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General Category => Yeast and Fermentation => Topic started by: klickitat jim on December 17, 2015, 03:20:22 am

Title: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 03:20:22 am
My new favorite lager yeast is a special release, so im kind of being forced into learning how to do this. I looked up a BYO article. A couple things come to mind.

1.How do you clean and sterilize a 10ml pipette?

2. Is a pipette really necessary? Couldn't I just eye-ball it and pour into my slant? So, one 3rd full of water-glycerin mixture, then another 3rd of yeast slurry?

3. The article claims gelatin works in place of agar for plates. True or not? Admittedly I won't need plates to streak my wyeast sample, but I figure if I'm going to do this it will probably lead to streaking plates. Someday...

4. If to get started, all I was going to do is keep that one yeast on hand, can't I just use only slants? Leave plates out of it all together for now? For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

5. Glycerine? Like from Walmart? Or is there a special kind? Anything to avoid?

6. Recipes... The article called for
Slants: 250ml h2o and 100ml glycerin boiled 15 minutes. Cool and fill slants 1/3 full. Add yeast till 2/3 full. Place in a small Styrofoam cooler with ice packs into the freezer. Store up to a year. Thaw 2 days in fridge, 1 day room temp. Propagate in 100ml 48hrs, then step to 200ml 48 hrs.

Plates: 250ml h2o and 20g DME boil 15 min. Stir in 3g agar or gelatin,  boil 15 min more. Cool, pour into plates, when set store upside down. Streak with a loop. When ready to propogate select singles and prop in 10ml wort 48 hrs, then 100ml 48 hrs, then 200ml 48 hrs.

Is anything in that way off?

Thanks
Jim


Title: Re: Yeast Slanting and Plating
Post by: RPIScotty on December 17, 2015, 05:48:35 am
"Paging Mark. Mark please report to the forum please...."
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 05:50:23 am
I anticipate he will not be a fan of the 15% glycerin method, but who knows. If it works it would be perfect for what I need to do.
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 17, 2015, 08:29:01 am
It sounds to me like you've got an article that's hybridizing slanting with freezing. I may be wrong here (wouldn't be the first time :D) but slants are generally stored at refrigeration temperatures so that glycerin is not needed. Glycerin is used when freezing yeast to reduce the sharp-end crystalline structure such that it doesn't pierce the cell walls of the yeast - leaving them for dead upon thawing.

1. Rubbing alcohol (95%) will clean and sterilize a pipette.

2. If you're freezing, then probably eyeballing is sufficient as long as you're a good eye-baller  :o. If you're slanting then you should only be piercing the slant with your innoculant so no eye-balling is necessary.

3. I've also read/heard that gelatin can work in place or agar for slanting and plates, but it's also my understanding that (basically) a lifetime supply of agar-agar can be bought for a few dollars so why not just make the initial purchase (amazon, asian market, health food store, etc).

4. Plates are for a different purpose. If you're not concerned with isolating individual colonies and propagating them up then I do not see the need to worry about plates. If you do happen to go the direction of agar slants with autoclave and the works then you may as well get some plates for playing with too. If nothing else, swab the inside of your mouth and plate it out - just for fun :D

5. USP Glycerine is food grade as long as there is no other ingredient but glycerine. I believe the USP is important.

6. Again, this seems like methods of freezing yeast. It sounds like you'd come out with an ~14% glycerine yeast slurry which is in the "ideal" range for glycerine to prevent cell death due to ice crystals. Freezing is the method I use since I am not concerned (read: haven't take the steps) with ensuring a 99.9% clean yeast stock. I propagate enough extra yeast from the initial vial/pouch purchase such that I can freeze a couple vials of yeast for future use, and it gives me one to use now. If I didn't repitch then I would have three uses per yeast purchase, but I almost always repitch a few times. I figure it works out to be about 7-10 pitches per yeast purchase on the low end most times.

As you can tell, I'm not overly familiar with slants, plating, and agar-agar related processing or isolating yeast colonies. One day probably, but not today.

