Nerdity – Page 8 – The Big Room (and the little things in it)

Chunky Bacon Agar, and Expired Jell-O™ again

I’m still working on the “Taxonomy of Yogurt” post which I currently plan to do next, but I’m overdue for a post already – therefore, here’s a brief one to keep my legion of adoring fans appeased until the next long post, here’s a short one.

Part 1: I got an interesting search-query hit recently – looks like (I’m guessing) a technician working at a famous pharmaceutical/healthcare-product company ran into the same problem I did during my current Bacterial Virology lab – “chunky microbiology agar in microwave”.

Agar is nifty stuff to use for microbiology. Dried, it’s a lumpy powder. To use it, you dump around 1-2% w/v (more or less, depending on the consistency of agar that you need) into water and heat it up to dissolve it. It’s basically seaweed-JellO� – except it’s not actually affiliated with Kraft Foods nor made of gelatin. Anyway – once it’s dissolved, it’ll cool into a gel.

The nice thing is, you can make up a bottle of this stuff and let it solidify, and store it (sealed) for quite a while. When you want to use it, you can just stick it in a microwave oven to melt it back down. It has to get pretty hot for this, but it then stays liquid until it gets down nearer to room temperature, so you’ve got plenty of time to pour it into plates or tubes or whatever.

For bacterial virology purposes, we make up a “soft agar” (about 0.8% agar, as I recall) to make an “overlay” – after mixing bacteria and virus together into a small amount of melted [but mostly cooled, so it doesn’t fry the bacteria] agar, we pour the soft agar in a thin layer over the top of a regular layer of nutrient agar in a plate. (The idea is that then wherever there is a virus that can infect and kill bacteria, it’ll wipe out all the bacteria growing in a particular part of the overlay, leaving a cleared “plaque” – you can then count how many plaques there are to find out how many virus were in the original sample, for example).

Earlier this semester, we had a fair amount of trouble with this. We’d go to pour the overlay and it’d come out chunky, even though it looked completely melted when we prepared it. Fortunately, the problem is simple and easily solved – you just need to nuke the heck out of the stuff, frequently swirling the container to make sure it’s completely mixed. What seems to be happening is that a few bits of agar remain unmelted but hard to see if you’re not careful, and those bits allow the melted agar to coagulate around them more readily. In short, the trick is to make really sure that all of the agar is completely melted.

Note that you have to be careful while doing this – lots of bubbles end up coming out of the agar when you swirl it, and it can easily foam out of the container and burn your hand. (Oh, obviously you also need to leave the lid a little loose to let off the pressure.) Of course, the stuff will be really hot when you’re done with the microwave, but as mentioned before, it’ll stay liquid until it is much cooler before it solidifies. If you set the bottle in a warm-water bath (~50�C or so) you can basically walk away for hours, leave it overnight, or whatever, and it should still be completely liquid and smoothly pourable – not to mention cool enough to handle with bare hands – when you get back.

And on the subject of gelled material – the fact that I mentioned all the hits about expired Jell-O� in the previous post seems to have substantially increased the number of “expired-JellO�-related” hits I’ve gotten, so here’s a slightly more expanded update.

Assuming one is referring to the “instant gelatin” powders (regardless of brand), as far as I can tell they ought to be safe to use almost indefinitely. Officially, Kraft Foods, the owners of the Jell-O� trademark say that the expected shelf-life is 2 years (“24 months”). I still think, personally (Note – Your Mileage May Vary, Do Not Try This At Home, and other standard disclaimers apply here) just like sugar, that it is probably safe to use practically forever as long as it doesn’t get wet (and isn’t stored in humid conditions). I don’t think anything of consequence would be able to grow on the dry powder, and I find it unlikely that the normal flavorings would be prone to suddenly become poisonous as a result of ordinary aging. The only thing you might have to worry about is maybe some of the flavoring compounds getting slowly oxidized by the air, so maybe the result wouldn’t taste quite the same. As far as I am concerned, so long as there weren’t fuzzy clumps growing in it, if the contents of the packet were still flaky/powdery, I’d most likely go ahead and use it, and not expect to suffer any ill effects.

