Libel! Blasphemy! Slander!…

Injustice! Perfidy! HUMBUG!

Periodically, someone puts up a “could you pass a grade-school science class” quiz. The one linked to the image below goes to one that I just broke down and took, purely out of curiousity. Take a look at this outrage!:

JustSayHi - Science Quiz

Oh, sure, it LOOKS good, but what you don’t see is that it only gave me a 96%, implying that I missed one (it was a short quiz)! Sure, the quiz was very much in the modern fashion for “standardized testing” (aka the “No Child Left Awake” project) where the emphasis is on memorizing stuff for a test rather than actual comprehension. So, I thought, maybe I hadn’t correctly memorized which word was correct for one of the word-memorization questions. But, no, according to the “answer sheet”, the one I supposedly got wrong was this one:

(Note: If you’re planning to actually take that quiz, do so now before you read on and I give away one of the answers…)

“How do mammals respire?”

The options were:

  • Aerobically
  • Anaerobically
  • Both aerobically and anaerobically

Come on, I may hardly ever concern myself with perverse eukaryotic systems but…never mind just “mammals”, as far as I know, all eukaryotes (animals, plants, and fungi) only possess aerobic (oxygen-requiring) respiratory systems.

However, the “answer sheet” for the quiz claims that the answer is “Both aerobically and anaerobically”.

So….they’re wrong. I’m pretty sure what what they were intending to ask, given this answer, is “what kind of metabolism do mammals have?”, in which case their answer is correct.

See, “respiration” is only one part of the cellular energy-generating system. Specifically, it’s our friend, the Electron Transport Chain, which (to grossly oversimplify) harnesses the energy of oxygen sucking electrons off the end of the chain various biochemicals to recharge molecules of ATP. That’s not the only way a cell can get ATP, though. What the quiz authors are presumably alluding to is that there are non-oxygen-requiring biochemical pathways that animal cells can take to make energy – such as the one your muscles use when they can’t get enough oxygen, which involves production of lactic acid, which in turn gets blamed for the “burn” sensation you get when you work your muscles hard.

So, the authors of this quiz are bad, bad people, besmirching my reputation and harming my precious self-esteem by giving me less than 100% on that quiz!

On a related subject: breathing causes cancer in Sprague-Dawley™ rats!

No, seriously, it’s true – try raising one group of Sprague-Dawley™ rats with air, and one group with no air, and examing both populations 150 days later. I guarantee you’ll find many more cancerous growths in the “with air” group than in the group that was denied air to breathe…

What brought this outburst on? It was this blog article. “No, It’s for Real: Aspartame Causes Cancer”, the post proclaims. They’re talking about This study(pdf). Go ahead, take a look, but in particular, look at the tables of actual data, not the paper’s abstract. In particular, take a look at Figure 1, especially “D” and “E” (showing survival rates for the different groups of Sprague-Dawley™ rats as the study progressed), and at the number of “tumor-bearing animals” in Table 2.

Notice that at around 120 days on the survival graphs, the groups with the highest percentage of members still alive were the groups receiving the most aspartame in their feed. It’s worth noting that the highest-Aspartame group there was getting roughly the equivalent of a human drinking <em>thousands</em> of cans of diet soda every day. Also note, in fairness, that both graphs seem to show little difference between the groups, so rather than assuming that Aspartame makes Sprague-Dawley™ rats live longer, I would tend to assume that there’s really not much difference.

Notice also that in terms of the percentage of Sprague-Dawley™ rats that developed one or more tumors, there were fewer of them in the group that got the equivalent of 500 mg/kg of aspartame: which scaled up to human terms means about 200-250 cans of diet soda EVERY DAY worth of aspartame.

You may be wondering why I keep mentioning Sprague-Dawley™. It’s because this is a particular commercially-bred strain of rat that’s popular with labs for this kind of thing. One point that isn’t always mentioned is this: Sprague-Dawley™ rats are known to be prone to developing cancer spontaneously. This can be handy if you’re doing studies of “borderline” carcinogens. The hope is that if something has even a tiny ability to cause cancer, you’ll be able to measure the effect in a population of critters known to get cancer at the drop of a metaphorical hat, when in a human population the incidence might be so rare that you can’t distinguish it from random chance. To my admittedly-not-big-on-the-biochemistry-of-perverse-eukaryotes mind, this study really seems to show that there’s little or no effect – and certainly no dose-dependent effect – of aspartame even on cancer-prone lab rats.

I don’t know what it is, but “artificial sweeteners”, and especially aspartame, seem to generate such passionate hatred in some people. It reminds me a great deal of people’s reactions to “genetically modified” crops. People just really want to hate it. The authors of this paper are obviously trying REALLY hard to show somehow that aspartame is a dangerous poison, despite the inconclusive-appearing actual results. Though I suppose one could argue that they showed Aspartame to be at least as much of a deadly poison as Expired JellO®.

