Table 10.4 Characteristics of Some Important Biochemical Tests

Biochemical Test

Principle of the Test

Positive Reaction


Detects the activity of the enzyme catalase, which causes the breakdown of hydrogen peroxide to produce O2 and water.



Determines whether or not citrate can be used as a sole carbon source.

Growth, which is usually accompanied by the color change of a pH indicator.


Detects enzymatic breakdown of gelatin to polypeptides.

The solid gelatin is converted to liquid.

Hydrogen Sulfide Production

Detects H2S liberated as a result of the degradation of sulfur-containing amino acids.

A black precipitate forms due to the reaction of H2S with iron salts in the medium.


Detects the enzymatic removal of the amino group from tryptophan.

The product, indole, reacts with a chemical reagent that is added, turning the reagent a deep red color.

Lysine Decarboxylase

Detects the enzymatic removal of the carboxyl group from lysine.

The medium becomes more alkaline, causing a pH indicator to change color.

Methyl Red

Detects mixed acids, the characteristic end products of a particular fermentation pathway. ■ mixed acids, p. 153

The medium becomes acidic (pH < 4.5); a red color develops upon the addition of a pH indicator.


Detects the activity of cytochrome c oxidase, a component of the electron transport chain of specific organisms. ■ cytochrome c, p. 148

A dark color develops upon the addition of a specific reagent.

Phenylalanine Deaminase

Detects the enzymatic removal of the amino group from phenylalanine.

The product of the reaction, phenylpyruvic acid, reacts with ferric chloride to give the medium a green color.

Sugar Fermentation

Detects the acidity resulting from fermentation of the sugar incorporated into the medium. Also detects gas production.

The color of a pH indicator incorporated into the medium changes if acid if produced. An inverted tube traps any gas that is made.


Detects the enzymatic degradation of urea to carbon dioxide and ammonia.

The medium becomes alkaline, causing a pH indicator to change color.


Detects acetoin, an intermediate of the fermentation pathway that leads to the production of a 2, 3-butandiol. ■ 2,3-butanediol, p. 152

A red color develops upon addition of chemicals that detect acetoin.

Commercial Modifications of Traditional Biochemical Tests

Several less labor-intensive commercial modifications of traditional biochemical tests are available (figure 10.6). For example, the API™ system utilizes a strip holding a series of cupules that contain dehydrated media. A liquid suspension of the isolated test bacterium is inoculated into each of the compartments, thus rehydrating the media. Because the formulations of the media are similar in composition to those used in traditional tests, the positive results give rise to similar color changes. After a 16-hour incubation of the inoculated test strip, the results are read manually. The pattern of results is converted to a numerical score, which can then be entered into a computer to identify the organism. A similar system is the Enterotube™, a tube with small compartments each containing a different type of medium. One end of a metal rod that runs through the tube is used to touch a bacterial colony. When the rod is withdrawn it inoculates each of the compartments. A system by Biolog uses a microtiter plate, a small tray containing 96 wells, to assay simultaneously an organism's abil ity to use a wide variety of carbon sources. Modifications of these plates enable a researcher to characterize the metabolic capabilities of microbial communities, such as those in soil, water, or wastewater.

Highly automated systems also are available. The Vitek™ system uses a miniature card that contains multiple wells with different formulations of dehydrated media. A computer then reads the growth pattern in the wells after a relatively short incubation period.

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