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Direct plating of phages from environmental samples

Objective

To isolate phages from an environmental sample that has been filter sterilized

Rationale

This is the fastest method to find phages in environmental samples. The filter sterilized sample is directly mixed with host bacteria and grown on an agar plate using the plaque assay. Any phages that are in the sample will infect and kill the immobilized bacteria in the agar, resulting in a hole in the bacterial lawn: a “plaque”. This method gives you a quick snapshot of the phages present in your environmental sample that can infect your host bacteria, but any phages present in the sample at very low abundance may be missed using this approach.


Before you start:

  • You need a growing liquid culture of your bacterial host(s).
  • Retrieve your filtered sample(s) from the 4 ℃ fridge.
  • You need to have a tube of molten LB top agar in your bead bath.
  • Add salts to the top agar to facilitate phage adsorption. (10 mM CaCl2) *

Materials for direct plating

  • Processed environmental samples that have been filtered to remove all biomass.
  • LB agar plates (as many plates as samples to be plated + 1 for no-sample control)
  • Culture tubes (as many tubes as samples + 1 for no-sample control)
  • LB top agar (4mL per number of plates you are using)
  • Serological pipettes

Procedure

  1. 🖊️ For each sample you are plating, label LB agar plates with today’s date, your name, your host bacteria, and your sample ID. Label an additional LB agar plate as your no-sample control plate.
  2. 🧪 Set out as many culture tubes as samples you will be plating. Label one tube: no-sample control, and label the rest of the tubes with the corresponding sample ID.

    You should have one plate and one tube per filtered sample, plus one tube and one plate for your no-sample control.

  3. ➕ Add the appropriate bacterial host culture(s) to each of the sample tubes.
    • Team Coryne:
    • 250 µL of C. glu
    • Team plasmid-dependent:
    • 100 µL of E.coli - pRP4
    • 100 µL of P. putida - pRP4
  4. ➕ Add 250 µL of each filter-sterilized sample to the tubes.
  5. 🌪 Gently vortex the tube.
  6. ⏳ Let the tubes sit on the benchtop undisturbed for 10 minutes to allow phage adsorption.
  7. ➕ Using a sterile serological pipette, gently add 4 mL molten top agar supplemented with CaCl2 to a single tube, and immediately suck up the top agar/sample/bacteria mixture and transfer it to the appropriately labeled plates. Be sure to avoid air bubbles, as they can look like plaques on plates.
  8. ↔️↔️ Quickly tilt the plate in multiple directions until the top agar mixture evenly coats the agar plate. Once top agar has totally coated the surface, cover the plates with the lid and leave to set.
  9. Repeat this process for all of your samples, including the no-sample control.
  10. ⏳ Let the plates sit undisturbed for 20 minutes on the benchtop to allow the top agar to fully solidify.
  11. 🌡️ Incubate the plates overnight in the 30℃ incubator.

  12. ▶ Continue to Protocol 6: Phage purification