Freezing is the method of choice for me. I'm able to maintain a large collection of yeast in my frosty (not frost-free) deep freeze at about -10 to -20F. I have found the method to be sufficient for my needs and low-tech enough that I can deal with it without creating a complete yeast lab experience. I recently used a white labs PC yeast (3726-PC) that had been frozen for 2.5 years. It took about 24 hours to "wake up" in the starter, and after that was done propagating in another 24 hours. The resulting beer tastes like what I expect to get from that strain so I call it a success. The biggest thing I have noticed in resuscitating frozen yeast is the variable "wake up" time which can be from 1-3 days. My experience has been that the quicker the "wake up" the more likely the yeast is to perform as expected. Those that took 3 days to "wake up" were less stellar fermentations than those that only took a day. I think an important key in ensuring that the samples have a good dormancy is how they are treated just prior to freezing. Quick propagation without letting them overspin (stirplate here), and then quick crash, and then directly into freezer has worked best for me and shown to give the best "wake up" times.

Edit: I should add that when pulling a frozen vial from the freezer for use I will have the starter it's going into all ready (boiled, cooled, aerated, etc). Then I'll quickly thaw the yeast in warm (100F) water until the slush has just melted (about 5 minutes). Then it's immediately pitched into the starter. It's my understanding that as soon as those yeast cells come out of dormancy they will start using their reserves causing even more stress. I've opted for this method and it has worked for me.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 09:33:04 am
It sounds to me like you've got an article that's hybridizing slanting with freezing. I may be wrong here (wouldn't be the first time :D) but slants are generally stored at refrigeration temperatures so that glycerin is not needed. Glycerin is used when freezing yeast to reduce the sharp-end crystalline structure such that it doesn't pierce the cell walls of the yeast - leaving them for dead upon thawing.

1. Rubbing alcohol (95%) will clean and sterilize a pipette.

2. If you're freezing, then probably eyeballing is sufficient as long as you're a good eye-baller  :o. If you're slanting then you should only be piercing the slant with your innoculant so no eye-balling is necessary.

3. I've also read/heard that gelatin can work in place or agar for slanting and plates, but it's also my understanding that (basically) a lifetime supply of agar-agar can be bought for a few dollars so why not just make the initial purchase (amazon, asian market, health food store, etc).

4. Plates are for a different purpose. If you're not concerned with isolating individual colonies and propagating them up then I do not see the need to worry about plates. If you do happen to go the direction of agar slants with autoclave and the works then you may as well get some plates for playing with too. If nothing else, swab the inside of your mouth and plate it out - just for fun :D

5. USP Glycerine is food grade as long as there is no other ingredient but glycerine. I believe the USP is important.

6. Again, this seems like methods of freezing yeast. It sounds like you'd come out with an ~14% glycerine yeast slurry which is in the "ideal" range for glycerine to prevent cell death due to ice crystals. Freezing is the method I use since I am not concerned (read: haven't take the steps) with ensuring a 99.9% clean yeast stock. I propagate enough extra yeast from the initial vial/pouch purchase such that I can freeze a couple vials of yeast for future use, and it gives me one to use now. If I didn't repitch then I would have three uses per yeast purchase, but I almost always repitch a few times. I figure it works out to be about 7-10 pitches per yeast purchase on the low end most times.

As you can tell, I'm not overly familiar with slants, plating, and agar-agar related processing or isolating yeast colonies. One day probably, but not today.

Freezing is the method of choice for me. I'm able to maintain a large collection of yeast in my frosty (not frost-free) deep freeze at about -10 to -20F. I have found the method to be sufficient for my needs and low-tech enough that I can deal with it without creating a complete yeast lab experience. I recently used a white labs PC yeast (3726-PC) that had been frozen for 2.5 years. It took about 24 hours to "wake up" in the starter, and after that was done propagating in another 24 hours. The resulting beer tastes like what I expect to get from that strain so I call it a success. The biggest thing I have noticed in resuscitating frozen yeast is the variable "wake up" time which can be from 1-3 days. My experience has been that the quicker the "wake up" the more likely the yeast is to perform as expected. Those that took 3 days to "wake up" were less stellar fermentations than those that only took a day. I think an important key in ensuring that the samples have a good dormancy is how they are treated just prior to freezing. Quick propagation without letting them overspin (stirplate here), and then quick crash, and then directly into freezer has worked best for me and shown to give the best "wake up" times.

