Environmental Chemistry Field Trip – Day 1, part 2

Our next stop was Appolinaris Spring, which seems to be an uncommon thing in Yellowstone National Park: ordinary springwater. No sulfuric acid, no steam, just plain old water that sinks into the ground and then comes back up later. For most of the park’s history, it seems like this used to be a popular place to stop to get a drink of water.

water emerging from small copper pipes
Although the signs around the spring now all suggest that you really shouldn’t drink it, at least not without filtering it first, I’m kind of kicking myself now for not having tasted it. Perhaps I’ll have to go back on my own time and try it.

Our on-site tests showed a pH of 5.9 (slightly acidic: milk is normally around 6.8 or so, Root Beer somewhere around a more acidic 4.0, cola beverages around 3.0, for reference…), relatively low TDS of about 100ppm, coming out of the ground cool (about 7°C, or 43°F), with very little dissolved Oxygen (about 6.0ppm) and faintly carbonated (300ppm CO2). It reportedly didn’t taste too good, but having foolishly missed out on tasting it, I don’t know why.

There were hints that perhaps contamination from surface water – like rain trickling through bison poo – but quite some time ago they sealed the spring up to protect it from that kind of thing. This is the actual spring now:

Appolinaris Spring is a concrete box in the ground with locked metal tops...
Even so, the signs still try to discourage people from drinking the water coming from the pipes that lead out of the spring, which I take to be the park service covering themselves just in case someone claims to get sick from it. (“Hey, we TOLD you not to drink it!”).

Appolinaris: This spring water has been used by visitors since early days of the park.  However modern water tests show periodic contamination.  Park waters, even though clear and running are subject to pollution by wildlife.  As with all untreated water, purify before drinking.
Periodic pollution by wildlife? What the…

The northern end of a south-bound bisonOh, right. Natural bottled-spring-water flavor. Hey, it’s natural, it’s got to be good for you, right?

And to end this post on a complete and totally baffling non-sequitur: the student lounge I’m sitting in right now has a television constantly tuned to some cheesy mass-media channel. Today it’s “E!®”. I overheard something on it just now that made me sit up and take notice: Evidently “Leprechaun” made a profit. Wow.

One never knows what kind of amazing things one might learn at college…

Environmental Chemistry Field Trip – Day 1, part 1

I can think of a number of things to complain about with regards to living where I do. However, it is nice that we live near enough to Yellowstone to day-trip there. In fact, it’s close enough for my local college to take field-trips there – which we did.

Environmental Chemistry spent the weekend there, examining the area, discussing the chemistry of the natural waters and geothermal features, and collecting samples (yes, we had a permit for this…).

We started with a stop by the side of the Madison River to collect a sample of the surface water. Clear, cool (12°C, or about 55°F), mildly basic (pH of about 8.0), and a TDS reading of about 300ppm, which is roughly the same as mildly to moderately hard tapwater, I suppose.

sampling water from the Madison river

The sampling device -seen being hurled over the water here – is kind of interesting – it’s a hollow tube (a bit of plastic pipe) with two spring-loaded balls that slam shut on either end to trap the water inside when you tug on the string. That lets you throw the device out and trigger it when it gets to the precise spot that you want to take a sample from.

We made a brief stop at Beryl Spring afterwards. We didn’t do any sampling here, but we did talk about acid-sulfate water systems. “Reduced” sulfur – as Hydrogen Sulfide gas – comes boiling out from underground along with steam, and ends up being oxidized by oxygen from the air to become sulfate in the end – combining with the water and forming sulfuric acid.

Sulfur-encrusted pipe at Beryl Spring

Of course, it doesn’t go from sulfide to sulfate all at once. There’s a stop along the way as elemental sulfur. The whitish-yellow stuff here is crystals of elemental sulfur. The black stuff you see is…also crystals of elemental sulfur. The difference is just how the atoms of sulfur collect together. The black form is actually a little less stable than the yellow, so it tends to form first, but then slowly convert to the yellow form over time as the sulfur atoms settle into a more stable arrangement. Being a chemistry class, we didn’t really discuss the possible microbial activity that might be involved here. Note the small patch of dark-green there. I suppose this could be a “Green Sulfur Bacteria“, which does something like photosynthesis except that it makes sulfur instead of oxygen in the process. These are normally anaerobic but perhaps the concentration of hydrogen sulfide (H2S) and carbon dioxide gas coming out of the ground right there is enough to crowd out the oxygen. Alternatively, it could just be a heat-loving cyanobacterium or something.

I really wish I wasn’t too poor to buy a good field microscope to go along with the good lab microscope that I am also too poor to buy…

The last two stops of the day – Appolinaris Spring and Narrow Gauge Spring – will be in the next post…

This weekend should be worth at least one decent post…

This weekend, one of the two of this semester’s classes that I have not yet used for a “what I learned in school today” post took a field trip.

Yes, Our “Environmental Chemistry” lab went to Yellowstone National Park and (legally – we had a permit and everything) did some water sampling. We got some on-site lectures about the types of water systems in the park, considerations involved in sampling things, and so on. All in all, I thought it was pretty interesting, but after spending the entire weekend either driving to or from the park or walking around in the park I’m a bit exhaustipated. Plus, bummed out that I can’t afford a good portable field microscope to go with the regular microscope which I also can’t afford. Woe unto me. I imagine the permit we had would have allowed me to also dangle some slides in the water to look at.

I did record a GPS track of both days field-trips, I got ICBM addresses for our sampling sites, and a number of photographs with my cheap and ancient digital camera along the way. Give me some time and I’ll get at least one real post out of it.

