Category: Why Does It Work?

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

The Gram Stain Post to End All Gram Stain Posts

Gram stain, Gram stain, Gram stain! Bah. I think it’s time Microbiology grew up and moved out of Medicine’s basement.

Sure, the Gram stain[1] has its uses, but the procedure is grossly over-hyped. “[…]the most important stain in microbiology[…]”[2]! “[…]it is almost essential in identifying an unknown bacterium to know first whether it is Gram-positive or Gram-negative.”![3] “The Gram Stain reaction is an especially useful differentiating characteristic.[…]The Gram reaction turns out to be a property of fundamental importance for classifying bacteria phylogenetically as well as taxonomically.”![4] “[…]differentiates bacteria into two fundamental varieties of cells.”![5] “The Key to Microbiology“![6] [emphasis added…]

Bah! Sure, the Gram stain has its uses, but the hype it gets (even 125 years after its invention) is ridiculous. It’s worse than Harry Potter!

You really want to know what the Gram reaction tells you? Really? Okay, here it is:

A “Gram Positive” reaction tells you that your cells have relatively thick and intact cell walls

A “Gram Negative” reaction tells you that they don’t.

That’s it. That’s about all you can reliably infer from the Gram stain.

Previously, I put up a post describing what was my understanding of the conventional view of why the Gram stain works. Today, I’ll give you a much more detailed – and more correct – explanation of why it works as well as what its real significance is to identification of microbes. But first, a brief one-paragraph rant on why I think the Gram stain has such a hold on microbiology teaching.

I blame the fact that microbiology education is still largely in the shadow of medical technology education. When you artificially exclude the 99+% of organisms that aren’t associated with human diseases, the tiny number left do, indeed, seem to largely separate into two phylogenetic categories. Judging by what I’ve encountered thus far, it seems you get a lot of Proteobacteria (especially ?-Proteobacteria, like E.coli), which are “Gram-negative”. You also get a lot of Firmicutes (Bacillus, Streptococcus, Staphylococcus, etc.), and a couple of scattered Actinobacteria (Mycobacterium, for tuberculosis and leprosy, Corynebacterium for diptheria…). Both of these are considered “Gram-positive” (although if you use the standard procedure these days, the Mycobacteria may show no reaction at all). That’s, what, 3 phyla out of about 25 eubacterial and archael phyla? If we throw in Syphilis and Chlamydia, that’s still only 20% or so of the currently recognized prokaryotic phyla. If your microbiology classes assume everybody is training to be a medical technologist or clinical microbiologist, then the Gram stain becomes inflated in importance.

Enough of that – here’s a quick review of how the Gram stain works. Solutions of “Crystal Violet” (a purple dye) and Iodine are applied to cells fixed to a slide, where they soak in and precipitate in the cells. A “decolorizer” (usually ethanol) is applied to see if it will wash this dye precipitate out of the cells. A different, lighter-colored dye (such as safranin) is added so that the cells which DO have their dye washed out can be seen as well. In the end, “Gram positive” cells are a dark purple from the crystal violet/iodine that was not washed away, and “Gram negative” cells are not dark purple. (Usually they are pink, from the safranin, assuming that’s the dye used as the counterstain.)

Note that this does not differentiate cells into “two fundamental types” as is often claimed. You actually get four types: Groups of cells that are normally always “Gram positive”, Groups of cells that are normally always “Gram negative”, Groups of cells that are normally sometimes “Gram positive” and sometimes “Gram negative” (“Indeterminate”, or as I like to call it, “Gram-biguous”), and groups of cells that are normally NEITHER Gram-positive nor Gram-negative, like Mycoplasma, which aren’t dyed at all by the process. Incidentally, phylogenetically speaking, Mycoplasma is one of the “Gram positive” Firmicutes, just like Bacillus and Staphylococcus.