‘course, if you read my obituary someday and it notes that I died of expired-gelatin-poisoning, you’ll know I was wrong…

UPDATE: I empirically test the toxicity of of expired JellO� on my own body! The saga begins here!

Search Queries That Came To This Site, Part 2: Odd but coherent

There have been a few queries that somehow led to this blog which weren’t actually bizarre, as such, but which were kind of unexpected…

Someone in Maryland was trying to find out “why does alcohol rub work?”, for example. The answer is, it’s a “counter-irritant”. The effect is somewhat similar to rubbing a sore spot, stretching a sore muscle, or scratching an itch. The mild “burning” of the alcohol helps deaden the soreness. Beyond that, it’s some medical/physiology-of-freakish-gigantic-multicellular-eukaryotes thing, so I’ll defer a detailed explanation to someone who’s more of an expert on such things.

Someone in New Jersey wanted to know “what does a gram of nutmeg look like”. Well, I’ve never actually measured it but I’d guess a gram of ground nutmeg is probably about half-a-teaspoon of coarse (sand-grain-sized) light-brown-and-tan bits.

Pakistan wanted “total molecules of universe”. Doesn’t everyone know this? It’s exactly 2.379×10^{some-really-big-number}. To 4 significant figures, of course. Oh, and the last digit of pi is “3”.

Colorado (I think) consulted the oracle of Google for “Why does immersion oil work”. It’s a refraction thing. Simplistically, when light goes from one substance to another – like the glass of the slide, to your sample material, to the air, to the objective lens of the microscope – it’s direction gets bent slightly. Different substances cause a different amount of bend. The immersion oil causes less bend than air does, and when you’re operating at really high magnification, it’s important to keep as much of the light as possible getting into the lens of the microscope instead of ending up “bent” away from it. Unless I’ve badly mangled my understanding of it, this is also part of why the light in the microscope seems to get dimmer as you increase the magnification.

Possibly from Florida came a query on “Scientific How Enzymes work to get stains out of carpet”. Even more simplistically than the previous explanation: Everything wants to fall apart, but is usually too lazy to just do so, so a little bit of extra energy (the “activation energy”) needs to be added to push-start it. Enzymes are catalysts – they each make a specific kind of reaction able to happen with less activation energy. If the enzyme is good enough, you reach the point where the ordinary ambient heat supplies enough energy to get things going – like breaking down those large, ugly, dark-colored chunks of protein and stuff in stains into smaller, invisible bits.

California asked Google about “eugenol clove isolation”. I suspect that if you want some kind of extreme high purity thing, you might be better off just approaching it as an Organic Synthesis problem. Otherwise, why cut out any other components of the clove buds that may add subtle flavors to the mix? If you’re just trying to separate the clove flavor from the chunks of dried evergreen-bush-flower-buds, though, there are a few ways to do it. Eugenol’s a phenol-like compound, and it’s soluble in ethanol. Go to the local liquor store and buy some vodka or EverClear�, soak the clove buds in it for a while, and pour it off. You could conceivably also try distilling it directly from the buds, as some people do to extract perfume oils from flowers.

Luxembourg sought “+homebrew +LED +Flashlight”. If anybody’s interested in that, it deserves a separate post, but it’s pretty easy. I’ve been planning to make an infrared one to do some IR digital photography, and to modify an 8-white-LED flashlight to turn it into a UV flashlight anyway.

Somewhere in Michigan, some concerned soul wanted to know “does beer have red dye in it”? Well, I’d argue that definitely, no real beer does. It’s possible that some mass-market commercial swill does, though I suspect even then it might only happen as A)a ‘novelty’ beer (like Green beer on St. Patrick’s day) or B)places like China or any other country where there seems to be a lot of unnecessary prettification of alleged foodstuffs. Now, I’m no Rheinheitsgebot zealot or anything, but beer ought to at least be reasonably “natural”…

There were a couple of queries on why Bunsen burners work, for some reason. Well, they’re essentially just tiny little carburetors for making variable flames instead of feeding a combustion engine.

