May 2008 – The Big Room (and the little things in it)

“Antibiotic Susceptibility Testing by a Standardized Single-Disk Method”

Okay, one last post in the Classic Science Papers challenge before my time’s up:

Bauer AW, Kirby WM, Sherris JC, Turck M :”Antibiotic susceptibility testing by a standardized single disk method.” Am J Clin Pathol. 1966 Apr;45(4):493-6.

The “Kirby-Bauer” antibiotic susceptibility test is another standard method that you should cover in microbiology class. The method involves getting a pure culture of the bacteria you want to treat, and then growing it in a petri dish. By putting paper disks soaked with various anti-bacterial substances, you can identify which ones are most effective at killing (or at least stopping) the bacteria in question – for example if you’re trying to figure out what kind of antibiotic to give to the guy coughing up some unknown plague in your doctor’s office… The anti-bacterial substance that the paper is soaked in slowly diffuses into the area around it on the petri dish, getting more dilute the further it gets from the paper. You can then estimate how powerfully anti-bacterial the stuff is by how far from the paper the bacteria stop growing.

The authors here didn’t invent this trick. Not all antibiotic-susceptibility tests are “Kirby-Bauer” tests (the blurry picture there is of an experiment I did involving the beer ingredient hops, and is not a Kirby-Bauer test. Click the picture to go to my “Beer Cures Anthrax” post from long ago…). What this paper describes is a method that finally standardized this test. Instead of having to use multiple paper disks with different amounts of the same substance, the “Kirby-Bauer” test prescribes specific concentrations of each antibiotic, and specific nutrient agar formulations, and so forth, so that determining which antibiotic your mystery bug is best treated with can be done in a way that gives consistent results regardless of who is performing the test.

The method is regularly updated to account for new antibiotics, but is still referred to as the “Kirby-Bauer” antibiotic susceptibility test to this day. Incidentally, the American Society for Microbiology kindly hosts a reprint of this paper as a .pdf file, so you can read it yourself if you’d like.

(UPDATE 20110328: new URL for the reprint of the paper. Thanks, Alex S!)

“A simplified method of staining endospores”

One more for the “classic papers” challenge:

Schaeffer AB, Fulton MD: “A Simplified Method of Staining Endospores”; 1933; Science; 77; pg 194

If you take a microbiology lab, this is the endospore staining technique (or “technic” as they used to spell it) that you’ll practice. This is a nice, simple, one-page paper. Alice B. Schaeffer and co-author Mac Donald Fulton describe a few of the other variations on endospore staining techniques, then describe how they’ve further simplified what they felt was previously the simplest one, described by a Mr. Wirtz in 1908.

“Endospores” are a sort of “escape pod” for certain specific kinds of bacteria. Unlike spores formed by yeasts and molds, these are not reproductive – each bacterium only produces one thick-coated spore, into which it shoves it’s genetic material and a few vital enzymes to get itself going again later when the spore finds itself in favorable conditions.

Since only a few kinds of bacteria produce these endospores, if you see endospores in your unknown bacterial culture it goes a long way towards helping to identify the bacterial species, so having a simple method for staining your bacteria so that endospores are obvious under a microscope is helpful. (Of course, these days most of us would rather just get a 16s rDNA sequence with PCR, but never mind that for now…)

Evidently, Wirtz’s original method involved using Osmium Tetraoxide (“osmic acid”) to stick the bacteria to the slide before staining. Not only is that stuff poisonous, it’s also expensive. I found a site selling sealed glass ampoules containing 1 gram each of this stuff for $35.00 each. Schaeffer and Fulton’s method does away with this in favor of much cheaper and easier heat-fixing (just as is done with the Gram stain and others). They use the dye “Malachite Green” for the initial stain, and steam-heat the dye-covered bacterial slide a few times to sort of “cook” the dye into the thick-walled endospores if they are there. Rinsing then washes the dye out of everything but the endospores, and a light red dye (safranin) is added as a counterstain. The end result is that under the microscope you’ll see light-red bacteria. If any of them form endospores, you’ll be able to see them as smaller green dots – sometimes still bulging inside of bacterial cells, sometimes floating around freely having escaped from the now-empty bacterial cell.

The “Schaeffer-Fulton Endospore Stain” is pretty easy to do, though the occasionally messy steambath part can be annoying. The method is pretty resistant to errors, so it’s not too hard to get good results even if you’ve never done it before.

