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Guide to Making a Frozen Yeast Bank

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FlyGuy

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I really like the variety and quality of liquid yeasts that are available today, but I sure don’t like paying $10 for a smack pack of yeast that I will only use once. It is by far my most expensive ingredient. Also, fresh liquid yeast can be tough to find at my LHBS, so I really wanted to have a bank of yeast on hand for whenever I needed it. Some quick research on the internet revealed that building a yeast bank in your freezer is a pretty easy thing to do, and the equipment required is minimal. The advantage is that you can easily cut the cost of liquid yeast by at least 90%, and you can have a good variety of yeast on hand whenever you need it. Below is the method that I have been using with great success.

Here is a picture illustrating the items I use (clockwise from top):
- 2000 mL Pyrex flask and stir-plate for propagating yeast (optional)
- pressure cooker (poor man's autoclave) for sterilizing equipment
- 500 mL beaker for holding glass vials
- 15 mL (1/2 oz) flat-bottomed glass vials with autoclavable screw tops
- small glass graduated cylinder
- glycerine
- autoclavable pipette (not shown)
- spray bottle filled with Star San solution (not shown)

 
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FlyGuy

FlyGuy

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Obviously, the first step in the process is to prepare some yeast for freezing. It is best to freeze concentrated yeast from the bottom of a starter, so I have been building a big (1.5 – 2 quart) starter on my stir plate from a new pack of liquid yeast. If you don’t have a stir plate, make your starter in two rounds. For example, start with 3 cups of wort, ferment it out, and then add another 3 or 4 cups of wort to really build up the yeast. Aerate the heck out of the wort at each addition to ensure the yeast get lots of oxygen required for reproduction. An oxygenation system is handy for this, but not necessary.

Once your starter is built up and the yeast have fully fermented the beer, crash cool your yeast in the fridge to drop the yeast out of suspension. If you like, you can also pour off the beer on top, but this isn’t necessary. In Step 4 you will be drawing yeast off the bottom of the flask, and you want it to be as concentrated as possible, so make sure that the yeast have sedimented well to the bottom of the flask.

 
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FlyGuy

FlyGuy

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To freeze yeast successfully, you need to do so without killing the yeast. Yeast can recover from sub-zero temperatures, but frozen water can kill them. Frozen water crystallizes and can puncture yeast cell walls, but two things can be done to prevent this. First, use a small volume of water (hence the small vials) and leave a bit of room for expansion. Second, a bit of glycerine, also known as glycerol (but NOT glycol!!) helps prevent freeze damage. I buy glycerine from my LHBS or pharmacy. Just be sure to get the type that is safe to ingest (it is harmless at these quantities).

I use a small graduated cylinder to measure and pour glycerine into the test tubes. You want less than one-third of the solution in the test tubes to be glycerine, so I shoot for about 2-3 ml in each tube (15%). I do 10 to 16 tubes at a time, so I just measure my total amount necessary in the graduated cylinder, and distribute it approximately evenly among all the tubes. You don't need to be absolutely precise.

 
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FlyGuy

FlyGuy

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It is essential to be as sanitary as possible when storing yeast for a long period of time. I use my pressure cooker (doubles as my steam infusion system, see here) as a home autoclave to sterilize my equipment. Pressure cookers are great because they will heat up to about 250 degrees, which is enough to kill everything inside. Boiling water works pretty well, too, but it only gets up to about 212 degrees (depending on your elevation), which kills all living things but not wild yeast spores.



Very loosely cap your vials (now partially full with glycerine) and put them in a Pyrex beaker or other heat-resistant container inside the pressure cooker. Also put in your pipette, eye dropper, stainless steel turkey baster, etc. that you will use to extract yeast from your starter flask. Note that in this picture I am using a SS turkey baster because it can be sterilized easily, but I have since switched to a pipette and bulb. Don’t put the bulbs in the pressure cooker unless you are certain they can withstand the heat! (I just sanitize mine with Star San.)

Heat the pressure cooker to steam pressure and hold for 15 mins. Turn off the cooker and let it cool without opening the lid (will take at least an hour or two).

If you don’t have a pressure cooker, just boil your items for at least 20 minutes to sanitize them. Alternatively, if you start with clean vials, a no-rinse sanitizer like Star San should also do an acceptable job and would be much quicker.
 
