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.

Phlogiston and Aether

No, that’s not the name of an obscure pair of mythological Greek characters. Nor do they really have much to do with microbiology, either, but they’re kind of interesting, now-obsolete scientific theories. And since I mentioned them both in the last post, I may as well say something about them.

It’s worth mentioning that being a “theory” is a big deal, scientifically. I means something that’s been examined and tested repeatedly and still appears to correctly describe and predict natural events.

Phlogiston seems really bizarre in a modern context. Phlogiston was supposed to be a sort of “fluid” that came out of things when they burned. It was invented to explain the observation that if you burn something in a sealed container, it will only burn so much and then stop, until you give it fresh air. The theory held that the air could only hold so much of the phlogiston, and when it was saturated with it (“phlogisticated”) it simply wouldn’t support combustion any more.

You can test this, of course. If this hypothesis is accurate, then if you have a lot of air but just a LITTLE bit of material that you burn in it, then the air will still be partly “dephlogisticated” and if you then add more fuel it, too, will still be able to burn. Naturally if you actually try this, that’s exactly what you see – the theory accurately predicted what would happen.

Of course, modern chemistry knows that what’s really happening is that oxygen in the air is combining with the fuel being burned – “stuff” is going from the air to the fuel. (That’s not the first time a popular scientific theory turned out to be exactly the opposite of correct.)As chemists did more experiments they were able to show more cases where phlogiston theory no longer predicted correctly, and it was abandoned.

Aether (I spell it that way rather than “ether” do better distinguish it from the modern meaning, which is used in organic chemistry) has to do with light. Leaving out all the historical details around it, since light seems to be a wave (okay, AND a particle, but never mind that for now), and since waves have to travel through some kind of medium (like sound travelling through air, or a wave travelling through water in the ocean), there must be some kind of “stuff” that light waves are going through, right? Well, that “stuff” was referred to as “luminiferous aether”.

This one is somewhat trickier, but basically, fancy experiments with lasers which ought to have shown some effects of “aether” if it existed didn’t show anything. I’m not a physicist, so I’ll stop here before I badly butcher an attempted explanation of what was tested and how it failed to demonstrate existence of “aether”. If you’re interested, the relevant experiment is the Michelson-Morley experiment. Of course, if the universe actually IS geocentric, then the test results merely demonstrate that the aether is fixed with respect to the Earth…

I’m not even going to attempt to explain the whole “it’s a wave AND at the same time it’s a particle” thing. It’s kind of like how Schroedinger’s Cat is both dead AND alive at the same time until somebody checks on it. I think to really explain it you have to fluently speak a specialized made-up nerd language because normal speech doesn’t seem to handle it very well…

Actually, in the unlikely but not impossible event that someday, a physicist happens to read this who can explain “wave-particle duality” comprehensibly using only plain English prose I’d be interested (I ‘get’ how light is both a wave and a particle simultaneously, I just don’t get why.)

Obsolescent terminology: “Schizomycete”

I couldn’t resist doing a bit of research to track down what “schizomycete” meant. I can’t help it, it’s fun, and in this case also was both informative and amusing. Don’t tell the Psych department, though – I think the MMPI probably formally classifies this kind of thing as a perversion…

In any case, here’s what I’ve come up with.

First, a simple Google search turned up some online “medical dictionary” sites. Where they had a definition of the term, they just said it was “a class of bacteria” (or something similarly vague). However, neither the NCBI Taxonomy Browser nor the RDP Heirarchy Browser seemed to have any kind of category called “schizomycete” (or “Schizomycetales” or similar variant). Obviously, the term is no longer in use.

Although my academic interests are specifically not medical in nature, I do casually collect old medical (and scientific) books. So, I dug out my handy 1953 “Stedman’s Shorter Medical Dictionary (“Revised and Enlarged”)” and looked up the term. Here’s what it said:

“A class of vegetable organisms which reproduce by fission; fission-fungi or bacteria.”

Fission-fungi“???Okay, I can excuse such an obviously archaic term in a dictionary from 1953 – at that point, Watson, Crick, and Franklin hadn’t even puzzled out the structure of DNA yet (in fact there aren’t even entries for DNA or Deoxyribonucleic acid in this edition). What’s funny, though, is that I stopped in on the campus library and looked at the current edition of Stedman’s Medical Dictionary and the famous Physician’s Desk Reference. The current (2006) Stedman’s still has an entry for “Schizomycete”, as well as an entry for “schizomycetic”. “Schizomyces” now says:

“Member of class schizomycetes; a bacterium”

Even funnier, “Schizomycetic” says:

“Relating to or caused by fission-fungi (bacteria)”

Okay, the fact that they’re happily referencing a bacterial “class” that was rendered obsolete and nonexistent decades ago is funny enough…but they STILL refer to “fission-fungi”? Unless I’m mistaken, that would be like looking up a current “Dictionary of Chemistry and Physics” and seeing an entry that describes some optical phenomenon in such a way as to make reference to “the Aether”.

