Yeast Master and Working Stocks Question

I’ve just stated to delve into the world of yeast management. So far I’ve washed yeast out of a primary and am storing it in the fridge for a brew in a few weeks. Also, mostly as an experiment, I’ve mixed some with 10%glycerin to freeze. I haven’t used any of my saved yeast in a brew yet, so we’ll see how that goes in the future (fingers crossed!). For now I’m contemplating if/how to go about getting into proper yeast banking/ranching.

My question is about and “master” and “working” stocks (using agar slants). Basically I’m wondering what precisely theses are, and how they work in practice? I recall reading somewhere that its common practice to maintain a “master stock” that is only used for re-propagating masters and inoculating the working stocks. The working stocks are then only used for inoculating your starters. Is that the correct workflow?

If so, is a master stock normally just one vial of each strain? or should you keep two or three to reduce risk of losing the strain?

How many vials of the working stock? I assume this probably depends on how often you use the strain within the life of the slant? What do people here use personally?

When propagating the master, do you simply inoculate a new slant and then call it a master, or should you go thru the intermediate step of streaking a plate? (I assume the only reason to streak a plate is to ensure you’re only propagating pure cultures?)

What about using frozen slurries as you’re master? Is there a problem in thawing and re-freezing every time you want to inoculate new working slants?

Lots of questions... I know! Looking forward to feedback from the amazing wealth of knowledge in this community!

Cheers!

steveahol said:

My question is about and “master” and “working” stocks (using agar slants). Basically I’m wondering what precisely theses are, and how they work in practice? I recall reading somewhere that its common practice to maintain a “master stock” that is only used for re-propagating masters and inoculating the working stocks. The working stocks are then only used for inoculating your starters. Is that the correct workflow?

If so, is a master stock normally just one vial of each strain? or should you keep two or three to reduce risk of losing the strain?

Click to expand...

We usually streak between 5-10 slants to maintain frozen master stocks of one particular strain. More info below...

steveahol said:

When propagating the master, do you simply inoculate a new slant and then call it a master, or should you go thru the intermediate step of streaking a plate? (I assume the only reason to streak a plate is to ensure you’re only propagating pure cultures?)

Click to expand...

An original yeast sample (from whatever source) is first plated to ensure purity, and confirmed with microscopy and staining. Colonies from this plate are used to inoculate slants for long term storage (master stocks), and used to inoculate a series of stepped up cultures in order to acquire a large quantity of cells. The large culture is divided (aliquoted) into 50ml working stocks. When the yeast is required, one working stock is used to inoculate a starter culture.

We are less concerned about #'s of working stocks (although you get many stocks from a 10L batch culture), and instead keep careful record of passages. A passage is when the batch of cells is required to reproduce. A working stock sample is added to a starter (which at this point is usually passage 3), the cells are then pitched into wort (passage 4), after fermentation, the yeast is harvested and repitched (passage 5), etc. Different strains can be passaged more/less times and still retain their characteristics.

steveahol said:

What about using frozen slurries as you’re master? Is there a problem in thawing and re-freezing every time you want to inoculate new working slants?

Click to expand...

You should not use a slurry as long term storage unless you wash it. You want to remove the healthy yeast from the trub. Freeze/thaw cycles reduce viability, which is why we aliquot our samples. You do not want to constantly freeze/thaw your master, you just want to thaw one working stock and step it up.

Yeast libraries are fun and useful, but also require very careful sterile technique. If you contaminate your master, everything down the line will be contaminated. Also, frozen stocks need to be kept as consistently cold as possible. -80C is industry standard, although I know homebrewers can't normally achieve this. Frost-free freezers have a warm-up cycle to remove the frost, this is not ideal for frozen cultures. Good luck,

Thanks for that great insight ColoHox! Sounds like you’re brewing commercially. Is that right?

ColoHox said:

We usually streak between 5-10 slants to maintain frozen master stocks of one particular strain.

Click to expand...

Thats interesting. I havn’t read anywere about freezing slants. How does that work? With frozen slurries I understand you mix in some glycerol as a cryoprotectant. How do you do that for a slant?

How long do frozen slants last?

steveahol said:

Thanks for that great insight ColoHox! Sounds like you’re brewing commercially. Is that right?

Thats interesting. I havn’t read anywere about freezing slants. How does that work? With frozen slurries I understand you mix in some glycerol as a cryoprotectant. How do you do that for a slant?

How long do frozen slants last?

Click to expand...

Sorry, that was misleading. That should have read "and" maintain frozen stocks. The slants are refrigerated, while a few are scraped into a cryovial (with glycerol of course) and frozen.