Author Topic: Yeast Slanting and Plating  (Read 5141 times)

Offline klickitat jim

  • I must live here
  • **********
  • Posts: 8604
Re: Yeast Slanting and Plating
« Reply #30 on: December 18, 2015, 05:21:59 PM »
Stpug, what volume are you freezing and what thickness of slurry? I'm considering using 250ml canning jars. I would mix 70ml water with 30ml glycerine per jar and pressure cook them 15min at pressure. I could let them cool while staying sterile that way. Then I plant to pitch one wyeast pack to 1L oxygenated wort and let that ferment out. When all done I'd swirl up the starter so its homogeneous and add 100ml to each water/glycerine jar, close lid finger tight and freeze. Once frozen I'd tighten the lids. This figures to 10 frozen cultures in 15% glycerine. Id store them in the freezer inside a small, thick walled Styrofoam cooler I have with a couple gell packs, to protect against the defrost cycle.

Does that sound right?

I've heard two thaw methods. Quick in 100F bath and pitch quickly. Or slow thaw two days in fridge then 1 day at room temp. I know you've said you use the quick method. Have you tried the other? If so what were results?

I also figured I would do a two step revival starter,  probably 500ml till fully fermented,  decant and then do my normal 1L oxygenated high krausen pitch. Sound about right?

I freeze in 50ml centrifuge tubes with collar (collar helps them stand). My process is fairly simple. Prepare a mix of 50/50 glycerin/water and sterilize to your personal requirements, and chill. Centrifuge tube is filled with ~31ml yeast slurry (fairly thick; assumed 90-100billion cells) and 13.5ml glycerin solution to produce a WhiteLabs-equivalent vial of yeast at ~15% glycerin content and enough head space for freeze expansion. Agitate enough to ensure good mixing. Directly into deep freeze. I should also mention (because I had the same question back when I started), the frozen vials freeze solid - they do not remain liquid.

I chose the 50ml centrifuge tubes because, when refrigerating yeast under beer I quickly found out how much space it can take. Once I got into freezing the yeast I wanted to be able to keep several strains on hand for future use. Quite honestly, if I were to do it again I might opt for smaller 30ml tubes and just plan two-step starters for each use just to save even more space. The 2-step starter process you have planned is a good way to check for any problems with your frozen strain since you can judge performance and fermentation characteristics during that first step, and then decide if you should continue with that frozen culture or not.

If your freezer has thaw cycles then your styrofoam cooler is helpful. Folks will often times include containers of isopropyl alcohol in the foam cooler to help stabilize the temperature during the thaw cycle.

I've used both methods of thawing yeast: Quick and slow (2 day fridge thaw). I did not see much (or any really) visible difference in how this affected the yeast propagation/lag/etc. Since I saw no difference, and recognize that the yeast begin using their reserves once they thaw, I opted for the quick method. The other benefit is no prior planning two days before making a starter.

As far as "freezing not being proper", while not the most proper way of preserving yeast for future use, I can definitely say that it works and works well. I wouldn't continue to do it if it wasn't reliable.

Wow, you are really generous with the info and time. Thanks

One last question. Is the slurry thickness just for desired pitch count, or does it have something to do with making it work? My idea would be a lot thinner. Maybe I should decant that 1L down to about 300ml before swirling, and just make 3 jars?

Offline narcout

  • Brewmaster General
  • *******
  • Posts: 2101
  • Los Angeles, CA
Re: Yeast Slanting and Plating
« Reply #31 on: December 18, 2015, 05:28:38 PM »
Mark what is your plate/slant medium recipe?

Mark, what is a decent one stop shop for a dozen glass plates and slants, probably a loop and alc flame too?

I think you can find the answer to both of your questions above in this thread (see replies #5 and #26): https://www.homebrewersassociation.org/forum/index.php?topic=24596.0

Also, does 5% w/v mean 5g dme to 100ml water?

Yes.
« Last Edit: December 18, 2015, 05:30:11 PM by narcout »
Sometimes you just can't get enough - JAMC

Offline stpug

  • Brewmaster
  • *****
  • Posts: 727
Re: Yeast Slanting and Plating
« Reply #32 on: December 18, 2015, 05:50:42 PM »
Wow, you are really generous with the info and time. Thanks

One last question. Is the slurry thickness just for desired pitch count, or does it have something to do with making it work? My idea would be a lot thinner. Maybe I should decant that 1L down to about 300ml before swirling, and just make 3 jars?

