Starting a small yeast bank

[Jhedrick83]

I brew infrequently and small batches. Due to life events I haven’t brewed since the first weeks of the year. I just had a window to brew in and all I was lacking was the yeast. All my Pure Pitch packs expired in May. That got me thinking. I have all the gear for making a starter (that was supposed to be the next skill I was going to develop this year before life got in the way. Why don’t I take some time to make a small yeast bank of the strains I use the most. Honestly, probably just a lot of WLP 530 and some wlp540/wlp500. I’m thinking go following the guidelines set out in (Frozen Stock)

Maintaining A Healthy Yeast Bank Long Term

I do have a few questions though that I’m hoping you all can give some guidance on.

1. I’d be making the starter that gets split into the vials straight from a Pure Pitch pack. Would you adjust how big a started you make based on the age of the pack?

2. The cell counts of those starter vials that end up being frozen seem low for someone who is making high gravity Belgians. As long as the ratios stay the same, any issue with cranking up the volumes? Obviously means more math for me though.

3. Does it have to be frozen to -20? My wife keeps our freezer a bit warmer.

4. Any suggested places for the equipment like vials and glycerin?

 

[Sammy86]

1. I’d be making the starter that gets split into the vials straight from a Pure Pitch pack. Would you adjust how big a started you make based on the age of the pack?

I would make a starter and over build then separate into viles. I would use a yeast calculator like :Homebrew Dad's Online Yeast Starter Calculator

2. The cell counts of those starter vials that end up being frozen seem low for someone who is making high gravity Belgians. As long as the ratios stay the same, any issue with cranking up the volumes? Obviously means more math for me though.

No clue, sorry I can't help.

3. Does it have to be frozen to -20? My wife keeps our freezer a bit warmer.

Colder the better!

4. Any suggested places for the equipment like vials and glycerin?

Amazon I know has viles, as far as glycerin I'm not sure.

 

[MajorJC]

I'm planning on the same thing after reading that same article. I have already received all of the supplies from Amazon except for the glycerin. The gallon jug of glycerin has gone missing in UPS system twice so far. I have placed a third order and I have my fingers crossed.

I ordered the vials that he suggested in the article from Amazon. Globe Scientific 6285 Polypropylene Centrifuge Tube with Attached Blue Flat Top Screw Cap, Sterile, Printed Graduation, Bag Pack, 15mL Capacity (Case of 500): Science Lab Centrifuge Tubes: Amazon.com: Industrial & Scientific

I'm actually planning on inoculating some wort/agar petri dishes from my yeast pack on brew day to grow some colonies and then build them up incrementally like in the article.

To grow a starter from the plate, take the sterilized loop and scoop a sample from the slant, twist it around in 2-5 ml of wort. Grow the wort for a couple of days, and incrementally build that volume into your target starter volume, not exceeding a 1:10 dilution (for example, take the 5ml starter, dump it into a 50ml starter and grow, and dump that into a 500ml starter, etc).

Then I'll use this purified yeast culture to fill my sterile centrifuge tubes with half yeast/half glycerin for the freezer. I've got some vials to make slant tubes with also, just don't know if there would be any benefit to making slants also.

This will be my first time to try anything like this.

3. Does it have to be frozen to -20? My wife keeps our freezer a bit warmer.

I do know that a frost free freezer will not be good for your frozen yeast. As far as the optimal storage temperature, I would tend to believe Pete Geisen, click on his name and look at his references on LinkedIn. I checked my deep freeze and in the bottom my temp is -24.5°C (-12.2°F) and at the top level it is -20.8°C (-5.5°F).

 

[Jhedrick83]

Offhand, does anyone know the volume in a pure pitch pack? Just thinking of the alternative of taking a pure pitch pack, doing a 2L starter, splitting it in half and freezing each half as a “pitch able” amount for each brew. I typically need 1.5-2 pure pitch packs for each brew day. I’d have to double check the math on rough cell count but that may simplify things for me since I don’t need tons and tons of yeast in the bank.

 

[Sammy86]

Offhand, does anyone know the volume in a pure pitch pack? Just thinking of the alternative of taking a pure pitch pack, doing a 2L starter, splitting it in half and freezing each half as a “pitch able” amount for each brew. I typically need 1.5-2 pure pitch packs for each brew day. I’d have to double check the math on rough cell count but that may simplify things for me since I don’t need tons and tons of yeast in the bank.