Meanwhile, a bit of trivia: “The Microsoft Network” search system is pretty Fupped Duck. I do get the occasional obviously relevant hit from one of their searches, but the great majority seems to be “hits” from random one-word searches, many of which seem to refer to words that appear nowhere on the site (and others of which are so broad I have no idea how many pages some MSN user would have to click through before hitting my site. For example, while I like to think I’m making a reasonable effort to do interesting science blogging, I’m having trouble imagining that this blog would show up in the first few pages for a search consisting solely of the word “science”…which one of the recent hits seemed to show.

Actually, this probably has less to do with users than with Microsoft itself – the hits for this don’t appear to be loading real views (it pulls one page and doesn’t reference, for example, images) though it is coming from “The Microsoft Network” addresses. Perhaps Microsoft has one of their bots masquerading as a real user (the user-agent string looks like regular “Internet Explorer 7″)…even the IP address resolves to a bogus name ” bl2sch1082217.phx.gbl.”, for example) which doesn’t resolve back the other way. Of course, it’s also possible the hit is ENTIRELY bogus and the “referer” tag that seems to indicate this is also faked. Perhaps it’s time to start blocking Microsoft…or maybe just messing with them. This apparent standards abuse and obfuscation of what exactly it is that they’re trying to do with my blog (and messing up my logs!) kind of bugs me. (Moral of the story is probably “Everybody should just use Google“…)

Sure “Cardboard Sarcophagus Instructions” is a pretty weird search, too, coming from Google, but at least I know why THAT one got here. I doubt the searcher – possibly from the Memphis, Tennessee area – was really searching for metaphors for expired JellO boxes.

If I Win It…

One topic that I have hoped to emphasize much more on this blog is amateur science, and in particular (given my educational background) amateur Microbiology.

Don’t be dissuaded by my use of the word “amateur” here. I don’t mean “not really” science (i.e. the microbiological equivalent of the “baking soda volcano”). Rather, here I’m using “amateur” in its proper etymological sense – science done for the love of it. I don’t just mean my brief series of experiments on the toxicology of expired JellO®. I mean actual microbiology with potential practical application as well as educational value. Unfortunately, there are a few bits of equipment for this that I can’t reasonably cobble together out of spare parts or repurposed household appliances. A microscope, for instance. Or a dry-ice maker.

Being a full-time college student, I’m poor, and can’t afford a microscope. A decent ordinary “brightfield” microscope appears to cost about $400. Bonus materials like a “darkfield” condensor are extra, unless I think I can rig up an equivalent on my own. A nicer digital camera to take pictures with to share with you, my loyal reader(s) would add some more to the cost. Even in the case of equipment and supplies improvised from more ordinary and readily-available materials (pressure-cooker=”autoclave”), there is still a cost. Woe unto me, what shall I do?!?!?

For the moment, I shall revert to the time-honored traditions of “begging” and “hoping”…

You see, there appears to be a scholarship available for bloggers who are full-time college students. Why, what a coincidence! I blog…and I’m a full-time college student! What luck!

There appears to be a US$10,000 (that’s almost 10000 CANADIAN dollars!). It’s not explicitly stated but last year they also had $1,000 “runner-up” awards as well. Here, then, is my pledge to you all.

Should I be selected as a finalist for this scholarship competition, I will eat 2-year-old JellO! Furthermore, if I were to actually be selected to win a $1000 scholarship, I will buy a real microscope and be able to blog my microbiology experiments and studies much more vividly. I will also blog the design and construction of my own amateur microbiology lab, to the extent that I can afford. (Well, I was ALSO going to do this anyway, but with a scholarship I’d actually be able to start doing it…)

Were I to be selected to win the full $10,000 scholarship I propose to go absolutely Nucking Futs, with a microscope, a nice new digital camera, dry-ice maker and plenty of CO2, perhaps some dedicated hosting for this blog, and a complete collection of useful microbiology equipment (mostly improvised still, but that’s half of the education right there…). Furthermore, should my readers demand it, I might even be persuaded to drink a cup of fresh Lysogeny Broth!

Come on, who needs this money and attention more – me, or some wealthy (compared to me) graduate student over on scienceblogs.com? I bet none of them would eat 2-year-old JellO or drink E.coli Chow for it, would they?

10 Finalists are to be announced October 7th, from what I understand…wish me [good] luck…
UPDATE: I made the finals, though my fame doesn’t seem to be carrying along a rose-petal-strewn path to victory yet…

Why I blog, and the Office of Technology Assessment

Via a post over on the Aetiology blog (and Retrospectacle) I happened upon a survey being taking about science blogging. It got me thinking a bit about why I’m doing this – aside from the masses of screaming groupies I have.

Aside from just being fun (I like to write), I set up this particular blog as a platform to practice communicating scientific topics. It’s a skill that really isn’t emphasized much in science education as far as I can tell, and regardless of where my career may go post-graduation I’m sure the ability to articulate scientific and technical topics will be beneficial to me.

In fact, I can see two different ways I could go with a career either during or after graduate school. Obviously, I could end up employed in a capacity where I’m officially “doing” science, which could be anything from “brewmeister” to curating a culture collection to academic research to being a lab grunt. I could also see myself pursuing a policy or science communication angle as well, though. This could be anything from Public Relations for a scientific or technical company to science writing to scientific advising…which brings me to the Office of Technology Assessment.

A post over on the “Denialism Blog” at Scienceblogs.com started a stream of “Bring Back the Office of Technology Assessment” posts around the net. Now, there’s a dream job. I would personally love to have a job like that. Make an enjoyable and comfortable living from whatever talent I have at explaining scientific and technical topics, and directly and substantially benefit my country in the process? Sign me up! Of course, even when the OTA existed, it only had a small number of employees, and presumably they were all Ph.D.’s with backgrounds in science and public policy, so the odds of me getting hired there (specifically) would probably be comparatively slim. Still, I can dream, and perhaps if we luck out and my wife (a Ph.D. Geologist with a background in borehole geophysics, petroleum geology, nuclear technology, and a variety of other areas – anybody out on the East coast in the general vicinity of Washington D.C. need anybody like that?…) and I have the opportunity to move somewhere with a good “science and public policy” graduate program I may have a chance.

