Is it in the bottle? You can just build up the dregs. I did this with allagash yeast. Yum.
You don’t really need a lab to do that, to be honest. Do the smart thing by ‘contracting out’ the lab work (for free!) to White Labs, Wyeast, etc., and just buy your preferred strains then aliquot them straight from the packs into sterile 1.5ml cryovials or similar. Store them in a fridge. To propagate, add the contents of a 1.5ml vial to 50ml sterile wort. Step up when it’s done culturing. Repeat step up procedure until you have enough cells to pitch into FV wort. You don’t even need a microscope. Nice-to-haves include a pressure cooker, Bunsen burner, mechanical pipette with tips and 1.5ml tube racks. This is the most reliable and cost effective way for home brewers to store and culture yeast.
Sounds like you need a little more practice. Did you use a pressure cooker to autoclave?
I use my electric breville and just up the time 5mins.
There are a number of electric pressure cookers FB marketplace, but I guess I didn't realize they went as high as 15 psi. How much capacity do you have?
They do 12.5 with spikes in the 13s, good enough for me. That’s why I do a little longer time. Mine can hold 3 canning jars carefully but has no issue with my beakers and glass vials. Breville fast slow.
Thanks.
Well some instapots do 11.6 it looks like.
Read the link I posted, it’s from a microbiologist. Yeast do very well being frozen as long as the ice crystals don’t destroy the cells. That’s why you add glycerin and freeze them slowly.
) that they really need QC on an agar plate with healthy-looking colonies being selected for propagation. Mutation is going to occur at a much higher rate when freezing compared with ‘cosy’ refrigeration temperature. The most vulnerable bits of DNA are the mitochondrial genomes (hello petit!) and the strands of DNA exposed on the surface of chromosomes. Then there’s the impact of freezing on the mRNA, the transcriptome and proteome, the ‘memory’ of the cells. I routinely QC yeast on plates when they come out of the freezer, after an overnight culture. I can say yeast kept in small aliquots in the fridge have noticeably fewer suspect-looking colonies on agar plates. Sure, much longer term, freezing is probably better, but I’m really not confined that time frame is relevant for most home brewers for something like yeast. There are clearly much simpler alternatives that are more reliable and more accessible to home brewers generally, imho.
Awesome. Thanks. Wasn't aware of that germination cycle on spores, so presumed it's 250 or face the consequences (potentially serious ones).To be brutally honest, I wouldn’t employ that ‘microbiologist’ in my lab. Some glaring mistakes there, frankly. Freezing (and thawing) is so much more stressful on yeast cells (especially using glycerol, not just at a low conc and without incubation time to replace enough H2O molecules in and around membranes, cell and organelles
As far as pressure cookers go, definitely use one if you have one, but don’t worry if you din’t. Double boil. ‘Simmer’ temp with lid on really. After the first boil, let it cook then leave it overnight. Any viable spores present ‘germinate’. Boil again to kill them. This is wort, it’s highly unlikely there’s going to be nasty thermophilic bugs in there. Focus on sterility when aliquoting and propagating 10-50ml cultures. After that, just follow good home brew practices. It’s not a problem to start treating it a little bit like food. It’s kind of what it is at this point, right? Best thing we can do really is pitch sufficient healthy yeast to get ethanol production going as soon as, to protect the wort from infection. That cooled kettle wort ain’t sterilised. It’s only been sanitised. Like most things at this stage, if we’re thorough.
He does mention to leave in the fridge for a few days before freezing to increase vitality but doesn’t say exactly why…To be brutally honest, I wouldn’t employ that ‘microbiologist’ in my lab. Some glaring mistakes there, frankly. Freezing (and thawing) is so much more stressful on yeast cells (especially using glycerol, not just at a low conc and without incubation time to replace enough H2O molecules in and around membranes, cell and organelles
As far as pressure cookers go, definitely use one if you have one, but don’t worry if you din’t. Double boil. ‘Simmer’ temp with lid on really. After the first boil, let it cook then leave it overnight. Any viable spores present ‘germinate’. Boil again to kill them. This is wort, it’s highly unlikely there’s going to be nasty thermophilic bugs in there. Focus on sterility when aliquoting and propagating 10-50ml cultures. After that, just follow good home brew practices. It’s not a problem to start treating it a little bit like food. It’s kind of what it is at this point, right? Best thing we can do really is pitch sufficient healthy yeast to get ethanol production going as soon as, to protect the wort from infection. That cooled kettle wort ain’t sterilised. It’s only been sanitised. Like most things at this stage, if we’re thorough.
