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How can I avoid precipitation when adding 1 M CaCl2 solution to TSB?
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+ Calcium phosphates
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Posted by
Keith Bronstrup
How can I avoid precipitation when adding 1 M CaCl2 solution to TSB?
Thanks for your answer.
Unfortunately, even when I only add 0.5 mL instead of 10 mL of 1 M CaCl2 I can see precipitation forming. Immediately after a drop of CaCl2 solution touches the medium it looks like “fog” or “dust” forming…
The reason why I would like to use 5 – 10 mM CaCl2 in the medium is because it is written in the phage protocol I have received (it says recommended medium of host + 5–10 mM CaCl2; for my host bacterium TSB is recommended). They say the phage needs CaCl2 to attach to the bacteria cells. Also, I have seen several papers / protocols that used TSB / TSA supplemented with 1 – 10 mM CaCl2, but nothing special was mentioned how to prepare it... So, I’d assume it must have somehow worked for them?
e.g.
https://www.researchgate.net/publication/23642155_Enumeration_of_Bacteriophages_by_Double_Agar_Overlay_Plaque_Assay/stats
Unfortunately, even when I only add 0.5 mL instead of 10 mL of 1 M CaCl2 I can see precipitation forming. Immediately after a drop of CaCl2 solution touches the medium it looks like “fog” or “dust” forming… The reason why I would like to use 5 – 10 mM CaCl2 in the medium is because it is written in the phage protocol I have received (it says recommended medium of host + 5–10 mM CaCl2; for my host bacterium TSB is recommended). They say the phage needs CaCl2 to attach to the bacteria cells. Also, I have seen several papers / protocols that used TSB / TSA supplemented with 1 – 10 mM CaCl2, but nothing special was mentioned how to prepare it... So, I’d assume it must have somehow worked for them? e.g.https://www.researchgate.net/publication/23642155_Enumeration_of_Bacteriophages_by_Double_Agar_Overlay_Plaque_Assay/stats
Dear Sandra, The formation of a "fog" of precipitate is probably due to the fact that you create a local sursaturation of calcium phosphate. I would suggest that you try slowly adding CaCl2 from a 100 mM solution instead of 1 M while mixing. Besides, if the final concentrations of Ca2+ and PO4 are such that their product remains below the solubility product, the precipitate should eventually redissolve.
Dear Sandra, The formation of a "fog" of precipitate is probably due to the fact that you create a local sursaturation of calcium phosphate. I would suggest that you try slowly adding CaCl2 from a 100 mM solution instead of 1 M while mixing. Besides, if the final concentrations of Ca2+ and PO4 are such that their product remains below the solubility product, the precipitate should eventually redissolve.
Sandra, You might consider a different pH buffer as suggested by Pierre. To assist your choices you could use a friendly chemical speciation program such as GEOCHEM-EZ, Paul.
Sandra, You might consider a different pH buffer as suggested by Pierre. To assist your choices you could use a friendly chemical speciation program such as GEOCHEM-EZ, Paul.
You are correct in identifying the precipitate as calcium phosphate. There is no way to avoid precipitation if you insist in adding such a high concentration of calcium (10 mM final) to your medium (I assume that you wish to keep the phosphate as a buffer). But I strongly doubt that it is necessary. Thus I would advise to reduce the concentration of Ca 10-fold. Most media recipes (e.g. M9) mention Ca concenrations in the range 0.1 to 0.5 mM.
You are correct in identifying the precipitate as calcium phosphate. There is no way to avoid precipitation if you insist in adding such a high concentration of calcium (10 mM final) to your medium (I assume that you wish to keep the phosphate as a buffer). But I strongly doubt that it is necessary. Thus I would advise to reduce the concentration of Ca 10-fold. Most media recipes (e.g. M9) mention Ca concenrations in the range 0.1 to 0.5 mM.
Thanks for your answer.