My personal desires aside, though, if there’s one thing the people who are supposed to be running the country seem to really need, it’s rational science and technology information. Since the disbanding of the OTA we’ve had the DMCA and the costly and predictable abuses it brought (such as DMCA lawsuits over printer ink refills and replacement garage door openers), minimally-rational ideological fights over things like stem cell research and global climate change, panic and “security theater” over technically improbable-to-impossible “terrorist” threats (like the possibility that a terrorist will blow up a plane with a “liquid bomb” made of 4 ounces of baby food and shampoo, or “blow up” the fuel depot at JFK airport) (Mayor Bloomberg’s “STFU and GBTW” style of response to the panic was a glimmer of hope to me that there was some rationality left among my fellow human beings). I will refrain from picking on Ted “Series of Tubes” Stevens other than bringing this up as another example of lack of good information for policy-setting congresspeople. All this disruptive fuss, largely over ignorance and misunderstanding, which seems to be what the Office of Technology Assessment was intended to address. I would definitely agree that the OTA or something like it appears to be an urgent need – either that or Congress should quit playing around and just formally declare a science-boosting ‘War on Science’.

There are one or two things I’d like to figure out before I start mailing letters to congresspeople and presidential candidates though. For one thing – what would be the difference between the Congressional Research Service’s Resources, Science, and Industry division? Would one group be more focussed on specific policy implications while the other deals with “just the facts”? Also, the one legitimate-sounding complaint that I’ve seen in some of the newspaper articles on the subject is that it would often take longer to come out with a report on a subject than congress had (that is, congress would end up having to assemble a law and vote on it before the reports were completed). Should whatever takes the place of the OTA be re-designed to focus more on getting quicker answers? Like, maybe, hiring a bunch more people? Including, say, eager and capable grad-students…Okay, I’ll stop begging…

More to follow on this and related topics. Oh, and advice on successfully pursuing this type of career would be welcome.

What I Learned In School Today: Mortals have Limits, and Socrates was a jerk…

…Or at least, Plato’s accounts of him make him seem that way.

We’ve been reading (as English translations) accounts of Socrates’ trial and related occurrences as written by Plato. It started off with a possibly fictional dialog before Socrates’ trial, between Socrates and some guy named Euthyphro, who is some kind of priest.

In short, they get to talking about why they are there at the court, and it turns out Euthyphro is there to accuse his own father, who has apparently committed what we in the modern U.S. would call “manslaughter”. Euthyphro’s father had caught a murderer, then tied him up and thrown him in a ditch while he sent somebody to ask the authorities what to do with the murderer. While waiting for the messenger to return with the answer, the murderer in the ditch had evidently died. Euthyphro says “piety” demands that his father be tried for the death of the murderer but complains that everybody seems to think that hauling his own father to court for this is “impious”. And now you have the backstory for a long, involved, and ultimately unsettled discussion of just what the heck “piety” is supposed to be.

That’s where you notice that Socrates is a sarcastic butthead who thinks he’s on a mission from The Gods™ to prove that everybody is an ignorant fool. He puts on a snide show, pretending that he expects Euthyphro will reveal The Secret Of What Piety Really Is (Socrates claims that the knowledge will be useful for his own defense at his trial later) all the while busily demonstrating that Euthyphro really can’t answer the question.

The argument dances around various definitions. They more or less settle on the notion that “piety” is something that pleases all of the Gods (and impiety, by definition, displeases all of the gods), and that The Gods™ love an action because it is pious rather than something being pious because it is loved by The Gods™. (There’s a bit of virtually Dickensian pedantry there involving whether something is “being carried” because someone is carrying it or whether people carry things because they are “being carried” objects.) I guess that means nobody would have to worry that they’d wake up one morning and discover that The Gods had decided on a whim that “piety” would mean raping puppies and eating babies for the next few days. They manage to reach agreement that “piety” has something to do with being like a servant to the gods, but are completely unable to come up with a definitive test by which they could define any particular act as “pious” or “impious”.

Euthyphro finally says (more or less) that hey, he’d love to stay and go around and around and around and around with this annoying little brain-teaser but he’s got places to be and things to do, and the dialog ends with one last bit of Socratic Sarcasm as Socrates wails about how he was hoping to show up at his trial and tell everyone he’d learned the divine secret of Piety from Euthyphro and therefore wouldn’t be accidentally corrupting the youth with his lack of wisdom any more…

I have to wonder if Plato wrote this dialog so that readers would understand why so many Athenians wanted to get rid of him…

Of course, as an (self-proclaimed) Applied Empirical Naturalist, I think they’re whole problem is that the knowledge they were seeking was defined as something that would only necessarily exist in the minds of Supernatural entities – since “piety” and “impiety” are entirely defined in terms of what The Gods thought, it’s not clear that “piety” can be known outside of the Invisible Giant People Up On Mount Olympus. Of course for Euthyphro, being a professional priest, admitting that he doesn’t – and maybe can’t – know what “piety” actually is and that he really has no clue what’s going on in the minds of The Gods would be a definite Career-Limiting Move, and Socrates doesn’t seem (to me) to actually care what it means so long as he gets to prove that Euthyphro doesn’t know either, so it’s no wonder that this point doesn’t come up.

