Got a microscope in the mail, but not the one I ordered. Ugh.

Things are looking a little up, I’m working things out with the scope supplier and lab promises to be interesting tonight. We inoculated some Pseudomonas aeruginosa last week on media that induces their characteristic blue-green pigment. I’ve been wanting to see this ever since I found of P. aeruginosa makes it;, the specific epithet even means ‘copper rust’. We’ve been using that bug all semester, but it doesn’t seem to want to express the pigment on TSA, so I’m looking forward to the pretty colors tonight.

I’m also going to see if I can get a whiff of the distinct odor (previous plates shared space with less-than-pleasant smelling bugs) and maybe even get use of the UV lamp to look for fluorescein.

I’ve realized that since my 30th birthday, I’ve had many more days where I’m growing bugs than days where I’m not. It may be silly, but this makes me pretty happy.

Edit: Blame it on stress during an exam.  The skin swabs went on MSA, not BA, which we’ll be using for throat swabs. All this makes sense, given the environments of skin and MSA. My beard came back with a higher than normal, but not super high bacterial count. Very nicely defined mannitol acid zones. No really scary bugs, just the usual Staphylococcus spp. suspects. Waiting on a trehalose test to narrow one down.

He’d bring in the same whiskey he just used to wash out the bullet hole received while laying down the law.  I grabbed a small amount of my Jameson Irish Whiskey (40% alc by volume) and headed out to the lab.

To test it, we divided two petri dishes into five sections.  One dish was for Pseudomonas aeruginosa, the other for Staphylococcus aureus. The first section of each plate was labelled ‘0′ and streaked with an undiluted culture of the appropriate bug.

Next, we diluted 0.5 mL of each broth with 5 mL of Jameson Irish Whiskey. At 2.5, 5, 10, and 20 minutes after dilution, we streaked a section of each plate.  (I finally got to use a nifty mechanical pipette, rather than the ABCDE rubber valve, YAY!)

We left them incubating at 37 Celsius until this evening. Here are the results, growth is ranked relative to the initial streak on a scale of 0(no growth) to 4(about the same):

* There was a single colony. Multiple groups working with S. aureus had an unexplained ‘no growth’ at 2.5. Let me know if you have ideas. Also apologies for the unstyled table, I’ll make it pretty after I finish studying.

This is fairly effective. Indeed, it was much more effective than Lysol against Pseudomonas, which didn’t even make a dent.  If you think 10 minutes is a long time, double check the instructions on your household cleaners.  Yeah, you’re not supposed to mop it up as soon as you spray it.  This is why.

Now, remember that this data is really only good for disinfecting flat surfaces, not as an antiseptic for actual cowboy bullet wounds, which have a lot of complicating factors. I also make no guarantees about effectiveness vs spore-formers. Finally, Jameson costs more than most similarly effective disinfectants and tastes good enough that it would be a pity to regularly use it in this manner.

P.S. If/when I start plating from home, I’ll take pictures, rather than just feed y’all tables.

We’re building up a toolbox of techniques we can use to work through a d-key and identify bugs in an unknown culture.

There’s three things I take away from all this.

It’s just incredibly awesome to see how flexible bacteria are when it comes to metabolism. Heck, some of the tests are time sensitive because a bug will rewire itself to take better advantage of the changing biochemical landscape. No more glucose? Ok, let me just turn on my lactose fermentation machinery.  It’s a pain in the ass for diagnostics, but really neat.  If it can be nommed, there’s a bug out there that can do it. Seriously, Pseudomonas aeruginosa can eat phenol. Not just degrade, but actually use it as the sole carbon source.  We usually use phenol to kill bugs, not feed them.

The manner of thinking used to create these tests and to work through a d-key is strikingly similar to that which I use when debugging code, it’s very comfortable and feels ‘right’.

