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.

Expired JellO®! Flee! FLEE FOR YOUR LIVES!!!!

Expired JellO®! Deadly Poison, or Merely Debilitating? Can a human being withstand the toxic load of an *entire box* of it? Would he suffer embarassingly loud and messy gastrointestinal distress, or would immediate organ failure set in before this could take place? STAY TUNED TO FIND OUT!…

Yes, loyal readers, as I type this I have subjected my own body to unthinkable risks to answer these very questions. That, dear readers, is how much I care about your health and welfare. You can thank me later…

…If I survive!

What does it mean to be an “Applied Empirical Naturalist”, anyway? As a naturalist, I look for natural explanations for natural observations. If I survive this ordeal, I will not explain it as being due to protection by supernatural forces, and conversely if I end up confined to an intensive care unit, my body ravaged by Expired-Gelatin-Syndrome, I will not seek to explain it as divine punishment for violating Kosher. As an Empirical naturalist, I investigate things by actual observation and direct testing wherever possible, rather than purely philosophical means. And – particularly important to me – Applied Empirical Naturalism is intended to convey that I am primarily interested in investigations with practical uses. Discovering the “Pineapple-Upside-Down Quark” with an umpty-brazillion-dollar particle accelerator and six months of supercomputer time to crunch the data wouldn’t do me, personally, much good. Knowing whether expired JellO® is safe to eat or not, however, has obvious practical application. Especially considering that I seem to have about 5 more boxes of the stuff in the pantry.

So, here I sit, perhaps writing my very last words ever before Expired-Gelatin-Shock causes my brains to swell up and explode messily and fatally from my ears like the popping of two superintelligent zits, in the service of Science. Here, then, is my story.

I begin by building my dire experiment around the following excessively-formal Valid Argument:

Upon expiration, JellO® becomes a deadly poison which causes great harm to those who dare ingest it
I prepare and consume an entire box of expired JellO®
Therefore, I suffer great harm due to its ingestion.

Last night, I plucked from the depths of my pantry an expired-2½-years-ago box of sugarless orange-flavored gelatin with which to begin this investigation. I blew the layer of dust off of the box, and carefully opened it, half-expecting to find some strange mutant gelatin-beast had developed in it over the years since expiration. One hand poised to protect myself should the creature leap from the box to eat my face in anger of being disturbed, I was both relieved and slightly disappointed to find nothing more than a foil packet containing what sounded like perfectly ordinary gelatin-powder. The packet proved to be intact, and the happy orange powder poured into a freshly-cleaned dish in a manner perfectly imitating that of wholesome non-expired gelatin. I dismissed the faint demonic snickering sound I seemed to hear as a figment of my fevered imagination and prepared the gelatin powder in the usual manner.

I took up my electric kettle, containing distilled water, and threw the switch. Seconds passed into minutes. Minutes passed into more minutes. Then, the water began boiling vigorously, and I applied one cup (8 fluid ounces) of this to the dish of powder, stirring it with a tablespoon. It seemed to take at least two minutes of continuous stirring, but the deceptively innocent-looking powder finally dissolved without the slightest scent of brimstone. As prescribed by the instructions on the box, I added a further 8 fluid ounces of cold water (from the tap of my kitchen sink), stirred briefly to mix, and placed the dish in the refrigerator to gel overnight.

I lay awake in bed for hours, wondering if I was doing the right thing. Was I insane? Did I not remember the tales of Jeckyll and Hyde? Of Doctor Frankenstein? Of Pons and Fleischman? What horrible fate was I setting myself up for? Finally, I dropped into a fitful slumber, disturbed only by dreams of amorphous orange demons stalking me to feast upon my soul…

Day broke, and this very afternoon I took the now solidified mass from the refrigerator. This was it. My last chance to avoid whatever hellish abuses this disturbingly orange substance had planned for me. But no…it was far too late to turn back now. I took up my spoon, and devoured every last bit of happy orange jiggliness.

This was approximately seven hours ago. In the intervening time, I have experienced the following symptoms: Occasional thirst, mild generalized anxiety about the near future, hunger, and an urge to write this blog post in a hyperbolic language more suited to an H.P. Lovecraft story than a scientific report. In other words…I appear to have been entirely unaffected, despite consuming an entire box of expired gelatin.

