Tasty Acids

The story so far – I’ve got 8 live bacterial cultures (and two yeasts) obtained from a bottle of Peach Lambic, imported from Belgium. I strongly suspect that 6 of the 8 are Pediococcus species, and the other two are in the Lactobacillus genus. It is also possible that some of them might turn out to Leuconostoc or some other genus, but I suspect them all to be in the Order Lactobacillales somewhere, anyway.

Hopefully I’ll be able to get good, definitive sequence data from the bacterial isolates later this week.

Lactic acid seems to be the predominant acid in Lambic ales, produced by the various bacteria which break down the sugars in the beer and spew out lactic acid as a waste product.

Pediococcus also shows up in wines, where it’s associated with “malolactic fermentation” – where it converts the harsher malic acid into the more mellow-tasting lactic acid.

Thinking about this led me to think about the other distinctive acids found in foods. Here’s a listing (in no particular order) of some, with foods associated with their distinct flavors:

  • Lactic acid = “Yogurt” acid (and Sour Cream.  And many types of pickles.)
  • Malic acid = “Apple” acid (“Green Apple” flavor)
  • Tartaric acid = “Grape” acid (Verjuice and “Grape flavor”)
  • Acetic acid = “Vinegar” acid
  • Citric acid = “Lemon/Lime” acid (or “Pixy Stix®” flavor)
  • Propionic acid = Swiss cheese acid

In other news, I need a real microscope of my own.

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…

More Lambic pictures

Ah, that’s better – a more traditional heat-fix/simple stain (using Methylene blue) shows my yeast isolates better:

Sally the maybe-Brettanomyces-type yeast
(“Sally”, a yeast that I suspect is a Brettanomyces-type yeast.)

Sam the...Saccharomyces-type yeast?
(“Sam” now looks awfully small…but more experienced observers than I am said that it could actually be a Saccharomyces-type yeast.)

Lucy the possibly-PediococcusI also got two more Coccoid-Cluster-type Gram-positive bacterial isolates. The look pretty much the same under the microscope, though one had gooey wet, slightly larger colonies than the other’s smaller, hard-lump colonies. I see another one of those tetrads in the hard-lump-colony microscope image.

All told, I now have 10 isolates to check out. I’ve been given the go-ahead to try sequencing on the 8 bacterial isolates so hopefully I’ll be able to get a clear identity for Fred, Sid, Lisa, Lucy, BillyBob, JimBob, BettySue, and MarySue. Sally and Sam will have to wait for now, though I’m looking into ways to characterize them, too.

“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.

Chunky Bacon Agar, and Expired Jell-O™ again

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Why MSN loses to Google and Yahoo:

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

Just plain “Huh????”

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


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

Kinda Scary

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

And, perhaps scariest of all:

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

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

Short Low-content (but relevant) post

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

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

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

Sorry about the recent case of blogstipation…

So, here I am blogging from the hospital…

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

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

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

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

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

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

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

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

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

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

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

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

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

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