I was perversely even more amused to see that, as I recall, the famous “Physician’s Desk Reference” happily parroted the definition word-for-word.

Judging by the occurrence of the term in Pubmed, the term “Schizomycete” disappeared sometime in the mid-1980’s, and even then was used almost exclusively in Italian journals (presumably it was just slower to drop out of the language than in English). I did find, however, a series of articles on bacterial taxonomy which started in 1916, and included an entire article from 1917 on “Schizomycetes”[1]. Being 90 years old, this is a mature public-domain work so you can make, print, and share all the copies you want (Even in the U.S., believe it or not). Pubmed has a copy here if you’re interested.

In the end, though, here’s the summary:

Even when it was in common use, it seems like “Schizomycete” was an unreliable term. It seems to have been in use during a time when there was a lot of argument over how to categorize microbes. The 1917 article has a whole section discussing the historical meanings of the term and where various researchers drew the lines of what was a “schizomycete” or not. In general, it seems to have usually meant any relatively “ordinary” bacteria that didn’t produce chlorophyll (that is, wasn’t some kind of “algae“)

In honor of the completion of this batch of grueling and difficult research, I hereby declare by the power vested in me that henceforth “Schizomycetic” shall be defined as “pertaining to any relatively unremarkable-looking prokaryotic organism” and that all microbiology-related professionals should be compelled to use the term regularly. I further decree that the terms “aerobic” and “anaerobic” shall be replaced by “dephlogisticated” and “phlogisticated”, respectively.

Incidentally – Hello, “Tangled Bank” readers! Comments, suggestions, and corrections on any of my postings (or this blog in general, for that matter) are welcome and encouraged, in case that isn’t clear.

[1] – Buchanan RE, “Studies in the Nomenclature and Classification of the Bacteria: II. The Primary Subdivisions of the Schizomycetes.” Journal of Bacteriology. 1917 Mar; 2(2):155-64

Ye Olde Science Paper?

As I finished up the report for the second Pathogenic Microbiology lab, I found myself – again – wanting access to a classic scientific paper.

I mean really classic. I mean, all professional microbiologists know what a Gram stain is, but how many have actually read Gram’s “Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten” Published in “Fortschritte der Medizin” in 1884.

I wonder what would happen if I asked the college library to get me a copy via Inter-Library Loan?

A short post on what the heck “Schizomycete” means may be in order later, too.

Ah, a good-natured rant

One of the sources of my daily metaphorical firehose of information that I try to take in is the RSS feed for the blogs at Scienceblogs.

One of the sources of my nearly-daily metaphorical itchy rash of irritation that I encounter along my studies is the fact that as far as most people are concerned (including, most importantly, the people responsible for the required “Microbiology” degree curriculum), “Microbiology” is essentially the study of diseases.

These two collided today when a professor of Epidemiology who is a blogger on Scienceblogs posted offering “Everything you want to know about Microbiology and Epidemiology[…]”. A perfectly reasonable post (downright laudable, actually – it’s part of an effort by several of the bloggers there to be try to get posts going offering explanations of basic concepts in their respective fields). Nonetheless, I couldn’t resist mentioning that what I wanted to know was what it would take to get people to quit assuming microbiology was about diseases (while acknowledging that an epidemiologist was probably not the most sympathetic person to ask…). Hopefully the mini-rant came across in as good-natured a manner as I’d intended.

Those bored enough to care can read it here (the main post is here).

(At least) one of the other organisms…

Which I think I shall designate as Ulysses (the Ubiquitous). This sample came from what appeared to be an individual colony on an agar plate made with ~8% salinity diluted Great Salt Lake water. I tried to ‘pick’ a colony with the tip of a hypodermic and ‘poke’ it into the anaerobic tubes. Guess it may have worked, at least with a few cells. (This was done 6 months ago, so if I even got only a few cells over, it’s had plenty of time to grow…) The picture’s a little overexposed (and I tweaked the contrast) though…

