Jul 20, 2015
Version 1
Iron Chloride Precipitation of Viruses from Seawater V.1
- Seth John1,
- Bonnie Poulos1,
- Christine Schirmer1
- 1Matthew Sullivan Lab, University of Arizona, Ohio State University
- VERVE Net
- Sullivan Lab
Protocol Citation: Seth John, Bonnie Poulos, Christine Schirmer 2015. Iron Chloride Precipitation of Viruses from Seawater. protocols.io https://dx.doi.org/10.17504/protocols.io.c2wyfd
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
Created: June 15, 2015
Last Modified: July 09, 2018
Protocol Integer ID: 822
Abstract
This protocol describes a technique to recover viruses from natural waters using iron- based flocculation and large-pore-size filtration, followed by resuspension of virus- containing precipitates in a pH 6.5 buffer. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (> 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered if using oxalic acid in the resuspension buffer. Particles lose infectivity if resuspended with ascorbic acid in the buffer.
Guidelines
PCR inhibition: Note, that due to the likely issue with the EDTA in the resuspension buffer interacting with the MgCl in a PCR reaction, direct PCR is ineffective. To circumvent this, extract DNA using Wizard columns from Promega (see Tucson Marine Phage Lab’s DNA Extraction of Viruses Using Wizard Columns protocol).
Materials:
- Seawater
- Tris-base (FW=121.14)
- Na2-EDTA dihydrate (FW=372.24)
- MgCl2 hexahydrate (FW=203.3)
- Ascorbic acid (FW=176.12) and/or Oxalic acid (FW=126.07)
- NaOH, 5N
- HCl
- FeCl3·6H2O (FW=270.3)
- MilliQ water
- pH paper
- Whatman glass fiber filter GF/A (cat. 1820-150; 1.6μm retention; 150mm
diameter)
- Millipore Express PLUS filter (cat. GPWP14250; 142mm, 0.22μm)
- Pall Supor-800 filter (cat. 60114; 142mm, 0.8um)
- GE Polycarbonate Membrane filter (cat. K10CP14220; 142mm, 1.0μm),
Available through Midland Scientific Inc. (cat. MAINE 1216611)
- pH meter
- 142mm filtering towers (Need 2 for efficiency)
- Peristaltic pump with a pressure gauge
Notes:
Alternative Pre-filtering Method: An alternative method for preparing seawater is to pre-filter the seawater through a Whatman GF/D glass fiber filter (cat. 1823-150; 2.7μm retention; 142mm diameter) to remove large particles. The filtrate is filtered again using a Millipore Steripak GP10 or GP20 (cat. SPGPM10RJ or SPGPM20RJ; 0.22μm). The GP 10 works for <10L volumes and GP20 works for <20L volumes. Larger capsule filters will easily filter 100-200 liters: Pall Acropak 200, 500, 1000 and 1500. The cellular fraction is retained on the filter while the virus fraction is in the filtrate.
Filtration should be done at a maximum pressure of 15 psi. A peristaltic pump applies the pressure and a stainless steel Millipore filter rig (PN YY3014236) holds filters. For less expense, use an acrylic or polycarbonate 142 mm filter holder (Geotech Environmental Equipment, acrylic or PC) and pressurize carboy with an air pump. Most lab vacuum pumps have an air-pressure outlet on the other side that can easily be adjusted to provide 15 psi pressure. Nalgene sells caps for their 20L carboys with fittings in the top that can be used for pressurization.
FeCl3 Formulation: The solution of ferric chloride hexahydrate is calculated based on the amount of iron, not on the amount of the salt. The stock solution has 10g Fe per liter. For precipitation, the final optimal concentration of Fe in seawater (SW) is 1mg Fe per liter SW which is equal to 2.9mg FeCl3 per liter SW or to 4.83mg FeCl3·6H2O per liter SW.
Equipment Care: Carboys and collection bottles should be washed with 1N HCl and rinsed with MilliQ water before use. Checking tubing before use and replace if worn. Always rinse stainless steel filter rigs with MilliQ water after use and air dry to prevent corrosion.