And now that I have exposed my readers to several times the Recommended Daily Allowance of Humbug, I bid you all a good night – I have Art History and Philosophy to attend in the morning…

The Unbearable Limeness of Being

I awaken. Am I alive? The temperature is neither extremely hot nor extremely cold, so I’m apparently not in some punishment-afterlife. And there’s no beer volcano or stripper-factory, so this obviously isn’t heaven. On the other hand, I am experiencing the usual persistent discomfort involved with waking up early in the morning. On the assumption that Catholic “purgatory” would be more dull, I will assume I am still alive, and had better get up and get to class.

Since my previous experiment, I have obviously had to revise my original hypothesis. Since the last caused me no ill effects, I had to abandon the notion that expired gelatin products become a deadly poison. Instead, as I consume this batch of official, non-sugarless Jell-O®-brand Gelatin (Lime flavored), I operate on a new hypothesis:

“Expired instant gelatin products from intact packaging will not harm me if I eat it.”

My precious stock of expired JellO® is depleted by one more box, the packet ripped from its cardboard sarcophagus, the contents prepared according to the standard instructions, and consumed hastily last night (the animation from the previous post is the actual container of prepared Lime JellO® made from digital photographs taken between helpings.). You can see the old-style date code on the box. According to Carolyn Wyman’s “JELL-O: A Biography”, the code indicates that it was packaged in 2003 (the “3” at the beginning of the code), on the 343rd day of the year, in the San Leandro (California) packaging facility. Although there is no official “expiration date” shown, given the “expected shelf life” of 24 months, this package is approximately 2 years out of date. And I ate it. I appear to have suffered no ill effects. Not even a decent sugar-rush: the entire box contains 320 calories, barely equivalent to a package of Twinkies®. The flavor even appeared to be perfectly normal. Mmmmmm, Lime JellO…

When I took it out to eat it, I did spot a beautiful if alarming sight, though:

The crystalline-appearing sheets of growth from the edge of the bowl into the gelatin was slightly disturbing. Was I crystallizing something odd out of the gelatin/sugar/flavor solution? The growth resembled infiltration of mold into the gelatin medium enough to slightly worry me. But only slightly.

In fact, as I had most suspected, these turned out to be ice crystals. Quite pretty, but they started slowly melting away after the bowl was allowed to sit at room temperature for fifteen minutes or so – plus, they crunched when I ate them just like ice. Thus encouraged, I ate the gelatin and went to bed. And here I am (sitting in the student lounge between “History of Western Art” and “Introduction to Philosophy”) happily blogging away, apparently unharmed.

Does this prove that expired instant gelatin is harmless? Well, no, not exactly. Scientists never really “prove” anything. Instead, we attempt to “falsify” our hypotheses and theories as best we can. This is where the concept of the “null hypothesis” comes in.

The “Null Hypothesis” here is the situation that, if true, falsifies my hypothesis. In this case, it would be “Expired instant gelatin products from intact packaging will harm me if I eat it.”. However, I did eat expired gelatin products from an intact package and was NOT harmed. Therefore I must “reject the Null Hypothesis”…and therefore my experimental evidence does not fail to support my hypothesis! SUCCESS!

If we are unable to find a condition which renders our hypothesis or theory incorrect after many and varied tests, ideally by several different researchers, then we can be confident that our hypothesis or theory is correct, but we don’t necessarily KNOW that there isn’t some odd undiscovered exception that we don’t know about.

Two samples (this one and the previous sugarless-orange one) is hardly a large number of trials. This doesn’t prove that expired JellO® is always safe, but since I know of no plausible way by which an intact package of instant gelatin could become hazardous I feel pretty comfortable that expired gelatin from intact packaging won’t harm me.

If the package is not intact and contains a fuzzy green lump instead of the usual powder, then it’s a whole other situation, obviously…

I do still have three or four more boxes of the sugarless generic expired gelatin – perhaps I can come up with some more tests. Meanwhile, I do hope that my incredibly brave, life-threatening experiments here will relax nervous expired-JellO eaters everywhere…

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 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!

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

[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.

I, for one, WELCOME our new radiation-eating fungal overlords…

Though I am getting a little annoyed at the breathless prose about how it’s “like photosynthesis” and might be a way to sustain astronauts during long space flights and so on.

The story’s about a fungus they found growing (thriving, even) inside the reactor at Chernobyl, despite all the radiation the fungus is exposed to in there.

What the original paper – which you can find here from PubMed Central (and where you can find what the study actually shows, rather than the somewhat lower-content hype found in most news reports on it) – seems to show based on my hasty undergraduate-level reading is that the fungi do grow faster when exposed to “ionizing radiation”, and that it appears to be due to melanin in the plant getting energy from the radiation (and passing that energy on to the fungus to use for growth).

