The bacterium Streptococcus pneumoniae, commonly known as pneumococcus, is a frequent human pathogen that causes illnesses, including pneumonia, meningitis, and middle ear infections. Classification relies heavily on its reaction when grown on a specialized medium containing blood. This reaction, known as hemolysis, provides an initial, observable characteristic that helps microbiologists categorize the species. S. pneumoniae is definitively alpha hemolytic, but this observation is only the first step in positive identification.
Defining Hemolysis The Basis for Classification
Hemolysis is a method used in microbiology to classify bacteria, particularly those in the genus Streptococcus. The process involves culturing the bacteria on a blood agar plate, a nutrient medium enriched with five percent sheep or horse blood. Bacterial enzymes called hemolysins interact with the red blood cells, and the resulting reaction is categorized into three distinct types.
Alpha hemolysis is the partial breakdown of red blood cells, resulting in a greenish discoloration of the agar surrounding the colonies. This color change is an incomplete or “green” hemolysis, not a true clearing of the blood. Beta hemolysis represents the complete destruction of red blood cells, creating a clear, transparent zone around the colonies.
Gamma hemolysis describes the absence of any hemolytic activity, where the bacteria grow with no visible change or discoloration to the surrounding medium. These reactions serve as a rapid, observable tool to place unknown bacteria into broad groups. However, the alpha-hemolytic category contains multiple species, making further testing necessary for precise identification.
The Alpha Hemolysis of Streptococcus pneumoniae
Streptococcus pneumoniae is classified as an alpha-hemolytic organism because it produces a greenish halo around its colonies on blood agar plates. This color results from the bacteria’s metabolic activity, which causes a chemical change to the hemoglobin inside the red blood cells. The organism produces hydrogen peroxide as a byproduct of its metabolism in the presence of oxygen.
Hydrogen peroxide acts as an oxidizing agent, converting the red oxyhemoglobin into a greenish-brown compound called methemoglobin. This oxidation is the mechanism behind the incomplete hemolysis seen with S. pneumoniae, rather than the complete structural breakdown of the red blood cell. The visual result is a zone of greening, indicating the partial effect on the blood components.
When grown for an extended period, S. pneumoniae colonies often display a specific physical trait known as the “draughtsman” or “carrom coin” morphology. Initially, the colonies appear small and dome-shaped, but as the culture ages, they begin to flatten in the center. This central collapse is due to the activation of the bacterium’s own autolytic enzymes, which break down the bacterial cells themselves.
Key Tests for Positive Identification
Alpha hemolysis is insufficient for definitive identification because other common, often non-pathogenic bacteria like the Viridans group streptococci, also exhibit this reaction. Clinical microbiology laboratories rely on two additional biochemical tests to differentiate pathogenic S. pneumoniae from these other alpha-hemolytic organisms. The first is the Optochin sensitivity test, which utilizes the chemical ethylhydrocupreine hydrochloride.
S. pneumoniae is susceptible to Optochin, which causes the bacterial cell to lyse due to changes in surface tension. The test involves placing an Optochin-impregnated disk onto the inoculated blood agar plate. If the organism is S. pneumoniae, a clear zone of inhibition—a space where the bacteria cannot grow—will appear around the disk after incubation.
A zone of 14 millimeters or greater around a 6-millimeter disk is considered a positive result, confirming sensitivity. The second confirmatory test is the bile solubility test, which takes advantage of the organism’s autolytic enzyme system.
When exposed to bile salts, such as sodium deoxycholate, the autolytic enzymes of S. pneumoniae are rapidly activated, leading to the dissolution of the bacterial cells. A positive result is observed when a suspension of the bacteria becomes visibly clear after the addition of the bile salt solution. Other alpha-hemolytic streptococci lack this accelerated autolytic response and remain insoluble.
Together, a positive alpha-hemolysis reaction combined with susceptibility to Optochin and solubility in bile provides the necessary evidence to confirm the presence of Streptococcus pneumoniae.