It’s kind of interesting to me that the Gram stain reaction has been such a mystery up until a century after its invention. What is it that makes “Gram positive” cells retain the dye while “Gram negative” ones don’t? Along the way, it seems like nearly every part of the bacterial cell was hypothesized to be the reason for the Gram reaction – lipids, carbohydrates, nucleic acids, “Magnesium ribonucleates”, and so forth. Davies et al, 1983, includes a table listing many of these and referencing historical papers making the claims. The fact that the reaction had something to do with the cell wall seems to go back quite a while, though the “Magnesium ribonucleates” idea doesn’t seem to have been entirely abandoned until the mid-1960’s[7]. It was also hypothesized that the “Gram positive” cells simply absorb more dye and therefore take longer to “decolorize”.

It turns out that “Gram-positive” cells actually don’t, necessarily, take up more dye than Gram negative ones. This was tested by taking a set concentration of bacterial cells and adding them to a set concentration of dye. After letting them soak, the samples were centrifuged to remove the bacteria, and the amount of dye found to be missing from the liquid was taken as the amount absorbed by the cells. They found that some Gram negative cells actually took up more dye than the Gram positives did. So much for that idea.[8]

Even relatively recently, I’ve seen it written that the bacterial cell wall, specifically, is what holds onto the stain, but even that turns out not to be true. Although the cell wall is the structure that seems to be responsible for the Gram reaction, in the late 1950’s it was demonstrated that it was not actually the staining of the cell wall that caused the reaction, but rather the ability of the cell wall to keep the decolorizer out of the cell.[9]

Apparently, the Crystal Violet/Iodine complex itself doesn’t even play a vital role. The complex apparently dissolves again more or less instantly as soon as the decolorizer touches it[10], and it’s even possible to differentiate “Gram positive” and “Gram negative” with simple stains like methylene blue or malachite green, if you’re clever about it[11]. The latter authors set up a clever test with crushed cell material, dye, and paper chromatography. They had the decolorizer soak into the paper, past a spot where dye-soaked cell material from Gram-positive and Gram-negative cells was placed, and watched for obvious differences in the amount of time it took the dye to be carried out by the decolorizer. Incidentally, my quick examination of this paper makes it look like cheaper 100% isopropyl alcohol (“rubbing alcohol”) might be slightly better than the standard 95% ethanol for Gram stains.

– INTERLUDE –

So, here we are at 1970 or so, and we already know that the Gram reaction is entirely based on how well the cell wall structure prevents organic solvents (like ethanol) from soaking into the cell to dissolve the dye complex. Yes, the mystery of why the Gram stain works in normal cells was largely solved by the Nixon era.
A few corners of the mystery remained, though. Why do “old” cultures of “Gram positive” cells often end up staining “Gram negative”, for example? Why do some kinds of cells seem to be sometimes Gram positive and sometimes Gram negative in the same culture? What, exactly, is really happening to the cell, deep down, during the staining process?

In 1983, the Gram Stain made the great technological leap into the 1930’s, when a variation of the technique was devised which allowed the Gram Stain to be observed by electron microscopy[12]. Using a funky platinum compound in place of iodine, the electron microscope reveals exactly where the dye complex is at any particular stage of the Gram stain process. Using this technique, it was possible to see how the decolorizer disrupts the outer membrane of classically-Gram-negative organisms and to see that the decolorizer potentially damages the cell wall and interior membrane, possibly allowing cell material to leak out (or decolorizer to get in and dissolve the dye complex). It was also seen that the dye complex permeates the entire cell, not just the cell wall.[13]

If you’ve been wondering about the sometimes-Gram-positive-sometimes-Gram-negative cells, the same technique was also used to investigate this. As suspected, it turns out that the “old cultures become Gram negative” problem is due to the cell walls breaking down as the culture ages. Bacteria are continuously, simultaneously, building up and tearing down their cell walls, in order to be able to grow and divide. As nutrients run out, the bacteria run out of material to rebuild cell walls, while the cell-wall degrading enzymes keep on chugging. Breaks in the cell wall occur, and through these breaks the decolorizer can get in and rapidly dissolve the dye. Actinobacteria can have a similar problem, but rather than only being in “old” cultures, apparently weaknesses appear briefly during cell division, and if a particular cell happens to be at this stage of growth when you stick it on a slide, heat-fix, and Gram stain it, the weakness at the septum where the division is occuring can crack and allow the decolorizer in, resulting in a “Gram negative” response even while surrounding cells of the same kind might still be “Gram positive”.[14]