Edit: I should add that when pulling a frozen vial from the freezer for use I will have the starter it's going into all ready (boiled, cooled, aerated, etc). Then I'll quickly thaw the yeast in warm (100F) water until the slush has just melted (about 5 minutes). Then it's immediately pitched into the starter. It's my understanding that as soon as those yeast cells come out of dormancy they will start using their reserves causing even more stress. I've opted for this method and it has worked for me.
Just what I was looking for. I appreciate you taking the time. Im going to give this a shot.
Title: Re: Yeast Slanting and Plating
Post by: coolman26 on December 17, 2015, 09:48:30 am
What is your new favorite?  I really need to look into how to freeze yeast 
Title: Re: Yeast Slanting and Plating
Post by: charles1968 on December 17, 2015, 03:03:09 pm
For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

I'm no expert on this as I don't make yeast slants, but isn't the purpose of slants to step up or store tiny samples from a plate after isolating a pure culture? If you aren't isolating colonies from single cells from a streak, wouldn't it be simpler to store slurry under beer so that it remains anaerobic? I don't see any advantage in storing nonpurified yeast on agar, where it's exposed to air, rather than leaving it under beer. Except that slants take up less space than vials.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 04:17:20 pm
For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

I'm no expert on this as I don't make yeast slants, but isn't the purpose of slants to step up or store tiny samples from a plate after isolating a pure culture? If you aren't isolating colonies from single cells from a streak, wouldn't it be simpler to store slurry under beer so that it remains anaerobic? I don't see any advantage in storing nonpurified yeast on agar, where it's exposed to air, rather than leaving it under beer. Except that slants take up less space than vials.
Long term storage, like year or years
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 04:17:35 pm
What is your new favorite?  I really need to look into how to freeze yeast
2352
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 04:45:53 pm
The limited release ends soon so I ordered 5 packs today, fresh from Odell. I'm going to make a starter with one pack just to grow it up for storage and use this 15% glycerine method. I'll report back next fall on how well it worked.
Title: Re: Yeast Slanting and Plating
Post by: charles1968 on December 17, 2015, 05:11:55 pm
For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

I'm no expert on this as I don't make yeast slants, but isn't the purpose of slants to step up or store tiny samples from a plate after isolating a pure culture? If you aren't isolating colonies from single cells from a streak, wouldn't it be simpler to store slurry under beer so that it remains anaerobic? I don't see any advantage in storing nonpurified yeast on agar, where it's exposed to air, rather than leaving it under beer. Except that slants take up less space than vials.
Long term storage, like year or years

Year or years? Not very precise! I'm pretty sure yeast can survive a year in liquid. I can't think of any scientific reason why it would survive longer in agar.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 05:19:34 pm
For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

I'm no expert on this as I don't make yeast slants, but isn't the purpose of slants to step up or store tiny samples from a plate after isolating a pure culture? If you aren't isolating colonies from single cells from a streak, wouldn't it be simpler to store slurry under beer so that it remains anaerobic? I don't see any advantage in storing nonpurified yeast on agar, where it's exposed to air, rather than leaving it under beer. Except that slants take up less space than vials.
Long term storage, like year or years

Year or years? Not very precise! I'm pretty sure yeast can survive a year in liquid. I can't think of any scientific reason why it would survive longer in agar.
Not agar. Grycerine/water solution. Obviously, im not an expert on this because I haven't done it yet. Supposedly the 15% glycerine solution prevents damaging crystals from forming when frozen. I think the length of storage time depends on strain and conditions. If you have good luck storing your wyeast long term in the fridge thats awesome. Im going to give this a try for two reasons. 1 is already stated, the other is turning 1 smack pack into 10 pitches.

