Dilution plating to measure colony forming units (cfus)

General Notes:

• Determine how you will normalize the raw colony counts, and record relevant units.
  • CFUs/g plant tissue –> make sure to weigh plant tissue pre-homogenization. See Colonization of tomato stems protocol
• Estimate your cell density if possible. If you know the range of CFUs that you expect, you can save yourself a little effort & some plates. Plate 1 or 2 dilutions higher/lower than the dilution you expect to have countable colonies.
  • If dilution plating plant samples, plate the 10^0 to 10^-7 dilutions. Wilt-symptomatic plants have >5x10^8 cfu / g stem,
• When in doubt, plate a large range of dilutions. Make sure you catch the range of cfus.

Prepare your workspace

1. Check consumable levels in advance:
   • 200 ul tips (1 box per 12 samples) - autoclaved
   • 96-well round-bottom microplate (1 per 12 samples) - sterilized in 10% bleach 30 min, 4x thorough rinses with diH2O (ensure bleach is removed from plate), dried in 50C oven… or new microplate.
   • Agar plates (e.g CPG + TZC). Plates should be fairly dry (but not cracked & agar should not be completely dried to agar plate surface) or else the droplets will merge on the plate. If plates are poured same-day, let them dry in the laminar flow hood with lids ajar for … 1 hr?
2. Load microplate with dilution liquid
   • Pour diH2O (Ralstonia) or buffer (any bacteria) into a sterile reservoir (autoclaved glass petri dish or plastic petri dish).
   • Using multichannel P200, add 180 ul diH2O or buffer

Dilute samples:

• If laminar flow hoods are occupied, dilution and plating can usually be done on the open bench without significant contamination. Contaminating fungi usually originate from (plant) sample
  1. Add 20 ul of sample (10⁰ dilution) to 180 di H2O in row A (10^-1 dilution).
  • If sample is homogenized tissue, make sure 20 ul is transfered–it’s possible for plant debris to clog tip. Gently pipette to relieve clog. An accurate 20 ul will come to the 20 ul line without bubbles
    1. With multichannel P20, THOROUGHLY pipette to mix row A (~10 pipette pulses), and transfer 20 ul to row B. Mix THOROUGHLY again and discard tips.
    2. Continue down plate, discarding tips after mixing each new row.
  • Tip: If you lose your place, look at the microplate from the side for the row with 200 ul, which will be noticeably higher than the 180 ul rows.
  • Tip: If you are unsure your pipette-to-mix skill is adequate, practice with a colored solution like 0.1% crystal violet, DNA loading dye, or even chlorophyll-pigmented plant homogenate.
    1. Plate dilutions:
  • Decide how many samples you can fit onto an agar plate. You will be plating a rectangular array of dots onto a circle. Arrays that work well:
    • 3 x ??
    • 6 x 6
  • Orient agar plates in a consistent manner (helpful to diagnose technical errors with dilution plating).
    1. Moving from most dilute (e.g. row H) to least dilute (row A, then sample, if 10^0 sample should be plated), use the P20 multichannel to plate 8 ul.
    • Avoid letting droplets merge. If they do merge, plates are too wet. Discard the plate & try again with a drier plate.
    • If minor errors are made (innacurate volume pipetted or droplets merge), gouge the agar with the your tip at those spots & use agar area around that. 1. Allow drops to absorb into agar.
      1. Incubate 28 C for ~2 days (40 h is best) or 3 days on the bench before counting colonies.
  • The mucoid colony morphology can cause the colonies to merge; count colonies before that happens. A sweet spot of 1 day at 28 C & 1 day at room temp can be helpful