Category: Microbiology

Posts that are Microbiological in nature

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

How does one categorize prokaryotes?

If you’re taking or have taken only basic or “medical” microbiology courses, it may seem like bacteria are still classified by ancient 19th-century criteria. Are the bacteria Gram-positive, Gram-negative, Acid-fast? And then, are they round, straight, or bendy? Then, to categorize any further, there’s a bewildering array of culture tests you can go through – some of which seem archaically specific with quaint un-intuitive terminology for the results.

Take “hemolysis“, for example. Considering how wide-ranging the environments are in which different bacteria can live, going through all that effort to see if the bacteria secrete any special substances that have a special effect on sheep red blood cells seems less than helpful. Okay, in fairness, when you’re dealing specifically with disease bacteria with the potential to possibly get into somebody’s bloodstream this test is useful, but in the entire population of all bacteria, this is a ridiculously tiny number of species.
And then there’s the terminology for this one – if the blood cells in the media are popped open but not totally destroyed (the media underneath will be greenish from the iron compounds released from the popped cells) it’s called “?-hemolytic”. If they ARE essentially totally destroyed (the media underneath the bacteria clears completely) it’s “?-hemolytic”. Not bad so far, but what if the bacteria doesn’t visibly destroy the blood cells at all? Why, that’s “?-hemolytic”. No, not “non-hemolytic”, even though that’s what it means. And, no, I have no idea why this is. I’m glad whoever came up with this scheme wasn’t a legislator. Otherwise, people who were accused of being murderers but were proven to be innocent would be declared to have committed “?-homicide”…

Using these kinds of phenotypic tests does have some advantages – most of them have been around so long that they’ve been thoroughly tested, they’re usually pretty easy to do, and if you’re hiring people to do bacterial classification grunt-work, finding people who can handle “mix this stuff together and see if it gets clumpy” is easier and cheaper than finding people to do more complex molecular work. On the other hand, there are some major disadvantages.

Firstly, there doesn’t seem to be any publically-accessible data repository with useful information of this kind that I’ve been able to find, unlike, say, the genetic data readily accessible at Genbank or the RDP-II. Sure, you can sometimes find some of this kind of data in papers describing specific species or strains, but to use this kind of information you have to already have a good idea of what your microbe might be – you can’t just plug “it ‘ferments’ lactose, sucrose, and xylitol, but not sorbitol or fructose, it’s catalase-negative, liquifies gelatin, and grows happily even when there’s tetracycline in it’s culture media” into a decent online database anywhere that I’ve been able to find and get a listing of known possibilities.

The other problem is that a lot of these kinds of traits aren’t necessarily part of the bacterium’s own genome. Bacteria often inherit certain traits from “bonus” DNA that they can pick up from other bacteria (“plasmids”) or from a viral infection. Yes, bacteria can be infected by viruses. Resistance to antibiotics is commonly spread by plasmids (though not always – I seem to recall that Klebsiella pneumoniae, for example, has a penicillin-resistance ability that actually is part of its core genome.) A number of diseases are actually caused by bacteria getting infected with viruses. Maybe your case of Necrotizing Fasciitis isn’t because your Streptococcus pyogenes on your skin, but because your Strep. pyogenes is rabid…

The most reliable way available to work out who a microbe is involves comparing versions of a gene that essentially is never moved around between different microbes, but all microbes have. For prokaryotes, the gene in question is the “16s small subunit ribosomal RNA” gene sequence – or just called “16s“. This is what the taxonomy you can find at NCBI for bacteria and archaea is based on.

You know, when I started this post, I was just going to say that I wanted to get a more intuitive understanding of prokaryotic taxonomic groups and to that end was going to put together some posts on particular types of bacteria and archaea. I hope all the “bonus” background that seems to have come pouring forth from my overheated mind is useful.

Anyway, stay tuned this weekend for a post on firmicutes at some point.

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

I am filled with shame…

It’s been a long day away from home, and I’ve got nothing much prepared for tonight – the last day of “Just Science” week. (Not that I’m going to stop posting after today or anything…)

So, I’ll cop out, and instead post a question.

What textbook(s) are you currently using for Microbiology classes, and what do you think of them? My “Introductory Microbiology” class was over 8 years ago, but the textbook was Tortora, Funke, and Case – “Microbiology: An Introduction (sixth edition)”.

