Guide to Making a Frozen Yeast Bank
- Thread starter FlyGuy
- Start date
Help Support Homebrew Talk - Beer, Wine, Mead, & Cider Brewing Discussion Forum:
OP
OP
FlyGuy
Well-Known Member
That's really hard to guess at what went wrong. You can try a 10% glycerine solution instead. Also make sure that your glycerine solution is at room temp when you combine it with the yeast. One more thing to try is a small starter (100 - 200 mL is good), and ensure that the gravity of the wort is around 1.030.
One quick question -- how fast did you thaw the yeast? If you did it slowly, you can try quick-thawing it in some warm water.
Other than that, it is really hard to say what could have happened. Hopefully some trial and error will help you to determine what step in your process can be corrected.
TristanL
Well-Known Member
That's really hard to guess at what went wrong. You can try a 10% glycerine solution instead. Also make sure that your glycerine solution is at room temp when you combine it with the yeast. One more thing to try is a small starter (100 - 200 mL is good), and ensure that the gravity of the wort is around 1.030.
One quick question -- how fast did you thaw the yeast? If you did it slowly, you can try quick-thawing it in some warm water.
Other than that, it is really hard to say what could have happened. Hopefully some trial and error will help you to determine what step in your process can be corrected.
Thanks for the reply! I just tried a different sample in a 250 ML starter. I pulled the sample out, wrapped it in paper towel and let it thaw in the fridge all morning. That night I took it out and put it in the starter wort (which had also been in the fridge). I let it sit on the stir plate for 24 hours. I saw what looked like a krausen ring on the starter vessel. I just took a hydrometer sample. It only attenuated down to 1.044 from 1.048. It looks like there is a little more yeast than what I started with but it's really hard to tell. I assume the yeast weren't healthy enough to do the job.
I have the samples sitting in a soft cooler, a lunch bag type. It's got a thin layer of insulation and the samples are surrounded by ice packs. I'm thinking maybe this isn't insulated enough and the frost free cycle killed all my samples? From what I've read it would seem more likely that this would be the case.
I'm going to try using a 50ML starter at 1.020 and giving that a shot. I'll bump it up to 500ML and then give that another 24 hours and take a reading. I hope that some of the yeast is still viable.
If not, I'm going to let everything thaw, dump the samples, soak the tubes in PBW and try again with some more harvested yeast. This time inside a better cooler, like a foam type.
I don't feel this was a waste of start wort. I just put the wort into jars and put them in the pressure cooker to re-sterilize. I figure, the dead yeast will be good nutrients and the wort still has plenty of sugars in it!
mordantly
Well-Known Member
- Joined
- May 6, 2008
- Messages
- 3,863
- Reaction score
- 16
does anyone use beakers when building up the cells on a stir plate? im trying to figure out the best way to cover them in a sanitary fashioned. the first step-up i use a 250mL with a 1000mL over it, but once i step again, i don't have anything glass that can be autoclaved to keep on it.
OP
OP
FlyGuy
Well-Known Member
It sounds to me like your yeast viability coming out of the freezing process is too low/negligible. Try freezing a new batch and cut back on the amount of glycerine this time. Make sure to mix thoroughly. And try warming the frozen vials in some lukewarm water to quickly thaw them instead of slowly. All of that may help.
And yes, I think you are correct about the starter wort. Just re-can (i.e. re-sterilize) them and they can easily be reused. Good thinking.
OP
OP
FlyGuy
Well-Known Member
Just cover it in tin foil before you pop it in the pressure cooker. Leave a good length of tin foil to cover over the lip of the beaker, and you will be good to go.
conpewter
Well-Known Member
Alright so I stopped by the LHBS yesterday and finally got the sticker shock from the dry yeast price hike. Last batch I bought was $1.25 each, this time it was right around $3.00 each. I usually stock up by getting 6 or so of S-05 and same of S-04, not yesterday!
So what would be the best way to culture dry yeast? I know you shouldn't use a starter with dry yeast, but in this case I think you'd want to. I also could just put a little dry yeast in each vial, add water and glycerin but I don't think that would produce healthy yeast. I figure they need to have nutrients and food before getting frozen.
I could also just make a starter each time from 1/4 of a packet but I'd be worried about the yeast viability on an open package.
