SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 15 Oct, 2019 15:22
viknesh	UNHM Hello, We are having the exact same problem right now as you describe cellokiwi with A. globiformis. After incredible success in the first few several weeks with several of our students reaching high titer and others just needing to titer their likely high titer lysates, we have been met with a standstill for the last week and half unable to get an adequate lawn plate from our bacterial liquid culture(s). Most of our attempts at lawn plates have either given us NO lawn at all (after 24 hrs, our typical incubation at 30 degrees) or a very inconsistent/spotty lawn. We have restreaked from several glycerol stocks onto new PyCa plates and remade our liquid PyCa media and top agar. Any other thoughts would help as well for us in order to get our last students through the last steps and to the DNA extraction phase. Thank you, Kristen Kristen, Can you conform that you are starting from a single colony? Also, can I have you streak a plate your glycerol stock as well as from the liquid culture (which is misbehaving) and share with us what those streak plates looks like after 3 days of incubation at 30C? Vic

Link to this post | posted 15 Oct, 2019 15:22

UNHM
Hello,
We are having the exact same problem right now as you describe cellokiwi with A. globiformis.
After incredible success in the first few several weeks with several of our students reaching high titer and others just needing to titer their likely high titer lysates, we have been met with a standstill for the last week and half unable to get an adequate lawn plate from our bacterial liquid culture(s).
Most of our attempts at lawn plates have either given us NO lawn at all (after 24 hrs, our typical incubation at 30 degrees) or a very inconsistent/spotty lawn.
We have restreaked from several glycerol stocks onto new PyCa plates and remade our liquid PyCa media and top agar.
Any other thoughts would help as well for us in order to get our last students through the last steps and to the DNA extraction phase.
Thank you,
Kristen

Kristen,

Can you conform that you are starting from a single colony? Also, can I have you streak a plate your glycerol stock as well as from the liquid culture (which is misbehaving) and share with us what those streak plates looks like after 3 days of incubation at 30C?

Vic

Posted in: Arthrobacter → Success followed by failure?

Link to this post \| posted 11 Oct, 2019 17:36
viknesh	cellokiwi Streak Plate I asked for a picture of the streak plate because we've seen a contaminant that looks very much like A. globiformis colonies, except that they are more "white and dry" in appearance.We've accidentally picked these contaminants before, and as you can expect, all downstream experiments with a culture of the contaminant did not behave as expected. I noticed that there's quite a bit of condensation on your streak plate. Are you preparing cultures from colonies that are > 2 weeks old and have been stored in the fridge? If so, perhaps that is a source of variability. This might also explain what you're observing with your plaque assay plates in the fridge. It might be that the majority of your Arthrobacter lawn is dying with prolonged incubation in the fridge. You may want to streak plates weekly, and setup cultures from freshly formed colonies, which have never been stored in the fridge. Hope this is helpful. Vic Edited 11 Oct, 2019 17:37

Link to this post | posted 11 Oct, 2019 17:36

viknesh

cellokiwi
Streak Plate

I asked for a picture of the streak plate because we've seen a contaminant that looks very much like A. globiformis colonies, except that they are more "white and dry" in appearance.We've accidentally picked these contaminants before, and as you can expect, all downstream experiments with a culture of the contaminant did not behave as expected.

I noticed that there's quite a bit of condensation on your streak plate. Are you preparing cultures from colonies that are > 2 weeks old and have been stored in the fridge? If so, perhaps that is a source of variability. This might also explain what you're observing with your plaque assay plates in the fridge. It might be that the majority of your Arthrobacter lawn is dying with prolonged incubation in the fridge.

You may want to streak plates weekly, and setup cultures from freshly formed colonies, which have never been stored in the fridge.

Hope this is helpful.

Vic

Edited 11 Oct, 2019 17:37

Posted in: Arthrobacter → Success followed by failure?