Incidentally, you can buy Malachite Green at many pet stores – it’s still used as a treatment for “ick” (Ichthyophthirius infestation) in tropical fish.

Hmmm…still a couple of hours before it’s not longer May – Perhaps I can throw in one last post before time’s up…

I guess now I find out how good or bad Magellan, inc. is.

A couple of months ago I traded up from my Garmen etrex Legend to a Magellan eXplorist 600. I wanted to get away from Garmin due to a couple of irritating limitations that they seem to cling to (like losing your timestamps if you save an individual track, lack of SD card support, and dropping NMEA data support “to support [their] software” [see the review in Make #1]). So far, I’ve been pretty pleased with the shiny new Magellan. Though their software (as usual) only works on Microsoft Windows, I didn’t buy it for the software, and GPSBabel handles the data conversion of the files just fine.

So, naturally, now that I’ve just started blogging with the track data I’ve been recording with it, it has died on me. Yesterday – worked fine. Today – won’t turn on or respond in any way.

The good news is that I DID register the thing online at Magellan corporation’s website, and it’s supposed to have a 1-year warranty. Support’s only available Monday-Friday, though, so I’ll have to wait until Monday to see a response to my support query. Wish me luck. I’ll post updates in case anyone is interested in how Magellan responds.

In other news, “BigC” followed up on my previous queries about digital microscopes and Linux, saying that the manufacturer is now offering a Linux-compatible version of their AM311S model (the Linux-compatible one is the “AMU311S”) by special-order. Only goes up to 200X magnification, though, so it’s not real useful for my microbiological purposes. I can’t say the manufacturer of these digital scopes is impressing me much, but BigC certainly seems to be going out of their way to give good service.

There’ll be at least one other post later today – I wanted to get some more “classic” microbiology papers blogged before the day was up. Kirby-Bauer or Schaeffer-Fulton?…

“A small modification of Koch’s plating method.”

Only two more days for the Classic Papers Challenge, so if I’m going to get any more up, I’d better get my butt in gear.

Here’s a nice easy one:

Petri, R. J.:”Eine kleine Modification des Koch�schen Plattenverfahrens.” Centralblatt f�r Bacteriologie und Parasitenkunde; 1887; Vol. 1, pages 279-280.

The American Society for Microbiology has a translation available online. It’s only about a page-and-a-half of relatively large type – check it out.

There’s a trick we microbiologists use for growing bacteria. You make a solid (but wet) surface that contains whatever nutrients the microbe (bacteria, archaea, yeasts, mold spores…) you’re interested in need, and then you spread a diluted mixture of the microbe on it. The idea is that since the surface is solid the microbes can’t move around too much, and at any spot where a single cell starts initially, a whole pile of that cell and it’s genetically-identical (non-sexually-produced) clone-children will form until it gets big enough to see without a microscope. This cell-pile is called a “colony”, and you can poke or rub it with a sterile object, then stick the object into a sterile nutrient source. The end result is you have a “pure” culture of microbes that are effectively genetically identical. The solid material could be a lot of things – I’ve seen references to using slices of potato – though these days agar-agar gel mixed with nutrients is the preferred substance.

Koch (that is, Robert Koch of “Koch’s Postulates” fame, not Ed Koch the former mayor of New York City) used gelatin (so, hey, here’s another thing you can do with your expired Jell-O®). He apparently used to have a stack of shallow bowls, and had to use a special pouring device to carefully dump the gelatin into each stacked bowl in turn, then cover the works with a bell jar in order to keep stuff from falling into them from the air and contaminating them.

This was kind of a pain to work with, so some clever guy named Julius came up with a modification of this method in 1887, using pairs of shallow dishes, one slightly larger than the other so that it could be turned upside down to use as a lid. Then, you don’t necessarily need the bell jar, and you don’t need to stack them so they’re easier to pour.

Julius Robert Petri’s idea was so useful that we still use it today. Oh, yeah, and they named the dish-and-lid combination after him.

How’s that for a “classic” paper?

Meanwhile, my “Mountain Dew� Wine” project is turning out to be substantially more educational and fascinating than I’d hoped. There seems to be a decent amount of information available on how benzoic acid affects yeasts. I intend to turn that into a post later, but first I’ll try to find at least one more old paper to post before tomorrow is over…

Another “Open Thread”/quick followup

Time to take the special-needs kitty to the vet for boarding and gas up Flagella (yes, I named my car Flagella) and hit the road. By popular (i.e. more than zero people) demand, here’s my route (again, RSS readers will need to come to the website to see the map). Wish me (good) luck.