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FlyGuy

FlyGuy

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Once the pressure cooker and contents have cooled, clean your counter-top really well and spray with a no-rinse sanitizer like Star San, if you have some. Working quickly, open the pressure cooker and carefully remove your vials and line them up on your counter. Remove your sterile beaker and use it to hold your pipette/dropper/turkey baster. Put your yeast starter in easy reach.

Now, one at a time, uncap your vial and extract a sample of yeast slurry from the bottom of the flask. Inject enough into the vial to fill it about 80%. If the yeast is really thick at the bottom, you may have to fill it about 60% full and top off with some beer from the flask. You don’t want the mixture to be too viscous or the glycerine won’t mix in very well. Cap the vial immediately after filling, and move on to the next one.

Once all the vials are full, vigorously shake each one to thoroughly mix the glycerine with the yeast. You should still have plenty of yeast left in the flask to pitch into a batch of beer.

 
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FlyGuy

FlyGuy

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Your vials are now prepared and ready to be frozen. I use a small cooler lined with ice packs to store my yeast inside my freezer. The cooler and ice packs are actually essential for keeping the frozen yeast at a relatively constant temperature while in the freezer. This becomes especially important if you have a self-defrosting freezer: the defrost cycle will warm the fridge and potentially thaw and re-freeze the yeast repeatedly, which could kill them.

Be sure to label each vial very carefully. I like to use the Avery 06504 removable white ID labels. As a minimum, the following information should appear on each label:

- brand and strain of yeast (e.g., Wyeast 1028 London Ale)
- date of preparation
- generation # (e.g., 1 if frozen from a fresh pack of yeast, 2 if re-cultured from a vial, etc.)



EDIT: Just found a great tip here -- refrigerating your vials for 48 hours prior to freezing can double your yeast viability. I'll be doing this on my next batch.
 
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FlyGuy

FlyGuy

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When you are ready to use a vial of yeast, leave about 3 – 4 days lead time before you brew. First, you need to gently warm the yeast to room temperature. I usually take a vial out of the freezer in the morning, wrap it in a dry hand towel, and set it in the fridge to gently warm. That evening, I take it out of the fridge and warm it to room temperature while I prepare a small (1 cup/250 ml) starter. Allow the starter to cool to about 105 F/40 C, and then aerate it really well to ensure good yeast propagation.

When you pitch the vial, you want to be as sanitary as possible because you aren’t starting with much yeast and there is a real risk of infection. Open the cap and use a butane torch to flame the mouth of the vial to kill any nasties. Alternatively, spray it well with Star San. Pitch the yeast immediately.

The next morning, the small starter should have fermented out (look for a krausen ring as proof). Now use this yeast to build up a big starter (usually 1 to 2 quarts) the same way that you would use a fresh pack of liquid yeast. Give the big starter 1 or 2 days to propagate and ferment out completely. Before you pitch it, smell the starter or (using sanitary techniques) pour off some of the beer and give it a taste. If it smells or tastes bad, it may have developed an infection so don’t use it. Otherwise, you should have a healthy culture of yeast ready to work its magic for you in your next brew.


References:
http://www.schwedhelm.net/brew/yeast_harv_freeze.html
http://maltosefalcons.com/tech/MB_Raines_Guide_to_Yeast_Culturing.php
http://www.ipass.net/mpdixon/Homebrew/Freezing Yeast.htm
http://www.beertown.org/events/hbc/presentations/TSPresentation.pdf
http://www.mrmalty.com/starter_faq.htm
 

RadicalEd

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Very nice and thorough job! I'll be maintaining a few yeast stocks myself; I just can't justify $7 a pop for yeast. DIY stirplate here I come (electrical engineering student and computer geek so I only need to buy a stirbar :p).
 

McKBrew

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Excellent. Thanks for taking the time to detail the whole process.
 

thebikingengineer

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Any tips on sources for the vials? I'd like to start doing this, but I haven't been able to find these things except by the case.
 

Bellybuster

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I think this is really good that more HB'ers are doing this. I've been freezing yeast for a number of years now and prior to my recent move I had I think 7 different varieties in my "bank".
What I do is use the smack pack normally and freeze the bit that's left in the smack pack. I try to reuse the yeast cake whenever I can and if not I'll freeze 2 more vials from that. Each smack pack gives me 4 vials of frozen yeast and usually 3 batches on the yeast cake. That's 7 batches from the original $10 smack pack. Add in starter material and it works out to around $3 for each batch, a savings of around $7 every batch.
Once I had my yeast bank built up, I never again bought yeast :mug:
 

rdwj

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Quick question...

