Intentional food microbiology:
UNintentional food microbiology:
I still don’t feel like I got nearly enough productive stuff done this weekend, but I did manage to do a bit of microscopy – plus demonstrating to myself that I still remember how to do a “Gram stain”. Real Post with explanation and more pictures to follow Real Soon Now…
People usually assume soap gets rid of funky microbes that might grow on things, so I was very amused several months ago when I spotted something growing on top of the soap in one of the household hand-soap dispensers. As of today, it looks as pictured at left. That lumpy yellow and brown mass atop the the soap looked to me like some sort of soap-sodden mold, and have been saving the dispenser specifically in the hopes that someday I’d have a microscope and could take a look at it. Meanwhile, the mass spread, and slowly started releasing some kind of yellow pigment into the soap.
Incidentally, I kind of doubt this indicates some sort of failure on the part of the manufacturer of the soap. I don’t recall for certain, but I think I may have opened the dispenser at one point to transfer some of the soap to another nearly-empty dispenser. When the mass started growing originally, it was a single spot, which suggests a single spore or speck of dust floating in and landing on the surface. Hey, it happens. Anyway, I’ve therefore blanked out the name of the manufacturer since I don’t think they really have anything to do with this.
This mysterious growth upon my soap remained mysterious until today. Thanks to the Minister of Domestic Affairs and VWR (who managed to find me a really good deal), I finally got to actually get a close look at that lumpy mass. Meet my new friend Minnie (pictured at right). I could gaze into those eyes for hours. I couldn’t afford a darkfield condenser, and I sure as heck couldn’t afford to upgrade to phase-contrast gear, but I can add either one later if the opportunity presents itself. I also can’t afford the overpriced proprietary digital camera attachments either, though working around that is a whole other project. Until I identify an affordable model that plays well with Linux or work out how to modify a webcam into an ocular attachment,
I’ll have to settle for a trick…
It turns out if you take a digital camera and set it for close-up photos, you can actually stick the camera lens right up to the eyepiece and often get a serviceable picture.. Now, I had to subject the pictures I got today to moderately heavy processing to bring out the detail a bit better, but at least part of that is just me working on learning how to optimize the camera settings for this kind of use.
Equipped with some surplus slides and cover-slips donated by a kind professor who had some extra packages, I opened up the soap container and smeared a little of the yellow crud on a couple of them. One I just slapped a coverslip on for direct observation – the other I smeared over a slide and let dry with the intention of staining using the tiny, previously-unused vial of methylene blue left over from a very old plastic toy microscope. While the latter dried, I took a look at the wet mount hoping to finally see the mold mycelia that I had been expecting…
There wasn’t enough contrast to bother trying to get a photo, but it was obvious at 400x that what I was looking at was bacteria, not mold. Nerdly joy at learning something by looking in the microscope that I wouldn’t have otherwise known ensued, along with happiness as I realized this meant I had a perfect excuse to dig out my recent shipment from the Maker Shed – materials for doing a “Gram Stain”. Incidentally, the “Maker Shed” had the supplies on the way to me within hours of my ordering it, and they have lots and lots of cool stuff. I highly recommend it. Anyway, I got to do a “Gram Stain” for the first time in a couple of years (and the first time ever outside of a school lab). Want to see?
Here it is – the nasty yellow goo that infected my bottle of hand-soap. My staining technique was a little off since I’m out of practice – the way I interpret the results is that what I’ve got here is neither a member of the Firmicutes (i.e. “Gram positive”) nor – probably – Actinobacteria. I really can’t guess at more than that, though. I think the few “Gram-positive”-looking cells there are artifacts of insufficient decolorization. I know I still had a surplus of the purple “Crystal Violet” stain still on the slide at the end. (How did I know? I’ll show you at the end…). The irregular bluish bits towards the bottom are, I believe, just bits of stuff from the soap itself.
