An Introduction to
Bacterial Identification

Page 1: General Principles

• Page 1 – General Principles
• Page 2 – API-20E Enteric ID System
• Page 3 – Genotypic Identification

This page amplifies material discussed in our
UW-Madison Microbiology 102 laboratory course.

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" or "easy-to-grow" 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. To utilize Glucose O/F Medium as a routine test for glucose catabolism is unwise, as it was designed to differentiate gram-negative bacteria, and gram-positive bacteria happen to grow poorly in the medium. To assist in explaining the general principles of pH-based differential media, Glucose O/F Medium can be valuable in a demonstration of the relative effects of amino acid deamination (usually never considered important enough to mention), glucose respiration and glucose fermentation as shown here.

When running a test for "oxygen relationship" with Thioglycollate Medium, consider that (1) many organisms (including a lot of chemoheterotrophs!) cannot grow in this medium, and (2) anaerobic growth is only associated with glucose fermentation. (Anaerobic growth associated with anaerobic respiration or anoxygenic phototrophy would not be supported.) Also, (3) the test can be difficult to read, and the related, essential physiological information about the organism can be more clearly determined by running the catalase test (respirers tend to be positive), the glucose fermentation test, and whether the organism is oxygen tolerant. Just explaining the results of this test in terms of whether or not an organism "likes" oxygen – not considering the basic concepts of respiration and fermentation – is as anti-educational as ignoring the pervasive influence of amino acid deamination in pH-based differential media. So, the use of the relatively expensive Thioglycollate Medium can be considered redundant, of low priority and financially wasteful. Further criticism of this test can be found here, and a relatively objective discussion 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 appropriate use of Glucose Fermentation Broth and O/F Medium. The benzidine test which has been used effectively in our food microbiology 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.

The idea for the format of this 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 our beginning lab course for majors 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. Further tests (not indicated in the table) can then be done to determine positively the genus identification and also some likely species.

One can then go where the experts are and consult the latest editions of Bergey's Manual of Systematic Bacteriology and The Prokaryotes for more information. Bergey's Manual of Determinative Bacteriology is mainly used for identification, but the present 9th edition has become quite dated in that respect.

gram reaction
(young culture)
+ + + + + + + + + +
shape coccus
rod rod irreg.
rod rod rod rod rod rod rod rod coccus
aerobic growth + + + + + + + + + + + + + + +
anaerobic growth + + + + + + + + +
endospores + + +
(Motility Medium)
+ +
+ +
catalase reaction + + + + + + + + + + + +
benzidine reaction + + + + + + + + + + + +
oxidase reaction + +
+ + + +
glucose fermentation
to acid or to acid+gas
+ + + + + (or
+ + + +
Glucose O/F Medium                     O F F F O
Micrococcus X                              
Staphylococcus   X                            
Streptococcus     X                          
Lactococcus     X                          
Enterococcus     X                          
Leuconostoc     X                          
Pediococcus     X X                        
Aerococcus       X                        
Lactobacillus         X                      
Kurthia           X                    
Arthrobacter             X                  
Clostridium               X                
Bacillus                 X X            
Alcaligenes                     X          
Pseudomonas                       X        
Klebsiella                         X      
Shigella                         X      
Salmonella                           X    
Escherichia                           X    
most other
enteric genera
Aeromonas                             X  
Chromobacterium                             X  
Neisseria                               X

These general microbiology pages have copyright by John Lindquist
and found their permanent sanctuary on circa 2001.
Copies found elsewhere are neither authorized nor up to date.
Page content was substantially revised 8/11/09 at 9:45 AM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison