Surface Sterilization of Herbarium Specimens for DNA extraction

Our lab is fascinated by microbial communities, particularly fungal endophytes residing within the intercellular spaces of plant cells. These fungal endophytes form essential associations with plants, contributing significantly to their healthy growth and protection from herbivory. To isolate these endophytes accurately, we use a surface sterilization technique to eliminate any extraneous microbes present on the plant surface as follows:

  1. Cut plant material into segments of 2 mm2 each.
  2. Place segments into a 100mm x 15mm Petri dish with a lid.
  3. Wash the segments in 95% EtOH (Ethanol) for 30 seconds.
  4. Wash the segments in 0.5% NaOCl (Bleach) for 120 seconds.
  5. Wash the segments in 70% EtOH (Ethanol) for 120 seconds.
  6. Transfer the container to a biosafety cabinet or laminar flow hood without removing the lid.
  7. In the biosafety cabinet or laminar flow hood, remove the lid and let the remaining ethanol evaporate.

Adapted from: Daru, B.H., Bowman, E.A., Pfister, D.H. & Arnold, A.E. (2018) A novel proof-of-concept for capturing the diversity of endophytic fungi preserved in herbarium specimens. Philosophical Transactions of the Royal Society B 374: 20170395 doi: 10.1098/rstb.2017.0395.