Meanwhile, this pretty much satisfies my curiousity about the Mystery Soap-Infecting Microbe. There’s certainly a lot more I could investigate, but my developing Hillbilly Biotech lab is really intended to support my interest in intentional food microbiology and perhaps evenutally some small-scale non-food industrial microbiology. I have some remaining curiousity about the yellow pigment and whether or not it might be useful for something, but I’m doubting there is any food or beverage I might want to grow this stuff in and therefore don’t have much use for it. Still, I’ll keep the bottle around for a while before I throw it out in case I think of something fun to do with it. If I end up being really interested in the identity of the bug growing on it, I should be able to find a liquid that I can grow a big mess of it in, then run it through a simple DNA extraction process. Then all I need to do is find someone who can supply PCR primers, a thermocycler, and sequencing services cheap. It might sound like I’m being facetious, but I wouldn’t be surprised these days if I manage to find somewhere that’d do it for $20/sample or less. I may eventually do this will the Mystery Soap Bug anyway, since I hope to be running through this process with cultures of sourdough, yogurt, cheese, vinegar, and brewing microbes that I develop myself. For now, though, it’s just nice to be playing with microbiology equipment again. And now fully independently! Wheeeeeeee!!!!!!
Yes, I’m a nerd. And proud of it!
Now that I finally have a microscope, I no longer have any excuse for not getting to work on the rest of my Hillbilly Biotech lab. Just this weekend I was pricing out Hillbilly Autoclaves. I picked up a cheap air pump and air stone
for potentially building an aerobic bubble-column fermenter (for quick growth of yeast starters or a working model of a “Fring’s Acetator®”-style vinegar generator. I still want to build an ozone generator for sanitization and to get a pH meter. I’d like to also get my hands on some wheat, barley, and rye seeds to sanitize, sprout, and grow here as the first stage of developing a truly local sourdough culture, plus arrange to have several pounds of plain flour irradiated to sterilize it.
I’m also like summer to be over. Yes, I’m writing this in Winter, but it’s not until later in the summer to autumn that locally-grown fruits will start becoming available, and locally grown fruits ought to be an ideal source of local brewing and baking yeasts and bacteria. Finally, I’d like to find a wealthy patron (or matron, I’m no sexist…) who would sponsor me so I could just pursue food-microbe bioprospecting and research full-time…
Oh, yes, and I need to get around to finishing Episode 4 of my little podcast project, especially since episode 4’s topic is a fundamental microbiology technique.
Comments welcome below – thanks for reading!
Oh, and as a reward for getting all the way to the end, here’s a picture that I thought was pretty – crystals of “Crystal Violet” and iodine. I told you I had too much left on the slide…
Dr. Joseph over on the “(It’s a…) Micro World (…after all)” blog posted a list of his ten favorite microbes. After showing up in the comments of his post and being a wiseass about E.coli and Gram staining, the least I can do is participate here. Besides, it’s a great idea. Therefore here are ten of my current favorite microbes:
Let me pause now for a moment to review what I’ve learned so far:
No, seriously. I’ve previously reviewed their promiscuous sex lives,
their sexually-transmitted diseases, and their toiletry habits. Somehow, though I still want to do more brewing, so let’s continue.
The three major elements needed by pretty much every living thing for “food” are Carbon, Nitrogen (as reduced “amino” nitrogen), and phosphorus (as oxidized phosphate) (Reduced sulfur is also needed in small amounts for proteins). Glucose (“dextrose” or “corn sugar”), fructose, or sucrose (“table sugar”, each molecule of which is made of a molecule of glucose attached to a molecule of fructose) are all used as carbon sources by Saccharomyces yeasts. Possibly also Galactose under certain conditions[1]. Saccharomyces yeasts don’t appear to be able to use lactose (“milk sugar”, each molecule of which is made of a molecule of glucose and a molecule of galactose), so some recipes include lactose in order to ensure there is some residual “sugar” in the mix at the end, for flavor and “body”.