I was just looking for some feedback from people who do it and have success. Its clearly not something you want to try and thats ok. Im not trying to talk anyone into doing it.
Title: Re: Yeast Slanting and Plating
Post by: charles1968 on December 17, 2015, 05:31:57 pm
For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

I'm no expert on this as I don't make yeast slants, but isn't the purpose of slants to step up or store tiny samples from a plate after isolating a pure culture? If you aren't isolating colonies from single cells from a streak, wouldn't it be simpler to store slurry under beer so that it remains anaerobic? I don't see any advantage in storing nonpurified yeast on agar, where it's exposed to air, rather than leaving it under beer. Except that slants take up less space than vials.
Long term storage, like year or years

Year or years? Not very precise! I'm pretty sure yeast can survive a year in liquid. I can't think of any scientific reason why it would survive longer in agar.
Not agar. Grycerine/water solution. Obviously, im not an expert on this because I haven't done it yet. Supposedly the 15% glycerine solution prevents damaging crystals from forming when frozen. I think the length of storage time depends on strain and conditions. If you have good luck storing your wyeast long term in the fridge thats awesome. Im going to give this a try for two reasons. 1 is already stated, the other is turning 1 smack pack into 10 pitches.

I was just looking for some feedback from people who do it and have success. Its clearly not something you want to try and thats ok. Im not trying to talk anyone into doing it.

Sorry, I didn't spot that you're freezing the yeast. Yes that would extend storage time if enough cells survive the temperature. I don't bother ranching yeast as I bottle all my beer - I can always reculture yeast from the sediment.

I haven't made yeast slants but I've worked with bacteria a lot. I can see the logic of slanting a colony from a streak as you'll get the pure yeast strain, free of any bacteria or wild yeast. That makes total sense to me as a step in yeast management, with storage time being a bonus. .

Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 05:52:24 pm
For example, buy a pack of yeast, build a half dozen slants, divide up the pack slurry among the half dozen slants, freeze. Then when I need some just prop it up?

I'm no expert on this as I don't make yeast slants, but isn't the purpose of slants to step up or store tiny samples from a plate after isolating a pure culture? If you aren't isolating colonies from single cells from a streak, wouldn't it be simpler to store slurry under beer so that it remains anaerobic? I don't see any advantage in storing nonpurified yeast on agar, where it's exposed to air, rather than leaving it under beer. Except that slants take up less space than vials.
Long term storage, like year or years

Year or years? Not very precise! I'm pretty sure yeast can survive a year in liquid. I can't think of any scientific reason why it would survive longer in agar.
Not agar. Grycerine/water solution. Obviously, im not an expert on this because I haven't done it yet. Supposedly the 15% glycerine solution prevents damaging crystals from forming when frozen. I think the length of storage time depends on strain and conditions. If you have good luck storing your wyeast long term in the fridge thats awesome. Im going to give this a try for two reasons. 1 is already stated, the other is turning 1 smack pack into 10 pitches.

I was just looking for some feedback from people who do it and have success. Its clearly not something you want to try and thats ok. Im not trying to talk anyone into doing it.

Sorry, I didn't spot that you're freezing the yeast. Yes that would extend storage time if enough cells survive the temperature. I don't bother ranching yeast as I bottle all my beer - I can always reculture yeast from the sediment.