This is actually pretty spiffy, but really – so far – doesn’t look like “photosynthesis” at all. They’re not testing for any kind of carbon fixation, and I’m guessing that if there is any carbon fixation going on, that it doesn’t generate oxygen in the process. It also seems unlikely to me that even then, the fungus can grow autotrophically. This would seem to drastically reduce the possibility of this stuff ever being Purina® Astronaut Chow – you’d still need some other way to get the carbon dioxide out of the Astronaut’s air and put oxygen back in it. If you’re going to do that, you might as well just use plants (or cyanobacteria) and eat THEM.

Still, the implication that you could adapt some melanin-producing fungus to absorb “radiation” and turn it into useful materials of some kind is spiffy, even if it’s not going to allow us to turn nuclear fission plants and spent nuclear fuel depots into fungus-powered anti-global-warming-gas powerhouses.

One thing’s bugging me, though. I obviously don’t have enough understanding of how “ionizing radiation” behaves at a biochemical level, since I’m wondering if it’s proper for everyone to treat “radiation” (both from flying electrons and from high-energy light) as some sort of generic substance, whose only useful attribute is how much energy it has.

As far as I know, most of the “radiation” that the fungus inside the Chernobyl reactor is getting is Gamma-radiation – basically high-energy light (one step above “X-rays”, two steps above sunburn-causing Ultraviolet light). What the researchers are hitting their test-subjects with looks like it’s mainly “Beta”-radiation (which is to say – electrons)*. In both cases it’s “ionizing” radiation, which is to say (more or less) that the radiation knocks electrons off of atoms that it runs into in both cases, and in the ideal “spherical horse” world of a Physicist, the same amount of energy is going to knock the same amount of electrons loose from various molecules and therefore have the same effect, right?

Except I’m having trouble convincing myself that’s a valid assumption here. The results seem to show that exposure to radiation is somehow resulting in the melanin in the fungus being able to “reduce” a chemical (changing “NAD+” into “NADH”) that can potentially in turn dump electrons into the beginning of the Electron Transport Chain to in turn provide biological energy in the form of ATP…

Can one reasonably assume that the mechanism by which this happens would be the same regardless of the form of ionizing radiation? The big deal with melanin seems to be that it absorbs a wide range of light wavelengths (which is why it looks black to dark-brown, and why it protects skin from Ultraviolet radiation…) which implies that absorbing the gamma radiation is where the energy is coming from that makes the fungus thrive in the Chernobyl reactor building. I guess I’m just having trouble picturing how a much more massive, slower-moving electron could have precisely the same effect as a virtually massless, much faster photon. (Yes, I know that beta and gamma radiation are said to have the same amount of “effect” on living tissue per unit of energy…)

Is it possible that the melanin is directly “capturing” the beta particles (electrons), while gamma radiation is kicking electrons off of something ELSE, and melanin is then only indirectly taking up those? For that matter, is it possible that in both cases it’s just something silly like the radiation inducing hydrolysis of water, and it’s just hydrogen gas supplying the reducing power? Thinking about this is making me feel dumb – can anyone reading this explain what I’m missing here?…

I suppose I could just cheat and ask someone in the biology department. We’ve GOT a professor who ought to know – her research has specifically focussed on zapping prokaryotes with “ionizing radiation” (electrons from the college’s linear accelerator)…But that would rob my dear readers of the chance to participate here…

* – okay, it’s probably even more complicated than that. If I understand what the paper is describing and what my Minister Of Funky Physics Knowledge showed me, the source of the “ionizing radiation” for the experiments is radioactive Tungsten(W) and Rhenium (Re) (A “188Re/188W Isotope Generator”). W-188 gives off beta particles when it decays to Re-188. But Re-188 can go through some sort of funky subatomic rearrangement before it decays so that it can EITHER give off beta particles OR gamma-rays as it decays down to stable Osmium-188. I have no idea what the proportion between beta and gamma is at that step (the “conversion efficiency”) so it’s possible there’s enough gamma radiation coming out to do something, regardless of what the beta particles are doing. (The experiment doesn’t do any comparisons with “pure” gamma radiation, which I imagine is not simple to arrange…). So now I’m even MORE confused. Thanks, physics. Thanks a lot.

I am filled with shame…

It’s been a long day away from home, and I’ve got nothing much prepared for tonight – the last day of “Just Science” week. (Not that I’m going to stop posting after today or anything…)

So, I’ll cop out, and instead post a question.

What textbook(s) are you currently using for Microbiology classes, and what do you think of them? My “Introductory Microbiology” class was over 8 years ago, but the textbook was Tortora, Funke, and Case – “Microbiology: An Introduction (sixth edition)”.

