SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 30 Sep, 2024 17:11
viknesh	nietof Hi everyone Has anybody successfully isolated phages from soils stored frozen after sampling? Thank you Fernando Wanted to share that no faculty has shared with me that they've tried this.

Posted in: Phage Discovery/Isolation → Isolating from frozen soils

Link to this post \| posted 20 Sep, 2024 14:48
viknesh	bavina Hello, I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts? The precipitation could be problematic for at least two reasons: First, it is hard to tell if the media is contaminated and second, the precipitate itself, say calcium chloride, might have been a necessary component of the solution needed for infectivity. If you keep those two things in mind, you can still use your media. To address the possibility of contamonation, you'll want to include controls. For example, when using the top agar for a plaque assay, include a control where no host is added to the top agar. No lawn should form and the top agar should remain clear after 24 hours. To address the concern that precipitation might negatively impact infection, you'll want to pay attention to any change in infectivity for phages that you've isolated. In the longer term, it would be good to change the media components. As we look to no longer use Fiher brand peptone, the HHMI SEA team hasn't had a chance to test alternatives to Bacto products that we know are quite expensive, but we plan to do so – more soon!

Link to this post | posted 20 Sep, 2024 14:48

viknesh

bavina
Hello,

I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts?

The precipitation could be problematic for at least two reasons: First, it is hard to tell if the media is contaminated and second, the precipitate itself, say calcium chloride, might have been a necessary component of the solution needed for infectivity. If you keep those two things in mind, you can still use your media. To address the possibility of contamonation, you'll want to include controls. For example, when using the top agar for a plaque assay, include a control where no host is added to the top agar. No lawn should form and the top agar should remain clear after 24 hours. To address the concern that precipitation might negatively impact infection, you'll want to pay attention to any change in infectivity for phages that you've isolated.

In the longer term, it would be good to change the media components. As we look to no longer use Fiher brand peptone, the HHMI SEA team hasn't had a chance to test alternatives to Bacto products that we know are quite expensive, but we plan to do so – more soon!

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 06 Sep, 2024 23:42
viknesh	serivas Hello all, Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar? It is likley that you'll be able to culture the bacteria we use for phage-hunting just as well using peptone or tryptone. However, for us to compare data from across the SEA program, I would recommend using peptone. If you use tryptone, it will be important for you to disclose this information (i.e. that you are not usingP PYCa) when contributing data to the program and when publishing elsewhere.

Link to this post | posted 06 Sep, 2024 23:42

viknesh

serivas
Hello all,

Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar?

It is likley that you'll be able to culture the bacteria we use for phage-hunting just as well using peptone or tryptone. However, for us to compare data from across the SEA program, I would recommend using peptone. If you use tryptone, it will be important for you to disclose this information (i.e. that you are not usingP PYCa) when contributing data to the program and when publishing elsewhere.

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 03 Sep, 2024 13:51
viknesh	engstrom Hi Folks, However, what Eric Engstrom should have written would be "we switched from Fisher Brand Peptone to Bacto Peptone." I should read my notes before posting anything. We did indeed try the agar switch first, but as I review this, that does not appear to have been the fix. The subsequent shift to Bacto Peptone is what saved our bacon. Heartfelt apologies for disseminating inaccurate information. I was traveling and relying on my memory, rather than my notebook. Never a good idea. Eric No apologies necessary, Eric. This is why this forum can be so valuable – thanks for so quickly responding to this post. We'll try to find additional ways to disseminate this info. Vic

Link to this post | posted 03 Sep, 2024 13:51

viknesh

engstrom
Hi Folks,

However, what Eric Engstrom should have written would be "we switched from Fisher Brand Peptone to Bacto Peptone." I should read my notes before posting anything. We did indeed try the agar switch first, but as I review this, that does not appear to have been the fix. The subsequent shift to Bacto Peptone is what saved our bacon.

Heartfelt apologies for disseminating inaccurate information. I was traveling and relying on my memory, rather than my notebook. Never a good idea.

Eric

No apologies necessary, Eric. This is why this forum can be so valuable – thanks for so quickly responding to this post. We'll try to find additional ways to disseminate this info.

