Slow Starters from Frozen yeast bank

[HiGravShawn]

So I stepped up to building a frozen yeast bank. I copied some steps from somewhere a while back (maybe HBT), but just tried harvesting from my last brew (Pacman). Basically I sanitize 15ml vials and lids, add about 3-4 ml glycerin. After racking off the cake I used a pipette to fill the vials to about 80% so I'm guessing maybe 8ml of slurry. Then freeze. To use, I pull it out in the morning to let it warm up 4-6 hours. Then I made a 1 cup starter with 2 oz DME (I only realized after the fact that it was 1.092 and I was supposed to do 1oz). Then after 24 hours I added 1L + 4.3oz DME (1.047). For a grand total of 1.055 which is still a bit high so maybe some stress there. I don't have a stirrer yet and just shake it regularly and keep a gel heating pack under it for warmth. The steps said to treat the larger starter like any other starter, so another 24-30 hours. After another 48 hours though, there is no krausen or bubbling in the airlock except when it's being shaken. There does seems to be an increasing white layer at the bottom when it settles.

Questions:
Is this sort of time table normal when coming from such a small amount of frozen yeast?
Could the high starter gravity cause a slower start?
Is there something I can do to improve speed this up without reducing quality?
How can you tell when your starter is ready?

 

[prosper]

some yeast seem to tolerate freezing better than others. 1968 seems to die pretty much instantly in the deep freezer, but 3724 appears entirely unaffected and comes out vital and healthy.

 

[Captain_Bigelow]

I have frozen several strains of yeast recently, but yet to unfreeze and use any. Based upon what I read I fully expect your starter to take longer. You will have a lot fewer cells in your vial, than when you purchase a regular vial of liquid yeast. I wouldn't be surprised if it were to take a week for your starter to be ready. But others who have experience doing so can chime in.

You can always add another vial to double your counts if you feel you are not getting much out of one vial.

 

[krops13]

OK, There are a few things I see you did wrong. First let me make it clear I have never frozen yeast or brought them back from the freezer. But were my qualification comes from many hours of talking to microbiologists, many many microbiologist. ( I work in a lab :rockin

Basically I sanitize 15ml vials and lids, add about 3-4 ml glycerin. After racking off the cake I used a pipette to fill the vials to about 80% so I'm guessing maybe 8ml of slurry.

I'm kinda of confused here. Are you adding 8ml of slurry to the 3-4 mls of glycerin, or are you adding 4ml of slurry to equal the total of 8mls. Ether way your using way to much glycerin. If its the first one your using 33% if the latter your using 50%. You want to ideally use 13%

To use, I pull it out in the morning to let it warm up 4-6 hours. Then I made a 1 cup starter with 2 oz DME (I only realized after the fact that it was 1.092 and I was supposed to do 1oz). Then after 24 hours I added 1L + 4.3oz DME (1.047). For a grand total of 1.055 which is still a bit high so maybe some stress there.

Your procedure is good just lower all your O.G. to 1.040. How would like to not eat for a month and then be told you have to eat an 12oz steak. You'd be sick. Also you didn't say you aerated your worts. This is a must, yeast use the oxygen to grow and reproduce. They don't grow during fermentation. I use a fish aquarium oxygen pump to diffusion stone in my flask, while on the stir plate.

I don't have a stirrer yet

Get one. Its not impossible to do so without one. But unrealistic.

and keep a gel heating pack under it for warmth.

Helps but unnecessary. And it only helps up to 86 degrees.

After another 48 hours though, there is no krausen or bubbling in the airlock except when it's being shaken.

Not really a bit deal you may have missed it. There fast. Like I said earlier. When I do mine I aerate it constantly while on the stir play. I don't get a krausen. After a pitch it takes about 2-3 hours for me to see signs of fermentation.

There does seems to be an increasing white layer at the bottom when it settles.

It shouldn't seem like an increasing layer. It should be just an increasing layer.

Sorry if I came off harsh. Your on the golf course, just using the wrong irons.

 

[HiGravShawn]

Thanks for the feedback, but while I do appreciate your help if you recognize you're coming off harsh before you post, why not just perform a bit of editing? And yes you sound a bit snippy there.

Anyway... I will try to reduce the amount of glycerin, the instructions I had said to add about 3ml of glycerin, keeping it less than a third of the way up the vial so you're 33% is a good estimate of what I shot for.

3ml glycerin + 20% expansion room left about 9ml for slurry.

It finally took off though. As of this morning it was very cloudy and while no real krausen there were lots of bubbles and when you swirl it tons more bubbles released so I went ahead and pitched. So that's about 4 days to start. I think you're right that simply hitting my gravity and getting a stir plate (SWMBO will love to here about that) will be enough to get it to more like 2 days. I just hope such an extensive propagation doesn't alter the yeast profile. Sounds like an experiment in the making! I'd like to brew the same beer with four different generations of a yeast to see how it differs.

