Extraction of Apoplastic Bacteria Using a Syringe-Based Pressure Method

Take an Arabidopsis plant with leaves pre-inoculated with bacteria to study plant-microbe interactions.

These bacteria proliferate in the apoplast, the intercellular space between plant cells, forming microcolonies.

Once apoplastic colonization is established, excise the leaves and place them into a needleless syringe.

Add distilled water to cover the tissue.

Insert the plunger, then hold the syringe upright, tap it, and adjust the plunger to release any trapped air.

Seal the tip with parafilm to create a closed chamber for apoplast extraction.

Apply positive pressure by pushing the plunger until the tissue turns dark, indicating compression and water entry into the intercellular spaces to dislodge bacteria.

Next, apply negative pressure by pulling the plunger to expand the tissue matrix and draw out fluid and bacteria.

Repeat the pressure cycles to generate shear forces that disrupt microcolonies, releasing bacteria without damaging the plant tissue.

Unseal the syringe and collect the apoplastic fluid containing dispersed bacteria for further analysis.

Four days after inoculation, cut either the aerial part of the Arabidopsis plant or the inoculated leaf from the bean plant and place it into a 20-milliliter syringe without a needle.

For bean leaves, roll the leaf on itself, leaving the abaxial face outward. Add enough volume of distilled water to cover the tissue. Then, insert the plunger holding the syringe in an upright position and remove the excess air and air bubbles inside the syringe by gently tapping the barrel until all the air is located near the tip, and, after removing the air, cover the tip of the syringe barrel with paraffin film.

Now carefully press the plunger to generate positive pressure until the tissue turns darker. Then, pull the plunger to generate negative pressure. Remove the paraffin film and the plunger and collect the fluid containing the apoplast-extracted bacteria.