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 (C. glu).
  • Your filtered sample(s) from the 4 ℃ fridge.
  • You need to have a tube of molten LB top agar in the bead bath with 10 mM CaCl2 and 0.4% Glucose added.

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 + glucose and CaCl2 (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 250µL of C. glu culture to each of the sample tubes.
  4. ➕ Add 250 µL of each filter-sterilized sample to their corresponding tubes.
  5. 🌪 Gently vortex the tube.
  6. ⏳ Let the tubes sit on the bench top undisturbed for 10 minutes to allow phage adsorption.
  7. ➕ Using a sterile serological pipette, gently add 4 mL molten top agar to a single tube.
  8. 🌪 Put the lid back on the tube and very gently vortex.
  9. 🧪 Immediately pour the contents of the tube on the corresponding plate. Be sure to avoid air bubbles, as they can look like plaques on plates.

    If you wait too long at this step, the agar may start to solidify in the tube. You don’t need to rush, but don’t let the tube sit for too long.

  10. ↔️↔️ 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.
  11. Repeat this process for all of your samples, including the no-sample control.
  12. ⏳ Let the plates sit undisturbed for 20 minutes on the bench top to allow the top agar to fully solidify.

  13. 🌡️ Incubate the plates overnight in the 30℃ incubator.

  14. ▶ Continue to Protocol 6: Phage purification