Guide to Making a Frozen Yeast Bank

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those are for RNA and DNA and pre-sterilized which makes them much more expensive and not needed for this application. If you have a pressure cooker just sterlize them yourself and order 02-681-258 from fisher (Pack of 500 for $23.83)
 
as a side not if you would not like to sterlize them yourself you can use these 02-681-5 they are sterile and individualy wrapped so you can use them as needed without special handeling. Pack of 250 for $18.13
 
those are for RNA and DNA and pre-sterilized which makes them much more expensive and not needed for this application. If you have a pressure cooker just sterlize them yourself and order 02-681-258 from fisher (Pack of 500 for $23.83)

THANK YOU! This is why i posted rather than just ordering...so these won't melt in the pressure cooker? I did a little reading and found the PET ones are not autoclavable. But these are polypro. Any issue with the snap caps instead of screw on caps? don't want them to snap open and spill...

500 for $23 is much better than $100 for 240 of the glass ones!
 
the snap caps do not open for common use. every science lab uses these tubes for various applications. When freezing in -80 your suppose to keep cells in screw cap Cryogenic Vials which are much more expensive. However, with these tubes if you store them at -20 C they will work perfect. If you store them at -80 C let them warm up slowly and you will be fine. Screw caps are designed to be used for fast thaw and freeze such as submersion in N2.
 
Thanks ekjohns, very much appreciated!

So i have another question..more about starters than anything else...
Looking at mr malty calculator it says I need 323 billion yeast cells & 5.23L for my starter for the beer i'm brewing this weekend (1.090 OG Belgian Noel from AHS)

the other beer we're brewing this weekend a DFH60min says i need 219billion cells & a 1L starter. How do i know i have enough cells?
 
if your are buying yeast from wyeast or white labs there is a date on each. wyeasts date is the date it was packaged, white labs date is 3 months after it was packaged (if the date is march 1st it was packaged Jan 1st). Use those dates to calculate how big of a starter you need. Dont worry about the actual number of cells
 
Newbie here.....haven't read through the 40 plus pages here

Would it be possible to store the yeast in used 12oz soft drink bottles? Would dunking them in starsan be enough sanitation?
 
Newbie here.....haven't read through the 40 plus pages here

Would it be possible to store the yeast in used 12oz soft drink bottles? Would dunking them in starsan be enough sanitation?

Nope. Because we are working with small amounts of yeast everything need to be sterlized to prevent bac taking hold. If your wanting large volumes use 12oz canning jars which can be sterilized
 
with these 2mL containers...how small of a starter do we need? Should we pitch two at a time? How much glycerin are you guys putting in the 2ml tubes prior to adding yeast? the OP was using 15mL tubes...and using 2mL of gylcerin
 
Not sure why the op would autoclave the smaller containers if the yeast is coming from a non-autoclaved starter. I'd probably suggest autoclaving that original starter. My current practice is to divide a store bought vial into ten or so sterile tubes. A few days before brewing, 100mL then 1L then whatever else I need for the batch. Sure you don't have the starter to make a batch from the get go, but you'd still have to make one for the remaining 9 of ten vials, and you don't have to divide up the original tube on a schedule that coincides with brewday.
 
with these 2mL containers...how small of a starter do we need? Should we pitch two at a time? How much glycerin are you guys putting in the 2ml tubes prior to adding yeast? the OP was using 15mL tubes...and using 2mL of gylcerin

Pitch one at a time to save your stocks. First step should be something like 50x-100x ratio. So for 1 ml of slurry in a 2 ml tube, somewhere between 50-100 ml starter on a stir plate for your first step and then go from there.

You should make a 50% dilution of glycerin with water and sterilize this in your pressure cooker. Then mix this solution 1:1 with the yeast slurry. Jamil/White also recommend 1% ascorbic acid in the glycerin solution. So in your final tube, you should have 50% Yeast Slurry, 24.75% water, 24.75% gycerin, and 0.5% ascorbic acid.
 
the snap caps do not open for common use. every science lab uses these tubes for various applications. When freezing in -80 your suppose to keep cells in screw cap Cryogenic Vials which are much more expensive. However, with these tubes if you store them at -20 C they will work perfect. If you store them at -80 C let them warm up slowly and you will be fine. Screw caps are designed to be used for fast thaw and freeze such as submersion in N2.

In the -20 you may have some slow gas pressure created in the tube which could force the tube open. It's your personal call on whether to take this risk or not in your own storage system. I wouldn't recommend the snap cap tubes without expressing that warning first.
 