I wasn’t kidding about the “thinks he’s on a mission from The Gods™ to prove that everybody is an ignorant fool” comment, either. In his “Apologia” (defense speech during his trial), as reported by Plato, Socrates describes how someone once went to the Oracle at Delphi and asked if anyone was wiser than Socrates, and was told that, no, nobody was wiser than Socrates. Socrates says he interprets this to mean that nobody is really wise and that this answer from the Oracle (who is just passing on messages from the Gods, after all) means that he has a sacred duty to go around demonstrating this fact – which is the basis of the famous “Nobody knows anything, but I know I don’t know anything, so I know more than anybody else” flippant description of this argument.

Oh, and one unrelated odd fact – the introduction to the translation says that Socrates is about 70 years old during this trial, but at one point during Socrates’ rambling defense speech he explains that he wouldn’t want to be like other people who show up in court and have their kids plead with the jury for mercy in order to avoid punishment. Socrates says he had three kids – one adolescent and two who are “children”. So, wait, he’s wandering around Athens unmarried, when suddenly he runs into some woman willing to shack up with a penniless, irritating old guy who’s almost sixty and they have three kids who survive childhood? What?

This isn’t discussed at all, really, it just struck me as a really odd circumstance…

What I Learned In School: “Valid” arguments

The new semester has begun on this, my last schedules semester as a mere old Undergraduate. This semester’s primary purpose is to fill in the two vitally important “general education” goals for my current Institute of higher learning: Art Appreciation and Philosophy.

I added a “What I Learned in School Today” category to the blog just because of this semester. My loyal readers (all 2-4 of you…) can look forward to occasional posts on other aspects of my Higher Education as the semester goes along, besides microbiology. On the metaphorical menu over the next 16 weeks: “Introduction to Philosophy” (today’s topic), “History of Western Art“, Applied Calculus, and finally I have a chance to take Environmental Chemistry.

Prior to reading some Plato for next week, we started out “Philosophy 101” with a discussion of “Valid” arguments. In Philosophy, this has a very specific meaning. If you make an argument in the general form of “This, and that, therefore something”, the argument is “valid” when if “This” and “that” are both true, then “something” must also be true.

The thing that most of the class seemed to have trouble with is that being “valid” has nothing to do with whether or not the argument is “sound“, or whether the statements in the argument are true.

An example from the class:

All mammals have lungs.
Whales have lungs.
(Therefore) all whales are mammals.

This is an invalid argument, despite the fact that every statement is actually true. The reason is simply that the fact that whales are mammals does not automatically follow from the fact that they have lungs. (Chickens have lungs, too. Does this mean chickens are mammals?…)

It took two class sessions before most of the class seemed to “get” this. I felt as though I was in Junior High again…though I think this had more to do with watching the freshman girls in front of me passing notes during the class. Come on, kids, grow up! We adults are using IM for that now! Sheesh. Kids today…

On the other hand:

You’ve got to be some kind of genius to attend college and blog at the same time.
I attend college and I blog at the same time.
I am, therefore, a genius.

is a valid argument. As written, if both of the first two statements are true, then the third statement must be true. This is where the value of valid arguments come in – if it turns out that the conclusion is false, then one of the premises must also be false. If anyone were to discover that I am, in fact, not a genius, then either it’s unnecessary to be a genius to blog and go to college at the same time, or perhaps I’m paying someone else to write this stuff for me.

Who cares, I’m a science major, not a philosophy major, right? Except: a properly designed scientific hypothesis should be a premise in a “valid argument”, and an experiment is merely a test to see if the argument is unsound. For example:

All lactic acid bacteria, grown in otherwise sterile milk, will make yogurt.(the underlying hypothesis being tested)
I inoculate sterile milk with a culture of Pediococcus damnosus(the test performed by the experiment)
(Therefore) I obtain yogurt. (Expected results and conclusion of the experiment)

This is (as far as I can tell) a completely valid argument. Now, I haven’t actually done this experiment, but let’s pretend I did, and the end result was a smelly mass that kind of looked like yogurt except it turned out to be slimy rather than firm. I cannot in fairness call it “yogurt”, so my conclusion in the argument is false. Thanks to the magic of Valid Arguments™, I know that either my assumption is wrong (maybe not all lactic acid bacteria turn sterile milk into yogurt after all), or there was a problem with the experiment (perhaps the milk was contaminated with something and wasn’t really sterile, or I grabbed a culture of something other than P.damnosus by mistake.)

Assuming I carefully recheck the materials and repeat the experiment to confirm that I really am inoculating actually-sterile milk with a definitely clean culture of P.damnosus and continue to get the same results, then my hypothesis – the first premise in the argument – must be false. I have to then go back and revise my hypothesis and test again, until I have a hypothesis that seems to consistently generate true conclusions. Thus, the “valid argument” is the basic tool which allows hypotheses to grow up and become theories.

Incidentally, some Pediococcus damnosus strains are a cause of “ropy” wine, which is why I chose that example. I don’t actually know what, if anything, it would do to pure, sterilized milk, though.

Coming up next: I picked up a 100-year-old microbiology book while on vacation!

The Gram Stain Post to End All Gram Stain Posts

Gram stain, Gram stain, Gram stain! Bah. I think it’s time Microbiology grew up and moved out of Medicine’s basement.

Sure, the Gram stain[1] has its uses, but the procedure is grossly over-hyped. “[…]the most important stain in microbiology[…]”[2]! “[…]it is almost essential in identifying an unknown bacterium to know first whether it is Gram-positive or Gram-negative.”![3] “The Gram Stain reaction is an especially useful differentiating characteristic.[…]The Gram reaction turns out to be a property of fundamental importance for classifying bacteria phylogenetically as well as taxonomically.”![4] “[…]differentiates bacteria into two fundamental varieties of cells.”![5] “The Key to Microbiology“![6] [emphasis added…]

Bah! Sure, the Gram stain has its uses, but the hype it gets (even 125 years after its invention) is ridiculous. It’s worse than Harry Potter!

