Acquiring and processing environmental samples

Table of contents
  1. Sample collection
  2. Sample processing

Objective

To collect samples from the environment that may contain phages that infect your host bacteria

Rationale

By collecting diverse environmental samples of soil, water, compost etc. you will aim to capture new phages. The golden rule in phage hunting is “where you find your host is where you will find its phages”, so bear in mind that environments that your specific host bacteria thrive in are where you are most likely to find phages. Secondly, the more diverse samples you can collect, the higher your chances of finding something. Phages are so small that just a tablespoon of soil could contain thousands of different phages, so it’s less important to take large volumes of sample, and more important to get a diversity of different samples.

Sample collection


Sample collection form

Materials for sample collection

  • 50 mL falcon tubes
  • Wooden tongue depressors (for scooping)
  • Sharpie pen for sample tube labeling
  • Smart phone or tablet for recording sample metadata

Procedure for sample collection

  1. 👀 Identify your chosen sample location
  2. 🖊️ Label your tube with a sample ID (your initial and sample number e.g. TB01)
  3. 🥄 Collect your sample:

    • Solid samples: scoop sample into tube until approx. the 10 mL mark. Be careful not to overfill the tube!

    • Liquid samples: Collect up to 45 mL of liquid samples. Tightly seal the tube cap.

  5. 📲 Enter for your sample metadata into the sample collection form, making note of any interesting details of the sampling location
  6. 📸 Photograph the sample location if you can and upload to the form.
  7. Clean the tube exterior with ethanol wipes before collecting another sample.

Sample processing and screening takes much more time than collecting the sample, so try to limit yourself to only the best looking samples.

Sample processing

Materials for sample processing

  • Liquid or resuspended-solid samples
  • Water (non-sterile)
  • Large centrifuge for 50mL falcon tubes
  • Serological pipettes
  • 50 mL falcon tubes
  • 50mL Steriflip 0.22 μm membrane vacuum filter units

Procedure for sample processing

  1. Organize sample tubes on the lab bench.
  2. ➕ Add 20 mL of water to all solid samples using serological pipettes.
  3. 🌪 Vortex rehydrated sample for at least 1 minute.
  4. ⏳ Transfer all samples to 4℃ overnight to allow phage particles to fully resuspend.

    Overnight resuspension is not necessary if you only have liquid samples, but all samples should be stored at 4°C regardless of composition.

  5. 💫 Transfer resuspended samples to the centrifuge and spin at 4,000 x g for at least 30 minutes. You may need to spin dense samples for longer.
  6. Carefully remove tubes from the centrifuge and observe the change in formation. All solid biomass should be pelleted at the bottom of the tube leaving a clarified liquid supernatant. Sometimes debris will float on top of the sample and won’t pellet- just try to avoid it when collecting the supernatant.
  7. Use a serological pipette to transfer 10 mL of supernatant to a clean 50 mL falcon tube taking care not to disturb the settled biomass at the bottom.

    Hint: loosen all the tube lids before you begin pipetting so that you can open the tubes one handed.

  8. Connect a 50mL Steriflip vacuum filter unit to the vacuum tube and label the collection tube with sample ID.
  9. Apply vacuum until the whole sample has filtered into the collection tube or until the sample has stopped passing through the filter (i.e. the filter is saturated).
  10. Your filtered sample is now free of bacteria and sterile. Practicing aseptic technique, remove the vacuum line and filter unit, close the tube and store your filtered samples at 4℃ until needed.
  11. ▶ Continue to Protocol 4: Direct plating of environmental samples or ▶ Continue to Protocol 5: Phage enrichment