Category: Why Does It Work?

How things work is usually much less interesting than why, in my experience.

Poisoning Prokaryotes in the Park

(Well, okay, it was a “Pathogenic Microbiology Lab”, not a “Park”, but whatever).

Antibiotic Susceptibility of a Poor, Innocent Microbe

Objective:Experience the awesome power of the mighty Antibiotic Susceptibility Test, wielded against an unsuspecting bacterial organism!

Introduction:

Every day, billions of innocent bacteria are ruthlessly slaughtered by antibiotic substances introduced into their callous, inconsiderate hosts. Condemned to death as nuisances due to nothing more than the potential inconvenience of debilitation, tissue necrosis, death, halitosis, and other minor problems, the unstoppable might of all medical science is focussed on our prokaryotic friends like medical professionals around the world focussing the rays of the sun through a thousand magnifying glasses to obliterate innocent prokaryotic “ants”.

The ?-lactam antibiotics – the blahblahcillins (Penicillin, Ampicillin, etc.) and the Cefablahblah compounds (Cephalosporin, Cephalothin, Cefuroxime, etc.) all interfere with the formation of the bacterial cell wall – loosely analogous to the human epidermis. This treatment viciously targets the hardest-working, actively-reproducing bacteria, spilling their guts as they attempt binary fission, while leaving the lazy, dormant microbes alone. Gram-negative bacteria are somewhat protected from this torture by their outer membrane, but some (such as ampicillin) can affect even some of them. Gram-positives, with their simple structure, are hardest hit. A few microbes have learned to counter this by secreting an enzyme which disables many of these drugs, though medical science has countered with clavulanic acid – an inhibitor of the ?-lactamase enzymes. Enzymes resistant to inhibition by clavulanic acid are being developed as part of this continuing arms race.

Chloramphenicol is an artificially manufactured bacteria-poisoning chemical synthesized in laboratories (though it was originally obtained during interrogation of a captured Streptomyces species) which interferes with protein synthesis at the 50s ribosome. Erythromycin has the same affect, by a slightly different mechanism, and is a macrolide – a class of large molecules with lactone rings which resemble in shape the poisoned ninja throwing-stars seen in movies. Both of these chemicals are bacteriostatic rather than bacteriocidal, but are broad-spectrum. Chloramphenicol’s devious action sometimes backfires on a small number of people, causing potentially fatal aplastic anemia.

The Sulfonamides are also bacteriostatic, and are competetive inhibitors of enzymes that convert the nutrient PABA into biochemical products vital for prokaryotic health. Much like a hypoglycemic person with diarrhea given a “cupcake” made entirely out of Olestra® and Sucralose, the microbial victim of this chemical ingests it but finds that it merely inconveniences the metabolic processes rather than feeding them.

Tetracycline (the first of the Tetracycline-type antibiotics) and Tobramycin (an Aminoglycoside) both jam the gears of protein synthesis, inhibiting the action of the ribosome in the former case, and actively causing erroneous protein formation in the latter. The latter effect is outright bacteriocidal, causing the poor bacterium’s protein assembly systems to make broken enzymes until the cell’s protein factory is bankrupt and has to lay all the enzymes off. Tetracyclines appear to only slow down the cell, but in the cutthroat competition for cellular activity in the human body, this stumbling can be a death sentence for the business of prokaryotic replication.

In order to determine which of these lethal agents to deploy against the oppressed bacteria, a medical professional may capture a microbe and torturously test various agents on it, watching without emotion to see which ones destroy the microbe most efficiently. This awful, coldly clinical process is standardized in the Medical Microbiologist Field Manuals on Interrogation as the “Kirby-Bauer”[1] antibiotic susceptibility test. In these tests, 6mm diameter paper disks soaked with various antibiotics in specific amounts, is pressed onto a growing young microbe culture to see which ones are most destructive, leaving desolate areas devoid of life in the culture…

Recently, we got to do this..

Materials and Methods:

A colony of Pseudomonas aeruginosa was lured into a culture tube with the promise of free candy. Happily replicating, this culture was transported to a secret location containing Mueller-Hinton agar. 100?l of this culture was told that it had won an all-expense-paid stay at a four-star hotel with room-service, and was plated onto the agar. The culture was then strapped down and tortured for its secrets by application of 12 antibiotic-soaked disks. The torture was performed at 37°C for 24 hours, and the results measured with a ruler.