Aside from sugars, this seems to be possibly the most important yeast nutrient. The most
“natural” source of this nutrient would seem to be amino acids or very short peptides (2-5 amino acids long). Apparently urea (carbamide) also makes a good yeast nutrient, but:
This could combine with the ethanol to make “ethyl carbamate”, which is considered
a probable carcinogen, at least if it’s present at a high enough level. Obviously if you use urea as a
yeast nutrient, that’s only going to increase the possibility of a problem.
Proteins can be a source of amino nitrogen (and carbon and sulfur), but like all real microbes, yeast cells cannot just “eat” chunks of protein. They have to be broken down into very small chains of amino acids or even as individual amino acid molecules before the yeast can suck them up and use them. Saccharomyces yeasts do not appear to normally excrete protein-digesting enzymes, so by themselves they cannot make any use of protein for nutrition[3].
Oxygen is necessary for making certain components of the cell membrane, in addition to it’s more obvious role in respiration. Without a way to replace used up membrane components, the yeast stop reproducing and eventually fall apart and die. There seems to be some suggestion that to a certain extent one can substitute some raw membrane material for oxygen here (either as “yeast hulls” or possibly even certain of the natural waxes on some fruits).
…or do they? Between the “Crabtree effect” (when there are high concentrations of glucose, alcohol production continues even in the presence of oxygen) and indications in scientific papers[2], it seems SMALL amounts of oxygen may not be a problem, and might very well be beneficial.
B1 (“Thiamine”) is commonly mentioned, though apparently the need for it varies from strain to strain. Also potentially important are Pantothenic Acid (B5), Niacin (Nicotinic Acid, Vitamin B3), Biotin, Inositol, as well as Potassium, Magnesium, and trace amounts of calcium and a few other minerals[4].
For one thing, it seems to be a general rule that you don’t want your brew sitting on the corpses of dead yeast (the “lees” of wine, or “trub” of beer), because that is a potential source of (insert dramatic music and crash of thunder here)Off-Flavors and Off-Odors. Yeast dying and falling apart is also a major source of urea being dumped into the brew, too. Some strains of yeast under certain conditions, such as insufficient pantothenic acid, may be prone to producing nasty-smelling sulfides as well.
So, in most cases what we want to do when brewing is keep our yeast as alive and happy as possible, and get them to hurry up and finish our primary fermentation before they start dying off. Coming up: My (as yet untested) plot for accomplishing this – without specialized scientific equipment or materials.
[1] Wilkinson JF: “The pathway of adaptive fermentation of galactose by yeast” Biochem J. 1949; 44(4): 460–467
[2] Nagodawithana TW, Castellano C, Steinkraus KH: “Effect of dissolved oxygen, temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in rapid fermentations.” Appl Microbiol. 1974 Sep;28(3):383-91.
[3] Bilinski CA, Russell I, Stewart GG: “Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects.” Appl Environ Microbiol. 1987 Mar;53(3):495-499.
[4] Fugelsang KG, Edwards CG: “Wine Microbiology: Practical Applications and Procedures” 2007; Springer Science+Business Media LLC, New York; pg 17
Just a brief pre-post before the main one I’ve got brewing now (which will be posted either later today or tomorrow).
Microbiology is the dominating topic of this particular blog, but I don’t think I’ve ever addressed what I consider to really count as “micro”biology. This isn’t necessarily an obvious topic. My old “Microbiology” book from 8 years ago, plus the textbook from last year’s “Pathogenic Microbiology” class both contained large sections discussing organisms that are visible without a microscope. Heck, the “Pathogenic Microbiology” text even had a whole section on spider and insect bites. And, tapeworms? Since when is “over 30 feet long” considered “micro”? As I like to say: It’s time for Microbiology to grow up and move out of Medicine’s basement.