In the -20 you may have some slow gas pressure created in the tube which could force the tube open. It's your personal call on whether to take this risk or not in your own storage system. I wouldn't recommend the snap cap tubes without expressing that warning first.

Gas pressure from where? If there are no sugars in the slurry, where would the gas come from?
 
Gas pressure from where? If there are no sugars in the slurry, where would the gas come from?

well i had one snap cap pop open on me, so there was obviously some sugars in there the yeast decided to eat, in such a small tube a little bit goes a long way.

I bought the 2ml tubes from fischer scientific, and i wouldn't buy them again for this use...the amounts are so miniscule i have a hard time believing i'll have enough yeast in my bank to actually get anything going. I guess we'll find out in a few weeks when i go to use one of these strains i have saved.
 
I work in a biochem lab and at any given time we have upwards of 500-1000 snap caps in the -80 any i never have one open. If you are freezing these they will not be active and no gas should form. If you are worried about it just wrap the top of the cap in some parafilm. If you dont have any just get some plastic food wrap and pull it tight around the cap. problem solved
 
Gas pressure from where? If there are no sugars in the slurry, where would the gas come from?

Yeast "slurry" is yeast + wort (which is mostly or nearly completely fermented) but there may still be some small amount of residual sugars and activity. They also are not frozen at -20 and it would not be entirely shocking if they were not completely in stationary phase when frozen given most methods I have seen here.

In the end you're trying to preserve the yeast as best as possible. Why run the risk with snap caps if you don't have to?
 
I work in a biochem lab and at any given time we have upwards of 500-1000 snap caps in the -80 any i never have one open. If you are freezing these they will not be active and no gas should form. If you are worried about it just wrap the top of the cap in some parafilm. If you dont have any just get some plastic food wrap and pull it tight around the cap. problem solved

-80 is a different animal than -20.

And SWMBO is not only in a biochem lab, but she is a yeast geneticist who does "yeast ranching" every day. They use screw caps for anything being stored longer than a few days.
 
I bought the 2ml tubes from fischer scientific, and i wouldn't buy them again for this use...the amounts are so miniscule i have a hard time believing i'll have enough yeast in my bank to actually get anything going.


You will be fine as long as you used good sanitation and storage practices and pitch into a healthy wort. If you are worried about it, start with a smaller initial starter like 50 ml.

Trust me, a 0.5-1.0ml pellet of yeast is more than enough to revive and build to a brewing starter in a reasonable period of time.
 
i would assume the gases would most likely come from trapped CO2. I still think that plastic wrap or parafilm will hold fine, but as always its up to the user to decide. FWIW i just checked all my frozen yeast in -80C in snap caps all still sealed. The ones Im using have a designed lid to help keep them closed. Your dirt cheap run of the mill tubes will not have a lip on the cap, but the ones mentioned on here to have such a cap
 
Trust me, a 0.5-1.0ml pellet of yeast is more than enough to revive and build to a brewing starter in a reasonable period of time.


It is general practice to use small colonies that are VERY small to grow up large amounts so I agree with Randar. However, as he also said with a 50X step up everything needs to be sterilzed. I personally have 14 ml screw cap vials I sterilze wort in as will pitch a 1 ml frozen vial into that to get the yeast going out of the freezer then do a 100ml to 1L
 
Chris White, in his recent book' suggests that lagering below 40F means your yeast won't be doing much. At 0F I'd suspect even less activity since they won't even survive that temp without special precautions. Post above made me wonder if it is possible that there is some differential contraction between the tube and the lid? Are these tubes with the lids coming off stored upright, thus requiring an internal force to move it?
 
ive stored mine both upright and on occasion when im lazy on their sides. Still have never had a problem with them. Personaly, I dont think it has anything to do with yeast activity. I think its just trapped CO2 that may get released with handeling. I take these same tubes with 1 ml in them heat the for 5 min at 100 C and I will get some to pop open cause if pressure so they are not impossible to get the open with pressure. I personally have never had one pop in the freezer but that may be just me.
 
ive stored mine both upright and on occasion when im lazy on their sides. Still have never had a problem with them. Personaly, I dont think it has anything to do with yeast activity. I think its just trapped CO2 that may get released with handeling. I take these same tubes with 1 ml in them heat the for 5 min at 100 C and I will get some to pop open cause if pressure so they are not impossible to get the open with pressure. I personally have never had one pop in the freezer but that may be just me.

