Monitoring Bacterial Colonization of Arabidopsis thaliana Roots in a Soilless System

Abstract

Source: Harris, S. L., et al. Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System. J. Vis. Exp. (2019)

This video demonstrates a protocol for monitoring the colonization of root-associated bacteria on Arabidopsis thaliana seedlings grown in a soilless system. Following colonization, bacteria are dislodged by sonication, then diluted, plated, and incubated to form colonies that reflect bacterial attachment and viability.

Protocol

Collection of bacteria for viable cell counts

NOTE: The number of bacteria per seedling root can be determined at any incubation timepoint. Colonization can be monitored between 0 h and 18 h, while maintenance can be monitored from 18 h onwards.

1. Remove the seedlings from the mesh (Figure 1, step 5). Gently place flame-sterilized forceps below the leaves (but on the leaf side of the mesh), and lightly pinch the stem. Wiggle the seedlings up and away from the mesh to dislodge the root without breaking it. If the root breaks, gently scrape the mesh bottom to collect the full length.
2. Remove bacteria from plant roots. Transfer the bacteria to wells of a 24-well plate containing 1 mL of ddH₂0. Sonicate the samples.
3. Quantify the bacteria on roots.
   1. Perform serial 10-fold dilutions of the sonicated samples up to a 10^-6 dilution in bacterial growth medium. Add 50 µL of each dilution to individual agar plates and spread with sterile glass beads. Incubate plates at the optimal temperature for bacteria until individual colonies are countable.

Results

Figure 1: Assay for bacterial colonization and maintenance on A. thaliana roots.<...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	BD Falcon	1801343
Flame
Forceps
Incubator			At optimal temperature for growth of specified bacteria
Sterile water
Ultrasonicator
24 Prong Ultrasonicator attachment			For sonicating multiple samples at once. Can be done individually
Glass beads
Multipetter/Repetter
Sterile 96-well plates			For serial dilutions. Can be replaced by eppendorf tubes
Arabidopsis thaliana seeds			We recommend Arabidopsis Biological Resource Center for seed stocks
Pseudomonas simiae WCS417r