Amateur Soap Microbiology and my new Friend

I thought soap was supposed to be *clean*!

lumpy yellow microbial colonies growing on the soap inside of a hand-soap dispenser
People usually assume soap gets rid of funky microbes that might grow on things, so I was very amused several months ago when I spotted something growing on top of the soap in one of the household hand-soap dispensers. As of today, it looks as pictured at left. That lumpy yellow and brown mass atop the the soap looked to me like some sort of soap-sodden mold, and have been saving the dispenser specifically in the hopes that someday I’d have a microscope and could take a look at it. Meanwhile, the mass spread, and slowly started releasing some kind of yellow pigment into the soap.

Incidentally, I kind of doubt this indicates some sort of failure on the part of the manufacturer of the soap. I don’t recall for certain, but I think I may have opened the dispenser at one point to transfer some of the soap to another nearly-empty dispenser. When the mass started growing originally, it was a single spot, which suggests a single spore or speck of dust floating in and landing on the surface. Hey, it happens. Anyway, I’ve therefore blanked out the name of the manufacturer since I don’t think they really have anything to do with this.

VWR VistaVision Microscope
This mysterious growth upon my soap remained mysterious until today. Thanks to the Minister of Domestic Affairs and VWR (who managed to find me a really good deal), I finally got to actually get a close look at that lumpy mass. Meet my new friend Minnie (pictured at right). I could gaze into those eyes for hours. I couldn’t afford a darkfield condenser, and I sure as heck couldn’t afford to upgrade to phase-contrast gear, but I can add either one later if the opportunity presents itself. I also can’t afford the overpriced proprietary digital camera attachments either, though working around that is a whole other project. Until I identify an affordable model that plays well with Linux or work out how to modify a webcam into an ocular attachment,
I’ll have to settle for a trick…

It turns out if you take a digital camera and set it for close-up photos, you can actually stick the camera lens right up to the eyepiece and often get a serviceable picture.. Now, I had to subject the pictures I got today to moderately heavy processing to bring out the detail a bit better, but at least part of that is just me working on learning how to optimize the camera settings for this kind of use.

Equipped with some surplus slides and cover-slips donated by a kind professor who had some extra packages, I opened up the soap container and smeared a little of the yellow crud on a couple of them. One I just slapped a coverslip on for direct observation – the other I smeared over a slide and let dry with the intention of staining using the tiny, previously-unused vial of methylene blue left over from a very old plastic toy microscope. While the latter dried, I took a look at the wet mount hoping to finally see the mold mycelia that I had been expecting…

There wasn’t enough contrast to bother trying to get a photo, but it was obvious at 400x that what I was looking at was bacteria, not mold. Nerdly joy at learning something by looking in the microscope that I wouldn’t have otherwise known ensued, along with happiness as I realized this meant I had a perfect excuse to dig out my recent shipment from the Maker Shed – materials for doing a “Gram Stain”. Incidentally, the “Maker Shed” had the supplies on the way to me within hours of my ordering it, and they have lots and lots of cool stuff. I highly recommend it. Anyway, I got to do a “Gram Stain” for the first time in a couple of years (and the first time ever outside of a school lab). Want to see?

Mystery Microbe, I see you!

Gram-stained bacteria
Here it is – the nasty yellow goo that infected my bottle of hand-soap. My staining technique was a little off since I’m out of practice – the way I interpret the results is that what I’ve got here is neither a member of the Firmicutes (i.e. “Gram positive”) nor – probably – Actinobacteria. I really can’t guess at more than that, though. I think the few “Gram-positive”-looking cells there are artifacts of insufficient decolorization. I know I still had a surplus of the purple “Crystal Violet” stain still on the slide at the end. (How did I know? I’ll show you at the end…). The irregular bluish bits towards the bottom are, I believe, just bits of stuff from the soap itself.

