Update

I was hoping to premiere DCY01 to interested parties at the January DC Homebrewers’ meeting. Since it’s this Wednesday, and I haven’t started culturing it yet, that’s not going to happen.

Every once in a while I realize that this is much more difficult than it looks, and I have the results to prove it. It starts to feel like I’m in way over my head as a total amateur. My past two efforts at growing the cultures have resulted in sour starters. I’m not quite sure at what point the contamination is setting in so it’s difficult to know what to change at this point. The plates and slants are clean, as far as I can tell, but they seem turn sour in the second step up. It turns cloudy with a less cloudy layer on top of the liquid. So far that looks like a telltale sign of infection.

One possibility that comes to mind is the fact that my building’s heat is quite intense right now and the radiator in the kitchen leaks a bit. The air is humid and probably teeming with  bacteria. It doesn’t take much to contaminate a tiny sample of wort with an initially tiny population of yeast. I’m not sure how much the flow from the single alcohol burner’s flame helps avoid things from falling in. It’s worth trying to do it in another space.

There are other possibilities I’m less likely to be able to work around. I remember reading somewhere (a forum post) that yeast colonies on plates are often contaminated with bacteria. I haven’t been able to find anything more on this in my limited research. It seems a bit surprising due to the idea that an isolated colony on a plate is supposed to be descended from a single cell, and on my plates there are no visible bacteria colonies. But this would support the claim I’ve seen in some places that plating is not effective at isolating yeast from bacteria without some sort of antibiotic media (out of my reach as an amateur, as far as I can tell). It doesn’t help that I don’t have access to a microscope to test this out or help identify the source of my problem.

In spite of all of this, I am working on what will hopefully be DCY02. One of the two step-up cultures I just talked about is this one, so I had to restart the process. Hopefully by next week, when I will be off work, it’ll be ready to go so I can brew a one-gallon batch with it.

Perhaps I should attempt more straightforward yeast ranching using known-good cultures. I have a slant of WLP001 I cultured out of the remnants of a tube. I should try stepping that up to a starter since it is more likely to be clean than my wild cultures.

Update: I have now seen this with cultures that were not infected with bacteria–at least not noticeably–so it is probably not a telltale sign of infection after all. In the most recent case it is just yeast that refuses to settle, even after refrigeration. It’s worth noting, though, that according to this document Pediococcus form a clear layer on top when cultured in liquid.

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7 thoughts on “Update

  1. eurekabrewing

    Hi there, cool another yeast rancher. May I ask you some questions about your technique to maybe help prevent sour starters? I had some sour starters on my own with yeasts from different sources (agar plates, slants, beers…). How do you close your flask with the starter, do you use a tin foil and some cotton? How do you sterilise the starter? What starter-volume do you use when adding a colony from a slant/plate whatever? Or do you add several colonies? How do you transfer the colonies to the starter? Since you have a stirring plate I guess you do aerate your starter, right? Sorry for all this questions, but I know how depressing it is to get a sour starter after some hard work. Cheers and keep us informed with your yeast ranching.

    Reply
  2. dcylab Post author

    Hi there. I checked out your blog–very cool stuff. I’ll add it to the links list.

    You are right–it is quite depressing to get a sour starter. I was starting to lose my momentum but fortunately I have a great fascination with these microorganisms. I definitely appreciate the help in tracking down the source of the problem.

    The method I am using is more or less the method outlined on BrauKaiser.com. He’s doing more straightforward yeast ranching with commercial yeasts so I have had to branch out a bit to do the stuff I want to do. But so far, the process is similar.

    I am sterilizing everything in a pressure canner around 15-17psi according to the dial. I usually let it go 20 minutes once it reaches pressure. Before that I let it flush out with steam for 10 minutes. After it cools and depressurizes I tighten the lids on the small vials I use for the first step and the repurposed glass cans I use for the larger steps. I move everything to a clean surface to dry, and then tape the plates shut. I have been taping the lids on the 12ml vials too but I am not so sure it’s necessary.

    I have not seen any evidence of contamination from bad sterilization except one or two plates that had some water trapped in the lid. Those grew mold after a few days at room temperature. The vials and glass jars appear to be fully sealed and “woosh” like normal cans do when you open them. I will most likely have to retire these repurposed glass jars once the seals wear out.

