Jan 04, 2016
Phage Tangential Flow Filtration
- Forest Rohwar1
- 1Matthew Sullivan Lab, University of Arizona/Ohio State University
- VERVE Net
- Sullivan Lab
Protocol Citation: Forest Rohwar 2016. Phage Tangential Flow Filtration. protocols.io https://dx.doi.org/10.17504/protocols.io.c7yzpv
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 26, 2015
Last Modified: March 16, 2018
Protocol Integer ID: 984
Abstract
In tangential flow filtration (TFF), the sample is run parallel to the filter. Particles that are smaller than the pore size are pushed out through the holes (the filtrate). Particles that are larger than the pore size are retained inside the filter (the retentate). TFF is most effective when a reservoir is used so that the sample can be recycled through numerous times.
Guidelines
We use 2 sizes of filters. All the filters are from Amersham Biosciences (1-800-526-3593):
Large filters: for concentrating 10 or more liters down to ~1 liter
-Large 0.2 µm filter: CFP-2-E-9A ($1306)
-Large 100 kD filter: UFP-100-C-9A ($1306)
Medium filters: for concentrating up to 10 liters to a final volume of ~100 ml
-Medium 0.2 µm filter: CFP-2-E-4A ($459)
-Medium 100 kD filter: UFP-100-C-4A ($444)
This picture is of the large sized filters – you can see the straw-like filters inside:
The input can go in either end of the filter, the retentate comes out the opposite end. The filtrate comes out the 2 side outlets – tubing can be attached to both of these, or one can be covered so you only use one filtrate output.
Tangential flow filtration allows you to filter large volumes and concentrate them into smaller volumes
The medium filter is shown here. The medium and large tangential flow filters work in the same way. The main difference is in the amount of surface area, which controls how much volume you can filter per time.
The input can go in either end of the filter, the retentate comes out the opposite end. The filtrate comes out the 2 side outlets – tubing can be attached to both of these, or one can be covered so you only use one filtrate output.
With a 0.2 µm filter, the viruses are in the filtrate
With a 100 kD filter, the viruses are in the retentate
For the medium filters, the tubing (size 15) can be attached directly to the TFF. The tubing is secured to the filter using cable ties. The large filters require a tubing adaptor kit – more information found later in the protocol.
Our lab setup is shown here. The reservoir can be any container ranging from a beaker to a large trash can. Attaching the filter to a ringstand makes things more manageable, but it doesn’t matter if the filter is vertical or horizontal.
In the example shown here, the viruses are being concentrated on a 100 kD filter. The water is being pushed out of the sample while the viruses are retained on the filter and in the retentate. The filter is run until there is very little volume left in the flask. At this point, the pressure on the retentate tube is released, allowing the sample to wash over the filter, freeing any attached viruses. The sample is then concentrated further – with the final step being removal of the input tube which runs air through the filter and forces all remaining sample out of the filter and tubing.
The peristaltic pump, tubing, and pressure gauges are all purchased from Cole Parmer (1-800-323-4340)
Tubing: MASTERFLEX!
Size 82 (large filters): U-96440-82 ($258 for 25 feet)
Size 15 (medium filters): U-96440-15 ($108 for 25 feet)
Size 73 tubing (to adapt medium filters to large pump heads): U-96440-73 ($196 for 25 feet)
Peristaltic pump:
Size 82 or 73 tubing (large filters):
Masterflex Pump, I/P Brushless Drive: EW-77410-10 ($1475)
Masterflex Pump head, Easy-load PSF/SS: FF-77601-10 ($490)
Mounting hardware for additional pump heads: FF-77601-96 ($22)
Size 15 tubing (medium filters):
Masterflex Pump, L/S Easy-load: FF-77521-40 ($865)
Masterflex Pump head, Easy-load: FF-07518-12 ($190)
Mounting hardware for additional pump heads: FF-07013-05 ($22)
In order to avoid having to buy 2 peristaltic pumps, you can use the large pump for both. In order to do this, thread the size 15 tubing through size 73 tubing where it goes into the pump head.
The pressure gauges and adaptors are purchased from Cole Parmer (1-800-323-4340)
Pressure Gauge and adaptor to connect it:
0-30 PSI Stainless steel gauges: P-68007-03 ($62)
Gauge guards U-07359-08 ($118)
¼ female-female teflon adaptor: P-06376-31 ($15)
Kynar T-adaptors: P-30604-65 ($30 for a pack of 10)
These gauges need to be filled with mineral oil (we use catalog # U07359-50 from Cole Parmer). The gauge guard prevents the oil from getting into the rest of the system.
