Do you know how to make a yeast starter? Then why not farm yeast and freeze it?

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BBL_Brewer

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There’s some information out there about freezing yeast, but I’m not sure how many people are actually doing it. I’ve read a lot of posts about yeast washing and harvesting from ferementors so I thought I’d do a pitch (no pun intended) on yeast farming/freezing as an alternative.

Freezing yeast is actually quite simple. You mix water, glycerine (glycerol), and yeast slurry together and throw it in the freezer. That’s all there is to it. Glycerine is the magic ingredient that protects the yeast during the freeze. The yeast will remain vaible like this for quite a long time. I’ve brought up yeast that was almost 2 years old and brewed with it. So here’s my pitch.

The number one reason that I freeze yeast is for convenience. Smack packs, vials, slurry in a mason jar all loose viability over time and eventually die while sitting in the fridge. Dry yeast lasts a lot longer but will also lose viability. A frozen jar of yeast has a much longer shelf life. This means you can brew whenever you want (with a little planning) and never have to worry about available, healthy yeast.

Another benefit is cost. It ends up costing me about the same price as a pack of dry yeast (or a little less) for a jar of frozen liquid yeast and that’s after pitching to a 5-gallon batch of beer.

The process

There are a couple of different approaches that you can take. The main thing is making sure you use enough glycerine. Unfortunately, there is no set number out there on how much glycerine to use. I use a 20% by volume concentration of glycerine. Others report using anywhere from 15% to 50% or over. Some like to add so much glycerine that the mixture does not actually freeze at all. Bottom line, pick what you think is best and run with it. If it works, continue on.

What glycerine to use?
You want to use food grade vegetable glycerine. I get mine from Nutrition Geeks. The brand I get is “NOW.” It’s made in the United States. Be careful and don’t buy glycerine made in China because there have been reports of the Chineese selling Ethylene Glycol (antifreeze) and passing it off as glycerine. The two compounds have similar properties except Ethylene Glycol will kill you and glycerine won’t.

How much yeast to freeze?

Most people I’ve read about out there are freezing small portions of yeast in vials or something similar. I like to freeze estimated portions of 100 billion cells. This way I have more viable cells after the freeze and can start with a larger starter instead of having to do small step ups.

As always, there is more than one way to skin a cat, but here’s how I do my process.

I like Wyeast so I always start with an activator smack pack. You can use whatever yeast you want. I buy one smack pack of every yeast strain I plan to use and then farm enough yeast of each to last me for about a year or a little over. I use the Yeast Calc pitching rate calculator, but you can pick the calculator of your choice (I know a lot of people like Mr. Malty). I make up an appropriately sized starter in steps so that the estimated final cell count is ~500 billion. I setlle out the yeast, siphon off the starter beer and add enough glycerine/water solution to bring the volume up to 500 milliliters (with a final glycerine contration of 20% by volume). Then I split the yeast into 5 equal portions and refridgerate them for 48 hours before freezing. If I want more jars than that of a particular strain I will take one of the portions and put it back through the process. You can do this over and over again until you have as much yeast as you want.

A few things that will help

A deep freezer. You want your yeast to remain frozen without the temperature fluctuating. It will have a longer shelf life this way. The best thing for this is a deep freezer. I like to store mine at the bottom of my chest freezer buried under a bunch of food. You don’t want to put the yeast in a self-defrosting freezer (like your refridgerator freezer). Self-defrosting freezers warm up once in a while and this is how they defrost. You don’t want your yeast warming and cooling all the time. If you don’t have a deep freeze, you can get around this by putting the frozen yeast in a small cooler with some ice packs and storing the whole cooler in the freezer. Freeze the yeast first and then throw the cooler in on the mix. I’ve read reports of this working well for folks but I’ve never done it myself.

A pressure cooker. I like to sterilize what materials that I can by pressure canning them. After all, it’s very important to practice excellent sanitation when farming yeast. The yeast has a long way to go before it gets to your prized wort and contamination from the start will have ample opportunity to magnify along the way. I pressure my starter wort, glycerine solution and the jars that I freeze the yeast in. It’s also more convenient this way because you can make up a bunch of this stuff all at one time and not have to worry about boiling/cooling/sanitizing every step of the way. I pressure everything at 15 psi for 15 minutes. For my freezer jars, I use 4 oz jelly jars. They hold about 120 milliters. I fill them up with tap water and pressure them. When it’s time to freeze yeast I pop the seal, dump the water and fill. Put the lid back on and tighten down the ring nice and snug. If you don’t have a pressure cooker, don’t fret, you could still do this. You would just have to boil and cool as usual and soak your jars in star san or something before filling.

