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[personal profile] fredrikegerman
As the lovely [ profile] desireearmfeldt and [ profile] kirisutogomen point out, it's been ages since last I posted.

I've got a really poorly-planned trip to Athens coming up in a week or so (we'll be talking programming languages, though, which I enjoy). Meanwhile [ profile] desireearmfeldt is working on her dissertation, and it's tool, tool, tool hereabouts.

But that's not what I came here to talk about. I came here to talk about yeast morphology.

Those of you who know me know I'm a huge fan of sour beers like lambic and berliner weiße. There's now (to my great joy) quite a burgeoning set of microbreweries making American Sour Beers, and events like the Extreme Beer Fest that showcase the emerging new styles.

What distinguishes these beers are the organisms used during fermentation. Most beer is fermented by one or another strain of Sacchromyces Cerevisiae (it's right there in the name – think cerveza). This is the same yeast you use to make bread and wine, and the one used in most genetic research on yeast.

Sour beers add one or more other organisms to the mix. The first is Lactobacillus. Strains of this bacterium occur naturally in grain – when we make beer, we normally boil it (killing the lactobacillus) and add hops (an antibacterial, preventing Lactobacillus from taking hold later on). You're already familiar with various strains of Lactobacillus – they make sourdough bread sour (another mix of Sacchromyces and Lactobacillus), and they are responsible for turning milk into yogurt, sour cream, or buttermilk. They've also become the all-important basis for fermented hipsterpickles of the sort made by bearded, pierced, and tattooed entrepreneurs in Brooklyn studio kitchens. Wherever they occur, they're converting sugars like lactose into a mix of lactic acid and alcohol. Generally we select for the acid.

The other, more intriguing addition are yeasts from the genus Brettanomyces. These "wild" yeast are much more aggressive than Sacchromyces – they'll chow down on long-chain dextrines and other complex sugars and generally ferment bone-dry. By contrast, Sacchromyces will give up while there's still some residual sugar (and we can deliberately sweeten a beer or add body by increasing the long-chain dextrines that don't get fermented). Brettanomyces tend to reproduce relatively slowly, so they'll get out-competed by Sacchromyces for smaller sugars. And when there's oxygen around they love to produce acetic acid – so they can turn your beer vinegary if you don't keep it under a fermentation lock. Also, these guys love to burrow into wood – they can actually digest cellulose to a very small degree. They were first discovered in barrels of English ale about a century ago; they were considered a big contributor to beer spoilage if you kept the beer in the barrel for too long. A quick glance at the Wikipedia page explains why (beyond the acetic acid, of course): when they munch on the by-products of Sacchromyces fermentation they produce flavors described as "barnyard", "mousy", or "horse blanket". Or often just "funky". When Brettanomyces was discovered most beer was getting drunk in a matter of a month or so after it went into the barrel, so this wasn't a big deal as it takes months for the flavors to really develop. What sour beer brewers tend to do is deliberately infect a barrel with Brettanomyces and then age beers in that barrel. You can keep reusing a barrel if you like the beer that's coming out of it.

A side note here is that warehousing that quantity of beer in barrels for a year or so turns out to be expensive – barrels take up a lot of space and require a lot of maintenance – so sour beers tend to be on the pricier side.

We're now also starting to explore fermentation using only Brettanomyces, with no Sacchromyces or Lactobacillus at all. Interestingly, it tends to yield fruity flavors rather than funky flavors when used on its own, especially if you don't drop the pH with the help of our friend Lactobacillus.

Quite apart from its funky tendencies, Brettanomyces has a distinctive morphology. If you've ever seen a picture of yeast – nice round cells with little buds becoming daughter cells – you've probably seen a picture of Sacchromyces Cerevisiae. Brettanomyces by contrast forms oval or long sausage-shaped cells.

When they're fermenting they also look pretty different. Sacchromyces forms a foamy krauzen on top of the beer (this is the foam you see if you've ever proofed baker's yeast). By contrast, Brettanomyces forms a pellicle – a thin, solid-looking mat on the surface of the beer. The pellicle is actually formed in response to oxygen, and it's theorized that it protects the beer from the oxygen – thus keeping out aerobic organisms like the vinegar-forming acetobacter that might muscle in on the territory.

