Dead frozen yeast bank?

[gmmoran]

First, apologies for the long post. TL/DR at the bottom.

I think I may be having issues with my frozen yeast bank. I started it a few months back and have built up a few strains before trying any of the vials. Three days ago I made a yeast starter and pulled a vial from the frozen bank. I took it out of the freezer and let it warm to room temp and then pitched into the starter. Two days later there were no signs of activity, no visible changes, no bubbles, no smell change, nothing. So, I pulled a second vial and put it into the fridge for a few hours and then up to room temp and pitched it, about 12hrs ago. As of this morning, still no change at all. My thinking with the second vial was maybe the first one warmed up too quickly. My next step is to thaw the remaining vials for this yeast strain and pitch them all to see if any of them survived.

At this point I’m thinking my yeast bank is dead and that something in my process killed the yeast. Here is my process for the freeing of the yeast (I’ve followed the Homebrew Challenge YouTube channel process). Any thoughts on where I went wrong are greatly appreciated. I have a few things that I think could be the issue. As a note, everything is sanitized thoroughly with star san through all the steps.

• Make a yeast starter from a store-bought pack of yeast. Cold crash and decant like normal. The starter is made from starter concentrate that is sterilized through pressure canning and store-bought distilled water to an OG of about 1.040.
• To a 10ml vial add 5ml of yeast slurry to 5ml of 30/70 glycerin-water mixture. The water is filtered fridge water and the glycerin-water mixture is sterilized through pressure canning along with my starter concentrate.
• Gently rock the vials back and forth to mix.
  • <I think I could have more thoroughly mixed the vials here by shanking it up.>
• Place sealed vials into a container filled with rubbing alcohol up to the liquid level of the vials. Put container of vials and rubbing alcohol into the freezer overnight.
  • <Should I put them into the fridge first for a few hours to slowly cool before going into the freezer?>
• The next day pull the vials out of the alcohol and place into long-term storage in the freezer.

And then when starting a new yeast starter:

• Pull vial out of freezer and let warm up to room temperature.
  • <Should I move from freezer to fridge then to room temp to more slowly warm up the vial?>

Those three points are the only places I can think of where the process failed and caused the yeast to die. I’ll try the other strains to see if maybe it was just a one-time issue but so far things are looking bleak.

Again, any help or thoughts you might have are greatly appreciated. Thank you!

TL/DR: My frozen yeast appears to be dead. Should I shake it like it owes me money? Or treat it all kind and gentle like?

 

[gmmoran]

I may how found the issue. I think my freezer has an auto-defrost "feature". That will kill the frozen yeast. I'm running a test with a glass of water and then will put a coin on it to see if the coin moves down over time... Shoot! Guess I'm starting the frozen bank over again.

 

[RM-MN]

I think all refrigerator freezers have an auto defrost and have had that feature for more than30 years. You probably need a chest freezer for a yeast bank. It will probably be more cost effective to buy new yeast every time you need it.

 

[cyberbackpacker]

An alternative to buying a chest freezer: put your frozen vials inside of a cooler with ice packs surrounding the vials inside of your current fridge/freezer combo. This mitigates the warming that occurs during the autodefrost cycle.

 

[Stand]

Your process looks fine. even if you have a freezer that auto-defrosts you can still just put vials in an insulated box/bag. That is a good idea to slow the cooling when you put them in as well.

I would ditch the water/glycerin mixture. I just fill the vials to 20% with glycerin and pressure cook them. Yeast is mostly water, so you will hit the right levels and it makes one less thing to sterilize.

How much wort are you pitching into? I do 50ml vials and I do 300ml starter and then step up to 3 liters. That works great.

I have have been freezing for a couple years, and it seems like sometimes the process just doesn't work right for me either.

Certain yeasts I have just pulled from my bank and tossed because they wouldn't come back.

 

[mashpaddled]

The auto-defrost is a problem. As the posts above say, you can employ other tools to maintain freezing. It's a real commitment to yeast banking to switch out ice packs regularly.

Do you have a friend with a chest freezer that might be willing to offer up a little space to you? That would be the easiest way to resolve this problem.

 

[Stand]

With extra thermal mass (I have some lunch box freezer blocks) and decent insulation (foam box etc.) you can easily spread out the spikes you will get from auto-defrosts.

 

[deuc224]

Frozen yeast can take a bit to start. Ive had a strain take 4 days to wake up and multiply, also dont thaw the yeast out, put it in a cup of room temp water and it should be ready in about 5 minutes. Thawing can kill the yeast if done slow.

 

[IslandLizard]

Frozen yeast can take a bit to start. Ive had a strain take 4 days to wake up and multiply

Yup, that^
Give it more time.

BTW, how much starter wort did you use to awake your 5 ml frozen yeast slurry?

In that light, your frozen yeast cultures may fair better by using a 1.010 starter wort (instead of 1.040) at an appropriately (small) volume for the first step.

It all depends on the condition of the yeast slurry, the number of yeast cells, age, partially defrosting/refreezing cycles while in frozen storage, etc.

 

[Brooothru]

An alternative to buying a chest freezer: put your frozen vials inside of a cooler with ice packs surrounding the vials inside of your current fridge/freezer combo. This mitigates the warming that occurs during the autodefrost cycle.

^^^THIS^^^

Exactly my process. I bought a small insulated lunch bag. After freezing the yeast/glycerin, put the vials in the lunch bag and surround them with frozen gel packs. The vial will stay frozen during defrost cycles. You can also place frozen packs (or frozen foods in the freezer) around the outside of the lunch bag to further keep it cold.

I've been ranching like this for 3-4 years, and it works. Don't forget to place something frozen underneath the lunch bag since the bottom of the freezer box is often defrosted (warmed) during the DF cycle.

Brooo Brother

 

[ba-brewer]

No yeast freezing experience but do have slants. If you froze only 5mL of yeast that is a pretty small amount of yeast to start with and even less assume some may die from the freeze and thaw.

Using slants I start off with a 20mL step of 1020 wort and I assume at the end I only 1B cells. Normally takes 12 to 18hours to really get going( manually shaken).

I then do a 100mL step of 1040 on a stir plate. I do further step on the stirplate after that and try to make sure the inoculation rate for those are between 25 and 50 million/ml to get good growth for the steps. Takes a few steps to get a pitchable amount but the smaller steps are good for judging if you have growth.

I like the Brewunited yeast calculator as it has best practices incorporated into the interface and will highlight items that are too big or small.

edit: I believe the rule of thumb for yeast slurry density is 4.5B/mL so 5mL would be maybe 20Bcells to start. Assuming you loose only half from freezing a first step of 200mL to 500mL would be reasonable. If freezing actually kills off more, than 100mL first step might be better.