ID to TX [height=480;width=560]

Another cheatin’ “Open Thread” and random stuff

No single topics to dominate a post today. I’m in a hurry (as usual lately) and have very little time. Tomorrow morning I’m back on the 1600-mile route back to Southeast Texas, hopefully to sign the closing paperwork on the house we’re trying to buy.

My Mountain Dew� Wine appears to be still sitting there after several hours. Either the benzoic acid is still inhibiting the fermentation (in which case it’ll go REALLY slowly) or the yeast is just in shock or something. We’ll see how it looks in the morning. I’ll leave it for a week or so anyway to see how it does. Meanwhile I’ll refrigerate the other batch of yeast culture until I get back. If I have to develop my own strain of “Mountain Dew Yeast” I will, dagnabbit!

I did get a chance to go for a quick walk in the Big Room on the way back from some errands yesterday, so it gives me an excuse to play with the wordpress map plugin again (RSS feed readers: the map doesn’t get inserted there. Please check out the interactive map at the blog’s website here.) Comments on the map (or anything else, really – I DID say “Open Thread” after all) are encouraged – what do you think? I’d like to do some audio content for points on a map at some point, too. Maybe some video.

Lava Rock Walk [height=560;width=560]

If anyone’s bored enough to want to see how I get from Southeast Idaho to Southeast Texas, I can post a map of that tomorrow, too…

I’m not a real hillbilly, but I play one on the internet…

My first “Will It Ferment” project is now in process.

See, I’m stuck with a somewhat unpredictable schedule and a need to travel frequently, cramped spaces to work in at the moment, and a streak of gustatory perversion that I just can’t help an urge to rebel against purists at the moment.

Quick bit of background on that latter statement: I’ve noticed that people seem to think there are only two-and-a-half kinds of “real” non-commercial fermented beverages. There’s beer (which is apparently defined as a strong tisane of hops, flavored by mixing it into fermented malt), there’s wine (which is always made of grapes of course), and out on the fringes of respectability is Mead (“Honey wine”) which seems to be slowly gaining some acceptance as a mildly exotic brew. The attitude is that anything else you might want to brew (say, a beer flavored and preserved with something besides hops, or a wine made out of anything but grapes) is probably some quaint “country” (i.e. hillbilly) thing for people who either live too far from civilization or are too poor to just buy a “real” beverage, or are too ignorant to know the difference. Either that, or it’s just some desperate attempt to make something to get drunk on. Might as well be making pruno.

The attitude kind of annoys me, so I’m trying to make a sort of “free person’s pruno”. I figure if the end result is as palatable as it could be, it’ll probably resemble “Zima�”.� Uh, no, I don’t expect it to be great – this is merely an experiment.� My must has an Original Specific Gravity (“O.G.”) of approximately 1.054, about the same as most lagers start out. As I write this, my gallon of must is on the stove in a stainless steel pot. I’m going to try to heat it to boiling and let it boil for a few minutes in hopes of driving off much of the benzoic acid in it, so as not to inhibit the yeast fermentation.

To your right, you should see the ingredients used in this project. Yes, those two bottles in the background are there on purpose. I’m trying to make…Mountain Dew� Wine.

Here’s the plan:

