Setting up a yeast lab?

[mac_1103]

I don't know about yeast but in the food industry the faster(colder) the food is frozen the less ice crystals form.

Completely different than freezing live cells.

 

[McMullan]

It's pretty well established that rapid freezing promotes higher rates of ice crystal formation in living yeast cells. Regardless of food processing and rock formation. The scientific literature goes back decades with more reviews you could shake a stick at. Ice crystals are not good for living cells either. Once frozen, the yeast cells are under stresses associated with dehydration too. In fact, thawed frozen yeast are in worse shape than hydrated dry yeast. They are knackered and need to be carefully selected for post thawing. This is why my view, for home brewers generally, at least those who want to store liquid yeast strains, is shifting towards storing small aliquots of high-quality yeast in the fridge. I can't say it's a viable alternative for long-term storage over years, I'm genuinely skeptical about that. But I can say it's a better, easier option for most home brewers, at least up to about 18 months so far. I stand by my original reply to the OP, that freezing yeast cells is technically demanding involving a lot more than simply freezing tens of mls of slurry with glycerol then thawing to pitch or make a large starter. That's not sound microbiology or good brewing practice at all therefore not something I can endorse, because it's simply not something I'd do myself.

 

[user 246304]

It's pretty well established that rapid freezing promotes higher rates of ice crystal formation in living yeast cells. Regardless of food processing and rock formation. The scientific literature goes back decades with more reviews you could shake a stick at. Ice crystals are not good for living cells either. Once frozen, the yeast cells are under stresses associated with dehydration too. In fact, thawed frozen yeast are in worse shape than hydrated dry yeast. They are knackered and need to be carefully selected for post thawing. This is why my view, for home brewers generally, at least those who want to store liquid yeast strains, is shifting towards storing small aliquots of high-quality yeast in the fridge. I can't say it's a viable alternative for long-term storage over years, I'm genuinely skeptical about that. But I can say it's a better, easier option for most home brewers, at least up to about 18 months so far. I stand by my original reply to the OP, that freezing yeast cells is technically demanding involving a lot more than simply freezing tens of mls of slurry with glycerol then thawing to pitch or make a large starter. That's not sound microbiology or good brewing practice at all therefore not something I can endorse, because it's simply not something I'd do myself.

I might have missed it McMullan, but are your aliquots simply good yeast under some amount of wort? Loose cap?

 

[McMullan]

I might have missed it McMullan, but are your aliquots simply good yeast under some amount of wort? Loose cap?

These ones are straight from a White Labs’ WLP007 pack. ‘Outsourcing’ the lab work, making it incredibly simple. They know what they’re doing. Or make a nice clean starter with your strain(s) of choice and store the same way. The cryovial caps are screwed on tight. They can handle a little pressure, but I haven’t noticed any building up. I recommend just pitching one vial in a 50-100ml first step starter, for simplicity, but I actually just use a loop’s worth to inoculate 10ml sterile wort then step up to 100ml, 500ml, for a half batch, or step up again to 2.5L for a whole (5G) batch. Half batch then repitch is best, if you have time to brew another batch soon enough.

 

[beren]

There are a lot of technical considerations when freezing and thawing yeast such as osmotic pressure of solution and speed of freezing and thawing. I have decent wet-lab education, training, and experience which carries over well to the home brewing. I haven't specifically measured my cell counts and viability, but get decent enough fermentation timecourses and flavor profiles in beer.

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So, it's likely not as simple as "freezing yeast doesn't work." There will be a heavy influence of the technical aspects of the lab procedures that one implements. I think one key aspect everyone could do is to freeze yeast slowly (about -1C/min) by placing in an insulated container in the freezer and thawing rapidly about about 30-35C.

Sadly those pdf links don't work. Maybe you could upload them directly? I'd love to do a better job keeping yeast alive, I just don't have practical experience so have to rely on what people post and hope it's correct. I'd be glad to learn better. The post I've been following does have the slow freezing/fast thaw info, but I have never heard of the sugar Trehalose, and looks like there's even newer research into improving things: https://www.sciencedirect.com/science/article/abs/pii/S0268005X22006129

Also, my frozen vials have about 5-6ml of slurry. Should this be pitched right into a 1l starter or a smaller one, and 1.040 ok or weaker at first?