Results:

The defiant Pseudomonas organism bravely withstood the application of Ampicillin, Cephalothin, Chloramphenicol, “Triple Sulfa”, Nafcillin, Cefazolin, Amoxicillin with clavulanic acid, Cefuroxime, and Penicillin G. Erythromycin seemed to cause the subject some discomfort. It was not quite able to grow to the edge of the antibiotic disk all the way around but was able to get within less than a millimeter of it, even touching it in a few spots. Tetracycline was unbearable to the subject, who was limited to a 11mm (diameter) zone of inhibition around the Tetracycline-containing disk. Finally, Tobramycin (zone of inhibition approximately 23mm) finished breaking of the subject, who then confessed to several murders of immunocompromised individuals, robbing a “Wal-Mart”, and once molesting an archeaean of the genus Thermoplasma. Investigations may already be underway to determine the authenticity of these confessions. Then again, they may not.

Conclusions and Discussion:

Pseudomonas aeruginosa is a hardy little bugger who can put up with a wide variety of antibiotic insults. Its weak point appears to be its ribosomal machinery, as this was the target of the three drugs which had any apparent effect. Should intelligence indicate the threat of attack by the terrorist Pseudomonas organization, ribosome-targeting, protein-synthesis-inhibiting agents should be deployed as a countermeasure.

References:

[1] – Bauer AW, Kirby WM, Sherris JC, Turck M, “Antibiotic susceptibility testing by a standardized single disk method” Am J Clin Pathol. 1966 Apr;45(4):493-6

[2] –Tortora GJ, Funke BR, and Case CL, “Microbiology – An Introduction (sixth edition)” 1997, Benjamin/Cummings Publishing Company, Menlo Park, CA

The “Electron Transport Chain”, Grossly Oversimplified

Why does breathing work, anyway? And can I possibly explain it in a couple of paragraphs? I don’t know, but I’m going to try…it leads into the subject that got me interested in majoring in Microbiology in the first place. It’s probably kind of foolish to try to cram in this explanation in the half-hour or so before midnight (and hence the informal deadline for getting a post up every day for “Just Science week”), but here goes:

First, a bit of really simplistic background. Since the fundamental principle of the universe is basically that stuff likes to fall apart (dang lazy molecules), in order for a cell (bacterial or otherwise) to make new proteins and strands of DNA and so on, it has to have some kind of energy that it can use to pay for the increasing orderliness that it’s causing. The chemical that’s usually used to provide this energy is ATP. The energy comes from a string of three Phosphate (PO4) groups that are attached to it. The third phosphate in the chain comes off really easily, releasing a bit of energy in the process, like a spring uncoiling. A lot of enzymes work by attaching to ATP, letting ATP fall apart (becoming the slightly more “relaxed” ADP in the process), and using the released energy to power some other process.

In order for this to work, the cell has to be constantly re-charging ADP, cramming that third phosphate back onto the end along with putting back the bit of chemical energy.

The point of this post is a major way that cells provide the energy to reassemble ATP. There are actually a number of ways, but one of the more effective is the “Electron Transport Chain”.

In simple terms, the cell takes an electron from a simple “food” molecule of some sort, and passes it along to a type of protein that reaches through the cell’s membrane. This protein passes the electron along to another protein, but in the process, it goes through a series of changes in shape that allows it to pump a few hydrogen ions (“protons”) from the inside of the cell’s membrane to the outside. Depending on how much energy (as “electrical potential”) was released along with the electron by the “food” molecule’s electron donation, there may be enough energy to shove the electron through up to three different proteins that do this “proton-pumping” trick with each electron.

This process causes there to be a buildup of protons outside of the cell membrane. Since the universe is lazy, it doesn’t want to hold all those crammed-together protons in place – it really wants to shove them back inside the cell so there’ll be an even concentration of them on both sides of the membrane.

The cell has a special sort of gate which lets the protons shove their way through back to the inside of the cell – but in the process, they make part of the ‘gate’ mechanism rotate. The rotating part essentially grabs ADP and loose phosphate and virtually crams them back into place – the energy to do this comes from the force of the protons shoving their way back into the cell.

