January 23, 2022

Basic Bacterial Identification

Kicking off the first post with an exciting topic (to me at least): bacterial identification! I actually learned this in my Essentials of Biotechnology class during my 10th grade year, but the applications course starts off with an identification lab to ease us back into microbiology. The lab itself was rather simple too; we were given 10 different unknown bacteria and information regarding 10 known species of bacteria. Over the course of a week, my lab team was able to formulate a plan of action and conduct tests in the most optimal path possible. The tests that were available to us are as follows: simple/gram stain, oxidase, catalase, MacConkeys agar, MSA, MR-VP, and thioglycollate broth. The purpose of this lab, of course, is to be able to handle and identify bacteria semi-autonomously and with as little teacher intervention as possible.

Simple Staining and Gram Staining

Gram (-) vs Gram (+) Bacteria - Microbiologics Blog

The simple stain is the most basic preliminary test of our toolset, yet it also happens to be the easiest to screw up. The purpose of the simple stain is to highlight the bacteria of interest and identify the morphology/arrangement. Going one step further, the gram stain tells us the relative thickness of the peptidoglycan in the cell wall. This is one of the most common indicators for bacteria and is often the first test conducted on an unknown species.


Oxidase (+) vs Oxidase (-) - Orbit Biotech

Oxidase tests for cytochrome c oxidase, an enzyme found in aerobic bacteria. It lies in the membrane and is the last enzyme of the electron transport chain. Its job, as you may infer from the name, is to take electrons from cytochrome c and transfer it to an oxygen molecule. This means bacteria with an oxidase (+) result are definitively aerobic as it uses oxygen in their respiration. However, bacteria with an oxidase (-) result are not necessarily obligate anaerobe as there are aerobic bacteria species that don't utilize cytochrome c oxidase.


Catalase (-) vs Catalase (+) - BiokiMicroki

Catalase tests for catalase (who would've guessed?), an enzyme responsible for converting hydrogen peroxide into a water molecule and oxygen atom. This test tells us whether a species is possibly aerobic or not. A catalase (+) result means that the species can do the conversion, and a catalase (-) result means it can't. Compared to oxidase, however, catalase is much less conclusive. While hydrogen peroxide is a byproduct of aerobic respiration, it doesn't necessarily mean anaerobic bacteria can't have catalase, let alone facultative bacteria.

MacConkey Agar

MacConkey Agar (+) - Microbe Online

Moving onto the media tests, MacConkey agar is a media that contains lactose. By streaking bacteria on the plate, we can learn whether the bacteria can ferment and process said sugar. A common confusion among students comes from the interpretation of the plate (and many other media tests). It's important to understand that a change in color or plate pH indicates lactose fermentation, not growth in general!

Mannitol Salt Agar (MSA)

MSA - ASM MicrobeLibrary

Mannitol Salt Agar is exactly what it sounds like, agar that contains a lot of salt and mannitol, a type of sugar. The salt is to have the media act as a high salinity environment for the selection of specific species of bacteria, often with a cocci morphology in clusters (streptococci). The mannitol is to check whether the species can ferment it. From the test alone, it can be determined whether a species can grow in high salinity and whether it can ferment mannitol.

Methyl Red - Voges Proskauer

MR (+) vs MR (-) - Microbiology Note

Methyl Red and Voges Proskauer are 2 different tests that test for a similar thing: glucose fermentation. Getting a positive result for either of these tube tests indicates the ability to ferment glucose. However, MR tests for a different pathway than VP does. MR does this by reacting with mixed acid byproducts, while VP reacts with an acetoin intermediate. Both are byproducts of the different metabolic pathways available to ferment glucose. Most bacteria are positive for only one of these tests, however, some may be positive for both.

Thioglycollate Broth

Thioglycollate Broth - John Lindquist

Thioglycollate is a test for respiration. What makes thioglycollate special is that it can differentiate between obligate aerobe, obligate anaerobe, facultative anaerobes, and aerotolerant. This is determined by the placement of colonies throughout the tube and how concentrated those colonies are. Thioglycollate is one of the most definitive tests for respiration and can even tell us if bacteria are aerotolerant. Aerotolerant anaerobes are bacteria that are anaerobic but can protect themselves from reactive oxygen.

Closing Remarks

One of the most important skills to master in microbiology is the ability to accurately conduct tests. While there are many tests out there for the multitude types cell/organism types, the 8 listed above are the first that I and many others learned. These only apply to bacteria but are a launch pad for other tests and skills. This becomes especially important when conducting more complex tests, where every step requires careful deliberation to ensure accuracy.