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Old 10-28-2009, 01:42 PM   #31
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I've used a simular process in the past. One problem I keep having, is after inoculating the slants and the yeast start to grow they produce a lot of C02. I vent the tubes but am always worried about contamination. When activity starts to die down (less gas to vent) I place the tubes in the fridge. But they still ferment a bit, and when I go to use the slant, I get a large puff of C02 and the agar actually starts to bubble up from the base of the tube and push its self slowly out of the tube.

Do you vent your culture tubes?

Have you ever had this problem?

Maybe I just used the wrong mix of agar/wort.

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Old 10-28-2009, 02:59 PM   #32
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No I incubate the tubes in a sanitized beaker covered with foil with the lids slightly loose so they can offgas. After 10-14 days I tighten them up and into the fridge. I have had one vial give off a slight puff of CO2 so far when opening it but it was very slight.

Yeast/bacteria/mold can only get in through dust which can't get through the threads under the cap, but moisture and CO2 can escape.

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Old 10-28-2009, 03:21 PM   #33
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i tested that theory, i sterilized (15 psi for 30 min and cooldown time) 500ml wort. left the flask for a week with an shimmed, inverted beaker.. to leave plenty of gap for airflow. no mold or anything visibly colonized it. so i agree. airborn bad guys cant fly up.

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Old 10-30-2009, 03:04 AM   #34
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There is enough yeast that a 250mL starter is actively fermenting on the stir plate after 24 hours. Assuming a growth rate of one generation every two hours, and a maximum yeast concentration of 80M cells per mL, there would have to be at least 5M cells in the slant. So a conservative estimate would be somewhere around 1M ~ 10M cells... but it's just a guess.

This is the best online resource IMO regarding yeast cell concentration and growth rates in wort:

http://www.maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices

If you read, you will see why I use a stir plate AND sterile wort for the first step; the method he gives you can use non-sterile wort safely but it requires adding 10mL of wort (which you could add directly to the slant vial) and wait 2-3 days before pitching the 250mL starter. Since I use sterile wort and am very very careful with my sanitation in the first step, I go straight from the slant into 250mL without any problems.

I'm just getting started in slanting, but I've been going from slant to ~200mL for the first step, then from there to ~1000mL, then higher if necessary. It's working fine so far.

Also, 'he is a 'she'. The author of that Maltose Falcons article is Maribeth Raines.


Here's a question i don't think I've seen an answer to...What makes a slant 'go bad'?
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Old 11-04-2009, 12:07 PM   #35
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A slant can go bad if it dries out, or if it becomes infected with mold, fungus, wild yeast, ect.

Another question, what do you do about the condensation that forms inside the slant tubes while setting? With the petri dishes it's fine, you just turn them upside down. With the slants, I store them upside down but then when I go to innoculate, a few drops of water leak around the cap. I blow torch this away, but just wondering how you handle it.

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Old 11-04-2009, 12:43 PM   #36
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To me this looks like an alternative to washing yeast and keeping it in 200ml jars but slants take up less space - a typical test tube stand could easily keep ten strains. Are there any other advantages to slanting as opposed to washing ?

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Old 11-04-2009, 05:51 PM   #37
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i got 12 ml vials . a little small. do you think i could work with those?

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Old 11-04-2009, 06:33 PM   #38
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A slant can go bad if it dries out, or if it becomes infected with mold, fungus, wild yeast, ect.
I have only had one slant 'go bad', it had visible green mold growth from a single spore that managed to sneak into the slant vial while I had the cap off.

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Another question, what do you do about the condensation that forms inside the slant tubes while setting? With the petri dishes it's fine, you just turn them upside down. With the slants, I store them upside down but then when I go to innoculate, a few drops of water leak around the cap. I blow torch this away, but just wondering how you handle it.
I haven't been that concerned about it, doesn't seem to effect anything.

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To me this looks like an alternative to washing yeast and keeping it in 200ml jars but slants take up less space - a typical test tube stand could easily keep ten strains. Are there any other advantages to slanting as opposed to washing ?
Washing yeast the viability drops off quickly so you have to re-use the yeast relatively soon, or re-wash and propagate the yeast prior to pitching. I have also had a few infection problems with washed yeast, whereas building up from a slant everything is sterile (you are always working with yeast which came from a pure culture rather than from a fermenter).

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i got 12 ml vials . a little small. do you think i could work with those?
Most folks use the 12ml vials for yeast banking. I went with the bigger ones because I think they are easier to handle.
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Old 11-04-2009, 06:52 PM   #39
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thanks for all the information Saccharomyces .

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Old 11-05-2009, 05:42 PM   #40
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i can only find gelatin in 1oz packaging. will regular gello work? thicker of course.

it looks like you penetrated the surface of the medium at innoculation time vs swiping the surface?

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