95% ethanol
0.5% NaOCl (bleach)
70% ethanol
Sterile DI water
Clean paper towels
Sterilized plastic containers to hold each solution
Vials
Collect roots in field, keep cold until ready to sterilize.
(1) Wash roots in tap water until soil is removed and roots are clean, pat dry on clean paper towels
(2) 5 sec in 95% ethanol -- acts as a wetting agent to make the bleach more effective
(3) 2 min in 0.5% NaOCl -- sterilizes the tissue
(4) 2 min in 70% ethanol -- removes the bleach
(5) 1 min in sterile DI water -- removes the ethanol
(6) Pat dry on clean paper towels
(7) Divide roots by cutting on sterilized surface
(8) To store roots for later root staining, add a portion of the roots to 15-20ml vials filled with 80% ethanol or isopropyl alcohol. For transport, vials should be sealed then wrapped with parafilm with both the vial and the cap labeled.
(9) To store roots for later DNA extraction, add a portion of roots to eppendorm tubes and freeze at -80C.
(10) For endophyte culturing, add root pieces to pre-made culture plates. To check that your surface sterilization procedure worked, always include some plates where you press the processed roots onto the agarose and remove them.
NOTE: The sterilization protocol is largely based on Betsy Arnold's papers and the literature on root sterilization for mycorrhizal characterization. Note that when you buy the bleach you need to make sure it actually tells you the bleach concentration - some brands do and some don't – and make sure it doesn’t have other additives.