P.S. Where is the best place to get a reasonable sized glass pipet (10 ml or so) with a bulb that an both be autoclaved (pressure cooked) My current setup is a graduated turkey baster, but it's plastic so it only gets a star-san bath, and is hard to fit in the flask at an angle.
So what would be the best way to culture dry yeast? I know you shouldn't use a starter with dry yeast, but in this case I think you'd want to. I also could just put a little dry yeast in each vial, add water and glycerin but I don't think that would produce healthy yeast. I figure they need to have nutrients and food before getting frozen.
I could also just make a starter each time from 1/4 of a packet but I'd be worried about the yeast viability on an open package.
P.S. Where is the best place to get a reasonable sized glass pipet (10 ml or so) with a bulb that an both be autoclaved (pressure cooked) My current setup is a graduated turkey baster, but it's plastic so it only gets a star-san bath, and is hard to fit in the flask at an angle.
Hey pewter, I built a bank of s-04 and their dry wheat as well. I used half of the package to make a 1L starter at 1.04 OG . When it was complete I chilled it down in the frig, poured off the wort, and stepped it up again with 1.04. I then followed the steps using water and glycerin. For making the bank I used 30ml glass vials with screwcaps. I didn't use a pipette. Instead I got a set of glass lab measuring cups and used them for pouring into the vials. I've made starters with both the S04- and with rogue pac man and had amazing results with the finished product. In fact, I've been able to take a 30ml vial and make three starters out of them.
My 2 cents
My 2 cents
OP
OP
FlyGuy
Well-Known Member
I haven't seen the answer to this anywhere, but I would tend to agree with you. Using yeast dry inside the vial probably won't work well at all. You need to start with healthy, rehydrated yeast and then add it to the vial with glycerine and water.
OR, skip all that, and just put a small amount of dry yeast in each sterile vial, and store that. Honestly, I think that would work just fine (and it would be a LOT easier!).
FlyGuy, great post! I have one question. I don't have the freezer space at this point. Instead of any cooler, do you think wrapping the refreezable ice bags around vials and wrap with a bugi cord will work?
OP
OP
FlyGuy
Well-Known Member
I don't think it would work as well as using a cooler, but I bet it would work. You just might not see the same length of yeast viability.
I did exactly this for a while. It will work, just make sure you get completely around the vials, including the top and bottom.
I don't think it would work as well as using a cooler, but I bet it would work. You just might not see the same length of yeast viability.
Appreciate it much, you too Scimmia
camiller
Well-Known Member
I don't think it would work as well as using a cooler, but I bet it would work. You just might not see the same length of yeast viability.
Appreciate it much, you too Scimmia
And I'd put the whole thing inside a ziplock, the bag will keep interaction with the air in the freezer during the defrost cycle to a minimum.
Hermit
fuddle
How cold can you freeze yeast without damaging them without taking special precautions?
OP
OP
FlyGuy
Well-Known Member
None. The transition to a frozen state is what damages them.
Once frozen, they stay preserved best at the coldest temperatures (e.g., labs store yeast at around -80).
Hermit
fuddle
Are you saying this is at 0C?
OP
OP
FlyGuy
Well-Known Member
Actually, I believe the most damage occurs just below that (by a few degrees). That is why allowing the yeast to freeze then thaw then freeze then thaw (e.g., with a frost-free freezer) you can really do them harm.
Hey FlyGuy,
What type of pipette do you use? I have everything except the yeast collector, I was thinking of getting a graduated glass pipette and bulb. The one I'm looking at (10mL) is about 14" long. Tough to fit in the pressure cooker.
Other than the turkey baster, what are people using for this?
What type of pipette do you use? I have everything except the yeast collector, I was thinking of getting a graduated glass pipette and bulb. The one I'm looking at (10mL) is about 14" long. Tough to fit in the pressure cooker.
Other than the turkey baster, what are people using for this?
OP
OP
FlyGuy
Well-Known Member
I am using a graduated glass pipette and a rubber bulb now. I have a big 5.5 gal pressure cooker now, and the pipette fits inside.
The turkey baster actually worked OK though.
The turkey baster actually worked OK though.
g-brewer
Member
I spent a long time lurking and learning from everyone on the forum. I've searched the 26 pages of this thread but still have a question!
