I've managed to isolate the phage for Clostridium perfringens, but whenever I tried to filter it even with the biggest pore size of 0.45 μm the phage didn't manage to pass through. I also have done the filtration using nylon-type filter and PES-type filter. Both filters yield negative results. Anyone found a similar problem and how do I solve this particular problem?
I doubt you would be able to see aggregating phage in a (light) microscope unless they were massively aggregated. But they could certainly could be aggregating to an apparent size larger than 0.45um. Or they could be sticky and are not releasing from the membrane.
What type of sample are you filtering? If it's a straight lysate there will be a lot of debris that will be difficult to filter out and will hold up the filter potentially blocking your phage from passing through. You will need to determine a method for clearing the lysate first and then proceed with filtering. Centrifugation usually works to clear a lysate enough for filtration.
If this isn't your problem then I suggest scraping a sample off the surface of the filter when filled with a bit of buffer and trying to use that to infect bacteria to determine if all your phage are adhering to the filter.
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