The interesting thing about the pellicle is that it starts to make Brettanomyces look very fungus-like indeed. It's not the only yeast in the family that has interesting large-scale morphology. Candida albicans is a cousin of Sacchromyces and Brettanomyces that lives in your mouth and gut and elsewhere on your body. Most of the time it's round and yeasty. It can also turn oval like Brettanomyces. But every now and then it decides to morph into a fungal filamentous form and invade the body. This is responsible for thrush and yeast infections. It's also one of the big killers of immunocompromised individuals like AIDS patients – without an immune response the invasion can continue unchecked. That's also the reason cancer patients get thrush during chemotherapy.

Why does this all come to mind right now? Genetics and morphology! One of the most popular "Brettanomyces" strains used in home brewing today is White Labs WLP644, Trois. This yeast is derived from lambics brewed by Drie Fonteinen in Belgium (Drie = 3 = Trois, geddit?) and was sold as a strain of Brettanomyces Bruxellensis, one of the characteristic lambic species. But there's been rumbling for a while that this yeast might not be Brettanomyces at all. But what is it? Its morphology often matches the oval shape of Brettanomyces, but sometimes it's the round shape of Sacchromyces Cerevisiae instead. It also produces flavors that are strongly reminiscent of Brettanomyces, and behaves in a Brettanomyces-like way during fermentation – though it's known as being rather more vigorous of a fermenter than Brettanomyces typically is.

Genetic sequencing has revealed that Trois is not Brettanomyces at all – but a subspecies of Sacchromyces, probably distinct from the ones that we've already classified. This resulted in a reclassification this week by White Labs. This has one potentially very interesting consequence. A number of competitions (such as the Great American Beer Festival) have defined categories for beers fermented with Brettanomyces, and a number of beers fermented with Trois have done quite well in those competitions! Are these relatively exotic fruity/funky beers worthy of living together in the same category now that we know how different the yeasts really are? This is interesting in part because these tend to be the most diverse categories in the competitions, and comparing the beers can be a bit like deciding whether you prefer apples or oranges (whereas most categories are just searching for the poodliest poodle). Ongoing genetic sequencing of the various strains revealed the miscategorization. Lots of great discussion of that can be found in the Sui Generis Brewing post on the subject.

So, another victory for modern genetics in teasing apart differences between species with highly flexible morphologies and apparently similar behaviors!

Date: 2015-04-12 02:15 am (UTC)
From: [identity profile]
By a funny coincidence (or maybe I just think so because I'm sleep-deped) one of my driving students told me today she's leaning toward 6-7: CS & Molecular Bio. Which I hadn't known was a thing. And here you've been intermingling the two fields for decades.

Date: 2015-04-12 12:48 pm (UTC)
From: [identity profile]
Well, my sophistication about bioinformatics is pretty low. I hear it's a great way to do interesting and deep software engineering and algorithms work while getting all the pay of a biologist and all the research recognition of a lab janitor. But then, some of my colleagues (former SCAdians) are a little bitter about the way Broad was treating programmers with Ph.D.s.

Date: 2015-04-12 01:07 pm (UTC)
From: [identity profile]
FYI, there's apparently a craft beer joint in Portland (OR) that specializes in sours. Possibly because there are 98537298 craft beer joints in Portland. At any rate, more support for your contention that hipsters like sours enough that soon they'll be widespread :)

Date: 2015-04-12 01:29 pm (UTC)
From: [identity profile]
I went to just such a place when I was in Seattle a couple of months back. And at the Extreme Beer Fest I met the head brewer of a sour-only brewery in Berkeley (he hosts a podcast on the subject).

Round here Cambridge Brewing has been doing sours about as long as anyone in the US, and is profiled in American Sour Beers. Their space is pretty limited, though. And a bunch of other local small breweries are in on the act – I got to see the barrels at Trillium brewing in Fort Point. The big player hereabouts is Allagash in Portland, ME, who have devoted their entire former brewery building to barrel aging to keep the spoilage organisms away from their non-sour beer.

Date: 2015-04-12 05:02 pm (UTC)
From: [identity profile]
There was that nice article I read - NYtimes? I forget - which is going around about sour beers. Most of it wasn't particularly new, but the thing I mainly learned was that White Labs has a tasting room where they have a bunch of beers brewed completely the same except for the yeast.

I do really like this direction. I've been doing an unpasturized cider every year for a while, sometimes with a hefe yeast, sometimes with a more conventional cider yeast, but you end up with some really interesting sour notes that develop over time, and some surprising accompanying color changes.

Do you have a sense of how difficult it would be to actually look at these yeasts [slash whatever the hell they are] under a microscope, like the $15 one I got at goodwill? Can I just stick some beer on a slide? Or trub, at least? Or do I have to stick it in a petri dish and the like to grow it up?