I’ve been taught that when hypothesis-testing, one considers the “null hypothesis”. That is, the hypothesis that would falsify the one that I’m starting with. In this case, it would be something to the effect of “I will suffer no harm whatsoever from eating expired JellO®”. Given the results in this experiment I must – in the tortured language of philosophical science – “fail to reject the null hypothesis”, because my results show no evidence whatsoever that I have suffered harm from eating expired gelatin. In other words, I cannot rationally cling to my original hypothesis as written, and must confess that perhaps expired instant gelatin still in intact packaging may, in fact, be harmless.

Ah, but I know what happens now. “Cad!”, you cry! “Fraud! Sham! This experiment is, like, totally bogus! This is not normal JellO® but a sugar-free impostor! And furthermore, this isn’t even JellO®-brand gelatin, but a cheap knock-off brand! How dare you, sir, feed us this crap, which proves nothing!”

I answer in two parts: Firstly, ladies and gentlemen who are my readers, I assure you that the contents of the less-famous brand and the official Kraft® Foods brand are essentially identical, and indeed, might conceivably have come from the same source. It’s common practice for one factory’s product to be shipped to multiple sellers who each offer it under their own label, as the wide variety of affected brands during the recent “salmonella peanut butter” scare demonstrated. And secondly: as it happens, I also have in my possession a box of JellO®-brand lime-flavored gelatin, WITH sugar, which although it lists no obvious “expiration date”, has a code stamped on the box indicating that it was originally packaged in late 2003, and therefore should have exceeded the expected 24-month shelf-life about the same time as today’s test subject did. I swear to you, dear readers, that I will repeat my experiment with this sample next.

Stay tuned: “Expired JellO II: Lime’s Revenge”, coming soon to a blog near you!

UPDATE: The Expired JellO® Saga continues here!

Okay, this is completely bogus…

The folks over at BBspot are callously spreading microbiological misinformation (click for full-size):

This is just plain irresponsible and utterly wrong. Surely everyone can see the obvious problem here, right?

When this happens, the flame is blue, not orange.

Uh, or so I’ve heard.

(If it isn’t obvious – what you do in this situation is calmly take the spreader out and set it on your nice, flameproof benchtop, and set something non-fragile and non-flammable on top of the flaming jar of alcohol, which will then go out quickly as the oxygen gets used up. All the labs I’ve been in lately use “canning” jars for the alcohol in this application, complete with lids which can be set on top to extinguish the flame.)

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…

The Oldest Microbiology Book (that I own)

There’s this thing that some people do sometimes when they’ve been getting stressed out in one place for a while. I hadn’t done it in so long I can’t remember what it’s called. You know, where you Leave the area and then avoid it for a while. Oh, yes, that was it, a vacate-shun. Anyway, leaving the barren desert wastelands of the West, we headed east, and spent a few days admiring the area around the midpoint of the Appalachian Trail: Harpers Ferry, West Virginia. (Incidentally, I can recommend the “Angler’s Inn” Bed and Breakfast there, and the whole time there was incredibly delightful to me. I think I’d love to move to the area.).

I was delighted to note that there was an Old Book store in downtown Harpers Ferry. One thing about the Eastern US is that it’s been settled by book-using folks for somewhat longer than the West, so it would seem it’s easier to find really good Old Books. I found a publication of a 110-year-old microbiology book. In decent condition, for just over $20, no less! Not counting the (relatively modern) reprint of Micrographia that I picked up from a library sale, this makes it by far the oldest microbiology book I own now.

Oh, yes, did I mention I collect (casually) old books, especially old scientific and technical books?

The book in question, published in 1897, is “Story of Germ Life”, by Herbert William Conn. Not to be confused with Harold Joel Conn of “Conn’s Biological Stains” fame…who happens to be Herbert William Conn’s son. To be fair, the book *I* got was actually a republication from 1904, so only 103 years old…back when copyright was more rational (7 years, plus an OPTIONAL 7 more years. Thus explaining why my republication came out 7 years after the original.) It appears to have been part of a series called “Library of Valuable Knowledge”. The bookstore actually had another one of them, but I don’t remember what its topic was.