Vic

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 03 Sep, 2024 11:46
viknesh	Hi all, Eric Engstrom recently shared the following: "Our problems finally went away when we switched from Fisher Brand Agar to Bacto Agar. With Bacto Agar, we did not need to drop the CaCl2 level and we could return to using our regular DI water. The stuff is expensive, so we have a container of Bacto Agar labeled "Top-agar Only" and we still use the Fisher Agar with no issue for our plate media.

Link to this post | posted 03 Sep, 2024 11:46

viknesh

Hi all,

Eric Engstrom recently shared the following:

"Our problems finally went away when we switched from Fisher Brand Agar to Bacto Agar. With Bacto Agar, we did not need to drop the CaCl2 level and we could return to using our regular DI water. The stuff is expensive, so we have a container of Bacto Agar labeled "Top-agar Only" and we still use the Fisher Agar with no issue for our plate media.

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 20 Aug, 2024 01:37
viknesh	Ah, I didnt see the image you attached. Very odd looking. Is each row a dilution series for a different phage? And is this on the isolation host or a different bacterium you are testing for host range? Do you have a photo of the second plate?

Posted in: Phage Discovery/Isolation → Plaques appear on spot test, don't appear in titer

Link to this post \| posted 20 Aug, 2024 01:04
viknesh	JustinA Hey, Ryan. Did you ever figure this out? We're seeing "spots" forming when we do spot titers and host-preference assays, but then no real plaques forming. This actually seems to be a random issue on our plates, where we have a clear spot in our host lawns that don't yield any plaques when picked. But we're also seeing it very clearly on spot titers, where the spots clear, but there definitely are not plaques. It seems to be less bad when we use PYCa for dilutions, but still obvious. Attached is a spot titer plate showing the spots we get. Thanks! Justin Hi Justin and Ryan, One explanation for what you are observing is called "lysis from without". This happens when phages that are not able to successfully infect and replicate in particular bacteria are nevertheless still able to lyse those bacterial cells when they are applied to cells at high concentrations. Such lysis is not the result of replication and lysis from within the cells but instead is happeneing from the outside of cells, hence the term "lysis from without". Such lysis is likely due to the mass action of lots of hydrolase enzymes that phages typically have as part of their tails and that are used to locally depolymerase the cell wall in order to deliver the phage genome into the cell. At some point, there is too much of these "local" depolymerization activity that the cells are overwhelmed and lyse as a result. Because lysis from without only happens at high concentrations, you will not see lysis when the phage sample is diluted. Because lysis from without does not involve phage replication, you will not see individuals plaques form. When performing host-range assays, it is typical to observe clearing of spots at high phage concentrations but no individual plaques at the lower concentrations. Such results are regularly interpreted as phage not being able to infect that particular host. I hope this helps explain some of your observations.

Link to this post | posted 20 Aug, 2024 01:04

viknesh

JustinA
Hey, Ryan.

Did you ever figure this out? We're seeing "spots" forming when we do spot titers and host-preference assays, but then no real plaques forming. This actually seems to be a random issue on our plates, where we have a clear spot in our host lawns that don't yield any plaques when picked. But we're also seeing it very clearly on spot titers, where the spots clear, but there definitely are not plaques. It seems to be less bad when we use PYCa for dilutions, but still obvious.

Attached is a spot titer plate showing the spots we get.

Thanks!
Justin

Hi Justin and Ryan,

One explanation for what you are observing is called "lysis from without". This happens when phages that are not able to successfully infect and replicate in particular bacteria are nevertheless still able to lyse those bacterial cells when they are applied to cells at high concentrations. Such lysis is not the result of replication and lysis from within the cells but instead is happeneing from the outside of cells, hence the term "lysis from without". Such lysis is likely due to the mass action of lots of hydrolase enzymes that phages typically have as part of their tails and that are used to locally depolymerase the cell wall in order to deliver the phage genome into the cell. At some point, there is too much of these "local" depolymerization activity that the cells are overwhelmed and lyse as a result.

Because lysis from without only happens at high concentrations, you will not see lysis when the phage sample is diluted. Because lysis from without does not involve phage replication, you will not see individuals plaques form.

When performing host-range assays, it is typical to observe clearing of spots at high phage concentrations but no individual plaques at the lower concentrations. Such results are regularly interpreted as phage not being able to infect that particular host.

I hope this helps explain some of your observations.

