Author Topic: Yeast Slanting and Plating  (Read 2746 times)

Offline klickitat jim

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Yeast Slanting and Plating
« on: December 17, 2015, 10:20:22 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



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Re: Yeast Slanting and Plating
« Reply #1 on: December 17, 2015, 12:48:35 PM »
"Paging Mark. Mark please report to the forum please...."

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #2 on: December 17, 2015, 12:50:23 PM »
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.

Offline stpug

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Re: Yeast Slanting and Plating
« Reply #3 on: December 17, 2015, 03:29:01 PM »
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.
« Last Edit: December 17, 2015, 03:35:58 PM by stpug »

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #4 on: December 17, 2015, 04:33:04 PM »
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.

Offline coolman26

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Re: Yeast Slanting and Plating
« Reply #5 on: December 17, 2015, 04:48:30 PM »
What is your new favorite?  I really need to look into how to freeze yeast 
Jeff B

Offline charles1968

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Re: Yeast Slanting and Plating
« Reply #6 on: December 17, 2015, 10:03:09 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.
« Last Edit: December 17, 2015, 10:05:54 PM by charles1968 »

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #7 on: December 17, 2015, 11:17:20 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

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #8 on: December 17, 2015, 11:17:35 PM »
What is your new favorite?  I really need to look into how to freeze yeast
2352

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #9 on: December 17, 2015, 11:45:53 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.

Offline charles1968

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Re: Yeast Slanting and Plating
« Reply #10 on: December 18, 2015, 12:11:55 AM »
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.

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #11 on: December 18, 2015, 12:19:34 AM »
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.
« Last Edit: December 18, 2015, 12:22:46 AM by klickitat jim »

Offline charles1968

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Re: Yeast Slanting and Plating
« Reply #12 on: December 18, 2015, 12:31:57 AM »
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. .


Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #13 on: December 18, 2015, 12:52:24 AM »
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.

S. cerevisiae

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Re: Yeast Slanting and Plating
« Reply #14 on: December 18, 2015, 02:11:55 AM »
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




Inoculated slants



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.




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




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).
« Last Edit: December 18, 2015, 03:45:48 AM by S. cerevisiae »