Tests for Identification of Bacteria

Differential Bacterial Stains, Specialized Media and the API-20E

Jul 16, 2008
Tami Port

Blood Agar Medium Showing Alpha-hemolysis. - Tami Port

Gram, Acid Fast and Endospore stains; MacConkey's, Mannitol Salt, and Blood Agar media as well as the API-20 test strip all provide information to identify microbes.

Most of these are procedures taught in introductory microbiology courses, and are useful in sorting bacteria into broad categories rather than identifying bacteria to the species level.

Differential Stains

Differential bacterial stains are the application of a series of dyes that result in some organisms turning one color while other physically or chemically distinct bacteria retain a contrasting color. Gram stain, Acid Fast-stain and Endospore stain are differential staining procedures used to classify bacteria into general groups.

Gram stain distinguishes Gram+ from Gram– bacteria based on differences in bacterial cell wall structure. The Acid-fast stain protocol helps differentiate bacteria that have a waxy cell wall from those that do not. The Endospore stain procedure is useful in determining whether or not the bacteria in question produces tough, dormant spores.

Differential & Selective Media

Media is an agar-based substance used to grow bacteria in the laboratory environment. Some types of agar will grow just about anything, whereas others are differential or selective media.

Selective bacterial growth media excludes some types of bacteria, whereas differential agars allows for a color change that reveals bacteria that have specific metabolic capabilities. MacConkey’s Agar (MAC) grows only Gram-negative bacteria and contains a differential pH indicator that changes the color of bacteria that can ferment the sugar lactose.

Mannitol Salt (MSA) is a medium with high sodium chloride (NaCl) content, selective for halophile (salt-loving) bacteria. Mannitol salt also has a pH indicator that changes color when there are bacteria present that can ferment mannitol, a sugar alcohol also known as a polyol. Pathogenic Staphylococcus aureus grows well on this medium and produces the color change of a mannitol fermenter.

Blood Agar (BAP) is nutrient-rich and will grow many different types of bacteria, but the presence of sheep’s blood in the agar allows for a differential distinction between organisms possessing hemolytic enzymes that can cut up red blood cells. This medium is used to test for beta-hemolytic bacteria, like the pathogenic Streptococcus pyogenes. S. pyogenes completely destroys the red blood cells in BAP, resulting in clear halos around its bacterial colonies and, if obtained from a throat culture, a diagnosis of strep throat.

API-20E

The API-20E test strip, a very specific means of bacterial identification, is a collection of mini test tubes, each with a reagent that test for a different aspect of bacterial metabolism. After incubation with an unknown Gram-negative bacterium, the interpretation of positive and negative tests allows for identification to the species level.