Link to this post \| posted 09 Oct, 2019 01:43
viknesh	cellokiwi Hello Hive Mind, We are having a rather weird problem that has persisted for a few weeks. We started the semester with Arthrobacter growing well. It formed beautiful lawns, we had the majority of students with plaques, some progressing as far as flooding a webbed plate. They just needed to start titering. Then things suddenly stopped working. We can't get a nice lawn anymore to save our lives. We have tried refreshing the LB plates, the top agar, the CaCl2, all of it. We went back to the first streak plate (that worked initially), didn't work. Went back to the sample sent from Pitt, didn't work. I'm running out of ideas as to why something that worked beautifully before is suddenly crashing and burning. Weird thing #2: Some students that put their plates with plaques in the refrigerator (wrapped in parafilm) went back to check them and the lawn was disintegrated to the point that you could no longer see the plaques. What is UP with this organism? Any and all tips/help is GREATLY appreciated! Could I have you attach/post some photos of your streak plates and perhaps the odd lawn form the fridge? Vic

Link to this post | posted 09 Oct, 2019 01:43

viknesh

cellokiwi
Hello Hive Mind,
We are having a rather weird problem that has persisted for a few weeks. We started the semester with Arthrobacter growing well. It formed beautiful lawns, we had the majority of students with plaques, some progressing as far as flooding a webbed plate. They just needed to start titering. Then things suddenly stopped working. We can't get a nice lawn anymore to save our lives. We have tried refreshing the LB plates, the top agar, the CaCl2, all of it. We went back to the first streak plate (that worked initially), didn't work. Went back to the sample sent from Pitt, didn't work. I'm running out of ideas as to why something that worked beautifully before is suddenly crashing and burning. Weird thing #2: Some students that put their plates with plaques in the refrigerator (wrapped in parafilm) went back to check them and the lawn was disintegrated to the point that you could no longer see the plaques. What is UP with this organism? Any and all tips/help is GREATLY appreciated!

Could I have you attach/post some photos of your streak plates and perhaps the odd lawn form the fridge?

Vic

Posted in: Arthrobacter → Success followed by failure?

Link to this post \| posted 22 Sep, 2019 22:40
viknesh	fogartym1 Does anyone have a general protocol for growing phage in culture? We need to increase titers for a few of our phage and would love a protocol to get us started. Thanks Hi Marie, The Enriched Isolation protocol is essentially a modification for preparing liquid lysates. There's a commonly used protocol for amplifying phages of E. coli in liquid, which I provide below. Please also see the caveats, below. 1. To 1 ml of a saturated bacterial culture, add 10 ul of a phage lysate. Note: Include a control that replaces phage with just phage buffer. 2. Allow to sit on bench, undisturbed, for 10 min. 3. Add the bacteria+phage mix to 10 - 15 ml of fresh media (e.g. PYCa, or 7H9 liquid media complete, depending on the bacterial host). 4. Incubate, with shaking, for 24 - 48 hrs. 5. Spin the culture, and filter the supernatant. Then determine the titer of your lysate. Caveat: 1. Like preparing webbed plates, it is important that not all the host bacteria is lysed early. It is therefore important that you setup the experiment early in the day and check on the culture before you leave in the evening. If the culture is clear (and the control is cloudy), then you've run out of host bacteria for phage amplification. 2. Because it is hard to know how much phage to add, it is worth setting up at least one additional culture, using a 1/10 dilution of phage lysate. (The same logic for bracketing when preparing webbed plates). Hope this is helpful. Good luck.

Link to this post | posted 22 Sep, 2019 22:40

viknesh

fogartym1
Does anyone have a general protocol for growing phage in culture? We need to increase titers for a few of our phage and would love a protocol to get us started.
Thanks

Hi Marie,

The Enriched Isolation protocol is essentially a modification for preparing liquid lysates. There's a commonly used protocol for amplifying phages of E. coli in liquid, which I provide below. Please also see the caveats, below.

1. To 1 ml of a saturated bacterial culture, add 10 ul of a phage lysate.
Note: Include a control that replaces phage with just phage buffer.
2. Allow to sit on bench, undisturbed, for 10 min.
3. Add the bacteria+phage mix to 10 - 15 ml of fresh media (e.g. PYCa, or 7H9 liquid media complete, depending on the bacterial host).
4. Incubate, with shaking, for 24 - 48 hrs.
5. Spin the culture, and filter the supernatant. Then determine the titer of your lysate.

Caveat:
1. Like preparing webbed plates, it is important that not all the host bacteria is lysed early. It is therefore important that you setup the experiment early in the day and check on the culture before you leave in the evening. If the culture is clear (and the control is cloudy), then you've run out of host bacteria for phage amplification.
2. Because it is hard to know how much phage to add, it is worth setting up at least one additional culture, using a 1/10 dilution of phage lysate. (The same logic for bracketing when preparing webbed plates).