Meanwhile, this pretty much satisfies my curiousity about the Mystery Soap-Infecting Microbe. There’s certainly a lot more I could investigate, but my developing Hillbilly Biotech lab is really intended to support my interest in intentional food microbiology and perhaps evenutally some small-scale non-food industrial microbiology. I have some remaining curiousity about the yellow pigment and whether or not it might be useful for something, but I’m doubting there is any food or beverage I might want to grow this stuff in and therefore don’t have much use for it. Still, I’ll keep the bottle around for a while before I throw it out in case I think of something fun to do with it. If I end up being really interested in the identity of the bug growing on it, I should be able to find a liquid that I can grow a big mess of it in, then run it through a simple DNA extraction process. Then all I need to do is find someone who can supply PCR primers, a thermocycler, and sequencing services cheap. It might sound like I’m being facetious, but I wouldn’t be surprised these days if I manage to find somewhere that’d do it for $20/sample or less. I may eventually do this will the Mystery Soap Bug anyway, since I hope to be running through this process with cultures of sourdough, yogurt, cheese, vinegar, and brewing microbes that I develop myself. For now, though, it’s just nice to be playing with microbiology equipment again. And now fully independently! Wheeeeeeee!!!!!!

Yes, I’m a nerd. And proud of it!

What’s next?

Now that I finally have a microscope, I no longer have any excuse for not getting to work on the rest of my Hillbilly Biotech lab. Just this weekend I was pricing out Hillbilly Autoclaves. I picked up a cheap air pump and air stone
for potentially building an aerobic bubble-column fermenter (for quick growth of yeast starters or a working model of a “Fring’s Acetator®”-style vinegar generator. I still want to build an ozone generator for sanitization and to get a pH meter. I’d like to also get my hands on some wheat, barley, and rye seeds to sanitize, sprout, and grow here as the first stage of developing a truly local sourdough culture, plus arrange to have several pounds of plain flour irradiated to sterilize it.

I’m also like summer to be over. Yes, I’m writing this in Winter, but it’s not until later in the summer to autumn that locally-grown fruits will start becoming available, and locally grown fruits ought to be an ideal source of local brewing and baking yeasts and bacteria. Finally, I’d like to find a wealthy patron (or matron, I’m no sexist…) who would sponsor me so I could just pursue food-microbe bioprospecting and research full-time…

Oh, yes, and I need to get around to finishing Episode 4 of my little podcast project, especially since episode 4’s topic is a fundamental microbiology technique.

Comments welcome below – thanks for reading!

Oh, and as a reward for getting all the way to the end, here’s a picture that I thought was pretty – crystals of “Crystal Violet” and iodine. I told you I had too much left on the slide…
Crystallized dye left on the slide

“Untersuchungen über Bacterien”

Cultures of Blastomyces dermitiditis, showing how it grows like a mold at one temperature and like a yeast at another.Once again I’m down to the last minute, trying to juggle too many things and almost missing this month’s “Giant’s Shoulders” blog carnival. Almost.

Today we go once again all the way back to the Victorian era, to see that if you thought bacterial taxonomy was difficult now, imagine what it was like over 130 years ago:
Cohn F:”Untersuchungen über Bacterien”; Beiträge zur Biologie der Pflanzen; 1875, vol 1; pp 127-222
(Or “Researches regarding Bacteria”, in “Contributions towards the Biology of Plants”)

This paper is an overview of the problem of categorizing bacteria among the types of living things, and makes some early suggestions. I don’t think it’ll spoil too much of the punchline to point out that not only is the journal about the biology of plants, but the paper also starts out with Cohn describing how he came to work at the “plant physiological institute”. Cohn’s assertion that bacteria are definitely a form of plant actually stuck for at least another three-quarters of a century or so – I have a copy of a 1945 book on bacteriology that actually has a short discussion on why bacteria are categorized as plants rather than animals (or “animalcules”, even). That’s only part of what’s interesting about this paper, though.