I've tried this twice now, and each time I get far less yeast in the vial than I think I should. There was so little the first time I dumped the vials. I have a batch in the pre-freeze refrigeration period now that looks marginally better, but I'm only seeing about 1/8 inch in most (I'm using 16ml vials). There still appears to be some yeast suspended, but I dont think there's going to be a significant increase.
I've been using a pipette to draw from the bottom of my starter flask. It looked good in the pipette, and it all stayed suspended in the vial initially.
I don't want to freeze a bunch of flat beer and glycerin holding a useless amount of yeast. I'm not clear on this: Should I be seeing a significant layer of yeast settling on the bottom like I do on my crash-cooled starters, and if so how deep might I expect it to be?
Thanks - not just for this, but for all the great info everyone shares!
I've tried this twice now, and each time I get far less yeast in the vial than I think I should. There was so little the first time I dumped the vials. I have a batch in the pre-freeze refrigeration period now that looks marginally better, but I'm only seeing about 1/8 inch in most (I'm using 16ml vials). There still appears to be some yeast suspended, but I dont think there's going to be a significant increase.
I've been using a pipette to draw from the bottom of my starter flask. It looked good in the pipette, and it all stayed suspended in the vial initially.
I don't want to freeze a bunch of flat beer and glycerin holding a useless amount of yeast. I'm not clear on this: Should I be seeing a significant layer of yeast settling on the bottom like I do on my crash-cooled starters, and if so how deep might I expect it to be?
Thanks - not just for this, but for all the great info everyone shares!
Yep, you won't be able to "overfreeze" them without some special steps. The freeze/thaw cycle just exposes them to more of the cell-wall bursting impact of water in the solution. This is why we use glycerin in the "preservation" steps prior to freezing. Additionally, once you get it frozen, as long as you keep it frozen, it will last just about forever. Long-term storage in -80C freezers is what labs generally do, Now, you won't get anywhere near that in a home chest freezer or side-by-side.
You want to take any and all steps to keep it from thawing during the auto-defrost cycles. Use heavy duty styrofoam insulated coolers and it's best if you can get your hands on the lab/medical grade ones. You can usually find them around hospitals, labs, etc and you can usually get them without much convincing.
Hey Guys,
First off, Flyguy you rock! Thank you so much for the great information.
I wanted to say that I followed the steps and made a successful frozen yeast bank. I just thawed and made my first starter and was wondering something. I went straight from my vial to about 1 litre of wort and put it on my stir plate and let it go. It fermented fantastically and once pitched it seems to be working great.
I was wondering if I made a mistake by going straight from the vial to 1 litre of wort rather than incrementing up. I read that someone else said it was alright and I realize I should have asked before I did it. I know I can just wait a couple weeks til bottling time and figure it out for myself I just thought I would see everyone else's experience and if I can just keep pitching from vial in the future.
They are 16ml vials btw.
Cheers
First off, Flyguy you rock! Thank you so much for the great information.
I wanted to say that I followed the steps and made a successful frozen yeast bank. I just thawed and made my first starter and was wondering something. I went straight from my vial to about 1 litre of wort and put it on my stir plate and let it go. It fermented fantastically and once pitched it seems to be working great.
I was wondering if I made a mistake by going straight from the vial to 1 litre of wort rather than incrementing up. I read that someone else said it was alright and I realize I should have asked before I did it. I know I can just wait a couple weeks til bottling time and figure it out for myself I just thought I would see everyone else's experience and if I can just keep pitching from vial in the future.
They are 16ml vials btw.
Cheers
archiefl98
Well-Known Member
groosh:
I use 30ml vials and pitch them directly into 1L-2L of wort on a stir plate. I have no science for you, just saying that I've never had a problem with it. I'm not really sure what stepping up allows me. I figure if I'm continually aerating the wort on a plate, then the yeast are going to reproduce and eat sugar -- why not feed 'em a bunch of it?
Perhaps I need to find more info on stepping up a starter myself.
I use 30ml vials and pitch them directly into 1L-2L of wort on a stir plate. I have no science for you, just saying that I've never had a problem with it. I'm not really sure what stepping up allows me. I figure if I'm continually aerating the wort on a plate, then the yeast are going to reproduce and eat sugar -- why not feed 'em a bunch of it?
Perhaps I need to find more info on stepping up a starter myself.
From a vial, start with less wort. I wouldn't start with more than 0.5 L. Get the alcohol content built up as fast as possible in that starter to protect from bacterial infection. Larger wort = longer time to 2% alcohol = more risk. Step up, repeat until desired volume is reached.