“Story of Germ Life” isn’t really a textbook so much as an overview of the subject of “Bacteriology” (as understood in 1897) for otherwise well-educated people – the kind of book I don’t think there are enough of these days. The Gutenbook project actually has a plain-text-only version of the book online here. Of course, then you miss out on the incredibly useful illustrations:

I always find it interesting to go back and see the earlier stages of scientific endeavors – especially as relates to my own interests. There always seem to be things that have since been forgotten, abandoned, or glossed over in them.

H.W. Conn seems to have been most interested in dairy microbiology, so there is a substantial amount of space devoted to it. I’ve heard of “blue milk” before (Yummy!….Pseudomonas?), but not Red or Yellow milk. He also devotes space to discussing the affect of “good” (and “bad”) bacterial cultures on butter, cream, and cheeses. I’m not even sure if butter is cultured these days, or if they just churn it up fresh and cold with minimal growth. Dangit, one of these days we’re just going to have to move somewhere we can keep a miniature dairy cow so I can do some experimentation with real unpasteurized fresh milk.

Bacterial phylogeny was so quaint back then. “Bacillus acidi lacti.” Ha! I love it. Interestingly, the term “Schizomycete” doesn’t appear anywhere in the text, though that may or may not be because it was considered unnecessarily technical for the intended audience. There’s actually very little about microbiological methods, too, which is the one major disappointment for me. Oh well, still interesting stuff. Conn actually mentions various “industrial” uses of bacteria including retting (soaking fibrous plants like flax or hemp so that bacteria eat the softer plant material to free the fibers), the roles of different bacterial cultures in curing tobacco, and even a fermentation in the production of opium (which Conn says is fungal rather than bacterial).

Also, much to my approval, the first 2/3 of the book is not about diseases. Only the last third of the book discusses “parasitic bacteria” and related topics. I leave you with this quote from the book’s 1897 Preface, which I think is still relevant today:

“Few people who read could be found to-day who have not some little idea of these organisms and their relation to disease. It is, however, unfortunately a fact that it is only their relation to disease which has been impressed upon the public. The very word bacteria, or microbe, conveys to most people an idea of evil. The last few years have above all things emphasized the importance of these organisms in many relations entirely independent of disease, but this side of the subject has not yet attracted very general attention, nor does it yet appeal to the reader with any special force. It is the purpose of the following pages to give a brief outline of our knowledge of bacteria and their importance in the world, including not only their well-known agency in causing disease, but their even greater importance as agents in other natural phenomena. It is hoped that the result may be to show that these organisms are to be regarded not primarily in the light of enemies, but as friends, and thus to correct some of the very general but erroneous idea concerning their relation to our life.” — April 1, 1897

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.

– INTERLUDE –

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.

[8] BARTHOLOMEW JW, FINKELSTEIN H:”CRYSTAL VIOLET BINDING CAPACITY AND THE GRAM REACTION OF BACTERIAL CELLS.” J Bacteriol. 1954 Jun;67(6):689-91.

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

[10] LAMANNA C, MALLETTE MF. “CHROMATOGRAPHIC ANALYSIS OF THE STATE OF ASSOCIATION OF THE DYE-IODINE COMPLEX IN DECOLORIZATION SOLVENTS OF THE GRAM STAIN.” J Bacteriol. 1964 Apr;87:965-6.

[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?…



“Teh Deth Kitteh!”

Run! Itz teh Deth Kitteh!

The prestigious New England Journal of Medicine reports (Dosa, DM: New England Journal of Medicine; 2007; 357:4; pp 328-329 ) on the case of a single eukaryotic organism – a specimen of Felis catus – who is reported to identify People Who Are About To Die (insert ominous thundercrash here).

It is presented in a tone that is a mixture of “OOOo, spooky, mysterious!” and standard issue “Human Interest Story“, as though it was a baffling or unexplainable phenomenon. Honestly, didn’t modern science explain this long ago?