This brings us to archaea and some eukaryotes (i.e. yeasts). Yeasts stain “Gram positive” normally. Although their cell walls are completely different chemically than bacterial cell walls, they are quite thick (microbially speaking). Poor, neglected Archaea seem to be all over the place in terms of Gram reaction. Since their Gram reaction doesn’t tend to correlate to any particular phylogenetic grouping[15], it seems nobody really pays much attention to their Gram stain reaction. On the other hand, and on the subject of “Gram-biguity”, I thought the investigation of Methanospirillum hungatei[16] was interesting. M.hungatei is an archaen that grows in chains. When Gram-stained, the cells on the ends of the chains are “Gram positive”, while the others have no Gram reaction at all. It turns out that the chains are covered by a sheath, and the only contact with the outside world is through thick “plugs” in the cells at the ends of the chains. These “plugs” act like thick cell walls, allowing the Gram stain dye material to soak in but excluding the decolorizer, while the sheath keeps the rest of the cells from soaking up any stain at all.

There you have it – a relatively detailed history and explanation for the Gram stain, and you didn’t even have to get through some obnoxious paywall to read it. Aren’t you lucky?

Comments, suggestions, and corrections, as always, are welcome.

[1] Gram, HC.”Ueber die isolirte Faerbung der Schizomyceten in Schnitt-und Trockenpraeparaten.” Fortschitte der Medicin. 1884 Vol. 2, pp 185-189.

[2] Popescu A, Doyle RJ. “The Gram stain after more than a century.” Biotech Histochem. 1996 May;71(3):145-51.

[3] Brock TD, Madigan MT, Martinko JM, Parker J. “Biology of Microorganisms (7th Edition).” 1994. Prentice Hall, Englewood Cliffs, NJ pg. 46

[4] ibid, pg. 715

[5] Beveridge TJ.”Use of the gram stain in microbiology.” Biotech Histochem. 2001 May;76(3):111-8.

[6] McClelland, Rosemary. “Gram’s stain: The key to microbiology – isolate identification method – Tutorial” Retrieved 20070810 from http://findarticles.com/p/articles/mi_m3230/is_4_33/ai_74268506/print

[7] Normore WM, Umbreit WW.”Ribonucleates and the Gram stain.” J Bacteriol. 1965 Nov;90(5):1500.

[8] BARTHOLOMEW JW, FINKELSTEIN H:”CRYSTAL VIOLET BINDING CAPACITY AND THE GRAM REACTION OF BACTERIAL CELLS.” J Bacteriol. 1954 Jun;67(6):689-91.

[9] BARTHOLOMEW JW, FINKELSTEIN H.”Relationship of cell wall staining to gram differentiation.” J Bacteriol. 1958 Jan;75(1):77-84.

[10] LAMANNA C, MALLETTE MF. “CHROMATOGRAPHIC ANALYSIS OF THE STATE OF ASSOCIATION OF THE DYE-IODINE COMPLEX IN DECOLORIZATION SOLVENTS OF THE GRAM STAIN.” J Bacteriol. 1964 Apr;87:965-6.

[11] Bartholomew JW, Cromwell T, Gan R.”Analysis of the Mechanism of Gram Differentiation by Use of a Filter-Paper
Chromatographic Technique.” J Bacteriol. 1965 Sep;90(3):766-77.

[12] Davies JA, Anderson GK, Beveridge TJ, Clark HC.”Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain.” J Bacteriol. 1983 Nov;156(2):837-45.

[13] Beveridge TJ, Davies JA.”Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain.” J Bacteriol. 1983 Nov;156(2):846-58.

[14] Beveridge TJ. “Mechanism of Gram Variability in Select Bacteria.” J Bacteriol. 1990 Mar;172(3):1609-20.

