Microbiology – Page 4 – The Big Room (and the little things in it)

It’s comin’ right for us!

(Click image for Wunderground.com’s interactive “Wundermap”…)

Actually, looking more closely at the picture there I think we may be slightly further west than it indicates, but it’s still very likely we’ll end up losing power for a while sometime tonight, but I still am going to try to get a post up for the Giant’s Shoulders blog carnival – I’ve got the paper picked out and read, I just need to do a write-up before I go to bed tonight. Currently it looks like the winds shouldn’t get TOO bad until late tonight/early-tomorrow-morning so we ought to have power long enough…

Yes, there’s a disturbingly severe hurricane “Comin’ right for us!” and I’m worrying about posting about a classic scientific paper before the blog-carnival deadline while I still have power. How’s THAT for dedication?

First we need to move stuff inside, though. I don’t want The Ascospore getting blown away…

Yeast needs to breathe

As readers may have guessed from previous posts, my brewing interests are minimally conventional. Fortunately, the Basic Brewing Radio podcast seems to regularly expand well beyond the usual “fermented malt flavored with a tisane of hops” thing (I need to try to make my own “Ginger Beer Plant” from scratch one of these days…). A couple of weeks ago, they did an episode covering an experiment on aeration methods which was very interesting. It does my ego good to know that I correctly guessed how the results would turn out. You can get a copy of the nice write-up of the experiment itself here, but here’s the simple version:

Continue reading Yeast needs to breathe

Aqua-pedestrianism and Ice Cream Yeast

Today’s batch of blog-based Stir-Fried Random includes another interactive map of a lake-spanking expedition, a very brief musing on search engines, and a return to “intentional food microbiology” discussion. To preview: you can get pizza without ever getting out of the water on Lake Conroe, “spanking” is amusingly popular for search engines, and no, there is not normally any yeast in ice cream, but perhaps there could be. Read on, please…

Continue reading Aqua-pedestrianism and Ice Cream Yeast

FoodTV’s new “Food Detectives” show…

That’s all I can stands, I can’t stands no more! I had intended to try to come up with another post for this month’s “The Giant’s Shoulders” anthology, but I’ve just encountered such an appalling concentration of disappointing un-science that I cannot restrain myself any further. Guess I’ll have to settle for one post in the anthology this month.

FoodTV’s new “Food Detectives” show sounded so promising. I thought to myself “‘MythBusters’ meets ‘Good Eats’!?!? That would be pure, refined, pharmaceutical-grade WIN!” Then I saw their premier episode. The “experiments” appeared blatantly and badly staged, and in some cases shockingly badly designed. For example, their “experiment” with refrigerator deodorants involved showing a guy sticking his face into a ‘fridge allegedly full of smelly stuff and filming him making faces while they timed how long he pretended to be willing to keep his face in there.

Continue reading FoodTV’s new “Food Detectives” show…

“Ueber die isolirte Faerbung der Schizomyceten in Schnitt- und Trockenpraeparaten”

the 1884, #6 edition of 'Medical Progress', containing Hans Christian Gram's invention of the 'Gram Stain' The Giant’s Shoulders blog carnival is coming up in two days, and I just realized I still haven’t gotten a post up for it yet. So, here it is.

I put up some quick reviews of several classic microbiology-methods papers for the previous edition of this blog carnival, but didn’t actually get around to putting up the one for what is almost certainly the most well-known microbiology technique: “The Gram Stain”. So, this post is about it:

Gram HC: “Ueber die isolirte Faerbung der Schizomyceten in Schnitt- und Trockenpraeparaten”; Fortschritte der Medicin; 1884; vol 2, pp 185-189

That’s “Regarding the Isolational(?) Coloring of Schizomycetes in Cut- [i.e. tissue sections] and Dried Preparations” in “Medical Progress”. The translation hosted by the American Society for Microbiology uses the word “Differential” where I’ve put “Isolational” – which is probably not quite right either but it’ll have to do for now – but I’ll get to that in a moment.

