How to grow Pseudomonas syringae for dip inoculations

Growth of P. syringae strains for Silwet L-77 Dip Inoculation, Kunkel Lab, Washington University

For inoculating A. thaliana you will need approximately 1 L of cells per flat of 3″ pots.

  1. The afternoon prior to inoculating your plants streak out the strains that you will be using in the following manner:
    • With a sterile inoculating stick or loop scoop up a large clump of P. syringae cells (approximately the size of a grain of rice) off of a relatively fresh KB or NYGA plate.
    • Streak out the bacteria evenly over the entire surface of the plate (either KB or NYGA is fine).
    • You will need 5 to 6 plates full of bacteria to make up 1 L of cells at the appropriate cell density (2 – 4 x 108 cells/ml, or an OD600 of ~ 0.2 -0.4)
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  2. Incubate the freshly patched out cells overnight at 28-30oC.
  3. When you are ready to start your inoculations, flood each plate with ~10 ml 10mM MgCl2.
  4. Using a sterile spreading stick or glass pipette, scrape up the cells, so that they begin to resuspend in the MgCl2.
  5. Remove the cells from the plates by aspirating the cells into a sterile pipette.
  6. Add the cells from all 5-6 plates to 1 L of 10 mM MgCl2, and mix well.
  7. Check the cell density with a spectrophotemeter, and adjust the suspension if needed by diluting with 10mM MgCl2, so that you end up with an OD600 of ~0.5.