SEA-PHAGES | Low plate titer issues

Link to this post \| posted 11 Apr, 2022 21:29
anesb7940	I have found and isolated a phage that I named Ascela. It hasn’t been confirmed yet, but I’m fairly certain that Ascela is a temperate phage. I have been trying to get Ascela to a higher titer, but I can’t seem to get good plated titers. The current titer that I calculated was 7.0x10^6. As I looked back through pictures of my full plate titers, I noticed that there are three plates present in every dilution that have a rapid decline in plaques present. I will go from a web plate and then suddenly have a significant drop in plaques (normally occurs in the 10^-1 to 10^-4 plates). This has been a reoccurring issue from the very beginning of isolating my phage and has been replicated in multiple different series of plates. I’ve made sure that I am vortexing them well before I plate them, but it’s not helping. I recently did another full plate titer, and it did the same thing it normally does. I wiped out the first two plates then had a web plate on 10^-2, but then on 10^-4 I only had sixteen plaques. There was nothing on 10^-5, 10^-6, or 10^-7, but then suddenly there was one plaque on the 10^-8 plate. I also did a DNA extraction following the SEAPhages protocol and got 226.8 ng/uL with 1.88 purity. So, I'm pretty sure that there is a fairly good titer in my lysate, but how do I get Ascela to titer well on a plate? Has anyone else had this issue where their plaques significantly drop after a web plate? If so, did you figure out how to fix it? Is there a different protocol that I can try to get a better full plate titer? Thank you in advance for your help.

Link to this post | posted 11 Apr, 2022 21:29

I have found and isolated a phage that I named Ascela. It hasn’t been confirmed yet, but I’m fairly certain that Ascela is a temperate phage. I have been trying to get Ascela to a higher titer, but I can’t seem to get good plated titers. The current titer that I calculated was 7.0x10^6. As I looked back through pictures of my full plate titers, I noticed that there are three plates present in every dilution that have a rapid decline in plaques present. I will go from a web plate and then suddenly have a significant drop in plaques (normally occurs in the 10^-1 to 10^-4 plates). This has been a reoccurring issue from the very beginning of isolating my phage and has been replicated in multiple different series of plates. I’ve made sure that I am vortexing them well before I plate them, but it’s not helping. I recently did another full plate titer, and it did the same thing it normally does. I wiped out the first two plates then had a web plate on 10^-2, but then on 10^-4 I only had sixteen plaques. There was nothing on 10^-5, 10^-6, or 10^-7, but then suddenly there was one plaque on the 10^-8 plate. I also did a DNA extraction following the SEAPhages protocol and got 226.8 ng/uL with 1.88 purity. So, I'm pretty sure that there is a fairly good titer in my lysate, but how do I get Ascela to titer well on a plate? Has anyone else had this issue where their plaques significantly drop after a web plate? If so, did you figure out how to fix it? Is there a different protocol that I can try to get a better full plate titer? Thank you in advance for your help.

Link to this post \| posted 31 Mar, 2023 14:10
Thiel	I've had this same issue with a new turbid plaque, PhriedEgg. I'm not sure of an answer but if anyone has suggestions then that would be great. Thanks.

Link to this post \| posted 31 Mar, 2023 14:13
Thiel	I've had this same issue with a new turbid plaque, PhriedEgg. I'm not sure of an answer but if anyone has suggestions then that would be great. Thanks.

Link to this post \| posted 31 Mar, 2023 14:37
viknesh	Thiel I've had this same issue with a new turbid plaque, PhriedEgg. I'm not sure of an answer but if anyone has suggestions then that would be great. Thanks. Hi Sarah, Would you be able to share photos of what you are seeing? Vic

Link to this post \| posted 06 Apr, 2023 16:48
Thiel	Hi, I can share a photo but Kristen shared a great suggestion with me that worked. We put 150uL of phage buffer in a tube and then picked 15-20 plaques from the same plate. We had done 4 purifications so we were confident that it was pure. We put the 15-20 plaques in the 150uL of phage buffer. We then did serial dilutions, infected the bacteria and plated as normal the 10^0 through 10^-4 and the 10^-3 was very nicely webbed.

Link to this post | posted 06 Apr, 2023 16:48

Thiel

Hi, I can share a photo but Kristen shared a great suggestion with me that worked. We put 150uL of phage buffer in a tube and then picked 15-20 plaques from the same plate. We had done 4 purifications so we were confident that it was pure. We put the 15-20 plaques in the 150uL of phage buffer. We then did serial dilutions, infected the bacteria and plated as normal the 10^0 through 10^-4 and the 10^-3 was very nicely webbed.

Link to this post \| posted 06 Apr, 2023 17:27
viknesh	Thiel Hi, I can share a photo but Kristen shared a great suggestion with me that worked. We put 150uL of phage buffer in a tube and then picked 15-20 plaques from the same plate. We had done 4 purifications so we were confident that it was pure. We put the 15-20 plaques in the 150uL of phage buffer. We then did serial dilutions, infected the bacteria and plated as normal the 10^0 through 10^-4 and the 10^-3 was very nicely webbed. Great!

Link to this post | posted 06 Apr, 2023 17:27

viknesh

Thiel
Hi, I can share a photo but Kristen shared a great suggestion with me that worked. We put 150uL of phage buffer in a tube and then picked 15-20 plaques from the same plate. We had done 4 purifications so we were confident that it was pure. We put the 15-20 plaques in the 150uL of phage buffer. We then did serial dilutions, infected the bacteria and plated as normal the 10^0 through 10^-4 and the 10^-3 was very nicely webbed.

Great!

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Low plate titer issues