A stir plate. You can grow a lot more yeast a lot quicker and with less wort with a stir plate. However, a simple starter would work just fine as long as you make one big enough to hit your target cell count. What this means is that you will probably have to make more starters to get the same job done.

Make sure you freeze healthy yeast. I go the extra mile when I’m farming yeast to be frozen. I make sure the yeast have plenty of nutrients and give shots of pure oxygen at every wort addition. When the starter is done I settle out the yeast as fast as possible and refridgerate for 48 hours before freezing. I want to make sure that the yeast have healthy cell walls and a full belly of reserves before I freeze.

How to mix up your glycerine solution. I've been doing mine in % by volume.

Scenario 1
You have 150 ml of yeast slurry. You want to know how much glycerine to add so that the final concentration is 20% by volume. Working with percents is kind of weird. We're shooting for 20% glycerine, so we have to divide the volume of slurry we have by 80% to find out what the final volume would be with 20% glycerine mixed in. If we wanted 30% glycerine, then we would divide by 70%, Here is the equation.
Volume of Yeast slurry / 0.8 = final volume. We have 150 ml of slurry. 150 / 0.8 = 187.5 ml. Now that we know the final volume, we can subtract the amount of slurry we already have (150 ml) to determine how much glycerine we need. 187.5 - 150 = 37.5. So, you add 37.5 ml of glycerine to the 150 ml of slurry. Final concentration of glycerine - 20%. 37.5 / 187.5 = 0.2

Scenario 2
You have 150 ml of slurry. You want to use this slurry to fill (5) 100 ml vials. You want a final concentration of glycerine of 20% by volume. In this instance, we know what the final volume is going to be, 500 ml, becasue that is what we need to fill all the vials. Here is the equation for this one.
Desired final volume * desired glycerine concentration % = glycerine needed. 500 ml * .20 = 100. So, we add 100 ml of glycerine to the yeast slurry we already have and then add water to top it off at 500 ml.

Scenario 3
You have 150 ml of yeast slurry. You want to use this slurry to fill (5) 100 ml vials. You want a final concentration of glycerine of 20% by volume. You have a pre-sterillized jar of 60% by volume glycerine solution. You want to know how much of the 60% solution to add to your yeast slurry. Here is the equation.
(Desired final volume * desired glycerine concentration %) / concetration of existing glycerine solution = glycerine solution needed

Our final volume is going to be 500 ml because that's what we need to fill the vials. We want 20% glycerine. Our pre-sterilzed glycerine solution is 60% by volume. (500 ml * 0.20) / 0.60 = ~167 ml. So, we add 167 ml of glycerine solution to the 150 ml of yeast slurry that we already have. Then we top off with water to get 500 ml.


Thawing the yeast and brewing with it

Ahh, the fruits of your labor. Thaw the yeast as quickly as possible with a water bath and pitch strait into a starter. It appears that viability after the freeze is on the low side (around 25%). Again, I freeze an estimated 100 billion cells. When I thaw out a jar I use the Wyeast pitch rate calculator, select the propagator pack and then make an appropriately sized starter for the gravity beer that I’m brewing. Since propagators have 25 billion cells this works out nicely since 25 billion cells should make it through the freeze. Sometimes the frozen yeast exhibit an unusually long lag time on the first step of a starter. This is likely due to a low viable cell count. After the first step is complete, the yeast chew through whatever I throw at em in 12 hours or less. At any rate, once the starter gets going, the yeast act as normal as ever, so be patient your first couple times doing this and don’t be alarmed by an extended lag time on the first step. I recently updated the orginal post to include some process changes. I made the edits in red. I will be getting some lab equipment in the near future and running some tests on cell counts and viability after the freeze. As soon as I have some consistent results I will be sure to post them and update the post accordingly.

