SEA-PHAGES | Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 05 Oct, 2023 14:52
engstrom	Hi Community, We have been stopped in our tracks here at Monmouth, by a very strange issue. Our PYCa top-agar is useless as our CaCl2 is consistently precipitating. We made a new 1M stock of course. We made new dextrose as well. We played with our autoclaving times. And we are not having any trouble with plate agar. At a loss. My question–can we use top-agar for plaque assays if we omit the CaCl2? This would seem to be the quickest route to solving our problem and we will make a test of it soon. Other suggestions? Thanks!

Link to this post | posted 05 Oct, 2023 14:52

Hi Community,

We have been stopped in our tracks here at Monmouth, by a very strange issue. Our PYCa top-agar is useless as our CaCl2 is consistently precipitating. We made a new 1M stock of course. We made new dextrose as well. We played with our autoclaving times. And we are not having any trouble with plate agar. At a loss. My question–can we use top-agar for plaque assays if we omit the CaCl2? This would seem to be the quickest route to solving our problem and we will make a test of it soon. Other suggestions?

Thanks!

Link to this post \| posted 05 Oct, 2023 15:16
debbie	Eric, Yep. Calcium does precipitate out of solution at high temps. You can omit Ca in the autoclaved recipe and then add it back as you use the agar. You can also play with the concentrations. What you can't do is omit calcium. Many phages need calcium for adsorption, infection and stability. Make sense? debbie

Link to this post \| posted 05 Oct, 2023 15:42
viknesh	engstrom Hi Community, We have been stopped in our tracks here at Monmouth, by a very strange issue. Our PYCa top-agar is useless as our CaCl2 is consistently precipitating. We made a new 1M stock of course. We made new dextrose as well. We played with our autoclaving times. And we are not having any trouble with plate agar. At a loss. My question–can we use top-agar for plaque assays if we omit the CaCl2? This would seem to be the quickest route to solving our problem and we will make a test of it soon. Other suggestions? Thanks! Eric, thanks for sharing. We too were stumped this year with calcium precipitating in our PYCa top agar. It is incredibly frustrating – so sorry you too are experiencing this. I believe I heard from one other person about this too. I wonder if it has to do with more recent batches of media components. I'm not sure what the best way forward is, but one thing we've found that minimizes the likelihood of precipitation is to first autoclave the media without calcium chloride (and we all typically do), then allow it too cool all the way do about 60C (before the top agar begins to set), then add the calcium chloride (and in our case, dextrose too), swirl, and place it in the water bath. Allowing it to solidify and melting it in the microwave seems to promote the precipitation. Out of 20 x 100 ml bottles of top agar prepared as a single batch, all at once, we find that a majority will not crash for at least 2 - 3 days. We have noticed that in those that do not crash, we see flaky crystals forming, presumably calcium precipitating in a more ordered fashion. The randomness of this phenomenon across the aliquots suggests that we're at some tipping point, and that very slight variations can trigger precipitation. I suspect pH can have done nothing to verify this. We'll see what we can learn. in the meanwhile, good luck and keep us up to date. Thanks. Edited 05 Oct, 2023 15:46

Link to this post | posted 05 Oct, 2023 15:42

viknesh

engstrom
Hi Community,

We have been stopped in our tracks here at Monmouth, by a very strange issue. Our PYCa top-agar is useless as our CaCl2 is consistently precipitating. We made a new 1M stock of course. We made new dextrose as well. We played with our autoclaving times. And we are not having any trouble with plate agar. At a loss. My question–can we use top-agar for plaque assays if we omit the CaCl2? This would seem to be the quickest route to solving our problem and we will make a test of it soon. Other suggestions?

Thanks!

Eric, thanks for sharing. We too were stumped this year with calcium precipitating in our PYCa top agar. It is incredibly frustrating – so sorry you too are experiencing this. I believe I heard from one other person about this too. I wonder if it has to do with more recent batches of media components. I'm not sure what the best way forward is, but one thing we've found that minimizes the likelihood of precipitation is to first autoclave the media without calcium chloride (and we all typically do), then allow it too cool all the way do about 60C (before the top agar begins to set), then add the calcium chloride (and in our case, dextrose too), swirl, and place it in the water bath. Allowing it to solidify and melting it in the microwave seems to promote the precipitation. Out of 20 x 100 ml bottles of top agar prepared as a single batch, all at once, we find that a majority will not crash for at least 2 - 3 days. We have noticed that in those that do not crash, we see flaky crystals forming, presumably calcium precipitating in a more ordered fashion. The randomness of this phenomenon across the aliquots suggests that we're at some tipping point, and that very slight variations can trigger precipitation. I suspect pH can have done nothing to verify this. We'll see what we can learn. in the meanwhile, good luck and keep us up to date. Thanks.

Edited 05 Oct, 2023 15:46

Link to this post \| posted 05 Oct, 2023 17:48
debbie	Hi all, I chatted with Graham about this and here's what i have gleaned from the conversation. Temperature and pH can affect how well the calcium goes into the solution. Maybe try a different source of peptone or yeast extract to see if it improves. best, debbie

Link to this post \| posted 06 Oct, 2023 15:12
engstrom	Thanks Everyone! We MAY have achieved a breakthrough yesterday by using Millipore water. Haven't run a plaque assay yet, but the appearance of the TA is encouraging. Looks like our DI system may need some maintenance. I'll report on the assays after next week. Eric

Link to this post \| posted 03 Sep, 2024 09:34
sklambet@ncsu.edu	I am also having this problem with top agar. I am going to remake this morning and let the media cool more before adding the CaCl2 as suggested. Is there any more information about this since last year? Thanks!

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.

Link to this post \| posted 03 Sep, 2024 13:33
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

Link to this post | posted 03 Sep, 2024 13:33

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

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

Link to this post \| posted 03 Sep, 2024 14:12
sklambet@ncsu.edu	Thank you all so much!!! This is super helpful. Also no apologies needed this response was surprising fast. Stephanie Edited 03 Sep, 2024 14:12

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Precipitation of CaCl2 in PYCA top-agar