Category: Microbiology

Posts that are Microbiological in nature

Environmental Chemistry Field Trip – Day 1, part 1

I can think of a number of things to complain about with regards to living where I do. However, it is nice that we live near enough to Yellowstone to day-trip there. In fact, it’s close enough for my local college to take field-trips there – which we did.

Environmental Chemistry spent the weekend there, examining the area, discussing the chemistry of the natural waters and geothermal features, and collecting samples (yes, we had a permit for this…).

We started with a stop by the side of the Madison River to collect a sample of the surface water. Clear, cool (12°C, or about 55°F), mildly basic (pH of about 8.0), and a TDS reading of about 300ppm, which is roughly the same as mildly to moderately hard tapwater, I suppose.

sampling water from the Madison river

The sampling device -seen being hurled over the water here – is kind of interesting – it’s a hollow tube (a bit of plastic pipe) with two spring-loaded balls that slam shut on either end to trap the water inside when you tug on the string. That lets you throw the device out and trigger it when it gets to the precise spot that you want to take a sample from.

We made a brief stop at Beryl Spring afterwards. We didn’t do any sampling here, but we did talk about acid-sulfate water systems. “Reduced” sulfur – as Hydrogen Sulfide gas – comes boiling out from underground along with steam, and ends up being oxidized by oxygen from the air to become sulfate in the end – combining with the water and forming sulfuric acid.

Sulfur-encrusted pipe at Beryl Spring

Of course, it doesn’t go from sulfide to sulfate all at once. There’s a stop along the way as elemental sulfur. The whitish-yellow stuff here is crystals of elemental sulfur. The black stuff you see is…also crystals of elemental sulfur. The difference is just how the atoms of sulfur collect together. The black form is actually a little less stable than the yellow, so it tends to form first, but then slowly convert to the yellow form over time as the sulfur atoms settle into a more stable arrangement. Being a chemistry class, we didn’t really discuss the possible microbial activity that might be involved here. Note the small patch of dark-green there. I suppose this could be a “Green Sulfur Bacteria“, which does something like photosynthesis except that it makes sulfur instead of oxygen in the process. These are normally anaerobic but perhaps the concentration of hydrogen sulfide (H2S) and carbon dioxide gas coming out of the ground right there is enough to crowd out the oxygen. Alternatively, it could just be a heat-loving cyanobacterium or something.

I really wish I wasn’t too poor to buy a good field microscope to go along with the good lab microscope that I am also too poor to buy…

The last two stops of the day – Appolinaris Spring and Narrow Gauge Spring – will be in the next post…

If I Win It…

One topic that I have hoped to emphasize much more on this blog is amateur science, and in particular (given my educational background) amateur Microbiology.

Don’t be dissuaded by my use of the word “amateur” here. I don’t mean “not really” science (i.e. the microbiological equivalent of the “baking soda volcano”). Rather, here I’m using “amateur” in its proper etymological sense – science done for the love of it. I don’t just mean my brief series of experiments on the toxicology of expired JellO®. I mean actual microbiology with potential practical application as well as educational value. Unfortunately, there are a few bits of equipment for this that I can’t reasonably cobble together out of spare parts or repurposed household appliances. A microscope, for instance. Or a dry-ice maker.

Being a full-time college student, I’m poor, and can’t afford a microscope. A decent ordinary “brightfield” microscope appears to cost about $400. Bonus materials like a “darkfield” condensor are extra, unless I think I can rig up an equivalent on my own. A nicer digital camera to take pictures with to share with you, my loyal reader(s) would add some more to the cost. Even in the case of equipment and supplies improvised from more ordinary and readily-available materials (pressure-cooker=”autoclave”), there is still a cost. Woe unto me, what shall I do?!?!?

For the moment, I shall revert to the time-honored traditions of “begging” and “hoping”…

You see, there appears to be a scholarship available for bloggers who are full-time college students. Why, what a coincidence! I blog…and I’m a full-time college student! What luck!

There appears to be a US$10,000 (that’s almost 10000 CANADIAN dollars!). It’s not explicitly stated but last year they also had $1,000 “runner-up” awards as well. Here, then, is my pledge to you all.

Should I be selected as a finalist for this scholarship competition, I will eat 2-year-old JellO! Furthermore, if I were to actually be selected to win a $1000 scholarship, I will buy a real microscope and be able to blog my microbiology experiments and studies much more vividly. I will also blog the design and construction of my own amateur microbiology lab, to the extent that I can afford. (Well, I was ALSO going to do this anyway, but with a scholarship I’d actually be able to start doing it…)

Were I to be selected to win the full $10,000 scholarship I propose to go absolutely Nucking Futs, with a microscope, a nice new digital camera, dry-ice maker and plenty of CO2, perhaps some dedicated hosting for this blog, and a complete collection of useful microbiology equipment (mostly improvised still, but that’s half of the education right there…). Furthermore, should my readers demand it, I might even be persuaded to drink a cup of fresh Lysogeny Broth!