Well, there you have it. If you decide to give this a try, I think you’ll find farming yeast and freezing it to be very convenient, cost effective, and relatively easy to accomplish. Anyone who makes yeast starters on a regular basis can do it. You’ll always have ample yeast on hand to brew with and you’ll never have to wash yeast again. For further reading I’ve posted some sources at the bottom.


Cheers



https://www.homebrewtalk.com/f13/guide-making-frozen-yeast-bank-35891/

https://www.homebrewtalk.com/f128/glycerin-water-ratio-freezing-yeast-215319/

http://www.dow.com/glycerine/resources/freezept.htm

http://www.schwedhelm.net/brew/yeast_harv_freeze.html

http://www.bodensatz.com/staticpages/index.php?page=yeast-culture-FAQ

http://maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices
 
BBL Brewer. I'm have a lab and I can do viability assays. I'd be happy to follow your procedure and then do an assay. I'd like to use this approach myself rather than having to start a starter every time. I have tease recovered from microbrews that I have been storing in glycerol in small amounts to grow up starters but I haven't done large amounts. I think storing at -80c is best but not accessible to most brewers. I would think viability at -20c is part of the problem.
 
BBL Brewer. I'm have a lab and I can do viability assays. I'd be happy to follow your procedure and then do an assay. I'd like to use this approach myself rather than having to start a starter every time. I have tease recovered from microbrews that I have been storing in glycerol in small amounts to grow up starters but I haven't done large amounts. I think storing at -80c is best but not accessible to most brewers. I would think viability at -20c is part of the problem.

That would be awesome! I'd love to see some data on this. It would be great actually if you could test the viability of a very small portion and a large portion. I've often wondered if smaller portions freeze faster while keeping more yeast in suspension and therefore surviving the freeze better. You may be right about temperature as well. As you said though, we have to get by with what we have. Any input that you could contribute would be greatly appreciated.
 
That would be awesome! I'd love to see some data on this. It would be great actually if you could test the viability of a very small portion and a large portion. I've often wondered if smaller portions freeze faster while keeping more yeast in suspension and therefore surviving the freeze better. You may be right about temperature as well. As you said though, we have to get by with what we have. Any input that you could contribute would be greatly appreciated.

I'll second the desire for some data! It sounds like I go about opposite of you, small amounts (5ml) at high enough levels to keep from freezing (40-50% glycerol). I normally just fill up some 5ml tubes about halfway with glycerol and add about 2.5ml of yeast from a decanted starter and toss it inside a styrofoam container with a few ice packs to help with the defrosting. I've got to step it up a couple of times to get to a useable amount for a batch and have always wondered what my viability was coming out of the freezer. On a side note, how cool would it be to have a tabletop -80 for homebrew yeast cultures :D.
 
I have some yeast cultures frozen in my parents' chest freezer. I haven't pulled one to see if it is viable but I assume as long as they never thawed they should be fine. I'll have to pull a sample next time I visit and see how it works out.
 
I have some yeast cultures frozen in my parents' chest freezer. I haven't pulled one to see if it is viable but I assume as long as they never thawed they should be fine. I'll have to pull a sample next time I visit and see how it works out.

I'm willing to bet that they are still alive.
 
Just a small update. I just got done freezing my yeast for the year and decided to do a little qualitative testing while I was at it. I used three different strenght glycerine solutions this time. 35% 50% and 60%. My plan is to see if different levels of glycerine affect the lag time when making the first step of starter from frozen yeast. If more glycerine = shorter lag time then I will assume that more glycerine = more viable yeast surviving the freeze. If I observe no appreciable difference, then I will stay with my current assumption that the yeast are just sluggish after freezing and need more time to regain activity. If Brewitt does some viability assays for us and contributes that information, then we should have a pretty good idea of what to expect from freezing yeast at home.
 
Got my pressure cooker this week with some glycerine.
Just finished testing it by canning 7 qts of water and pressure cooking it (I will now have sterile water on hand for a while). I also did a 50:50 glycerine/water in 2x 4oz jars (only filled halfway, so yeast can occupy the other half). Next weekend I will be bottling and I plan on freezing washed yeast.

Will 25% glycerin be enough to keep them viable?
 
Try it and find out. I'm guessing it will since I've heard reports of people using 15%.
 
If Brewitt does some viability assays for us and contributes that information, then we should have a pretty good idea of what to expect from freezing yeast at home.