Yes, 1.5-2.0ml aliquots direct from a fresh yeast pack. That’s the simplest way. Next would be making a small sterile culture/starter to aliquot. No additives. I’m experimenting with trehalose, but not enough data yet. Scaling things down regardless, to 1-2ml is one of the tricks, fridge or freezer. It forces us to start from little and step up multiple times, promoting a better pitchable population. For lagers, my final ‘starter’ (stepped up from 2L) is actually a half batch lager 10L) with harvested yeast re pitched into a full batch or another half batch, which ferments out within 5 days or so. I find it the best way to build up a decent quantity of really health yeast, with the benefit of getting a beer out of it. Better than wasting 4-5L on a starter.
It doesn’t take that long to replace a significant amount of H2O, but it is advisable to allow the cells to condition themselves (increase tolerance) before prepping them for freezing. Hours rather than days, for glycerol to work its way in. And trehalose is much better than glycerol, if it’s easier to get hold of.
So usp glycerin doesn’t work? And I have been freezing about 5-6ml of slurry per vial.
Not as well as trehalose, which has been recognised as a much better cryoprotectant for years. I think once samples go above about 2ml, controlling freeze and thaw rates starts to get a bit sketchy with diminishing returns, wasting space and resources. When it comes to propagating from a frozen sample, ideally, only a tiny scrape with a needle is required to inoculate a 10ml mini prep/starter. There seems to be a deeply flawed approach recommended by home-brew protocols where the whole vial, up to 50ml or more gets tossed into an oversized starter, or even pitched directly. These freshly-thawed yeast are pretty much knackered mainly and are much better cultured up from something small. It might well ‘work’ according to an individual’s expectations, and that’s absolutely fine, but compared to what? Wouldn’t you want to make it work noticeably better, possibly with less effort and lower cost? I’m not trying to sell you anything, just offering better advice for free. It gives me a buzz to help people with what I know. If I charged lots of money, would it convince you more?
No I love Quality free advice. I have about 15 strains in the freezer and would love to know how I can do things better in the future. Would you consider doing an updated post with your recommendations?Not as well as trehalose, which has been recognised as a much better cryoprotectant for years. I think once samples go above about 2ml, controlling freeze and thaw rates starts to get a bit sketchy with diminishing returns, wasting space and resources. When it comes to propagating from a frozen sample, ideally, only a tiny scrape with a needle is required to inoculate a 10ml mini prep/starter. There seems to be a deeply flawed approach recommended by home-brew protocols where the whole vial, up to 50ml or more gets tossed into an oversized starter, or even pitched directly. These freshly-thawed yeast are pretty much knackered mainly and are much better cultured up from something small. It might well ‘work’ according to an individual’s expectations, and that’s absolutely fine, but compared to what? Wouldn’t you want to make it work noticeably better, possibly with less effort and lower cost? I’m not trying to sell you anything, just offering better advice for free. It gives me a buzz to help people with what I know. If I charged lots of money, would it convince you more?
From frozen, I’ll add a scratch to a 10ml overnight culture, plate a loop then add loop, collected from a few healthy-looking colonies, into 10ml, 100ml, 500ml, 2.5L then 10L (half batch of lager) then start repitching. Adding more frozen slurry to a bigger 1st step starter is increasing the chance of problems and could promote under pitching, even if the recommended number of cells are being pitched, because so many are potentially aberrant/knackered. It might work better simply due to Lady Luck, but it won’t be a reliable strategy. Otherwise White Labs and Wyeast, etc., would be selling packs of frozen wet yeast.
