Lecture Notes on TSI, IMViC, Selective and Differential Media, Enterobacteriacea, MIO, LIA, Oxidase Test


Many intestinal bacteria are classified in the tribe Enterobacteriaceae. These are gram negative rods which are able to ferment glucose.

Enterobacteriacieae which are also capable of fermenting lactose are called coliforms. Coliforms are generally considered to be opportunistic pathogens and non-pathogens, however there are many instances of severe, life-threatening infections caused by coliforms. Coliforms are very important as INDICATORS OF FECAL CONTAMINATION. Some common coliforms are:

Recently a number of pathogenic strains of E. coli have been recognized. A number of non-coliform Enterobacteriaciae are important pathogens. Since these non-coliforms are LACTOSE NEGATIVE, lactose fermentation tests are important in trying to identify them.  

There are a number of non-Enterobacteriaceae which are important intestinal pathogens.


A number of selective isolation techniques are used for fecal organisms.

SELENITE BROTH is used mainly to enrich for Salmonella and to some extent Shigella. This broth is called an enrichment broth because it suppresses the growth of most intestinal organisms while allowing the growth of the pathogens thereby increasing the chances of finding the pathogens.


Plating media such as:

TRIPLE SUGAR IRON (TSI) agar is used to help identify the biochemistry of the Enterobacteriaceae. This medium contains three sugars and a pH indicator: This medium also contains iron salts which will react with any H2S produced by the growing organism to form a black precipitate.



The INDOLE test measures the ability of the microorganism to degrade tryptophan into indole, ammonia and pyruvic acid:


The METHYL RED - VOGUES PROSKAUER test measures the types of products produced in the fermentation of glucose.


The CITRATE test determines whether an organism is capable of using citrate as a sole carbon and energy source. Such organisms will produce alkaline end products in the presence of oxygen and these can be measured with a pH indicator. Bromthymol blue is green at neutral pH and blue at alkaline pH, therefore blue represents a positive test.



This medium allows you to test

The possible color reactions after 24-48 hours of incubation are: If the organism is producing hydrogen sulfide a black precipitate will be visible. H2S production is usually not as strong in this medium as it is with TSI.



This medium allows you to

Hazy growth or growth spreading away from the line of inoculation indicates motility.


The decarboxyation of ornithine will cause the medium to become alkaline, therefore:

The indole test can be performed on this medium by dripping several drops of Kovac's reagent onto the top of the medium. A bright red color is positive for indole.


The oxidase test will determine if the organism possesses cytochrome c, one of the electron carrier proteins found in the electron transport chain. (Even though many bacteria are capable of respiration and therefore have an electron transport chain, not all bacteria have the same cytochrome proteins in the chain. For instance, E. coli and the other Enterobacteriaceae lack cytochrome c but have cytochrome o and cytochrome d instead. On the other hand, Pseudomonas aeruginosa, a number of other organisms as well as our mitochondria use cytochrome c in aerobic respiration.)

The test uses a compound called tetramethyl-p-phenylenediamine which is clear in the reduced (electron rich) state and purple in the oxidized (electron poor) state. In the presence of an organism with cytochrome c, the reduced tetramethyl-p-phenylenediamine will donate its electrons to the cytochrome c and become purple colored.

©2008 by John M. Berestecky
All Rights Reserved