I found it annoyingly heavy on the “disease-listing” and way too sparse on the rest of the microbial world – though they did have a couple of chapters on applied/industrial type microbiology.

Please leave comments…

My posts will likely be pretty sparse until after Wednesday, when I have back-to-back Microbial Genetics and Pathogenic Microbiology exams. Ick. As you can imagine, I’ll be studying a lot for the next few days to make sure I’ve learned what I’m supposed to up to this point.

Curse you, public library!

Tonight’s post will be an eclectic one…

I made the mistake today of heading for what passes for a “large city” in my local area in a general need to go somewhere besides my house and the college. I figured I could browse the local discount bookstore and see if they had anything interesting.

I happened to notice a sign advertising a book sale at the local library.

Why did they have to do this to me? Have they no decency? Have they no shame? Have they no MERCY?

As I previously mentioned, I actually do collect old science (and medical) books. Unfortunately, I ended up walking out of the library with a whole mess of microbiology books (and one Botany book that I picked up just because it was old – 1930’s). Fortunately, they were cheap.
I was just perusing one of the books I picked up: an old “Bacteriology” book[1] from the late 1940’s. It’s fascinating and instructive to see what scientists used to believe was true and what observations led them to believe it.

The introductory chapters of the book include a discussion of taxonomy and the place of “Schizomycetes” (meaning bacteria that aren’t photosynthetic) in the overall scheme of things. There’s a discussion that, given what information was available at the time, is perfectly reasonable and explains why bacteria are “plants”, just like other fungi (Fungi, you see, are just plants that aren’t photosynthetic – or so they explain). The author gives a classification scheme for plants that divide them into three categories, which roughly equate to “normal” plants (with stems and leaves), moss-type plants, and plants that don’t have roots, leaves, stems, or flowers. This latter category he broke into two sub-categories – Algae (including “Blue-green” algae, which we now know are actually bacteria) and Fungi. “Bacteria” are listed as one of the categories of Fungi.

The discussion justifying this categorization makes some interesting claims – some of which are startling to me. The author claims that some bacteria – “Acetobacter xylinum” have cell-walls that consist of cellulose, just like plants. (Actually, it would appear this bacterium does make cellulose, though I don’t think it’s actually a component of the cell wall – this is a standard “Gram-negative” type ?-proteobacterium). I had no idea up to this point that there were cellulose-producing bacteria. Interestingly, the author also states

“Some bacteria are said to possess cell walls of chitin, a distinctly animal substance which is the material of horn, hair, hoof, and insect shell”

which is completely wrong on every count except for the part about insect shells. (Horn, hair, and hoof (and fingernail) material is Keratin, which is a type of tough protein. Chitin is actually a polysaccharide…and it is what most fungal cell walls are made of.There are some interesting statements in the section on microscopy as well. The author claims:

“There seems no doubt that the gram-positive material in bacteria is ribonucleic acid. Bartholomew and Umbreit[2] have shown that it can be removed by soaking the gram-positive cells in sodium choleate. It may be replaced by treating them with magnesium ribonucleate. Normally gram-negative species will not accept the applied coating. The specificity of these reactions is shown by the fact that an enzyme, ribonuclease, will remove the gram-positive character (ribonucleic acid) of the cells very quickly.”

What the heck?… Now I have an urge to see if I can sneak a culture of some kind of Bacillus and some RNAse and see how much of this explanation actually matches observation. (Perhaps I can dig up Bartholomew and Umbreit’s paper as well). The author also mentions that nobody has managed to get a good image of a bacterial nucleus, either, which of course is because they don’t actually have one…
One other thing I’d never heard of: Proton Microscopy. According to the author, this technique, apparently first implemented in France in 1948, could theoretically give substantially better resolution than electron microscopy.

Some quick poking around seems to show that this is partly true, and there actually are proton microscopes that get used for some kinds of studies. However, protons are a heck of a lot harder to “focus” and they don’t seem to have caught on for microbiological work. They do evidently have some useful properties for doing analysis of what specific elements are in a sample, though[3].

I noticed some other apparent differences in style between the older textbooks and current ones, but I’ll save that for another time.

I will also at some point go back and re-write the Schizomycete article to include some of the information I’ve picked up in the last couple of weeks. Meanwhile – one more day of “Just Science” week! Looks like I should survive it after all.

[1] – Frobisher, Martin Jr. “Fundamentals of Bacteriology (Fourth Edition)”, 1949, W.B Saunders Company, Philadelphia
[2] Bartholemew JW, Umbreit WW, “Ribonucleic Acid and the Gram Stain”, J. Bacteriol. 1946, 48:567
[3] “Microscopy with Protons” (visited 2007-02-10)