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, but before the development of necrotic lesions. For each genotype, prepare six grinding tubes.

Place one stainless steel grinding ball and add 500 microliters of sterile 10 millimolar magnesium chloride into each tube. Next, detach the infected leaf from the plant and punch a leaf disc with a one hole paper punch. Using forceps, randomly place two leaf discs from two different plants into each grinding tube. Seal the tubes. Homogenize the tissue with a high throughput homogenizer at maximum speed for 10 minutes.

Repeat this process if needed until the tissue is well homogenized and the solutions turn green due to chlorophyll release from the infected leaves. While waiting for the homogenization, fill each well in the first six rows of a 96 well culture plate with 180 microliters of 10 millimolar magnesium chloride. Transfer 20 microliters of the ground tissue suspension into each well of the first row of the 96 well plate and mix by repeatedly pipetting the liquid up and down.

If small fragments of tissue clogged the tip, clip it by two to three millimeters to help acquire the correct volume of the solution. To provide enough space for the droplet on the top of the plate, space the tissue from different genotypes in alternative rows. To prepare a tenfold serial dilution, transfer 20 microliters of tissue suspension into the second row and repeat this procedure until the sixth dilution. Using divided pipette tips, transfer 20 microliters of the solution from the 96 well plate onto the 150 millimeter by 15 millimeter KB plate.

Work from the most dilute suspension to the most concentrated so it is unnecessary to change pipette tips between the dilution. Dry the plate at room temperature with the lid cracked. Once no more liquid can be observed on the plate surface, close the lid, invert and incubate the plate at room temperature.

Incubate plates for 40 to 60 hours until the colonies become visible. Confirm that the bacteria on the plates reflects the predictable tenfold drop in colony forming units.

Count the bacteria before they overgrow and colonies fuse. Determine the number of bacteria in the lowest dilution that does not have overlapping colonies. Usually the preferred dilution to be counted will contain between 10 and 50 colonies, and it may vary among technical replicates.

To calculate the levels of bacterial proliferation, determine the number of colony forming unit per leaf disc using this formula, where R is the number of the row and T is the number of the bacteria within each technical replicate. The data is then used to produce a graph. Each data point is represented as the mean of six technical replicates on a logarithmic scale.

Error bars represent the 95% confidence interval of the mean.