Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Our research focuses on the interaction of beneficial rhizomal bacteria with plant roots. We are looking for the key mechanisms to enhance the colonization of beneficial bacteria, thus improving the efficiency and the stability of microbial fertilizers in the field. The methods for bacterial colonization of plant roots have been extensively studied, but there are still problems.

For example, it's hard to measure the colonization on different sites distinctively. In the future, we think applying microscope and high throughput counting will help to solve the problems. Our protocol is highly applicable with good repeatability, and it is more suitable for accurate rhizosphere bacteria colonization determination.

To prepare the culture medium for Arabidopsis thaliana seedlings, mix half strength Murashige and Skoog or MS Medium with 2%sucrose and 0.9%agar. After sterilization, pour the medium into a square Petri dish, ensuring to avoid air drying to maintain humidity. Immerse Arabidopsis seeds in a two milliliter microcentrifuge tube containing one milliliter of sterile water at four degrees Celsius for over 12 hours.

Then sterilize the seeds with one milliliter of 2%sodium hypochlorite solution for two minutes. Thoroughly wash the seeds six times with sterile water to remove the sodium hypochlorite solution. Using a sterile one milliliter tip, sow the seeds on the MS agar dish.

Seal the Petri dish with breathable medical tape and place it vertically in a light incubator for one week at 28 degrees Celsius with humidity between 40 to 60%Make five millimeter holes on a 40 micron pore size cell strainer and sterilize it at 115 degrees Celsius for 30 minutes. After drawing, transplant three seven-day old A thaliana plants through the holes of a cell strainer and put them in a six well plate containing three milliliters of half strength MS medium with 0.5%sucrose. Inoculate Bacillus velezensis cells into sterile Luria Bertani medium, and incubate at 37 degrees Celsius with shaking at 170 RPM until an optical density at 600 nanometers reaches 0.8 21.2.

Centrifuge the cell suspension at 6, 000 G for two minutes. And resuspend the pellet in PBS buffer. After the last wash resuspend the pellet in a fresh half-strength MS medium to a final optical density of one at 600 Nanometers.

Dilute the bacterial culture 100 times until optical density reaches 0.01. Inoculate the diluted bacterial suspension to the six well plate containing Arabidopsis thaliana seedlings and cultivate for two days in the light incubator. To begin, weigh two milliliter tubes and record the initial weight as W0.Harvest the co-cultured Arabidopsis thaliana root tissues and wash them three times in sterile water.

Place the root on filter paper to remove the excess water. Transfer the roots into the pre-weighed microcentrifuge tube. Weigh the roots with the tube and record it as W1.Next, add one milliliter of PBS to the tube and Vortex for eight minutes at maximum speed.

Dilute the suspensions containing the root colonized bacterial cells from one by 10 to the negative first to one by 10 to the negative fifth. Spread the diluted bacterial suspensions on Luria Bertani agar plates, and incubate at 37 degrees Celsius for 10 hours. After incubation, count the bacterial colonies on Luria Bertani agar plates.

Calculate the colony forming units according to the corresponding dilution, and normalize the data with the root fresh weight to get a count of bacterial cells colonized per gram of root. At two days post inoculation, the colonization of Bacillus velezensis wild type sqr nine and a derived mutant, delta eight MCP, demonstrated significant root colonization reduction of delta eight MCP, suggesting the effectiveness of this condition and method in measuring bacteria colonization.