 

[Jhedrick83]

Thanks, I kept finding the next gen sizes. Then if I got 100ml vials, that’s theoretically enough for nearly the volume of two packs plus the 12.5% glycerin mix.

 

[CascadesBrewer]

I brew infrequently and small batches.

Have you thought about using more dry yeasts? After many years of using mostly liquid yeast strains, in 2021 I started a deep dive into using dry yeast. I have been really impressed with the quality and selection of dry yeasts on the market these days. I don't have an issue with stocking up on packs of dry yeast that I will use a year or more later. For small batches, I will use a partial pack and reseal the rest.

WLP530 is one of the liquid yeasts that I have been using (along with WLP565...I often harvest and repitch yeast for a dozen or so batches). I want to try out more Belgian dry yeasts, but I was very happy with the Belgian Blond that I made with Lallemand Abbaye. A split batch to compare WLP530 vs Lallemand Abbaye is on my to do list, but I am pretty confident I could make a solid Trappist style beer with Lallemand Abbaye.

 

[Jhedrick83]

Have you thought about using more dry yeasts? After many years of using mostly liquid yeast strains, in 2021 I started a deep dive into using dry yeast. I have been really impressed with the quality and selection of dry yeasts on the market these days. I don't have an issue with stocking up on packs of dry yeast that I will use a year or more later. For small batches, I will use a partial pack and reseal the rest.

WLP530 is one of the liquid yeasts that I have been using (along with WLP565...I often harvest and repitch yeast for a dozen or so batches). I want to try out more Belgian dry yeasts, but I was very happy with the Belgian Blond that I made with Lallemand Abbaye. A split batch to compare WLP530 vs Lallemand Abbaye is on my to do list, but I am pretty confident I could make a solid Trappist style beer with Lallemand Abbaye.

I have thought about trying the Lallemand Abbaye but was unsure how it compared to the 530. I’m so used to how my beers taste with 530 I’m hesitant to try something new. Creature of habit I guess. For all I know though, I may like it better. A question that may merit its own thread.

 

[IslandLizard]

Just thinking of the alternative of taking a pure pitch pack, doing a 2L starter, splitting it in half and freezing each half as a “pitch able” amount for each brew.

Thanks, I kept finding the next gen sizes. Then if I got 100ml vials, that’s theoretically enough for nearly the volume of two packs plus the 12.5% glycerin mix.

I'm pretty sure you cannot freeze such large amounts of the yeast/glycerine mixture in a single vial. IIRC, 10-15 ml total is about the maximum volume you want to freeze per vial. You would then build them back up to pitchable amounts, in 3 or 4 steps.

Although the yeast will propagate fine from a single 10-15 ml vial, you could use 3 or 4 of those vials, to get started with a higher initial cell count. But in the end it doesn't make all that much difference compared to starting with only a single vial. Do the math.

From what I gathered it's also best to pack all your vials together inside a styrofoam box or better yet, in a box lined with freezer packs, to prevent (large) temp swings, or worse, defrost then refreeze again. Such a box is probably mostly useful when storing in an auto-defrost freezer. In the bottom of a chest freezer temp swings are usually nonexistent, so any box will be fine.

 

[Jhedrick83]

I'm pretty sure you cannot freeze such large amounts of the yeast/glycerine mixture in a single vial. IIRC, 10-15 ml total is about the maximum volume you want to freeze per vial. You would then build them back up to pitchable amounts, in 3 or 4 steps.

Although the yeast will propagate fine from a single 10-15 ml vial, you could use 3 or 4 of those vials, to get started with a higher initial cell count. But in the end it doesn't make all that much difference compared to starting with only a single vial. Do the math.

From what I gathered it's also best to pack all your vials together inside a styrofoam box or better yet, in a box lined with freezer packs, to prevent (large) temp swings, or worse, defrost then refreeze again. Such a box is probably mostly useful when storing in an auto-defrost freezer. In the bottom of a chest freezer temp swings are usually nonexistent, so any box will be fine.

Ah. Well that may/may not be a problem as what I run in to is that I can’t really plan a brew day more than maybe a couple days in advance. So the time to take a small vial through a few starter rounds may end up just being a waste of time. I was hoping to do larger vial and be able to thaw 1-2 a day or two in advance to drop.

 

[IslandLizard]

I was hoping to do larger vial and be able to thaw 1-2 a day or two in advance to drop.

