Simple Yeast Banking with Sodium Chloride

[SanPancho]

Oxigen oxidizes, for one. But im sure you already knew that from google.

 

[RPh_Guy]

Yeast don't oxidize from a little DO.

There's no mention of purging headspace by anyone using this method, and I'm unaware of any evidence to suggest that oxygen is harmful when storing yeast in NS.

I think 2+ years viability speaks for itself.

 

[SanPancho]

@beervoid so ihate to add more variables, but was just talking to biochem/macrobiologist and mentioned isotonic storage. He says if you can get your hands on it something called pbs would also control the ph during storage as well as the osmotic pressure. Phosphate (?) buffering solution i believe was the name.

And he said yes, if you can remove the oxygen you should. Especially in a long term nonbuffered solution as dead yeast cells and oxygen are gonna be no bueno. Ph gets affected more when the yeast cells lyse with nothing to buffer it like in beer or wort.

But he also said as a homebrewer wont have a laboratory to work in or swipe chems from, just do what you can thats reasonable and repeatable.

Hope that helps.

 

[SanPancho]

Yeast don't oxidize from a little DO.

There's no mention of purging headspace by anyone using this method, and I'm unaware of any evidence to suggest that oxygen is harmful when storing yeast in NS.

I think 2+ years viability speaks for itself.

Well if you are unaware of evidence (proving a negative?) im torn as to who i should believe. A guy who just signed $$ to consult on a plant creating bioengineered enzymes in south africa, or analong/fleshbot version of google search button.

Decisions.
Decisions.
Decisions.

 

[beervoid]

@beervoid so ihate to add more variables, but was just talking to biochem/macrobiologist and mentioned isotonic storage. He says if you can get your hands on it something called pbs would also control the ph during storage as well as the osmotic pressure. Phosphate (?) buffering solution i believe was the name.

And he said yes, if you can remove the oxygen you should. Especially in a long term nonbuffered solution as dead yeast cells and oxygen are gonna be no bueno. Ph gets affected more when the yeast cells lyse with nothing to buffer it like in beer or wort.

But he also said as a homebrewer wont have a laboratory to work in or swipe chems from, just do what you can thats reasonable and repeatable.

Hope that helps.

Great stuff, this thread is getting better and better.. There will always be perfectionists that want to optimize the simple methods...

Did he mention how much PBS to add? Or does this solution replace the sodium chloride solution entirely?

 

[NeverDie]

Sounds like maybe it is this: https://en.wikipedia.org/wiki/Phosphate-buffered_saline

but now it seems like maybe we're sailing into uncharted waters regarding yeast preservation?

 

[isomerization]

Keep it simple, storing yeast under fermented beer (preferably from an overbuilt starter) will work just fine.

Yes, there are “better” solutions, but I’d imagine that the overwhelming majority of them cause an increased risk of failure for homebrewers.

PBS = phosphate buffered saline (typically between pH 7.2 to 7.4 and composed of 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 at 1X strength; can be purchased at 10X too).

 

[beervoid]

Keep it simple, storing yeast under fermented beer (preferably from an overbuilt starter) will work just fine.

Yes, there are “better” solutions, but I’d imagine that the overwhelming majority of them cause an increased risk of failure for homebrewers.

PBS = phosphate buffered saline (typically between pH 7.2 to 7.4 and composed of 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 at 1X strength; can be purchased at 10X too).

Are you saying the difference in stability between storing yeast under leftover beer vs sodium chloride is neglectable?
I've succesfully brewed with 3+ month old yeast stored under beer but the extra stability seems attractive.

 

[NeverDie]

Keep it simple, storing yeast under fermented beer (preferably from an overbuilt starter) will work just fine.

Yes, there are “better” solutions, but I’d imagine that the overwhelming majority of them cause an increased risk of failure for homebrewers.

Doesn't using a yeast slurry kept under beer for more than 3 months also carry an increased risk of failure? And isn't that risk of failure extremely high if it's stored under beer for one or two years?