No worries. If I have information to share then I'll gladly do it, unlike some other folks (cough **rabeb** cough). I also recognize that I'm not as knowledgeable as Mark when it comes to maintaining lab-quality yeast cultures, and while the world of yeast isolation and culture purifying intrigues me, I'm just not willing to take the necessary steps to implement it in a way that I would be content with. I'm certain, Mark's methods of yeast handling are far superior to mine. I just look at my methods as "no worse than pitching slurry from one batch into another" (and probably better is some regards).

The slurry thickness I aim for is just to try to end up in the ballpark of 100billion cells ± 50billion :D. Sometimes I just want a single step starter and starting with near 100billion can get me where I want to be in one step.  There is nothing magic about the thickness of the slurry that makes it work/fail. The glycerin content will keep the ice crystals from forming sharp points thus rupturing the cell walls. I have a few vials (propagated from bottle dregs) that I estimate at much lower cell count (i.e. thinner slurry).

As far as what is "best" in terms of slurry thickness, decanting, etc..., I think it just comes down to your own wants and requirements. For me, it was about space savings and keeping many strains on hand. If you're only ever planning on a few frozen jars then there's no harm in using larger jars with thinner slurry, IMO.

If you want to test the freezing idea/method out before "pot committing" yourself then you can easily do so with a little bit of yeast slurry from any batch of beer. You don't even need to keep the process sterile since it's just a "proof of concept" test. Just pull enough from a yeastcake after racking your beer and put in a clean but non-sterile canning jar. Add some premix glycerin solution to it just like you would if you were doing it for real (again, no need to sterilize). Freeze it. Wait a week or four. Make up a small batch of starter wort and use your frozen yeast. Judge how it performs after being frozen (taking into account the lack of sterilization of the process). Again, just a proof of concept test if you will.

Offline Stevie

  • Official Poobah of No Life. (I Got Ban Hammered by Drew)
  • *********
  • Posts: 6858
Re: Yeast Slanting and Plating
« Reply #33 on: December 18, 2015, 05:57:49 PM »

If I have information to share then I'll gladly do it, unlike some other folks (cough **rabeb** cough).
Come on bro, don't stir the pot.

Offline stpug

  • Brewmaster
  • *****
  • Posts: 727
Re: Yeast Slanting and Plating
« Reply #34 on: December 18, 2015, 06:12:18 PM »

If I have information to share then I'll gladly do it, unlike some other folks (cough **rabeb** cough).
Come on bro, don't stir the pot.
Well, it's true. I'm happy to freely share my information, like Mark, and unlike rabab. Think of it however you like.

Offline Stevie

  • Official Poobah of No Life. (I Got Ban Hammered by Drew)
  • *********
  • Posts: 6858
Re: Yeast Slanting and Plating
« Reply #35 on: December 18, 2015, 06:20:10 PM »
That's great, but you don't need to call him out. That noise happened weeks ago, might as well have been years ago. Like me, I'm sure many had already dumped it from their memories.

Your yeast collection is pretty nuts. How many strains is that?

Offline stpug

  • Brewmaster
  • *****
  • Posts: 727
Re: Yeast Slanting and Plating
« Reply #36 on: December 18, 2015, 06:51:16 PM »
It's a matter of perspective I suppose. I see your point, but still....  :-X  ;D

It's 23 strains ATM. I culled the first ones I ever froze (US05 and S04) due to inconsistent fermentation end products (poor handling on my part), and there were a few more I culled that I initially held onto that I knew I'd never use again. It's probably time to pull a few more I don't think I'll ever get back to (MJ Newcastle Dark [terrible attenuator], Westmalle [would prefer to replace with 3787]). Nothing too spectacular in the collection since it just came to fruition a little more than three years ago (mostly WL, WY, MJack, Danstar strains plus a few from bottles). One of my favorites is Odell yeast harvested from a bottle of Mercenary IIPA about three years ago (amazing top cropping powdery strain that behaves much like WLP029 but much better for top cropping from); beers remain a bit hazy but worth it IMO.

Offline klickitat jim

  • I must live here
  • **********
  • Posts: 8604
Re: Yeast Slanting and Plating
« Reply #37 on: December 18, 2015, 07:02:14 PM »
Wow, you are really generous with the info and time. Thanks

One last question. Is the slurry thickness just for desired pitch count, or does it have something to do with making it work? My idea would be a lot thinner. Maybe I should decant that 1L down to about 300ml before swirling, and just make 3 jars?

No worries. If I have information to share then I'll gladly do it, unlike some other folks (cough **rabeb** cough). I also recognize that I'm not as knowledgeable as Mark when it comes to maintaining lab-quality yeast cultures, and while the world of yeast isolation and culture purifying intrigues me, I'm just not willing to take the necessary steps to implement it in a way that I would be content with. I'm certain, Mark's methods of yeast handling are far superior to mine. I just look at my methods as "no worse than pitching slurry from one batch into another" (and probably better is some regards).