I found it annoyingly heavy on the “disease-listing” and way too sparse on the rest of the microbial world – though they did have a couple of chapters on applied/industrial type microbiology.

Please leave comments…

My posts will likely be pretty sparse until after Wednesday, when I have back-to-back Microbial Genetics and Pathogenic Microbiology exams. Ick. As you can imagine, I’ll be studying a lot for the next few days to make sure I’ve learned what I’m supposed to up to this point.

Curse you, public library!

Tonight’s post will be an eclectic one…

I made the mistake today of heading for what passes for a “large city” in my local area in a general need to go somewhere besides my house and the college. I figured I could browse the local discount bookstore and see if they had anything interesting.

I happened to notice a sign advertising a book sale at the local library.

Why did they have to do this to me? Have they no decency? Have they no shame? Have they no MERCY?

As I previously mentioned, I actually do collect old science (and medical) books. Unfortunately, I ended up walking out of the library with a whole mess of microbiology books (and one Botany book that I picked up just because it was old – 1930’s). Fortunately, they were cheap.
I was just perusing one of the books I picked up: an old “Bacteriology” book[1] from the late 1940’s. It’s fascinating and instructive to see what scientists used to believe was true and what observations led them to believe it.

The introductory chapters of the book include a discussion of taxonomy and the place of “Schizomycetes” (meaning bacteria that aren’t photosynthetic) in the overall scheme of things. There’s a discussion that, given what information was available at the time, is perfectly reasonable and explains why bacteria are “plants”, just like other fungi (Fungi, you see, are just plants that aren’t photosynthetic – or so they explain). The author gives a classification scheme for plants that divide them into three categories, which roughly equate to “normal” plants (with stems and leaves), moss-type plants, and plants that don’t have roots, leaves, stems, or flowers. This latter category he broke into two sub-categories – Algae (including “Blue-green” algae, which we now know are actually bacteria) and Fungi. “Bacteria” are listed as one of the categories of Fungi.

The discussion justifying this categorization makes some interesting claims – some of which are startling to me. The author claims that some bacteria – “Acetobacter xylinum” have cell-walls that consist of cellulose, just like plants. (Actually, it would appear this bacterium does make cellulose, though I don’t think it’s actually a component of the cell wall – this is a standard “Gram-negative” type ?-proteobacterium). I had no idea up to this point that there were cellulose-producing bacteria. Interestingly, the author also states

“Some bacteria are said to possess cell walls of chitin, a distinctly animal substance which is the material of horn, hair, hoof, and insect shell”

which is completely wrong on every count except for the part about insect shells. (Horn, hair, and hoof (and fingernail) material is Keratin, which is a type of tough protein. Chitin is actually a polysaccharide…and it is what most fungal cell walls are made of.There are some interesting statements in the section on microscopy as well. The author claims:

“There seems no doubt that the gram-positive material in bacteria is ribonucleic acid. Bartholomew and Umbreit[2] have shown that it can be removed by soaking the gram-positive cells in sodium choleate. It may be replaced by treating them with magnesium ribonucleate. Normally gram-negative species will not accept the applied coating. The specificity of these reactions is shown by the fact that an enzyme, ribonuclease, will remove the gram-positive character (ribonucleic acid) of the cells very quickly.”

What the heck?… Now I have an urge to see if I can sneak a culture of some kind of Bacillus and some RNAse and see how much of this explanation actually matches observation. (Perhaps I can dig up Bartholomew and Umbreit’s paper as well). The author also mentions that nobody has managed to get a good image of a bacterial nucleus, either, which of course is because they don’t actually have one…
One other thing I’d never heard of: Proton Microscopy. According to the author, this technique, apparently first implemented in France in 1948, could theoretically give substantially better resolution than electron microscopy.

Some quick poking around seems to show that this is partly true, and there actually are proton microscopes that get used for some kinds of studies. However, protons are a heck of a lot harder to “focus” and they don’t seem to have caught on for microbiological work. They do evidently have some useful properties for doing analysis of what specific elements are in a sample, though[3].

I noticed some other apparent differences in style between the older textbooks and current ones, but I’ll save that for another time.

I will also at some point go back and re-write the Schizomycete article to include some of the information I’ve picked up in the last couple of weeks. Meanwhile – one more day of “Just Science” week! Looks like I should survive it after all.