Unfortunately, even when I only add 0.5 mL instead of 10 mL of 1 M CaCl2 I can see precipitation forming. Immediately after a drop of CaCl2 solution touches the medium it looks like “fog” or “dust” forming…
The reason why I would like to use 5 – 10 mM CaCl2 in the medium is because it is written in the phage protocol I have received (it says recommended medium of host + 5–10 mM CaCl2; for my host bacterium TSB is recommended). They say the phage needs CaCl2 to attach to the bacteria cells. Also, I have seen several papers / protocols that used TSB / TSA supplemented with 1 – 10 mM CaCl2, but nothing special was mentioned how to prepare it... So, I’d assume it must have somehow worked for them? e.g. https://www.researchgate.net/publication/23642155_Enumeration_of_Bacteriophages_by_Double_Agar_Overlay_Plaque_Assay/stats
https://iai.asm.org/content/iai/48/3/782.full.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711990/
https://www.atcc.org/~/media/0871A5EC67CF499B81EE09DDE1906E3F.ashx
Thanks for your answer.
Unfortunately, even when I only add 0.5 mL instead of 10 mL of 1 M CaCl2 I can see precipitation forming. Immediately after a drop of CaCl2 solution touches the medium it looks like “fog” or “dust” forming…
The reason why I would like to use 5 – 10 mM CaCl2 in the medium is because it is written in the phage protocol I have received (it says recommended medium of host + 5–10 mM CaCl2; for my host bacterium TSB is recommended). They say the phage needs CaCl2 to attach to the bacteria cells. Also, I have seen several papers / protocols that used TSB / TSA supplemented with 1 – 10 mM CaCl2, but nothing special was mentioned how to prepare it... So, I’d assume it must have somehow worked for them? e.g.https://www.researchgate.net/publication/23642155_Enumeration_of_Bacteriophages_by_Double_Agar_Overlay_Plaque_Assay/stats
https://iai.asm.org/content/iai/48/3/782.full.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711990/
https://www.atcc.org/~/media/0871A5EC67CF499B81EE09DDE1906E3F.ashx
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Dear Sandra,
The formation of a "fog" of precipitate is probably due to the fact that you create a local sursaturation of calcium phosphate. I would suggest that you try slowly adding CaCl2 from a 100 mM solution instead of 1 M while mixing. Besides, if the final concentrations of Ca2+ and PO4 are such that their product remains below the solubility product, the precipitate should eventually redissolve.
Dear Sandra,
The formation of a "fog" of precipitate is probably due to the fact that you create a local sursaturation of calcium phosphate. I would suggest that you try slowly adding CaCl2 from a 100 mM solution instead of 1 M while mixing. Besides, if the final concentrations of Ca2+ and PO4 are such that their product remains below the solubility product, the precipitate should eventually redissolve.
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Sandra, You might consider a different pH buffer as suggested by Pierre. To assist your choices you could use a friendly chemical speciation program such as GEOCHEM-EZ, Paul.
Sandra, You might consider a different pH buffer as suggested by Pierre. To assist your choices you could use a friendly chemical speciation program such as GEOCHEM-EZ, Paul.
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Hi, I am having the same issue, did you find any solution for propagating the phi6?I appreciate your help :)
Thanks
Hi, I am having the same issue, did you find any solution for propagating the phi6?I appreciate your help :)
Thanks
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You are correct in identifying the precipitate as calcium phosphate. There is no way to avoid precipitation if you insist in adding such a high concentration of calcium (10 mM final) to your medium (I assume that you wish to keep the phosphate as a buffer). But I strongly doubt that it is necessary. Thus I would advise to reduce the concentration of Ca 10-fold. Most media recipes (e.g. M9) mention Ca concenrations in the range 0.1 to 0.5 mM.
You are correct in identifying the precipitate as calcium phosphate. There is no way to avoid precipitation if you insist in adding such a high concentration of calcium (10 mM final) to your medium (I assume that you wish to keep the phosphate as a buffer). But I strongly doubt that it is necessary. Thus I would advise to reduce the concentration of Ca 10-fold. Most media recipes (e.g. M9) mention Ca concenrations in the range 0.1 to 0.5 mM.
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Thanks for your replies.
I will try these alternatives and check whether I can revitalize and proliferate Phi6 in the adjusted medium.
Thanks for your replies.
I will try these alternatives and check whether I can revitalize and proliferate Phi6 in the adjusted medium.
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