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Old 11-16-2013, 06:10 PM   #561
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Originally Posted by BoShimTang View Post
Sterile technique is critical. Of the two books I listed, I most highly recommend Rajotte's as it is thorough but brief. Assuming you also have access to a pressure cooker with a gauge, you should have no problem with contamination. The selection of an isolated colony ensures that you have a pure culture derived from a single cell.
I'll have to get that book. I do have a pressure cooker, I use it to make my slants and also to can my starter wort, so sterile technique isn't an issue.

I was more concerned the fact that I can't pressure cook the petri dishes like I can the slants. Also about airborne stuff when I plate the yeast, then again when I transfer it to the slant.

Finally, last time I tried to make plates, I failed and didn't end up with any single cell colonies, just thick smears of yeast. I did use small petri dishes for that though, now I have some larger ones.

I have a bunch of empty slants I need to make an agar medium for though, so I will definitely fill some petri dishes as well and give it a try.
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Old 11-22-2013, 01:00 AM   #562
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Here are my 1st couple of slants. The yeast looks nice and white, but for some reason my slants are accumulating fluid at the bottom of the vials. Even the newer ones I have made with more agar are doing this. When putting the yeast in the vial, I poured out any liquid that had accumulated by condensation, so I'm not sure what is going on and whether it's a problem.

Why are you using vials? The proper tool for the job is a piece of lab glassware known as a screw-cap culture tube. A screw-cap culture tube looks like a test tube with a screw-on cap that has an autoclavable rubber liner. What kind of liner does that vial have?

Here's a photo that I took a few months ago when I made plates and slants:



Additionally, you should dump the Asian food store-grade agar. It's junk. Ideally, you should use lab-grade powdered agar. eBay seller solared2011 is selling 100 grams of lab-grade powered agar for $12.99 + $0.86 shipping (http://www.ebay.com/itm/100-Grams-0-...item20ce3e94ee), which is a very good deal because 100 grams of lab-grade agar will make 6.67 liters of malt agar. That's enough media to last an active amateur brewer ten years because 250ml of malt agar media will make twenty 20x125mm culture tube-based slants and six 100x10mm plates. If you must use non-lab-grade agar, then look for agar flakes in a health food store. Agar flakes, while not as pure as lab-grade powered agar, work very well.

Lab-grade agar is so pure that one only needs to 1.5% agar weight/volume when making malt agar media. A simple recipe for malt agar is 10% light dry malt extract (DME), 1.5% agar, and 0.1% yeast extract (yeast energizer also works). Putting those ratios into practice, in order to make one liter of malt agar, we need to use 100 grams of DME, 15 grams of agar, and 1 gram of yeast nutrient. I make up 250mls of malt agar media at a time, so I use 25 grams of DME, 3.5 grams of powdered agar, and 1/8th teaspoon of yeast extract.

With that said, it looks like your media is breaking down. How much agar did you use? How long did you autoclave your media? One only needs to autoclave media for 15 minutes at 100C/212F @ 15 PSI. While a gauge is nice to have, any quality pressure cooker will hit this temperature and pressure without a gauge. I have been using the same T-FAL/SEB 3125 stainless pressure cooker to sterilize media at home for over twenty years without single infection that could be traced back to the pressure cooker.

Finally, the proper tool to use when inoculating slants is a nichrome loop. A decent nichrome loop can be had for a few dollars. A nichrome loop is sterlized by heating it until it is red hot. Also, ALL LIQUID YEAST CULTURES should be plated for "singles" before transferring well isolated colonies to slants. Contrary to what you may believe, packages of Wyeast yeast and preforms of White Labs yeast are not 100% pure yeast. Plating for singles and using well isolated colonies that exhibit good morphology to inoculate your slants using aseptic technique will ensure that nothing else grows on your slants.
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Old 11-22-2013, 04:25 AM   #563
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Why are you using vials? The proper tool for the job is a piece of lab glassware known as a screw-cap culture tube. A screw-cap culture tube looks like a test tube with a screw-on cap that has an autoclavable rubber liner. What kind of liner does that vial have?

Here's a photo that I took a few months ago when I made plates and slants:



Additionally, you should dump the Asian food store-grade agar. It's junk. Ideally, you should use lab-grade powdered agar. eBay seller solared2011 is selling 100 grams of lab-grade powered agar for $12.99 + $0.86 shipping (http://www.ebay.com/itm/100-Grams-0-...item20ce3e94ee), which is a very good deal because 100 grams of lab-grade agar will make 6.67 liters of malt agar. That's enough media to last an active amateur brewer ten years because 250ml of malt agar media will make twenty 20x125mm culture tube-based slants and six 100x10mm plates. If you must use non-lab-grade agar, then look for agar flakes in a health food store. Agar flakes, while not as pure as lab-grade powered agar, work very well.

