14.2: The genus Streptococcus

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There are two genera of bacteria that can appear as a streptococcus arrangement that we will take up in the lab: the genus Streptococcus and the genus Enterococcus . Both are Gram-positive cocci 0.5-1.0 µm in diameter, typically occurring in pairs and chains of varying length when grown in a liquid medium, and often occurring singly, in pairs, short chains, and clusters when taken from an agar culture. As learned in Lab 8, they are both catalase-negative.

Fig \(\PageIndex{1}\): Scanning Electron Micrograph of Streptococcus pyogenes	Fig. \(\PageIndex{2}\): Scanning Electron Micrograph of Streptococcus pneumoniae

By National Institutes of Health (NIH) (National Institutes of Health (NIH)) [Public domain], via Wikimedia Commons	By Content Providers(s): CDC/ Janice Haney Carr [Public domain] Courtesy of the Centers for Disease Control and Prevention.

The streptococci are usually isolated on Blood agar. Blood agar is one of the most commonly used media in a clinical lab. It consists of an enriched agar base (Tryptic Soy agar) to which 5% sheep red blood cells have been added. Blood agar is commonly used to isolate not only streptococci, but also staphylococci and many other pathogens. Besides providing enrichments for the growth of fastidious pathogens, Blood agar can be used to detect hemolytic properties.

Note

Videos reviewing techniques used in this lab:

Hemolysis refers to is the lysis of the red blood cells in the agar surrounding bacterial colonies and is a result of bacterial enzymes called hemolysins. Although hemolysis can often be observed with the naked eye, ideally it should be examined microscopically using low power magnification, especially in cases of doubtful hemolysis. Reactions on blood agar are said to be beta, alpha, gamma, or double-zone:

1. Beta hemolysis (see Fig. 3A, Fig. 3B, and Fig. 3C) refers to a clear, red blood cell-free zone surrounding the colony, where a complete lysis of the red blood cells by the bacterial hemolysins has occurred. This is best seen in subsurface colonies where the agar has been stabbed since some bacterial hemolysins, like streptolysin O, are inactivated by oxygen.

Fig \(\PageIndex{3A}\): Beta Hemolysis on Blood Agar (Indirect Lighting)	Fig. \(\PageIndex{3B}\): Beta Hemolysis on Blood Agar (Indirect Lighting)	Fig. \(\PageIndex{3C}\): Beta Hemolysis on Blood Agar)

Note clear, colorless zone surrounding colonies where complete lysis of the red blood cells by the hemolysins has occurred.	Note clear, colorless zone surrounding colonies where complete lysis of the red blood cells by the hemolysins has occurred. You can actually read text through an area of beta hemolysis.	Note clear, colorless zone surrounding colonies where complete lysis of the red blood cells by the hemolysins has occurred.
Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0	By HansN. (Own work) [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons.	Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

2. Alpha hemolysis (see Fig. \(\PageIndex{4A}\ and Fig. \(\PageIndex{4AB}\) appears as a zone of partial hemolysis surrounding the colony, often accompanied by a greenish discoloration of the agar. This is also best seen in subsurface colonies where the agar has been stabbed.

Fig \(\PageIndex{4A}\): Alpha Hemolysis on Blood Agar (Indirect Lighting)	Fig. \(\PageIndex{4B}\): Streptococcus pneumoniae on Blood Agar Showing Alpha Hemolysis

Note the partial hemolysis accompanied by a greenish discolorization of the agar around the growth.	Note the mucoid, transluscent colonies and the alpha hemolysis (partial hemolysis typically accompanied by a greenish discolorization of the agar around and under the growth).
hotograph from MicrobeLibrary.org Courtesy of Rebecca Buxton, University of Utah

3. Gamma reaction (see Fig. \(\PageIndex{5}\)) refers to no hemolysis or discoloration of the agar surrounding the colony.

Photograph showing gamma reaction (no hemolysis) on blood agar. — Figure \(\PageIndex{5}\): **Gamma Reaction on Blood Agar.** Note there is no hemolysis and no change in the blood agar. (Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

4. Double-zone hemolysis refers to both a beta and an alpha zone of hemolysis surrounding the colony.

See Fig \(\PageIndex{8}\ to view a photograph showing alpha, beta, and gamma hemolysis on blood agar.

See Fig. \(\PageIndex{9}\) for a blood agar plate of a throat culture showing possible Streptococcus pyogenes.

Fig \(\PageIndex{6}\): A Plate of Blood Agar Showing Alpha, Beta, and Gamma Hemolysis (Indirect Lighting)	Fig. \(\PageIndex{7}\): A Blood Agar Plate of a Throat Culture Showing Possible Streptococcus pyogenes.

Alpha-, beta-, and gamma-hemolytic bacteria were streaked to form of the Greek letters alpha, beta, and gamma.	Note beta colonies mixed in with the normal alpha and gamma colonies of the viridans streptococci that normally inhabit the throat.
By Y tambe (Y tambe's file) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 (https://creativecommons.org/licenses...sa/2.5-2.0-1.0)], via Wikimedia Commons	Photograph from MicrobeLibrary.org Courtesy of Rebecca Buxton, University of Utah

Many of the streptococci can also be classified under the Lancefield system. In this case, they are divided into 19 different serologic groups on the basis of carbohydrate antigens in their cell wall. These antigenic groups are designated by the letters A to H, K to M, and O to V. Lancefield serologic groups A, B, C, D, F, and G are the ones that normally infect humans, however, not all pathogenic streptococci can be identified by Lancefield typing (e.g., Streptococcus pneumoniae). Serologic typing to identify microorganisms will be discussed in more detail later in Lab 17. Single-stranded DNA probes complementary to species-specific r-RNA sequences of streptococci and enterococci are also being used now to identify these organisms.

Contributors and Attributions

Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)