If you’ve ever been exposed to microbiology labwork before, you’ve almost certainly done or at least watched a procedure referred to as a “Gram stain”. In brief, you smear your sample with bacteria on a glass slide and bake it on, then you dump some purple stuff on it, them some brown stuff, then you rinse it briefly with alcohol, then you dump on some pink stuff, and then rinse it in water and look at it under a microscope. Bacteria that stay the original dark purple-blue color of the original purple/brown stuff are considered “Gram Positive”, and those that don’t instead appear the pink color of the last stain, and are considered “Gram Negative”. Many textbook authors and microbiology instructors will breathlessly proclaim that the Gram Stain reveals two “fundamental” categories of bacteria, but I’ll spare you my rant about that.

Properly speaking, this isn’t actually Gram’s stain, as described in his original paper. The modern variations that we’re all taught in microbiology class were developed later, and I believe they are nowadays based mainly on Victor Burke’s 1922 paper on the subject[1].

Regarding the title of the paper: “schizomycete” is what they used to call most kinds of bacteria. “Mycete” meaning “fungus”, as bacteria were assumed to be “plants without chlorophyll” just like molds and mushrooms, and “Schizo-” meaning “split in two”, since bacteria reproduce by splitting into two cells rather than by producing spores like “other” fungi. I say “most” because things like cyanobacteria (“blue-green algae”) or Green Sulfur Bacteria would have been referred to as “Schizophyta” (“fission-plants”). What Gram was originally trying to do wasn’t to differentiate one kind of bacteria from another, either, but to make it easy to tell bacteria from from the nuclei of cells in bacteria-infected tissue.

For that matter, Gram was really metaphorically standing on the shoulders of Koch and Erhlich, as he was building on their technique for staining “tubercle bacteria” – that is, tuberculosis-causing members of the genus Mycobacterium. Gram mentions that you need to stain this type of bacteria for the “usual” 12-24 hours to make this work, incidentally, as opposed to a few minutes for other “schizomycetes”. This suggests that you are expected to have some idea of what you’re going to find before you use the stain, as opposed to the modern implementation which is supposed to tell you something about what kind of bacteria you’re finding.

Still, Gram does report that some bacteria take the stain and some don’t, giving us a preview of the “differential” character of the modern version. He specifically notes typhoid and some causes of bronchial pneumonia fail to hold the stain. Given that Typhoid Fever is caused by a strain of the “Gram-negative” butt-bacter Salmonella enterica, and there are a number of “Gram negative” bacteria as well as “Gram positive” that can cause pneumonia, this makes sense. He also does mention the use of Bismarck Brown R a.k.a. Vesuvine as a counterstain in order to make the nuclei of the infected cells brown in contrast to the dark blue of the infectious bacteria in the tissue.

For much of the century-and-a-quarter since Gram’s publication, the question of why the Gram stain works was thoroughly investigated, and even today I occasionally hear or read assertions to the effect that the Gram Stain isn’t well understood. I disagree with this just as I think its importance to bacterial identification is grossly overblown, and if you want to know why, I have a previous post all about why the Gram stain works and how we know. You may or may not also be interested in an older post regarding whether or not “acid-fast” bacteria like the ones that cause tuberculosis (which don’t stain at all when using the modern version of the Gram stain) are “Gram Positive” or not. As always, if you spot any errors or have any questions, please let me know…

[1] Burke V: “Notes on the Gram Stain with Description of a New Method.” J Bacteriol. 1922 Mar;7(2):159-82.

Benzoic Acid Part 2: “Sour Stuff”

Okay, now that the boring review is over with…

Consider the cell. It doesn’t matter what kind of cell – bacterial, archael, fungal, animal, whatever. It’s still a tiny droplet of slightly salty water, thickened by a bunch of enzymes, other proteins, and various other substances floating around in the water. There’s also one other component that makes this a “cell” rather than soup: a bubble made of fatty material that the droplet is wrapped in, called the cell membrane. Depending on what kind of cell you’re thinking of, there may or may not be a “cell wall” made of some sort of rigid material, with the cell membrane inside of it. There may also be more than one membrane as is the case with the classic “Gram negative” style of bacterium, which has a second “outer” membrane wrapped around its cell wall. If it’s a eukaryotic cell, it’ll even have tiny little “organelles” inside itself wrapped in their own little membranes…but whatever. It’s the innermost one, inside of whatever cell wall may be there but wrapped around the cell’s guts, that we’re concerned with here.

