole (DAPI) at 2 mg/ml
  7. Incubate for 30 minutes in room temperature
  8. Centrifuge for 3 minutes at 1600 x g and discard the supernatant
  9. Homogenize the pellet in H2O and centrifuge for 3 minutes at 1600 x g and discard the supernatant
  10. Add 50 ml H2O and apply aliquots if 15 ml onto glass slides
  11. Allow to dry in room temperature and view under oil in a fluorescent microscope

Dead spores are identified as yellow-green ovals through the 470- to 490-nm excitation wavelength filter, and living spores are coloured with turquoise ovals through the 395- to 415-nm excitation wavelength filter. To differentiate extruded spores not visible by either Sytox green or DAPI staining, white-light microscopy, where extrudes polar filaments can be seen is employed during the viewing (Fenoy et al., 2009). The colouration test has not yet been evaluated with the ultimate test - in vivo tests in live bees.