This weekend I got around to setting up some agar slants and petri dishes in an effort to culture up some yeast. I have a slurry of Cal Ale yeast that has gone through several pitches and I'm going to streak a petri dish to isolate colonies of what hopefully will be my "house" yeast strain. I've been re-pitching this slurry at a temperature slightly lower than what is recommended for the strain (65° as opposed to 68°), so this is an experiment to see if the yeast has adapted to a slightly different environment and taken on a different character (i.e. produces a different tasting beer).
There are definite risks in this experiment. For example, since this slurry has been re-pitched several times, I'm quite sure that it's not 100% sanitary. I could easily wind up isolating a colony (or colonies) of wild yeast, bacteria, a mutated strain, who knows what... So what I'll probably wind up doing from a high level is:
1) Streak the petri dish to isolate colonies
2) Ferment three small samples of wort with three separate colonies
3) Taste the fermented wort and hopefully find that one of them is acceptable
4a) Inoculate an agar slant from the best sample
4b) Culture up a starter for a full 5 gallon brew from the best sample and hope that the resulting beer is awesome
Here are the steps that I followed to make create the culture medium for the agar slants and petri dish:
1) Wort - I usually save my last runnings from a brew day and boil them down to around 10 brix on the refractometer. Saves me from having to buy malt extract.
2) Agar - 1 tbsp per cup of wort. This stuff has to be simmered for a little while to dissolve before pouring into the petri dish or test tubes. You can find it in most health food stores - it's a vegan gelatin substitute.
3) Yeast nutrient - I add a pinch per cup of wort.
4) Five Star 5.2 - Since I'm boiling down last runnings, I like to make sure that the pH of my medium is stable, so I add a pinch of 5.2 to the wort before simmering.
5) Glass petri dish
6) Test tubes with screw on caps for agar slants - I get these. There's an option for autoclavable caps, which is really important.
7) Small funnel for pouring medium into slants
8) Test tube grabber - don't want to handle hot test tubes if you don't have to
9) Inoculating loop
10) Pyrex dish with autoclavable lid - I was lucky to just have this lying around the house. When you culture the petri dish, you want it to grow in a sanitary environment. So you need to enclose it in something. Pyrex glass is perfectly autoclavable, but the lid can be tricky. I tried my darndest to get Pyrex to tell me whether or not this lid would hold up in a pressure cooker at 15lbs. Of course they wouldn't give me a definitive yes, so I just went for it and when the lid didn't melt, I knew I was in business.
11) Pressure cooker - I think this is a 12 quart model, not sure. It's made by Mirro and I got it on eBay for a pretty reasonable price. Also great for canning stock!
12) Yeast to streak the petri dish with
13) Propane torch to flame the inoculating loop
14) Sterile distilled water in a small pyrex measuring cup, covered with foil - to cool the inoculating loop
15) Small stainless steel sauce pan to simmer the wort
And here are the steps that have been taken so far:
1) Set up the pressure cooker - Put the bottom down and add in the recommended amount of water. Place the glass pyrex dish in the center. Put the bottom half of the petri dish in the pyrex dish. Put the distilled water in the pyrex measuring cup next to - not in - the pyrex dish.
2) Simmer the wort, yeast nutrient, 5.2, and agar (this is the culture medium) for a few minutes in a small stainless steel sauce pan until the agar is all dissolved. Not too long though, because supposedly under too much heat you can denature the agar and it won't congeal.
3) Pour culture medium into the bottom half of the petri dish to a depth of about 1/8 to 1/4".
4) Place the plastic lid on the pyrex dish - I put it on kind of half cocked, so that it will vent, but just stable enough that I can put stuff on top of it, like....
5) Put the top half of the petri dish on top of the plastic lid, open side up.
6) Using the funnel and grabber, fill the test tubes with culture medium to about 75%. Be careful, they fill up quickly.
7) Screw the caps on the tubes very loosely and lay them down inside the top half of the petri dish. The idea is to keep them on an angle so that when they cool and the medium congeals it's in a slanted configuration. It just so happens for me that the top half of the petri dish is perfect for this.
8) Seal the pressure cooker according to its instructions.
9) Process on 15lbs for 15 minutes, as per its instructions. On mine, I keep it on high heat until steam starts to come out of the vent, then put the 15lb weight on it. Once the weight starts rocking gently, reduce heat to maintain and turn the heat off after 15 minutes.
10) Allow the pressure cooker to cool to room temperature - this takes a good few hours.
11) Set up to inoculate the petri dish - have your yeast, flame, and inoculating loop ready. I also wear a dust mask so I don't breath my filthy germs into my culture medium. This should be done in the cleanest, most draft-free area of your house.
12) Open the lid to the pressure cooker. Seal the caps on the slants and close the lid to the pyrex dish as quickly as possible. Note: I seal the slants in a sanitized sandwich bag and keep them in my fermentation fridge until I'm ready to use them. Also note: You should see that your culture medium has congealed and is solid. If it's not, (i.e. you pick up an agar slant and it's still liquid) something went wrong and you need to start over. You either denatured the agar, didn't dissolve it all the way, or didn't use enough. Also also note: There will be condensation in the slants and in the pyrex dish. I haven't figured out how to deal with that yet.
13) Bring the pyrex dish, top half of the petri dish, and distilled water over to where your inoculating area is set up. Peel back the foil on the distilled water. Open up your yeast sample.
14) Flame the inoculating loop to sterilize it and cool it in the distilled water.
15) Quickly dip the sterilized inoculating loop in the yeast sample.
16) Open the lid to the pyrex dish and streak the culture medium in the bottom half of the petri dish. The standard method of doing this is to run a zig-zag pattern in the four corners of the plate (I know, a circle doesn't have corners), dragging the fourth one into the center. The idea is that by the time you've swiped the sample across the four outer quadrants of the plate, you're down to single cells by the time you get into the middle. This is where your single cell colonies will be found. Something like this:
17) At this point I sprayed sanitizer in the top half of the petri dish, then quickly flamed it to dry it. There's probably a better way to keep the top half of the petri dish sterilized.
18) Put the lid on the petri dish, then put the lid on the pyrex dish.
19) Keep the whole thing in the same clean, draft-free area if you can. I can't, so I keep it in the most clean, draft-free area that is also inconspicuous. Periodically burp the lid as the yeast grows on the medium.
After a few days, I should have small colonies of yeast in the middle of the petri dish that have grown up from single cells. These will be used to make small samples of test beer as described above. When I get to that next step (hopefully tomorrow) I'll post more.
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