    I am using 7ml of ~1.020 wort in a 12ml vial for the first step. I let that go a few days with the lid loose until I see visible sediment. For the second step I open up one of the glass jars with about 50ml of ~1.020 wort. Following BrauKaiser’s method I open it up to let some air in, immediately tighten the lid again and shake up a bit to dissolve some air. I forgot to do this one time and still got some sourness (pH in the 3.x range) so I’m likely messing up somewhere else. I have been flaming the tip of the 12ml vial before pouring it in. Perhaps I am not doing it sufficiently long or covering enough of the surface. I am just putting the lid back on but keeping it loose. This is a normal metal screw lid (I’ve been using olive jars) with a gasket for sealing. I hold it while transferring and keep the jar open maybe one or two seconds total.

    After that ferments, I step it to 250ml of stronger ~1.035 wort in an Erlenmeyer flask. I was boiling it at first but now I am running it through the pressure canner using Fermcap to avoid boil-overs in there. I also have the stir bar in the flask so it gets sterilized too. I leave foil loosely on top while it’s in there and then tighten it down right when I open up the lid after it’s cooled down. I’ve tried both sterile alcohol wipes on the outside of the olive jar and flaming the rim but have had problems with both. In any case, I then seal with the foil again (keeping it pointed down at all times) and put on the stir plate.

    I am not using hopped wort at any point.

    I am in the middle of a step-up right now that I got from a freshly-streaked plate. I was a lot more careful with the environment this time, and wiped the whole counter down with alcohol first and worked between two alcohol lamps in an attempt to keep things from falling in. I could see only yeast colonies but the max I have is a magnifying glass. I am a bit worried because I had a bit of sediment from the 12ml vial that didn’t transfer and a pH strip looked like it was in the lower end of the 4.x range. It tasted like it had a bit of sourness but I think I am bad at detecting small levels of sourness. There is also some sweetness which makes it more difficult. The second stage tasted similar but the pH was a little bit higher. It’s on the stir plate right now so I should be able to tell tomorrow.

    Reply
  3. eurekabrewing

    Well, let me tell you. You have done your homework! Wow… Your technique seems very sophisticated to me.
    Just some thoughts:

    – Sterilisation: I see no problem here. Molds in plates just happen. Even with the best methods. We pour our plates with a very advanced machine in the lab and we even get molds sometimes. I am not sure how they get in there.

    – First transfer: The volumes are ok. I asked the volume of the starter because some tend to use way too large starter. The flaming and transferring are ok as well. So far so good.

    – Step up: See no problem here as well. Just one suggestion: Try to stick some cotton in the opening of the flask and close it as usual with tin foil. The cotton kind of improves the sealing of the flask. There are cotton stoppers available for lab purposes but normal cotton works just fine. One disadvantage of the cotton is to get it out after sterilisation to add the last starter. Alcohol for sanitisation of the glass jar is okay. I transfer as following with a cotton plug: Sanitise the glass with the previous starter, then take the foil of the flask and cross it through the flame and place it on the table where the part which was on the outside before faces the table. Then I use tweezers and remove the cotton plug and place it on the foil on the table. Then flame the opening of the flask and dump the content of the glass jar into the flask, place the cotton plug back in the flask, flame the tin foil once again and reseal the flask.

    – Hopped wort: I would not suggest using hopped wort. It would be totally okay, but as long as you have a problem to get a pure starter, I guess a hopped wort would be the wrong approach trying to fix the contamination problem.

    – Moist plates: Just looked at your last post with the Schneider yeast. You mentioned to use malt agar plates, right? One easy thing to prevent the plates to dry out is closing them with tape. I guess you could close your plates with a tape maybe an hour after sterilising. And use another tape for incubation. I guess there should be enough oxygen for the growth of the yeasts.

    – Schneider yeast: Your cells on the plate seem to me like yeast cells. But please don’t rely on my guess. How do the plates smell like? Sometimes the smell is a very good indicator about the bugs on the plate. If the plate smells kind of bready or yeasty (like the yeast you use for baking) then the chances are hight that you got yourself some yeasts. Does it smell acidic, rotten, sulphury, well lets say anything different than yeasty, then there might be something different than yeasts. Tasting the starter is quite difficult. I remember tasting a starter of an Irish stout yeast and it had some sourness as well. Microscopy told me that the yeast was pure…

    – What is the problem now? I guess the problem is even before the first transfer. I guess you got yourself a contamination while trying to purify the yeast strain. When and how is very difficult to say. You already mentioned that you had some molds… Might be an indicator for further contamination?

    How to test the purity of the yeast without a microscope seems very difficult to me. Why don’t you plate the original DCY-01 again using a dilution streak technique (there are some videos on U-Tube) and look carefully at the morphology and the smell of the plate?

    Let me say it again. Your technique is very, very good. And streaking without destroying the agar just needs some practice 🙂
    Hang in there, you will master it. I am pretty sure about that.

