An Introduction to
Bacterial Identification

Through the early part of the twentieth century, there appeared to be a general feeling that the same battery of observations and tests could be used to characterize and identify any kind of bacterium. But as different, "exotic" types of bacteria were discovered, it was found that they would tend not to grow in the standard test media nor even in the usual conditions of incubation. Obligate parasites and strict anaerobes were among the emerging groups of bacteria needing special methods for growth and characterization. By the 1930s, a standard descriptive chart was developed for uniformity in recording the characteristics of the "aerobic saprophytes" (which are equivalent to what we call the "commonly-found chemoheterotrophs" in our general courses today). Click here and here for both sides of the standard chart constructed in 1934. Notice that much weight was given at the time to determining cultural characteristics such as colony detail and the appearance of growth in gelatin and broth.

As we now know, a huge battery of tests done at once to identify an unknown organism would result in a lot of media and time being wasted dealing with irrelevant tests. (Time and media are money!) Thus we would like to proceed in stages, running those tests which are applicable to what basic knowledge we have about our unknown. That is, a very different set of tests would be run on a gram-negative rod compared to a gram-positive coccus.

There is no medium (differential or otherwise) that can possibly support the growth of all of the different species of bacteria. As an example, many different formulations exist for media to detect glucose fermentation, based on nutrient requirements of various groups of bacteria. Also, when running the standard test for oxygen relationship with Thioglycollate Medium, consider that (1) many organisms (including a lot of chemoheterotrophs!) cannot grow in this medium and (2) the medium does not allow for anaerobic growth which is due to phototrophy (more specifically, metabolism in the presence of light as performed by the non-oxygen-evolving photosynthetic bacteria) or anaerobic respiration (the use of alternate electron acceptors such as nitrate and sulfate). A discussion of this test is on our oxygen relationship page.

As genotypic characterization (determination of the DNA and RNA characteristics of our bacteria) is becoming more widely practiced, we may soon be back to one standard of characterizing and identifying bacteria. This time it will be universally applicable as all bacterial genera and species become uniformly defined according to genotypic uniqueness. We hope that the results of the phenotypic tests we run will correlate with the genotypic characteristics and bring about accurate and useful identification of our organisms.

In the table below, a few commonly-found and easily-grown chemoheterotrophic genera are sorted out based on various "primary tests" which include the use of Glucose Fermentation Broth and O/F Medium. The benzidine test which has been used effectively in the Bacteriology 324 course tests for the presence of iron-porphyrin compounds such as cytochromes and the true catalase enzyme. Some organisms possess the enzyme cytochrome a₃ oxidase as part of the electron transport system in respiration; this enzyme is responsible for a positive reaction in the oxidase test where the dye tetramethyl-p- phenylenediamine is reduced to a purple compound.

Further tests (not indicated) would then be done to determine positively the genus identification and also the likely species. You can go where the experts are and consult the latest editions of Bergey's Manual of Systematic Bacteriology and Bergey's Manual of Determinative Bacteriology for more information. Bergey's Manual of Systematic Bacteriology is a multi-volume set, and the first volume of the new, 2nd edition is out now but may not be specifically helpful for the organisms listed in the table below. Bergey's Manual of Determinative Bacteriology is mainly used for identification, but the present 9th edition has become quite dated in that respect. You should try out the on-line edition of The Prokaryotes for valuable, updated information on isolation and identification of bacteria. For example, type "Alcaligenes AND identification" in the search box (without the quotes) and see what comes up.

The idea for the format of the following table comes from the classic Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd edition, revised by S. T. Cowan (1974, Cambridge University Press). This table of often-isolated chemoheterotrophic bacteria was put together with the Bacteriology 304 course in mind as a guide in targeting likely names of genera to pin on the "nature isolates." An X marks the place where a certain pattern of characteristics matches up with a possible genus. Considering additional characteristics of the isolate, one can consult Bergey's Manual or The Prokaryotes for this genus and related genera (on nearby pages) for a more definitive identification.