Original Author(s): Seth John, Bonnie Poulos
Revision Author(s): Bonnie Poulos, Christine Schirmer
Citation: John, S.G., Mendez, C.B., Deng, L., Poulos, B., Kauffman, A.K.M., Kern, S., Brum, J., Polz, M.F., Boyle, E.A., & Sullivan, M.B. (2011). A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environ Microbiol Rep. 3(2), 195-202. doi:10.1111/j.1758-2229.2010.00208.x.
Materials
MATERIALS
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
Pall Supor-800 filterCatalog #60114
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
STEP MATERIALS
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Protocol materials
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
Pall Supor-800 filterCatalog #60114
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Safety warnings
Do NOT let the filters dry out before the precipitated viruses are resuspended! This will cause a loss in yield. - It is important that the filters remain moist until resuspension. The excess liquid on top of the filter can provide enough humidity if the tubes are well sealed with a tight-fitting cap and parafilm. One or two ml of sterile water or 0.2um filtered SW from the same source can be added if necessary to keep the filters moist. - Keep filters at 4°C until processed, and processing is best if done soon after collection (although we have successfully resuspended particles after several years if stored properly).
Virus Precipitation
Virus Precipitation
Assemble two 142mm filtering towers and attach the filter apparatus to a peristaltic pump with a pressure gauge (maximum pressure = 15 psi)
Note
See Guidelines note for an alternative pre-filtration method.
Pre-filter 20L of seawater using a 150mm Whatman GF/A filter.
Note
Wear gloves and use forceps for handling all filters.
Filter again to remove bacteria using a 0.22um, 142mm Millipore Express Plus filter.
Note
See note in guidelines for alternative pre-filtration method.
Note
Wear gloves and use forceps for handling all filters.
Millipore Express PLUS filter 0.22 µm, 142 mmCatalog #GPWP14250
Treat the virus fraction (0.22μm filtrate) with FeCl3 to precipitate the viruses by adding 1ml of 10g/L Fe Stock Solution to each 20L of filtrate. Shake vigorously for 1 minute.
1 mL
00:01:00
Note
Solution is acidic and should be handled with care.
Discard solution if a cloudy precipitate forms. Do not dilute solution as iron hydroxide precipitate will form quickly.
For use on board ship, it is best to pre-weigh iron chloride into 50ml centrifuge tubes and add water as needed during the cruise: pre-weigh 0.966g FeCl3·6H2O into several 50ml tubes. Add 20ml MQ-H2O when ready to precipitate viruses in seawater.
Protocol
NAME
10g/L Fe Stock SolutionCREATED BY
Bonnie Poulos
Measure out 4.83g FeCl3·6H2O.
5 g
Iron(III) chloride hexahydrateCatalog #236489
Dissolve FeCl3·6H2O in 100ml MilliQ water.
100 mL
Store at room temperature or 4°C.
Add an additional 1ml of 10g/L Fe stock solution to each 20L of filtrate (for a total of 2ml Fe Stock Solution per 20L filtrate). Shake vigorously for 1 minute.
1 mL
00:01:00
Note
For use on board ship, it is best to pre-weigh iron chloride into 50ml centrifuge tubes and add water as needed during the cruise: pre-weigh 0.966g FeCl3-6H2O into several 50ml tubes. Add 20ml MQ-H2O when ready to precipitate viruses in seawater.
Note
Discard solution if a cloudy precipitate forms. Do not dilute solution as iron hydroxide precipitate will form quickly.
Note
Solution is acidic and should be handled with care.
Protocol
NAME
10g/L Fe Stock SolutionCREATED BY
Bonnie Poulos
Measure out 4.83g FeCl3·6H2O.
5 g
Iron(III) chloride hexahydrateCatalog #236489
Dissolve FeCl3·6H2O in 100ml MilliQ water.
100 mL
Store at room temperature or 4°C.