It really depends on how many viable cells are in that vial. How long has it been in storage and how many viable cells were in the original sampling (and quality of your technique)...
Step in measures of no more than 10x volume until you reach .5L or so and then in steps of 4x.
As pawn said, you want to get a good active fermentation going ASAP and don't overwhelm the yeast (and give bacteria a chance to gain a foothold).
With my method of using just scrapings of frozen cells, I have to go from a tiny starter (10 ml) and then step up from there.
If the samples are pretty fresh with a decent amount of yeast, I go from the vial to a 500 ml starter to whatever starter size I need. If they've been stored a while, I put in an extra step with a 150 ml starter.
OP
OP
FlyGuy
Well-Known Member
I have had great success starting with a 125 ml (1/2 cup) starter from a frozen vial, then adding 500 ml more of wort, then another 1 L or so to get it to pitching strength.
The exact reason is not clear (I have asked around and not received a clear response, anyways), but you get a lot more yeast by stepping up in the manner above than by pitching a frozen vial directly into 1625 ml of fresh wort. You also make it more difficult for contaminants to take hold by stepping up, as well.
The exact reason is not clear (I have asked around and not received a clear response, anyways), but you get a lot more yeast by stepping up in the manner above than by pitching a frozen vial directly into 1625 ml of fresh wort. You also make it more difficult for contaminants to take hold by stepping up, as well.
Hermit
fuddle
I saw a reference to stepping up once that claimed something like 10x jumps until a certain point. Then it had to slow down some.
OP
OP
FlyGuy
Well-Known Member
Yep, it is four posts up on this page!
Seriously, that is what I had heard as well. This is apparently an empirical relationship, however, because I have yet to find an adequate explanation for it anywhere (by that, I mean an explanation of the mechanics of the process from which this pattern arises).
Hermit
fuddle
Yep, it is four posts up on this page!
Seriously, that is what I had heard as well. This is apparently an empirical relationship, however, because I have yet to find an adequate explanation for it anywhere (by that, I mean an explanation of the mechanics of the process from which this pattern arises).
I glanced over that and missed it. Opps.
Have you tried emailing White or Wyeast? They may be willing to give some insight. I don't remember seeing much on their web sites about that but that has been a while, and as the above shows, I'm not that sharp today.
Yep, it is four posts up on this page!
Seriously, that is what I had heard as well. This is apparently an empirical relationship, however, because I have yet to find an adequate explanation for it anywhere (by that, I mean an explanation of the mechanics of the process from which this pattern arises).
This, from the very excellent paper by MB Raines on the Maltose Falcons website:
"Although a 100-fold dilution or step-up seems reasonable for yeast propagation, most breweries (or at least what is taught by brewing schools) advocate even smaller dilutions. The Siebel Institute recommends that yeast be stepped up in 8-fold increments, the British Brewing schools recommend 10-fold increments, and the German Brewing schools recommend 4-fold increments."
Read the paper for a lot of insight into the step-up sizes, about halfway down.
http://www.maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices
Hermit
fuddle
OP
OP
FlyGuy
Well-Known Member
Yep. I emailed Wyeast, White Labs, and even Jamil Zainasheff who is a (self-proclaimed) yeast expert. None really had a good explanation -- all they could quote are the empirical relations like what you mentioned in your next post. No substantive explanation for why, though.
My curiosity eventually died out and I dropped it.
I'll ask the resident yeast geneticist when I get home tonight and see if she has any explanation.
OK, had a good talk with the YGWMBO (Yeast Geneticist Who Must be Obeyed) tonight about this topic. I got some of the same vague responses alluded to above, but I think we got down to the heart of the issue:
The stepped growing methodology is used for both yeast and bacterial growing techniques in labs (I hadn't considered growing bacteria for some reason, but it seems to be universal and it makes sense in isolated strains, so thought I'd mention it). You're trying to grow a single strain genetic colony and avoid infection from wild type, infection, or to have a colony that outcompetes any mutation.
From a practical homebrew/brewery standpoint, the key is to keep a healthy density of active cells (and high enough alcohol content) in order to not only prevent infection via alcohol content but also for the yeast to out-compete any incoming nasties.
