DCambic Plating

Last week Mike the Mad Fermentationist gave me a sample of his spontaneously-fermented DCambic. I had just made some brilliant green agar plates, from BKyeast’s excellent post on media. This media is antibiotic, killing most types of bacteria while allowing yeast to grow. My first thought was to plate some sort of lambic, hopefully being able to isolate the yeasts present. I figured what better lambic than a local one, and Mike was kindly willing to give me a sample.

For this plating I did my first serial dilution. I had previously only done streaking using a needle, whereas this involves placing a small amount of dilute liquid and spreading it around the whole plate. I diluted it 1:1000. I picked that factor somewhat randomly. I was planning on doing at least 10^6 as I had read somewhere, but since I was starting with a clear beer sample and not the dregs, I figured it wouldn’t need anywhere as much dilution. I had previously prepared vials with 9mL of distilled water, so I just had to pick three of these vials. I added a small amount to a brilliant green agar plate and a simple malt extract agar plate, and spread it around with a cell spreader (“hockey stick”). At the same time I also added about 5mL to 50mL of sterile wort to see what would grow in liquid media.

I did this on Saturday, and as of today, Tuesday, there is nothing growing yet. The plates are clean, and the liquid sample is still clear. I am afraid I made a dumb move and tried to do this after the sample had settled in the fridge, without shaking it up. If nothing grows soon I will try plating again. I already added another 5mL of the shaken-up sample to the liquid. Hopefully there will be some growth there soon. In any case, I will definitely post updates as this goes on. Who knows, maybe I’ll be able to isolate an interesting Brett from it. I would also love to isolate the saccharomyces that did most of the fermenting, though there might not be any more in suspension at this point.

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Things I’ve Learned

Six months ago I bought a bunch of lab equipment and took up yeast ranching. At this point it’s almost a parallel hobby on its own that happens to be connected to my interests in baking and brewing. I have learned a lot in these six months so it seems appropriate to share. Hopefully this will help those just starting out. Many thanks to Dmitri from BKYeast, Samuel from Eureka Brewing and jaapie for their help and suggestions.

  1. Use a jeweler’s scale for weighing out your ingredients for media. My regular kitchen scale was only accurate down to one gram, and even then it didn’t seem particularly accurate weighing small amounts. I had a lot of problems weighing out agar in particular, which is one of the more expensive ingredients. Using the right amount will help prevent you from making mushy or hard media.
  2. Malt-based media only need a tiny amount of malt extract. BKYeast suggests around 1.002 gravity wort. My last malt plates used only (I think) 2 grams per 100mL and yeast grew just fine on them. Using stronger wort makes it harder for the agar to solidify. I had to use twice as much before and still had a strange consistency. So, save your DME and agar.
  3. Potato-based media are cheap and easy and work well. Unlike malt extract media you can change the sugar, which lets you try to select for certain bugs.
  4. Prepare your medium in a flask, sterilize it and then pour into sterile plates. This is the standard way of pouring plates. I instead started using the method some homebrewers recommend, which is to pour the plates prior to sterilizing. I figured this saved me from having to open the plates in my kitchen and thus helped prevent contamination, but what happened instead was that media seeped into the sides while boiling, and this caused mold growth in storage.
  5. Plates will dry out, so store in a plastic Ziploc bag. I store them upside down and taped shut inside the bag. Lately I have stored them in the fridge until I am ready to use them. This also applies to incubating plates. Incubate upside down and inside a bag. Keeping them upside down keeps stuff from falling onto the media on top of the desired bugs.
  6. Even inside of a bag the plates will dry out, at least in the fridge. So, store your desired yeast strains in slants that you can tightly seal. After a few months the plates I had stored inside a tightly sealed bag in the fridge had dried out colonies and media, while an older slant was fine.
  7. It is possible to separate yeast from bacteria from a mixed culture at home. I had read something on a forum indicating that it’d be foolish to try this without antibiotic media, but even using standard media I have isolated yeast from mixed cultures like my sourdough starters.
  8. When preparing yeast for a batch make two in parallel or at least do it well in advance, because things can go wrong. I ended up brewing a batch with a contaminated, slightly sour starter. I got lucky and got clean beer out of it but don’t count on that!
  9. It can be hard to tell when a yeast colony is contaminated with bacteria without a microscope, so if you end up with sour starters it may be the slant or plate at fault, not the step-up technique. That’s what led me to get the contamination mentioned in #8.
  10. Glass plates are nice in that you can reuse them, but now that I pour plates the correct way I’m strongly considering switching to sterile plastic plates. For one thing, if you get something suspect growing inside you can just toss the plate without having to clean it up.
  11. It is easier to use an inoculating loop than a needle for streaking. I switched after watching a few YouTube videos where they only used a loop. The needle scratched the media more and provided no benefit that I could tell.