Lab-grade agar is so pure that one only needs to 1.5% agar weight/volume when making malt agar media. A simple recipe for malt agar is 10% light dry malt extract (DME), 1.5% agar, and 0.1% yeast extract (yeast energizer also works). Putting those ratios into practice, in order to make one liter of malt agar, we need to use 100 grams of DME, 15 grams of agar, and 1 gram of yeast nutrient. I make up 250mls of malt agar media at a time, so I use 25 grams of DME, 3.5 grams of powdered agar, and 1/8th teaspoon of yeast extract.

With that said, it looks like your media is breaking down. How much agar did you use? How long did you autoclave your media? One only needs to autoclave media for 15 minutes at 100C/212F @ 15 PSI. While a gauge is nice to have, any quality pressure cooker will hit this temperature and pressure without a gauge. I have been using the same T-FAL/SEB 3125 stainless pressure cooker to sterilize media at home for over twenty years without single infection that could be traced back to the pressure cooker.

Finally, the proper tool to use when inoculating slants is a nichrome loop. A decent nichrome loop can be had for a few dollars. A nichrome loop is sterlized by heating it until it is red hot. Also, ALL LIQUID YEAST CULTURES should be plated for "singles" before transferring well isolated colonies to slants. Contrary to what you may believe, packages of Wyeast yeast and preforms of White Labs yeast are not 100% pure yeast. Plating for singles and using well isolated colonies that exhibit good morphology to inoculate your slants using aseptic technique will ensure that nothing else grows on your slants.
Whew! That is a whole lot of words to tell us that the way the original poster described his slanting technique has led us all astray! I'm glad I didn't read this first, or I would have never started slanting!
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Old 11-23-2013, 03:25 AM   #564
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Whew! That is a whole lot of words to tell us that the way the original poster described his slanting technique has led us all astray! I'm glad I didn't read this first, or I would have never started slanting!
Yes... And thank you for your contribution as well.
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Old 11-24-2013, 01:36 AM   #565
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Whew! That is a whole lot of words to tell us that the way the original poster described his slanting technique has led us all astray! I'm glad I didn't read this first, or I would have never started slanting!
You can look at it that way, or you can look at my posting as a clarification of the process. There is a lot of rigor involved in maintaining a healthy yeast bank. One needs to ensure that the only microflora on one's slants are the reference cultures, and the only way to ensure that level of purity is to plate every liquid culture before transferring it to one or more slants. All commercial yeast cultures contain low levels of contamination. It is not economically feasible for the yeast manufacturers to ship lab-grade cultures at home brew trade price points, but that's exactly what one needs to plate if one wants to bank a culture for an indefinite amount of time.

Here's a plate that I made from a culture that I grew from a bottle of commercial bottle-conditioned beer:



The plated culture shown above was clean enough to use, but I wanted to ensure that I was banking the reference culture and not any wild microflora that made it into the bottle during bottling. That's how one ends up with mold and/or bacteria laden slants. The well-isolated colonies in the red rectangle are each the offspring of a single yeast cell; therefore, they are single-cell isolates (a.k.a. pure cultures). The colonies all demonstrate good morphology, that is, they are round, dome shaped, and creamy white, which makes them good candidates for transfer to slants. One can be assured that the slants containing these colonies will remain clean if one practices good aseptic transfer technique when performing plate to slant, slant to slant, and slant to starter transfers.
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Old 11-29-2013, 06:09 PM   #566
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I've got bubbles in all of my slants... probably from pouring/agitation. Any reason I can't just crack the caps and pop them all back in the pressure cooker for another 15min?

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Old 12-01-2013, 07:12 PM   #567
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I've got bubbles in all of my slants... probably from pouring/agitation. Any reason I can't just crack the caps and pop them all back in the pressure cooker for another 15min?
No problem, the media might darken a bit more but that's about it.
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Old 12-01-2013, 09:31 PM   #568
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no problem, the media might darken a bit more but that's about it.
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Old 12-26-2013, 01:54 PM   #569
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What is the amount of DME needed and what is the cost of bringing a slant to a White Labs vial size? Trying to convince myself to buy the equipment

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Old 12-26-2013, 02:07 PM   #570
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you won't be able to slant in a white labs vial (the caps are not able to be put into a pressure cooker). I'm sure they have a more sophisticated way of sterilizing them than with steam under pressure. The proper vials are very cheap - about $1 each and re-usable.

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