 

[soccerdad]

Ball makes a short little half pint jar like the one pictured. It's 8 ounces or 250 ml. It has one marking at 125ml. Seems easy enough to use 25ml yeast slurry and fill to 125 line with saline. Or, if concerned about o2 in the head space go 50ml slurry and fill er up. Three of these stacked take up about the same space as a jar of spaghetti sauce. If stacking them I would make a little slip sheet to go between jars .. They don't have a snug top to bottom fit. Edited .. Forgot pic

 

[SanPancho]

Sounds like maybe it is this: https://en.wikipedia.org/wiki/Phosphate-buffered_saline

but now it seems like maybe we're sailing into uncharted waters regarding yeast preservation?

Id agree. Despite being the one to bring up zinc and pbs and veer off into variables, id say just stick to original plan. Its reasonably expected to work well(based on others experience), and its repeatable in the sense you’re following an existing protocol.

If you’re gonna wait a year for results, maybe playing safe is the most sensible path to take.

 

[isomerization]

Are you saying the difference in stability between storing yeast under leftover beer vs sodium chloride is neglectable?
I've succesfully brewed with 3+ month old yeast stored under beer but the extra stability seems attractive.

Both cases will require you to build back up to a pitchable amount of yeast. I have stored yeast this way for over a year and brought it back (requires stepping up after a certain length of time or you have longer lag and increased risk). Anything that minimizes the number of user steps will likely decrease risk of failure.

Doesn't using a yeast slurry kept under beer for more than 3 months also carry an increased risk of failure? And isn't that risk of failure extremely high if it's stored under beer for one or two years?

Are you saying direct pitch? I thought both scenarios were for maintaining a reservoir of yeast for future inoculation. As said above, I feel comfortable with 1 year, but would be cool to see viability at 2 years with either approach. Regardless, you will have visual confirmation of yeast activity when stepping up.

 

[NeverDie]

Both cases will require you to build back up to a pitchable amount of yeast. I have stored yeast this way for over a year and brought it back (requires stepping up after a certain length of time or you have longer lag and increased risk). Anything that minimizes the number of user steps will likely decrease risk of failure.



Are you saying direct pitch? I thought both scenarios were for maintaining a reservoir of yeast for future inoculation. As said above, I feel comfortable with 1 year, but would be cool to see viability at 2 years with either approach. Regardless, you will have visual confirmation of yeast activity when stepping up.

Good question! I didn't know that it was possible to reliably keep yeast under beer in the refrigerator for an entire year. That's new information, at least to me. My reading had suggested that 3 months was about all I could count on or even hope for without freezing or the saline approach.

I had been thinking to keep about 10ml of yeast cream per vial, so then I'd only need to step it up once or twice but not three times, saving me some time. However, keeping a single bigger jar and only extracting say, 1ml of yeast cream each time via sterilized pipette , as you and Rph_guy seem to be suggesting would be a different way that would stretch the yeast even further. Or, I suppose one could do both approaches and maybe get the best of both worlds by saving one of the stepped-up starters (effectively generation 1) and dividing it up into 10ml's of yeast cream per vial but keeping the single big jar of generation 0 to draw upon as needed? Thinking about it now, that seems like the way to go. Is that how you do it?

 

[ba-brewer]

This guy revived some yeast slurry from a 6 year old bottle of beer, so I think storage in beer should get you well beyond 3 months if you build up a starter.
https://www.homebrewtalk.com/forum/...re-stingo-bottle-harvest.404511/#post-8523549

I used to save slurry from overbuilt starter and had revived yeast that was 12months old or older. I switched to slants to reduce the amount of space for storage. If you are looking to save yeast you should have at least one backup per strain. This approach seem a bit easier than slants but I think the larger vials might use more space for storage.
​

 

[NeverDie]

OK, how about this? Do both. Keep one sample under beer, on the theory that it's less likely to get infected but may possibly require the patience of a saint to revive after a year. Keep the other in saline, which, if not infected, may be more vital when you try to revive it.

 

[isomerization]

Good question! I didn't know that it was possible to reliably keep yeast under beer in the refrigerator for an entire year. That's new information, at least to me. My reading had suggested that 3 months was about all I could count on or even hope for without freezing or the saline approach.