I think that’s it for now. I may amend this post if I remember anything else.

A Quick Update

I have had very little time to do experiments, so there’s nothing really new to post. I finally had a chance to make plates this past weekend so I am hoping to get some new things done soon. I had several plates go moldy after storing them in a plastic bag at room temperature, so I started keeping them in the fridge until ready to use.

I also plan on having some vials of DCY01 at the March DC Homebrewers’ meeting. Let me know if you are interested. Scratch that, I won’t be able to make it to the meeting.

Unusual Flocculation

This past weekend I made plates using potato sucrose agar for the first time. The composition is given on BKYeast as well as the reason for using sucrose instead of dextrose. In my case I was less worried about being able to grow larger colonies of lactobacillus but I figured I might as well save some more expensive dextrose and use cheap, plentiful table sugar. Aside from a fumble with the pressure cooker that made me run out of water 5 minutes after it pressurized and thus having to start all over again after it cooled (can’t open a pressure cooker until it cools down fully), things went OK. It seems the additional time at high temperature caused the agar to get a little flimsier, because a plate I sterilized later for only 15 minutes was more firm.

I also made a smaller second batch I left as liquid. I’m seeing what happens trying to step up a culture from a plate using this broth instead of wort. I ended up not picking from a plate but taking a small amount  (~3mL) of sediment from a tej yeast 50mL culture I had growing for a few days. It looked rather unusual: large clumps formed that were swimming around. Swirling it around broke some of those and allowed them to settle, but some remained. I at first attributed it to the fact that I had used Whirlfloc when preparing the wort. It was somewhat successful because the wort was clearer but it had the troublesome effect of making larger clumps of break material than would normally be. Since some more break material formed during sterilization it was not too helpful.

Anyway, I brought it up in the potato sucrose broth for a few days. It was probably too small of a sample to begin with, so it took a while to show any signs of activity. But it did, and after three days I turned the stir plate off and saw this:

Loose, lumpy flocs from the tej yeast on PSB. The yellow part is the stir bar.

These flocs were very loose and lumpy, not unlike what was in the previous culture in wort. I did not see any of the larger clumps like I saw in the wort, but that may be due to the yeast clumping with the protein there. After I left it to settle overnight it began to compact somewhat to the point where it didn’t stir up the second I picked up the bottle (part of that is the stir bar moving around) which means it may still be useful for brewing. I will leave it for a little while longer to see how much settling takes place eventually. If it stays too loose it will be more difficult to transfer after fermentation.

So what’s going on here? Without a microscope I can only speculate (well, even then I would still be speculating) but this is a great reason for me to study more on flocculation.

First, I am fairly sure the culture is pure yeast, since at least in the previous step the pH was normal (~4.4 after fermentation) and there was no sour smell or taste. It tasted quite nice in the wort, actually (although it looked kind of scary the lack of any unusual smell finally convinced me to try it). It was not viscous or turbid; when looked against a light, the part above the clumps looked very clear.

In Brewing Yeast and Fermentation the authors cite a 1951 study dividing flocculant brewing yeast into flour classes:

  • Class I – compact sediment which on resuspending in 0.5 ml is completely homogeneous
  • Class II – compact sediment which on resuspending is distinctly granular
  • Class III – sediment peels away in large flakes/dense round clumps which will not disperse
  • Class IV – sediment consists of loose flakes/clumps of yeast

(Chris Boulton and David Quain. Brewing Yeast and Fermentation (Blackwell, 2006), 178)

The fourth class sounds a lot like what I’m seeing. I haven’t tried looking for it, but the study is by R.B. Gilliland, “The Flocculation Characteristics of Brewing Yeasts During Fermentation,” if you are so inclined. Some other species do flocculate, but I have not been able to get much on that yet as only a few studies exist. The one I am looking for doesn’t appear to be on any of the big online journal databases.

So it seems this is nothing too out of the ordinary for brewing yeast. I’m not sure that this is Saccharomyces but a lot of factors are pointing in that direction. For one thing, it ferments maltose, which Brewing Yeast and Fermentation seems to indicate is actually somewhat rare in the yeast world. Harry Kloman also cites an old Italian study that found Saccharomyces ellipsoideus to be the primary fermentation agent. A cursory Googling shows that it might be an older classification that today would be under S. cerevisiae.