 

[user 246304]

These ones are straight from a White Labs’ WLP007 pack. ‘Outsourcing’ the lab work, making it incredibly simple. They know what they’re doing. Or make a nice clean starter with your strain(s) of choice and store the same way. The cryovial caps are screwed on tight. They can handle a little pressure, but I haven’t noticed any building up. I recommend just pitching one vial in a 50-100ml first step starter, for simplicity, but I actually just use a loop’s worth to inoculate 10ml sterile wort then step up to 100ml, 500ml, for a half batch, or step up again to 2.5L for a whole (5G) batch. Half batch then repitch is best, if you have time to brew another batch soon enough.

Great, fantastic. Many thanks.

 

[user 246304]

These ones are straight from a White Labs’ WLP007 pack. ‘Outsourcing’ the lab work, making it incredibly simple. They know what they’re doing. Or make a nice clean starter with your strain(s) of choice and store the same way. The cryovial caps are screwed on tight. They can handle a little pressure, but I haven’t noticed any building up. I recommend just pitching one vial in a 50-100ml first step starter, for simplicity, but I actually just use a loop’s worth to inoculate 10ml sterile wort then step up to 100ml, 500ml, for a half batch, or step up again to 2.5L for a whole (5G) batch. Half batch then repitch is best, if you have time to brew another batch soon enough.

Sorry, neglected to ask. The aliqouts themselves - they are literally just the Wyeast pack or WL vial, etc., themselves, simply poured into the cryovials - the yeast and wort in the packs, then pitch into 50-100 ml 1st step starter, etc.?

 

[Bassman2003]

Thanks for all of the info on this thread. While I will continue to judge my freezing program, I will also order some 2ml vials and start a parallel fridge program. Just one more step than I usually do from freezing will not be that difficult. (50ml 1.020 to 250ml 1.020 to 2L 10.40)

 

[McMullan]

Sorry, neglected to ask. The aliqouts themselves - they are literally just the Wyeast pack or WL vial, etc., themselves, simply poured into the cryovials - the yeast and wort in the packs, then pitch into 50-100 ml 1st step starter, etc.?

Yes, mixed then transferred with a pipette to sterile cryovials. Ideally working in front of a Bunsen burner (or little bleuet burner). Eppendorf tubes are fine too. I just happen to have a box of cryovials hanging around not doing much and I like the more secure screw caps.

 

[Bassman2003]

This thread has got me thinking about my yeast process and handling which is always good. In the area of a portable yeast lab, what does everybody think of one of these to work in? Might seem like overkill but for those of us who have pets it might be an easy way to create a solid workspace and save time with prep and cleanup. Also a CO2 environment could be easily created which would be best for repitching. Thoughts?

https://www.coleparmer.com/p/glove-bag-inflatable-glove-chambers/49332

 

[mac_1103]

Thoughts?

Pretty expensive and not sterile.

 

[Bassman2003]

Not sterile but compared to our countertops, it seems like an improvement! Is this really a drawback or prerequisite?

 

[hottpeper13]

I do my transfers under a propane flame. If my overbuild saved yeast is over a year I use a loop to get the middle portion and have 15 ml screw top tubes of 1.030 wort I pressure can. I do the 15,150,1500 ml steps.

 

[mac_1103]

Is this really a drawback or prerequisite?

No, just pointing it out. And not real sure about the bang for the buck. We did sometimes use something similar in the BSL-3 lab for "containment within containment" but we were spending somebody else's money.

 

[Bassman2003]

Along the lines of the original post, just trying to picture how I can improve my home workspace. As a non-biologist, I am kind of blind to industry practices somewhat. Since I do not have the space to dedicate a clean room or area, what is the next best alternative? Easy and repeatable spring to mind along with compact in size. Sterile seems to = expensive. Where on the sterile vs open air spectrum do us homebrewers need to fall?

I like the repeatability and possible CO2 environment creation of a bag. Filters could probably improve the quality of the inside of the bag compared to the house environment.

 

[mac_1103]

Since I do not have the space to dedicate a clean room or area, what is the next best alternative?

Low traffic. Low air flow. Sanitize all surfaces.

Not meaning to dis the glove bag. Keeps dust out and dust carries organisms that can contaminate your work. If I was going to fill one with CO2 or N2 I'd probably use a sanitary air filter in the gas line.

