Biochemical Testing: Identifying Bacteria by Process of Elimination

Before we were able to identify bacteria with gene sequencing, we had to rely on our observations to figure out what species bacteria we were working with. These observations including things like the bacteria's morphology, growth habits, different staining method results, and tests that differentiate bacteria based on their phenotype called biochemical tests. Biochemical tests expose bacteria to certain conditions that the bacteria will have different reactions to based on what phenotypes they express. The results of these tests can then be used to identify bacteria by eliminating all of the species that don't match the test results until there only one left.

There are many different types of biochemical tests that bacteria can be run through to help identify them. Some of the most simple and easy biochemical tests to run is the oxidase test and catalyze tests. The oxidase test invokes smearing some of you bacteria on a test strip covered in a chemical called Kovács reagent that changes color when exposed to an enzyme produces by some bacteria called cytochrome c oxidase. The catalyze test is just as easy because to do it all you need to do is put a drop of hydrogen peroxide on some bacteria and see if it starts to bubble. If it does bubble it mean that the bacteria is producing the catalase enzyme. Oxidase and catalase are both produced by bacteria that use aerobic respiration so if these two test are positive it means that you can eliminate bacteria that primarily rely on anaerobic respiration.

There are also biochemical tests that involve culturing bacteria on special media to see how it effect their growth. There are some tests that are used to determine if the bacteria is able to tolerate certain environmental factors called selective tests. If the bacteria is able to tolerate the conditions it will grow on the media but if it can't there will be no growth. An example of a selective test is growing bacteria on mannitol salt agar media which will select for bacteria that are able to tolerate an environment. Other examples of selective media include eosin methylene blue agar which selects for gram negative bacteria and hektoen enteric agar which is selective for specific bacteria genera.

Most biochemical tests that involve growing bacteria on media have multiple purposes to give you more information about the bacteria with a single plate. Specialized media can be both selective for certain types of bacteria and show different results based on how the bacteria interacts with the media which is called a differential test. Differential media have indicators that will change the color of the media or bacteria depending on certain condition. For example, sheep blood agar will show if the bacteria is able to lyse red blood cells. If the bacteria can, it is considered beta hemolytic and there will be clear zones around where it grows, if it partially lyse blood cells it is alpha hemolytic and there will be cloudy greenish areas around it, it it can't lyse blood cells it is gamma hemolytic and the media around the bacteria won't change. Other examples of differential media are mannitol salt agar that will change from red to yellow if the bacteria is able to ferment the mannitol in the agar or stay red if it can't and SIM media that can show hydrogen sulfide production, indole production, and if the bacteria is motile or not.

By performing enough biochemical tests on an isolated bacteria and eliminating what you know it can't be based on the results you can eventually narrow down it down to a known species or find that it doesn't match any identified species.