[15] Beveridge TJ, Schultze-Lam S. “The response of selected members of the archaea to the gram stain.” Microbiology. 1996 Oct;142 ( Pt 10):2887-95. (Abstract)

[16] Beveridge TJ, Sprott GD, Whippey P. “Ultrastructure, inferred porosity, and gram-staining character of Methanospirillum hungatei filament termini describe a unique cell permeability for this archaeobacterium.” J Bacteriol. 1991 Jan;173(1):130-40.

A Government “War on Science” is GREAT for this country!

They say that politics and controversial statements are ways to encourage traffic on a blog, so here’s some. Comments welcome, of course.

I have cause to celebrate the future potential for science in the U.S. Here’s a bit of simple history (Update – added the “War on Poverty” to the list 20070810):

1964: Lyndon Baines Johnson declares a “War on Poverty” Today: the gap between the Rich and the Poor in the US is widening and economic mobility is stagnant.

1971: President Nixon declares a “War on Drugs”. Today: “Drugs” are widely used, even among kids, who appear to be losing their fear of drugs. Market innovations (blatantly illegal and of questionable morality, but innovations nonetheless) such as crack cocaine, MDMA (“ecstasy”), and “ice” (crystal meth) seem to be in the news a lot. People growing illegal plants in their closets and basements or brewing up complex chemical stimulants in the backs of minivans seems to be an almost daily topic of the news.

2001: President George W. Bush declares a “War on Terror”. Today: A majority of Americans feel that there is a greater threat of terrorism than before, which seems to be true, at least as far as “Jihadist” terrorists go, if the declassified portions of the government report paint an accurate picture of the situation. Heck, when the president invaded Iraq in 2003, major terrorist organizations didn’t even seem to be there. And now, it seems like EVERYONE we’re fighting in Iraq is Al Qaeda, and we’re treated to frequent vague but earnest-sounding warnings of impending terroristic doom.

Given these historical precedents, if there really is a government-run War on Science, then we’re in for a huge increase in scientific activity here.

I’m picturing a virtual underground Scientific Renaissance, where, like much of the late 1700’s and 1800’s, “citizen science” becomes a fashionable pursuit. People secretly building science labs in their basements and attics and performing legitimate, useful scientific research in them. Kids hanging out in abandoned parking lots at night, doing complex calculus problems in chalk on the ground and experimenting with broadcast power. Anonymous rebel scientists developing methods to cheaply and effectively convert lawn clippings into fuel ethanol and plastic grocery bags and soda bottles into biodiesel. Ignorant politicians assume home biology labs are marijuana-growing operations, that home chemistry labs are making methamphetamines, and that home physics labs are building radioactive “dirty bombs”. A multibillion-dollar new agency, the Science Enforcement Agency is hastily assembled and laws are badly written to restrict scientific activity to carefully-regulated government-controlled settings only.

Public science devolves into (when Republicans are in control) attempts to “debunk” global warming and evolution, “cure” homosexuality, develop ridiculously expensive military-grade weaponry, and silly projects that just plain won’t work but happen to be run by buddies of a senator or (when Democrats are in control) multimillion dollar projects to study “self-esteem”, research on “psychic powers”, development of homeopathic “medicine”, and silly projects that just plain won’t work but happen to be run by buddies of a senator. Disgusted underground scientists are only egged on by this state of affairs.

Within a few years, a cautious exchange of money in a public restroom will buy disease-curing doses of novel, effective, but non-FDA-approved antibiotics that cure drug-resistant Staphylococcus aureus or Tuberculosis. A backyard moonshiner-like biotech lab somewhere in the rural west secretly sets aside part of their flock of chickens, genetically engineering them to produce HIV vaccines with billions of dollars in “street” value. Someone with a closet chemistry lab develops an illicit catalyst that facilitates hydrolysis of water to produce hydrogen with no more energy input than ordinary body heat, while another develops an illegal strain of cyanobacteria that turns atmospheric carbon dioxide into a plastic substance which can either be used for building or is easily converted to biodiesel at such a rate that the developer has to rapidly build a huge, secret underground complex to hide the vast quantities of material produced overnight….