So: Here are the defining features of what I consider to be a “microbe”, at least for purposes of what I tend to discuss here on the blog:
It’s that last point that prompted me to write this post, mainly because it’s such an important part of why microbes work and how they affect their surroundings, especially when it comes to food microbes. What I mean by “do not eat” is that they are incapable of taking large (microbially speaking) chunks of material into themselves to use. Any cell nutrient for a microbe must be in the form of small molecules, like sugars, small peptides or individual amino acids, and so on that can be easily transported across the cell membranes and through the cell wall where applicable.
The importance of this is that for a microbe to grow on a complicated substance like meat or bread (for example), they have to excrete specialized enzymes that break down the substances out in the environment into simpler components like sugars or small peptides. If a microbe cannot secrete a protein-digesting “protease” enzyme, it can be surrounded by tasty, nutritious proteins and still starve to death. If a microbe can’t secrete an amylase (starch-digesting) enzyme, it doesn’t matter that starch is made of nice yummy glucose molecules because they’re all wadded up into long chains of starch that the microbe can’t get at.
And that, finally, is important because it brings up issues of growing multiple microbes together to accomplish something. Sake, for example, is made by fermenting rice, but rice is made primarily of starch. Saccharomyces yeasts don’t make amylases, so in order to make sake, you also have to add a kind of mold (Aspergillus oryzae, one of the types of white-mold-with-little-black-specks that you may see growing on the bread you’ve left sitting around for too long). A. oryzae is also a microbe and therefore can’t “eat”, but it does produce amylase. Since the amylase is breaking down the starches outside of the cells, this means the released glucose is also available for the yeast to use.
Admittedly, my definition above isn’t perfect. On the one hand, it leaves out protozoa (like amoebae and the well-known Paramecium, both of which actually do take in “chunks” of food, but both of which most people would normally consider to be “microbes”. It also leaves IN things like mushrooms, which are not usually thought of as being “microbes” by people who aren’t microbiologists. And, of course, it leaves me with no excuse not to go and learn something about eukaryotic (“plant”) algae (as opposed to bacteria-algae, a.k.a. cyanobacteria) and diatoms. Suggestions for updating my definition may be left in the comments…
Just something that came up while I was assembling what will be the next post. Stay tuned.
My parents, apparently comfortable with being microbiology “enablers“, ran into a digital microscope (pictured at right) that they pointed me to. It looks pretty nifty for the price, except for one issue: it only comes with three objectives, topping out at 40X. I can find marketing materials for this microscope, but no technical information beyond what’s on the company’s website (click image to reach that). Therefore, I can’t tell if the objectives are replaceable or not. If they are, the idea of picking up an inexpensive surplus 100X oil-immersion lens and ending up with a decent microscope with which to watch the Yeast Porn and such – on a nice digital screen, no less, rather than squinting into an eyepiece – has a certain appeal to it. The fact that the digital camera takes the pictures itself and only needs a computer connection to transfer files – and not even then if you use an SD card – means that I wouldn’t have to worry about ending up with something that requires Microsoft® Windows® just to look at some microbes (or post glamour shots of them to this blog).
However, I don’t think trying to view conjugating yeast cells or, say, Lactobacillus or Gluconobacter/Acetobacter bacterial cells at 400X would be very rewarding, so an oil-immersion lens is a necessity. And don’t let the “up to 1600 Power with Digital Zoom” thing fool you – it just makes the picture bigger and blockier, not actually more detailed.
So….do I WANT, or not? I sent off an email to Celestron asking about whether I could swap out objective lenses on this model or not. I got back the form “we got your email” email – we’ll see if I get a real reply from them or not. If so, I’ll update this post.
Speaking of which – still no reply from Bristol Brewing Company. Guess they’re either just not “nerd-friendly”, or the person who handles email queries is on vacation or something.
P.S. Dear Celestron – if you were to send me an evaluation model, I’d be happy to review it here on my blog for the thousands hundreds dozens pairs of people who read my elegant prose incoherent babbling…
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