I haven't made yeast slants but I've worked with bacteria a lot. I can see the logic of slanting a colony from a streak as you'll get the pure yeast strain, free of any bacteria or wild yeast. That makes total sense to me as a step in yeast management, with storage time being a bonus. .
Right on. Im not ready yet to dive into full on yeast lab stuff, but this seems really easy. Im planning on using 1/2 pint mason jars, autoclaved. I'll prop up a smackpack into 1L of thin slurry and divide that up into 10 jars. I'll brew with some in about 3 months just cuz. And then see how it works out next fall in the beginning of lager season for me. I think I will keep a couple for two years just as a viability test.
Title: Re: Yeast Slanting and Plating
Post by: S. cerevisiae on December 17, 2015, 07:11:55 pm
Unless you are interpreting the article incorrectly, the person who wrote the article is mistaken.  The author is describing cryostorage, not slanting.  Glycerine (a.k.a. glycerol) is added to the medium when freezing yeast cultures to prevent ice crystals from forming inside of the cells; thereby, causing the cells to rupture.

Slants are called slants because solidified media is cooled at an angle.  The culture grows on the surface of the media inside of a slant.   Slanting the tube increases the surface area.

Blank slants cooling after being autoclaved

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/slants_zpsd8559e74.jpg)


Inoculated slants

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/CCyeast1_zpsdc754fa7.jpg)

Agar is the preferred solidifier.  Agar remains solid at room temperature.  The only application where gelatin is the preferred solidifier is giant cell morphology.

Plating is a requirement if one seeks purity.  Liquid cultures are not 100% pure, and they become less pure every time that they are subcultured.  Cultures that are stored using cryopreservation are revived and plated for singles before use.   Liquid cultures that are transferred to slant are plated for singles.  Plating for singles (a.k.a. colonies or colony-forming units) is a fundamental technique in yeast management.   Slant-to-slant subculturing (i.e., inoculating a blank slant from an inoculated slant) is usually performed without plating, that is, as long as the donor slant is the result of a previous isolation event and all transfers are aseptic.

The well-isolated colonies shown in the red rectangle shown below are candidates for transfer to slant.

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/PlatedYeast_zps10c1ab8c.jpg)


The round well-isolated colonies in the right-hand corner of the plate shown below are candidates for transfer to slant. 

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/SandNYeast_zpsc0067d33.jpg)


While the process of plating and slanting a yeast culture appears to be complex, it becomes second nature with practice. 

My process after the culture has been plated for singles and well-isolated colonies have been allowed to grow to the point where the cells cover the entire surface of the slant is to store until needed.  When I am ready to use a slant, I subculture a new slant before starting a small amount of autoclaved 5% w/v wort. The original slant is then discarded.  If I am planning to brew a series of beers with the same culture or reuse the culture within 6 weeks or so, I take and store crops.   

One last thing, a home freezer does not get anywhere remotely close to being cold enough to properly store yeast cultures.  Non-critical cultures are frozen at -80C.  Cultures where the depositor wants the cells to remain stable indefinitely are stored at 77 Kelvin (-196C).
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 17, 2015, 07:27:17 pm
One last thing, a home freezer does not get anywhere remotely close to being cold enough to properly store yeast cultures.  Non-critical cultures are frozen at -80C.  Cultures where the depositor wants the cells to remain stable indefinitely are stored at 77 Kelvin (-196C).

I can say, from experience, that a home freezer does store yeast cultures "properly enough" to reproduce those beers a homebrewer may want. Is it up to lab standards? Not even remotely. Does it work? Absolutely.
Title: Re: Yeast Slanting and Plating
Post by: S. cerevisiae on December 17, 2015, 08:35:16 pm
I can say, from experience, that a home freezer does store yeast cultures "properly enough" to reproduce those beers a homebrewer may want. Is it up to lab standards? Not even remotely. Does it work? Absolutely.

Everyone says that until they start performing viability tests.  The reason why your cultures take so long to start is because they have low viability, not because they were frozen.  A yeast culture will easily perform equally well when stored under low-to-mid ABV beer.   
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 08:39:56 pm
Unless you are interpreting the article incorrectly, the person who wrote the article is mistaken.  The author is describing cryostorage, not slanting.  Glycerin is added to the medium when freezing yeast cultures to prevent ice crystals from forming inside of the cells; thereby, causing the cells to rupture.

Slants are called slants because solidified media is cooled at an angle.  The culture grows on the surface of the media inside of a slant.   Slanting the tube increases the surface area.