Since stuff that will dissolve readily in water doesn’t tend to dissolve well into fats, and vice-versa, the cell membrane not only prevents stuff dissolved in the water inside the cell from leaking out, it also prevents stuff in the water outside from getting in. This lets a cell maintain itself at near neutral pH even if it happens to live in a very acidic environment, or an appropriate level of, say, sodium salts even if it lives in the Great Salt Lake.

This brings us back again to benzoic acid, which you should recall from the previous post alternates between a dissociated hydrogen-ion-and-benzoate-ion form and a combined, netural form in water. You may have noticed that foods preserved with benzoates tend to be sour, like fruit juices or soda. That’s because “sour” is the flavor of acid, and benzoic acid’s ability to be a preservative is only good in acidic environments

Useless Knowledge Break: the German word for acid is “Saurstoff”. Yes, that is pronounced like “sour stuff”, and no, that is not a coincidence.

An acidic environment means lots of extra hydrogen ions (“protons”) floating around. That also means that when a molecule of benzoic acid splits into a hydrogen ion and benzoate ion, it takes less time before another hydrogen ion comes by and the molecule can recombine again and therefore a bigger majority of the benzoate floating around at any moment is in the combined, somewhat fat-soluble neutral form. In that form, it can soak into a cell membrane if it encounters one.

If that molecule drifts through the membrane and gets to the inside of the cell, it may touch the less acidic watery environment there and dissociate into ions again and be unable to return through the membrane. The released hydrogen ions mean the inside of the cell becomes more acidic. As of today (20080806), the Wikipedia entry for Sodium Benzoate cites a single paper from the early 1980’s saying that when the inside of a yeast cell gets acidic enough, it prevents a specific step in the energy-generating process from working. This may be true, but there’s more to the story than this.

Obviously the membrane can’t totally seal the cell off from the outside, or the cell would be unable to excrete wastes or take in food molecules, so there are numerous specialized “transport” proteins that stick through the membrane to allow specific kinds of molecules in and out. Lots of biochemical reactions release hydrogen ions, so there are transport proteins that can shove hydrogen ions out of the cell and into the cell’s surroundings. The problem is that all substances naturally diffuse from areas of higher concentration to areas of lower concentration, so in an acidic environment the natural direction that hydrogen ions “want” to flow is into the more neutral cell. These transport proteins can shove the hydrogen ions in the opposite direction, but like pushing a boulder uphill it costs energy. This seems to be the primary reason that benzoic acid prevents bacteria and yeasts from growing – it makes them waste energy that they would be using for growth just to keep taking the hydrogen ions that the benzoic acid helps leak in through the cell membrane and shoving them back outside. The figure above is linked to a page at Helsinki university that discusses this type of preservative action in more detail.

Simple and elegant, and this seems to have been assumed to be the whole explanation for some time. But what happens to the benzoate ion when its hydrogen ion gets pumped away? Does it do anything?

Coming up next: Endocannibalism!

(Tap Tap Tap) Is this thing on?…

Move is underway. Here’s a quick update / test.

Cornelia and Monk(eymutt), the Laser Dogs

On the way home (to Texas, that is, with a load of furniture from House v1.0 in Idaho) we adopted a co-dog. Now Cornelia isn’t the only official dog in the house.

Yeah, I know, pretty frivolous stuff. What do you want – this is mainly a test post. You guys ARE seeing this post, right? (Please comment and let me know). I’ve finally found and jumped through the hoops necessary to migrate the bigroom.org website over to the new host. It’s also running on the old server in House v1.0 on the DSL line as well, but this post isn’t getting put on it. If I’ve done everything correctly, hosting for bigroom.org should now be handled by Eskimo North. Just a few other minor tweaks and I’ll be set to return to House v1.0 and shut down the old server so I can move it down here to Texas.

I’ve gone from about 4400ft elevation down to about 250ft, but the plastic bottles of Mountain Dew� Wine seem to have positive pressure, so it would appear there is still some live yeast left in there and the priming sugar is slowly doing its job. I’m pretty sure the benzoic acid is what’s slowing down the activity so drastically, but it hasn’t killed it off yet.

I’ve also got a couple of entries in mind already for the next The Giants’ Shoulders blog carnival, and hopefully some more crazed brewing project news to offer sometime. And, of course, the promised post on why benzoic acid works. Stay tuned.

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.