Leaf Surface Sterilization Protocol
Author
May Lab
Associated project
2017 QUMAFE pilot project
Updated
2017
Goal
This protocol documents surface sterilization of leaves for fungal endophytes assay using ethanol and bleach.
Materials
- Sterile Water.
- 70% Ethanol.
- 70% Bleach.
- 5 beakers (1 for each of the 5 treatments).
- Forceps.
- This should be performed in a BSL1 or BSL2/laminar flow hood to keep the leaves in a sterile environment.
Steps
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First wash: Rinse with sterile water to remove dusts.
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Second wash: Place the plant sample in 70% ethanol for 1 minute.
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Third wash: Place the plant sample in Bleach (50% or higher) for 2 minutes.
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Fourth wash: Place the plant sample in 70% ethanol for 1 minute.
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Fifth wash: Rinse the plant sample off in sterile water.
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Leaves are now surface sterilized and can be placed into sterile 50mL tubes,then into -80 storage.
Hazardous Waste Handling
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Ethanol and bleach wastes can be poured down the sink with large amounts of water.
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Any plant samples that are not used should be bagged and stored in freezer for further use or autoclaved and disposed of in the garbage.