Day: May 31, 2008

“Antibiotic Susceptibility Testing by a Standardized Single-Disk Method”

Okay, one last post in the Classic Science Papers challenge before my time’s up:

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.

Petri dishes containing bacteria, showing inhibition of growth by certain substancesThe “Kirby-Bauer” antibiotic susceptibility test is another standard method that you should cover in microbiology class. The method involves getting a pure culture of the bacteria you want to treat, and then growing it in a petri dish. By putting paper disks soaked with various anti-bacterial substances, you can identify which ones are most effective at killing (or at least stopping) the bacteria in question – for example if you’re trying to figure out what kind of antibiotic to give to the guy coughing up some unknown plague in your doctor’s office… The anti-bacterial substance that the paper is soaked in slowly diffuses into the area around it on the petri dish, getting more dilute the further it gets from the paper. You can then estimate how powerfully anti-bacterial the stuff is by how far from the paper the bacteria stop growing.

The authors here didn’t invent this trick. Not all antibiotic-susceptibility tests are “Kirby-Bauer” tests (the blurry picture there is of an experiment I did involving the beer ingredient hops, and is not a Kirby-Bauer test. Click the picture to go to my “Beer Cures Anthrax” post from long ago…). What this paper describes is a method that finally standardized this test. Instead of having to use multiple paper disks with different amounts of the same substance, the “Kirby-Bauer” test prescribes specific concentrations of each antibiotic, and specific nutrient agar formulations, and so forth, so that determining which antibiotic your mystery bug is best treated with can be done in a way that gives consistent results regardless of who is performing the test.

The method is regularly updated to account for new antibiotics, but is still referred to as the “Kirby-Bauer” antibiotic susceptibility test to this day. Incidentally, the American Society for Microbiology kindly hosts a reprint of this paper as a .pdf file, so you can read it yourself if you’d like.

(UPDATE 20110328: new URL for the reprint of the paper. Thanks, Alex S!)

“A simplified method of staining endospores”

One more for the “classic papers” challenge:

Schaeffer AB, Fulton MD: “A Simplified Method of Staining Endospores”; 1933; Science; 77; pg 194

If you take a microbiology lab, this is the endospore staining technique (or “technic” as they used to spell it) that you’ll practice. This is a nice, simple, one-page paper. Alice B. Schaeffer and co-author Mac Donald Fulton describe a few of the other variations on endospore staining techniques, then describe how they’ve further simplified what they felt was previously the simplest one, described by a Mr. Wirtz in 1908.

“Endospores” are a sort of “escape pod” for certain specific kinds of bacteria. Unlike spores formed by yeasts and molds, these are not reproductive – each bacterium only produces one thick-coated spore, into which it shoves it’s genetic material and a few vital enzymes to get itself going again later when the spore finds itself in favorable conditions.

Since only a few kinds of bacteria produce these endospores, if you see endospores in your unknown bacterial culture it goes a long way towards helping to identify the bacterial species, so having a simple method for staining your bacteria so that endospores are obvious under a microscope is helpful. (Of course, these days most of us would rather just get a 16s rDNA sequence with PCR, but never mind that for now…)

Endospore stain under a microscope (via Wikipedia)Evidently, Wirtz’s original method involved using Osmium Tetraoxide (“osmic acid”) to stick the bacteria to the slide before staining. Not only is that stuff poisonous, it’s also expensive. I found a site selling sealed glass ampoules containing 1 gram each of this stuff for $35.00 each. Schaeffer and Fulton’s method does away with this in favor of much cheaper and easier heat-fixing (just as is done with the Gram stain and others). They use the dye “Malachite Green” for the initial stain, and steam-heat the dye-covered bacterial slide a few times to sort of “cook” the dye into the thick-walled endospores if they are there. Rinsing then washes the dye out of everything but the endospores, and a light red dye (safranin) is added as a counterstain. The end result is that under the microscope you’ll see light-red bacteria. If any of them form endospores, you’ll be able to see them as smaller green dots – sometimes still bulging inside of bacterial cells, sometimes floating around freely having escaped from the now-empty bacterial cell.

The “Schaeffer-Fulton Endospore Stain” is pretty easy to do, though the occasionally messy steambath part can be annoying. The method is pretty resistant to errors, so it’s not too hard to get good results even if you’ve never done it before.

Incidentally, you can buy Malachite Green at many pet stores – it’s still used as a treatment for “ick” (Ichthyophthirius infestation) in tropical fish.

Hmmm…still a couple of hours before it’s not longer May – Perhaps I can throw in one last post before time’s up…

I guess now I find out how good or bad Magellan, inc. is.

A couple of months ago I traded up from my Garmen etrex Legend to a Magellan eXplorist 600. I wanted to get away from Garmin due to a couple of irritating limitations that they seem to cling to (like losing your timestamps if you save an individual track, lack of SD card support, and dropping NMEA data support “to support [their] software” [see the review in Make #1]). So far, I’ve been pretty pleased with the shiny new Magellan. Though their software (as usual) only works on Microsoft Windows, I didn’t buy it for the software, and GPSBabel handles the data conversion of the files just fine.

So, naturally, now that I’ve just started blogging with the track data I’ve been recording with it, it has died on me. Yesterday – worked fine. Today – won’t turn on or respond in any way.

The good news is that I DID register the thing online at Magellan corporation’s website, and it’s supposed to have a 1-year warranty. Support’s only available Monday-Friday, though, so I’ll have to wait until Monday to see a response to my support query. Wish me luck. I’ll post updates in case anyone is interested in how Magellan responds.

In other news, “BigC” followed up on my previous queries about digital microscopes and Linux, saying that the manufacturer is now offering a Linux-compatible version of their AM311S model (the Linux-compatible one is the “AMU311S”) by special-order. Only goes up to 200X magnification, though, so it’s not real useful for my microbiological purposes. I can’t say the manufacturer of these digital scopes is impressing me much, but BigC certainly seems to be going out of their way to give good service.

There’ll be at least one other post later today – I wanted to get some more “classic” microbiology papers blogged before the day was up. Kirby-Bauer or Schaeffer-Fulton?…