I had been thinking to keep about 10ml of yeast cream per vial, so then I'd only need to step it up once or twice but not three times, saving me some time. However, keeping a single bigger jar and only extracting say, 1ml of yeast cream each time via sterilized pipette , as you and Rph_guy seem to be suggesting would be a different way that would stretch the yeast even further. Or, I suppose one could do both approaches and maybe get the best of both worlds by saving one of the stepped-up starters (effectively generation 1) and dividing it up into 10ml's of yeast cream per vial but keeping the single big jar of generation 0 to draw upon as needed? Thinking about it now, that seems like the way to go. Is that how you do it?

I would consider how often you plan on using a strain in a given year (or two), and then make that many vials for propagation. Once you get down to the last one, propagate another round of vials. Worst case scenario, you buy another commercial pack, and start again.

 

[NeverDie]

Reporting back: I moved two strains over to the saline storage method today using my pressure canner'd homemade saline solution, so I guess time will tell how well it works out.

 

[Bigdaddyale]

Can you use an instant pot to sterilize a saline solution?

 

[NeverDie]

Can you use an instant pot to sterilize a saline solution?

No. It doesn't get hot enough.

 

[soccerdad]

Would it be wrong .. or a bad idea anyway .. to sterilize the jars, but rely on the saline solution to be ok after boiling a cup of distilled water and adding 1/2 teaspoon of salt to that boiled, distilled water? Or alternatively, to sterilize the jars and use store bought saline solution.

 

[NeverDie]

Would it be wrong .. or a bad idea anyway .. to sterilize the jars, but rely on the saline solution to be ok after boiling a cup of distilled water and adding 1/2 teaspoon of salt to that boiled, distilled water? Or alternatively, to sterilize the jars and use store bought saline solution.

Well, let's consider the worst case scenario: is it possible for botulism to get in there? I don't know the answer to that, but if the answer is yes, then I'd say it's a very, very bad idea, as botulism toxin can quickly kill you dead.

 

[soccerdad]

Well, let's consider the worst case scenario: is it possible for botulism to get in there? I don't know the answer to that, but if the answer is yes, then I'd say it's a very, very bad idea, as botulism toxin can quickly kill you dead.

I'm ok being killed - but I don't want to be killed DEAD !

No seriously , I have stored yeast slurry in the fridge under beer, and the process has been to sterile the jar, but not the mix of beer and slurry. I was just wondering if the risk is the same .. or greater.

 

[NeverDie]

I'm ok being killed - but I don't want to be killed DEAD !

No seriously , I have stored yeast slurry in the fridge under beer, and the process has been to sterile the jar, but not the mix of beer and slurry. I was just wondering if the risk is the same .. or greater.

From what I've read, storing it under beer is safe, because the acid pH prevents botulism. However, in our scenario the beer goes away and is replaced by saline, so there's no longer low pH to inhibit botuism. Thinking about this now: maybe it would be prudent to make the saline low pH to avoid this worst case scenario?

After all, my name is neverdie.

How best to do it? Phosphoric acid maybe?

 

[ba-brewer]

I thought botulism toxin was formed in anaerobic conditions but found this that said just low oxygen.
https://www.ndhealth.gov/Disease/Documents/faqs/Botulism.pdf

 

[NeverDie]

Maybe citric acid? Anyway, having an acid saline would probably help prevent bacteria infection, just as the beer does, so I think it would be a good idea.

 

[NeverDie]

Reporting back: I used a very tiny amount of diluted phosphoric acid to drive the pH in the saline solution housing the two yeast strains down to 3.2pH, as confirmed by my digital pH meter. I consider that the primary protection against botulism. The secondary protection is keeping my refrigerator at under 38F (which may not hold during the thaw cycle, so I may need to come up with a better way).

Anyhow, I'm not suggesting that anyone follow what I did per se, but that's what I did. Doing it after-the-fact was a hassle, and I would hope that collectively we could devise a better recipe for doing this in the future.

 

[RPh_Guy]

If there's oxygen in the headspace, Clostridium spp (e.g. botulinum) won't grow.

 

[NeverDie]

LOL. I love how all of the "Can I cut this corner and/or that corner?" type questions just suddenly halted.

 

[beervoid]

LOL. I love how all of the "Can I cut this corner and/or that corner?" type questions just suddenly halted.

Thread killed by botulism

 

[soccerdad]

I would respond if I were still alive.