But what about the electron? Well, at the end, there has to be something that will pull the electron off of the last protein. One of the best “electron acceptors” is oxygen. Oxygen is the second most electron-loving kind of atom there is. Half of an oxygen molecule (O2), a couple of spare protons, and two electrons make a nice, relaxed, stable molecule of H2O.

The reason I find this interesting is because some bacteria can use something besides oxygen, if oxygen isn’t available. They don’t get quite as much energy out of the process since these other “electron acceptors” don’t pull the electron out quite as hard at the end, but it’s better than suffocating. Sulfate-respiring bacteria, for example, can use sulfate (SO4) as the place to dump the electron, converting it to sulfite (SO3) in the process – and eventually converting it to plain “elemental” sulfur (just “S”) or even in some cases using the elemental sulfur in place of oxygen and making H2S – which is that ‘rotten-egg’ smelling gas.

There are some even more exotic “electron acceptors” that some bacteria can use…which will be the topic of another post.

(And, again, please let me know if you spot anything wrong here, and please ask questions if I’m not making any sense – I’m pretty sure I need the practice explaining this kind of thing…)

Are “Acid-Fast” bacteria Gram-positive or Gram-negative?

I was wondering what today’s post ought to be – but a Google™ search that reached the page posed an interesting question and made it easy.

Someone from a Miami, Florida college got to this site after asking Google:
“Assuming you could stain any cell, would an acid-fast [bacterium] be gram-positive or gram-negative?”

Therefore, today’s post will deal with some Microbial Physiology.

The easy answer is, of course, “no“.

A more useful answer, though, is that it depends on what you mean by “Gram-positive”.

If one takes the “Assuming you could stain any cell” part of the question to mean that you’ve done whatever it takes to get the Crystal Violet/Iodine into the cell wall, and you mean “will the cell still look purple instead of pink at the end of the Gram Stain process”, then I’m pretty sure the answer would be yes, that it would be “Gram Positive”. It actually IS possible to stain “acid-fast” bacteria with the Gram stain. The catch is that it takes 12-24 hours of staining (according to Gram’s original paper) rather than a minute or so. This still counts as Gram-positive, though, and in fact the whole Phylum of Actinobacteria (including the Mycobacterium genus) is considered “High G+C Gram-positive”. (If the query was for an exam or homework problem, this is probably the answer you’re looking for.)

On the other hand, if you mean “does the cell wall structure of an acid-fast bacterium better resemble Gram-positive or Gram-negative bacteria?”, you can make an argument that instead of a nice simple “inner membrane surrounded by a thick peptidoglycan-layer cell wall”, the “acid-fast” bacterial cell wall looks more like a complex gram-negative-type cell wall, having multiple layers, with special proteins that form channels through them to let substances in and out of the cell through the otherwise penetration-resistant outer layer, just as Gram-negative bacteria have through their outer membrane. (On yet another hand, those outer layers are tough like a gram-positive bacterium rather than fragile like a gram-negative bacterium’s outer membrane, and don’t dissolve in alcohol.)

Therefore, if you’re speaking in terms of taxonomy, and by “Gram-positive” you mean firmicutes which, as far as I know at the moment, are really the only class with the simple, officially-gram-positive-type cell wall structures and therefore are usually what is meant when someone says “Gram-positive” (someone please correct me if I’m wrong here), the obvious differences with “acid-fast” type cell walls can at least make a good argument that they are “not ‘Gram Positive’ bacteria”.

But if you put that on your homework or exam answers, don’t blame me if you get marked wrong…

I actually found a pretty nice illustration on the University of Capetown website showing the differences in cell wall structures here, which might help.

So, to summarize – Officially, they’re either “neither” or “Gram-positive”. Unofficially, you can probably argue either way. Hmmm. I should try to work in a post on bacterial taxonomy one of these days.

