The Big Room (and the little things in it) – Page 12 – Applied Empirical Naturalism on a Microbiological Scale

Mountain Dew® Wine: Disappointment strikes!

<whine excuse=”obligatory”>Have I ever mentioned what a huge hassle it is to relocate from one abode to another 1600 miles away?…</whine>

I’m finally back at House v1.0 where I can check on the progress of my Mountain Dew� Wine. It appears to have managed to ferment, in spite of the severe dose of preservatives in the stuff designed to prevent that from happening. It went somewhat slowly, but it’s gone from an original gravity of around 1.054 down to about 1.011 or so, suggesting about, say, 5-6% alcohol in the final product, which has faded to a pale, cloudy yellow color. Hopefully the cloudiness is from still-living yeast, which has now demonstrated that it is reasonably benzoic-acid-and-caffeine tolerant.

I fear I must report that the result is a crushing disappointment to me. It’s not very good. Worse yet, it’s not very bad, either. I was hoping that if it wasn’t surprisingly tasty that it would at least be shockingly awful in some interesting way so I’d have something entertaining to say about it here.

Actually, the adjective that comes to mind is “inoffensive”. If you would like to whip up a quick simulation of what I’ve got here, you might be able to do it like this: Take some citrus-flavored sparkling mineral water. Dilute it about half with (uncarbonated) distilled water. Then mix about 7 volumes of that with one volume of vodka. What I’ve got here is slightly sparkling, with a barely noticeable citrus flavor and little or no remaining sweetness. It’s a little surprising to me just how much of the flavor of Mountain Dew� apparently comes from its sweetness. Perhaps next time I try this (if there ever IS a “next time”…) I’ll have to mix regular and “diet” Mountain Dew� – with some bonus sugar to make up the difference, of course.

I’m not completely done here. I’m still going to dispense it into cleaned bottles with a little bit of sugar to prime it for full carbonation. Sanitized plastic soda-bottles of course – none of that snobby glass stuff for this here experimental drinkin’ substance! It may be high-class for “pruno”, but it’s sure as heck not Champagne�. (Besides, I want to evaluate reusing plastic bottles anyway – it’d be a lot easier to tell when there’s too much pressure and to let some of the pressure off if there is.) Plus, I need to take some of the still-live yeast and�keep it alive. No point in developing a benzoic-acid-tolerant yeast strain and not keeping it!

On a related note, an article was mentioned on fark.com, saying that back in 1955, a scientist “proved” that it is not normally possible to get drunk on beer. Of course, he seems to have been referring to dilute mass-market bladderwash and his reasoning was that a typical human stomach cannot contain enough 3.7%-alcohol beer for a typical human to achieve a dangerous blood-alcohol level.

As usual, the articles (see here and here) go for the “a scientist says this” part but never bother to say WHERE the scientist says it – usually a real scientific publication.

A quick search of pubmed turns up a likely candidate:

Greenberg LA: “The definition of an intoxicating beverage.” Q J Stud Alcohol. 1955 Jun;16(2):316-25 (link goes to the pubmed entry, which has little more information that this).

I do believe it is a moral imperative that I get a copy of this article somewhere so that I may reference it later. Is there anyone out there reading this who might be able to get a copy of this paper somewhere for me? Please?…

SHENANIGANS! Caffeine is our FRIEND!

Our new Asylum has real internet finally now and we’re getting settled in. The Houston area here is one of the most hot and humid areas of the US. All hot and sweaty. So of course I’ve been advised that my favorite psychotropic substance – 1,3,7-trimethylxanthine [“caffeine” for party-poopers who aren’t into the fancier names] – is no longer my friend, because it’s a diuretic that’ll dehydrate me, right?

NO! Shenanigans! Caffeine is our FRIEND! And that stuff about it being a diuretic? CRAP! LIES AND SLANDER!

