SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 05 Feb, 2021 01:34
viknesh	cheryl.brown@mnstate.edu I have another PYCa media question. Does the type of peptone you get affect the media? We have been using VWR 97064-330, but there is a non-animal, defatted soy peptone (97064-186) for less than half the price. Has anyone used this? Does the source of the proteins matter in this application? Hi Cheryl, This is a little tricky to answer. While peptones are generally composed of the same components (amino acids, peptides, carbs, salts, vitamins), the ratio of the components and pH of the peptone will vary based on the source material and the process used to generate the peptone. These diffrences have been shown to dramatically impact growth and the behaviour of some cell lines and bacteria but not others, so most people tend to stick with what they know to work. I'm hoping the Hatfull lab will chime in here, but I for one use the recommended peptone because I've not tested any other sources of peptones. If you are keen to give the soy-based peptone a try, then I would advise purchasing a small batch to do some growth comparisons. If you do, I would love to know what you discover. Vic Edited 05 Feb, 2021 01:34

Link to this post | posted 05 Feb, 2021 01:34

cheryl.brown@mnstate.edu
I have another PYCa media question. Does the type of peptone you get affect the media? We have been using VWR 97064-330, but there is a non-animal, defatted soy peptone (97064-186) for less than half the price. Has anyone used this?

Does the source of the proteins matter in this application?

Hi Cheryl,

This is a little tricky to answer. While peptones are generally composed of the same components (amino acids, peptides, carbs, salts, vitamins), the ratio of the components and pH of the peptone will vary based on the source material and the process used to generate the peptone. These diffrences have been shown to dramatically impact growth and the behaviour of some cell lines and bacteria but not others, so most people tend to stick with what they know to work. I'm hoping the Hatfull lab will chime in here, but I for one use the recommended peptone because I've not tested any other sources of peptones.

If you are keen to give the soy-based peptone a try, then I would advise purchasing a small batch to do some growth comparisons. If you do, I would love to know what you discover.

Vic

Edited 05 Feb, 2021 01:34

Posted in: Phage Discovery/Isolation → Peptone question

Link to this post \| posted 22 Jan, 2021 21:03
viknesh	cheryl.brown@mnstate.edu We have had some issues with our PYCa agar getting dark while being held at 45-50 C. I am trying to figure out if it could be one of the ingredients, as we did buy new yeast extract and peptone, or if this is normal and harmless. Hi Cheryl, At high temperatures, dextrose and amino acids can undergo the Maillard reaction, which results in a darkening of the media. The products of the Maillard reaction impacts the growth of some bacteria more than others. It may be fine for you to use the darkened media, but it may impact reproducibility of bacterial growth on your plates. The way to reduce the Maillard reaction is to keep dextrose and amino acids in as cool a solution as possible. This means waiting for freshly autoclaved/molten PYCa agar to cool to ~ 60C before adding dextrose, minimizing the number of time you reheat (e.g. in the microwave) PYCa agar once dextrose has been added, and not keeping molten dextrose in the warm bath for extended periods of time. In our lab, we add dextrose to molten PYCa agar at ~ 60C, then allow the media to solidify for storage. When we want to pour plates, we melt the solidified agar in the microwave, allow it to cool to 60C before adding supplements like cycloheximide, then pour the plates. We don't keep PYCa agar in the molten state for long. I hope this is helpful.

Link to this post | posted 22 Jan, 2021 21:03

viknesh

cheryl.brown@mnstate.edu
We have had some issues with our PYCa agar getting dark while being held at 45-50 C. I am trying to figure out if it could be one of the ingredients, as we did buy new yeast extract and peptone, or if this is normal and harmless.

Hi Cheryl,

At high temperatures, dextrose and amino acids can undergo the Maillard reaction, which results in a darkening of the media. The products of the Maillard reaction impacts the growth of some bacteria more than others. It may be fine for you to use the darkened media, but it may impact reproducibility of bacterial growth on your plates.

The way to reduce the Maillard reaction is to keep dextrose and amino acids in as cool a solution as possible. This means waiting for freshly autoclaved/molten PYCa agar to cool to ~ 60C before adding dextrose, minimizing the number of time you reheat (e.g. in the microwave) PYCa agar once dextrose has been added, and not keeping molten dextrose in the warm bath for extended periods of time.

In our lab, we add dextrose to molten PYCa agar at ~ 60C, then allow the media to solidify for storage. When we want to pour plates, we melt the solidified agar in the microwave, allow it to cool to 60C before adding supplements like cycloheximide, then pour the plates. We don't keep PYCa agar in the molten state for long.

I hope this is helpful.

Posted in: Phage Discovery/Isolation → PYCa media turning dark

Link to this post \| posted 10 Dec, 2020 18:23
viknesh	Hi Maria We have imaged phage using the TEM mode on an SEM. The quality and magnification are understandably lower. I've attached one of the better micrographs, and you can see that a lot of the usual detail we can visualize with TEM is lost. 1.64Mb

Posted in: Phage Discovery/Isolation → Electron Microscopes

Link to this post \| posted 19 Nov, 2020 18:14
viknesh	erin.windsor The only enzyme that cuts well for us in Gordonia rubripertincta is NspI. See attached. Thanks for sharing this, Erin. We too have found that NspI reliably cuts DNA of phages isolated on G. rubripertincta. We've also found that BamHI, HindIII, and SacII also cut a few of the G. rubripertincta phages we've isolated. Your results will defintiely help us get a better grasp of which enzymes are best to recommend moving forward. Vic

Link to this post | posted 19 Nov, 2020 18:14

viknesh

erin.windsor
The only enzyme that cuts well for us in Gordonia rubripertincta is NspI. See attached.