Come on, who needs this money and attention more – me, or some wealthy (compared to me) graduate student over on scienceblogs.com? I bet none of them would eat 2-year-old JellO or drink E.coli Chow for it, would they?

10 Finalists are to be announced October 7th, from what I understand…wish me [good] luck…
UPDATE: I made the finals, though my fame doesn’t seem to be carrying along a rose-petal-strewn path to victory yet…

Okay, this is completely bogus…

The folks over at BBspot are callously spreading microbiological misinformation (click for full-size):

This is just plain irresponsible and utterly wrong. Surely everyone can see the obvious problem here, right?

When this happens, the flame is blue, not orange.

Uh, or so I’ve heard.

(If it isn’t obvious – what you do in this situation is calmly take the spreader out and set it on your nice, flameproof benchtop, and set something non-fragile and non-flammable on top of the flaming jar of alcohol, which will then go out quickly as the oxygen gets used up. All the labs I’ve been in lately use “canning” jars for the alcohol in this application, complete with lids which can be set on top to extinguish the flame.)

The Oldest Microbiology Book (that I own)

There’s this thing that some people do sometimes when they’ve been getting stressed out in one place for a while. I hadn’t done it in so long I can’t remember what it’s called. You know, where you Leave the area and then avoid it for a while. Oh, yes, that was it, a vacate-shun. Anyway, leaving the barren desert wastelands of the West, we headed east, and spent a few days admiring the area around the midpoint of the Appalachian Trail: Harpers Ferry, West Virginia. (Incidentally, I can recommend the “Angler’s Inn” Bed and Breakfast there, and the whole time there was incredibly delightful to me. I think I’d love to move to the area.).

I was delighted to note that there was an Old Book store in downtown Harpers Ferry. One thing about the Eastern US is that it’s been settled by book-using folks for somewhat longer than the West, so it would seem it’s easier to find really good Old Books. I found a publication of a 110-year-old microbiology book. In decent condition, for just over $20, no less! Not counting the (relatively modern) reprint of Micrographia that I picked up from a library sale, this makes it by far the oldest microbiology book I own now.

Oh, yes, did I mention I collect (casually) old books, especially old scientific and technical books?

The book in question, published in 1897, is “Story of Germ Life”, by Herbert William Conn. Not to be confused with Harold Joel Conn of “Conn’s Biological Stains” fame…who happens to be Herbert William Conn’s son. To be fair, the book *I* got was actually a republication from 1904, so only 103 years old…back when copyright was more rational (7 years, plus an OPTIONAL 7 more years. Thus explaining why my republication came out 7 years after the original.) It appears to have been part of a series called “Library of Valuable Knowledge”. The bookstore actually had another one of them, but I don’t remember what its topic was.

“Story of Germ Life” isn’t really a textbook so much as an overview of the subject of “Bacteriology” (as understood in 1897) for otherwise well-educated people – the kind of book I don’t think there are enough of these days. The Gutenbook project actually has a plain-text-only version of the book online here. Of course, then you miss out on the incredibly useful illustrations:

I always find it interesting to go back and see the earlier stages of scientific endeavors – especially as relates to my own interests. There always seem to be things that have since been forgotten, abandoned, or glossed over in them.

H.W. Conn seems to have been most interested in dairy microbiology, so there is a substantial amount of space devoted to it. I’ve heard of “blue milk” before (Yummy!….Pseudomonas?), but not Red or Yellow milk. He also devotes space to discussing the affect of “good” (and “bad”) bacterial cultures on butter, cream, and cheeses. I’m not even sure if butter is cultured these days, or if they just churn it up fresh and cold with minimal growth. Dangit, one of these days we’re just going to have to move somewhere we can keep a miniature dairy cow so I can do some experimentation with real unpasteurized fresh milk.

Bacterial phylogeny was so quaint back then. “Bacillus acidi lacti.” Ha! I love it. Interestingly, the term “Schizomycete” doesn’t appear anywhere in the text, though that may or may not be because it was considered unnecessarily technical for the intended audience. There’s actually very little about microbiological methods, too, which is the one major disappointment for me. Oh well, still interesting stuff. Conn actually mentions various “industrial” uses of bacteria including retting (soaking fibrous plants like flax or hemp so that bacteria eat the softer plant material to free the fibers), the roles of different bacterial cultures in curing tobacco, and even a fermentation in the production of opium (which Conn says is fungal rather than bacterial).