Hope this is helpful.

Good luck.

Posted in: Phage Discovery/Isolation → Protocol to grow phage in culture?

Link to this post \| posted 22 Oct, 2018 13:13
viknesh	smolloy123 Hi All, We have been growing G. terrae in PYCa broth for four days with sterile glass beads in baffled flasks. They plate like buttah! I take the glass 3-mm beads we use for archiving (a bottle is super cheap), sterilize then in culture tubes so that I can easily pour them into flasks, and I do not use Tween ( I am too lazy to take time to sub-culture). After four days of growth, the cultures are smooth and produce perfect lawns! I wash beads and re-autoclave for re-use. Just don't get these re-used beads mixed up with the beads you would use for archiving as those should not be recycled beads. I meant to post this over the summer when I discovered this but forgot! Best, Sally That's a really neat trick, Sally. Do you know if it works for M. smeg too? I'll definitely give it a try and report back.

Link to this post | posted 22 Oct, 2018 13:13

viknesh

smolloy123
Hi All,
We have been growing G. terrae in PYCa broth for four days with sterile glass beads in baffled flasks. They plate like buttah! I take the glass 3-mm beads we use for archiving (a bottle is super cheap), sterilize then in culture tubes so that I can easily pour them into flasks, and I do not use Tween ( I am too lazy to take time to sub-culture). After four days of growth, the cultures are smooth and produce perfect lawns! I wash beads and re-autoclave for re-use. Just don't get these re-used beads mixed up with the beads you would use for archiving as those should not be recycled beads. I meant to post this over the summer when I discovered this but forgot!
Best,
Sally

That's a really neat trick, Sally. Do you know if it works for M. smeg too? I'll definitely give it a try and report back.

Posted in: Gordonia → Clumping in liquid cultures

Link to this post \| posted 14 Oct, 2018 16:34
viknesh	c.sunnen Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred. Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too! Nikki I edited my post above to reflect the correct concentration (should someone else refer to it).

Link to this post | posted 14 Oct, 2018 16:34

viknesh

c.sunnen
Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

I edited my post above to reflect the correct concentration (should someone else refer to it).

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 13 Oct, 2018 02:47
viknesh	c.sunnen Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred. Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too! Nikki Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.

Link to this post | posted 13 Oct, 2018 02:47

viknesh

c.sunnen
Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 12 Oct, 2018 17:50
viknesh	c.sunnen Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK? Ah, I now see that you intend to complete the protocol by precipitating DNA instead of using the Wizard columns. You are correct - you can go ahead and resuspend in TES, then add ProtK. In the zinc chloride precipitation protocol, we use EDTA instead of TES just to make things easier for those wanting to precipitate with zinc and continue with the old protocol (that is, they do not have to make TES). However, considering that you will use both zinc and protK, and therefore EDTA and SDS, using TES makes sense. The conc. of SDS recommended for ProtK is 0.5% w/v. Perhaps you could prepare your TES to contain 0.1M EDTA and 0.5% SDS. Edited 14 Oct, 2018 16:31

Link to this post | posted 12 Oct, 2018 17:50

viknesh

c.sunnen
Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK?

Ah, I now see that you intend to complete the protocol by precipitating DNA instead of using the Wizard columns. You are correct - you can go ahead and resuspend in TES, then add ProtK. In the zinc chloride precipitation protocol, we use EDTA instead of TES just to make things easier for those wanting to precipitate with zinc and continue with the old protocol (that is, they do not have to make TES). However, considering that you will use both zinc and protK, and therefore EDTA and SDS, using TES makes sense. The conc. of SDS recommended for ProtK is 0.5% w/v. Perhaps you could prepare your TES to contain 0.1M EDTA and 0.5% SDS.