You definitely need to make a starter from frozen yeast, it cannot be pitched directly, no matter how many vials you'd pitch.

Instead of using one or two big 100ml vials, you can make a starter using 10 or 20 (small) 10-15 ml vials, to build it up faster, possibly in one step.
Not sure if 2 days is enough time, though. The freezing process itself is hard on the yeast, there's significant cell loss. To reduce osmotic pressure it's recommended to use lower gravity, 1.010-1.020, starter wort for the first round coming from the freezer.

 

[Taket_al_Tauro]

I'm pretty sure you cannot freeze such large amounts of the yeast/glycerine mixture in a single vial. IIRC, 10-15 ml total is about the maximum volume you want to freeze per vial. You would then build them back up to pitchable amounts, in 3 or 4 steps.

Why shouldn't it work with volumes larger than 10-15 ml? I regularly freeze in small, 30 ml glass bottles and it works perfectly. That is about double your maximum stated volume. I chose to go with a slightly higher volume because I usually collect from undecanted starters, where cell density isn't very high. To make sure I have at least enough cells in my sample for freezing.
That said I cannot see the point of freezing much larger volumes either (such as 100 ml), since you'll need to perform a thorough step-up anyway...so that would be just a waste of yeast, glycerin and freezer space.
However, from a purely technical standpoint, why shouldn't this work? If I remember correctly, it is also recommended to freeze slowly, so freezing larger volumes could even be an advantage for that (at least theoretically).

 

[IslandLizard]

Why shouldn't it work with volumes larger than 10-15 ml?

I'm going by what I've read in Chris White's "Yeast" book and various online resources over the years. 15 ml vials are mentioned everywhere, filled to 80%.
Perhaps there are "mechanical complications," effectively reducing viability, when freezing culture sizes larger than 10-15 ml?

I regularly freeze in small, 30 ml glass bottles and it works perfectly.

Alright, that's good to know.

We have a large thread on freezing yeast cultures, worth reading/skimming:
https://www.homebrewtalk.com/threads/guide-to-making-a-frozen-yeast-bank.35891/

 

[mashpaddled]

The idea of a frozen yeast bank is that you're saving enough to grow back up, not saving enough for a pitch even in a small batch.

Honestly you could just save some of the pure pitch or a starter in a mason jar, keep it in your fridge and feed it a little wort three or four times a year to keep the cells alive. If you primarily only use two or three strains, it's probably more work building up the frozen slurry than maintaining a couple jars in your fridge.

 

[Jhedrick83]

The idea of a frozen yeast bank is that you're saving enough to grow back up, not saving enough for a pitch even in a small batch.

Honestly you could just save some of the pure pitch or a starter in a mason jar, keep it in your fridge and feed it a little wort three or four times a year to keep the cells alive. If you primarily only use two or three strains, it's probably more work building up the frozen slurry than maintaining a couple jars in your fridge.

A very good idea. Any guidance on feeding volumes? I assume the idea is to siphon off enough for a starter here and there as the colony grows. Is there a theoretical time limit where you start having a jar that’s more dead than alive?

I have that book and have it waiting for a long plane ride coming up.

 

[Taket_al_Tauro]

I'm going by what I've read in Chris White's "Yeast" book and various online resources over the years. 15 ml vials are mentioned everywhere, filled to 80%.
Perhaps there are "mechanical complications," effectively reducing viability, when freezing culture sizes larger than 10-15 ml?

Thanks for the clarifications. Just to be clear I did not want to be argumentative or anything. I was genuinely curious about the reasons as to why freezing volumes larger than 10-15 ml may be counterproductive. As said I had great results freezing about 30 ml but I'm open to reconsider my procedure if there are valid reasons to do so.
Yes that thread you linked to is definitely a great resource on this subject. I based myself a lot on that one, and a few other online blog articles when I originally got into freezing yeast samples.

 

[beren]

This is what I’m doing Maintaining A Healthy Yeast Bank Long Term and my first use worked well. I got glass vials as they were almost the same price and I can sterilize them. Must be careful they don’t break. One nice thing about this as you can get easily 10 or 20 batches per generation if you just wait until the last vial to make a new set.

 

[beren]

The idea of a frozen yeast bank is that you're saving enough to grow back up, not saving enough for a pitch even in a small batch.

Honestly you could just save some of the pure pitch or a starter in a mason jar, keep it in your fridge and feed it a little wort three or four times a year to keep the cells alive. If you primarily only use two or three strains, it's probably more work building up the frozen slurry than maintaining a couple jars in your fridge.

