SEA-PHAGES | All posts created by UNHM

Link to this post \| posted 30 Oct, 2019 13:28
UNHM	Sorry for the delay. Things started working again, with no explainable reason. But now they are not again! Perhaps its glassware issues? We always start with a single colony. We often just use an overnight culture of A. globiformis for our lawn plates and it was working just fine. We've recently switched to longer incubations, but have not seen a difference in lawn formation. We've been able to store the liquid culture for days/up to a week at room temperature and still use it for a lawn. But now this has not been consistent. Luckily we have good DNA preps from all but two students, and we were just going back to try to make a fresh webbed plate to get fresh lysates for doing microscopy next week. How fresh should these lysates be for microscopy? If we can't get fresh ones at the end of this week, some of ours are 1 month old.

Link to this post | posted 30 Oct, 2019 13:28

Sorry for the delay. Things started working again, with no explainable reason. But now they are not again!
Perhaps its glassware issues? We always start with a single colony. We often just use an overnight culture of A. globiformis for our lawn plates and it was working just fine. We've recently switched to longer incubations, but have not seen a difference in lawn formation. We've been able to store the liquid culture for days/up to a week at room temperature and still use it for a lawn. But now this has not been consistent.
Luckily we have good DNA preps from all but two students, and we were just going back to try to make a fresh webbed plate to get fresh lysates for doing microscopy next week.
How fresh should these lysates be for microscopy? If we can't get fresh ones at the end of this week, some of ours are 1 month old.

Posted in: Arthrobacter → Success followed by failure?

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

Posted in: Arthrobacter → Success followed by failure?

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

Posted in: Arthrobacter → Success followed by failure?

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