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

Link to this post \| posted 06 Sep, 2024 21:34
serivas	Hello all, Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar?

Link to this post \| posted 06 Sep, 2024 23:42
viknesh	serivas Hello all, Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar? It is likley that you'll be able to culture the bacteria we use for phage-hunting just as well using peptone or tryptone. However, for us to compare data from across the SEA program, I would recommend using peptone. If you use tryptone, it will be important for you to disclose this information (i.e. that you are not usingP PYCa) when contributing data to the program and when publishing elsewhere.

Link to this post | posted 06 Sep, 2024 23:42

viknesh

serivas
Hello all,

Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar?

It is likley that you'll be able to culture the bacteria we use for phage-hunting just as well using peptone or tryptone. However, for us to compare data from across the SEA program, I would recommend using peptone. If you use tryptone, it will be important for you to disclose this information (i.e. that you are not usingP PYCa) when contributing data to the program and when publishing elsewhere.

Link to this post \| posted 07 Sep, 2024 03:47
serivas	Thank you for your advice, I'll stick to peptone at least for the first semester trial. Thank you again for your timely response!

Link to this post \| posted 20 Sep, 2024 14:07
bavina	Hello, I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts? 3.14Mb

Link to this post | posted 20 Sep, 2024 14:07

bavina

Hello,

I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts?

Link to this post \| posted 20 Sep, 2024 14:48
viknesh	bavina Hello, I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts? The precipitation could be problematic for at least two reasons: First, it is hard to tell if the media is contaminated and second, the precipitate itself, say calcium chloride, might have been a necessary component of the solution needed for infectivity. If you keep those two things in mind, you can still use your media. To address the possibility of contamonation, you'll want to include controls. For example, when using the top agar for a plaque assay, include a control where no host is added to the top agar. No lawn should form and the top agar should remain clear after 24 hours. To address the concern that precipitation might negatively impact infection, you'll want to pay attention to any change in infectivity for phages that you've isolated. In the longer term, it would be good to change the media components. As we look to no longer use Fiher brand peptone, the HHMI SEA team hasn't had a chance to test alternatives to Bacto products that we know are quite expensive, but we plan to do so – more soon!

Link to this post | posted 20 Sep, 2024 14:48

viknesh

bavina
Hello,

I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts?

The precipitation could be problematic for at least two reasons: First, it is hard to tell if the media is contaminated and second, the precipitate itself, say calcium chloride, might have been a necessary component of the solution needed for infectivity. If you keep those two things in mind, you can still use your media. To address the possibility of contamonation, you'll want to include controls. For example, when using the top agar for a plaque assay, include a control where no host is added to the top agar. No lawn should form and the top agar should remain clear after 24 hours. To address the concern that precipitation might negatively impact infection, you'll want to pay attention to any change in infectivity for phages that you've isolated.

In the longer term, it would be good to change the media components. As we look to no longer use Fiher brand peptone, the HHMI SEA team hasn't had a chance to test alternatives to Bacto products that we know are quite expensive, but we plan to do so – more soon!

Link to this post \| posted 28 Jan, 2025 02:59
viknesh	Thanks to a collective effort across SEA, we have found that the Fisher brand of peptone is the culprit for the precipitation that many are observing in their PYCa media. In fact, there is another brand of peptone by Fisher called "peptone for science education" which make PYCa media crash immediately after it is sterilized in an autoclave. The SEA Team (thanks, Cole Jirsa!) has now tested multiple brands of peptone for precipitation, and we recommend any of the following 4 brands of peptone, which we've listed from most expensive to least expensive : Bacto Peptone (Difco) , MP Biomedical Casein Peptone, Neogen Peptone, and HIMEDIA Peptone. We also note that there are no observable differences in the efficiency of plating or plaque morphology for at least three different phages when plated on media made using any of these 4 brands of peptone. We'll try to include these brands of peptone in the Fisher quote as soon as possible. If you switch to any of these brands of peptone, please share with us any notable observations! Edited 28 Jan, 2025 03:00

Link to this post | posted 28 Jan, 2025 02:59

viknesh

Thanks to a collective effort across SEA, we have found that the Fisher brand of peptone is the culprit for the precipitation that many are observing in their PYCa media. In fact, there is another brand of peptone by Fisher called "peptone for science education" which make PYCa media crash immediately after it is sterilized in an autoclave.

The SEA Team (thanks, Cole Jirsa!) has now tested multiple brands of peptone for precipitation, and we recommend any of the following 4 brands of peptone, which we've listed from most expensive to least expensive :
Bacto Peptone (Difco) ,
MP Biomedical Casein Peptone,
Neogen Peptone, and
HIMEDIA Peptone.

We also note that there are no observable differences in the efficiency of plating or plaque morphology for at least three different phages when plated on media made using any of these 4 brands of peptone.

We'll try to include these brands of peptone in the Fisher quote as soon as possible. If you switch to any of these brands of peptone, please share with us any notable observations!

Edited 28 Jan, 2025 03:00

Link to this post \| posted 28 Jan, 2025 16:46
bavina	Hello, I wanted to share that switching to Bacto Peptone really helped with out CaCl2 issues! With this brand of peptone, our top agar was more stable and was able to remain in the water bath for around a week. It did eventually crash out of solution over the weekend, but that could have been due to the water level getting low and the bath overheating the agar.

Link to this post \| posted 28 Jan, 2025 18:20
sklambet@ncsu.edu	Bacto peptone also solved the precipitation issues last semester for me! Thank you Cole for working on this.

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Precipitation of CaCl2 in PYCA top-agar