In the end, as always, government goes utterly insane and bankrupts themselves (more, I mean) trying to stamp out Illegal Science, but in the meantime, anyone who’s scientifically inclined ends up making a fortune. On the other hand, the efforts drive a lot of the science out of the country and Mexico becomes the new world superpower with their fleet of antigravity flying armored space cars, zap death ray guns, and clusters of quantum-supercomputers. (Note to self: get back to learning to speak Spanish!). This doesn’t really slow the flow of science into the US, though, and “science tourists” can sneak to Mexico to undergo age-reversing and/or intelligence-boosting medical treatments or to obtain cures for cancer or obesity that actually work. People end up in jail for recovering from leukemia or losing weight.

Meanwhile, on a more personal note, people like me who actually think doing science is fun get a few publications in underground science-journal ‘zines, spend a few years developing something useful, make a huge pile of money, and then retire before The Man catches up to us, to live a life of luxury somewhere. Maybe living in a giant mansion in Mexico between stints as lab techs for Mexican scientists once in a while, done just for fun and extra pocket-money…

It’ll be glorious. So – write your legislators today, and tell them we NEED the “War on Science”. For the Children.

(My political opinion? Lets just say that my political fantasy right now is that the 2008 presidential race will come down to a run-off between a Bloomberg/Paul ticket and a Gravel/Kucinich ticket….)

There, is THAT enough controversy to get some new traffic here?…



“Teh Deth Kitteh!”

Run! Itz teh Deth Kitteh!

The prestigious New England Journal of Medicine reports (Dosa, DM: New England Journal of Medicine; 2007; 357:4; pp 328-329 ) on the case of a single eukaryotic organism – a specimen of Felis catus – who is reported to identify People Who Are About To Die (insert ominous thundercrash here).

It is presented in a tone that is a mixture of “OOOo, spooky, mysterious!” and standard issue “Human Interest Story“, as though it was a baffling or unexplainable phenomenon. Honestly, didn’t modern science explain this long ago?

Obviously, Oscar the Cat is simply waiting around to devour the souls of the departed as they are exhaled on the last breath. Or as “Mike, the Mad Biologist” puts it:

Shouldn’t the situation be obvious? I mean, come on, did ALL of these journalists sleep through Biology 101? Even if they did, surely at least some of them own cats and already know about this….

There are, of course, numerous examples in the scientific literature documenting the tendency of the cat to steal the breath of the living. See, for example, Bener A, Galadari I, Naser KA.”Pets, allergy and respiratory symptoms in children living in a desert country”;Allergie et immunologie;1995 Jun;27(6):190-5…

Interestingly, as I was trying to find a more explicit reference in PubMed to this folk-belief, I ended up stumbling upon an article entitled “Micturition and the Soul” [Holstege G.”Micturition and the Soul.” J. Comp. Neurol. 2005 Dec 5;493(1):15-20.]. I love browsing databases of scientific papers. Where else could you go looking for a folk belief and find an article about the neurology of peein’?

I know I don’t normally discuss freakish, perverted Eukaryotes on this blog – hey, CHILDREN could be reading this! – but I found the article (and the responses to it) interesting, and it serves as filler until I can finish putting together my “Gram Stain Article To End All Gram Stain Articles” post.

Simplify, Simplify…

DNA seems to have two main threats to its well-being once it’s extracted and purified.

  • Nucleases
  • Spontaneous Hydrolysis by water

Nucleases are the big one that everyone seems to mention. The seem to be fairly sturdy enzymes, and they’re everywhere (including fingertips – hence the need to wear gloves whenever you get near DNA samples…), and they “eat” DNA rapidly. Theoretically, you can destroy the enzymes with enough heat, but you still need to worry about them getting in every time you pop open your sample to get some out.

Apparently, DNA even in pure water can tend to slowly fall apart spontaneously. It doesn’t happen very fast, but bit by bit, it can undo the links between the individual nucleotides.

A common way to try to deal with nucleases is to add EDTA to the solution. Nucleases need magnesium ions dissolved in the water to do their job, and EDTA tightly binds to magnesium (and calcium). The idea is to “use up” any stray magnesium ions in the solution so that even if nucleases get in, they’re inactive because they have no magnesium available. That’s why you see EDTA in the recipes for so many DNA-related solutions. Of course – EDTA doesn’t permanently bind up all the magnesium – there’s always a tiny fraction that stays in the solution. So, although EDTA can drastically slow down any nucleases, it won’t actually stop them.