I really have no way of knowing for sure, but there does appear to be the same “mass of short rod-shaped bacteria attached to the iron oxide particles” in all of the samples I’ve “live/dead” stained and examined in the microscope so far. So far this is true in every salinity I’ve checked, from about what I estimate to be about 8% salinity (a little over twice as salty as regular ocean water) all the way up to about 27% (pretty much as much salt as you can PUT in water at room temperature and normal pressure). Through the entire range, I see what appears to be the same size-and-shape organism attached to the particles. That would actually be quite interesting. The highest salinity I’ve run into in the “literature” for an organism to be actively growing is about 20%, assuming I did the conversion from “g/L” correctly.
Speaking of which, it turns out the water in the Great Salt Lake is essentially the same as seawater, just saltier (that is, the minor salts in seawater besides “sodium chloride” are also nearly identical in proportion in the lake. There’s just more of it.). Assuming I did THOSE calculations correctly, the amount of sulfate in the water works out to about 0.22M. That’s roughly 8 times as much as in normal seawater – again, assuming my hasty formula-fondling didn’t have a lot of errors.

In any case, there’s a metric [pants]load of sulfate in the water. That means there’s PLENTY of it there for the microbes to breathe and turn into sulfide. No need for any of them to breathe the iron then.

I haven’t given up on that totally though – apparently molybdates can inhibit sulfate respiration, or so I’m told, so I can try making up a solution with some sodium molybdate added. Alternatively, now that I know the Great Salt Lake is very nearly just “concentrated seawater”, I can always make up some “artificial” sea salt, but substitute something else for most of the sulfate and see if things still grow on the iron oxides. That’s likely my next step as a last try to see if any of my Babies can actually breathe the iron. (This is in addition to trying to purify some DNA from Ulysses there, since I think that one is a pure culture.

“Bendy Benjamin” or “Flatulent Fred”?

I was doing a bit of poking around today, and now I’m wondering if the funky-looking spiral/bendy bacteria are of the genus Desulfovibrio. The cell morphology kind of fits – and there’s good evidence that there’s sulfate being reduced down to hydrogen sulfide in the samples (it’s possible there are no iron reducers at all, really, and that the iron reduction is just being caused by reaction with the hydrogen sulfide.) I should mention, though, that this is pretty audacious speculation, since it’s essentially impossible to identify anything based on just looking at it in a microscope…
If nothing else, though, if it IS a “desulfovibrio” type bacteria it might be the most halotolerant one described so far. I wouldn’t be surprised if I’ve actually got a few different sulfate-reducing bacteria growing in there.

That’s something I like about scientific research – even if you don’t get what you’re looking for, you can still find interesting new things (I’ll be a little disappointed if I can find no halophilic iron-reducing bacteria in the samples, but it would still be spiffy to just manage to isolate and identify some previously unknown bug. Or in my currently more-easily-pleased case, just to isolate an odd organism to add to the culture collection I’d like to start…

Hopefully more time at the microscope tomorrow, so maybe I’ll have more (and better) pictures.

“Where Was I?” Femto-episode 20070109 – Astronomy or Microscopy?

I got a chance to check out a bunch of my Gunnison Bay anaerobic iron-reducing cultures in the microscope.

I did a “Live/Dead stain and took a look:

Here you see an image from the Hubble Space telescope of a nebula called…oh, wait, that’s from the microscope. Never mind.

These are the (allegedly) “live” cells. The bright circles are out-of-focus cells – this is a “Wet mount” with huge chunks (from a microscopic point of view) of iron oxides in it, which makes the sample thick enough that it’s hard to focus on more than one part at a time. The vague blur that looks like a gas cloud in a nebula is what you can make out of the chunk of what is more-or-less just wet rust.

This particular specimen is from the positive control of my ongoing experiment (in what I estimate to be roughly 18% salinity, assuming my original Gunnison Bay samples were around 25-26% salinity). If you look close, you can barely make out the bendy/spiral shaped cells almost in focus in a few spots. I believe that this might be the iron-reducing organism that I’ve been trying to isolate. In one of the other samples I checked, I managed to spot one actually actively swimming around, corkscrewing its way around the slide.

I checked one of the experimental specimens that my hypothesis predicts will be growing at least one (and ideally ONLY one) kind of organism and it, too, has a lot of “live” cells growing on the rust-chunks.

I think I shall designate this as-yet-unidentified strain “Bendy Benjamin“…

Other samples from the same place (but not subjected to precisely the same conditions) also seem to have some long straight rod-shaped bugs and some short (straight) rods also still alive in them, so even in the pretty specialized high-salt/no-oxygen iron-oxyhydroxide-being-reduced environment I would guess I’ve got at least three different viable organisms that MIGHT be doing the iron reduction that I’m trying to track down the cause(s) of.

Any questions? No? Does this mean you’re all ready for the quiz?…