Actual Microbiology Post: some search queries

Before I embarass myself further trying to describe principles of Natural PhilosophyPhysics comprehensibly, I think I’ll do a post or two on things that I think I can more easily describe…

I will also remind everyone that I am merely an undergraduate, so if you happen to be speaking to a Ph.D. microbiologist and mention “some guy on the internet said…” and he or she tells you I’m full of it, I’d appreciate it if someone would tell me. (If they say “Wow, that guy’s a genius“, tell them I say “thanks” and ask if they’ve got any spare grant money or surplus equipment they could send me…)

I did some poking through the web server logs and noticed a few hits from search queries, looking for basic information about microbiology (and in particular, preparing and staining slides).

(I found it interesting that although most of the hits on this site are Mozilla Firefox, every single one of the search-engine-query hits were using Microsoft Internet Explorer. [Safari looks like it amounts to a little more than the number of MSIE hits.] Don’t know if it MEANS anything, but still interesting.)

Aside from a hit from someone looking for pictures of Gram-stained bacteria and, oddly, one person looking for information about the “No You Can’t Have (X), Not Yours” meme, here are the queries that have led to my page so far:

From somewhere in Kuwait: someone looking for “Gram (something) that stain red because”
From Toronto, Canada: “Gram Staining work”
Hopefully, I managed to explain whatever they were looking for back in my post about why the Gram Stain works.

The “Purpose of Heat-Fixing Bacteria on a Slide” query (posted about here) came from somewhere at a major technology company in Texas (and, coincidentally, today from someone at a college in Florida). And for Norfolk, Virginia (who just reached the site as I was typing this) – “Fixing” just means to keep something from moving – in this case, it means making the bacteria “stick” to the slide. Though I suspect he or she already figured that out from the previous “heat-fixing” post. On a related note, someone at a community college in Texas wanted to know “what would happen if too much heat were applied in heat-fixing”. To answer that one is (to the best of my knowledge), that it depends on how much is “too much”. Comparatively fragile bacteria like Mycobacteria would, I assume, tend to fall apart in the heat relatively easily. A bit more heat would probably fry the Gram-negative type bacteria, and a little more would finally destroy the Gram-positives (don’t quote me on this, I’m guessing here). One trick I’ve picked up is to hold the slide with my bare fingers (on the edges of the slide as far from the smear as possible) and slowly pass the slide into and out of the Bunsen burner flame until the slide gets uncomfortably hot (but stopping before it starts actually burning my fingers). That seems to do the job reasonably well.

Someone from the Fresno, California area wanted to find out how negative staining works.
A “Negative” stain is like a “negative” of a photograph – you’re staining everything BUT the bacteria on the slide (ideally). This is useful if something about the bacteria you’re looking at keeps it from being easily stained, or in particular if you want to see if the bacteria produces a capsule. If you stain the slide with India Ink, it’ll make the slide itself black, but leave a clear spot where the encapsulated (or, conceivably, any other bacteria that won’t soak up the ink) are, so you can find them. They also apparently do something similar in some kinds of electron microscopy.

Someone in New Jersey wanted to know what the purpose of “Simple Stains” were. A “Simple” stain just means you’re putting one kind of dye on the slide to color the bacteria, and you don’t really care about the color. Unlike differential stains (like the Gram stain) or a diagnostic stain (like an Endospore stain), it doesn’t really tell you anything about the bacteria other than what you can directly see in the microscope – but if the relative size, shape, and arrangement of the bacteria is all you’re interested in, a simple stain may be all you need. It doesn’t matter too much what kind of dye you use for this – I know methylene blue is a common one for this kind of thing.

Someone at a facility in Wyoming was trying to figure out what an alcohol wash did to bacterial cell walls. Presumably he got directed to this site because of my Gram Stain post. It’s probably worth mentioning that I believe the alcohol wash doesn’t actually do much to the cell wall – but it does seem to remove the outer membrane that is outside of the cell wall, if the bacteria have one.

Someone in the Chicago, Illinois area appeared to be searching for general information on choosing a dye for staining – that one probably deserves a post of its own, but I’ll try to put something together on it.

Another query was someone from the Phillipines specifically looking for an article on Schizomycetes. I just notices something about that post – I actually forgot to add one useful note about “Fission Fungi”: That’s what “Schizomycete” actually means (Greek: Schizo-: split in two -mycetes: relating to fungus). Also, for those photosynthetic bacteria (“Fission Algae”) the contemporary term was “Schizophyte”.