[1] – Frobisher, Martin Jr. “Fundamentals of Bacteriology (Fourth Edition)”, 1949, W.B Saunders Company, Philadelphia
[2] Bartholemew JW, Umbreit WW, “Ribonucleic Acid and the Gram Stain”, J. Bacteriol. 1946, 48:567
[3] “Microscopy with Protons” http://www.innovationmagazine.com/innovation/volumes/v7n1/coverstory3.shtml (visited 2007-02-10)

The “Electron Transport Chain”, Grossly Oversimplified

Why does breathing work, anyway? And can I possibly explain it in a couple of paragraphs? I don’t know, but I’m going to try…it leads into the subject that got me interested in majoring in Microbiology in the first place. It’s probably kind of foolish to try to cram in this explanation in the half-hour or so before midnight (and hence the informal deadline for getting a post up every day for “Just Science week”), but here goes:

First, a bit of really simplistic background. Since the fundamental principle of the universe is basically that stuff likes to fall apart (dang lazy molecules), in order for a cell (bacterial or otherwise) to make new proteins and strands of DNA and so on, it has to have some kind of energy that it can use to pay for the increasing orderliness that it’s causing. The chemical that’s usually used to provide this energy is ATP. The energy comes from a string of three Phosphate (PO4) groups that are attached to it. The third phosphate in the chain comes off really easily, releasing a bit of energy in the process, like a spring uncoiling. A lot of enzymes work by attaching to ATP, letting ATP fall apart (becoming the slightly more “relaxed” ADP in the process), and using the released energy to power some other process.

In order for this to work, the cell has to be constantly re-charging ADP, cramming that third phosphate back onto the end along with putting back the bit of chemical energy.

The point of this post is a major way that cells provide the energy to reassemble ATP. There are actually a number of ways, but one of the more effective is the “Electron Transport Chain”.

In simple terms, the cell takes an electron from a simple “food” molecule of some sort, and passes it along to a type of protein that reaches through the cell’s membrane. This protein passes the electron along to another protein, but in the process, it goes through a series of changes in shape that allows it to pump a few hydrogen ions (“protons”) from the inside of the cell’s membrane to the outside. Depending on how much energy (as “electrical potential”) was released along with the electron by the “food” molecule’s electron donation, there may be enough energy to shove the electron through up to three different proteins that do this “proton-pumping” trick with each electron.

This process causes there to be a buildup of protons outside of the cell membrane. Since the universe is lazy, it doesn’t want to hold all those crammed-together protons in place – it really wants to shove them back inside the cell so there’ll be an even concentration of them on both sides of the membrane.

The cell has a special sort of gate which lets the protons shove their way through back to the inside of the cell – but in the process, they make part of the ‘gate’ mechanism rotate. The rotating part essentially grabs ADP and loose phosphate and virtually crams them back into place – the energy to do this comes from the force of the protons shoving their way back into the cell.

But what about the electron? Well, at the end, there has to be something that will pull the electron off of the last protein. One of the best “electron acceptors” is oxygen. Oxygen is the second most electron-loving kind of atom there is. Half of an oxygen molecule (O2), a couple of spare protons, and two electrons make a nice, relaxed, stable molecule of H2O.

The reason I find this interesting is because some bacteria can use something besides oxygen, if oxygen isn’t available. They don’t get quite as much energy out of the process since these other “electron acceptors” don’t pull the electron out quite as hard at the end, but it’s better than suffocating. Sulfate-respiring bacteria, for example, can use sulfate (SO4) as the place to dump the electron, converting it to sulfite (SO3) in the process – and eventually converting it to plain “elemental” sulfur (just “S”) or even in some cases using the elemental sulfur in place of oxygen and making H2S – which is that ‘rotten-egg’ smelling gas.

There are some even more exotic “electron acceptors” that some bacteria can use…which will be the topic of another post.

(And, again, please let me know if you spot anything wrong here, and please ask questions if I’m not making any sense – I’m pretty sure I need the practice explaining this kind of thing…)

Cheap Miscellany

Tonight’s post will be a bit short, but I’ll try to make up for it tomorrow. (Three more days of Just Science week to go…)

First, a quick unsolicited plug for someone else’s site: Aetiology is doing a series of posts on “Normal Flora” – that is, the microbes that normally live on and in healthy people (as opposed to microbes that just cause disease). Since that’s an area where my own interests overlap with the more conventional “medical” microbiology, it seems appropriate to mention it here. As of right now, there is a Part 1 and a Part 2. Interesting and informative stuff.