I love that half the stuff is named So-and-so’s something; it feels like I’m acquiring old school D&D spells. Nerd. Quick, which is in the lab and which is in the AD&D Player’s Handbook ?  Tenser’s floating disc, Masson’s trichrome

• Ran out of time before finishing 3rd streak on my streak plate. I’ve got to learn to work a little more quickly, but still carefully. Mainly, I’ve got to be quicker with probe heating/cooling.
• I caught myself writing down a few brainfart mistakes. I’m hoping that I caught all the ones I made. I’ve got to stop tensing up over stuff I know.
• I can easily rattle off all sorts of terms for colony morphologies and cell morphology/arrangement. Unfortunately, I’ve been practicing with diagrams or picture-perfect textbook micrographs. In the real world, things can have a mostly entire margin, but be edging into undulate territory. Or, you’ll see definite cocci on a slide, but have to make a judgment call on whether they’re tetrads that occasionally broke apart into diplococci or diplococci that occasionally glommed together into tetrads. I should look at a bunch of non-ideal examples, make those judgement calls, and then compare them to commonly accepted answer.

I’m not too worried about my grade, and I’m all about making mistakes and moving on, but it would’ve been nice to continue batting 1000.

Oh, one part that gave me no trouble was using the scope to find bacteria on a slide I prepped. So, that’s a victory.

I’d been looking forward to doing a streak plate since before I even fully decided to take a class. Streaking is really basic and easy, but it elegantly solves one of the earliest problems in microbiology, “How do you isolate bacteria from the hodge podge that is their environment?”

The early attempts used error-prone dilution techniques, but that was not ideal. There were tons of problems with contamination, it’s fidgety, and it doesn’t isolate bugs that have a low population count with respect to the total number of microbes.

The streak method is simple and clever. You divide your plate into a number of partitions, usually 3 or 4. The first partition gets a bunch of streaks from an inoculated loop. Subsequent streaks are started with a sterilized loop that begins by briefly passing through the previous set of streaks. This essentially reduces the microbial load. By the last streak, the loop should be depositing either single bacteria or a small amount glommed together (basically when something like a diplococci or tetrad won’t break up). The colonies that arise from these colony forming units (CFU) pretty much contain clones of the isolated bugs.

Koch devised it after observing that colonies growing on some old potato slices in his lab had different morphologies from each other. He figured out that each colony must have arisen from a single airborne CFU landing on the slice and that streaking in the manner described would simulate this. Once again, Pasteur’s remark that fortune favors the prepared mind was borne out.

I’ve read about this multiple times, but the description that best captures how thrilling it must’ve been occurs in De Kruif’s Microbe Hunters, which is a must-read for anyone even passingly interested in the history of microbiology.

We got our plates back tonight. I was able to get isolated colonies of both species from our test sample, but my technique was really sloppy.  I took some time to practice on a blank plate. I think I’ve got it down, now. The ‘a-ha!’ moment came when I lifted my elbow off the bench and started streaking from the shoulder, rather than from the wrist. I was able to go slower, with a lot less jitter, and much  more control.

There was also a little messing about with differential media (EMB and MSA). Not much to mention here except that whole idea behind differential media deeply appeals to my analytical engineering side and that E. coli is really pretty on EMB, like even prettier in real life than in a photo. (Thanks to Wearn for putting up that image).

We also gram stained samples from the isolated colonies. I’m happy to report that my stains worked much better this time and I had absolutely no trouble using the scopes. I found out I prefer rotating the ocular tube and arranging the microscope so that the stage, rather than the arm, is facing me. I even had another student say that the E. coli under my scope looked like it came from a textbook, so I’m really happy!

Next lab session is our first practical exam. 3 minutes per station. I feel like my technique is respectable for someone who’s only been doing this a short while. I worry a little bit about the ‘over the shoulder factor’ causing me to stress and do something dumb. Wish me luck.

I crushed them, saw them driven before me, and heard the lamentation of their, uh, women.

I’m still not a master, but I had a much easier time and feel comfortable enough to not worry about them hindering me during a lab practical.

I determined my two biggest issues, aside from nerves.  I wasn’t selecting large enough clumps of interesting stuff; the scopes aren’t perfectly parcentered and I would end up magnifying the void.   I also had to develop a feel for just how thin the depth of field is at 1000x and how that affects fine adjustment –  there’s really no clues you’re almost in focus; either the specimen shows or doesn’t.

In addition to the fixed slides at the lab, I brought in some scrapings from the compost pile.  I had the most luck with water droplets off some seaweed.  Saw a few blobs with nuclei, something with chlorophyll, and maybe a Stentor. None of the expected fungi, oh well.

Not many people showed, but the prof was happy to help those that did.  It was a more relaxed atmosphere and lots of fun talking ensued.