The slurry thickness I aim for is just to try to end up in the ballpark of 100billion cells ± 50billion :D. Sometimes I just want a single step starter and starting with near 100billion can get me where I want to be in one step.  There is nothing magic about the thickness of the slurry that makes it work/fail. The glycerin content will keep the ice crystals from forming sharp points thus rupturing the cell walls. I have a few vials (propagated from bottle dregs) that I estimate at much lower cell count (i.e. thinner slurry).

As far as what is "best" in terms of slurry thickness, decanting, etc..., I think it just comes down to your own wants and requirements. For me, it was about space savings and keeping many strains on hand. If you're only ever planning on a few frozen jars then there's no harm in using larger jars with thinner slurry, IMO.

If you want to test the freezing idea/method out before "pot committing" yourself then you can easily do so with a little bit of yeast slurry from any batch of beer. You don't even need to keep the process sterile since it's just a "proof of concept" test. Just pull enough from a yeastcake after racking your beer and put in a clean but non-sterile canning jar. Add some premix glycerin solution to it just like you would if you were doing it for real (again, no need to sterilize). Freeze it. Wait a week or four. Make up a small batch of starter wort and use your frozen yeast. Judge how it performs after being frozen (taking into account the lack of sterilization of the process). Again, just a proof of concept test if you will.
Ok cool. Im going to do a side by side I think. One set of thick yeast cake slurry your method and one thin my way, seevif there's much diff other than I will need a baby step with the thin.

Anyone know how long normal robust strains are living on slants? I like this low tech freeze idea, but I can see that doing it the "real way" is in my future. I imagine they have to be streaked for singles, propped in a little wort and reslanted occasionally.

Disregard, found it on Marks thread...
« Last Edit: December 18, 2015, 07:47:36 PM by klickitat jim »

Offline klickitat jim

  • I must live here
  • **********
  • Posts: 8604
Re: Yeast Slanting and Plating
« Reply #38 on: December 18, 2015, 07:04:07 PM »
It am not crazy about the way that Bill suggests preparing plates.  Glass petri dishes should be dry sterilized in an oven at 177C/350F for 90 minutes with the media being autoclaved (pressure cooked), not boiled in a separate dish.  It makes no sense to autoclave the dishes and boil the media.   The media cannot be assumed to be vegetative cell or spore free going into the process.
Mark, what is a decent one stop shop for a dozen glass plates and slants, probably a loop and alc flame too?

I've been tempted to pick up something from these guys. They give some good information about harvesting wild yeast in your locale. They offer some lab equipment for plating yeast.

http://bootlegbiology.com/product-category/lab-equipment/
Took a peak. Their website gives me the heeby jeebies iD theft wise

Offline klickitat jim

  • I must live here
  • **********
  • Posts: 8604
Re: Yeast Slanting and Plating
« Reply #39 on: December 18, 2015, 07:46:19 PM »
Mark what is your plate/slant medium recipe?

Mark, what is a decent one stop shop for a dozen glass plates and slants, probably a loop and alc flame too?

I think you can find the answer to both of your questions above in this thread (see replies #5 and #26): https://www.homebrewersassociation.org/forum/index.php?topic=24596.0

Also, does 5% w/v mean 5g dme to 100ml water?

Yes.
Thanks! Found everything I need.

S. cerevisiae

  • Guest
Re: Yeast Slanting and Plating
« Reply #40 on: December 21, 2015, 02:52:31 AM »
How long a culture will live on slant is strain dependent.  My yeast bank was so large at one point that I routinely pushed things out to two years or more if I did not care if I lost the strain.   I spend so much on a yeast cultures today that I am unwilling to push the edge of the envelope.  One can afford to push things with cultures that cost $6.00.  However, one cannot afford to do so with cultures that cost at least $100.00 (I have three cultures in my bank that pushed $200.00 each by the time shipping and import duties were added to the equation).

With that said, I no longer collect and maintain yeast cultures that can be obtained via the home brew trade.  My time is more valuable than the cost of a commercial yeast culture.  In my humble opinion, the cultures that one should collect are those that are difficult or impossible to obtain.  For the home brewer who is unwilling to lay out the big bucks for culture collection cultures, strains from the vault and platinum/private collection strains are worth banking.  Brewery strains are also worth banking, and so are strains native to one's location.  Unless one lives in the absolute sticks where everything has to be ordered, banking standard production liquid cultures will get old quickly.
« Last Edit: December 21, 2015, 03:14:02 AM by S. cerevisiae »