Per your previous posts, you are only using a -80 (which few have access to), which will freeze them solid. At -20 (like most homebrewers have access to) they are not frozen solid.

Not sure if it makes a difference, but we need to keep the context for the normal yeast rancher at home.
 
Per your previous posts, you are only using a -80 (which few have access to), which will freeze them solid. At -20 (like most homebrewers have access to) they are not frozen solid.

Not sure if it makes a difference, but we need to keep the context for the normal yeast rancher at home.

good call! So add a $10,000 -80 C freezer to the shopping list (j/k). I still think if you give it some wraps with some parafilm ($5 for a huge roll) it will hold. Im even kind enough to link you to ebay for the parafilm... go ME!

http://compare.ebay.com/like/310211...fc506e5&itemid=310211166839&ff4=263602_309572
 
I really like the variety and quality of liquid yeasts that are available today, but I sure don’t like paying $10 for a smack pack of yeast that I will only use once. It is by far my most expensive ingredient. Also, fresh liquid yeast can be tough to find at my LHBS, so I really wanted to have a bank of yeast on hand for whenever I needed it. Some quick research on the internet revealed that building a yeast bank in your freezer is a pretty easy thing to do, and the equipment required is minimal. The advantage is that you can easily cut the cost of liquid yeast by at least 90%, and you can have a good variety of yeast on hand whenever you need it. Below is the method that I have been using with great success.

Here is a picture illustrating the items I use (clockwise from top):
- 2000 mL Pyrex flask and stir-plate for propagating yeast (optional)
- pressure cooker (poor man's autoclave) for sterilizing equipment
- 500 mL beaker for holding glass vials
- 15 mL (1/2 oz) flat-bottomed glass vials with autoclavable screw tops
- small glass graduated cylinder
- glycerine
- autoclavable pipette (not shown)
- spray bottle filled with Star San solution (not shown)

7346-Pict1531.jpg


Hi, I tried to PM you but your mailbox is full!

Hi, Thanks for the detailed explanation about the frozen yeast bank. I have 2 questions if you don't mind.
1- Where did you buy the 15 mL (1/2 oz) flat-bottomed glass vials with autoclavable screw tops? The ones I fond are very expensive, like $4-5 per vial, is that right?
2- Can I take the yeast sample from a fermented 5-gal primary instead of taking it out from the starter?

Thanks!
 
1- you can get them from scientific stores (www.cynmar.com #115-27908) or www.containerstore.com (look for coupons).

2 - Yes but there is a higher risk of infection since the fermenter was only sanitized and the amount of handleing is much greater to get yeast from a ferm. From the starter everything is cleaner and handled less. Save yourself even more time and when you buy yeast freeze down 10+ vials from the smack pack (or white labs tube) and you have sterilized everything with much less risk of infection and just make a bigger starter. What I do is use www.mrmalty.com to calculate the % viability then determine how many mL i can take out to freeze down while keeping my starter under 2L. Last time i did this i froze down 10 vials (1 ml of conc. yeast) of WLP028 and made a 1.5L starter.
 
1- you can get them from scientific stores (www.cynmar.com #115-27908) or www.containerstore.com (look for coupons).

2 - Yes but there is a higher risk of infection since the fermenter was only sanitized and the amount of handleing is much greater to get yeast from a ferm. From the starter everything is cleaner and handled less. Save yourself even more time and when you buy yeast freeze down 10+ vials from the smack pack (or white labs tube) and you have sterilized everything with much less risk of infection and just make a bigger starter. What I do is use www.mrmalty.com to calculate the % viability then determine how many mL i can take out to freeze down while keeping my starter under 2L. Last time i did this i froze down 10 vials (1 ml of conc. yeast) of WLP028 and made a 1.5L starter.


Thanks. The prices at cynmar are much cheaper compared to what I originally found! Excellent!

About question 2. The starter jar is just sanitized too just like the fermenter, but what you said makes sense. Less handling and less hassle, therefore lower chances of infection.

So, you froze down 10 vials with 1 ml of concentrated yeast per vial, with a total of 10 ml of concentrated yeast, did I get it correctly?

Thanks!
 
So, you froze down 10 vials with 1 ml of concentrated yeast per vial, with a total of 10 ml of concentrated yeast, did I get it correctly?


For frozen yeast storage there is really no reason to have 15 ml stocks. You can use microcentrifuge tubes (they make screw top ones) in the 1.5 to 2 ml range. In that way, you can take dense slurry culture straight from the smack pack or vial and into the glycerol solution/freezer.