There are also some interesting chemicals which can be added to destroy all proteins (including nuclease enzymes). Guanidine Thiocyanate is one rather nasty chemical that does this. 2-mercaptoethanol is another. Various other detergents like CTAB may also denature any proteins. Since they don’t harm the DNA in the process, you could keep the DNA sample dissolved in a solution with these chemicals…but then you can’t do PCR with the sample as it is, since the protein-denaturing chemicals will also destroy any enzymes that you WANT, like DNA Polymerase, when you try to mix it into your reaction.

I think the latter option will be great for collecting field samples (in fact, it’s papers specifically on the subject of preserving samples in the field with CTAB and Guanidine Thiocyanate based solutions that I’m adapting from), but isn’t going to be real useful once I’ve got my DNA relatively purified. What to do, what to do…

Actually, I think the answer’s simpler than I originally expected. I’ll just dry the purified DNA out. No water – no hydrolysis…and no nuclease activity, either.

I could actually just leave it as a dried pellet in the bottom of a microcentrifuge tube, but that leaves the problem of taking only a little bit of it for processing rather than taking the whole thing, and I want to avoid reconstituting it and re-drying it repeatedly. I think a variation of the “dry the DNA on a piece of paper” process will be in order – then I can just cut off a small strip of the paper to get a portion of the DNA. It appears that you can actually dunk the DNA-impregnated bit of paper right into whatever solution you’re using (like a buffered polymerase-and-primers solution for PCR) and go for it.

Among the several references I found on this, here are two:
Kawai J, Hayashizaki Y: “DNA Book”; Genome Res. 2003 13: 1488-1495
Burgoyne LA: U.S. Patent #5496562 “Solid medium and method for DNA storage” (1996); U.S. Patent and Trademark Office, Washington D.C.

I, for one, WELCOME our new radiation-eating fungal overlords…

Though I am getting a little annoyed at the breathless prose about how it’s “like photosynthesis” and might be a way to sustain astronauts during long space flights and so on.

The story’s about a fungus they found growing (thriving, even) inside the reactor at Chernobyl, despite all the radiation the fungus is exposed to in there.

What the original paper – which you can find here from PubMed Central (and where you can find what the study actually shows, rather than the somewhat lower-content hype found in most news reports on it) – seems to show based on my hasty undergraduate-level reading is that the fungi do grow faster when exposed to “ionizing radiation”, and that it appears to be due to melanin in the plant getting energy from the radiation (and passing that energy on to the fungus to use for growth).

This is actually pretty spiffy, but really – so far – doesn’t look like “photosynthesis” at all. They’re not testing for any kind of carbon fixation, and I’m guessing that if there is any carbon fixation going on, that it doesn’t generate oxygen in the process. It also seems unlikely to me that even then, the fungus can grow autotrophically. This would seem to drastically reduce the possibility of this stuff ever being PurinaŽ Astronaut Chow – you’d still need some other way to get the carbon dioxide out of the Astronaut’s air and put oxygen back in it. If you’re going to do that, you might as well just use plants (or cyanobacteria) and eat THEM.

Still, the implication that you could adapt some melanin-producing fungus to absorb “radiation” and turn it into useful materials of some kind is spiffy, even if it’s not going to allow us to turn nuclear fission plants and spent nuclear fuel depots into fungus-powered anti-global-warming-gas powerhouses.

One thing’s bugging me, though. I obviously don’t have enough understanding of how “ionizing radiation” behaves at a biochemical level, since I’m wondering if it’s proper for everyone to treat “radiation” (both from flying electrons and from high-energy light) as some sort of generic substance, whose only useful attribute is how much energy it has.

