SEA-PHAGES | Success followed by failure?

Link to this post \| posted 09 Oct, 2019 00:56
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!

Link to this post | posted 09 Oct, 2019 00:56

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!

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

Link to this post \| posted 09 Oct, 2019 01:44
cellokiwi	I will snap a picture of the streak plate in the am and post it.

Link to this post \| posted 11 Oct, 2019 15:35
cellokiwi	Hi Vic, Sorry for the delay. Attached is a picture that has the plate the day we first looked at it next to how it appeared after maybe 2 weeks in the fridge. It is so weird. It will only let me add one attachment to the post so I will put a second with a picture of the streak plate. 2.00Mb

Link to this post \| posted 11 Oct, 2019 15:36
cellokiwi	Streak Plate 90Kb

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

Link to this post \| posted 15 Oct, 2019 13:13
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

Link to this post | posted 15 Oct, 2019 13:13

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

Link to this post \| posted 15 Oct, 2019 15:00
cellokiwi	We finally had success again by doing 2 things. We let the culture get older in the shaking flask before plating and we doubled the amount of bacteria (from 250 uL to 500uL) when we plated. I still don't know what to do about the fridge plates degrading. Letting them sit at room temp just makes this microbiologist get a nervous tick… lol. Good luck! 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

Link to this post | posted 15 Oct, 2019 15:00

cellokiwi

We finally had success again by doing 2 things. We let the culture get older in the shaking flask before plating and we doubled the amount of bacteria (from 250 uL to 500uL) when we plated. I still don't know what to do about the fridge plates degrading. Letting them sit at room temp just makes this microbiologist get a nervous tick… lol. Good luck!

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

Link to this post \| posted 15 Oct, 2019 15:05
UNHM	Thank you for the advice. I let the culture grow over the weekend this time, and it did not make a difference. However, we will keep trying. I am going to change the incubation temp for all steps to 26 degrees to see if this makes a difference. The liquid culture looks nice and turbid, so I am not certain that the issue is that. I will also try to increase the volume of bacteria we use to create the lawn. To date, we have been using about 300 ul. Glad to hear that you have found success! cellokiwi We finally had success again by doing 2 things. We let the culture get older in the shaking flask before plating and we doubled the amount of bacteria (from 250 uL to 500uL) when we plated. I still don't know what to do about the fridge plates degrading. Letting them sit at room temp just makes this microbiologist get a nervous tick… lol. Good luck! 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

Link to this post | posted 15 Oct, 2019 15:05

UNHM

Thank you for the advice. I let the culture grow over the weekend this time, and it did not make a difference. However, we will keep trying. I am going to change the incubation temp for all steps to 26 degrees to see if this makes a difference. The liquid culture looks nice and turbid, so I am not certain that the issue is that. I will also try to increase the volume of bacteria we use to create the lawn. To date, we have been using about 300 ul.
Glad to hear that you have found success!

cellokiwi
We finally had success again by doing 2 things. We let the culture get older in the shaking flask before plating and we doubled the amount of bacteria (from 250 uL to 500uL) when we plated. I still don't know what to do about the fridge plates degrading. Letting them sit at room temp just makes this microbiologist get a nervous tick… lol. Good luck!
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

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

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Success followed by failure?