Thanks for sharing this, Erin. We too have found that NspI reliably cuts DNA of phages isolated on G. rubripertincta. We've also found that BamHI, HindIII, and SacII also cut a few of the G. rubripertincta phages we've isolated. Your results will defintiely help us get a better grasp of which enzymes are best to recommend moving forward.

Vic

Posted in: Gordonia → Gordonia DNA not cutting

Link to this post \| posted 19 Nov, 2020 16:58
viknesh	Hi Amy, Would you mind sharing a little bit more information about what you're using and seeing? For example: What restriction enzymes are you using? Did you also include DNA for your control phage? If so, what phage is it? Chidiebere? Could you share a gel image of what you are seeing? Thanks. Vic asprenkle Anyone have any trouble getting a good RE digest of Gordonia DNA? I've got good DNA from M. foliorum, and we're trying Gordonia rubripertincta for the first time this year. I've noticed it's a bit waxier than M. foliorum. I'm getting OK DNA concentrations with a new Wizard kit, but my Mf DNA cuts where my Gr does not. Any tips/tricks I'm missing? Thanks!

Link to this post | posted 19 Nov, 2020 16:58

viknesh

Hi Amy,

Would you mind sharing a little bit more information about what you're using and seeing? For example:

What restriction enzymes are you using? Did you also include DNA for your control phage? If so, what phage is it? Chidiebere? Could you share a gel image of what you are seeing?
Thanks.
Vic

asprenkle
Anyone have any trouble getting a good RE digest of Gordonia DNA? I've got good DNA from M. foliorum, and we're trying Gordonia rubripertincta for the first time this year. I've noticed it's a bit waxier than M. foliorum. I'm getting OK DNA concentrations with a new Wizard kit, but my Mf DNA cuts where my Gr does not. Any tips/tricks I'm missing?
Thanks!

Posted in: Gordonia → Gordonia DNA not cutting

Link to this post \| posted 21 Feb, 2020 20:51
viknesh	Hi Sarah, It's likely that the liquid culture was contaminated. As such, your lawn is composed of more than one bacterium - M. foliorum and other bacteria. Phages will still be able to infect M. foliorum, but plaques may not be apparent because contaminating bacteria, which cannot be infected by those same phages) are able to continue to grow evenly on the plate and therefore also within the plaque. The weird smell is likely due to the contaminant. Hope this helps. Vic Thiel Hello, I think that we are having a similar problem to the post above that says "D29 plaques barely visible on M. smeg" but I'm not sure. We are doing the phage isolation for the 2nd semester (so I'm not very experienced) and the students were on their 2nd round of purifications and starting to do the 3rd round and then make webbed plates. Our host is M. foliorum. I take new M. foliorum out of the -80C every 2 weeks, streak a plate and then shake it to make new stocks for them to use. The entire class started getting very similar looking plaques, that were difficult to see and smelled weird. (See image). The plates labeled "old" bacteria is what we had been using. The plates labeled "new" bacteria was when we picked a new colony from the same streak plate and it starting giving us the odd results. The difference in the incubation times was because we thought if we gave the 10^-3 plate a little more time- it would fully lyse, but it didn't. We picked a another new colony from the M. foliorum streak plate, grew it up and it seems to have fixed the problem (no pic). Just in case, I made a new M. foliorum streak plate from a different glycerol stock and will be growing that up for future use for the students. So we might have fixed the problem, but what do you think happened? Did we get lucky and pick a mutant colony? Any insight would be awesome. I'll let you know if the problem is not fixed.

Link to this post | posted 21 Feb, 2020 20:51

viknesh

Hi Sarah,

It's likely that the liquid culture was contaminated. As such, your lawn is composed of more than one bacterium - M. foliorum and other bacteria. Phages will still be able to infect M. foliorum, but plaques may not be apparent because contaminating bacteria, which cannot be infected by those same phages) are able to continue to grow evenly on the plate and therefore also within the plaque. The weird smell is likely due to the contaminant.

Hope this helps.

Vic

Thiel
Hello,
I think that we are having a similar problem to the post above that says "D29 plaques barely visible on M. smeg" but I'm not sure.
We are doing the phage isolation for the 2nd semester (so I'm not very experienced) and the students were on their 2nd round of purifications and starting to do the 3rd round and then make webbed plates. Our host is M. foliorum. I take new M. foliorum out of the -80C every 2 weeks, streak a plate and then shake it to make new stocks for them to use. The entire class started getting very similar looking plaques, that were difficult to see and smelled weird. (See image). The plates labeled "old" bacteria is what we had been using. The plates labeled "new" bacteria was when we picked a new colony from the same streak plate and it starting giving us the odd results. The difference in the incubation times was because we thought if we gave the 10^-3 plate a little more time- it would fully lyse, but it didn't.

We picked a another new colony from the M. foliorum streak plate, grew it up and it seems to have fixed the problem (no pic). Just in case, I made a new M. foliorum streak plate from a different glycerol stock and will be growing that up for future use for the students.

So we might have fixed the problem, but what do you think happened? Did we get lucky and pick a mutant colony? Any insight would be awesome. I'll let you know if the problem is not fixed.

Posted in: Phage Discovery/Isolation → Phages suddenly not fully lysing on M. foliorum

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

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

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?

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