    Cheers!
    Sam

    Reply
  4. DC Yeast Lab Post author

    Sam, many thanks for the help and nice words. I really appreciate it. A few questions:

    – I forgot to mention earlier I bought some autoclavable foam plugs for my flasks. They are supposed to allow flow without letting microbes pass so they would be an ideal replacement for the foil. I have not been confident using them because of moisture inside the plugs and on the edges against the glass. When I took the plug off it ran down into the starter, and again when I reinserted the plug. Have you ever worked with this sort of thing and/or have any suggestions? I hadn’t thought of flaming the foil.

    – I have been taping the plates shut both in storage and during incubation. I use two smaller pieces during incubation so I can see the surface. The plates are kind of cheap and have some flaws in the glass, so I think there’s still room for moisture to leave. Would storing them in a sealable bag help?

    – I am storing and incubating with the plates right side up (agar on the bottom) but I have read some things saying I should do both with the agar on top so that moisture doesn’t fall on the plate. My thought was that would encourage more contamination in the cracks of the plates which would get in while I am working on them. Any thoughts on the matter?

    I was planning on transferring the Schneider yeast to a slant for storage, so I will get a chance to smell that plate. I’ve had some plates smell very much like bread yeast but some smell a bit different. I have two plates in the fridge in a plastic bag, and when I open that bag it smells different. I am not sure if that’s a sign of mold or just a different concentration of that yeasty smell.

    Thanks again!
    Claudio

    Reply
    1. eurekabrewing

      Hi Claudio,
      some thoughts:
      – Plugs: They are very common in microbiology labs. (Common meaning in the microbiology labs I worked) And used them for every media I made: You just mix your media in a flask, seal it with a plug and foil and sterilise it in an autoclave. And they get moist due to the evaporation of some water from the media. But I see no problem here as the moisture is sterile as well. And I use cotton instead of plugs. And as mentioned, they may get really moist and kind of plug the flask. But I see no problem here as well.

      – Storage: I store my plates in a plastic bag in a refrigerator to keep them moist. I guess a bag could help.

      – Side up: I incubate and store my plates with the agar on top. Common lab practice as well. Normally, bacteria and yeast in the air tend to fall. Incubating with the agar on top makes it really, really hard for contaminants to get there as they should me able to fly I guess 🙂 But as you do so, it is not advisable to turn the plates at all with the cover on. There could be contaminants in the moisture on the cover and as you turn the plate, the moisture flows right into the agar… When you work with the plates, just remove the part with the agar in it and leave the cover on the table. Once again, some bugs may fall into the cover but when you don’t turn the plates, they stay there. I had some plates with molds in the cover and I just streak some bugs from the plate on a new one.

      I guess the nose is even better than a microscope. And what you describe is spot on what I meant. I it needn’t be just molds. Bacteria are very common contaminants.

      Let me emphasise it again, you did great research about the topic. You really know the stuff. It just needs some further tweaks. You need some time and practice to master it.

      Cheers from Switzerland
      Samuel

      Reply
  5. DC Yeast Lab Post author

    Could you clarify a bit on the incubation process? Do you leave the plates upside down without the covers on your surface while incubating? I want to get a glass surface to work on but I don’t think I have the room for that if I have to leave it out during incubation. My kitchen is very tiny.

    On a good note, I was able to step up a DCY01 culture from the restreaked plate up to the third step without contamination. However, it looked totally gross as I think I used too much Fermcap. There were streaks adhering to the side of the glass. My first thought was some sort of extreme ropy contamination but the pH was normal and the taste of a sample I pulled was normal, no rope. I think it was a combination of break material, Fermcap and perhaps Whirlfloc which I added as an experiment (trying to minimize break material). The stir plate got them to stick to the sides of the flask.

    Now, hopefully I didn’t contaminate it when taking the sample. I worked next to the flame on a clean surface and pulled with a sterile-wrapped plastic pipette.

    Reply
  6. eurekabrewing

    I leave the cover always on and stack them and then either close each of them with a piece of tape or use the bag which came from the plates and cover the tower of plates (I use disposable lab plates). And off they go into a cupboard which is near to the heater and therefore a bit warmer than room temperature. Take my picture of the plates: http://eurekabrewing.files.wordpress.com/2012/02/imag0303_2.jpg and imagine turning them upside down.

    I use a glass from an old refrigerator to work on…

    Good luck with your DCY01 starter. I can’t tell what Fermcap will do to your starter since I never used it before. I used Irish Moss one time but I couldn’t see a difference in clarity of the beer.

    Reply

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