Begin with Arabidopsis thaliana seedlings grown on low-density mesh supports that float at the surface of a liquid growth medium in a soilless system.
These seedlings have been pre-inoculated with bacteria that colonize the root and form a biofilm.
Hold the seedling, and gently pinch the stem below the leaves with forceps.
Then, lift the seedling from the mesh without damaging the root, preserving the colonized root structure.
Transfer the seedling to a multi-well plate containing double-distilled water.
Cover the plate with foil and sonicate the samples using a multipronged sonicator to disrupt the biofilm and dislodge bacteria from the root surface.
After sonication, use the sonicated bacterial suspension to prepare serial dilutions.
Plate the diluted samples onto agar plates and add glass beads to evenly spread the bacteria.
Remove the beads and incubate the plates to allow viable bacteria to form colonies.
Colony formation confirms successful root colonization by bacteria in the soilless system.
After the rinsing, remove the seedlings from the mesh by gently placing flame-sterilized forceps below the leaves on the leaf side of the mesh. Lightly pinch the stem, and wiggle the seedlings up and away from the mesh to dislodge the root without breaking it. To remove bacteria from plant roots, transfer seedlings from each mesh into individual wells of a 24-well sonication plate containing one milliliter of double-distilled water.
Sonicate all samples within the plate at once, using a multipronged sonicator as described in the manuscript. To quantify the bacteria, perform serial 10-fold dilutions of the sonicated samples in bacterial growth medium. Spread using sterile glass beads, and incubate at the optimal temperature for bacteria until individual colonies are countable.
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