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 95C 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.

“Live and active cultures” – of beer.

I’ve got a project going to isolate as many yeasts and bacteria as I can from the dregs of a bottle of relatively-famous-brand Lambic ale.

So far, I’ve got at LEAST 3 different types of bacteria and two different yeasts – all of which I suspect are “intentional” – that is, the bacteria are probably lactic-acid bacteria (Lactobacillus, Pediococcus, etc.) which are expected to grow there, and the yeasts I believe to be a Brettanomyces-type yeast and a Saccharomyces yeast (based purely on what I expect to find and the small amount of microscopy that I’ve been able to do so far.)

I have at least one and maybe two different “Gram-positive” rod cultures which I believe to probably be Lactobacillus species. I have several isolates of generic “clusters of Gram-positive coccoids” of which there are at least two different types (which look more or less identical in the microscope, but one of which seems to generate acid while eating mannitol and one that doesn’t).

I have so far named three isolates from Sabouraud agar: Sally, Sid, and Sam.

Sally the Yeast
Sally, the maybe-Brettanomyces-type yeast – 400X magnification (Lactophenol Cotton Blue stain)

Sam the Yeast
Sam, the maybe-Saccharomyces-type yeast – 400X magnification (Lactophenol Cotton Blue stain.)

Sid the [lacto?]bacillus-type-thing
Sid, presumably a Lactobacillus-type bacteria – 1000X magnification (Gram stain)

I’ve also collected four isolates (which may actually just be two different organisms) from an initial inoculation on MSA – BillyBob, JimBob, BettySue, and MarySue. MarySue is the one that seems to be “fermenting” the mannitol.

BillyBob, maybe a Pediococcus?
This is BillyBob (I clipped part of the image and moved it closer to the little “ruler”). The others look essentially the same when Gram-stained.

I’ve also got a bacillus-type (presumably Lactobacillus) critter that showed up on an initial BHI which may or may not be the same as Sid, and I got two more BillyBob/MarySue type colonies on another MRS agar plate.

Interestingly, when I did the original inoculations, it’s the ones that I added the LEAST amount of beer sediment to (20?l) that seems to get the growth – higher amounts may just add so much sugary solution (this stuff is quite sweet) that it inhibits growth.I really hope I can arrange to do molecular analysis (specifically, 16s rDNA sequences) on at least the bacteria, if not the yeast as well. I’d really like to get good identification of these. Assuming they’re real Lambic organisms, they’re probably already in the databases somewhere and should be readily identifiable – assuming someone will let me use up some supplies.

Beer cures flesh-eating bacteria, Staph, Strep, and Anthrax!*

* – These statements have not been evaluated by the Food and Drug Administration. Beer is not intended to diagnose, treat, cure, or prevent any disease, except for maybe hypobeeremia.

No, the title isn’t really true, exactly. However, it does appear to be true that a major component of modern beer – Hops (Humulus lupulus) flowers, really does appear to inhibit “Gram-positive” (Phylum firmicutes) bacteria.

The plates in the picture, clockwise from the upper-left, are inoculated with Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa (note the green pigment), and Staphylococcus aureus. ON the plates are 5 sterilized paper disks, each soaked with an extract of (again, clockwise from upper-left) Coriander, Hops flowers [Tettnanger], Cassia oil, Clove buds, and Ground Ginger root.

Except for the Oil of Cassia (“Cinnamon oil”), I took 2.5g of each ingredient, boiled it for 15 minutes in distilled water, soaked sterile paper disks in the water, then stuck the disks on top of plates inoculated with the bacteria in question. The cassia oil is about 10?l of the pure, full-strength oil as a sort of “positive control”. At that extreme concentration, it seemed to keep everything away.
The results are even more dramatic than I expected. For one thing, I expected at least some inhibition by the clove extract. The water was the color of a moderately strong tea and smelled strongly of clove, so I would have expected to have enough for some effect…but, no, it was just too feeble. (Had I used pure eugenol, I’d have probably seen the same effect as with the “cinnamon” oil.) Compared to the rest, a mere 15 minutes of boiling a comparatively mild variety of hops flower seems to very effectively prevent growth of certain types of bacteria – which would presumably include the varieties mentioned in the title of this post.

Hops skin-lotion to appear at hugely inflated prices on health-food-store shelves in 3…2…1…

Incidentally, if it does, I wouldn’t use it. “Gram positive” bacteria make up a substantial portion of the “normal flora” of healthy skin. Killing them off might easily leave room for other bacteria to take over and cause problems.

It does make me wonder about other possible uses of this effect, but I’ll save that for another time.

I’ll close by pointing out how useless allegedly “anti-bacterial” spices seem to be by comparison. Kind of puts the whole ridiculous notion of medieval cooks using spices to inhibit spoilage or to treat “rotten” food in its place, I’d say. It also implies that hops isn’t going to prevent “spoilage” of beer by itself, given that (for example) vinegar bacteria aren’t “gram-positive” types, nor are all the lovely ?-proteobacterial butt-bacter organisms like E.coli going to be affected…at least not by the hops. More experimentation to be performed at some later date.

This is just a simple experiment on the side of the main one I’m performing, where I attempt to isolate as many different viable organisms from a bottle of famous-brand Belgian Lambic ale as I can, hopefully for use in other foods (sourdough? Yogurt? And, of course, beer…) later.