Last night, I took a couple of clean glass 1 Qt milk bottles, rinsed them with iodophor solution to sanitize, let them dry. Then, I put about 12 ounces of tap water (unchlorinated) into one and dumped the contents of both yeast packets you see in the picture into it to rehydrate.
The yeast packets were opened over a year ago – I don’t even recall what I did with the tiny amount of yeast I poured out. They’ve been sitting in the ‘fridge since then. What’s more, they had expired in December of 2006 to begin with.
The two yeasts (Red Star Montrachet and Red Star Flor Sherry) were both mixed into the same container in hopes that I could encourage a yeast orgy, giving me as much genetic diversity as possible from the two strains and maximizing the chance that I’d be able to get a culture which can grow in flat Mountain Dew and whatever benzoic acid (from Sodium Benzoate) might remain in it. I had read that V8 juice was actually an excellent medium for inducing yeast sporulation. Since sporulation occurs as a result of yeast cells mating, I made a huge leap of logic towards thinking the V8 might maximize my chances of getting some yeast mating going on.
After a few minutes to rehydrate, I dumped the 12-ounce can of V8 into the bottle and shook well. I also crushed up a children’s chewable vitamin (see bottle at lower-left) and a small portion (perhaps 1/5) of a capsule of the L-Arginine as a nitrogen source and added them as well. I then poured it back and forth between the two bottles a few times to aerate, then split the mixture between the two bottles, capped loosely, and went to bed.
As of this morning, fermentation was obviously occurring in the mixture, so sitting in my fridge open for over a year hadn’t killed off ALL of the cells. I opened the bottles and swirled to re-aerate, and added about a tablespoon of “corn sugar” (glucose a.k.a. dextrose) to wake the yeast cells back up. Since then, I’ve been pouring a bit of Mountain Dew right out of a third bottle into each of the yeast starter bottles every couple of hours. The amount of bubbling I see suggests to me that fermentation is still going on (and is not just from the carbonation of the Mountain Dew). Hopefully this will help the yeast culture acclimate a bit to the benzoic acid.
I dumped the other 2 2L bottles of Mountain Dew into a stainless steel 8-qt pot and heated to boiling, whisking with a steel whisk frequently to help get the dissolved gases to bubble out (hopefully along with some benzoic acid in the steam). At this moment, it’s up to 75�C (about 165�F) according to the thermometer I have stuck in it. It’s steaming a bit, and I can smell some of the citrusy aroma boiling off, unfortunately. I was afraid that’d happen. UPDATE: our ancient stove with one working burner seems to have trouble getting this much liquid above 200�F without cranking it up all the way and risking the bottom getting too hot. Since there’s a substantial amount of hot water vapor coming off, I’m going to hope that’s carrying away some benzoic acid and just let it start cooling down. I’ll stir it vigorously and frequently with the whisk until the temperature drops to about 160�F and then I’ll cover it for the night.
Meanwhile, before I go to bed, I’m going to recombine as much of the two yeast culture bottles as I can into one bottle, then rinse out the other. In that one, I’ll mix up about 12 ounces of tap water and enough corn sugar to reach a gravity of about 1.050-1.055. I’ll crush up another chewable vitamin and about half of the remaining arginine capsule into it, mix well, and warm it with a quick spin in the microwave (no exact measurements, just until it’s “obviously warm” to the touch after mixing). Then I’ll shake the V8 culture well to mix, and splash about a tablespoon’s worth into the new bottle.
In the morning, I’ll re-aerate the new culture and add a tablespoon of corn sugar to wake it back up, then once it’s going, I’ll start adding the now-cooled cooked flat Mountain Dew to it in small increments. Assuming it keeps going, I’ll dump in the remainder of the L-Arginine capsule, crush in one last chewable vitamin, and them combine the new culture with the rest of the cooked flat Mountain Dew in a nice plastic 2-gallon “water” container that I picked up (already rinsed with iodophor and dried). Cap it with a latex glove attached with a rubber band as described in the “Pruno” entry in Leon Kania’s “Alaskan Bootlegger’s Bible” (Click image for link) just because I thought it was the funniest airlock design I’d ever run into. Yes, I am easily amused.
If I’m lucky, there’ll be so much live and active yeast in the second culture at that point that the fermentation will finish quickly, because I’m driving to Texas the following day. Alternatively, I may be lucky and the remaining benzoic acid will slow the yeast down, so I can safely leave it fermenting (sitting in my sink in case of overflow while I’m gone) for the week that I’ll be gone.

If I’m UNlucky, either it won’t ferment at all (and I can then use it to try to develop a Mountain Dew Tolerant strain of yeast), or it’ll ferment but taste utterly disgusting (in which case I can use it to try to obtain some Gluconobacter strains and make Mountain Dew Vinegar), or it’ll be “infected” and will already BE Mountain Dew Vinegar, which would also be pretty funny anyway. So, other than completely unforseen results, this experiment can’t be a TOTAL waste.

[Update 20080727: Preliminary results of this perverse project may be read In this more recent post…]

“They laughed at me! But I’ll show them all! AH, HAHAHAHA!”

Another T-shirt to add to my list of T-shirts I want.