Sorry for the delay in doing some experimentation on this. Work has been quite busy and I have had no time for side projects. I will prep a starter this week and set up some frozen preparations at a few glycerin concentrations from 15 to 60% and at -30C and -80C. It will probably be a couple weeks before I know anything about viability since it requires cell counts and colony counts.
 
That would be great!
I had good results with 30% glycerin solution, and I think that success with smaller concentration will depend on how well is glycerin mixed with slurry.. but experiment will give more relevant informations about that.
 
Sorry for the delay in doing some experimentation on this. Work has been quite busy and I have had no time for side projects. I will prep a starter this week and set up some frozen preparations at a few glycerin concentrations from 15 to 60% and at -30C and -80C. It will probably be a couple weeks before I know anything about viability since it requires cell counts and colony counts.

No worries! Take your time. Very cool of you to go to all that trouble in the first place. Can't wait to see the results though :mug:
 
Although I haven't had time to prep cells for this analysis, I did do some reading regarding yeast preservation conditions and found some results that appear reliable but were surprising to me. The results seem to indicate that freezing yeast at a rate of less than 1 degree C per minute in 10% glycerol and then thawing at 37 degree C yielded viabilities on the order of 50% whereas faster freezing and slower thawing led to significantly low viabilities. The one thing that did not replicate home freezing methods was that the storage was in liquid nitrogen (-196C) after freezing to -80C. However, I presume that the slow freezing and rapid thawing will still be advantageous. So, cooling by putting a large volume in the refrigerator and then moving to a standard freezer sounds like it might be a good approach and then thawing at around 100F with agitation (to prevent heating of the thawed portion) until it is completely thawed sounds like a good idea. Let's see.
 
You wouldn't think more glycerol would be a bad thing. Slow freezing I can see. Fast thawing seems odd, but if it works all the better (cuts down on lead time). Your results will be very interesting to see. Thanks Brewitt!
 
BBL Brewer - great post. I need to set up some time and farm some yeast for the year as you did.

Brewitt - you have me excited to see the results of the testing. This is a thread worth saving.
 
My mother always chilled ice cream before mixing in the cranker claiming it made better Ice Cream. I've seen more of this as I've gotten older and the explanation typically used is smaller ice crystals. Smaller crystals would be less likely to rupture the yeast cells so that makes sense. I can also buy the fast cooling thing but the extra agitation part doesn't quite make sense to me. Even with glycerine absorption, there will still be some small amount of water inside the cells that crystalized as you can't have a perfect mix inside the cells. That being the case it seems to me at least logically that it would increase the chance of damaging the cell. On the other hand it would also make more sense that it would be quicker to get up to speed (aka cell agitation already out of the way to get rid of the lethargy).

That said, I can't help but wonder if jars might not be the best medium to store them in at least in the traditional sense. While the vacuum isn't really necessary, I wonder if vacuum bags wouldn't function better as you could still store them inside a jar filled with water preventing a lot of the need for the cooler setup, and the surface area would be greater for thawing quicker with less potential cellular damage.

That said if you want to thaw them extremely quickly with agitation turn the jar sideways (I've not done it with 1/2 cup jars but 1c work fabulously) in a bowl and run cool tap water over it. It'll thaw in a matter of minutes. We do the same thing with our babies bottles (with a bit of hotter water added into the mix) and you can go from frozen to warmed enough for consumption in about 3 minutes depending on volume. The agitation (spinning in the water filled bowl) will keep the temperature smooth throughout as well.

That said I still think I'm going to give this a try. I'll probably go the vial route though and lean towards the higher end of the spectrum glycerine-wise to be safe as I have far less freezer space to work with. :(
 
My mother always chilled ice cream before mixing in the cranker claiming it made better Ice Cream. I've seen more of this as I've gotten older and the explanation typically used is smaller ice crystals. Smaller crystals would be less likely to rupture the yeast cells so that makes sense. I can also buy the fast cooling thing but the extra agitation part doesn't quite make sense to me. Even with glycerine absorption, there will still be some small amount of water inside the cells that crystalized as you can't have a perfect mix inside the cells. That being the case it seems to me at least logically that it would increase the chance of damaging the cell. On the other hand it would also make more sense that it would be quicker to get up to speed (aka cell agitation already out of the way to get rid of the lethargy).