As far as I know, most of the “radiation” that the fungus inside the Chernobyl reactor is getting is Gamma-radiation – basically high-energy light (one step above “X-rays”, two steps above sunburn-causing Ultraviolet light). What the researchers are hitting their test-subjects with looks like it’s mainly “Beta”-radiation (which is to say – electrons)*. In both cases it’s “ionizing” radiation, which is to say (more or less) that the radiation knocks electrons off of atoms that it runs into in both cases, and in the ideal “spherical horse” world of a Physicist, the same amount of energy is going to knock the same amount of electrons loose from various molecules and therefore have the same effect, right?

Except I’m having trouble convincing myself that’s a valid assumption here. The results seem to show that exposure to radiation is somehow resulting in the melanin in the fungus being able to “reduce” a chemical (changing “NAD+” into “NADH”) that can potentially in turn dump electrons into the beginning of the Electron Transport Chain to in turn provide biological energy in the form of ATP…

Can one reasonably assume that the mechanism by which this happens would be the same regardless of the form of ionizing radiation? The big deal with melanin seems to be that it absorbs a wide range of light wavelengths (which is why it looks black to dark-brown, and why it protects skin from Ultraviolet radiation…) which implies that absorbing the gamma radiation is where the energy is coming from that makes the fungus thrive in the Chernobyl reactor building. I guess I’m just having trouble picturing how a much more massive, slower-moving electron could have precisely the same effect as a virtually massless, much faster photon. (Yes, I know that beta and gamma radiation are said to have the same amount of “effect” on living tissue per unit of energy…)

Is it possible that the melanin is directly “capturing” the beta particles (electrons), while gamma radiation is kicking electrons off of something ELSE, and melanin is then only indirectly taking up those? For that matter, is it possible that in both cases it’s just something silly like the radiation inducing hydrolysis of water, and it’s just hydrogen gas supplying the reducing power? Thinking about this is making me feel dumb – can anyone reading this explain what I’m missing here?…

I suppose I could just cheat and ask someone in the biology department. We’ve GOT a professor who ought to know – her research has specifically focussed on zapping prokaryotes with “ionizing radiation” (electrons from the college’s linear accelerator)…But that would rob my dear readers of the chance to participate here…

* – okay, it’s probably even more complicated than that. If I understand what the paper is describing and what my Minister Of Funky Physics Knowledge showed me, the source of the “ionizing radiation” for the experiments is radioactive Tungsten(W) and Rhenium (Re) (A “188Re/188W Isotope Generator”). W-188 gives off beta particles when it decays to Re-188. But Re-188 can go through some sort of funky subatomic rearrangement before it decays so that it can EITHER give off beta particles OR gamma-rays as it decays down to stable Osmium-188. I have no idea what the proportion between beta and gamma is at that step (the “conversion efficiency”) so it’s possible there’s enough gamma radiation coming out to do something, regardless of what the beta particles are doing. (The experiment doesn’t do any comparisons with “pure” gamma radiation, which I imagine is not simple to arrange…). So now I’m even MORE confused. Thanks, physics. Thanks a lot.

Colony PCR – because DNA extraction protocols suck.

If you’ve got a culture of a single type of bacteria and you want to identify it, the standard method is to figure out the sequence of one particular gene, the 16s rDNA gene. That is – it’s the gene which encodes the a piece of RNA that gets used by the ribosome in part of the process of “reading” which amino acids to link together to make a particular protein. This is something that every prokaryote known has, and parts of it are conserved, so they’re similar enough to compare, while other parts can vary a lot, providing enough “difference” to tell different organisms apart.

To figure out the sequence, you use PCR to “amplify” this particular gene, making lots of copies of it so that the sequencing machine can clearly see the signal from each part of the sequence. And before you can do that, you have to get the DNA out of the cell relatively intact.

That part can be a pain. There are lots of different ways people have come up with (and made special canned “kits” out of) – you can use chemicals to try to dissolve the cells and let all their guts (including the DNA) out, you can try to mash them up with tiny glass beads in a “bead-beating” machine, you can stick them in a blender, you can even just boil them for a while…then usually you go through several steps of centrifuging and mixing with different chemicals and then centrifuging again until you’ve hopefully finally got the DNA out and gotten rid of most of the other cell bits. And, hopefully, you haven’t accidentally chopped up the DNA too much to use in the process.