Someone in Ghana wanted to know “How to make something disappear scientifically?”. Well, you can’t. But you can change something into something else. You can’t (scientifically speaking) make water disappear, but you can turn it into a gas by heating it. You can’t make oxygen gas “disappear” but you can combine it with hydrogen so that you end up with water but no molecular oxygen gas. And so forth.

Illinois wanted advice on “what to cook when you are bored and sick”. I think it depends on what kind of sickness you’ve got and how soon you expect to recover, and whether you’re cooking because you’re bored or specifically because you want something to eat that won’t make you feel sicker. Make some yogurt. Bake some bread. Or mild ginger cookies. Or make some “Jell-O�” (or other brand of instant-flavored-gelatin, for that matter). All easy to digest stuff. Or, you could make some Pepto-Bismol� Ice Cream

Texas wanted to know “Food science- why chill the dough”. The details depend on the context (cookies? Pie crust?) but generally it seems to be to make sure the fats in the dough stay solid. In pie crust, chilling the dough makes sure the bits of butter or lard stay chunky instead of getting spread evenly throughout the dough – when they cook and melt, this leave little areas in the dough that aren’t solid, making the crust flaky. In cookies, this might help keep the dough from flattening too quick when you cook them.

Hmmm. More of these than I realized. For tonight I’ll stop on this one: Pennsylvania was trying to find out: “what does the Giant Microbe factory look like”? I don’t actually know the answer to this one, but since the Giant Microbes headquarters looks like it’s just an office in an office building, they probably contract out to someone else to actually make the giant plush microbes. I’m guessing they probably renew their contracts every so often, and maybe shop out different runs to different factories, so there isn’t necessarily a single “Giant Microbe” factory…

I suppose that’s enough for one evening. I’ve really got to catch up on my sleep…

Search Queries That Came To This Site: Part 1 – comic relief

But first – a quick notice: I just added a “rating” bar for posts. Feel free to vote – the more feedback I get, the more likely it is that I may eventually learn to write more consistently coherent and interesting things…

At this point, this little blog seems to get most of its meager traffic (by far) from search queries. The searches have been piling up, and I figure it’s about time to do some posts to try to address those searches.

For part 1 here, I believe I’ll start with the oddball searches which often don’t seem to have anything to do with microbiology or, indeed, sometimes anything coherent at all. It’s late, and I could use some comic relief. (In Part 2 I’ll discuss some of the unexpected-but-coherent searches that led to my blog, and in Part 3 I’ll post about the kinds of microbiology searches I kind of expect to see in the logs that I’ve gotten…)

Why MSN loses to Google and Yahoo:

Out of the 5 whole MSN queries that have led to this site, two of them are: “mazda” and “debt”. I have no idea why. (In fairness, the other three queries were perfectly plausible microbiology-type queries).

Just plain “Huh????”

Someone in San Jose got here by Googling the phrase: “Type of fruit makes balloon grow bigger”
Someone from Nairobi(?) got here by querying “death and nuisances”
From a Washington State school organization of some sort: “a powder that looks slimy looking when lemon juice is added”
From a Toronto school network: “how does pink solution work(remove stain)”. (Actually, they may have been looking for information on Eradasol�, which is a seriously nasty-smelling detergent/solvent of some kind which does a good job of removing microbiology-type dyes from floors, countertops, fingers, etc…)
From the UK: “in search engine type cell a room” (Uh…what?)
From Indonesia: “expired of natto” (are they trying to find out when you throw away Natto instead of eating it, or people who died from eating Natto?)
From the Department of Education in Orange County (California, presumably): “water ballon splater”[sic]
From the Department of Education in Queensland, Australia: “why does this material work for the room”
And finally, my personal favorite from (apparently) Google itself: “iron chef cheese balloon”

BLASPHEMY!

Both New Zealand and the UK got here trying to find out about how “mushrooms are evil”. This is completely unfounded – Mushrooms are our FRIENDS.