 

[McMullan]

This thread has got me thinking about my yeast process and handling which is always good. In the area of a portable yeast lab, what does everybody think of one of these to work in? Might seem like overkill but for those of us who have pets it might be an easy way to create a solid workspace and save time with prep and cleanup. Also a CO2 environment could be easily created which would be best for repitching. Thoughts?

https://www.coleparmer.com/p/glove-bag-inflatable-glove-chambers/49332

I’m not sure what that ‘glove box’ is supposed to be used for, tbh, but it isn’t brewer’s microbiology. I don’t care what they claim. It’s not me trying to sell one. The most you’d need, as a home brewer, is a Bunsen burner, which is a lot cheaper and is going to last a lot longer.

Try not to overthink things. Just be a brewer.

 

[beren]

What do you mean working in front of a Brunson burner? To create an updraft?

 

[matt_m]

Might have missed something but basically you pipette viable yeast into these, store them in the fridge, then do several stepped starters to prep them for use?

The potential problem here, with large volumes of stored slurry, as the slurry ages, is cell counts and assumptions ignore a crucial part of the puzzle, vitality, which declines too over time. This can result in under pitching even if enough cells have been pitched. A case where pitching fewer healthier cells ferments better than more unhealthy cells. By starting at very small volumes, like 10ml, you force things in your favour.

View attachment 837431

 

[McMullan]

Might have missed something but basically you pipette viable yeast into these, store them in the fridge, then do several stepped starters to prep them for use?

Yes. Viability seems to remain higher at smaller volumes. Presumably something to do with surface-to-volume ratio and less resistance to nutrient diffusion during storage compared to a whole pack or jar of harvested yeast cake, etc.

 

[McMullan]

What do you mean working in front of a Brunson burner? To create an updraft?

Yes, the updraft helps promote a sterile envelope to work under. And the flame is used to sterilse tools like loops.

 

[Bassman2003]

@McMullan - I appreciate all of your info on this thread. I wanted to ask you if you had an opinion on isotonic storage vs just yeast/wort in small vials (your method)? They are very close together, with one using distilled water + a little sodium. Thanks in advance.

 

[McMullan]

The spent growth medium the yeast are already in is probably better, in terms of what the cells are conditioned to and availability of nutrients. Distilled water works well for some groups of multicellular fungi, but not that great for yeast cells.

 

[Bassman2003]

Thanks.

 

[user 246304]

These ones are straight from a White Labs’ WLP007 pack. ‘Outsourcing’ the lab work, making it incredibly simple. They know what they’re doing. Or make a nice clean starter with your strain(s) of choice and store the same way. The cryovial caps are screwed on tight. They can handle a little pressure, but I haven’t noticed any building up. I recommend just pitching one vial in a 50-100ml first step starter, for simplicity, but I actually just use a loop’s worth to inoculate 10ml sterile wort then step up to 100ml, 500ml, for a half batch, or step up again to 2.5L for a whole (5G) batch. Half batch then repitch is best, if you have time to brew another batch soon enough.

More to guys in the U.S., I would think - bit seeing a lot of unknown makes on Amazon, or in bulk way beyond enough for 20 lifetimes on this level. Anyone in the U.S. do this, and what's your source?

 

[Bassman2003]

I am going to give this method a try. I ordered these from Amazon - Amazon.com

I already ran them through the pressure cooker and they seemed fine. A couple lost their little sealing ring, but that is probably not that big of a deal. For $9 it is pretty cheap to give it a go.

 

[user 246304]

I am going to give this method a try. I ordered these from Amazon - Amazon.com

I already ran them through the pressure cooker and they seemed fine. A couple lost their little sealing ring, but that is probably not that big of a deal. For $9 it is pretty cheap to give it a go.

Funny, I saw them and took a pass because they weren't listed as sterile. Wouldn't have thought they're autoclavable because they're PP. I see the temp rating is for 356F? Wow. Thanks.

 

[mac_1103]

That's minus 356F (-180C). As in the vapor phase of a liquid nitrogen freezer. They're cryo-vials after all.

 

[user 246304]

That's minus 356F (-180C). As in the vapor phase of a liquid nitrogen freezer. They're cryo-vials after all.

Hahahahah - OMG, I'm such an idiot. I wasn't even thinking....thought if they're rated to 356F, they could easily withstand 15 psi/250F. Which begs the question again - Bassman, how have you not ended up with a big puddle?

Really, I'm looking for sterile cryovials, in the limited quantities, for now.

 

[mac_1103]

Is 50 too many? Bonus: color-coded caps!