Finally, I find myself intensely curious about the very focussed query originating from a healthcare product company in New York, looking for information about Gram Staining of Bacillus atropheaus, specifically. Maybe Willy Bacillus has found his first fan…

The Entire Universe Explained Part 2: The Most Fundamental Observation

“The Universe is Powered by Laziness”

(I obviously need more practice – I’m still not sure how coherent this explanation is to anyone but me. Comments welcome here – or if you prefer, you can contact me via XMPP (“Jabber”, “Google Talk”) at XMPP:epicanis@enzymestew.dogphilosophy.net )

There you have it, the big secret that is at the heart of every single thing that happens in the natural world. Everything is the result of the Universe’s laziness. This is more or less what the Second Law of Thermodynamics says. In more proper language, it’s the observation that the total “disorder” in the universe is continually and unstoppably increasing.

I like to think of the universe as a big, fat, obnoxious sports fan. Picture him slouching in his couch. In one hand he’s holding a gigantic can of the most awful “lite beer” you can think of – you know, the one that only losers like – and in the other he’s got a giant foam hand with the logo of that team that only complete weenies like. He doesn’t even bother cheering – he just sits there, slouching as much as possible, maybe drooling a little, and wishing he could relax until he was nothing but an ever-spreading lump of flab…

So, what does this mean? Firstly, that there are always some “losses” whenever something happens. Basically, the universe can never manage to open a fresh can of that awful Lite Beer that it drinks without spilling at least a little bit of it on the floor. Secondly, that any bit of the universe you might happen to look at always wants to slouch a little further if it can.

That first part is what accounts for the “losses” in light of the “nothing magically disappears” observation previously mentioned. In the real world, no matter how carefully you build something, you can never quite get as much energy out of something as you put into it. You might get nearly all of it back out if you’re really careful, but no matter how carefully you hand 12 ounces of beer to the Universe, it always seems to end up with only 11.999999999 ounces of beer to drink. Or a lot less. It didn’t “disappear”, it just ended up soaked into the Universe’s filthy carpet where it is no longer available for anyone to drink. That beer-spillage is what physicists call “Entropy”. Or, “Heat no longer available to do Work“, if you want the proper physics definition.

The second part relates to the fact that there is a certain amount of “energy” inherent in the way any kind of matter is arranged. Matter, being part of the universe, is lazy, and doesn’t like having to hold onto all that energy. If you give it an opportunity, a piece of matter will tend to want to rearrange itself so that it’s not holding onto as much.

As an example, if you mix together some chemicals that will burn together if you light them, then seal them completely in a solid container, and set them off (by adding just a little bit of energy), you’ll find that the weight of the sealed container stays the same before and after…but it got really hot. That means there was energy released, and apparently a lot more than the little bit that started the whole thing – where did it come from?

The answer is that it was “built in” to the structure of the chemicals. Setting them off with a little bit of energy shoved the chemicals together just hard enough to let them recombine in a way that they didn’t have to hold on to all the energy they had up until then. All the energy the lazy chemicals let go showed up as heat. The little bit of energy you had to put in to get things started is what chemists call “activation energy”. It’s just there because those molecular slackers sure weren’t going to put out any extra effort to start rearranging – but once a couple of them are shoved together hard enough to make them get started, the energy they release is enough to shove a few more molecules together and get them to release more energy…and so on.

Because cool science types seem to avoid using whole words whenever possible, this energy that comes out is referred to as ?G. The total amount of energy that is “built in” to a particular molecule is referred to as “Gibbs’ Free Energy” (named after William Gibbs.)

So, for the last horrible analogy for now, let’s return to our big fat slob of a Universe slouching on his sofa. He’s been drinking that disgusting beer of his all night, and his bladder’s full. He’d really like to go to the bathroom but, eh, it’s too much effort to stand up. However, if you get behind him and shove hard enough to push him off of the sofa, then he figures while he’s up he might as well hit the bathroom before he sits back down. And, yes, I suppose that means it is my fault if next time you go camping, you end up waxing poetic and describing the nice, comfy campfire as “urinating heat and light on everybody”.