That said, I can't help but wonder if jars might not be the best medium to store them in at least in the traditional sense. While the vacuum isn't really necessary, I wonder if vacuum bags wouldn't function better as you could still store them inside a jar filled with water preventing a lot of the need for the cooler setup, and the surface area would be greater for thawing quicker with less potential cellular damage.

That said if you want to thaw them extremely quickly with agitation turn the jar sideways (I've not done it with 1/2 cup jars but 1c work fabulously) in a bowl and run cool tap water over it. It'll thaw in a matter of minutes. We do the same thing with our babies bottles (with a bit of hotter water added into the mix) and you can go from frozen to warmed enough for consumption in about 3 minutes depending on volume. The agitation (spinning in the water filled bowl) will keep the temperature smooth throughout as well.

That said I still think I'm going to give this a try. I'll probably go the vial route though and lean towards the higher end of the spectrum glycerine-wise to be safe as I have far less freezer space to work with. :(

Great points! The vacume bag inside a jar of water is a novel idea for those who have a self-defrosting freezer. It would also take up less space or at least be easier to work with functionally in a smaller freezer.

As for thawing, if a quick thaw is the ticket, you're right, water would be the way to go. That's how I thaw my frozen meat. Works like a charm and quick to boot. However, using my existing method, I would have to put the jars in a zip lock since the jars are not sealed upon freezing and the agitation from spinning in the bowl would not be an option. I don't plan to use the bag in a jar method becasue the 4 oz jars are so small and full boxes of them stack nicely in my chest freezer while taking up a relatively small amount of space. Personally, I think just water bathing period would be better than any other heating method even without agitation.

Thanks for your input :mug:
 
EDITED FOR CLARITY (hopefully):

I have initiated the freezing experiment using a blend of the approaches that have been discussed here. I started with half a liter and ended with samples of about 5 ml (much smaller volumes than you would have for a full starter) but I presume the results will be about the same. The goal was to try different concentrations of glycerol (15% and 30%) and different cooling regimens (Add glycerol at room temp then transfer to refrigerator temp and then to freezer temp or cool first, add cold glycerol and then go straight to freezer). Finally, I will thaw in refrigerator overnight or in the warm. Here is basic outline.

1. Grow half a liter of starter using 10X dilutions to full density in 1.040 wort with nutrient. Don't have a number yet but around 2x10e8 per ml.

2. Split, chill 1/2 to 4C (refrigerator temp) and let half settle at room temp overnight (assuming that none of you have centrifuges so gravity settle to concentrate).

3. Pour off wort, and resuspend cells in 15 ml of either 15% glycerol at room temperature or 4C depending on the state of the starting cells.

4. Split each into three portions and adjust one portion of each to 30% glycerol (cold or room temp).

5. Room temperature culture was moved to 4C for an hour after adding glycerol and then to freezer. Culture that was in cold before adding glycerol was simply moved to freezer after glycerol was added. A single sample of the later was also later moved to an ultracold freezer where we usually store yeast.

6. After a week or two (depending on my schedule) I will remove from freezer, either thaw in refrigerator or at 37 (body temp) and then do a viability assay (count cells and plate and see how many grow up). I will also try to put some in wort and see how long they take to recover. This is a lot of work so it's going to take me a bit.

Hopefully this will be informative.

I should say that for a scaled up process this would be the same as growing three liters of starter, letting it settle out, and bringing all the cells up in about 2/3 cup of glycerol. That could then be split into thirds to make three starters of about 2x10e11 cells each in about 1/3 cup total volume. Just a bit more dilute than a tube of White Labs yeast. Also, this will tell us about freezing and thawing but not about long term storage. If you have a non-frost-free freezer or a frost-free freezer with some ice packs around your tubes I would think the results I report should hold for at least several months to a year.
 
If you have any luck with the Brett group pls post it. I was thinking of harvesting from one and I'd swear I read someone saying it doesn't work. TMF maybe? Will have to go try and dig that link up. He did use equipment like a centrifuge tho iirc along with other niceties.

Froze a s05 (just cause I plan on using it relatively soon and have a backup) and wlp001 which I don't have a need for for at least 9mos. Have read the 05 has no problems usually anyway tho.
 
