Science Buddies: "Ask an Expert"

I am using sewage sample to isolate bacteriophage but after many rounds of enrichment, I am not able to get sufficient phage titre and the spot assay is not showing much clearance. Please suggest why

Hello!

Can you share the procedure that you used to isolate the bacteriophages? We can take a look at it and see if we can identify where it might be going wrong.

Sewage sample was collected. It was centrifuged at 4C at 10,000rpm. The supernatant transferred to host bacterium culture ( bacteria in lb broth).
Kept for overnight incubation. Again centrifuged and obtained supernatant.Supernatant was filtered using 0.22mm filter. This was spotted in LB plate which had a lawn of host bacteria. Kept for incubation overnight but plaque size is very low.

Hello,

What type of bacteria are you using for your experiments? There are all different kinds of bacteriophages that are each specific to a different type of bacteria. It's possible that the phages you isolated weren't specific to the bacteria you cultured them with and that's why you're not seeing the plaques. Did you culture the bacteria overnight at 37 degrees Celsius before adding the sewage sample? And after you added the sewage sample and cultured it with the bacteria overnight, did you perform that culture at 37 degrees Celsius with shaking? If you did, I would guess that either there were no phages in the sewage sample or they just don't infect the specific type of bacteria you cultured them with. Can you share your research question and the goal for your project? That way we can figure out the best way to achieve your goals!

After adding the sewage sample to bacterial culture I kept it for incubation at 37 degree C overnight but did not keep for shaking. The host bacteria is Enterococcus.

Which species of Enterococcus did you use?

Enterococcus faecium

If the bacteria seem to be growing fine, as evidenced by cloudy growth media in the liquid culture and growth on your agar plates, I would guess that the problem is just that the bacteriophages you isolated do not infect E. faecium. Each bacteriophage is very specific to a certain species of bacteria, so it's very possible that no E. faecium specific phages were in your sewage sample.

So I should try with other samples?

Yes, I think it would be worth it to try other samples. Once you've isolated E. faecium specific phages, what do you plan to do from there?

That's it.Thank you