Edited 14 Oct, 2018 16:31

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 12 Oct, 2018 15:22
viknesh	c.sunnen Vic, Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful. Thanks all! I love this community! Nikki Hi Nikki, A couple of things. For M. foliorum, it is important to use ProtK and SDS to remove residual nuclease activity from DNA preps. Residual nuclease is not a problem during the prep process itself, since EDTA is present. Once eluted in water, the lack of EDTA is still not a problem since the nucleases have no access to metal cofactors required for activity. However, any downstream analysis using buffers will activate the nucleases. If you can get away with the standard DNA extraction protocol (wihtout a precipitation step), I would encourage that because we've tested that protocol (with ProtK and SDS) extensively over the summer. We have some, but significantly less, experience using ProtK with the zinc precipitation protocol. If you do decide to continue with the zinc chloride precipitation protocol, here are some things to note. With the zinc chloride protocol, EDTA has to be added after the precipitation step, since EDTA will chelate the zinc that is needed for the precipitation step. Currently, the Instructors Guide suggests the following: Add nuclease to the lysate -> precipitate with zinc chloride -> resuspend in EDTA -> add ProtK + SDS to the lysate -> continue with standard prep. One issue with this workflow is that the precipitation step results in a significant fraction of particles releasing DNA during the resuspension step. It is therefore critical that EDTA be used for resuspension, and that you work quickly to add the EDTA. Otherwise, any residual nuclease will rapidly degrade DNA that is released during pellet resuspension. In the Instructors Guide, I recommed using the "alternative" strategy for Step D1. Once resuspended in EDTA, you can then add ProtK and SDS to remove residual nuclease. Another workflow would be as follows: nuclease-treat the lysate -> add ProtK + SDS to the lysate -> precipitate with ZnCl -> resuspend in EDTA -> continue with standard prep. I've not tested this workflow. The hope here is that ProtK will remove all nuclease activity prior to precipitation, allowing you to work more slowly during the resuspension step. However, my fear here is that SDS might destabilize some fraction of phages, in a phage-dependent manner, and you'll end up losing a significant fraction of DNA to the supernatant during the precipitation step. If you have a student that has extra time and wants to help test this, let me know Vic Edited 12 Oct, 2018 15:27

Link to this post | posted 12 Oct, 2018 15:22

viknesh

c.sunnen
Vic,

Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful.

Thanks all! I love this community!
Nikki

Hi Nikki,

A couple of things. For M. foliorum, it is important to use ProtK and SDS to remove residual nuclease activity from DNA preps. Residual nuclease is not a problem during the prep process itself, since EDTA is present. Once eluted in water, the lack of EDTA is still not a problem since the nucleases have no access to metal cofactors required for activity. However, any downstream analysis using buffers will activate the nucleases.

If you can get away with the standard DNA extraction protocol (wihtout a precipitation step), I would encourage that because we've tested that protocol (with ProtK and SDS) extensively over the summer. We have some, but significantly less, experience using ProtK with the zinc precipitation protocol. If you do decide to continue with the zinc chloride precipitation protocol, here are some things to note.

With the zinc chloride protocol, EDTA has to be added after the precipitation step, since EDTA will chelate the zinc that is needed for the precipitation step.

Currently, the Instructors Guide suggests the following:
Add nuclease to the lysate -> precipitate with zinc chloride -> resuspend in EDTA -> add ProtK + SDS to the lysate -> continue with standard prep.
One issue with this workflow is that the precipitation step results in a significant fraction of particles releasing DNA during the resuspension step. It is therefore critical that EDTA be used for resuspension, and that you work quickly to add the EDTA. Otherwise, any residual nuclease will rapidly degrade DNA that is released during pellet resuspension. In the Instructors Guide, I recommed using the "alternative" strategy for Step D1. Once resuspended in EDTA, you can then add ProtK and SDS to remove residual nuclease.

Another workflow would be as follows:
nuclease-treat the lysate -> add ProtK + SDS to the lysate -> precipitate with ZnCl -> resuspend in EDTA -> continue with standard prep.
I've not tested this workflow. The hope here is that ProtK will remove all nuclease activity prior to precipitation, allowing you to work more slowly during the resuspension step. However, my fear here is that SDS might destabilize some fraction of phages, in a phage-dependent manner, and you'll end up losing a significant fraction of DNA to the supernatant during the precipitation step. If you have a student that has extra time and wants to help test this, let me know smile

Vic

Edited 12 Oct, 2018 15:27

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 10 Sep, 2018 15:39
viknesh	Hi Jake - thanks for writing. The app is down at the moment, and will likely not be back up again for a while. We suggest that you have your students record the information in their lab notebooks, and use their smartphones or computers to determine the GPS location of their collection sites. We've removed instructions for using the app from the Phage Discovery Guide. Edited 10 Sep, 2018 15:41

Posted in: SEA-PHAGES App → a work in progress

Recent Activity

All posts created by viknesh