Let the FeCl3 treated filtrate sit for 1 hour at room temperature.
01:00:00
Filter the FeCl3 treated filtrate using a 1.0μm, 142mm, polycarbonate (PC) membrane filter on top of a 0.8μm, 142mm, Supor support filter and attaching the filter apparatus to a peristaltic pump with a pressure gauge (maximum pressure = 15 psi).
Note
The FeCl3 precipitate will be captured on the PC filter; the Supor is just for support in the 142mm stainless steel filtration apparatus. Although the 20L can be collected on a single 142mm PC membrane, it is faster to change the PC membrane one or two times during the filtration; normally 3 PC membranes and 1 Supor membrane are used per 20L seawater.
GE Polycarbonate Membrane filter Catalog #MAINE 1216611
Place all of the polycarbonate filters from the 20L into a 50ml centrifuge tube being careful not to scrape off any of the FeCl3 on the edge of the tube (having precipitate facing out aids in dissolving the precipitate).
Discard the Supor support filter.
Note
The filters can be stored in the 50ml tubes at 4°C until ready to resuspend the precipitated viruses. Be sure the caps are on securely so that the filters do not dry out.
Resuspension
Resuspension
Prepare fresh 0.1M EDTA-0.2M MgCl2-0.2M Ascorbate Buffer, pH6.5.
Protocol
NAME
0.1M EDTA-0.2M MgCl2-0.2M Ascorbate BufferCREATED BY
Bonnie Poulos
Dissolve 1.51g Tris-base in 80ml Milli Q water.
Dissolve 3.72g Na2-EDTA dihydrate into solution.
Note
pH will be ~10.0
Once EDTA is in solution, dissolve 4.07g MgCl2.
Note
pH will drop to ~8.0
Add 3ml of NaOH.
Note
This will drop the pH to ~4.5 and the solution will become cloudy which indicates that the EDTA is coming out of solution.
Dissolve the reductant (3.52g of ascorbic acid or 2.52g of oxalic acid).
Note
The pH will increase to ~8.3 and the solution will clear up.
Once the reductant is in solution, add the last 1ml of NaOH.
Check the pH using pH paper (the buffer should be at pH 6.0 - 6.5)
Note
The solution may need some minor adjusting with NaOH or HCl to achieve a pH of 6.0.
Note
pH 6.0 is ideal for good recovery of viruses.
Check the volume and add MilliQ water for a total volume of 100ml.
Store the buffer in the dark (bottle wrapped in foil) and visually inspect prior to use. It should be clear without precipitates.
Note
At this point, 10-15ml of buffer can be sacrificed for a final pH check using a pH meter.
Note
The buffer will start to change color after about 24 hours. It is okay to use if slightly discolored, but do not use after about 36 hours (eventually the buffer will turn almost orange!).
Add 1ml buffer for each 1mg Fe, which is 20ml of 1x buffer for the 20L seawater precipitate (10ml of a 2X concentrated buffer). Shake the tube vigorously.
Place tubes on a rotator and rotate at 4°C overnight.
18:00:00
Note
You will see the solution change colors a number of times – that is okay. However, if precipitates form, there is a possibility that viruses are trapped or adhered to the particles. Centrifuge gently to pellet the precipitate, and try using additional buffer to get the pellet back into solution. Although it may not go back into solution, the pellets will have been washed and may recover some viruses. 13. Leave stored at 4°C until ready to analyze or further purify the viral fraction.
Leave stored at 4°C until ready to analyze or further purify the viral fraction.
To collect the resuspended precipitate, open the tube and collect liquid into a fresh tube.
Using a sterile pipet, applicator stick, or bleached and rinsed forceps pull an edge of the filter onto the tube edge so it is caught in the lid when secured.
Centrifuge at low speed (less than 1000 rpm) for 3-5 minutes to recover more buffer.
00:05:00
If the filter has precipitate on it, add more buffer, incubate again for a couple hours, and collect liquid as stated above.