Kinda Scary

From the Vancouver area: “world’s best bathrooms, microbiologically” (Ah, but best for what purpose?…)
I got two different queries (both from Pennsylvania?) for “eating expired jello” (Actually, as far as I know, so long as the stuff remains dried in its sealed pouches, it’s probably safe to eat almost indefinitely. I’d be a little leery of expired pre-made gelatin, though – that stuff’s a relatively simple protein mixed with lots of water and, often, sugar. Sounds like very attractive food for microbes of all kinds, including some that might make you sick…)
Speaking of which, someone at University of Michigan was looking for “eating expired bread spore”
Someone from Illinois was looking for “old interrogation room pictures”(?!) on Yahoo…
Someone on a military base in Ohio somehow got here looking for “solicitation can be released at least how many days”

And, perhaps scariest of all:

someone in Alabama had an odd search phrase: “organism +I*”

Why is this scary? Everyone remembers Isaac Asimov, who (while he was a live organism) wrote “I, Robot”, right? Well, obviously this means that a secret cabal of government agents managed to steal Asimov’s brain and upload it into a computer, thus creating a Robot Isaac Asimov (and this searcher wanted to know when Robo-Asimov would be publishing “I, Organism”.) Obviously, government “working” as well as it does, their Robo-Asimov still uses “Reverse Polish Notation”, hence the reverse-entry of “Organism I”…Okay, enough silliness for one evening. More – hopefully – tomorrow.

Short Low-content (but relevant) post

Mainly to remind myself, but in case anyone’s interested:

I’m going to have to do another “Searches that led to pages on this blog” post soon – there are some interesting ones.

If I have my way, I’ll also be in a position to do some posts on bacterial virology, yogurt, and the microbiology of Belgian Lambic ales. (For the Bacterial Virology lab, I’m going to see if I can play with temperate (“lysogenic“) phages in yogurt, and for the “food microbiology” portion of the Pathogenic Microbiology lab, I’m going to see if I can talk them into letting me try to isolate [normal] bacteria from Lambic [assuming there are any still living in there].)

Sorry about the recent case of blogstipation…

So, here I am blogging from the hospital…

What? Oh, no, I’m fine, it’s just right across the street from where all of my classes are this semester, and they have a fairly decent cheap cafeteria. Plus, if I get this particular table, I can just barely get enough of a signal with my laptop’s external antenna to connect to my college network account.

Last week was spring break. Although I probably SHOULD have spent it drunk and naked, according to common wisdom, I instead spent it trying to catch up on sleep and doing a bit of culturally and educationally enlightening travel.

Aside from yesterday’s trip to the Opera, we managed to get out to visit Lehman Caves. As one might guess, I was hoping I’d get to find out something about the microbiology of the cave (in addition to ogling the impressive mineral stuff.)

As far as the microbiology goes, I was quite disappointed. One of the small books in the visitor’s center mentioned the existence of chemolithoautotrophic bacteria. In one paragraph. The entire content of which I just summarized here. Not even an identification of what kind of bacteria they are. The guide for the cave tour only knew that the bacteria in the cave were “harmless” (well, yeah, I kind of imagined they would be). There were also cyanobacteria happily if slowly growing near the lights, which nobody seemed to know too much about either.

I did get the name of the person responsible for issuing research permits – I’m seriously considering trying to make the cave one of the sites for my Senior Thesis study.

I did some other things, too, but I’ve got to pack up and head for class now. In case anyone besides my immediate family is reading this regularly (please comment if you are!) I will try to post a lot more often now – the last couple of weeks have just been a major distraction.

I’ve got DNA! I’ve got DNA! I’ve got DNA!…

As you can see, I’ve got DNA. I’ve been trying to get this stuff successfully extracted and the 16s rDNA amplified for months (off and on) now. Looks like doing the whole-genome-amplification step first did the trick – this is from a set of mixed halophiles in a phlogisticated environment growing in approximately 18% salt solution, and they grow very slowly. It’s hard to get enough DNA extracted from such a small population to do useful work with. image of electrophoresis of 16s DNA amplicons

The gel “bands” you see to the right of the image are (or at least should be) made of copies of the DNA which codes for various “16s small-subunit ribosomal RNA” sequences for the one-or-more different kinds of prokaryotes living in my culture. The brighter the band, the more DNA is there.