 

[Revvy]

Actually you are looking for signs of starter activity that more often than not, isn't even there.

Just like in the fermenter, starter fermentation isn't always dynamic...It doesn't matter one blip in your fermenter or your starter flask if the airlock bubbles or not (if you are using an airlock and not tinfoil,) or if you see a krauzen. In fact starter fermentation are some of the fastest or slowest but most importantly, the most boring fermentations out there. Usually it's done withing a few hours of yeast pitch...usually overnight when we are sleeping, and the starter looks like nothing ever happened...except for the little band at the bottom. Or it can take awhile...but either way there's often no "activity" whatsoever....

I usually run my stirplate for the first 24 hours, then shut it down, if you are spinning your starter it is really hard to get a krausen to form anyway, since it's all spinning, and there's often a head of foam on it from the movement.


All that really matters is that creamy band o yeast at the bottom.

This is a chilled sample so it's flocculated, but even with an unchilled sample you should see a band of yeast at the bottom.

As it is I've only ever seen two or three krausens actually on my starter (one blew off a bunch of krausen and knocked the tinfoil off the flask,) and the evidence of one on the flask at the "waterline" once. But I've never not had a starter take off.

Look for the yeast at the bottom, don't worry what it looks like on top.

 

[HiGravShawn]

Good to know, thanks! When doing a starter from Smack pack I always had an active fermentation in the flask with a fairly obvious krausen and bubbling. I never use that to tell for normal fermentation I use the hydrometer, but only had the amount for starter I needed and didn't want to pull some for a reading.

 

[ArcaneXor]

It takes me about 5-6 days or so to get a full, large starter going from washed or frozen yeast - I generally step them up at least once to end up with a 2-l starter, which takes some time to ferment out. But it can take 2 days or so from taking the yeast out of the fridge/freezer until the first starter really shows activity.

 

[passedpawn]

I agree with everybody. There, that was easy. And get/build a stir plate.

 

[pjj2ba]

Most microbes don't start to grow real fast when pulled out of the freezer and put directly into liquid culture. In the lab, we take a freezer stock, and quickly take a small sample and streak it onto a plate. We don't let the sample thaw, that will lead to eventual death of the culture if done too often. One could also just as easily streak it out onto a slant tube if you don't have plates.

Let the culture grow on solid media for 2 days, and then step it up to a liquid culture. Since I work in a lab, what I actually do is I will keep the yeast on a plate (in the fridge) for a couple of months and work from that. I'll take a loop and grab a few colonies off the plate and inoculate a test tube containing about 5 ml of media. I'll grow that overnight on a shaker and the next day pour the tubes contents into my large flask. 24-48 hrs later my yeast are ready to go.

Every few months. I'll re-streak out a new plate. Yeast are much happier going from freezer to solid media to liquid media. It is fine to go from freezer to liquid, it just takes longer for the yeast to get situated and happy.

 

[krops13]

Yea I thought I did. It was a lot of info and my son was tuggin at my a leg, and swmbo needed help getting him ready to go. I was trying to squeeze it in real fast. Sorry for the smugness. not my intention.

I wish I had room in the freezer to switch my stock. I have everything but room. Stupid side by side.

Is this how you usually make a starter? Straight from frozen? I was thinking about doing like pjj2ba was saying. streak to a plate and use the frozen as a "master" stock. Less thawing and defrosting hopefully equaling less death. I love those little guys.

 

[Hasheronon]

When freeze/thawing: freeze slowly. There have been reports of increased viability by letting your yeast sit in the fridge for 4 days, then add glycerol (someone reported 13%, I've always heard 25% final - that would be 1ml glycerol to 3ml yeast culture). Then freeze. This allows the yeast to produce some chemicals that will help them to tolerate freezing better.

When thawing - thaw quickly. Take it out of the freezer and immediately into a warm water bath (not hot) and then pitch right away as soon as thawed.

Someone made a good point that they take colonies directly off of culture plates. That is a good idea. I don't think we need to subclone here though. Just make patches, and take a swipe. I could be wrong on this, but unless you know that the yeast you have on your streaks (subcloned) is exactly what you want, then patches would be better. Take a flat sterile toothpick and just smear your yeast on the plate where ever you want it. When ready to use, use a flat sterile toothpick to pick up a nice sized patch of cells (will look like a small booger on the end of the toothpick) and innoculate a SMALL culture. Ideally 10-50ml. Then go up from there.

If I ever get around to using that expensive liquid yeast, I plan on trying this stuff. (I also work in a biology science lab.) For now, I like Nottingham.