Most people say you can only repitch between five and eight generations though.

 

[IslandLizard]

Thanks for the clarifications. Just to be clear I did not want to be argumentative or anything.

YVW! You have a very valid question, whether you could freeze larger volumes of yeast with similar chances of success compared to the typically smaller volumes referred to in the "literature."

Maybe freeze some smaller amounts, as well as a few larger ones and perhaps compare the results? Would be awesome to report back on that experiment.

The main issue in the yeast freezing equation is to prevent the water it contains from freezing, creating crystals which damage the cell walls. The glycol component prevents that from happening, dropping the freezing temps, so the mixture remains a slush at -20°C. Not sure if it still applies when it gets chilled down to -60°C, which is the preferred storage temp, but perhaps it does.

 

[Taket_al_Tauro]

Maybe freeze some smaller amounts, as well as a few larger ones and perhaps compare the results? Would be awesome to report back on that experiment.

I do have both 20 ml and 30 ml vials at the moment in the freezer. However, not from the same yeast strains/batches. I may think about doing such a comparison next time I freeze yeast, but that won't happen very soon... I am planning on using my own cultures for a while now.

The main issue in the yeast freezing equation is to prevent the water it contains from freezing, creating crystals which damage the cell walls. The glycol component prevents that from happening, dropping the freezing temps, so the mixture remains a slush at -20°C. Not sure if it still applies when it gets chilled down to -60°C, which is the preferred storage temp, but perhaps it does.

I use a concentration of about 10 to 12 % glycerin in the final sample. At this concentration, my samples freeze solid in my normal household freezer (roughly -18 to -20 °C). Still, the glycerin must be doing its job of preventing the formation of ice crystals quite well, because up to now, I did not have a single sample failing me.
From what I read, the professional microbiologists with their -80 °C freezers go much higher on the glycerin concentration, something like at least 40 - 50 %, I believe. And at such concentrations, the culture may indeed not freeze solid even at those temperatures. If I recall correctly, a microbiologist friend of mine once told me that their cultures have roughly the consistency of a paste when they take them out of the freezer.
I chose the 10-12% concentration range because I read in one (or more) of those outlines that this is the preferred range if you are freezing in normal household freezers.
Maybe it would work also at much different concentrations, but given my results up to now, I was not willing to experiment any further...never change a running system, isn't it

 

[mashpaddled]

A very good idea. Any guidance on feeding volumes? I assume the idea is to siphon off enough for a starter here and there as the colony grows. Is there a theoretical time limit where you start having a jar that’s more dead than alive?

I have that book and have it waiting for a long plane ride coming up.

I feed mine like 10-20% volume with fresh wort like every 4-6 months. I shake up the mason jar with the lid on, then take the lid off, flame the mouth and pour out an amount equal to what I'm adding. If I'm pulling yeast to make a starter I take more and put more fresh wort in the jar. I leave the jars out of the fridge for a few days with the rings not completely sealed so gas can escape, then seal it and put it back in the fridge.

You will get some dying or dead cells but who cares. If cells rupture and give off nutrients to the living cells all the better.

Most people say you can only repitch between five and eight generations though.

But you're not repitching as much as maintaining the original pitch. The reasons repitching becomes an issue are primarily (1) picking up low level infections from imperfect cleaning and sanitation and (2) the yeast acclimating to your brewing system and practices. Here the yeast don't leave the jar and aren't getting enough of a change in environment to worry about either.

 

[jtgoral]

I'm pretty sure you cannot freeze such large amounts of the yeast/glycerine mixture in a single vial. IIRC, 10-15 ml total is about the maximum volume you want to freeze per vial. You would then build them back up to pitchable amounts, in 3 or 4 steps.

Although the yeast will propagate fine from a single 10-15 ml vial, you could use 3 or 4 of those vials, to get started with a higher initial cell count. But in the end it doesn't make all that much difference compared to starting with only a single vial. Do the math.

From what I gathered it's also best to pack all your vials together inside a styrofoam box or better yet, in a box lined with freezer packs, to prevent (large) temp swings, or worse, defrost then refreeze again. Such a box is probably mostly useful when storing in an auto-defrost freezer. In the bottom of a chest freezer temp swings are usually nonexistent, so any box will be fine.

I make a 1qt starter using 2x15ml vials. The starter is ready on 3rd day.