Obviously, Oscar the Cat is simply waiting around to devour the souls of the departed as they are exhaled on the last breath. Or as “Mike, the Mad Biologist” puts it:

Shouldn’t the situation be obvious? I mean, come on, did ALL of these journalists sleep through Biology 101? Even if they did, surely at least some of them own cats and already know about this….

There are, of course, numerous examples in the scientific literature documenting the tendency of the cat to steal the breath of the living. See, for example, Bener A, Galadari I, Naser KA.”Pets, allergy and respiratory symptoms in children living in a desert country”;Allergie et immunologie;1995 Jun;27(6):190-5…

Interestingly, as I was trying to find a more explicit reference in PubMed to this folk-belief, I ended up stumbling upon an article entitled “Micturition and the Soul” [Holstege G.”Micturition and the Soul.” J. Comp. Neurol. 2005 Dec 5;493(1):15-20.]. I love browsing databases of scientific papers. Where else could you go looking for a folk belief and find an article about the neurology of peein’?

I know I don’t normally discuss freakish, perverted Eukaryotes on this blog – hey, CHILDREN could be reading this! – but I found the article (and the responses to it) interesting, and it serves as filler until I can finish putting together my “Gram Stain Article To End All Gram Stain Articles” post.

Simplify, Simplify…

DNA seems to have two main threats to its well-being once it’s extracted and purified.

  • Nucleases
  • Spontaneous Hydrolysis by water

Nucleases are the big one that everyone seems to mention. The seem to be fairly sturdy enzymes, and they’re everywhere (including fingertips – hence the need to wear gloves whenever you get near DNA samples…), and they “eat” DNA rapidly. Theoretically, you can destroy the enzymes with enough heat, but you still need to worry about them getting in every time you pop open your sample to get some out.

Apparently, DNA even in pure water can tend to slowly fall apart spontaneously. It doesn’t happen very fast, but bit by bit, it can undo the links between the individual nucleotides.

A common way to try to deal with nucleases is to add EDTA to the solution. Nucleases need magnesium ions dissolved in the water to do their job, and EDTA tightly binds to magnesium (and calcium). The idea is to “use up” any stray magnesium ions in the solution so that even if nucleases get in, they’re inactive because they have no magnesium available. That’s why you see EDTA in the recipes for so many DNA-related solutions. Of course – EDTA doesn’t permanently bind up all the magnesium – there’s always a tiny fraction that stays in the solution. So, although EDTA can drastically slow down any nucleases, it won’t actually stop them.

There are also some interesting chemicals which can be added to destroy all proteins (including nuclease enzymes). Guanidine Thiocyanate is one rather nasty chemical that does this. 2-mercaptoethanol is another. Various other detergents like CTAB may also denature any proteins. Since they don’t harm the DNA in the process, you could keep the DNA sample dissolved in a solution with these chemicals…but then you can’t do PCR with the sample as it is, since the protein-denaturing chemicals will also destroy any enzymes that you WANT, like DNA Polymerase, when you try to mix it into your reaction.

I think the latter option will be great for collecting field samples (in fact, it’s papers specifically on the subject of preserving samples in the field with CTAB and Guanidine Thiocyanate based solutions that I’m adapting from), but isn’t going to be real useful once I’ve got my DNA relatively purified. What to do, what to do…

Actually, I think the answer’s simpler than I originally expected. I’ll just dry the purified DNA out. No water – no hydrolysis…and no nuclease activity, either.

I could actually just leave it as a dried pellet in the bottom of a microcentrifuge tube, but that leaves the problem of taking only a little bit of it for processing rather than taking the whole thing, and I want to avoid reconstituting it and re-drying it repeatedly. I think a variation of the “dry the DNA on a piece of paper” process will be in order – then I can just cut off a small strip of the paper to get a portion of the DNA. It appears that you can actually dunk the DNA-impregnated bit of paper right into whatever solution you’re using (like a buffered polymerase-and-primers solution for PCR) and go for it.

Among the several references I found on this, here are two:
Kawai J, Hayashizaki Y: “DNA Book”; Genome Res. 2003 13: 1488-1495
Burgoyne LA: U.S. Patent #5496562 “Solid medium and method for DNA storage” (1996); U.S. Patent and Trademark Office, Washington D.C.