Duckweed sterilization procedure - Cuddington-Lab/documents Wiki

Procedure to transfer field-collected duckweeds to lab conditions (sterilization)

  1. Wash duckweed fronds with deionized (DI) water

  2. Place in 0.5% v.v. sodium hypochlorite (bleach) solution for a period of time between 30 seconds and 5 minutes (OECD/OCDE guidelines; EPS Canada, 2007)

Household bleach = 5 to 6 % sodium hypochlorite (confirm the expired date of the product we used is within 6 months) (https://www.uwo.ca/animal-research/doc/bleach-sop.pdf)

0.5% v.v. sodium hypochlorite solution: Add 900 mL of water Add 100 mL of household bleach Ensure the contents are mixed thoroughly (https://www.northeastern.edu/ehs/wp-content/uploads/2014/12/Bleach-Fact-Sheet-Draft.ejc2_.pdf)

  1. Rinse again in DI water for several times: properly sterilized plants will have a small green area in the bud zone along the center of the frond; estimated rate of survival is 10% (EPS Canada, 2007)

  2. Aseptic transfer (creation of a new source culture): in the laminar flow room; autoclaved beakers (large and/or medium); 100mL of Hoagland solution; Place surviving fronds into beaker using our standard lab techniques; consider a number of fronds that does not result in crowing, ie., plants should not cover more than ⅔ of medium surface (EPS Canada, 2007)

  3. Before starting experiment: acclimation time options: 28 days (Armitage & Jones, 2019); 7 to 10 days (EPS Canada, 2007)

Alternative procedure to maintain field-collected non-axenic cultures of duckweeds

This procedure is to be applied to the remaining collected fronds that will not be sterilized. Maintain duckweeds in black containers to allow for light penetration only through the top (to avoid further algae proliferation). Change the pond water twice weekly (https://www.jstor.org/stable/43236327?seq=2#metadata_info_tab_contents). Keep duckweed containers under the light (growing cart).