I have not done anything with Brett. I have never used it and don't have any to work with.
 
This experiment sounds great, I am highly interested about the results.
Great job Brewitt!
 
Wow, I can't wait for the results Brewitt! This is the kind of experiment that homebrewers will quote to each other for years.

Science: it works.
 
I haven't done anything toward plating out the cells and counting yet. Probably be next month now given the way time is going. However, I did freeze up about 1/3 liter worth of cells in 30 ml 15% glycerol by chilling to refrigerator, then freezer, then ultracold freezer (-80C) temperature. I thawed that out last night and used it to start a 1.5 liter starter. This morning it is full density and cold crashing. I will keep that cold, dump the slurry of settled cells into 1.5 liter of fresh wort Sunday morning and use it to start my fermentation.

I think this is probably an equally good plan for the general user. Grow up a gallon of starter, split it into 8 half liter portions, cold crash, freeze the cells in 15% glycerol and use one of those to start an overnight starter of up to a gallon as needed. I will do the same without the ultracold step next time and see whether I get equally satisfying results for the home brewer. As long as there is not a significant lag, it should be great. Always good to wake up your cells for a bit before pitching anyway so the overnight is a good idea.
 
Wow... one of the coolest threads I've seen on here... as an engineer, this is very fascinating! Great work guys, I can't wait to see the final results! AWESOME!!!
 
I don't have the -80 degree freezer to work with but I did tests with some of the extra washed yeast (washed twice, separated out, pouring off the excess, and resuspending for division into glasses which was done by weight) I knew I wasn't going to use. With ~30% by volume glycerine I did three batches (WLP001). One went into the Freezer immediately after mixing and shaking, the other two were placed into the fridge and one was shaken vigorously every day for three days.

They were left in the freezer for 2 weeks (+ 3 days for the straight to freezer one) and all three brought out and added to Malta based starters (I had extra bottles, sue me). The one put straight into the freezer was actively started within just under 60 hours. The other two were active within ~30 hours. The shaken daily while refrigerated before freezing one was active *MAYBE* 4-6 hours faster, but just over 30 hours later they seemed equal so I don't know if that was coincidental or not.

All three were done in 1 cup freezer safe jars (Ball) and pitched to 1L flasks for starting (@~750ml of wort) and ran on stirplates with a 1.5" stirbar until my curiousity was satisfied. All three seem to have washable yeast that looked quite healthy and the starters tasted pretty much identical to my tastebuds. I know the above is far from scientific but I didn't have fancy things like microscopes and cyclotrons (centrifuge but cyclotron sounds so much cooler!) to work with.
 
All three seem to have washable yeast that looked quite healthy and the starters tasted pretty much identical to my tastebuds. I know the above is far from scientific but I didn't have fancy things like microscopes and cyclotrons (centrifuge but cyclotron sounds so much cooler!) to work with.

That's awesome, thanks for the results! So refrigerate for a few days before going to the freezer? Sounds like they need some time to get their parkas on.

And hey, if they taste and look good (well as good as a starter can), who needs fancy microscopes?
 
The chilling at refrigerator temperatures for several hours seems to make a significant difference. Not sure if giving them several days should make a difference. My culture grew very well and was at full density after overnight with shaking. Remember, if you start with a good dense slurry of yeast you are not asking for very much growth to reach a maximum density starter. The cells only need to go through a few rounds of division and they are there. I do think there is a very substantial advantage to getting your starter going overnight rather than just pitching it. My fermentation that is just finishing up was actively bubbling away in 4 hours after pitching with couple liter overnight starter made with my tube of glycerol frozen yeast, thawed in 100 F water with shaking until just thawed. Based upon this experience I think this is a very good approach and the one I will be using. I am going to make up half a dozen tubes of frozen cells of a couple different yeasts over the next couple weeks and keep them for exactly this purpose.
 
I agree but my question is does the refrigeration thing only chill it to reduce the size of the resulting ice crystals or do the yeast cells have more time to imbibe more glycerine into their cells preventing even further cell damage? At least that was my impression of how the glycerine aids it if it isn't in high enough volume to completely prevent freezing...
 
Oh, and I should probably mention mine was from a self defrosting freezer. If I go library this route I will use icepacks but did not this time.
 
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