Author Topic: Yeast Slanting and Plating  (Read 5158 times)

Offline stpug

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Re: Yeast Slanting and Plating
« Reply #15 on: December 18, 2015, 02:27:17 AM »
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.

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Re: Yeast Slanting and Plating
« Reply #16 on: December 18, 2015, 03:35:16 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.   

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #17 on: December 18, 2015, 03:39:56 AM »
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




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).
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.

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Re: Yeast Slanting and Plating
« Reply #18 on: December 18, 2015, 03:51:10 AM »
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.

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #19 on: December 18, 2015, 04:15:24 AM »
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

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Re: Yeast Slanting and Plating
« Reply #20 on: December 18, 2015, 12:18:13 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.

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #21 on: December 18, 2015, 02:39:25 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.
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.

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #22 on: December 18, 2015, 02:46:51 PM »
Mark what is your plate/slant medium recipe?

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

Offline stpug

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Re: Yeast Slanting and Plating
« Reply #23 on: December 18, 2015, 02:54:45 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.

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.

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #24 on: December 18, 2015, 03:39:42 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.

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?
« Last Edit: December 18, 2015, 03:42:06 PM by klickitat jim »

Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #25 on: December 18, 2015, 04:02:34 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?

Offline reverseapachemaster

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Re: Yeast Slanting and Plating
« Reply #26 on: December 18, 2015, 04:03:34 PM »
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.
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Offline klickitat jim

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Re: Yeast Slanting and Plating
« Reply #27 on: December 18, 2015, 04:14:58 PM »
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?

Offline troybinso

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Re: Yeast Slanting and Plating
« Reply #28 on: December 18, 2015, 04:37:37 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/

Offline stpug

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Re: Yeast Slanting and Plating
« Reply #29 on: December 18, 2015, 04:49:50 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.
« Last Edit: December 18, 2015, 04:54:22 PM by stpug »