Cohn discusses a number of problems with the nature of bacteria in his time. For one thing, he says there had been little real effort to even come up with a coherent scheme for classifying bacteria at that point. He does mention one previous attempt to come up with a system, but on the whole it seems everyone is just coming up with terminology on the fly – even taking Pasteur himself to task for throwing around a variety of terms related to microbes without distinguishing what the terms actually refer to. The reason for this, really, is just that figuring out pretty much anything in detail about bacteria was a seriously difficult problem at the time. Cohn explains why; how it is really impossible to make out more than general shape and size from microscopic examination, and how the lack of any detectable sexual reproduction makes it impossible to do positively identify members of the same species. In fact, even very obvious differences in appearance might not be definitive. It was suspected (and later demonstrated) that some of what appeared to be completely different fungi were actually just different life-stages of the same fungus. (Hopefully you can see the picture at upper right, with the bacteria/yeast-like growth on one tube and the obvious and very different fuzzy mold-type growth on the other. Both are actually the fungus Blastomyces dermitiditis.) Just as some of Cohn’s contemporaries considered that perhaps all molds and yeasts were really just different stages of life of the same organism, perhaps the same might also be true of bacteria?

Cohn does, after all, promote the notion of bacteria as a type of fungus. You may even remember an old word for bacteria: Schizomycetes, that is “fission-fungi” (that is, fungi that reproduce by splitting in half rather than producing spores). This makes sense if you consider that bacteria are more like plants and algae than animals, and fungi were considered to be plants that lacked chlorophyll. Although lamenting that it was not feasible to really separate out individual bacteria to determine whether they ever changed form – this was still three years before Joseph Lister actually did so – Cohn unwaveringly felt that bacteria were in fact made up of several different genera and species, and set out an early attempt at classification.

Once again, Our Friend the American Society for Microbiology hosts a translation of this paper, complete with a couple of paragraphs of more modern editorial commentary at the end. It’s well worth a look.

A photographic portrait of Ferdinand Julius CohnUnfortunately, I don’t think Ferdinand Cohn’s hairstyle is nearly as spiffy as Eduard Buchner’s cool “Colonel Sanders Guest Stars on Miami Vice” look. I think he looks kind of like a slightly-better-fed Sigmund Freud with a bad comb-over. But that’s just me.

Obscure scientific papers, Mad Science, Travel, and other randomness

First – an amazingly astute observation that I’m ashamed to have not previously noticed myself (click image to go to it’s original site and see it full-size…):
Most 'Mad Scientists' are actually just 'mad engineers'...

I’m proud to say that I think testing Mad Hypotheses is great, and will continue to try to be a Mad Scientist. And a “Dirty Old Man” someday, but that’s a whole separate issue.

Second – I am really loving the perks of my new job – namely access to the college library system. I had previously mentioned (see last couple of paragraphs) a certain article that I wanted to get my hands on:

Greenberg LA:”The Definition of an Intoxicating Beverage”;Q J Stud Alcohol. 1955 Jun;16(2):316-25

Not only does the medical library have copies of a Czechoslovakian microbiology journal, the main library had a set of this old journal, too. I have my bedtime reading for tonight…

Thirdly – Another Giant’s Shoulders carnival has come and gone. I now believe that Eduard Buchner had hit upon not only a useful truth of living systems, but also a nifty alternative “mad scientist” hairstyle. Now I need to come up with one for next month. It’s been getting me thinking, though. That blog carnival is intended for “Classic” papers. Implied is that the papers are somehow important to the development of some scientific field or other. I’d like to see a variation on the “old papers” theme focussing on random old papers (where “old” might mean a few years or decades, depending on the subject) that people have found useful or interesting. Stuff that isn’t necessarily ground-breaking and has perhaps been forgotten or lost to obscurity but still has useful things to teach us. Naturally, I’m thinking especially Microbiology (and especially Microbiology other than Medicine) and Food Science. The Carnival could be called something like “Second Chance Science” or something of the sort. Just a thought.

Fourth – speaking of “Microbiology Other Than Medicine” and Food Science, apparently The National Academies of Science want to know what scientific topics people most want to read about. As usual, “microbiology” appears to have been relegated in their breakdown to merely a subset of either medicine/diseases, “biology”, and perhaps a small subset of “energy” and “Feeding the World” (no, seriously). The survey includes space to tell them what they’re missing – I heartily encourage anyone who cares to make sure you take the survey, and mention industrial and environmental microbiology and food science as subjects they shouldn’t continue to neglect.

