#1 Agar plates (BFM, 3 Fonteinen, Girardin)

Eureka, after some recipe posting now a post that is right down my alley. I did some agar pouring, streaking and finally some microscopy analysis of different colonies. So this is the first post out of five about bugs. Today, pictures of bugs from commercial sour beers.

Plated bugs

01/30/2012: I plated the following bugs on Sabouraud plates: BFM La Torpille dregs, 3 Fonteinen dregs, Girardin dregs, Water Kefir liquid, Lactobacillus from a apple juice starter for a future Berliner Weisse, my Heidelberger Kellerbier yeast, #1056 from a starter for an IPA batch, Flanders Red, Kombucha liquid, #3787 Trappist yeast from 2011 and 2010 and I put the last plate outdoors for about six hours to collect some wild yeasts.

The plates were stored at room temperature for approximately three days until colonies appeared. The following pictures were taken after three days of inoculation.

I can already tell, that I did not collect any kind of wild yeast with my last plate, just some beautiful molds…

BFM La Torpille dregs:

I dumped some La Torpille dregs in a blended Belgian style beer (#39 Another Reason to Live) a while ago and poured myself a sample from the fermenter to taste and streak.

BFM bugs on Sabouraud agar

Well, I could observe just one kind of colonies: Off-white color, even, circular, glossy, convex, 2 mm diameter.

I guess it is normal brewers yeast due to the size, morphology and time the colonies needed to grow. If this is a yeast, then it must be #3522 Belgian Ardennes or/and #3787 Trappist High Gravity.

Microscopy picture BFM colony

And the microscopy picture above proves my guess. These colonies are cells of Saccaromyces cerevisiae. Unfortunately not Brettanomyces as hoped… Will use a different media next time to inhibit the growth of brewers yeasts.

3 Fonteinen dregs:

Isolated bugs from dregs of a 3 Fonteinen Oude Gueuze and streaked them as well to see what I got. The wort I used for isolating the bugs smelled very acidic and was kind of sticky. The stickiness might come from the work of Pediococcus.

3 Fonteinen bugs on Sabouraud agar

And I could observe just one kind of colonies again: Off-white, wavy, irregular, not glossy, 5 mm diameter. I was already aware that these might be something different than brewers yeast because they tend to grow in circular colonies and not wavy-ones.

Microscopy picture 3 Fonteinen colony

And the microscopy revealed that these colonies are in fact bacteria. Some sort of Bacillus. My guess is that these are maybe Acetobacter due to the acidic smell of the wort. I have to apologize for the poor quality of the pictures, it is quite tricky to get some pictures with my camera of bacteria due to their very small size. Well, no yeasts or Brettanomyces.

Girardin Gueuze dregs:

Girardin bugs on Sabouraud agar

And at last, some bugs from a Girardin Gueuze. The starter was sticky, like the 3 Fonteinen one as well, but the acidic note was subtle. Once again, just one kind of colonies:

Off-white color, even, circular, glossy, flat, 2 mm diameter. Darker than colonies from BFM dregs.

These colonies were circular again but had a different color than the ones from the BFM dregs (which were Saccaromyces cerevisiae). Maybe Brettanomyces this time?

Microscopy picture Girardin colony

And once again, these colonies are bacteria not yeasts. And they look very similar to the ones from the 3 Fonteinen colonies.

Although I could not detect any colonies of Brettanomyces so far, I learned, that I have to use a different kind of agar to prevent the growth of Saccaromyces and bacteria to give the Brettanomyces a chance. And I already have a different media in mind…

That’s all so far about the bugs from the dregs. Next post about agar plates will be about the water kefir culture and Lactobacillus. And the third one about some common yeasts, the fourth about the #3763 Roeselare Blend from Wyeast and the last one about Kombucha cultures. I can already tell that some of the results were quite unexpected and really interesting and to finish: spoiler alert: I could isolate some Brettanomyces!

Advertisements

6 thoughts on “#1 Agar plates (BFM, 3 Fonteinen, Girardin)

  1. I would suggest you diluting dregs in sterile water about 1:100 and plating 100uL of that (spread it around with a hokey stick for a more even distribution). Pouring them into a beer or starter is not the best idea, in my opinion, for a few reasons.
    1. If there are bacteria, they’ll wake up and overtake the entire culture WAY before yeast has any chance of waking up.
    2. Brett is a slower grower so Sacch will appear faster in my experience.
    3. Let the plates sit for at least a week. You’ll first see bacteria, then Sacch, and then Brett will pop out.

    Also, give bromocresol green a try since it kills Lacto for me. You can also try lactose in your plates while removing most other nutrients like I did since that’ll limit growth of a lot of bugs while allowing some Brett strains and Lacto ones to grow much quicker than other organisms.

  2. Thank you very much for your ideas. I really appreciate them. Just some thoughts.
    – I expect that platting with a drigalski spatula or hokey stick would not do the trick. The problem still are the bugs in the samples which grow faster than yeasts and Bretts. And I do not have any kind of micro pipette at home.
    – I can’t change the carbon source of the media as it is a complex one and already mixed. I would have to make my own media out of scratch and that would be way too expensive. I have to mention that I do my experiments at home, not in a lab. I just did normal yeast ranching with Sabouraud and this worked very well. I wanted to find out, if Sabouraud could be used for culturing some bugs from dregs. The answer is yes, but sometimes not the kind I expected.

    – Your reasons why not to dump the dregs in a starter seem very reasonable to me. The easiest way would have been to plate the dregs right after drinking the beer. But still, Sabouraud would not have been successful to prevent any other bugs from growing. The Bretts would have lost again. This time not in the starter but on the agar plate.

    – And the plates are still incubating. Maybe some other colonies arise? But I expect that the bacteria will have taken over the plate by then. I will see.

    My next step is to find a media which enables the growth of Brettanomyces and prevent bacteria and Saccaromyces. And there comes your potato media which I will try. Maybe buy some bromokresol green as well.

    • Yeah I also do all my experiments at home 🙂 Thus my search for cheap and effective medium hahaha.
      My point about hockey stick is that diluting and spreading over the plate will give a more even separation so that bugs won’t grow over each other like they do in the initial streaks with the loop. So if you only have 1 viable cell of a particular bug in the sample there is less chance that it’ll get choked out. And if they are slow starters, they’ll have their own space to grow in.

      Diluting is also good. I recently plated straight 100uL of dregs from New Glarus Cranbic because the sediment was MINUTE (like 30uL in 10mL) so I though that’s dilute enough. Not so. My plate looks like a lawn with probably 2000 colonies (brett colonies are clearly seen). So dilute away 🙂

      I don’t have micropipetters at home because that’s just way too rich for my blood. Use sterile transfer pipettes. You can get like 500 for 10 bucks or you can just reuse the same one by washing it with alcohol.

      Stay tuned for the selective media post. I think it’ll be something interesting for you 🙂

  3. I absolutely agree on your point about spreading the bugs over a plate. The growth is way better. But I use a Z-streak technique and only the first streaks (shown in the picture) are overcrowded with bugs. The next two will be less crowded but need further time to grow. I hope I’ll get a good example to show it in a post. As mentioned, the plates are still incubating.
    I am quite fascinated about the fact that you could identify Bretts out of nearly 2000 colonies. Well done!
    I use a syringe for dilutions. One disadvantage is the big sample volume you need but it does the job. And I will wait for your post….

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s