SEA-PHAGES | One Step Growth Curve

Link to this post \| posted 13 Jan, 2020 21:01
cellokiwi	Hello Hive Mind I'm hoping I can get some help with a one step growth curve. My students have tried two different protocols, one of which works successfully in another mycobacteriophage lab, and we have not been able to get a nice S-curve. There is a trend that we see every time, high titer initially that continues to drop. The earliest time point we have tried (other than 0 min which has no plaques) is 10 min and I honestly refuse to believe that they are completing a replication cycle that quickly… So why do we have essentially inverse data to what you would expect? Again, this is highly repeatable; we have tried many times. I'm attaching a representative graph of what we are seeing. Thanks for any and all help we can get! 8Kb

Link to this post | posted 13 Jan, 2020 21:01

Hello Hive Mind

I'm hoping I can get some help with a one step growth curve. My students have tried two different protocols, one of which works successfully in another mycobacteriophage lab, and we have not been able to get a nice S-curve. There is a trend that we see every time, high titer initially that continues to drop. The earliest time point we have tried (other than 0 min which has no plaques) is 10 min and I honestly refuse to believe that they are completing a replication cycle that quickly… So why do we have essentially inverse data to what you would expect? Again, this is highly repeatable; we have tried many times. I'm attaching a representative graph of what we are seeing. Thanks for any and all help we can get!

Link to this post \| posted 14 Jan, 2020 17:55
welkin	I wish I knew! I have yet to come across a protocol for a one-step growth curve in Actinobacteriophages that is accurate and repeatable. in general, it takes about three hours to get from infection to burst, but the adsorption does not seem as synchronous as it should be, and so rather than a clear latent/burst curve, I see somewhat tiered bursts over many hours. I would be interested to see your protocol that is supposed to work.

Link to this post | posted 14 Jan, 2020 17:55

welkin

I wish I knew!
I have yet to come across a protocol for a one-step growth curve in Actinobacteriophages that is accurate and repeatable.
in general, it takes about three hours to get from infection to burst, but the adsorption does not seem as synchronous as it should be, and so rather than a clear latent/burst curve, I see somewhat tiered bursts over many hours.
I would be interested to see your protocol that is supposed to work.

Link to this post \| posted 17 Jan, 2020 17:21
stukey	Hello - I've attached a protocol that we have worked up and use for our one-step assays. Consistent with Welkin's observation, we also find some phages work better (giving nice definitive latent period signal and obvious burst plateau) than others. But overall, we have had success in producing what I would say is good, clean data that can be interpreted and that is generally reproducible. Joe 83Kb

Link to this post | posted 17 Jan, 2020 17:21

stukey

Hello -

I've attached a protocol that we have worked up and use for our one-step assays. Consistent with Welkin's observation, we also find some phages work better (giving nice definitive latent period signal and obvious burst plateau) than others. But overall, we have had success in producing what I would say is good, clean data that can be interpreted and that is generally reproducible.

Joe

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One Step Growth Curve