And, finally – next week I need to make a very-long-overdue run back up to Idaho to grab some things from the old house and make sure it’s still standing, the water’s really turned off, nothing unnecessary is running, etc. 1600 miles of driving each way. Ugh. Anybody got any good recommendations for things to listen to on the trip? Other than having a chance to finally grab some things that I am missing, maybe I’ll at least have a chance to visit New Belgium Brewing Company again, since my route goes right past it. So long as I’m not driving by on Christmas day (when I assume they’ll be closed) I may have a chance.

Saccharomyces cerevisiae – Shameless Libertine!

I’ve been wondering about starting my own little yeast-breeding operation. I haven’t yet figured out where you can by the necessary teeny, tiny yeast-sized versions of the Implements of Extremely Impolite Probing that breeders of other species use, but even before that, I need to understand yeast reproduction better in the first place.

I had gotten an impression from some of the stuff that I’d read in a Genetics textbook and online that yeasts were normally haploid, and only became diploid briefly during mating (you see, when an “?” haploid yeast cell and an “a” haploid yeast cell fall in love, sometimes they’ll…). On the other hand, while reading a review of yeast virology[1], the author explicitly wrote that yeast cells are normally diploid. How to resolve this issue? Plus, I was wondering how, if I happened to have a pure culture of a haploid yeast, how could I tell if it was “a” or “?”?

I recently realized that there was one Dr. Bryk in the department where I work who specifically researches yeast chromosomes…so I asked her…

Continue reading Saccharomyces cerevisiae – Shameless Libertine!

Nerd Reading Spasm!

Did I mention the place I work has some amazingly spiffy perks for a nerd like me?

Last night, I was poking around pubmed looking for references to yeast and erythritol (namely, do yeast interact with it, and will they metabolize it?) I found precisely one relevant reference. From 1975. In a Czechoslavokian microbiology journal. A no-longer-existent Czechoslovakian microbiology journal. Even though it was a journal published in English, I didn’t figure I’d be able to find the article I was looking for. It did turn out that the greedy (insert long string of profanity here) anti-open-access “SpringerLink®” Netherlands organization has an electronic copy of the article…which I can get limited access to for a short time for a mere $34.00. Not going to happen, obviously.

Just in case the college had a subscription that would let me get to the article at no extra cost, I checked. No such luck. But…

…The campus medical science library just two buildings over from where I work has dead-tree editions of essentially the entire journal! Im name des Nudelmonster! Instead of paying $34.00, I got a photocopy of the article for about $0.50. Bonus: As I had hoped, the article[1] reports that erythritol is not metabolized by yeasts, although it is taken up to a small extent. That means I can add erythritol (or xylitol or sorbitol or whatever) to must or wort, and it’ll still be there when the yeast finish, leaving the resulting beverage still sweet. Hooray!

Plus, I was also able to get access to an electronic copy of a review of the uses of poly-?-glutamate[2], which I was bemoaning not having access to over on an interesting Small Things Considered post recently.

Speaking of reading, one thing I really could use are any worthwhile books on the general subject of applied/industrial microbiology, bioprocess engineering, fermentation, and so on. “Worthwhile” here means practical texts that are A)primarily about microbiological processes (as opposed to, say, bioengineering of plants) B)Reasonably technical, and C)Either “not very old” or “very old indeed” (I collect old science books).

I’m not a fan of’s abuses of the patent system, but I’m in a hurry since it’s past my bedtime already. Therefore, purely as a sampling of the kinds of books that sounded interesting to me, here is a selection in more or less random order of books that came up in a quick search on Anybody out there have any other suggestions?

Continue reading Nerd Reading Spasm!

