Recent content by Forkhead

I thawed some yeast that had been frozen in either 7.5%, 15%, or 30% final concentration of glycerol (v/v). 15% had the best viability and 7.5 & 30% had significantly worse viability. I would strongly recommend everyone to be using 15% v/v final concentration of glycerol.

That's a beautiful paper that agrees with what we've seen experimentally. Slow freezing, fast thawing is the best for viability. The difference between w/v and v/v is not very large. I doubt it will make a substantial difference. W/v will always be a higher % glycerol than v/v because...

Haha, yeah it's all part of the process. Smooth sailing from here on out (fingers crossed).

Apparently styrofoam coolers are NOT a good choice for holding isopropyl alcohol. I had a styrofoam cooler filled with isopropanol in the freezer since the weekend and when I came back to it today all the isopropanol had leaked out and made a huge mess. I had a somewhat similar thing happen...

I've been using v/v

I think that's a safe assumption as long as you're starting your culture at a concentration higher than 20e6/ml, as illustrated when you express the data as below.:mug:

I agree that if you're doing a starter, it shouldn't matter much. However, I've been doing some playing around with the Wyeast calculator and have generated the following graph on the top showing the 'Expected Fold Expansion' vs. 'Starting Cell Concentration'. I think this is the most useful...

Very nice write-up. Thanks for spending the time and effort! I've only done the freezing once, but I was surprised to read that you can get 100 billion cells to fit into a volume of 25ml. When I centrifuged my slurry to pellet the cells, 30 billion took up 15 ml. I would've guessed 100...

What's the consensus on the best way scale up cultures? Should I just add fresh wort to the culture, or is it better to let the yeast drop out and dump the supernatant first? I feel like it's better to drop the yeast and dump the supernatant, but if so, is it best to put it in the fridge or...

OK, thanks for the info! I'm growing up another culture to repeat the freeze/thaw. I'd like to optimize the procedure a little bit. I'll try water+glycerol instead of wort+glycerol. I'm also going to test a few other parameters.

What are people mixing the glycerol with to freeze? I used fresh wort because I was worried about osmotic stress, but it was a pain because the yeast was making CO2 when they warmed up and made things very messy. I actually had a yeast bomb go off in my apartment!!! What a disaster! Will...

Sound like your food fridge is perfect. Getting a cooler filled with isopropanol will help protect the cells from temperature fluctuations.

It's possible they froze faster. I'm thinking it'd be best to freeze them at -20C for a few days then move them to -80C. Alternatively, it's possible the colder temperature can more thoroughly freeze the tubes and may cause more ice crystals to form. That was actually predicted by that paper I...

When I did my freezing, I actually centrifuged the cells and removed the liquid before adding the glycerol solutions. As a result the yeast wasn't really a slurry and the glycerol wasn't diluted very much by residual water. If your yeast is actually a slurry then it might be better to use a...

If you're going to use 15% glycerol then you'll want to let the cells settle out and decant as much of the liquid off the top as possible. If you're slurry is quite watery, then you'll want to increase the % glycerol.