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Old 11-03-2010, 01:34 AM   #1
poolshark021
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Default Streaking plates from slurry

I just bought a bunch of equipment for making yeast slants and plates. I want to get some practice with my new toys so I figured I will make some plates and streak them with yeast from my 2 fermenters once I rack the beer out of them. I will probably wash the rest of the slurry and save it, but I am more interested in growing colonies.

Once the plates are inoculated I will harvest a couple colonies and put them in slants for long term storage (6 months or so). Does anyone see any problem with this or have any advise for me?

I know this isn't the easiest or cheapest way to re-use yeast, but I am more concerned with getting my sterilization and technique down.

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Old 11-03-2010, 05:55 AM   #2
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Keep slants @ RT. I wouldnt keep them for that long tho. If you want to freeze, add glycerol (14-20% is fine) to a liquid culture.

But I feel you are better off pulling a single colony from a plate to inoculate a small (1-10ml) starter and scale up from there. Once colonies appear on a plate, keep them in the fridge to ensure they dont grow too much. Restreak often.

What media are you streaking on? I recommend 0.1X ME with 1.5% agar as it allows only the strongest colonies to emerge first. YMMV.

You dont need much yeast to streak - simply touch your sterile loop to it and streak.

Unless you have access to an autoclave and laminar flow hood, sterilization is improbable and unneccesary. Just keep things sanitized.

Google 'Streaking Bacterial Stocks' for general techniques.

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Old 11-03-2010, 04:29 PM   #3
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I am basing most of my info off the yeast slanting sticky in this fourm, and also the links provided at the beginning of that thread. As I understand it if you sterilize everything right with a pressure canner you can store incubated yeast slants in the refrigerator for up to a year, maybe more. Also 15 min. in a pressure canner at 12-15 psi will do the same thing as an autoclave.

For media I was planning on using DME to make 1.040 wort and add 3% agar. I forget where I read that. Do you mean that a smaller amount of agar will keep weaker colonies from growing as fast?

I will also be sterilizing some of the wort without agar to make the intial 10-50mL starters as needed.

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Old 02-16-2012, 07:07 PM   #4
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Quote:
Originally Posted by poolshark021 View Post
I am basing most of my info off the yeast slanting sticky in this fourm, and also the links provided at the beginning of that thread. As I understand it if you sterilize everything right with a pressure canner you can store incubated yeast slants in the refrigerator for up to a year, maybe more. Also 15 min. in a pressure canner at 12-15 psi will do the same thing as an autoclave.

For media I was planning on using DME to make 1.040 wort and add 3% agar. I forget where I read that. Do you mean that a smaller amount of agar will keep weaker colonies from growing as fast?

I will also be sterilizing some of the wort without agar to make the intial 10-50mL starters as needed.

I would generally pressure cook my media for 1 hour at 15 PSI.
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Old 02-16-2012, 07:17 PM   #5
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3% agar is probably also just a waste of agar. I work in a yeast lab, and all of our plates are made with 2% or less agar. I would be wary about using year old yeast from the fridge because of mutation, especially if you are picking single colonies (greater chance your mutation will proliferate). I would say a few months would be ok, much better to freeze down and make a yeast library that you can collect from though.

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Old 02-17-2012, 05:41 PM   #6
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3% agar is probably also just a waste of agar. I work in a yeast lab, and all of our plates are made with 2% or less agar. I would be wary about using year old yeast from the fridge because of mutation, especially if you are picking single colonies (greater chance your mutation will proliferate). I would say a few months would be ok, much better to freeze down and make a yeast library that you can collect from though.
I think a lot of people here recommend processing the whole slant rather than picking an individual colony for this very reason. I have been making my 20% glycerol stocks in 1ml aliquots, and just using the whole thing to innoculate.

I'm in the same field as you, I work on directed evolution in yeast, and not going from single colony is foreign to me! It makes sense though, because having mixed background allows competition to do its thing.
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