Date: 2015-04-13 02:44 am (UTC)
From: [identity profile]
Tasting room? Something to add to the list next time I'm in San Diego I imagine.

The microscope question is an interesting one. I know there are folks who do this sort of thing with amateur equipment, but I think you do at least want to mount it on a slide. I suspect you can use actively fermenting beer or post-fermentation yeast, but stay away from the trub as it probably contains too much precipitated grain matter. They make gridded slips for cell counting, which is a thing.

Don't remember if you need to stain, but I think not if your light is good.

Lactobacillus is a different matter I think as bacterial cells tend to be much smaller and require staining. I think identification is often done by treating the media and seeing where propagates thrive. I've read a bit here and there about this but retained little I fear.

At least some of the wikipedia images had scale bars in both cases, but I've lost track of whether those are specific species pages or up at the genus or family levels.

Date: 2015-04-14 07:39 pm (UTC)
From: [identity profile]
See, I knew it would be a good idea to lure you back.

Bacterial sizes are usually on the order of a micron. Yeast are surprisingly small for eukaryotes, often on the order of 5 microns in diameter. Given that visible light wavelengths are ~0.4--0.7 microns, you're not going to be able to see bacteria usefully, and yeast are going to be right at the limits of an optical microscope. I would guess (without really having looked into it) that with yeast if you're insisting on visible light you'd need a fairly sophisticated set of equipment.

This is one of the nicer illustrations of relative sizes I've seen recently. In fact, the whole "Learn Genetics" site is pretty nice and we've made good use of it at the Museum of Science.

Date: 2015-04-15 02:06 pm (UTC)
From: [identity profile]
Fun, but not mobile accessible. :-/

Here's an example blog post on doing yeast cell counting:

General recommendation here and at White Labs is to use a 400x or better microscope. Other advice I've seen in completely disjoint discussions suggests big-ass heavy microscopes are better because stuff doesn't move around on the stage when you're using them. Apparently the problem with most microscopes sold for amateur use isn't the magnification, which is 1000x or better usually, but ability to get enough light and keep the sample still and in focus.

I'm guessing a used scope will do just great with sunlight.

But hey, I don't own a scope myself, so this is all hearsay on my part.

Date: 2015-04-15 03:04 pm (UTC)
From: [identity profile]
So I guess I wasn't thinking too precisely about how much detail we're looking for in looking at the yeast. If we are just counting cells, or just want to be able to say "I see a yeast!" then a decent optical microscope is probably plenty for our needs. I was probably implicitly thinking about seeing enough detail to distinguish Zygosaccharomyces from Torulaspora from Pichia. Not that I would be able to actually do that with any microscope, but I assume someone who knew what they were doing would.

Amateur microscopes do tend to have really uselessly high magnification. It's like megapixels on cameras -- people get lured by the notion that they're going to be able to judge the quality of the hardware on a single number, and the manufacturers find that they sell more equipment if they have 1000x magnification and skimp on everything else. Unless you have some sort of expensively stabilized foundation, 1000x is useless; every time a car drives by your house the vibrations will blur your image into a smeary mess.

I would not expect sunlight to be sufficient; it's easy to forget just how little light there is in a square millimetre, but you'll realize it when you try to use ambient light (even sunlight) at those scales. Also, if you look at that picture in the Eureka post you linked, you can see that the thick cell walls create a lot of diffraction, and I'd hate to try to deal with the interference patterns if I had light coming from all directions.

Keeping the sample still and in focus is also definitely a key concern. Actually maybe more so than light, especially with cheap microscopes. Although....with modern digital image processing, you can do some pretty amazing things that feel to me like they're bordering on black magic. Before Pioneer 10, all our Earth-based telescopic pictures of Jupiter looked like circles with a red spot, including pictures from 100+ inch reflectors. Now with wavelet processing and other stuff I don't understand, an amateur with an 8-inch reflector can get nicer pictures of Jupiter than the 100+ inch telescopes could get in 1973.
Edited Date: 2015-04-15 03:04 pm (UTC)

Date: 2015-04-13 04:45 am (UTC)
From: [identity profile]
I recommend the acropolis if you haven't been there before and can make time to get there.

Date: 2015-04-13 02:06 pm (UTC)
From: [identity profile]
That's pretty much the one certainty in an otherwise wildly unplanned trip. :-)

Date: 2015-04-13 09:35 pm (UTC)
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From: [personal profile] siderea!


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