Why Benzoic Acid Works: Part 1 – “Some Boring Review Material”

It’s about time I got to the long-promised post about benzoic acid. The thing is, I don’t want to assume everybody reading this is well-versed in chemistry or anything, so after much thought I’m going to split this into three posts. This first one is a bit of chemistry review for some topics that are important to how benzoic acid acts as a preservative. People who are bored by this or know more about it than I do are welcome to either wait for the next post or leave corrections or questions in the comments as you see fit. (Brief note to people reading this from the RSS feed – I’ve noticed that the stylesheet information doesn’t transfer with the RSS, so you won’t see where the web page view would indicate that there is additional information available for some of the terms here. Try hovering over various words and phrases in this post, though, and the information should pop up if it’s there…or just pop in at the main site and post questions if you have any.)

There are several ways people separate types of molecules into opposites. For example, ionic vs. covalent, polar vs. non-polar, or hydrophilic vs. hydrophobic. Although these three categories are each a little different from each other, they all relate to the same thing. As with all other chemistry, it all has to do with what the electrons are doing.

When atoms react with each other, they have a big fight over each other’s electrons. The reaction “finishes” (reaches equilibrium) when this custody battle is concluded. Each of the three categories above relate to how equitable the electron-sharing arrangment ends up being. Once the molecule’s atoms arrange themselves, if the custody of the electrons is distributed fairly evenly around the entire molecule, the molecule is considered “non-polar”. On the other hand, if the atoms at some corner of the molecule end up with more custody of the electrons than the other areas, the molecule ends up having an end that’s slightly more negatively charged (remember electrons are arbitrarily defined as being “negative”) than the others, and the molecule is “polar”. If you dissolve that polar molecule in water and the atoms remain together stubbornly clinging to the shared electrons, the molecule is considered “covalent” (“valence” refers to the area around atoms that electrons “orbit”), whereas if one or more of the atoms readily gains or gives up complete custody of one or more electrons and drifts away from the rest of the molecule, the molecule is considered “ionic”. (It amuses me to think of these latter two terms as “homoelectrical” and “heteroelectrical”. Yes, I am easily amused, why do you ask?) Plain old table salt is what you get when atoms of Sodium (“Na“) and Chlorine (“Cl”) get into one of these electron fights. If you were to look at a Periodic Table of Elements, take a look at the column way over on the left, with Sodium (Na) and Potassium (K) and so on. All of these have one electron that they just don’t really give a crap about. Way over on the other side of the table, one column over from the far right, you’ll see Fluorine (F), Chlorine(Cl) and so on. All of THOSE desperately want an extra electron (Chlorine is the third most electron-greedy – “electronegative” – atom, behind Fluorine and Oxygen). Stick Sodium Chloride in water, and Chlorine says “MINE!”, and Sodium says “Ah, whatever, who needs it?” and the gentle pull of the water molecules around them easily overcome the electric charge based attraction of the now positively charged sodium ion and the now negatively charged chlorine ion, and the two atoms drift apart.

This brings us to “hydrophobic” and “hydrophilic”. There’s a truism in chemistry that “like dissolves like”. Polar substances tend to dissolve well in other polar substances, and non-polar substances tend to dissolve well in other non-polar substances, but polar and non-polar substances don’t mix well at all. Water is a polar substance – it’s got an electron-greedy oxygen atom in between two comparatively electron-apathetic hydrogen atoms. What’s more, the two hydrogen atoms aren’t on exactly opposite sides of the oxygen atom. The “H-O-H” arrangement is actually bent (at just over 104°, if you care), so a water molecule ends up being slightly triangular, with one corner being a little bit negative (where the oxygen atom clings more to the electrons) and two corners with the hydrogens being a little bit positive. Any other molecule with a slightly-positive or slightly-negative part will find that part attracted to one side or the other of water molecules, and as a result will tend to be pulled out into the water as the molecules bounce around [i.e. it will dissolve]. On the opposite end of the scale, molecules with their electrons relatively evenly spread over them tend not to be soluble in water. Large molecules like fats are in this category, which is why fat floats on top of water rather than dissolving in it.

