Assessing Pseudomonas syringae Infection Load in Arabidopsis thaliana Leaves

Take leaves from an Arabidopsis thaliana plant infected with the pathogenic bacterium Pseudomonas syringae.

Cut small leaf discs from the collected leaves.

Place two leaf discs in a grinding tube containing a metal grinding ball and a salt solution.

Seal the tube and initiate homogenization.

During homogenization, the metal ball mechanically lyses the leaf tissue, releasing the cellular contents and the bacteria.

Serially dilute the homogenate in a multiwell plate.

Plate a droplet from each dilution on a nutrient-rich agar plate.

Allow the droplets to be absorbed, and then place a lid on the plate.

Invert and incubate under optimal conditions to promote bacterial colony formation.

Count the number of colonies from the lowest dilution to avoid overlapping colonies.

Determine the number of colony-forming units or CFU per leaf disc to evaluate the bacterial load of the infected leaf.

One day before quantifying pathogen growth. Pre-dry the 150 millimeter by 15 millimeter KB medium plates.

Dry the plates by keeping them at room temperature for about 24 hours. Process the infected tissue after the emergence of chlorosis indicated by the yellowing of the infected tissue in the susceptible genotypes,