How often do you find yourself changing out the ice packs in your cooler? Would using a cooler small enough to fit in a freezer, and perhaps packing that with ice packs, stabalize the sample's temp enough to avoid the self-defrosting freezer issues?

..oh, and any idea how long a frozen sample will remain viable?

Thanks for the tutorial; it's very well done.
 
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FlyGuy

FlyGuy

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rdwj said:
Quick question...

How often do you find yourself changing out the ice packs in your cooler? Would using a cooler small enough to fit in a freezer, and perhaps packing that with ice packs, stabalize the sample's temp enough to avoid the self-defrosting freezer issues?

Thanks for the tutorial; it's very well done.
My pleasure on the write-up -- I have been promising it for a while now on a few previous threads where people have asked what I do.

Sorry, if it wasn't clear but the whole cooler goes into the freezer. The ice-packs are only there for temperature stabilization when the defrost cycle kicks in, as you suggest.

(I'll go back and edit that step just so it is really clear.)
 

rdwj

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FlyGuy said:
My pleasure on the write-up -- I have been promising it for a while now on a few previous threads where people have asked what I do.

Sorry, if it wasn't clear but the whole cooler goes into the freezer. The ice-packs are only there for temperature stabilization when the defrost cycle kicks in, as you suggest.

(I'll go back and edit that step just so it is really clear.)

Ah, good! I'm only a few pieces of equipment away from doing exactly what you're showing here and I've been wanting to set up a steam injection system anyway. Now I have one more reason to get started.
 
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FlyGuy

FlyGuy

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thebikingengineer said:
Any tips on sources for the vials? I'd like to start doing this, but I haven't been able to find these things except by the case.
Some good deals can often be found on eBay. I almost bought some there. Actually, I got mine from my local university. You can phone their Chemistry Department and ask to speak to the person running their Chemistry Stores shop. Sometimes they will sell items to the public.

You shouldn't have to pay more than about 50 cents per vial. This may seem like a lot if you buy 30 or 40 of them, but if you get the glass autoclavable ones, they clean up easily and and you can sterilize them in the pressure cooker over and over. You can also find much cheaper vials out there, but they probably won't last as long.
 

fifelee

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The best write up I have seen. Thanks. I have been apprehensive in the past because I couldn't work out some of the details, but I actually feel like I can do it now.
 

98EXL

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very nice writeup! I've started more or less doing the same thing....just refrigerating for now...need some more stuff to freeze my babies
 

andyp

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Hey FlyGuy.

I've never cultured yeast, but I have cultured many other cell types. In every case I have always encountered slow freezing and fast thaw to minimize cell damage. I would revise your thaw steps such that you thaw in room temp water and immediately pitch into your starter at the same temp.

However, maybe I should look into yeast culturing and see if this is the case. Let me check on it and if I find a discrepancy I'll edit this post. Thanks for the methodology. Very solid home cell banking!

Edit:
Ok. I've looked at a couple of papers. For home freezer temps it doesn't look like thawing rates has much of an impact. Also, one paper basically proves the importance of high levels of osmotic pressure (more glycerol) to help preserve cell viability in the cooling, and optimizing (essentially faster) warming rates to prevent death in the warming. So, basically, slow cool with lots of glycerol and fast warming (even 37C or higher to thaw).

http://aem.asm.org/cgi/reprint/72/2/1330.pdf

"Thus, we found that conservation of cell viability following freezing could be improved by increasing the osmotic pressure of the medium and the warming rate should be optimized."

http://aem.asm.org/cgi/reprint/63/10/3818.pdf
 

l3lackEyedAngels

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I think I've mentioned this in other posts, but in freezing my yeast, I omitted the glycerine. My girlfriend centrifuged the vials and poured off the water. I've used two of the vials thus far, the last one having been in the freezer for about nine months. Not everyone has access to a centrifuge, but I thought I would point out a slightly different procedure. Excellent write-up, btw.
 

fifelee

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l3lackEyedAngels said:
I think I've mentioned this in other posts, but in freezing my yeast, I omitted the glycerine. My girlfriend centrifuged the vials and poured off the water. I've used two of the vials thus far, the last one having been in the freezer for about nine months. Not everyone has access to a centrifuge, but I thought I would point out a slightly different procedure. Excellent write-up, btw.
Could you explain that a little more? Do you just spin it, pour off the water, then freeze? Any other steps? Is the theory that the yeast cells are almost dry and therefore there is no ice crystals to damage the cells? Seem that even if spun there would be enough water remaining in the cells to still cause damage.