Microbiologically, that means that for a microbe to be able to live on some kind of “food”, it’s got to be possible to convert the food into substances with less energy built in, in such a way that it can capture and store some of the released energy in the process for its own use. It also means that if the microbe needs to make more of, say, some kind of enzyme (a more “ordered” combination of smaller molecules) it’ll have to shove in some energy to make up for the increased “order” while it assembles the parts.

Finally, what enzymes (or any other kinds of catalysts) do is reduce “activation energy” for a particular reaction, so bacteria don’t have to burst into flame in order to “burn” sugars for energy (for example).

And now I think I ought to go back to Microbiology posts before I get myself lynched by angry chemists and physicists for making a mess of this explanation…

Search Engines appear to have found me…

Quick update: I just checked my logs – I just realized there have been a number of hits from Google searches in the last couple of days, from people trying to find information about staining and such.  I’ll try to throw in a post on search query topics that showed up in the logs in the next day or two as well.  Stay tuned…

The Entire Universe Explained: Part 1. The Most Important Observation

This is just a downright flippant Gross Oversimplification™ of some things that I’ve noticed over the years which seem to come up in every science class, though occasionally they are described in slightly different ways or under different names. The more I’ve thought about it, the more I find these principles always buried, somewhere, in every observation of the natural world.

I present them here mainly for people who don’t have a background in science, though I’m hoping those who do will at least find this mildly amusing. Essentially, I just want to mention these things now on the assumption that it might help some people understand some of the basic reliable assumptions that science uses. Since they also then explain the underlying principles that drive the biochemistry that’s important to the microbiology that I want to get into, and I’d like as many people as possible to understand what I’m talking about when I do.

Therefore, I’m going to attempt to describe, using my Super Undergraduate Writing Skills, in very brief terms, everything you need to know to understand basic biochemical processes that I plan to try to explain to the best of my current ability later. I apologize in advance to any serious “hard science” types who may suffer blurred vision, seizures, dizziness, upset stomach, or psychiatric anaphylaxis from reading this.

(As always, if I get something outright wrong here, please say so…)

Part 1. The Most Important Observation of the Natural World: “You can’t get something from nothing – or vice versa”. Or in other words, nothing ever magically appears or disappears. If something appears where there previously wasn’t something, it came from somewhere. If something disappears, it went somewhere – it hasn’t simply ceased to exist. This is more or less all that is meant by “The First Law of Thermodynamics” in physics. In practice, this is what’s being invoked whenever you hear someone describing a scientific “Conservation of [whatever]”. You can still chemically combine things to make completely different substances, melt solids into liquids, bubble gasses away or dissolve solids, turn one kind of energy into another (like turning electrical force into light), and so forth, but in the end the amount of material and energy at the end should be the same as when you started. (It’s even possible to turn material directly into energy and energy into particles of matter, but as far as I know this only actually happens in conditions that matter to people like high-energy physicists and such, not in biological systems.)

This is a handy principle – it lets you measure things indirectly. If, for example, you’re studying some kind of chemical reaction that makes hydrogen gas, you can measure just the amount of hydrogen that comes out when you mix the chemicals together and be confident that this will tell you exactly how much hydrogen was chemically combined into the original ingredients that reacted. Or if you put a three-ounce cricket in a cage with a 12-ounce snake, then come back a few minutes later and discover that the cricket has “disappeared” but the snake now weighs 15 ounces, it should be obvious what’s happened without having to give the snake an MRI.

I’ll pause here for comments before I get to parts 2 (regarding the 2nd law of thermodynamics) and 3 (tying the first two parts together to make Equilibrium and such), both of which I’ll try to get to tomorrow, unless it turns out that I’m completely butchering this whole explanation and have to fill in with something else while I rework it…

Just a little over an hour…

“Just Science” blogging week starts tomorrow, which is to say, in a little over an hour where I am. Starting sometime in the next 24 hours or so, all of the participants are supposed to post at least once each day, and only about scientific topics, for the whole week. Of course, I intend to do my part.

I haven’t decided what to start with yet, though. Depending on how much time I can afford to devote to it, I may either make my ambitious attempt to, in one post, explain the workings of the entire natural world (in Grossly Oversimplified form, of course – e.g. “The Universe is Powered by Laziness“), or I may just start out with a simple post or two on staining methods or how to cheat and quickly identify the “Pseudomonas” cultures when you have to do the “Unknown” identifications in basic microbiology labs, or something like that.