You really want to know what the Gram reaction tells you? Really? Okay, here it is:

A “Gram Positive” reaction tells you that your cells have relatively thick and intact cell walls

A “Gram Negative” reaction tells you that they don’t.

That’s it. That’s about all you can reliably infer from the Gram stain.

Previously, I put up a post describing what was my understanding of the conventional view of why the Gram stain works. Today, I’ll give you a much more detailed – and more correct – explanation of why it works as well as what its real significance is to identification of microbes. But first, a brief one-paragraph rant on why I think the Gram stain has such a hold on microbiology teaching.

I blame the fact that microbiology education is still largely in the shadow of medical technology education. When you artificially exclude the 99+% of organisms that aren’t associated with human diseases, the tiny number left do, indeed, seem to largely separate into two phylogenetic categories. Judging by what I’ve encountered thus far, it seems you get a lot of Proteobacteria (especially ?-Proteobacteria, like E.coli), which are “Gram-negative”. You also get a lot of Firmicutes (Bacillus, Streptococcus, Staphylococcus, etc.), and a couple of scattered Actinobacteria (Mycobacterium, for tuberculosis and leprosy, Corynebacterium for diptheria…). Both of these are considered “Gram-positive” (although if you use the standard procedure these days, the Mycobacteria may show no reaction at all). That’s, what, 3 phyla out of about 25 eubacterial and archael phyla? If we throw in Syphilis and Chlamydia, that’s still only 20% or so of the currently recognized prokaryotic phyla. If your microbiology classes assume everybody is training to be a medical technologist or clinical microbiologist, then the Gram stain becomes inflated in importance.

Enough of that – here’s a quick review of how the Gram stain works. Solutions of “Crystal Violet” (a purple dye) and Iodine are applied to cells fixed to a slide, where they soak in and precipitate in the cells. A “decolorizer” (usually ethanol) is applied to see if it will wash this dye precipitate out of the cells. A different, lighter-colored dye (such as safranin) is added so that the cells which DO have their dye washed out can be seen as well. In the end, “Gram positive” cells are a dark purple from the crystal violet/iodine that was not washed away, and “Gram negative” cells are not dark purple. (Usually they are pink, from the safranin, assuming that’s the dye used as the counterstain.)

Note that this does not differentiate cells into “two fundamental types” as is often claimed. You actually get four types: Groups of cells that are normally always “Gram positive”, Groups of cells that are normally always “Gram negative”, Groups of cells that are normally sometimes “Gram positive” and sometimes “Gram negative” (“Indeterminate”, or as I like to call it, “Gram-biguous”), and groups of cells that are normally NEITHER Gram-positive nor Gram-negative, like Mycoplasma, which aren’t dyed at all by the process. Incidentally, phylogenetically speaking, Mycoplasma is one of the “Gram positive” Firmicutes, just like Bacillus and Staphylococcus.

It’s kind of interesting to me that the Gram stain reaction has been such a mystery up until a century after its invention. What is it that makes “Gram positive” cells retain the dye while “Gram negative” ones don’t? Along the way, it seems like nearly every part of the bacterial cell was hypothesized to be the reason for the Gram reaction – lipids, carbohydrates, nucleic acids, “Magnesium ribonucleates”, and so forth. Davies et al, 1983, includes a table listing many of these and referencing historical papers making the claims. The fact that the reaction had something to do with the cell wall seems to go back quite a while, though the “Magnesium ribonucleates” idea doesn’t seem to have been entirely abandoned until the mid-1960’s[7]. It was also hypothesized that the “Gram positive” cells simply absorb more dye and therefore take longer to “decolorize”.

It turns out that “Gram-positive” cells actually don’t, necessarily, take up more dye than Gram negative ones. This was tested by taking a set concentration of bacterial cells and adding them to a set concentration of dye. After letting them soak, the samples were centrifuged to remove the bacteria, and the amount of dye found to be missing from the liquid was taken as the amount absorbed by the cells. They found that some Gram negative cells actually took up more dye than the Gram positives did. So much for that idea.[8]

Even relatively recently, I’ve seen it written that the bacterial cell wall, specifically, is what holds onto the stain, but even that turns out not to be true. Although the cell wall is the structure that seems to be responsible for the Gram reaction, in the late 1950’s it was demonstrated that it was not actually the staining of the cell wall that caused the reaction, but rather the ability of the cell wall to keep the decolorizer out of the cell.[9]

Apparently, the Crystal Violet/Iodine complex itself doesn’t even play a vital role. The complex apparently dissolves again more or less instantly as soon as the decolorizer touches it[10], and it’s even possible to differentiate “Gram positive” and “Gram negative” with simple stains like methylene blue or malachite green, if you’re clever about it[11]. The latter authors set up a clever test with crushed cell material, dye, and paper chromatography. They had the decolorizer soak into the paper, past a spot where dye-soaked cell material from Gram-positive and Gram-negative cells was placed, and watched for obvious differences in the amount of time it took the dye to be carried out by the decolorizer. Incidentally, my quick examination of this paper makes it look like cheaper 100% isopropyl alcohol (“rubbing alcohol”) might be slightly better than the standard 95% ethanol for Gram stains.


So, here we are at 1970 or so, and we already know that the Gram reaction is entirely based on how well the cell wall structure prevents organic solvents (like ethanol) from soaking into the cell to dissolve the dye complex. Yes, the mystery of why the Gram stain works in normal cells was largely solved by the Nixon era.
A few corners of the mystery remained, though. Why do “old” cultures of “Gram positive” cells often end up staining “Gram negative”, for example? Why do some kinds of cells seem to be sometimes Gram positive and sometimes Gram negative in the same culture? What, exactly, is really happening to the cell, deep down, during the staining process?