Since all of the samples were processed exactly the same way, then, the brightness of the band should, at least indirectly, indicate how many bacteria were in each sample to begin with. This isn’t necessarily true – there can be variation in how many copies of the gene each kind of bacteria has, so if the populations are very different the results could be misleading. Still, it’s gratifying that my little ‘proof-of-concept’ experiment not only finally gave me some DNA but even shows exactly the kind of difference I originally hoped for. (The second “lane” from the top with the brightest band was SUPPOSED to be enriched for certain types of bacteria, according to my hypothesis. The first “lane” should have had less, and it does. The third lane is my “positive control”, growing without special influences, and the fourth lane with no DNA visible is my negative control, which I hoped would have little or no DNA (indicating little or no bacteria growing in it) – and that’s what I see.

It doesn’t prove anything at this point, but finally getting results and having them turn out to look the way I’d hoped is a good start. I wonder if I can get them into a clone library, separated, and sequenced before next weekend?

I’ll have to remember to thank last semester’s “Senior Seminar in Microbiology” instructor for assigning me that paper[1] – I thought some of the technology described in it sounded like it’d be useful to me personally.

Anybody else going to the Northwest Regional ASM meeting next weekend?…

[1] Wu L, Liu X, Schadt CW, Zhou J: “Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification.” Appl Environ Microbiol. 2006 Jul;72(7):4931-41.

Officially “Gram-positive” – the Firmicutes

In a typical student microbiology lab, it seems whenever you get your hands on some bacteria the first thing you do is check to see if it’s “Gram-positive” or “Gram-negative”. But does this old Victorian-era test still mean anything useful in the context of modern bacterial taxonomy?

It would seem that it actually does. In my admittedly limited experience, an un-ambiguous Gram-positive result using the standard procedure nearly always indicates bacteria in the phylum firmicutes, sometimes also referred to as the “low G+C gram positives”.

The “low G+C” part has to do with the chemical characteristics of the DNA. If you’re already familiar with DNA’s structure then you probably already know what this means, but for everyone else, in brief:
DNA is made of strings of four different chemical “bases” chained together. The bases are “Adenine”, “Guanine”, “Cytosine”, and “Thymine” (abbreviated as A, G, C, and T). These bases make up a sort of chemical “alphabet”, which encode how to make various proteins. These chemical bases each have an opposite base that they are attracted to – Adenine to Thymine, Cytosine to Guanine, so DNA’s strings end up matched with an “opposite” string, which together form the easily recognized “double helix” shape of the overall DNA molecule. The relevance here is that the attraction between the Guanine and Cytosine (“G+C”) is stronger than the attraction between Adenine and Thymine, so you can estimate relatively how much Guanine and Cytosine is in an organism’s DNA by how tightly the two strands of DNA stick together.

And, yes, there is a “high G+C” gram-positive group, with a larger proportion of the G and C bases as compared to the A and T. That’s the “Actinobacteria”, which includes the “acid-fast” Mycobacterium group. But that’s a topic for another post.

The firmicutes, with the exception of one group that I know of, all have a distinctive, simple outer structure. It’s something like this:

Imagine a water balloon filled with lime Jell-O�. Now, tightly wrap the water balloon with a piece of thick, padded packing blanket. The thick layer of packing blanket is the cell wall. The balloon is the inner cell membrane. The Jell-O� is the cytoplasm. (It’s Lime merely because I like lime Jell-O�.) It might be worth noting that since this group of bacteria relies so much on it’s cell wall, they are more likely to be killed off by the ?-lactam antibiotics (which specifically attack bacterial cell wall generation) than other types of bacteria.

There is one exception to this structure – the class of firmicutes known as the mollicutes. There’s one example of this group that gets mentioned in basic medical-centric microbiology classes: the genus Mycoplasma (as in Mycoplasma pneumoniae). Members of this class have lost their ability to make cell walls entirely.

