I have access to a flow hood, sterile 2 ml flip cap tubes, and sterile lab equipment. So would it work to take my smack pack or white labs tube and and some glycerine (should be purchased sterlie) and transfer lets say 1 ml of the pure yeast slury and 300 ul of glycerine into the tubes and freeze in the -80? I should be able to get atleast 20 tubes of pure yeast with plenetly left over for that weeks brew (using a starter). Then when i need the yeast just thaw and pitch that into 10 ml tubes of sterile wort and step up from there to 50ml then 2L? Anyone see a problem with this?
alternative method for forzen yeast bank
- Thread starter ekjohns
- Start date
Help Support Homebrew Talk - Beer, Wine, Mead, & Cider Brewing Discussion Forum:
spareparts
Well-Known Member
I go ahead and sterilize the 10, 50, and 500 ml in either an autoclave or pressure cooker. Boil the 2000 ml or use pressure canned wort. Has worked great for me for years. If I was only relying on boiling for 10 ml and 50 ml steps I'm not sure that I would even culture yeast. There's nasties that can survive boiling and they'd have alot of opportunity to multiply in the culturing process.
devilishprune
Well-Known Member
- Joined
- Mar 26, 2010
- Messages
- 1,241
- Reaction score
- 15
Would your lab be cool with you using yeast in the hood? I don't know if you work in a yeast lab or what. We had a string of yeast infections in my lab lately, and I bet they would have been PO'ed had I suggested this.
More power to you though, I wish I could access my lab stuff for beer brewing!
More power to you though, I wish I could access my lab stuff for beer brewing!
yeah good point maybe ill work outside the hood. I was planning on doing it over the weekend where the hood would not be used for 24 hrs with the UV light on but stuff does have a habbit hidding out of uvc range
I do the same as you: I freeze 10 tubes straight from the smack pack. The rest goes into a starter for the current beer.
I do the 10ml to 4L jump all the time. Stir plate and sane step-ups. No problem. Takes at least a week, tho, to build a nice starter for 10g batch. Eats up some DME, too.
10ml
250ml
2L
4L
decant, repeat
I do the 10ml to 4L jump all the time. Stir plate and sane step-ups. No problem. Takes at least a week, tho, to build a nice starter for 10g batch. Eats up some DME, too.
10ml
250ml
2L
4L
decant, repeat
No. I really really wish I could, but I don't have the space to freeze a bunch of wort, and I don't have a pressure canner (man, I've got to get one of those). So, it's boiled DME for me.
I do a lot of work with yeast in cloning/inserting, if you're going to store at -80 I would try to make it a 50% glycerol especially if you are planning any long term storage (>12 months).
A 1 ml spike in to 10ml would be an overkill inoculation. I get away with 100ul into 10ml using YPD or sabouraud, I would assume DME would fall into the same lines? I use a shaker at 200rpm @ 30C , I'll get enough growth to go up to 250-500ml within 5-8 hours.
My experience is all in the research lab, I have no idea how well DME works as a starter as I'm browsing here to learn about this.
Also if your hood has a UV, just nuke it for 5 minutes if your worried about yeast contamination, but I would say that is very rare occurrence. Its a lvl 1 spp, you don't really need a hood if you use decent sterile technique.
Let me know how it turns out! I work in a lvl 3 lab, I've thought about doing it, but if I walk out with a vial of anything I wouldn't have a job....
A 1 ml spike in to 10ml would be an overkill inoculation. I get away with 100ul into 10ml using YPD or sabouraud, I would assume DME would fall into the same lines? I use a shaker at 200rpm @ 30C , I'll get enough growth to go up to 250-500ml within 5-8 hours.
My experience is all in the research lab, I have no idea how well DME works as a starter as I'm browsing here to learn about this.
Also if your hood has a UV, just nuke it for 5 minutes if your worried about yeast contamination, but I would say that is very rare occurrence. Its a lvl 1 spp, you don't really need a hood if you use decent sterile technique.
Let me know how it turns out! I work in a lvl 3 lab, I've thought about doing it, but if I walk out with a vial of anything I wouldn't have a job....