In 1983, the Gram Stain made the great technological leap into the 1930’s, when a variation of the technique was devised which allowed the Gram Stain to be observed by electron microscopy[12]. Using a funky platinum compound in place of iodine, the electron microscope reveals exactly where the dye complex is at any particular stage of the Gram stain process. Using this technique, it was possible to see how the decolorizer disrupts the outer membrane of classically-Gram-negative organisms and to see that the decolorizer potentially damages the cell wall and interior membrane, possibly allowing cell material to leak out (or decolorizer to get in and dissolve the dye complex). It was also seen that the dye complex permeates the entire cell, not just the cell wall.[13]

If you’ve been wondering about the sometimes-Gram-positive-sometimes-Gram-negative cells, the same technique was also used to investigate this. As suspected, it turns out that the “old cultures become Gram negative” problem is due to the cell walls breaking down as the culture ages. Bacteria are continuously, simultaneously, building up and tearing down their cell walls, in order to be able to grow and divide. As nutrients run out, the bacteria run out of material to rebuild cell walls, while the cell-wall degrading enzymes keep on chugging. Breaks in the cell wall occur, and through these breaks the decolorizer can get in and rapidly dissolve the dye. Actinobacteria can have a similar problem, but rather than only being in “old” cultures, apparently weaknesses appear briefly during cell division, and if a particular cell happens to be at this stage of growth when you stick it on a slide, heat-fix, and Gram stain it, the weakness at the septum where the division is occuring can crack and allow the decolorizer in, resulting in a “Gram negative” response even while surrounding cells of the same kind might still be “Gram positive”.[14]

This brings us to archaea and some eukaryotes (i.e. yeasts). Yeasts stain “Gram positive” normally. Although their cell walls are completely different chemically than bacterial cell walls, they are quite thick (microbially speaking). Poor, neglected Archaea seem to be all over the place in terms of Gram reaction. Since their Gram reaction doesn’t tend to correlate to any particular phylogenetic grouping[15], it seems nobody really pays much attention to their Gram stain reaction. On the other hand, and on the subject of “Gram-biguity”, I thought the investigation of Methanospirillum hungatei[16] was interesting. M.hungatei is an archaen that grows in chains. When Gram-stained, the cells on the ends of the chains are “Gram positive”, while the others have no Gram reaction at all. It turns out that the chains are covered by a sheath, and the only contact with the outside world is through thick “plugs” in the cells at the ends of the chains. These “plugs” act like thick cell walls, allowing the Gram stain dye material to soak in but excluding the decolorizer, while the sheath keeps the rest of the cells from soaking up any stain at all.

There you have it – a relatively detailed history and explanation for the Gram stain, and you didn’t even have to get through some obnoxious paywall to read it. Aren’t you lucky?

Comments, suggestions, and corrections, as always, are welcome.

[1] Gram, HC.”Ueber die isolirte Faerbung der Schizomyceten in Schnitt-und Trockenpraeparaten.” Fortschitte der Medicin. 1884 Vol. 2, pp 185-189.

[2] Popescu A, Doyle RJ. “The Gram stain after more than a century.” Biotech Histochem. 1996 May;71(3):145-51.

[3] Brock TD, Madigan MT, Martinko JM, Parker J. “Biology of Microorganisms (7th Edition).” 1994. Prentice Hall, Englewood Cliffs, NJ pg. 46

[4] ibid, pg. 715

[5] Beveridge TJ.”Use of the gram stain in microbiology.” Biotech Histochem. 2001 May;76(3):111-8.

[6] McClelland, Rosemary. “Gram’s stain: The key to microbiology – isolate identification method – Tutorial” Retrieved 20070810 from http://findarticles.com/p/articles/mi_m3230/is_4_33/ai_74268506/print

[7] Normore WM, Umbreit WW.”Ribonucleates and the Gram stain.” J Bacteriol. 1965 Nov;90(5):1500.


[9] BARTHOLOMEW JW, FINKELSTEIN H.”Relationship of cell wall staining to gram differentiation.” J Bacteriol. 1958 Jan;75(1):77-84.


[11] Bartholomew JW, Cromwell T, Gan R.”Analysis of the Mechanism of Gram Differentiation by Use of a Filter-Paper
Chromatographic Technique.” J Bacteriol. 1965 Sep;90(3):766-77.

[12] Davies JA, Anderson GK, Beveridge TJ, Clark HC.”Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain.” J Bacteriol. 1983 Nov;156(2):837-45.

[13] Beveridge TJ, Davies JA.”Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain.” J Bacteriol. 1983 Nov;156(2):846-58.

[14] Beveridge TJ. “Mechanism of Gram Variability in Select Bacteria.” J Bacteriol. 1990 Mar;172(3):1609-20.

[15] Beveridge TJ, Schultze-Lam S. “The response of selected members of the archaea to the gram stain.” Microbiology. 1996 Oct;142 ( Pt 10):2887-95. (Abstract)

[16] Beveridge TJ, Sprott GD, Whippey P. “Ultrastructure, inferred porosity, and gram-staining character of Methanospirillum hungatei filament termini describe a unique cell permeability for this archaeobacterium.” J Bacteriol. 1991 Jan;173(1):130-40.

A Government “War on Science” is GREAT for this country!

They say that politics and controversial statements are ways to encourage traffic on a blog, so here’s some. Comments welcome, of course.

I have cause to celebrate the future potential for science in the U.S. Here’s a bit of simple history (Update – added the “War on Poverty” to the list 20070810):

1964: Lyndon Baines Johnson declares a “War on Poverty” Today: the gap between the Rich and the Poor in the US is widening and economic mobility is stagnant.

