SEA-PHAGES | Our finnicky phages either grow too much or not at all

Link to this post \| posted 03 Apr, 2019 02:20
agm32	My research project consists of using a new bacterial host to hunt for phages. So far we know that phages can infect the bacterial strain we're using, so I don't think that's the problem here. We've been trying just to get to the purification stage, but we keep encountering problems. It's 2 steps forward, 3 steps back every time! Our first round of environmental samples yielded seemingly temperate phages which would not grow consistently. We assayed 500uL of these lysates as per the SEA-PHAGES discovery guide protocols, but no plaques formed. Then we assayed 1mL, and the little buggers lysed the whole plate. We tried finding a lysate concentration that would yield isolated plaques by assaying 250, 500, and 750uL of lysate, but again these formed no plaques. So we flooded the 1mL assay and tried to use that lysate for a plaque assay. Nothing. Our second round of environmental samples yielded lytic phages. We again assayed 1mL of lysate and got isolated plaques! We picked 3 plaques and serially diluted them. We assayed the serial dilutions. 2 of the lysates yielded no plaques. The other formed ~3 plaques on the 10^0 plate, and ~23 plaques on the 10^-1 plate. That doesn't make any sense… We've tried to serial dilute and plaque assay multiple lysates at least 4 times now, and not a single one has worked. Plaques either do not form, or they behave in odd ways like I mentioned above. Generally it's the former. I'm really at my wits end here. I've been going in the same circle for MONTHS with no success. I'm desperate to get this to work…How do I get these phages to do what I want? – UWF B.S. of Marine Biology 2019 SEA-PHAGES Cohort 8.2

Link to this post | posted 03 Apr, 2019 02:20

My research project consists of using a new bacterial host to hunt for phages. So far we know that phages can infect the bacterial strain we're using, so I don't think that's the problem here. We've been trying just to get to the purification stage, but we keep encountering problems. It's 2 steps forward, 3 steps back every time!

Our first round of environmental samples yielded seemingly temperate phages which would not grow consistently. We assayed 500uL of these lysates as per the SEA-PHAGES discovery guide protocols, but no plaques formed. Then we assayed 1mL, and the little buggers lysed the whole plate. We tried finding a lysate concentration that would yield isolated plaques by assaying 250, 500, and 750uL of lysate, but again these formed no plaques. So we flooded the 1mL assay and tried to use that lysate for a plaque assay. Nothing.

Our second round of environmental samples yielded lytic phages. We again assayed 1mL of lysate and got isolated plaques! We picked 3 plaques and serially diluted them. We assayed the serial dilutions. 2 of the lysates yielded no plaques. The other formed ~3 plaques on the 10^0 plate, and ~23 plaques on the 10^-1 plate. That doesn't make any sense…

We've tried to serial dilute and plaque assay multiple lysates at least 4 times now, and not a single one has worked. Plaques either do not form, or they behave in odd ways like I mentioned above. Generally it's the former.

I'm really at my wits end here. I've been going in the same circle for MONTHS with no success. I'm desperate to get this to work…How do I get these phages to do what I want?

–
UWF B.S. of Marine Biology 2019
SEA-PHAGES Cohort 8.2

Link to this post \| posted 03 Apr, 2019 02:23
debbie	Hi! Wow! More details are needed. Would you provide who you are, Where you are from, What host you are using? Once I get some basics, I may have more questions.

Link to this post \| posted 03 Apr, 2019 02:25
agm32	Debbie Jacobs-Sera Hi! Wow! More details are needed. Would you provide who you are, Where you are from, What host you are using? Once I get some basics, I may have more questions. I'm a student at the University of West Florida in Pensacola. I was in SEA-PHAGES two years ago and am continuing research with the same professor. We're using Psychrobacter nivimaris as our host. – UWF B.S. of Marine Biology 2019 SEA-PHAGES Cohort 8.2

Link to this post | posted 03 Apr, 2019 02:25

agm32

Debbie Jacobs-Sera
Hi! Wow! More details are needed. Would you provide who you are, Where you are from, What host you are using? Once I get some basics, I may have more questions.

I'm a student at the University of West Florida in Pensacola. I was in SEA-PHAGES two years ago and am continuing research with the same professor. We're using Psychrobacter nivimaris as our host.

–
UWF B.S. of Marine Biology 2019
SEA-PHAGES Cohort 8.2

Link to this post \| posted 04 Apr, 2019 03:25
dbolliva	One trick we have used when starting with a new host bacterium is to do spot plating with the dilution series. If we have a lysate we would make a lawn and then spot 10 ul of the lysate onto the plate. You can also create a serial dilution series and spot 10 ul of each of the dilutions. We have had good success with this and if you do the serial dilutions you will get to a concentration where it appears there are plaques. If you are using marine agar or salt containing medium, I have also seen problems with top agar getting too high in salt if we melt it too many times in the microwave. Sometimes when isolating phages we have also run into is the growth of a different bacterium on the plate that produces a compound inhibiting growth of our host. This can be possible if the bacterium that produces the plaque like appearance is a spore-forming bacterium.

Link to this post | posted 04 Apr, 2019 03:25

dbolliva

One trick we have used when starting with a new host bacterium is to do spot plating with the dilution series. If we have a lysate we would make a lawn and then spot 10 ul of the lysate onto the plate. You can also create a serial dilution series and spot 10 ul of each of the dilutions. We have had good success with this and if you do the serial dilutions you will get to a concentration where it appears there are plaques.

If you are using marine agar or salt containing medium, I have also seen problems with top agar getting too high in salt if we melt it too many times in the microwave.

Sometimes when isolating phages we have also run into is the growth of a different bacterium on the plate that produces a compound inhibiting growth of our host. This can be possible if the bacterium that produces the plaque like appearance is a spore-forming bacterium.

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Our finnicky phages either grow too much or not at all