Nonetheless, a centrifuge seem like a great hombrew project.
 

l3lackEyedAngels

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Originally Posted by fifelee
Could you explain that a little more? Do you just spin it, pour off the water, then freeze? Any other steps? Is the theory that the yeast cells are almost dry and therefore there is no ice crystals to damage the cells? Seem that even if spun there would be enough water remaining in the cells to still cause damage.

Nonetheless, a centrifuge seem like a great hombrew project.
I forget the size of the vials, but they were plastic, four inches long, and about one inch in diameter with conical bottoms. I think they were 50mL vials. Anyway, they were filled with yeast slurry, capped, and then spun down. I forget the size of the rotor or the RPMs, but duration couldn't have been more than 10 minutes. After being centrifuged, the yeast formed what my girlfriend called a pellet on the bottom one sixth of the vials with almost crystal clear water on top. I uncapped the vials, poured off the liquid, flamed the caps and rims, and recapped. Then I brought them home and put them in the freezer. I don't know what the theory is. As for water content, when I thawed the yeast out, it wasn't dry. It was a much thicker slurry than what I started with. So, there was some water present. Damage to the yeast may have occurred, for all I know, but the beer they made tasted fine.

Yes, a homemade centrifuge would be sweet, but hilarity/carnage could ensue.
 

andyp

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From a technical aspect you're probably losing a lot of yeast by freezing them down without any cryopreservative. However, as long as you're pitching enough viable yeast, which would be greatly improved by preparing a starter, it's probably fine.

I don't know if the amount of lysed yeast would be detectable by taste...probably not that much, but that's just another thing to consider.

Writeup looks great on the wiki.

I would amend the part about the slow thaw though...if we're trying to stack the deck in our favour, every little bit helps.
 

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rdwj said:
Ah, good! I'm only a few pieces of equipment away from doing exactly what you're showing here and I've been wanting to set up a steam injection system anyway. Now I have one more reason to get started.
At first, I was all excited thinking "Snizzle Yeast Farm!" but then I remembered that with gas prices today, it's cheaper to buy yeast than to drive to your place to pick up some, even if it was free.

Still, this is a great idea. I've got some 1056 currently fermenting my AIPA. I might pull the sludge from the secondary and see if I can revive it to the point of being able to freeze some for the future.
 
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FlyGuy

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andyp said:
Edit:
Ok. I've looked at a couple of papers. For home freezer temps it doesn't look like thawing rates has much of an impact. Also, one paper basically proves the importance of high levels of osmotic pressure (more glycerol) to help preserve cell viability in the cooling, and optimizing (essentially faster) warming rates to prevent death in the warming. So, basically, slow cool with lots of glycerol and fast warming (even 37C or higher to thaw).

http://aem.asm.org/cgi/reprint/72/2/1330.pdf

"Thus, we found that conservation of cell viability following freezing could be improved by increasing the osmotic pressure of the medium and the warming rate should be optimized."

http://aem.asm.org/cgi/reprint/63/10/3818.pdf
Thanks for the good info, Andy. Much appreciated.

I already modified my original post some time ago to recommend a 48 cooling period prior to freezing. This isn't quite the same as the slow freezing method, but perhaps more practical for homebrewers. The method has been researched by Tom Schmidlin and presented at the most recent NHC. Essentially, the theory is that cooling the yeast in the fridge prior to freezing stimulates a stationary phase where yeast accumulate glucose and trehalose, which appears to promote higher viability during freeze-thaw cycles (see also Kandror et al., 2004). I presume that the cooling period prior to freezing could produce similar results to the slow freezing method you were advocating. Interestingly, Schmidlin also reports that a study by Park et al. (1997) found little difference in viability between frozen yeast warmed rapidly at room temperature vs. those warmed at temperatures just above freezing.