1971: President Nixon declares a “War on Drugs”. Today: “Drugs” are widely used, even among kids, who appear to be losing their fear of drugs. Market innovations (blatantly illegal and of questionable morality, but innovations nonetheless) such as crack cocaine, MDMA (“ecstasy”), and “ice” (crystal meth) seem to be in the news a lot. People growing illegal plants in their closets and basements or brewing up complex chemical stimulants in the backs of minivans seems to be an almost daily topic of the news.

2001: President George W. Bush declares a “War on Terror”. Today: A majority of Americans feel that there is a greater threat of terrorism than before, which seems to be true, at least as far as “Jihadist” terrorists go, if the declassified portions of the government report paint an accurate picture of the situation. Heck, when the president invaded Iraq in 2003, major terrorist organizations didn’t even seem to be there. And now, it seems like EVERYONE we’re fighting in Iraq is Al Qaeda, and we’re treated to frequent vague but earnest-sounding warnings of impending terroristic doom.

Given these historical precedents, if there really is a government-run War on Science, then we’re in for a huge increase in scientific activity here.

I’m picturing a virtual underground Scientific Renaissance, where, like much of the late 1700’s and 1800’s, “citizen science” becomes a fashionable pursuit. People secretly building science labs in their basements and attics and performing legitimate, useful scientific research in them. Kids hanging out in abandoned parking lots at night, doing complex calculus problems in chalk on the ground and experimenting with broadcast power. Anonymous rebel scientists developing methods to cheaply and effectively convert lawn clippings into fuel ethanol and plastic grocery bags and soda bottles into biodiesel. Ignorant politicians assume home biology labs are marijuana-growing operations, that home chemistry labs are making methamphetamines, and that home physics labs are building radioactive “dirty bombs”. A multibillion-dollar new agency, the Science Enforcement Agency is hastily assembled and laws are badly written to restrict scientific activity to carefully-regulated government-controlled settings only.

Public science devolves into (when Republicans are in control) attempts to “debunk” global warming and evolution, “cure” homosexuality, develop ridiculously expensive military-grade weaponry, and silly projects that just plain won’t work but happen to be run by buddies of a senator or (when Democrats are in control) multimillion dollar projects to study “self-esteem”, research on “psychic powers”, development of homeopathic “medicine”, and silly projects that just plain won’t work but happen to be run by buddies of a senator. Disgusted underground scientists are only egged on by this state of affairs.

Within a few years, a cautious exchange of money in a public restroom will buy disease-curing doses of novel, effective, but non-FDA-approved antibiotics that cure drug-resistant Staphylococcus aureus or Tuberculosis. A backyard moonshiner-like biotech lab somewhere in the rural west secretly sets aside part of their flock of chickens, genetically engineering them to produce HIV vaccines with billions of dollars in “street” value. Someone with a closet chemistry lab develops an illicit catalyst that facilitates hydrolysis of water to produce hydrogen with no more energy input than ordinary body heat, while another develops an illegal strain of cyanobacteria that turns atmospheric carbon dioxide into a plastic substance which can either be used for building or is easily converted to biodiesel at such a rate that the developer has to rapidly build a huge, secret underground complex to hide the vast quantities of material produced overnight….

In the end, as always, government goes utterly insane and bankrupts themselves (more, I mean) trying to stamp out Illegal Science, but in the meantime, anyone who’s scientifically inclined ends up making a fortune. On the other hand, the efforts drive a lot of the science out of the country and Mexico becomes the new world superpower with their fleet of antigravity flying armored space cars, zap death ray guns, and clusters of quantum-supercomputers. (Note to self: get back to learning to speak Spanish!). This doesn’t really slow the flow of science into the US, though, and “science tourists” can sneak to Mexico to undergo age-reversing and/or intelligence-boosting medical treatments or to obtain cures for cancer or obesity that actually work. People end up in jail for recovering from leukemia or losing weight.

Meanwhile, on a more personal note, people like me who actually think doing science is fun get a few publications in underground science-journal ‘zines, spend a few years developing something useful, make a huge pile of money, and then retire before The Man catches up to us, to live a life of luxury somewhere. Maybe living in a giant mansion in Mexico between stints as lab techs for Mexican scientists once in a while, done just for fun and extra pocket-money…

It’ll be glorious. So – write your legislators today, and tell them we NEED the “War on Science”. For the Children.

(My political opinion? Lets just say that my political fantasy right now is that the 2008 presidential race will come down to a run-off between a Bloomberg/Paul ticket and a Gravel/Kucinich ticket….)

There, is THAT enough controversy to get some new traffic here?…

Officially “Gram-positive” – the Firmicutes

In a typical student microbiology lab, it seems whenever you get your hands on some bacteria the first thing you do is check to see if it’s “Gram-positive” or “Gram-negative”. But does this old Victorian-era test still mean anything useful in the context of modern bacterial taxonomy?

It would seem that it actually does. In my admittedly limited experience, an un-ambiguous Gram-positive result using the standard procedure nearly always indicates bacteria in the phylum firmicutes, sometimes also referred to as the “low G+C gram positives”.

The “low G+C” part has to do with the chemical characteristics of the DNA. If you’re already familiar with DNA’s structure then you probably already know what this means, but for everyone else, in brief:
DNA is made of strings of four different chemical “bases” chained together. The bases are “Adenine”, “Guanine”, “Cytosine”, and “Thymine” (abbreviated as A, G, C, and T). These bases make up a sort of chemical “alphabet”, which encode how to make various proteins. These chemical bases each have an opposite base that they are attracted to – Adenine to Thymine, Cytosine to Guanine, so DNA’s strings end up matched with an “opposite” string, which together form the easily recognized “double helix” shape of the overall DNA molecule. The relevance here is that the attraction between the Guanine and Cytosine (“G+C”) is stronger than the attraction between Adenine and Thymine, so you can estimate relatively how much Guanine and Cytosine is in an organism’s DNA by how tightly the two strands of DNA stick together.

