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Old 06-15-2010, 04:25 PM   #121
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I have heard of people boiling w/ some luck, but i think you're going to get more contam's and headaches then is worth it. Even working with fully sterilized agar can be quite discouraging if your handling procedure is not fully sterile, aka good glove box (or better yet flow hood).

The real big issue will be if there is a small contam you cannot see, and it makes it into your batch of beer. No good.

Really there is no getting around buying a pressure cooker if you want to work with agar/slants. But a small pressure cooker, enough for a beaker of vials, is not a large investment.

 
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Old 06-15-2010, 04:39 PM   #122
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Quote:
Originally Posted by pbarning View Post
Would I be correct in assuming that the pressure cooker provides a means of sterilizing both the media and vials?

I am thinking about boiling the media and funneling it into the vials. Then I'll put the filled and partially capped vials into a beaker that will be placed into a boiling water bath. I will cover the bath (pot) so as to allow the steam to make contact with the vials. This should be sufficient for sterilizing the vials, right?

-or-

Could I simply boil the media and funnel it into the vials and then place the partially capped vials into a stove for a period of time? Like a ghetto autoclave?

Bottom line is that I do not own a pressure cooker, but there should be alternative means of successfully sterilizing the media and equipment.
Boiling will not sterilize. It will kill most things, so you may get lucky, but you never know.

Truethfully, though, most pressure cookers only get to 10 PSI (240F) which will kill just about everything, but not quite. To really sterilize, you need a canner that gets to 15 PSI (250F). I wonder how many people are using 10PSI with success. You may be ok at 212F, but I wouldn't count on it.

 
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Old 06-15-2010, 07:42 PM   #123
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It isn't the steam making contact, it's the temperature of the steam. At normal atmospheric pressure, water boils at around 212F. The steam is no hotter than that. It takes about 15 PSI above atmospheric pressure to raise the boiling point of water to 250F.

This is why it is necessary to have a pressure cooker to sterilize...
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Old 06-16-2010, 07:18 AM   #124
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Quote:
Originally Posted by EricMc View Post
I have heard of people boiling w/ some luck, but i think you're going to get more contam's and headaches then is worth it. Even working with fully sterilized agar can be quite discouraging if your handling procedure is not fully sterile, aka good glove box (or better yet flow hood).
I don't believe you need a glove box or flow hood for any of this kind of stuff unless you have a really unclean environment.

I do all of my yeast work on my washing machine which I clean the top of and sanitize. I make sure there are no drafts (windows or heat/air running). I use a simple alcohol burner right there and make sure not to move quickly or breathe in the direction of an open container.

Unless I was testing procedures, I've never had a contaminated plate/slant. Thinking through your process and planning out what you are going to do before you open anything is a big help.

I also will wipe everything I'm going to use with rubbing alcohol while setting it into my clean area.

If you have long hair, tie it back. Scrub up your hands and arms before starting.

 
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Old 06-16-2010, 09:49 AM   #125
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Edit: doh! didn't realize you were saying you were having to by a PC. You can buy per-sterilized agar, but its expensive for the shipping costs if you purchase it in liquid form.

Don't be discouraged! I don't believe you need a flow hood at all. That's over kill. What you need is a nice small room (ie. bathroom) and the air is not actively circulating via air conditioning. Airborne spores of molds and bacteria are carried through the vent ducts which can cause problems contaminating your slants. That being said there are good precautions you can use to try to prevent contamination.

Practice Sterile Technique

-Pick up an alcohol wick candle and some 90% ethanol(or better yet Grain Alcohol!). High % Ethanol will help sterilize the equipment by killing most microscoping living organisms. The flame helps insure that no contaminants transfer between different samples you may work with during that one sit down time. NOTEeep each item ~1 foot from one another at all times when using them together.

- Every time you change samples (ie. Slanting Pacman and US-05 in one sitdown) you should flame your dip your tool in Ethanol for a few seconds. Let the ethanol evaporate - don't shake near the flames. When dry flame your tool for a quick second - if its metal get it red for a second then let cool.