There are two other random facts that I need to wedge in here somewhere. First, the line between “covalent” and “ionic” is actually kind of arbitrary. Water is considered “covalent”, but a very small fraction of the times that two water molecules run into each other, they’ll hit just right so that the slightly-negative oxygen atom on one of them manages to attract one of the slightly-positive hydrogen atoms enough to make it leave an electron behind and jump over. When that happens, you end up briefly with a positively-charged “hydronium” ion (“H3O+“) and a negatively-charged “hydroxide” (OH) ion. It doesn’t take too long for a “hydronium” to find a “hydroxide” again and rearrange back into two water molecules, but in pure water at “standard temperature and pressure” (defined as 25°C and one atmosphere of pressure) at any time there are about 620,000,000,000,000,000 hydroniums and hydroxides floating around in a liter of water – assuming I didn’t screw up my math there.

And, finally: a classical definition of an “acid” is something that “donates protons” (that is, hydrogen ions). In water, that means a molecule that provides extra available hydrogen atoms that water can pull off to form “hydronium” ions more often that water alone does.

And now, at last, we reach the subject of the preservative known as “benzoic acid”. If you read the ingredients lists of the food and drink you buy, you’ll probably never actually see “benzoic acid” on the label. Instead, you’ll see “sodium benzoate” or “potassium benzoate”. If you remember, sodium and potassium don’t really care about one of their electrons, so when you dump “sodium benzoate” in water, the sodium goes floating off to play with the water, leaving behind a negatively-charged benzoate ion with its electron. The extra electron hangs out around the part of the benzoate ion where the electron-greedy oxygen atoms are, making the molecule quite polar. Along comes a new “hydronium” ion, carrying a hydrogen that decides it misses its electron after all, and it jumps over to take over partial custody of the electron that the sodium left behind. In short, you’re going from Sodium + Benzoate + Hydronium + Hydroxide to Sodium + Hydroxide +…Benzoic Acid. (Plus a molecule of water, which is traditionally left out of these kinds of equations, which used to be the “hydronium”.) With the hydrogen attached and sharing the electron, benzoic acid no longer has so much of a charge imbalance and is a lot less polar. Being an acid, Benzoic Acid can also give that hydrogen ion back up again to a molecule of water – exactly the reverse of the reaction that formed it.

That’s the punchline to this: in water, a molecule of benzoic acid might at any one time be without it’s hydrogen and therefore charged/polar and hydrophilic, or it might have the attached hydrogen and be uncharged, relatively non-polar, and be comparatively hydrophobic…or “fat-soluble”.

Next post: So what?

“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…

“A small modification of Koch’s plating method.”

Only two more days for the Classic Papers Challenge, so if I’m going to get any more up, I’d better get my butt in gear.

Here’s a nice easy one:

Petri, R. J.:”Eine kleine Modification des Koch’schen Plattenverfahrens.” Centralblatt für Bacteriologie und Parasitenkunde; 1887; Vol. 1, pages 279-280.

The American Society for Microbiology has a translation available online. It’s only about a page-and-a-half of relatively large type – check it out.

There’s a trick we microbiologists use for growing bacteria. You make a solid (but wet) surface that contains whatever nutrients the microbe (bacteria, archaea, yeasts, mold spores…) you’re interested in need, and then you spread a diluted mixture of the microbe on it. The idea is that since the surface is solid the microbes can’t move around too much, and at any spot where a single cell starts initially, a whole pile of that cell and it’s genetically-identical (non-sexually-produced) clone-children will form until it gets big enough to see without a microscope. This cell-pile is called a “colony”, and you can poke or rub it with a sterile object, then stick the object into a sterile nutrient source. The end result is you have a “pure” culture of microbes that are effectively genetically identical. The solid material could be a lot of things – I’ve seen references to using slices of potato – though these days agar-agar gel mixed with nutrients is the preferred substance.

Koch (that is, Robert Koch of “Koch’s Postulates” fame, not Ed Koch the former mayor of New York City) used gelatin (so, hey, here’s another thing you can do with your expired Jell-O®). He apparently used to have a stack of shallow bowls, and had to use a special pouring device to carefully dump the gelatin into each stacked bowl in turn, then cover the works with a bell jar in order to keep stuff from falling into them from the air and contaminating them.