Since it did eventually compact a little bit, it should be useful if not ideal for brewing. It might be an interesting study of flocculation trends over time.

The First DCY01 Beer

Uncarbonated Sample

This is the beer I brewed in December, the first with the isolated yeast. As I mentioned in an earlier post the starter for this beer was mildly sour and turbid, but tasted good. I ended up brewing it anyway since I had not realized this about the starter until after the beer was finished. I now keep dry yeast in the fridge just in case, but this ended up being a good experiment anyway.

Despite my interest in the more obscure aspects of fermentation I am quite new to brewing, having started in August of 2011 (my interest in yeast began when I was baking bread). I’ve done lots of experiments but not many actual batches (this was #4, I think) so I am still a nervous klutz during the process. Brewing 1 gallon is great if you screw up but it also is a bit wasteful, since I end up losing so much if I want to avoid the sediment and take hydrometer readings. So, as a result of both of these conditions I ended up with only 6 bottles and two glasses to take sample and take readings with.

I didn’t measure the OG, but it was calculated at 1.062. It finished around 1.011 which is about what I was hoping for.

To my surprise the pH was in normal beer range, in the lower-mid 4.x range. There is a slight sourness but it’s not pronounced. I fined it with gelatin to clear up but some turbidity remained. I don’t think gelatin does much for bacteria but whatever it cleared out, it helped.

Fairly clear. The clumps are stuck to the sides, not floating around.

I don’t know how much yeast was left so I added a small amount of EC-1118 champagne yeast. I have no idea if that was a good choice but I had it, and thought it’d pick up easily. I may have used way too much dextrose for priming. We’ll know in a week whether or not any of this worked.

As for the taste… as I said it wasn’t very sour at all. Banana esters were very prominent with fermentation temperatures in the mid 60’s. There was some sweetness and very little hop bitterness. Even though it was fairly heavy (assuming the OG really was around 1.062) there was little alcohol evident. I wonder what those yeast were doing (or did the bacteria chomp through some of that?) It was not a great beer but it was not awful. We’ll see what carbonation does to it. I look forward to a less klutzy and problem-ridden re-brewing of this later.

I got the recipe from a post by KingBrianI on HomeBrewTalk.

1 lb. light DME

0.25 lb. wheat DME

0.125 lb. table sugar

60 minute boil with:

0.7 oz Hallertau (3%) @ 20 min. for 27 IBU

Pinch Wyeast nutrient @ 15 min.

Brewed 12/23/2011, cold-crashed and fined with gelatin in primary 1/30/2012, bottled 2/6/2012.

Plates

Schneider Weisse and Tej yeasts

I have been plating some stuff for practice and fun, trying to work out some of my problems. I think I am leaving the plates out too long at room temperature before streaking them, so they are drying out. You can see the cracks, which are not necessarily where I streaked. I still have a bit of a heavy hand with the needle while streaking so those tend to break the agar in a few days, too. I am usually only making 100ml of media so it’s hard to measure the agar consistently with my regular scale. I need a jeweler’s scale, and perhaps lab grade agar powder instead of the flakes I bought at Whole Foods. This latest batch of media was made with bakers’ type malt extract since I had run out of the brewing stuff. It is very dark and probably not all that great for this. It seems to be giving a hue to the colonies themselves (upper left on the tej plate; is that possible?) in some places. The media is just extract, Wyeast brewing nutrient and agar.

I had a bottle of Schneider Weisse, which is supposed to be bottle conditioned with the fermentation strain.  Some searching showed it’s not the easiest to culture, probably due to age and harsh shipping conditions from Europe. But with some careful sanitation I diluted the dregs in sterile wort and streaked a few days later. I see only yeast on the plate. It was quite slow to grow but it did. I haven’t tried to culture it in liquid.

Tej plate

As mentioned in the Tej post I streaked out the yeast during active fermentation. I saw only yeast at first though after looking at this picture I see what looks to be some type of bacteria colony right in the middle. There is some tang to the tej so I was actually looking for some LAB. I’m still working out how to take close-up shots without too much reflection, so pardon the quality. The cracks in the agar don’t help. There are spots on the outside that kind of look like they’re inside, but they are just hard water stains.

Tej, Ethiopian Honey Wine

One of the staff in my building told me about tej when she saw a brewing catalog in my mail. I had never heard of it before despite there being a heavy Ethiopian presence in DC, so I was quite interested. To complement what she told me I found this great site with pretty much everything you need to know about it, and much to my delight she gave me some gesho so I could try to make some of my own.