- You can use sterilized tooth picks to do this too, but they're a bit harder to find sterilized than to buy a single metal inoculation loop. Its far easier to sterilize and can be reused. Also if you use wood, don't do the ethanol step. Just do the flame step.

- Open container 1 obtain your yeast from container 1, flame the glass mouth in the flame for 1-2 seconds while rotating the bottle to get all edges. Then close. Open slant 1, flame top if its glass,streak it on the agar slant, and close slant tube quickly after inoculation. Either toss your wooden spread stick for another or dip your metal loop in the Ethanol to start back over.


- Also be sure not to breath on the agar surface if you can help it rather breathe away from it. Its really not that bad when you get used to it. You just need some patience and a pair of latex gloves.

- Dont' allow any tool that will touch the yeast un-clean surfaces without being sterilized again. Bacteria are ubiquitous in the environment regardless of how clean you think it may be. Those same bacteria can easily utilize the same agar for quicker growth than the yeast can handle. Thats why I suggest the sterile technique.

Trust me, it's do-able. I work in a microbiology lab where we constantly culture bacteria on all types of agar. We deal with the same issues, but we find ways where we don't have to use the 1 hood available. The name of the game is being sterile and careful. Best of luck!

You guys should take a note from people who grow mushrooms. those people really know how to culture and keep around their fungus. you guys have it easy compared.


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Old 07-01-2010, 02:47 PM   #126
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Any tips on handling a pure brett strain on agar? I have just acquired lambicus and it grows insanely on the plates I have just innoculated. I can tell already its a totally different behavior. Should it get different treatment/media/agar type for long term storage? Maybe something to retard its growth... Also I'm hoping water storage also works well for lambicus.
It is truly frightening.
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Old 07-15-2010, 11:57 PM   #127
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i have an opportunity to pick up a nice 4.2 qt pressure cooker for $10 that is in good shape. It is kind of small but would it still work? I dont brew too much so i will only have a few slants of each strain so i wont be making more than 1o ready to go slants at a time? Just not sure it its a waste of money and i should wait for a deal on a bigger pressure cooker

 
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Old 07-21-2010, 04:22 PM   #128
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Eric, just wanted to give a big thanks to you for this tutorial. As my brewing's come along, I've started buying bulk grain and hops, but it's still a chore to head to the LHBS every time I want to brew to snag the yeast - and not cheap at ~$7 a pop. (And with the nearest LHBS a 2 hour round trip from me, it's hugely inconvenient, I'm usually unable to get out there until brew day, which means I always have to get at least two packs, since I don't have time for a starter). Having slants in my arsenal would be outstanding.

I will say, I'm still terrified by the thought, though. Just a couple of little stray cells in one of these can happily kill a batch of beer - but only way to try is to... try!

 
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Old 07-24-2010, 05:30 PM   #129
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so if i made 12 ml slants and was preparing with a stir plate could i follow these steps?

1 - add canned wort to slant and start a 100ml starter for 1 day
2 - day 2 pitch the 100 ml into boiled 1L or 2L started
3 - pitch into fermenter

How does one know how big of a final starter to use without counting? Is there a general rule of thumb that the yeast should be 100% viable so a 100ml into a 1L starter would be fine for a beer with OG under 1.060. My biggest concern is i use to just pitch the white labs vials and i got alot of bad off flavors from under pitching

 
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Old 07-24-2010, 06:41 PM   #130
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ek, that's one of the issues (in my mind) that's stopping me from using slants. From what I've read, the time from slant to pitching is about a week - I believe what I read was along the lines of "if you get your starter going Monday, it should be ready for Saturday - start a couple days earlier if your OG is over 1.060."

You'd probably want to start with a 50ml starter (36 hours?), stepping to 250ml (2 days), then 1000ml (2 days) for a beer, under 1.060. If it's higher than that, add another 2 days of a 2000ml starter.

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