By contrast, a typical “Gram-negative” cell has a more complicated outer makeup. In brief, start with the same lime Jell-O� balloon, but instead of a thick packing blanket, wrap it with a single thin layer of cloth, and then stuff the whole thing inside of another lime Jell-O� balloon. You can also imagine a bunch of valves stuck through the outer balloon to let stuff in and out, but then you end up imagining the Jell-O� spurting out all over the place and the whole analogy breaks down. If it helps, you can also imagine that if you punched out a section of a gram-negative outer structure with a cookie-cutter, you’d get something kind of like an inverted Oreo� cookie, with easily-dissolved filling on either side of a thin layer of cookie. Anyway, the outer balloon represents the outer cell membrane, the cloth is the (much smaller) cell wall, and the layer of lime Jell-O� between the outer balloon and the cell wall is called the “periplasmic space”.

Interestingly, despite this difference in complexity, the molecular evidence[1,2] seems to indicate that the simpler firmicutes diverged evolutionarily from the more complex “gram-negative” types, not the other way around. The lineage suggested by the 16s gene sequences implies that both types of Gram-positives split off from the Gram-negative type bacteria as a single ancestral group, and some time later the two types diverged into separate groups. If I had to bet, I’d personally put my money on the evolutionary sequence going Gram-negative-type->actinobacteria->firmicutes.

Another characteristic of some (but not all) of the firmicutes is the production of “endospores”. Unlike the spores of molds or Myxobacteria, endospores aren’t reproductive. Instead, they’re a like a lifeboat, or perhaps a metaphorical bomb-shelter in the cells’ also-metaphorical basement. If environmental conditions get unpleasant, the bacteria essentially pull their DNA and a few necessary enzymes into a small, thick, multilayered compartment – the endospore – where they can wait, protected and dormant, until conditions become comfortable again.

The special “Endospore stain[3]” uses a dye called Malachite Green and works somewhat similarly to the Gram and “Acid-fast” stains – the extra-thick spore coats retain the green stain (once it’s been driven in by some extra heat) while the decolorization rinse washes it out of everything else. I still need to try using a microwave oven for the heating step…but that, too, is a topic for another post.

If your microbiology class was typical, when you think of the firmicutes, you may think of little more than “Strep throat”, “Anthrax”, and “MRSA“. If so, though, you’re missing out on the useful ones.

Since Gluttony is my second most favorite “Deadly Sin�”, I tend to think of food-related possibilities here. And, no, I don’t mean Botulism.

Yogurt and Kumiss and Kefir, Sauerkraut and Kimchi, Sourdough bread, Salami, and Belgian Lambic ales all involve (at least in part) growth of various lactic acid producing firmicutes. Mostly members of the Lactobacillus genus, though if you take a look at the labels of some of the “Live and Active Cultures” yogurt you may also spot a close relative of the ‘Strep throat’ bacterium in the list. These kinds of bacteria can be happy members of the “Normal Flora” of the healthy human gut.

Another, more obscure example is “Natto”. A strain of Bacillus subtilis, originally derived from rice straw, is allowed to ferment soybeans. The end result is a pungent mass of beans, covered with gooey slime, and having an odor vaguely resembling old cheese. I’ve actually eaten it, and they aren’t nearly as bad as this makes them sound…though I personally still haven’t acquired a taste for them. You may have seen this stuff if you watch Iron Chef – it was used as the Secret Ingredient in one episode. There does appear to be some real potential for it as a “health food”, though, which you can see if you poke around Google or PubMed searching for “Natto”. Maybe next time you end up in the basic Microbiology lab and you’re given some B.subtilis to look at you can whip out a jar of soybeans to inoculate while you’re at it. (Note that I wouldn’t actually recommend eating the results in this case…)

Comments and corrections, as always, are welcome.