If you have access to an autoclave or pressure cooker, sterilize it yourself, while you're sterilizing your other stuff. It'll be cheaper to buy that way.
Also, it's easier to work with if you have a 50/50 volume/volume solution (or even 70/30), which you can sterilize yourself
Most of my -80 yeast stocks are from YPD grown, very dense, cultures. I 'skipped a few steps' and added glycerol to smack pack contents about 10 years ago. Those don't have the best viability right now. That should be OK for short term use, though.
Paul, 50% glycerol isn't good for long term yeast storage. 15%-25% is way better. A buddy of mine tried 50% back in the 90's. His tubes were in the same box as mine in the -80. His crapped out after a year; I just made a beer with London Ale that was frozen in 1992 at 25% final glycerol.
That, sir, is amazing. I'd like to solicit your services in about 40 years. I will have my glycerol koolaid ready.
Sorry that happened.
I also have yeast strains from the 80-90s that are all in 50% and their viability is fine. I stand by my suggestion of 50% for -80. You can get away with 15% for bacteria, and we sell material with 15% simply because it expires and it generates more sales for us. Kind of an inside tip there.... All of my research materials and stocks are in 50%.
Preserving microbes is part of what I do for a living; though my focus is DNA/molecular.
Are use using 50% final volume/volume? Or are you adding 50% to a culture?
The story of my friend's yeast jives with experiences I've had with E. coli cultures I've received in 50% glycerol; they died after 6-12 months. I never used 50% for two reasons: first, because I'm not so adventurous, I just follow standard protocols, such as:
In the 1986 Cold Spring Harbor Methods in Yeast Genetics Course Manual, by Sherman, Fink and Hicks, appendix D (looking at my paper copy) it says to use 15% v/v final.
The 2005 version by Amberg, Burke, and Strathern says to use 25% v/v final (appendix B, you can see it on Google Books)
Molecular Cloning: A Laboratory Manual by Maniatis, Fritsch and Sambrook says to use 15% for bacteria. Most likely, E. coli or Salmonella.
Most Agrobacterium protocols I've read say to use 10% glycerol v/v, though Chapter 1 by Wise, Liu and Binns, in Agrobacterium protocols, Volume 1 Edited By Kan Wang says to use 25%.
and second because it's so easy to handle 50% glycerol v/v in water and just mix it 50:50 with a liquid culture to make 25% final.
You mean human ERC detection? Or maybe External RNA Control? Either way, nope.
Most of the PCR I do is to clone something from plant cDNA or to add/modify the ends of something already cloned in order to clone it some other way. No need for external controls there; I either get my clones or I don't. There's another group for any quantitative stuff.
Most of the PCR I do is to clone something from plant cDNA or to add/modify the ends of something already cloned in order to clone it some other way. No need for external controls there; I either get my clones or I don't. There's another group for any quantitative stuff.
Well dude, you just go ahead at use your 15% then. I tried to talk sense in to you.
Same back at you, with literature references even.
You quoted bacterial references. Re-read my posts I already defined the concentrations for bacteria. I started to ignore you then. I have no patience for ignorance.
Seriously, I offered advice, I've been preserving everything from RBCs to virus for over 25 years. I do this for a living and I get paid very well for it. Seriously, you have no idea what an external run control is and you do PCR?
I deal with kids at the university everyday as I help at the cloning lab. I have to deal with it there because I get paid for it. I will not put up with it while looking into my hobby, I enjoy coming here because I like talking about beer.
I offered advice, take it or leave it.
Read again Mr. Patience. The Cold Spring Harbor reference is for...yeast. From a National Academy Member, no less. Do you know what that means?
I do have an idea what an external control is, I was simply asking if that was what you were referring to. If you are really the expert you claim, you'd know that cloning PCR doesn't require external controls.
Yes, you did, while claiming
Alright seriously I'm done with you. I gotta unsub from this thread because you sir are an idiot.
Yell, get emotional, start calling names, you lose. sorry, that's how it works.
Similar threads