Regardless, I have now seen two suggestions now that faster warming is beneficial, so I will definitely try that. Thanks for the tip. I'll make sure to modify my thread/wiki article too (probably in a couple weeks when I am back from work travel).

Cheers! :mug:
 

andyp

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Great. Yeah, there's probably a benefit to putting them in the fridge before the freezer. Often, when freezing bacteria, I would stick them straight into the -20C freezer first for 4h to overnight, then to -80C. As long as you don't leave them lying around at room temp in the glycerol for too long...4C is likely helpful. Sounds like you've nailed down a good system.
 

jdoiv

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Quick question: What section of the pharmacy or grocery store will have the glycerin? The local walgreens didn't carry it. The pharmacy at the grocery store had the kind that was indigestible. Would it be in the vitamin or health food section?
Thanks
 

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jdoiv said:
Quick question: What section of the pharmacy or grocery store will have the glycerin? The local walgreens didn't carry it. The pharmacy at the grocery store had the kind that was indigestible. Would it be in the vitamin or health food section?
Thanks
All Seasons has a couple different varieties. Alternatively, if we ever get together to brew, I can give you some for free.
 

macs

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thebikingengineer said:
Any tips on sources for the vials? I'd like to start doing this, but I haven't been able to find these things except by the case.
I got some from an Ebay store last year. :)
 

jdoiv

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PseudoChef said:
All Seasons has a couple different varieties. Alternatively, if we ever get together to brew, I can give you some for free.
Cool. I should of thought to look there last weekend but forgot. I got all my vials and beakers in today. Just need to get a pressure cooker and the glycerin and I'm good to go.

Another question. Instead of the pressure cooker, can you bake the glassware in the oven instead? Or is that not going to be a sterile enough environment?
 

PseudoChef

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jdoiv said:
Cool. I should of thought to look there last weekend but forgot. I got all my vials and beakers in today. Just need to get a pressure cooker and the glycerin and I'm good to go.

Another question. Instead of the pressure cooker, can you bake the glassware in the oven instead? Or is that not going to be a sterile enough environment?
You can, but you need to bake for like 12 hours or something crazy like that. Pressure cooker's gonna run you what? $20-$30?
 
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what do you folks think about washing the yeast as per the instructions in other posts before "bottling" and freezing. would it be beneficial to get rid of the trub and break. i have a big half gallon starter that i made with wyeast 1010 that i'll be preparing to freeze on friday when my vials get here. it started as a 1000ml starter, then half gallon, and then today i poured off the beer and added another half gallon of fresh wort on top of the yeast so i've built up quite a bit of trub.

i also have about 100ml of washed yeast in a pint jar in the fridge that i harvested from a 5g batch of wheat beer. its been in there for about a month, maybe 6 weeks. its a different strain so i might go ahead and freeze a couple vials of this yeast as well. then maybe i can make a starter with it in a few months just to see how viable it is having been washed and then sat in the fridge for a while. comments?
 

macs

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If you guys see the book..."First Steps in Yeast Culture" by Pierre Rajotte, buy it!!
It is an awesome book on this subject and is full of procedures on sterilizing, transferring, storing and other methods of manipulating yeast.
 

Llarian

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SenorWanderer said:
what do you folks think about washing the yeast as per the instructions in other posts before "bottling" and freezing. would it be beneficial to get rid of the trub and break. i have a big half gallon starter that i made with wyeast 1010 that i'll be preparing to freeze on friday when my vials get here. it started as a 1000ml starter, then half gallon, and then today i poured off the beer and added another half gallon of fresh wort on top of the yeast so i've built up quite a bit of trub.

i also have about 100ml of washed yeast in a pint jar in the fridge that i harvested from a 5g batch of wheat beer. its been in there for about a month, maybe 6 weeks. its a different strain so i might go ahead and freeze a couple vials of this yeast as well. then maybe i can make a starter with it in a few months just to see how viable it is having been washed and then sat in the fridge for a while. comments?
There is no trub in a starter to speak of. If you take your washed yeast and pump it up to a half gallon starter, you can just pull the yeast slurry from the bottom and freeze that without worrying about any further washing.

-D
 

jdoiv

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Ok, so I went by the LHBS this morning and picked up what I hope is the right stuff. If was Glycerin USP Finishing Formula from JD Carlson. Is this the right stuff?
 
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