Posted in: Phage Discovery/Isolation → Plaques appear on spot test, don't appear in titer

Link to this post \| posted 04 Jun, 2024 01:12
viknesh	This is the first time I'm seeing this request. I would leave it at the Class level, and include the morphology.

Posted in: General Message Board → Classificiation with ICTV guidelines

Link to this post \| posted 03 Apr, 2024 12:04
viknesh	Applications for BioInteractive’s Ambassador Academy are now open! We’re looking for educators with diverse backgrounds and teaching contexts to apply to this multi-year professional development program to join our Ambassador Community. Applications consist of two sections: completion of eligibility questions and completion of an application package. To apply, educators must complete the eligibility questions by May 7, 2024. The remainder of the application package is due by September 12, 2024. More about the Academy, including eligibility criteria, benefits of participation, and a link to the application, can be found on the Academy webpage. Edited 03 Apr, 2024 12:06

Link to this post | posted 03 Apr, 2024 12:04

viknesh

Applications for BioInteractive’s Ambassador Academy are now open! We’re looking for educators with diverse backgrounds and teaching contexts to apply to this multi-year professional development program to join our Ambassador Community.

Applications consist of two sections: completion of eligibility questions and completion of an application package. To apply, educators must complete the eligibility questions by May 7, 2024. The remainder of the application package is due by September 12, 2024.

More about the Academy, including eligibility criteria, benefits of participation, and a link to the application, can be found on the Academy webpage.

Edited 03 Apr, 2024 12:06

Posted in: General Message Board → BioInteractive Ambassador Eligiblity is Open until May 7, 2024. Apply Now

Link to this post \| posted 31 Mar, 2024 02:59
viknesh	tperezmo Hi all, I recently switched one section to work on A. atrocyaneus while the others work on A. globiformis. For A. globiformis, we always have really good DNA yields 40 - 100 ng per ul. However, for A. atrocyaneus, we are not hitting even 20 ng / ul. I have used the original protocol with nucleases, SDS, EDTA, Proteinase K. The results are for all students in the class, for 2 semesters now. When trying to do the restriction digests, we can barely see a band in the controls. I have looked at the Microbacterium forum but there it seems it's a lysate concentrations. All my students have at least 1 x 10^8 or more. Any thoughts? Thank you! Hi Tiara, I don't have experience working with A. atrocyaneus, but a couple of things. 1. Have you considered using ZnCl2 precicptation? It seems to do the trick for folks with low yield. Also, if your titers are generally around 10^8, you might want to see if your titer is the issue. Do you still get low DNA yield from a lysate with a titer of 5 x 10^9 pfu/ml? 2. A quick search on phagesDB shows that Pitt and UCLA have submitted DNA for a few phages isolated on this host last year. I'd recommend pinging SEA faculty Kristen Butela and Amanda Friese for input (and have the respond on the forum so we capture it here for others too). Thanks. Vic

Link to this post | posted 31 Mar, 2024 02:59

viknesh

tperezmo
Hi all,

I recently switched one section to work on A. atrocyaneus while the others work on A. globiformis. For A. globiformis, we always have really good DNA yields 40 - 100 ng per ul. However, for A. atrocyaneus, we are not hitting even 20 ng / ul. I have used the original protocol with nucleases, SDS, EDTA, Proteinase K. The results are for all students in the class, for 2 semesters now.
When trying to do the restriction digests, we can barely see a band in the controls.
I have looked at the Microbacterium forum but there it seems it's a lysate concentrations. All my students have at least 1 x 10^8 or more.
Any thoughts? Thank you!

Hi Tiara,

I don't have experience working with A. atrocyaneus, but a couple of things.

1. Have you considered using ZnCl2 precicptation? It seems to do the trick for folks with low yield. Also, if your titers are generally around 10^8, you might want to see if your titer is the issue. Do you still get low DNA yield from a lysate with a titer of 5 x 10^9 pfu/ml?

2. A quick search on phagesDB shows that Pitt and UCLA have submitted DNA for a few phages isolated on this host last year. I'd recommend pinging SEA faculty Kristen Butela and Amanda Friese for input (and have the respond on the forum so we capture it here for others too).

Thanks.
Vic

Posted in: Phage Discovery/Isolation → A atrocyaneus DNA extraction

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