Second, a small addendum to the previous post – I mention that I think when people say “Gram-Positive” they generally really mean Firmicutes, but I just realized there’s one exception to that. The Firmicutes actually include Mycoplasma and related bacteria – which have no cell wall at all. I would guess their ancestors were normal “Gram-Positive”-type firmicutes but somewhere along the way “lost” function of a key gene involved in making the thick cell wall. (There’s a similar group of Archaea – the Thermoplasmata. ) These don’t stain Gram-positive (and perhaps don’t stain gram-negative, either – seems like such fragile things would be destroyed by the staining procedure). In order to see these in the microscope, the standard method seems to be to use a chemical that actually stains DNA instead. Instead of staining the outer surface of the cell like most of the classical stains, this stains the inside of the bacteria (which tend to have their DNA spread more or less throughout the entire cell in one form or another, since they have no nucleus to pack it into) with a fluorescent material, which you can then see in the microscope with the right kind of light. You can also use this kind of technique for other bacteria, too. The “Live/Dead” stain I previously mentioned works this way, if I remember correctly.

Since you probably don’t want to try heat-fixing Mycoplasma, you have to use a “fixative” (a preservative made of alcohol and pure acetic acid) and then air-dry the slides.

This leads to one last correction – in a previous post I suggested that it was unlikely that you could “glue” your smear to the slide rather than heat-fix (assuming that the “glue” would interfere with the subsequent staining and viewing of the sample) – this is actually not completely correct. I heard today of a protocol for doing endospore and acid-fast stains which called for mixing the culture sample with serum on the slide to make it stick, so there are at least a few ways to “stick” cells to the slide and still look at them.

Are “Acid-Fast” bacteria Gram-positive or Gram-negative?

I was wondering what today’s post ought to be – but a Google™ search that reached the page posed an interesting question and made it easy.

Someone from a Miami, Florida college got to this site after asking Google:
“Assuming you could stain any cell, would an acid-fast [bacterium] be gram-positive or gram-negative?”

Therefore, today’s post will deal with some Microbial Physiology.

The easy answer is, of course, “no“.

A more useful answer, though, is that it depends on what you mean by “Gram-positive”.

If one takes the “Assuming you could stain any cell” part of the question to mean that you’ve done whatever it takes to get the Crystal Violet/Iodine into the cell wall, and you mean “will the cell still look purple instead of pink at the end of the Gram Stain process”, then I’m pretty sure the answer would be yes, that it would be “Gram Positive”. It actually IS possible to stain “acid-fast” bacteria with the Gram stain. The catch is that it takes 12-24 hours of staining (according to Gram’s original paper) rather than a minute or so. This still counts as Gram-positive, though, and in fact the whole Phylum of Actinobacteria (including the Mycobacterium genus) is considered “High G+C Gram-positive”. (If the query was for an exam or homework problem, this is probably the answer you’re looking for.)

On the other hand, if you mean “does the cell wall structure of an acid-fast bacterium better resemble Gram-positive or Gram-negative bacteria?”, you can make an argument that instead of a nice simple “inner membrane surrounded by a thick peptidoglycan-layer cell wall”, the “acid-fast” bacterial cell wall looks more like a complex gram-negative-type cell wall, having multiple layers, with special proteins that form channels through them to let substances in and out of the cell through the otherwise penetration-resistant outer layer, just as Gram-negative bacteria have through their outer membrane. (On yet another hand, those outer layers are tough like a gram-positive bacterium rather than fragile like a gram-negative bacterium’s outer membrane, and don’t dissolve in alcohol.)

Therefore, if you’re speaking in terms of taxonomy, and by “Gram-positive” you mean firmicutes which, as far as I know at the moment, are really the only class with the simple, officially-gram-positive-type cell wall structures and therefore are usually what is meant when someone says “Gram-positive” (someone please correct me if I’m wrong here), the obvious differences with “acid-fast” type cell walls can at least make a good argument that they are “not ‘Gram Positive’ bacteria”.

But if you put that on your homework or exam answers, don’t blame me if you get marked wrong…

I actually found a pretty nice illustration on the University of Capetown website showing the differences in cell wall structures here, which might help.

So, to summarize – Officially, they’re either “neither” or “Gram-positive”. Unofficially, you can probably argue either way. Hmmm. I should try to work in a post on bacterial taxonomy one of these days.