Oct 04, 2021

Public workspaceSterilizing the Surface of Seeds by Chlorine Gas V.2

This protocol is a draft, published without a DOI.
  • 1Realizing Increased Photosynthetic Efficiency (RIPE)
  • GEGC lab UIUC
  • UIUC Long Lab
Lynn Doran
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Protocol Citation: Lynn Doran 2021. Sterilizing the Surface of Seeds by Chlorine Gas. protocols.io https://protocols.io/view/sterilizing-the-surface-of-seeds-by-chlorine-gas-bynppvdnVersion created by Lynn Doran
Manuscript citation:
Lindsey III, B. E., Rivero, L., Calhoun, C. S., Grotewold, E., Brkljacic, J. Standardized Method for High-throughput Sterilization of Arabidopsis Seeds. J. Vis. Exp. (128), e56587, doi:10.3791/56587 (2017).
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 29, 2021
Last Modified: July 11, 2023
Protocol Integer ID: 53679
Abstract
This protocol sterilizes the surface of seeds for use in clean culture conditions without damaging the seeds germination or growth ability.
Materials
Safety warnings
This protocol uses both chemical fume hoods and biological safety. Understand the difference and how to safely and appropriately use both before performing the protocol.

This protocol intentionally produces chlorine gas which is toxic to human health. Please read all manufacturer safety data sheets before handling. UIUC personnel performing this protocol should be current on "Laboratory Safety", "Chemical Safety- An Introduction", and "Chemical Spills" Division of Research Safety training modules before performing this protocol.

Dispose of chemicals per your institutions hazard waste policy. For UIUC, solution can be neutralized to between pH 5 and 10 using 10M Sodium Hydroxide solution and discarded normally.
Before start
The volumes of reagents in this protocol were calculated for 6% chlorine gas in a 11,150 mL container (11" W X 13" H X 9" D).

If a different size container is used or a different percent gas is desired, adjust volumes of bleach and hydrochloric acid using the worksheet provided in Lindsey et al, 2017.

Download Worksheet for calculating chlorine gas concentration Lindsey et al. 2017.xlsxWorksheet for calculating chlorine gas concentration Lindsey et al. 2017.xlsx

Move an aliquot of seeds to be sterilized to a 2 mL microcentrifuge tube. Label clearly.

Note
If too many seeds are sterilized at once, seeds on the bottom layers may not be exposed to as much gas as top layers.

Document the seed vials identifications and order via photograph or notetaking.
Note
Exposure to chlorine gas can cause some types of ink, both marker and printed, to fade.

Place a desiccator in the chemical fume hood. Adjust the fume hood sash to a safe height.
Place the seed rack in a desiccator with no desiccant. Open the tube lids.
Fill a 250 mL beaker with about Amount160 mL of bleach, using graduations on the side of the beaker is sufficiently accurate for this purpose.


Place the beaker of bleach in the desiccator.


Quickly pipet Amount5 mL of hydrochloric acid into the beaker of bleach. Pipet all 5 mLs in one volume to avoid losing chlorine gas.

Note
The reaction of bleach and hydrochloric acid will form chlorine gas. Do not breath in vapors. Ensure fume hood is in good operation. Wear appropriate PPE when handling hydrochloric acid.

Toxic
Close the lid of the desiccator. If the desiccator contains any vacuum lines or vents, ensure that those valves are all in the closed position.
Allow the seeds to be exposed to the chlorine gas for a minimum of 3 hours.
Move the sealed desiccator to a biological safety cabinet that has been properly sterilized.

Safety information
Do not use a laminar flow hood. The biological safety cabinet will both keep the seeds sterile and protect you from any residual chlorine gas. A laminar flow hood will keep the seeds sterile but will blow residual chlorine gas back out at the operator.

Open the desiccator slowly.
Toxic
Close the seed vials. Seed vials can be stored outside the biological safety cabinet but should only be opened in properly sterilized biological safety cabinets or laminar flow hoods to ensure that they remain sterile.