This was kind of a pain to work with, so some clever guy named Julius came up with a modification of this method in 1887, using pairs of shallow dishes, one slightly larger than the other so that it could be turned upside down to use as a lid. Then, you don’t necessarily need the bell jar, and you don’t need to stack them so they’re easier to pour.

Julius Robert Petri’s idea was so useful that we still use it today. Oh, yeah, and they named the dish-and-lid combination after him.

How’s that for a “classic” paper?

Meanwhile, my “Mountain Dew® Wine” project is turning out to be substantially more educational and fascinating than I’d hoped. There seems to be a decent amount of information available on how benzoic acid affects yeasts. I intend to turn that into a post later, but first I’ll try to find at least one more old paper to post before tomorrow is over…

What really counts as a “microbe”?

Just a brief pre-post before the main one I’ve got brewing now (which will be posted either later today or tomorrow).

A tapeworm: Since when does 30-36 feet long count as 'micro'???Microbiology is the dominating topic of this particular blog, but I don’t think I’ve ever addressed what I consider to really count as “micro”biology. This isn’t necessarily an obvious topic. My old “Microbiology” book from 8 years ago, plus the textbook from last year’s “Pathogenic Microbiology” class both contained large sections discussing organisms that are visible without a microscope. Heck, the “Pathogenic Microbiology” text even had a whole section on spider and insect bites. And, tapeworms? Since when is “over 30 feet long” considered “micro”? As I like to say: It’s time for Microbiology to grow up and move out of Medicine’s basement.

So: Here are the defining features of what I consider to be a “microbe”, at least for purposes of what I tend to discuss here on the blog:

  • Obvious: the organism cannot be effectively examined visually without a microscope and individual organisms can virtually never be observed by the “naked eye”.
  • In nature, a full normal population of a microbe can and will develop from a single live cell, and isolated individual cells are reasonably commonly observed.
  • Microbes do not “eat”.

It’s that last point that prompted me to write this post, mainly because it’s such an important part of why microbes work and how they affect their surroundings, especially when it comes to food microbes. What I mean by “do not eat” is that they are incapable of taking large (microbially speaking) chunks of material into themselves to use. Any cell nutrient for a microbe must be in the form of small molecules, like sugars, small peptides or individual amino acids, and so on that can be easily transported across the cell membranes and through the cell wall where applicable.

The importance of this is that for a microbe to grow on a complicated substance like meat or bread (for example), they have to excrete specialized enzymes that break down the substances out in the environment into simpler components like sugars or small peptides. If a microbe cannot secrete a protein-digesting “protease” enzyme, it can be surrounded by tasty, nutritious proteins and still starve to death. If a microbe can’t secrete an amylase (starch-digesting) enzyme, it doesn’t matter that starch is made of nice yummy glucose molecules because they’re all wadded up into long chains of starch that the microbe can’t get at.

And that, finally, is important because it brings up issues of growing multiple microbes together to accomplish something. Sake, for example, is made by fermenting rice, but rice is made primarily of starch. Saccharomyces yeasts don’t make amylases, so in order to make sake, you also have to add a kind of mold (Aspergillus oryzae, one of the types of white-mold-with-little-black-specks that you may see growing on the bread you’ve left sitting around for too long). A. oryzae is also a microbe and therefore can’t “eat”, but it does produce amylase. Since the amylase is breaking down the starches outside of the cells, this means the released glucose is also available for the yeast to use.

Admittedly, my definition above isn’t perfect. On the one hand, it leaves out protozoa (like amoebae and the well-known Paramecium, both of which actually do take in “chunks” of food, but both of which most people would normally consider to be “microbes”. It also leaves IN things like mushrooms, which are not usually thought of as being “microbes” by people who aren’t microbiologists. And, of course, it leaves me with no excuse not to go and learn something about eukaryotic (“plant”) algae (as opposed to bacteria-algae, a.k.a. cyanobacteria) and diatoms. Suggestions for updating my definition may be left in the comments…

Just something that came up while I was assembling what will be the next post. Stay tuned.