Since this thread started, a great book called... wait for it... "YEAST" by Chris White and Jamil has been written and has all the info and methods for doing this at home. It gives more of the biology than what is covered in this thread (and is probably much more concise given the length of this thread)
 
For frozen yeast storage there is really no reason to have 15 ml stocks. You can use microcentrifuge tubes (they make screw top ones) in the 1.5 to 2 ml range. In that way, you can take dense slurry culture straight from the smack pack or vial and into the glycerol solution/freezer.

Since this thread started, a great book called... wait for it... "YEAST" by Chris White and Jamil has been written and has all the info and methods for doing this at home. It gives more of the biology than what is covered in this thread (and is probably much more concise given the length of this thread)

Really? Straight from the smack pack? That would make the process a lot easier, but I wonder how many mls each smack pack contains. I wonder how many 2 ml microtubes of yeast can be taken while allowing enough yeast to make a viable starter. Are you sure about this?

Besides, once your smart pack is gone, how can I keep a stock of the yeast frozen always available? At some point, I would have to culture the contents of the microtubes into a starter to increase the amount of yeast anyway.
 
10, 1 ml vials of yeast. I think the white labs tubes are 35 ml. Therefore If i have 100% viability (via mrmalty) and I take out 10ml I have in theory 57% viability left (20/35). So I freeze down 10 ml and calculate my starter size off of 57% viability.
 
Taking from the starter would probably increase the health/viability of what you store, no? Wouldn't the larger sample size also make a viable sample more likely the longer you kept it?
 
wyeast smack packs are 125ml (im assuming after the nutrient pack as been broken). Follow my instructions above for calculating size of starter needed after removing the amount to freeze down.
 
wyeast smack packs are 125ml (im assuming after the nutrient pack as been broken). Follow my instructions above for calculating size of starter needed after removing the amount to freeze down.

I see. I think I got it. So, you freeze 1ml of yeast in each vial taken straight from the smack pack or other liquid yeast. Then, you enter whatever you have left in the smack pack at Mrmalty to calculate the volume of starter. So, you don't realy take your yeast samples from the starter, you take them from the smack pack! Is that correct?
 
I see value in both methods (taking from smack pack or taking from starter).

In reality, the WL and WY yeast that is stored in those packs is fully uncontaminated and in stationary phase with trehalose built up. This is the perfect "state" you want the yeast in when you are going to freeze/store them. But you also have no idea how long they have been in there or what the storage/shipping conditions did to them before you got them, so in that regard, making a starter and rebuilding a suitable "active" culture has value. Also, capturing 8 or 10 1ml samples off a WY or WL smack pack has its drawbacks of reducing "what is left" for your initial brewday (although you should be making a starter so it's should be that big an issue if you adjust starter sizes/steps/etc)

On the other, you are running the risk of contamination by doing a starter and freezing off this sample. You are also not going to be centrifuging down the samples to get very high density cultures (like what is in the original WY and WL packets)...

So there are pros and cons of both methods, IMO. With WL I prefer to take straight from the dense slurry. For WYeast I prefer to smack the pack, let it grow out to completion, and then put in fridge for 2 days to allow trehalose and stationary phase to be back to suitable points and then open and capture yeast for the frozen vials.
 
I see. I think I got it. So, you freeze 1ml of yeast in each vial taken straight from the smack pack or other liquid yeast. Then, you enter whatever you have left in the smack pack at Mrmalty to calculate the volume of starter. So, you don't realy take your yeast samples from the starter, you take them from the smack pack! Is that correct?

Yes.

1 ml of yeast slurry goes into tube. Then I add 1ml of the sterilized glycerol/water/ascorbic acid solution as prescribed in the book. Shake the hell out of it and into the freezer they go.
 
Yes.

1 ml of yeast slurry goes into tube. Then I add 1ml of the sterilized glycerol/water/ascorbic acid solution as prescribed in the book. Shake the hell out of it and into the freezer they go.

Great, I will definitively consider buying the book.

What I don't understand tough is this:

Supposed I take 10 mls out of the 125 ml smack pack and freeze them into 10 separate tubes containing 1ml of yeast each (plus 1 ml of the solution, which acts like a vehicle/buffer to keep the yeast viable I think).

So, the remaining 115 mls in the smack pack can easily be propagated in a fresh starter before pitching, I got that.

But, how about the 10 tubes of frozen yeast? Even if you combine them, you end up with only 10 mls of yeast, which seems like it would never have enough viable cells to ignite a starter? Does that make sense?
 

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