But don’t just take my word for it. After all, humans are a bunch of freakish multicellular soft-celled eukaryotes, and I normally focus on normal organisms like bacteria, archaea, and yeasts. So, let’s ask some real human-physiology type scientists and check out their official peer-reviewed findings:

Armstrong LE, Pumerantz AC, Roti MW, Judelson DA, Watson G, Dias JC, Sokmen B, Casa DJ, Maresh CM, Lieberman H, Kellogg M: “Fluid, electrolyte, and renal indices of hydration during 11 days of controlled caffeine consumption.”; Int J Sport Nutr Exerc Metab. 2005 Jun;15(3):252-65.

“[…]The following variables were unaffected (P > 0.05) by different caffeine doses on days 1, 3, 6, 9, and 11 and were within normal clinical ranges: body mass, urine osmolality, urine specific gravity, urine color, 24-h urine volume, 24-h Na+ and K+ excretion, 24-h creatinine, blood urea nitrogen, serum Na+ and K+, serum osmolality, hematocrit, and total plasma protein. Therefore, C0, C3, and C6 exhibited no evidence of hypohydration.[…]”

Abstract on Pubmed

Armstrong LE, Casa DJ, Maresh CM, Ganio MS: “Caffeine, fluid-electrolyte balance, temperature regulation, and exercise-heat tolerance.” Exerc Sport Sci Rev. 2007 Jul;35(3):135-40.

“[…]This review, contrary to popular beliefs, proposes that caffeine consumption does not result in the following: (a) water-electrolyte imbalances or hyperthermia and (b) reduced exercise-heat tolerance.”

(Review article, apparently – Abstract on Pubmed)

Del Coso J, Estevez E, Mora-Rodriguez R: “Caffeine effects on short-term performance during prolonged exercise in the heat.” Med Sci Sports Exerc. 2008 Apr;40(4):744-51.

“[…]RESULTS: Without fluid replacement (NF and NF + CAFF), subjects were dehydrated by 3.8 +/- 0.3%[…]CONCLUSION: During prolonged exercise in the heat, caffeine ingestion (6 mg.kg body weight) maintains MVC and increases PMAX despite dehydration and hyperthermia. When combined with water and carbohydrate, caffeine ingestion increases maximal leg force by increasing VA (i.e., reducing central fatigue).”

(“NF” = “No Fluid replacement” – the “dehydration” mentioned here is due to exercising in the heat, and doesn’t appear to be related to whether the test subjects consumed caffeine or not)

Abstract on Pubmed

Scott D, Rycroft JA, Aspen J, Chapman C, Brown B:”The effect of drinking tea at high altitude on hydration status and mood.” Eur J Appl Physiol. 2004 Apr;91(4):493-8. Epub 2004 Feb 11.

“[…]Several markers of hydration status were also taken immediately pre and post each condition, including measures of urine specific gravity, urine electrolyte balance (K+, Na+), and urine colour. None of these measures indicated a difference in hydration status as a result of the dietary intervention in either the control or tea condition.[…]”

(In this study, the tea was the only caffeine-containing substance involved. The study group’s caffeine came solely from the tea. The control group got no caffeine at all.)

Abstract on pubmed

Paluska SA: “Caffeine and exercise.” Curr Sports Med Rep. 2003 Aug;2(4):213-9.

“[…]It[caffeine] is relatively safe and has no known negative performance effects, nor does it cause significant dehydration or electrolyte imbalance during exercise.[…]”

Abstract on Pubmed

Grandjean AC, Reimers KJ, Bannick KE, Haven MC.: “The effect of caffeinated, non-caffeinated, caloric and non-caloric beverages on hydration.” J Am Coll Nutr. 2000 Oct;19(5):591-600.

“[…]This preliminary study found no significant differences in the effect of various combinations of beverages on hydration status of healthy adult males.[…]”

Pubmed entry – full text available

See? Oh, I know what you’re going to say next – “But, like, dude! When I drink my Venti Mocha Crappucino [note: Link goes to “Foamy the Squirrel”, who is a bit of a pottymouth, ranting about the “Tall/Grande/Venti” nonsense.� It amused me.] or a can of Jolt Ultra I have to take a major whiz a little while later! Isn’t that ‘cuz of the caffeine?” Well, no, it isn’t. It’s because you just drank a bunch of liquid. Duh.

