Finding Waldo: A Systematic Approach to Isolating and Identifying Bacteria

By: Joseph Maa

Date: 6/5/2017

Hi everyone, Joseph here:

Today I wanted to talk about something near and dear to my heart: finding and isolating bacteria. Now, the process of isolating single pure colonies (all the same species) of bacteria is important for a couple reasons.

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Figure 1: Finding Waldo is akin to isolating a single bacterium. In order to grow them, you have to find them first, among their many brethren. [1]
For one, having a lot of colonies is amenable to extensive testing. If I wanted to run multiple tests when I have loads and loads of the same bacteria to choose from, I’m less stressed about running experiments. Furthermore, isolating rare, low concentration products from bacteria is most effectively done in large batches (e.g. vitamin B12).

Of course, a recurrent problem in this isolation method is the choice of growth medium. Normal procedure is a culture-dependent method, that is, a method that has a selected “soup” of different chemicals that bacteria can live in. As anyone whose ever played a farming simulator could guess, not all animals like the conditions you put them in, and in the case of bacteria, may refuse to grow at all. This is problematic as the bacteria will not even appear in culture. For all intents and purposes, if we didn’t identify this problem, we wouldn’t even consider their existence.

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Figure: A plate of legionella, streaked so that we can get single colonies. (apparent on the left side) Legionella is known to grow in warm water, a bacteria that has really taken advantage of human’s technologic advances [2]
Fortunately, we have found ways to work around the problem. Contemporary microbiologists have utilized clever techniques to find certain species of bacteria. For example FISH, (fluorescence in situ hybridization) and all it’s related friends (clasi- FISH, Flow- FISH, etc.) is a technique that uses little bits of DNA tossed into the environment, with a shiny colored molecule on it that only binds to bacteria that we care about. [3] Of course, like any science, the method has it’s flaws, mainly that the little bits of DNA that we engineer may be indiscriminate, binding to different bacteria, or the probes may not even be able to bind to the protein/DNA of interest if the membranes are not permeable enough (able to let molecules through). [4]

Consequently, alternative methods of isolating single cell bacterium have been developed to separate bacteria of different species and one of the most frequently used is flow cytometry, a method where bacteria pass single file through a stream of liquid with unique identifier tags. However, just like FISH, flow cytometry comes with its own set of problems, not including, but up to the operators of the machinery who are very touchy about what goes into their machine. After all, theoretically it could only take one sample to destroy a million dollar piece of equipment, especially one as sensitive as this one. [5]

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Figure: A simplified model of a working flow cytometer. Notice the cells leaving via the bottom of the picture. The colored cells can be separated on the basis of color, much like one would organize MnM’s into different piles. [6]
Strangely, if we go back to first simple technique, growing bacteria on a plate, it seems that all this work is entirely unnecessary. However, the problem of not seeing bacteria on a plate is more prevalent than you would expect. Dubbed the “Great Plate Count Anomaly” by authors in Nature|Methods and authors from a different blog here: [7], many different types of bacteria have gone missing under our radar. Hopefully as we develop different media and different recipes for keeping bacteria alive, just like Koch and Pasteur in the past have done, we may unearth different unknown families of bacteria, that can be used to the benefit of humankind.

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Figure: Picture from the same blog as above, co-opted into this article. It really is a great figure that lends credence to the idea that many bacteria have been hidden away from our scrutiny due to an inability to grow them. [8]
So in conclusion, finding bacteria, as straightforward as it sounds, is not a meaningless and trivial task. In fact, the future of microbiology may change drastically as different techniques for characterizing bacteria emerge. In practice, microbiologists are akin to simple farmers, trying to grow bacteria by whatever methods they can come across, but the gist of the  bacterial message is that finding Waldo is was never more important.

Your friend,


p.s. Just as a final aside, I’ve been thinking about making videos to either accompany the blog, or stand as a different medium for me to talk about bacteria and friends more frequently, as I’ve been getting more crunched for time. You can find the pilot project here:

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