Mar 19, 2025

Public workspaceViral extraction from Sediment V.1

DOI
dx.doi.org/10.17504/protocols.io.6qpvr97zbvmk/v1
  • 1California Institute of Technology
  • Aditi K. Narayanan: ORCID: 0000-0003-0627-1859
  • Alon Philosof: ORCID: 0000-0003-2684-8678
  • Orphan Lab
Victoria J Orphan
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr97zbvmk/v1
Protocol Citation: Aditi K. Narayanan, Alon Philosof 2025. Viral extraction from Sediment. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr97zbvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 16, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 118534
Funders Acknowledgements:
NOMIS Foundation
U.S. Department of Energy
Grant ID: DE-SC0020373; DE-SC0022991
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Abstract
Viruses are often undercounted in subsurface sediments due to their propensity to stick to surfaces. This protocol outlines a method for detaching viruses from sediment particles via a combination of chemical and physical agitation followed by density separation.
Aditi K. Narayanan and Alon Philosof
Aditi K. Narayanan and Alon Philosof
Based on An Improved Method for Extracting Viruses From Sediment: Detection of Far More Viruses in the Subseafloor Than Previously Reported
 
https://doi.org/10.3389/fmicb.2019.00878 (Pan et al 2019, Frontiers in Microbiology)
Materials
Materials
  1. 20mM tetrasodium pyrophosphate solution (0.532g in water for 100mL final volume), CAS #7722-88-5, Millipore-Sigma Cat. #P8010
  2. 10% NaCl solution (10g in water for 100mL final volume), CAS #7647-14-5
  3. 30% Nycodenz solution (4.5g in water for 15mL final volume). Nycodenz is also called Histodenz or Iohexol), CAS #66108-95-0, Millipore-Sigma Cat. #D2158
  4. 50% Nycodenz solution (7.5g in water for 15mL final volume)
  5. 15mL Falcon tubes or similar (e.g. Fiusher Scientific Cat. #4-959-49B), capable of withstanding ~4000xg
  6. Sonication wand (e.g. QSonica CL-188)
  7. Centrifuge capable of holding 15mL Falcon Tubes (e.g. Beckman Coulter Allegra X-15R Centrifuge with SX4570 swinging bucket rotor)
  8. Syringes, various volumes
  9. Hypodermic needles, between preferably 3-4 inches in length if possible (otherwise 1.5 inches will do)
  10. Glutaraldehyde solution (optional, for fixation)
  11. Liquid nitrogen (optional, for storage after fixation)
Protocol
Protocol
Take 0.1-0.33cm3 of sample (or 1mL of slurry) and mix enough NaCl solution and tetrasodium pyrophosphate solution to get to a final concentration of 2.5% NaCl and 5mM tetrasodium pyrophosphate in a 6mL volume. Your stock solutions under the Materials section should be 4X concentrations. Get rid of any clumps by shaking. Works best in a 15mL Falcon tube.

For example, to each 1mL of slurry, add 1.5mL of 4X tetrasodium pyrophosphate, 1.5mL of 4X NaCl, and 2mL of virus-free water.
Sonicate on ice at 25 amps for 10 seconds. Rest for another 10 seconds. Repeat 2 more times for a total of 30 seconds of sonication. Our sonicator is a QSonica CL-188. 
Create Nycodenz 30% and 50% gradient in a 15mL falcon tube using the underlay method:
  1. Fill a syringe with 1mL of 30% solution. Attach a needle to the end.
  2. Tap out any air bubbles in the syringe
  3. Dispense the 30% solution into the very bottom of the Falcon tube
  4. Repeat the above steps 1-2 with the 50% solution
  5. Push the needle of the syringe containing the 50% solution all the way to the bottom of the Falcon tube
  6. CAREFULLY dispense the 50% solution under the 30% solution, allowing the 30% layer to rise above it
Layer the sonicated sample over the gradient and centrifuge at 2900xg for 30min in a swinging bucket rotor.
Use a needle and syringe to remove the topwater, 30% fraction, and liquid portion of the 50% fraction. These fractions will contain your viruses, though you will need to stain with SYBR Gold to determine which of your fractions carries the largest number.
Filter the viral fraction through a 0.2µm membrane and store. If you choose to fix before storing, mix enough glutaraldehyde with the sample for a final concentration of 0.1% glutaraldehyde. Allow the samples to sit at 4˚C for 30 minutes, then flash freeze and store at -80˚C.
If you choose, you can repeat the pyrophosphate, sonication, and gradient steps on the sediment pellet to ensure maximum extraction efficiency. 
  1. Add 5-6mL of 1:1 solution (final concetration 5mM tetrasodium pyrophosphate and 2.5% NaCl)
  2. Repeat steps 2-6
Protocol references
https://doi.org/10.3389/fmicb.2019.00878 (Pan et al 2019, Frontiers in Microbiology)