I followed the basic process on Kloman’s page. I mixed generic grocery store clover honey, water and some sticks of gesho in a jar and let it sit. It took a little while to start fermenting, and I occasionally stirred it. Although I used a clean jar and utensils I made no effort at sanitizing, so it’s anyone’s guess whether the (raw, unwashed) gesho provided the yeast (as Kloman and others suggest) or if I introduced something else.

It turns out I took the gesho out too early, so there was rather little flavor from it. It mainly contributed a surprisingly tobacco-like aroma (like a cigar store, not a burning cigarette). My building’s attendant suggested I boil the gesho in some water to extract the flavor, allow that to cool and put it back in the already-fermenting honey water. Others suggest just letting it sit for a while longer. I confess I took it out because I saw some rather gross-looking gelatinous substance coming out the ends of a few. I strained them out and just kept the vigorously-fermenting honey water. I introduced quite a bit of oxygen at this point by straining it into another (clean, but not sanitized) jar.

Between the fact that it wasn’t bitter enough and the sulfur I got from fermenting in the 80’s it wasn’t quite a success, but it wasn’t awful either. It only fermented a week or so and there was no yeast nutrient, so I don’t think I got much alcohol. It certainly was not strong.  I streaked out the yeast from the dregs. It looks like standard yeast on the plate (malt extract agar). I actually should have kept those dregs to use in another batch but instead I made bread with it. It rose just fine but there was too much unfermented honey left over in the watered down dregs. It was too sweet for a basic bread.

Next time I will make a small starter using just a bit of gesho and step it up once it’s going. It’s not quite traditional but I like the idea. (I understand it’s traditional to use the dregs, but not in the step-up fashion) It’s not supposed to be strong but I figure a healthier fermentation with some yeast nutrient can’t hurt. I’ll also switch to raw honey with the wax intact, which I’m told will help it look more authentic.

Some Progress

I think I’ve traced the bacterial infection problem back to the slant I was culturing from. I tried to culture it once again this week only to end up with the same problem once I had it up to 250ml. This was from the slant of what I’m calling DCY01-B, which I picked from a plate streaked from the finished beer. I realized all of the infected ones had been from that slant. So, I’m trying again with the other slant, DCY01-A, and restreaking the other sample to see if there’s any bacteria present.

Also, the sour DCY01 1-gallon batch is probably ready for bottling. I’m going to cold-crash it for a few days to see if I can get any more stuff to settle out of it and perhaps try fining with gelatin. I’ll take a taste sample soon to see if any of that is worth the effort.

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.

DCY01: The Beginning

The yeast in DCY01 came from a glass of pasteurized cider left out for a few days. The purpose was actually to test the pectic enzyme I had just bought, so I didn’t bother to sanitize the glass. Once I saw it was fermenting, I kept an eye on it and smelled it every day. It went from slightly funky to straightforward, which could perhaps indicate different yeasts at work that died off or slowed down as the alcohol level increased.

When it looked like active fermentation was mostly over I took some of the yeast sediment to a bit of DME wort. To my delight it fermented. I then brewed a very small one-liter batch from DME and some old Cascade hops in my fridge. I bottled with a very small amount of corn sugar a few weeks later, still fearing bottle bombs a bit due to the unknown yeast. Fortunately that didn’t happen, and I got instead a lightly-carbonated but quite good beer. The aroma was somewhere between some of the yeastier Belgian beers and a cloudy hefeweizen. To my surprise there was no noticeable sourness or indication of bacterial contamination.

My first project in yeast culturing involved isolating this yeast, both from the cider sample I had behind and from the finished beer. There was some mold in it, apparently, but I got some isolated yeast growth from both samples, which I then propagated. I’m not 100% sure if there are any differences between the two strains so I am calling them DCY01-A and DCY01-B pending further taste, alcohol tolerance/attenuation and flocculation testing.

Characteristics

Flavor and aroma: slightly spicy, phenolic. Low-medium flocculation leaves behind some yeast flavor in the beer, but not any significant cloudiness.

I will hopefully be able to expand this if I can convince other, more experienced brewers and tasters to brew with this yeast.

Growth: colonies are white and initially spherical, but later form peaks in the middle.

Attenuation: currently untested (the small size of my 1 liter beer made me reluctant to take any readings). Leaves behind some sweetness in a few tests I’ve done so far. More testing is necessary to see whether this is related to poor fermentation of higher sugars like maltotriose.

Origin and species: completely unknown at the moment. I doubt I’ll ever know either of these. The origin is unknown because as noted above I used an unsanitized glass which could have held other beers I’ve drank, bread yeast or sourdough cultures. None of those had been done recently so I don’t think this is the likely origin.