Blank slants cooling after being autoclaved

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/slants_zpsd8559e74.jpg)


Inoculated slants

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/CCyeast1_zpsdc754fa7.jpg)

Agar is the preferred solidifier.  Agar remains solid at room temperature.  The only application where gelatin is the preferred solidifier is giant cell morphology.

Plating is a requirement if one seeks purity.  Liquid cultures are not 100% pure, and they become less pure every time that they are subcultured.  Cultures that are stored using cryopreservation are revived and plated for singles before use.   Liquid cultures that are transferred to slant are plated for singles.  Plating for singles (a.k.a. colonies or colony-forming units) is a fundamental technique in yeast management.   Slant-to-slant subculturing (i.e., inoculating a blank slant from an inoculated slant) is usually performed without plating, that is, as long as the donor slant is the result of a previous isolation event and all transfers are aseptic.

The well-isolated colonies shown in the red rectangle shown below are candidates for transfer to slant.

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/PlatedYeast_zps10c1ab8c.jpg)


The round well-isolated colonies in the right-hand corner of the plate shown below are candidates for transfer to slant. 

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/SandNYeast_zpsc0067d33.jpg)


While the process of plating and slanting a yeast culture appears to be complex, it becomes second nature with practice. 

My process after the culture has been plated for singles and well-isolated colonies have been allowed to grow to the point where the cells cover the entire surface of the slant is to store until needed.  When I am ready to use a slant, I subculture a new slant before starting a small amount of autoclaved 5% w/v wort. The original slant is then discarded.  If I am planning to brew a series of beers with the same culture or reuse the culture within 6 weeks or so, I take and store crops.   

One last thing, a home freezer does not get anywhere remotely close to being cold enough to properly store yeast cultures.  Non-critical cultures are frozen at -80C.  Cultures where the depositor wants the cells to remain stable indefinitely are stored at 77 Kelvin (-196C).
Thanks Mark!

I'm going to give this freeze thing a go. Worst case scenario is I end up not having any of the yeast, which is where I would be anyway.

In the future at some point I do plan to take up the propper plate n slant methods. I'm just not ready yet, but getting there.
Title: Re: Yeast Slanting and Plating
Post by: S. cerevisiae on December 17, 2015, 08:51:10 pm
It used to be much easier to get people started with plating and slanting when BrewTek was around.  BrewTek used to offer ready-made blank slants, plates, sterile first-level starter wort, and cultures on mini-slants, which allowed a new yeast farmer to be able to concentrate on mastering aseptic transfer technique before having to tackle sterile media preparation.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 17, 2015, 09:15:24 pm
It used to be much easier to get people started with plating and slanting when BrewTek was around.  BrewTek used to offer ready-made blank slants, plates, sterile first-level starter wort, and cultures on mini-slants, which allowed a new yeast farmer to be able to concentrate on mastering aseptic transfer technique before having to tackle sterile media preparation.
I think I would enjoy the whole thing, it's just going to be a couple more months till I'm ready to take the plunge.

By the way, the article was by Bill Pierce BYO Jan Feb 2005
Title: Re: Yeast Slanting and Plating
Post by: S. cerevisiae on December 18, 2015, 05:18:13 am
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.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 07:39:25 am
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.
Makes sense that if you are going for sterile then go all the way. Not sure but I think 50% sterile plus 50% sanitary equals 100% sanitary.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 07:46:51 am
Mark what is your plate/slant medium recipe?

Also, does 5% w/v mean 5g dme to 100ml water?
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 18, 2015, 07:54:45 am
I can say, from experience, that a home freezer does store yeast cultures "properly enough" to reproduce those beers a homebrewer may want. Is it up to lab standards? Not even remotely. Does it work? Absolutely.

Everyone says that until they start performing viability tests.  The reason why your cultures take so long to start is because they have low viability, not because they were frozen.  A yeast culture will easily perform equally well when stored under low-to-mid ABV beer.