In order to do this, you can't just pitch 1 ml of active cells in dilution into 1L of wort. The yeast has a lag phase that is generally the same no matter what the size of the starter is, so that phase can generally be ignored in the process since it depends on the viability of your sample and how "off" it has been turned by your storage technique and length of time. (Lag phase is the time between exposing the cells to the growth media and the point at which exponential colony growth is occurring)
In order to meet an optical density and alcohol content to keep out infection, you need to build in stages. Each stage keeps a solid base yeast density and builds to something that totally wipes out the competition and you start again. This will help you get to the desired number of cells for your pitch target without a huge lag time between the end of the cell colony's lag phase and the point at which it has reached a viable colony density and alcohol content to push out it's competition.
Hope this helps in the discussion.
Oh, and for background info and to make us brewers feel better, even in lab settings where sterile practices are maintained and everything is autoclaved, done in updraft hoods, etc, infections still occurs. Additionally, mutation is something that we should generally not be concerned with. Mutations happen all the time in a colony and the occurrence of a mutation that is both viable and able to compete with the primary strain is something SO rare that you're more likely to win the lottery. For this reason, I think some of the concern about using 4th, 5th, etc generation yeast cultures for brewing is overblown and irrelevant as long as you keep a healthy number of viable cells and you're able to store properly.
The stepped growing methodology is used for both yeast and bacterial growing techniques in labs (I hadn't considered growing bacteria for some reason, but it seems to be universal and it makes sense in isolated strains, so thought I'd mention it). You're trying to grow a single strain genetic colony and avoid infection from wild type, infection, or to have a colony that outcompetes any mutation.
From a practical homebrew/brewery standpoint, the key is to keep a healthy density of active cells (and high enough alcohol content) in order to not only prevent infection via alcohol content but also for the yeast to out-compete any incoming nasties.
In order to do this, you can't just pitch 1 ml of active cells in dilution into 1L of wort. The yeast has a lag phase that is generally the same no matter what the size of the starter is, so that phase can generally be ignored in the process since it depends on the viability of your sample and how "off" it has been turned by your storage technique and length of time. (Lag phase is the time between exposing the cells to the growth media and the point at which exponential colony growth is occurring)
In order to meet an optical density and alcohol content to keep out infection, you need to build in stages. Each stage keeps a solid base yeast density and builds to something that totally wipes out the competition and you start again. This will help you get to the desired number of cells for your pitch target without a huge lag time between the end of the cell colony's lag phase and the point at which it has reached a viable colony density and alcohol content to push out it's competition.
Hope this helps in the discussion.
Oh, and for background info and to make us brewers feel better, even in lab settings where sterile practices are maintained and everything is autoclaved, done in updraft hoods, etc, infections still occurs. Additionally, mutation is something that we should generally not be concerned with. Mutations happen all the time in a colony and the occurrence of a mutation that is both viable and able to compete with the primary strain is something SO rare that you're more likely to win the lottery. For this reason, I think some of the concern about using 4th, 5th, etc generation yeast cultures for brewing is overblown and irrelevant as long as you keep a healthy number of viable cells and you're able to store properly.
Hermit
fuddle
So the numbers are basically what have worked for others and is largely dependent on sanitation techniques?
Roughly, yes. I don't think there is a "right" number and it is simply a matter of keeping the colony healthy and exponentially growing in a contaminant-free media.
I think if you use a stir plate and practice good sanitation there is no fear to be had in continual 8-10x steps.
OP
OP
FlyGuy
Well-Known Member
Thanks Randa, but I don't think this really answers the question. I believe most that pitching an appropriate amount of yeast, relative to the volume of wort you are fermenting, is necessary to avoid infection.
The original question that has yet to be answered is why a stepped regime of yeast growth will produce MORE yeast than one single pitch in the same (total) volume of wort? It is possible to grow yeast from a single pitch in a large volume without detectable contamination, so I doubt that competition for resources with other bugs/yeast has anything to do with it.
The original question that has yet to be answered is why a stepped regime of yeast growth will produce MORE yeast than one single pitch in the same (total) volume of wort? It is possible to grow yeast from a single pitch in a large volume without detectable contamination, so I doubt that competition for resources with other bugs/yeast has anything to do with it.
brewmatersmike
Well-Known Member
If anyone is interested, I just got a bunch of 16 x 125mm (15ml) glass tubes with caps. I ordered WAY more than I really need. Give me a PM if you are interested.
Similar threads