And, yes, there is a “high G+C” gram-positive group, with a larger proportion of the G and C bases as compared to the A and T. That’s the “Actinobacteria”, which includes the “acid-fast” Mycobacterium group. But that’s a topic for another post.

The firmicutes, with the exception of one group that I know of, all have a distinctive, simple outer structure. It’s something like this:

Imagine a water balloon filled with lime Jell-O®. Now, tightly wrap the water balloon with a piece of thick, padded packing blanket. The thick layer of packing blanket is the cell wall. The balloon is the inner cell membrane. The Jell-O® is the cytoplasm. (It’s Lime merely because I like lime Jell-O®.) It might be worth noting that since this group of bacteria relies so much on it’s cell wall, they are more likely to be killed off by the ?-lactam antibiotics (which specifically attack bacterial cell wall generation) than other types of bacteria.

There is one exception to this structure – the class of firmicutes known as the mollicutes. There’s one example of this group that gets mentioned in basic medical-centric microbiology classes: the genus Mycoplasma (as in Mycoplasma pneumoniae). Members of this class have lost their ability to make cell walls entirely.

By contrast, a typical “Gram-negative” cell has a more complicated outer makeup. In brief, start with the same lime Jell-O® balloon, but instead of a thick packing blanket, wrap it with a single thin layer of cloth, and then stuff the whole thing inside of another lime Jell-O® balloon. You can also imagine a bunch of valves stuck through the outer balloon to let stuff in and out, but then you end up imagining the Jell-O® spurting out all over the place and the whole analogy breaks down. If it helps, you can also imagine that if you punched out a section of a gram-negative outer structure with a cookie-cutter, you’d get something kind of like an inverted Oreo® cookie, with easily-dissolved filling on either side of a thin layer of cookie. Anyway, the outer balloon represents the outer cell membrane, the cloth is the (much smaller) cell wall, and the layer of lime Jell-O® between the outer balloon and the cell wall is called the “periplasmic space”.

Interestingly, despite this difference in complexity, the molecular evidence[1,2] seems to indicate that the simpler firmicutes diverged evolutionarily from the more complex “gram-negative” types, not the other way around. The lineage suggested by the 16s gene sequences implies that both types of Gram-positives split off from the Gram-negative type bacteria as a single ancestral group, and some time later the two types diverged into separate groups. If I had to bet, I’d personally put my money on the evolutionary sequence going Gram-negative-type->actinobacteria->firmicutes.

Another characteristic of some (but not all) of the firmicutes is the production of “endospores”. Unlike the spores of molds or Myxobacteria, endospores aren’t reproductive. Instead, they’re a like a lifeboat, or perhaps a metaphorical bomb-shelter in the cells’ also-metaphorical basement. If environmental conditions get unpleasant, the bacteria essentially pull their DNA and a few necessary enzymes into a small, thick, multilayered compartment – the endospore – where they can wait, protected and dormant, until conditions become comfortable again.

The special “Endospore stain[3]” uses a dye called Malachite Green and works somewhat similarly to the Gram and “Acid-fast” stains – the extra-thick spore coats retain the green stain (once it’s been driven in by some extra heat) while the decolorization rinse washes it out of everything else. I still need to try using a microwave oven for the heating step…but that, too, is a topic for another post.

If your microbiology class was typical, when you think of the firmicutes, you may think of little more than “Strep throat”, “Anthrax”, and “MRSA“. If so, though, you’re missing out on the useful ones.

Since Gluttony is my second most favorite “Deadly Sin™”, I tend to think of food-related possibilities here. And, no, I don’t mean Botulism.

Yogurt and Kumiss and Kefir, Sauerkraut and Kimchi, Sourdough bread, Salami, and Belgian Lambic ales all involve (at least in part) growth of various lactic acid producing firmicutes. Mostly members of the Lactobacillus genus, though if you take a look at the labels of some of the “Live and Active Cultures” yogurt you may also spot a close relative of the ‘Strep throat’ bacterium in the list. These kinds of bacteria can be happy members of the “Normal Flora” of the healthy human gut.

Another, more obscure example is “Natto”. A strain of Bacillus subtilis, originally derived from rice straw, is allowed to ferment soybeans. The end result is a pungent mass of beans, covered with gooey slime, and having an odor vaguely resembling old cheese. I’ve actually eaten it, and they aren’t nearly as bad as this makes them sound…though I personally still haven’t acquired a taste for them. You may have seen this stuff if you watch Iron Chef – it was used as the Secret Ingredient in one episode. There does appear to be some real potential for it as a “health food”, though, which you can see if you poke around Google or PubMed searching for “Natto”. Maybe next time you end up in the basic Microbiology lab and you’re given some B.subtilis to look at you can whip out a jar of soybeans to inoculate while you’re at it. (Note that I wouldn’t actually recommend eating the results in this case…)

Comments and corrections, as always, are welcome.

P.S. no affiliation with any of the trademarks mentioned should be inferred here – I just figured the trademarked names would be more recognized than terms like “gelatin” or “sandwich-cookie”…

[1]Sheridan PP, Freeman KH, Brenchley JE: “Estimated minimal divergence times of the major bacterial and archaeal phyla.” Geomicrobiol J 2003, 20:1-14.
[2]Battistuzzi1 FU, Feijao A, Hedges SB: “A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land.” BMC Evolutionary Biology 2004, 4:44
[3]Schaeffer AB, Fulton MD: “A simplified method for staining endospores.” Science 1933 77:194