DCambic Update #2

It looks like nothing else is going to grow on the plates, so I went ahead and streaked out one of the colonies on another plate and inoculated a small liquid sample. I will step that up as usual. The plate had a slightly funky smell unlike any yeast plates I’ve dealt with so far. The colonies also look bigger, glossier and more irregular than others I have dealt with. I have not yet worked with laboratory Bretts, though, so I can’t compare. In any case, given that the previous liquid sample grown from the raw beer developed an intense Bretty character I am quite sure that is what I’m dealing with here. I’m not too good at describing the various Brett flavors, so I’ll venture only that it is fruity and phenolic. Although there was no visible fermentation there is definitely alcohol in the aroma. I am imagining the new liquid sample from the pure culture will smell the same.

I had originally planned to streak out two plates, one with potato dextrose and one with potato lactose, but I ended up not having the time to prepare them this weekend. I went instead with another malt extract plate. This doesn’t help me get any new information on how the strain behaves but it’ll do for now. When I have the time I will prepare those potato plates with bromocresol green and restreak. One nice thing about potato media is that you can easily swap the sugars and make different types from the same batch of potato infusion.

Nothing at all grew on the brilliant green plate. My previous experiments with them had at least some yeast growth but it was very slow. This time I don’t see anything. It is safe to say my experiment with that medium is a failure. Good thing it was unnecessary for this experiment.


6 thoughts on “DCambic Update #2

  1. jeffreyecrane

    So why don’t you think anything grew on the Brilliant Green plate? And you are planning to not use Brillant Green going forward for isolating Brett or Sacc? It seems like malt agar is working just fine for you.

  2. DC Yeast Lab Post author

    I don’t really know why nothing grew on there. I think I noted somewhere else that I didn’t go after the various yeast extracts and instead went with yeast nutrient. I don’t understand enough about that stuff to know what that did. Given that I made some successful plates using only a sugar and yeast nutrient I don’t think that was the problem. I think that may have issues due to having very little buffering capacity, but it should grow things at first. The nutrient should provide the things the yeasts need to grow. Perhaps I just used too much brilliant green solution and it was too effective.

    I’m not sure I’ll try again unless I am having a really hard time getting the yeast to grow without bacteria ruining things. As you can see from the pictures since I diluted the sample so much, had there been bacteria in the sample it would most likely not have interfered with any yeast colonies. Remember that even if bacteria or mold or whatever is present on a plate you can still pick out a colony you want and restreak from that. At that point it should be pure. I’ve used that with good success, for example, for isolating the yeasts from my sourdough starters. I had lots of bacterial growth on those plates but by picking the right colonies I got just yeast on the second streak. All that I needed was to select colonies that looked like they grew in total isolation. A few colonies looked like they had both yeast and bacteria due to them growing on top of each other.

    On that point, it might be easier to do this using the spread plating technique I did here instead of streaking. You can still do it with streaking, though. See Sam’s posts on Eureka Brewing where he has isolated Bretts from lambic dregs using Sabouraud agar. I don’t think the type he’s using has any antibiotics in it.

    What would probably be more useful is bromocresol green, which can be used to tell Brett from Sacch. I haven’t yet done that but I know it is useful in that regard. I have no idea what it does for yeasts other than those two. There are some other minor yeasts in lambic, too. I don’t know how I would go about trying to identify those if I had them. Without a microscope I think my options are limited to colony appearance and characteristics when grown in wort. I think most of them can’t ferment sugar to alcohol, which is the reason I was happy that I had alcohol in the liquid.

    So, in short, no, it’s not necessary for this type of experiment and with some patience it can be done with regular media. I’ve been meaning to redo the sourdough yeast isolation, so when I do I’ll take pictures and make a post about it.

  3. eurekabrewing

    Congratulations on your successful isolation of Bretts. You diluted your samples before streaking, right? Somewhat around 1:1’000 and then streaked directly on the plate if I remember correctly?

    You are right, my Sabouraud agar has no antibiotics in it. I just streak the undiluted dregs from a bottle directly on a plate and wait. I use a dilution streak method and therefore do not need any additional dilutions prior to streaking. One good way to do it is shown here: http://www.youtube.com/watch?v=yW56Ho1nEWQ. Although you do not necessarily need a hairnet for that in my opinion… 🙂 If there are a lot of other bugs on the plate I just pick a colony and do as you mentioned above.

    Please let me know if you have found an easy way to identify Bretts and other yeasts. I would be really interested in such a method. A microscope might help you do identify some of the bugs. Although many of the yeasts look very similar to each other. Much like the colonies on the agar. So no big improvement with a microscope in my opinion.

    Totally agree, you do not need fancy media to isolate some bugs. Any regular media suitable for yeasts does the trick.

    1. DC Yeast Lab Post author

      Thanks! Yeah, I diluted 1:1000 and then spread a small (unmeasured) amount on each of the plates.

      I’m glad you said that about microscopes. I was strongly considering getting one but now I am not so sure. I certainly often wish to see what is there but you are right, looking at your posts it seems it’s not so easy to tell them apart just by looking at them. My method for identifying this yeast so far is basically “well, it smells like Brett, right?” so I am not dealing with anything too sophisticated.

      1. eurekabrewing

        A microscope is really helpful to just have a look at the cells for sure. Or even check if your yeast starter for an upcoming batch is clean. When it comes to identify the yeasts, it is a different story. I therefore share your opinion, it is not so easy… The cells look very similar in my opinion. You might have read my post about the fermentations tests which is basically the same as you do. In my opinion the easiest way to test for Bretts. I could even observe pellicles. There is a Brettanomyces Agar (check out sciencebrewer http://sciencebrewer.com/2012/04/10/wild-yeast-collaboration-with-brooklyn-brewery-new-selective-media/) used to identify Bretts. In these media there is p-coumaric acid that can be metabolised by the Bretts and form strong smelling compounds. At the end, you just smell the plates and basically do the same as with your fermentation tests. Nothing wrong with your fermentation test approach to identify Bretts. The nose is a very sophisticated analysis tool.

        Concerning the plates. In my opinion, the reason for the lacking colonies on the Brilliant Green plate is the low concentration due to the dilution. Your other plate just has three colonies (if all the colonies are shown in the picture above). This is an indication for me that the concentration of the bugs was pretty low. I would advise you to directly streak whatever sample you have at the beginning on a plate and use a dilution streak method (as shown in the video in my last comment). The first streak might be overcrowded. The second or third one should be fine. Even the thickest dregs I plated did not overcrowded the plate with colonies. One advantage here would be to physically have some colonies. Even if they are overcrowded. Then just pick any colonies you are interested in on a new plate, on a different plate etc. By doing dilutions you risk to have no colonies at all… or even the wrong ones (if you dilute out the ones you are interested in).

        To summarise, I would not be afraid to have overcrowded plates. I would be concerned to lose colonies by doing dilutions. It is much easier to separate the bugs from an overcrowded plate than having no colonies at all…

  4. Pingback: DCY02 & DCY03: DCambic Isolates | DC Yeast Lab

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