In the TFF set-up, you need a pressure gauge between the peristaltic pump and the TFF. Always keep the pressure below 10 PSI to avoid blowing up the viruses.
The large filters require a hose-adaptor kit in order to connect the tubing (KA124PS-NY; $179).
With the medium filters, the tubing attaches directly to the filter.
When you have finished concentrating your sample, release the pressure clamp and allow the sample to wash over the filter for several minutes.
We have used this setup in a variety of environments – the only place that may cause problems from the generator is high altitude.
Wash Solution
*Add 50g NaOH to 5 liters of water
*Mix until NaOH is in solution
*Add 1 ml bleach and mix
Starting a new filter:
When you start a new filter, it needs to be rinsed of the storage preservatives.
1)Run DI water through the filter for 30 minutes. Make sure the pressure is adjusted to ~5 PSI
2)Run wash solution through the filter for 1 hour (for a large filter, you will need a lot of wash solution)
3)Rinse filters with DI water until both the filtrate and retentate fractions have a neutral pH
Cleaning the filters:
Filters should be cleaned with wash solution before and after processing a sample. Run at least 2 liters of wash solution through the medium filteres, and 5-10 liters through the large filters. Then run DI water through the filters (several liters) until the pH is neutral in both the filtrate and retentate fractions.
Storing the filters:
Store the filters at room temperature, wet with DI water. Do not store filters in wash solution. Make sure all the attachments points are plugged with the red covers, or that the ends of the tubing are in DI water. It is best if you do not allow the filters to dry out.
Other Recommendations
* It is important to look at your filtrate and retentate each time you process a sample, to make sure that everything is working. You should also perform counts on your input sample so you can determine recovery rates.
* If you are filtering a "dirty" sample with lots of particles or other things that would clog the filter we recommend first putting the sample through Nitex mesh (poor size ~100 um).
* Make sure to NEVER expose your filters to organic solvents such as chloroform. These will ruin the filters.
You should always monitor the filtrate and retentate to ensure that the filters are working properly. We fix our samples with 2% paraformaldehyde (Electron Microscopy Services), filter onto a 0.02 µm Anodisc (Whatman), stain with SYBR Gold (Molecular Probes), and then count bacteria and viruses using epifluorescence microscopy.
Nitex mesh is from Coastal supplies (619-562-8880) – we use cat #3-123-70 (pore size ~136 µm)
Other random parts you might want:
*various tubing connectors
*cable ties
*spare fuses for the peristaltic pump
*Teflon tape
In Step 1, the retentate (red) contains Bacteria, which are larger than the pore size of 0.2 µm. This is recycled back into reservoir “B” until most of the volume has been 0.2 µm filtered. The small amount remaining in reservoir “B” is the bacterial concentrate. The filtrate (blue) contains the viruses (which are smaller than the 0.2 µm pore size), and is collected in reservoir “V” for future concentration (see Step 2)
In both Steps 1 and 2, pressure should be applied on the retentate tube (shown by the yellow pentagon). This will force water through the filter into the filtrate.
It is important to keep the overall pressure of the sample below 10 psi. Pressures above this can compromise the viral particles. The pressure is regulated by 1) the speed of filtration (pump speed) and 2) the amount of pressure applied to the retentate tube.
The filtrate from Step 1 (containing the viruses) is then concentrated in Step 2 using a 100 kD tangential flow filter. Since viruses are larger than 100 kD, they are in the retentate. The 100 kD filtrate is sterile water (with no microbes or viruses present).
If this set-up is run without any added pressure, essentially no water will come out the filtrate. To enable concentration of the sample, it is necessary to apply pressure on the retentate tube (shown by yellow pentagon).
The total amount of volume left at the end of the concentration process is determined by the void volume of the system (both of the filter and the tubing).
To save time with samples that are relatively clean (not many particles or debris), you can skip the 0.2 µm filtration of Step 1 and concentrate the whole water sample directly on the 100 kD filter. The concentrated sample can then be filtered through an impact filter to remove the bacteria.
Using a peristaltic pump with 2 heads, Steps 1 and 2 can be combined into 1 process.