I consider the batch of beer the strains ferment to be the ultimate judge as to whether or not they were successful. Additionally, it's said that any mutations that take place happen much more slowly at freezing temperatures (slower the lower the temp). While 3726-PC is a hardy strain, I would have tossed it long before 12 months had it been in the fridge under beer, whereas it performed splendidly in a 15 gallon batch after being frozen for 2.5 YEARS! and that's without any additional maintenance during that time. A 24 lag and (possibly) low viability during the starter is a small price to pay IMHO. Lastly, once the lag is over and yeast have propagated then I'm not dealing with lag or low viability anymore ;)

I get it. I'm not up to lab standards. I don't do what I'd do if I was a commercial brewery. Luckily, I'm neither of those things.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 08:39:42 am
I can say, from experience, that a home freezer does store yeast cultures "properly enough" to reproduce those beers a homebrewer may want. Is it up to lab standards? Not even remotely. Does it work? Absolutely.

Everyone says that until they start performing viability tests.  The reason why your cultures take so long to start is because they have low viability, not because they were frozen.  A yeast culture will easily perform equally well when stored under low-to-mid ABV beer.

I consider the batch of beer the strains ferment to be the ultimate judge as to whether or not they were successful. Additionally, it's said that any mutations that take place happen much more slowly at freezing temperatures (slower the lower the temp). While 3726-PC is a hardy strain, I would have tossed it long before 12 months had it been in the fridge under beer, whereas it performed splendidly in a 15 gallon batch after being frozen for 2.5 YEARS! and that's without any additional maintenance during that time. A 24 lag and (possibly) low viability during the starter is a small price to pay IMHO. Lastly, once the lag is over and yeast have propagated then I'm not dealing with lag or low viability anymore ;)

I get it. I'm not up to lab standards. I don't do what I'd do if I was a commercial brewery. Luckily, I'm neither of those things.
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?
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 09:02:34 am
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?
Title: Re: Yeast Slanting and Plating
Post by: reverseapachemaster on December 18, 2015, 09:03:34 am
This thread reminds me that I need to go get the frozen yeast from my parents' chest freezer on Christmas. I think that yeast is all 3-4 years old. Probably not much in the way of viability but worth some wort to give it a test.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 09:14:58 am
This thread reminds me that I need to go get the frozen yeast from my parents' chest freezer on Christmas. I think that yeast is all 3-4 years old. Probably not much in the way of viability but worth some wort to give it a test.
Awesome!  I read this and immediately put it juxtaposed in my mind to the new brewer wondering if he can bottle once bubbling slows to 1 a minute lol. Imagine the patience it takes to wait 4 years to brew with your cool new yeast?
Title: Re: Yeast Slanting and Plating
Post by: troybinso on December 18, 2015, 09:37:37 am
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/
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 18, 2015, 09:49:50 am
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.
(http://images.tapatalk-cdn.com/15/12/18/938fce20c1914ca5409fc567a85c940c.jpg)
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 10:21:59 am
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.
(http://images.tapatalk-cdn.com/15/12/18/938fce20c1914ca5409fc567a85c940c.jpg)
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?
Title: Re: Yeast Slanting and Plating
Post by: narcout on December 18, 2015, 10:28:38 am
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.
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 18, 2015, 10:50:42 am
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.
Title: Re: Yeast Slanting and Plating
Post by: Stevie on December 18, 2015, 10:57:49 am

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.
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 18, 2015, 11:12:18 am

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.
Title: Re: Yeast Slanting and Plating
Post by: Stevie on December 18, 2015, 11:20:10 am
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?
Title: Re: Yeast Slanting and Plating
Post by: stpug on December 18, 2015, 11:51:16 am
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.
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 12:02:14 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...
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 12:04:07 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
Title: Re: Yeast Slanting and Plating
Post by: klickitat jim on December 18, 2015, 12:46:19 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.
Title: Re: Yeast Slanting and Plating
Post by: S. cerevisiae on December 20, 2015, 07:52:31 pm
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.