Fortunately, there’s a trick you can sometimes use, referred to as “Colony PCR”. In it, you literally just touch the top of your colony of cells and shake them off directly into the PCR tube. Then you just include an extra 5-10 minutes of 95°C heating to hopefully cook open enough of the cells to release DNA (and cook the cell’s enzymes to death so they don’t degrade the DNA and interfere with the PCR).

Not real reliable if you’re trying to do anything quantitative, like trying to figure out how many copies of a gene are in each cell, or trying to get an accurate estimate of how many cells of one type or another are in a mixed culture, but if you just need as much of a particular bit of DNA as you can get – such as for sequencing – a lot of people use this.

I just tried it on my Lambic isolates. Two of the 8 bacterial cultures worked beautifully. I’m pretty sure the problem with the other 6 was just the sheer amount of bacteria I ended up adding to the reaction – too much seems to “swamp” the PCR process and keep it from working. I’ll try it again this week. But it does seem to indicate that it works, at least.

E.coli – the “Microsoft” of the biotech world?

…by which I mean, it’s not always the best tool for the job, but everyone insists on always using it anyway, and has a variety of excuses for doing so…

Honestly – I’m trying to set up a clone library of 16s rDNA sequences using this kit. Never mind which kit it is – it actually does seem to work. I was just struck by the amount of hassle involved in shipping and storing the kit and it’s supply of “competent cells”.

When you get them, take them out of the dry-ice they’re shipped in and put them in the -80°C freezer immediately or they’ll die! Only thaw them carefully just before you use them, and do it on ice or they’ll die! Don’t heat-shock them for more than exactly 30 seconds or they’ll die! Once you’ve got them growing, you have to keep moving them to fresh selective media frequently or they’ll die! Or, you can carefully place them in the -80°C freezer…or they’ll die! Don’t look directly at them or they’ll die! (Do Not Taunt HappyFunCell!…)…

Seriously, running those gigantic -80° freezers can’t be cheap. Wouldn’t it be more convenient if you could grow up your transformant as an ordinary culture and just add your DNA samples and some kind of inducer chemical to make them take it up? Surely there must be some other organism that might be made to work like that.

Actually, it seems a number of the “Gram-positive” (firmicutes) organisms can enter a state of “natural competence”, where they naturally take up double-stranded DNA molecules from the environment. Bacillus subtilis is one. I’ve even seen references to “natural-competence” based protocols for transforming B.subtilis (or other Bacillus species, presumably) but it only seems to be in an out-of-print, $400 book.

Wouldn’t that be more convenient (using B.subtilis that is, not the $400 book)? Plus, when you wanted to store your transformed culture for later use, you could just heat the culture up to, what, about 55°C for 15 minutes or so (as I recall) then let it dry. The spores will contain whatever “bonus” plasmid DNA you added (if spores didn’t keep plasmids, then anthrax wouldn’t be such a danger…) and will last practically forever at room temperature. Mix the spores with some dried nutrient powder and seal them in a foil packet. Instant transformants, just add water!

But NOOOOO…..”But, everybody else uses E.coli, so I have to.” “They only make ‘BogoGen SuperMiniUltraKlone Kit 2000’ with E.coli, and we have to use that!” “But, nobody knows that other stuff, but everybody’s already familiar with E.coli!” “I’m a BogoGen Certified E.Coli Engineer, and I say everything else is just a toy and doesn’t work!” “All the books and stuff are about E.coli…”

Bah! Pathetic excuses. Anybody got a huge wad of venture capital to throw at me? The more I think about this, the more I think ‘untapped niche’…Heck, the electricity savings on not having to run a -80°C freezer constantly alone ought to qualify for a good “Fight Global Warming – Say ‘No!’ to E.coli!” marketing campaign…

Bonus perk: All the natto you can eat…

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 2.379×10some-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…

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-OŽ. 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-OŽ is the cytoplasm. (It’s Lime merely because I like lime Jell-OŽ.) 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-OŽ 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-OŽ 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-OŽ 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 OreoŽ 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-OŽ 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.