If I can get through the Three Basic Observations that (I boldly claim) describe pretty much everything in the natural world, plus a Grossly Oversimplified explanation of how chemistry works (hint: it’s nothing more than the aforementioned Three Basic Observations plus electricity) then maybe I can get into things like the Electron Transport Chain, the Sulfur, Nitrogen, and Phosphorus cycles, why microbial fuel cells work, and things like that.

Any requests?

They did it!

Thanks to our library staff and the people at the University of Illinois at Urbana-Champaign (from whom this comes) I was able to get a copy of the original “Gram Stain” paper…

Beginning of the original Gram Stain paper

I don’t know whether to be annoyed (that someone else beat me to it) or happy (because now I’ll be able to double-check my translation) but it appears the ASM actually already has a translation of the paper online. Interestingly, the attached commentary states that the Gram stain even works on bacterial protoplasts. If that’s true, then all this stuff I keep getting told about the action of the Gram stain being due to the cell walls themselves is incorrect. I wonder, do Gram-positive bacteria also have thicker inner membranes? Or is the commentary full of it?

Why this article didn’t originally show up when I was poking around Google looking for it by the title, I don’t know.

Next request will be for the “Everything is Everywhere” paper…

“What’s the purpose of heat-fixing bacteria on a slide?”

I check my web server’s logs fairly regularly. Given that right now this is a brand spankin’ new blog with a pitifully small readership (Hi, mom!), seeing a new reader (even just a casual drive-by reader) is interesting to me.

I just noticed someone bounced by the site, having gotten here via a Yahoo search for the question above. Just in case they come back (or anyone else comes by and is interested, for that matter), here’s the answer – at least to the best of my knowledge.

Have you ever thrown a piece of meat into a hot pan or barbeque that wasn’t sufficiently greased? You notice how it sticks and doesn’t want to come off? That’s what heat-fixing is for. It basically “bakes” the bacteria to the surface of the slide, so that when you then soak the slide with stains and rinse it with water and/or alcohol and/or other substances (like the “acid alcohol” stuff used for the “acid-fast” stain for Mycobacteria) they don’t get washed off. This can be an issue, since some of the staining techniques have a whole series of “soak/rinse/soak/rinse/soak/rinse” kind of steps with different kinds of solvents, and it would be very annoying to go through all that work and find out you’ve rinsed the stuff you wanted to look at down the sink in the process. It’s also nice if you want to look at the slide with an oil-immersion lens.

You can’t really “glue” the bacteria to the slide with some sort of chemical, either, since anything you “glue” them with might cover them and interfere with stains that you’re trying to soak them with so you can see them in the microscope.

Actually, it’s probably worth mentioning that since most sources seem to unfortunately assume that “microbiology” just means the tiny fraction of a percent of microbes that cause diseases, a lot of sites will also say that the heat-fixing process is also “to kill the bacteria” (so that if you are overcome by an uncontrollable urge to lick the slide later or rub it on an open wound for good luck or something, you hopefully still won’t get the disease). While it’s true that heat-fixing ought to kill just about any microbe on your slide, I suspect that most of us who are looking at things that aren’t disease-related probably don’t consider this a “purpose” of the heat-fixing process. Still, if you’re answering a question like this on a “General (medical-centric) Microbiology”-type exam, you may want to mention this as well.
(UPDATE 2010-08-25 I dug up some Real Science™ on this “killing the bacteria” idea in “Stir-Fried Stochasticity Episode 4: TuberculosisBurgers“, which is an amateur podcasting project I’m dabbling in. I’d very much appreciate feedback on it!)

Hopefully that information will be of interest or use to someone…

Coming up next – some commentary on the history of staining bacteria, and why it seems like all of the classic techniques of microbiology – most of which seem to still be in common use – seem to have been invented entirely in or near Victorian-era Germany…

Also possibly coming soon: Discussion of the “Everything is Everywhere” concept, making chemicals with bacteria, and (if I can manage to get the thought into some organized form) a simple discussion of the concept of speciation, using science itself as an example. And various other things as I think of them and get time to type them up.