So, you see, caffeine really is our friend. Be nice to caffeine. But don’t feed it to your yeast in the presence of benzoic acid because it’ll kill them. See? I managed to turn this into a segue back to the stuff I was talking about before the whole “buy a house in Texas” thing started interfering. Stay tuned…

Very brief post…

Sorry about having another brief bout of blogstipation. We’ve finally managed to close on a house and now we’re probationary Texans (y’all). I’ve been spending the last week+ driving back to SE Idaho (by way of Best Friends, since we now have room to hire a second dog to hopefully keep Cornelia the Laser Dog company. I’ve got to lug 3 cats (and one goldfish in a small fish tank) a distance of 1600 miles or so starting in about 6-8 hours. Wish me (good) luck.

After the weekend, I should have some time to get back to the real posts. The Mountain Dew Wine was virtually unfermented when I returned, even after sitting there 10 days, but since I’ve gotten back it’s started going. Still slowly – the bubbler spits out 3-5 bubbles ever 40 seconds or so – but it’s going. It’ll be interesting to see what I end up with. I wanted to do 2-3 posts on the effects of benzoic acid on yeasts (that’s the preservative in Mountain Dew®), and I would swear I had one or two others in mind. Oh, yes, and an update on getting a Magellan GPS replacement that can actually be used – they seem to have located a slightly lower-end model eXplorist that they can send me. I sent them back their “walled garden”-based Triton 500, so the replacement unit ought to show up next week, I think. Their service has been pretty good, at least.

Was it a mistake to buy Magellan GPS? Stay tuned…

A dead Magellan eXplorist 600 GPS unit Well, the good news is that after calling their support line and trying the three-button-reset ritual that I hadn’t known about, my Magellan eXplorist 600 is still completely dead. They did not give me any hassle about replacing the unit, they just gave me an RMA number. My dead eXplorist was sent to their “Repair” center in Fort Worth, TX this afternoon. A replacement should arrive in about a week to a week-and-a-half.

The bad news is that Magellan has discontinued the $350 eXplorist 600 (and the rest of the eXplorist line) and insists that they can only offer their newer $250 Triton 500 as a replacement. I’ll withhold judgement on whether to be ticked off at the $100 of retail value that is being lost here until I see the specifications. It looks at first glance that the basic capabilities really are pretty much the same, so it may turn out okay…except for the most important feature by far, which I originally chose the eXplorist for in the first place: documented data formats.

The Triton series GPS’s appear to use a bizarre, undocumented file format, completely different from the documented format of the Meridian and eXplorist models. This means that as of right now, a Magellan Triton owner is not permitted to work with their OWN DATA without going through a proprietary, Microsoft-Windows-only GUI package, which ironically apparently uses GPSBabel to do file conversion. GPSBabel doesn’t support the Triton formats since there is no information available on how to read and write them yet. Since I have no Microsoft Windows machines anywhere, this means the shiny new Triton 500 (which I – seriously – can’t stop thinking of now as the “Magellan Vista”) will be nothing more than a highway map that requires batteries unless their proprietary “VantagePoint” software will run under WINE.

I’m hoping that they’ve merely been busy and will soon get around to adding this units specifications to their “Interface Solutions” information. This is where the openly-published file format (and communication protocol) specifications which were highly (and rightfully) praised by the GPSBabel project are made available for the now-abandoned Meridian and eXplorist units. If the GPS data I work so hard to obtain remains locked inside the proprietary format, only accessible at the whims of Magellan and Microsoft, I’m going to be extremely peeved. If, on the other hand, GPSBabel soon gets the information necessary to add support for the Magellan Triton line, the only serious complaint I have with all this will go away. Honestly, if I can at least get enough documentation to write my own simple waypoint, track, and route reader for their Triton files I’ll be happy.

I think a real, old-fashioned letter, printed on dead tree and everything, mailed to their corporate HQ is called for…

Anybody out there have any experience dealing with Magellan corporation? I’d like to think they want to do the right thing…

“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…