Also, much to my approval, the first 2/3 of the book is not about diseases. Only the last third of the book discusses “parasitic bacteria” and related topics. I leave you with this quote from the book’s 1897 Preface, which I think is still relevant today:

“Few people who read could be found to-day who have not some little idea of these organisms and their relation to disease. It is, however, unfortunately a fact that it is only their relation to disease which has been impressed upon the public. The very word bacteria, or microbe, conveys to most people an idea of evil. The last few years have above all things emphasized the importance of these organisms in many relations entirely independent of disease, but this side of the subject has not yet attracted very general attention, nor does it yet appeal to the reader with any special force. It is the purpose of the following pages to give a brief outline of our knowledge of bacteria and their importance in the world, including not only their well-known agency in causing disease, but their even greater importance as agents in other natural phenomena. It is hoped that the result may be to show that these organisms are to be regarded not primarily in the light of enemies, but as friends, and thus to correct some of the very general but erroneous idea concerning their relation to our life.” — April 1, 1897

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.

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.

Is GFP GRAS?

Woohoo!

One of my longstanding questions has been, is Green Fluorescent Protein safe to put in food?

I always figured that it SHOULD be – it comes from jellyfish, which I know people eat in some cultures (though I’m not sure if any of the ones eaten actually express GFP).

Well, it seems someone in 2003 did a proper test…Check it out!

Sure, this is still some way before the FDA declares it “Generally Regarded As Safe” but it’s one step closer.

Soon, my dream of genetically-engineered “Glogurt®” will become reality! AH, HA HA HA HA HA!

Okay then…

My summer classes are finally over. Got an “A” in immunology (go, me). Now I just need to make sure everything’s done next semester. I’ve already signed up for the last two Underwater-Basket-Weaving-type “General education” classes required at this college: Intro to Philosophy and “History of Western Art”. I also went ahead and signed up for Environmental Chemistry, too – it’s not required, but it’s one of the last “not required but useful if I have time for it” classes on my list.

Meanwhile – is it just me, or is DNA some obnoxiously fragile stuff when you don’t want it to be? Sure, leave a few flakes of skin or hair follicles at a crime scene and they’ll nail you weeks or months later, but try to “gel purify” some DNA and it just falls apart…

The samples from my last post, about the colony PCR of my Lactic Acid beer-bacteria, I cut the bright bands of presumably-16s rDNA out of the gel and ran them through one of those canned “gel purification kit” processes. Then I froze them until I had a chance to finish my classes and play with them.

Yes, I was wearing gloves. No, I didn’t lick the gel. I think I must have looked at them too closely or something and they just disintegrated out of spite. In any case, my attempt at a restriction enzyme digest turned up NOTHING (other than the “ladder” lanes) on the gel.

I’m beginning to really distrust canned kits. On the upside, that means I get to learn some more in the process of developing my own replacement protocols.

I will probably try re-amplifying DNA from the frozen samples and see if there’s anything at all left in there that can be saved. Otherwise, I’ll also check and see if the plates I made a few days ago still grew okay.

In other news – I’m toying with the idea of literally begging for my own microscope and home-microbiology lab equipment. As in, actually putting on a lab coat, taking an old hat, and sitting outside of scientific meetings and such with a cardboard sign saying “want my own microscope – please help”. Of course, I’d have to report any donations as “income” for tax purposes – I doubt they’d let me form a 501(c)(3) corporation dedicated to just buying me toystools for my own microbiological amusement.

I haven’t decided, but it’s under active consideration. It’d make for some interesting blogging (and I promise in return that I’d account on the blog for any money donated, and blog all uses of the equipment under Creative Commons terms so everyone can use it). It’d presumably take a while for this to get anywhere if it ever did – it seems it’ll cost about $400-$500 just for a (good) basic light microscope, plus another few hundred for a darkfield condenser and related upgrades. Plus, of course, me wanting to build some LED-based lighting for fluorescence microscopy ($500 canned commercial upgrade? Bah!). Incidentally, it seems Green Fluorescent Protein fluoresces best right around the wavelength of a typical, inexpensive, off-the-shelf ultraviolet LED…

And then of course I need a pressure cooker and one or more incubator setups and some petri dishes and trips to the grocery store for growth media and staining supplies and slides and… well, anyway, as much stuff as I can arrange to get. But the microscope is the one component that is unavoidably expensive.

Oh, yeah, and some space to keep cheese and beer culture organisms and such for later use…

Comments, anyone? Suggestions?

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 Purina® 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.