P.S. no affiliation with any of the trademarks mentioned should be inferred here – I just figured the trademarked names would be more recognized than terms like “gelatin” or “sandwich-cookie”…

[1]Sheridan PP, Freeman KH, Brenchley JE: “Estimated minimal divergence times of the major bacterial and archaeal phyla.” Geomicrobiol J 2003, 20:1-14.
[2]Battistuzzi1 FU, Feijao A, Hedges SB: “A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land.” BMC Evolutionary Biology 2004, 4:44
[3]Schaeffer AB, Fulton MD: “A simplified method for staining endospores.” Science 1933 77:194

Making the great leap out of the 19th Century…”Acid-Fast” staining

The “standard” acid-fast differential staining process for the “High G+C Gram-Positive” bacteria[1] as we learned it is pretty archaic.

It goes something like this:

Smear and heat-fix the slide
flood the slide with Carbolfuschin
Heat the slide for 5 minutes in steam over a boiling water bath
Rinse with “Acid Alcohol“
Stain for a minute with Methylene blue.

At the end, anything “Acid-Fast” (having a “waxy” outer layer) will show up red, anything else will be blue.

My objection is the messy and time-consuming steam-bath.

Today’s lab included one “unknown” which we suspected to be a Mycobacterium, so while one of us was going through the tedious 19th-century-style procedure, I decided to try something.

The steambath heat is just intended to (I believe) slightly “melt” the waxy layer of the cell and otherwise help “drive” the dye into it. So, instead of dealing with the time to make a water bath, heat until it steams, and then wait for the slide to sit there and hope the bubbling bath doesn’t splatter the slide with crud, I just stuck the flooded slide in the lab microwave and cooked it for 20 seconds.

It worked. Quite well, actually (other than letting the slide dry out, leaving some crystals on the slide) – the bright red mycobacterial cells showed up nicely. I’m annoyed that my ‘stick the camera up to the eyepiece’ technique came out slightly out of focus (I may see if I can enhance it later – if so, I’ll post it.). Somebody commented that it looked as good as a “textbook” example, which was nice for my ego…

Unfortunately, I guess I’m far from the first person to think of this. I don’t know if anyone’s done this exactly the same way, but This procedure describes directly heating the Carbolfuschin in the microwave and soaking the slides directly in it. There is also apparently an old Lancet[2] article which I don’t currently have access to – I’ll have to check it out later.

Next thing to do is try the endospore stain this way. Behold, the miracles of applying last century’s technologies to the problems of century-before-last!

[1] Ehrlich P. Zur Fa�rbung der Tuberkelbakterien. Aus dem Verein fu�r innere Medizin zu Berlin. Deutsche Med Wochenschr 1882; 8:269?270
[2] Hafiz, S., R. C. Spencer, M. Lee, H. Gooch, and B. I. Duerden. 1984 . Rapid Ziehl-Neelsen staining by use of microwave oven. Lancet ii:1046.

This is your brain. This is your brain on Microbiology…

In an effort to eat a relatively healthy diet, I occasionally eat pieces of wholesome, natural fruit.

There, I’ve admitted it. I can no longer live the lie that I only eat junk-food. Of course, to maintain some appearance of having a normal mainstream type of diet, I at least tend to go for the pre-cut fruit mixtures – I’ve got way too much going on to have time to prepare cut fruit salad from scratch.

Anyway, a few weeks ago I had a platter of these, still sealed in their plastic container, and didn’t get around to eating them in time. About a week after it had expired, when I went to throw it away, I saw a few little white lumps growing all over the pieces of fruit. Non-fuzzy, so I expected they were bacterial rather than fungal. Of course, there’s only one thing I could think when I saw that.

“Oh, wow! It looks like there were only about 5-8 bacterial CELLS on each piece of fruit when I got them! Those things were really CLEAN!”

(Of course, once you can SEE the colonies growing, there are a lot more than a few cells there. Each visible colony’s probably got millions of the little buggers, but each colony starts as a single cell.)

This was, of course, followed by me lamenting that I didn’t have some culture supplies and a microscope of my own to examine them with. Sigh. Anybody out there have any extra microbiology equipment you’d like to donate to a good cause?

So, what’s next? Should I try to start a series of bacterial taxonomy posts? Searches for “what’s a gram-positive?” and “what’s a gram-negative?” sorts of questions seem to be popular ways to reach this blog…

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