I’m spending more hours shoveling my way through the books and papers and crap we’ve got up here at House v1.0, since if all goes well I’ll be making a brief run back down to Southeast Texas so we can sign the papers for House v2.0 down there, at which point we’ll be able to start actually moving. I sure hope this one goes through. Not only is it our third attempt to buy a house down there, but I’ve already identified a convenient location to build my “Intentional Food Microbiology” brewlab in it.

Since there’s no way I can afford to buy a -80�F freezer, I have an obvious interest in alternate means of preserving the yeast, mold, and bacterial cultures that I want to keep. To me, drying seems like the most desirable method when it’s feasible, since dried cultures should require the least amount of maintenance. After a several-month delay, I’ve finally gotten around to getting back in touch with the archivist at Brewer’s Digest to see about getting an old article on the viability of dried yeast cultures[1].

Speaking of old but useful scientific papers, there’s an extremely nifty challenge going on through the month of May (deadline: May 31st) over at “Skulls in the Stars” blog: find a classic scientific paper, read it, and blog about it.

“My �challenge�, for those sciencebloggers who choose to accept it, is this: read and research an old, classic scientific paper and write a blog post about it. I recommend choosing something pre- World War II, as that was the era of hand-crafted, �in your basement�-style science. There�s a lot to learn not only about the ingenuity of researchers in an era when materials were not readily available, but also about the problems and concerns of scientists of that era, often things we take for granted now!”

I think this is a brilliant idea – the classic papers often seem to be forgotten and often explain things that people seem to take for granted these days. I already mentioned my post about the Gram Stain (original paper published in 1884), though that post really talks more about what has happened with the Gram Stain over the last 125 years rather than only being about the original paper. There are a couple of other classic microbiology papers that I’m going to try to get to if I have time before the May 31st deadline arrives.

I also need to get some yeast activated and get my must processed – I’m hoping a brief boil will reduce the amount of a yeast-inhibiting substance in it. I’ll post more detail after I get it going.

[1] Wickerham LJ, AND Flickinger MH:”Viability of yeast preserved two years by
the lyophile process.” 1946; Brewers Digest, 21, 55-59; 65.

I finally got to Bristol Brewing Company…

Signage in front of the Bristol Brewing Company After spotting the company’s mention in Jeff Sparrow’s “Wildbrews“, I’ve been wanting to visit Bristol Brewing Company, particularly since they may be the only such brewery using local yeasts and bacteria that they isolated themselves from their local environment.

Since we’re moving from Southeastern Idaho to Southeastern Texas, I’ve been going back and forth between the two location. It just so happens that along with New Belgium Brewing Company, Bristol Brewing is actually right along a convenient route between the two locations. (Let’s see if I can figure out how to work the Google Maps plugin):

How’s that look? How does it work? (Those of you reading this on the RSS feed: The interactive map only appears on the website, it would seem. Please check it out here.) I made the KML file myself…

Bristol Brewing is a cozy little brewpub, and the people there are encouragingly helpful. They were in the middle of bottling, so there were no tours. Also, there were not currently any of the skull-‘n-bones beers available. However, I did get an educational series of tastes of their current brews on tap (thank you to the employee I spent most of the time talking to whose name I’ve embarrassingly forgotten, but who I believe was Tad Davis judging from the photos on the web site). I also lucked out and their microbiologist, Ken Andrews, happened to be there. I asked about their native Colorado brewing flora. Turns out Bristol Brewing isn’t quite as bold as I originally thought. They’re still doing their primary fermentation with “normal” brewing yeasts. What they’ve done is inoculated some wine barrels with the locally-isolated yeasts and bacteria, and they use the barrels for a secondary fermentation and aging instead. Much safer if you have to worry about having a drinkable product at the end, and of course it makes me feel like more of a crazed rebel for wanting to isolate my own local bugs for the main fermentation. So, a great visit overall.

Incidentally, it seems they’ll be tapping a new Skull-‘n-Bones brew in a couple of weeks, at 5pm on Tuesday, May 27th (2008). It sounds like they’ll probably have some available for a few days before it all disappears, so I’m hoping my return to Texas can be timed such that I can swing by and at least get a taste.

“Open Thread”

Too tired to do a real post at the moment. I’ve seen other bloggers do this “Open Thread” thing, inviting readers to post comments about whatever the heck they want so as to make the blog look more active without actually having to post anything. So what the heck. Real post in the next 24 hours hopefully, but in the meantime, what’s on everyone’s minds?

Oh, yeah, and am I right that typical cola drinks are about 3 mM phosphate (as phosphoric acid)?