Sterile Protocol for Seed Sterilization

Introduction

• This protocol outlines the steps for sterilizing seeds in a biohood.
• Focus on proper technique to avoid contamination.

Materials Needed

• 1000 microliter pipette
• 100 microliter pipette
• Sterilization solution
• 100% Ethanol
• Liquid waste container
• Labeled rack for drying seeds

Steps

1. Initial Setup

   • Ensure that seeds are sitting at the bottom of the sterilization solution.
   • Grab the 1000 microliter pipette.

2. Drawing Up Sterilization Solution

   • Draw up and expel a small amount of the sterilization solution.
   • This action will force the seeds up into the solution without grabbing them.
   • Tip: Remember to remove the cap of the liquid waste container before starting to avoid spills.
   • Eject sterilization solution into the liquid waste container.

3. Switch Tips

   • Use a new pipette tip for cleanliness.

4. Washing with Ethanol

   • Draw up 1 milliliter of 100% ethanol and add it to the tube.
   • Place the ethanol back in the holder after use.
   • Repeat the following steps to wash seeds:
     • Draw up a small amount of ethanol and expel it back into the tube, forcing seeds up.
     • Tilt the tube to a 45-degree angle so seeds rest against the side.
     • Draw up ethanol while leaving seeds in the tube.
   • Repeat this process two more times (total of 3 washes).

5. Final Wash

   • Switch to a 100 microliter pipette to draw up the final bits of ethanol.
   • Place the pipette tip to the bottom of the tube; move it around slightly to ensure all ethanol is drawn up.
   • Discard the tip used for this last step in tip waste.

6. Allow Seeds to Dry

   • Leave the tube open in the biohood.
   • Place the tube in the labeled rack and allow seeds to dry for at least 3 hours.

Important Reminders

• Always switch pipette tips between different solutions to avoid cross-contamination.
• Handle all materials carefully and maintain a sterile environment throughout the procedure.

Conclusion

• Proper adherence to these steps is crucial for successful seed sterilization.