Microbiology Diagnosis -- Streptococcus pneumoniae


Streptococcus pneumoniae bacteria are gram-positive cocci arranged in chains and pairs (diplococci) on microscopic examination. A green, α-hemolytic, zone surrounds S. pneumoniae colonies on blood-agar plates. Pneumococci can be differentiated from other catalase-negative viridans streptococci by their susceptibility to Optochin and solubility in bile salts. Molecular methods for detection of S. pneumoniae, many of which are PCR-based, can also be used. 1, 2


Streptococcus pneumoniae colonies vary in appearance depending on the degree of encapsulation of the organism. Heavily encapsulated strains can have large colonies, several millimeters in diameter, which appear gray and very mucoid, while less heavily encapsulated organisms usually have smaller colonies. 1

Pneumococci produce pneumolysin, which breaks down hemoglobin into a green pigment that can be observed as a large green zone surrounding S. pneumoniae colonies growing on blood-agar plates. This property is still termed α-hemolysis even though lysis of red blood cells is not responsible for it; green or yellow-green zones can also be seen around S. pneumoniae colonies growing on chocolate agar, a medium in which all red blood cells are lysed during preparation. 2

The opacity variance of S. pneumoniae colonies appears to reflect differences in the organism's pathogenesis and virulence. Animal model studies showed that transparent variants are more able to colonize the nasopharynx of infant rats, while opaque variants are more virulent in the intraperitoneal model of infection. The organism spontaneously alternates between transparent and opaque phases, each of which has different structural characteristics. For example, transparent variants are associated with increased amounts of cell-wall-associated teichoic acid, while opaque variants are associated with enhanced production of capsular polysaccharide. 2, 3


Ethyl hydrocupreine hydrochloride (Optochin) is a quinine derivative that is used to differentiate pneumococci from other viridans streptococci, with a sensitivity of greater than 95%, because of its ability to selectively inhibit the growth of S. pneumoniae on blood agar plates at very low concentrations (≤5g/mL).

The Optochin test is performed on a blood-agar medium using a disk diffusion principle. A few well-isolated colonies of the organism in question are streaked onto a blood-agar plate and a filter paper disk, impregnated with Optochin, is placed in the streaked area. The plate is incubated and examined after 18 to 24 hours. Pneumococci surrounding the disk are lysed, due to changes in surface tension, creating an inhibition zone (Figure 5).

An inhibition zone of 14 mm or more, around a 6-mm disk, allows for identification of the viridans streptococcus in question as Streptococcus pneumoniae. If the inhibition zone is less than 14 mm, further testing (bile solubility or serology) is indicated for the identification of S. pneumoniae.

The diagnostic reliance on a minimum diameter of the inhibition zone is due to the discovery of Optochin-resistant pneumococcal strains, and the ability of some non-pneumococcal viridans streptococci to show small zones of inhibition on the Optochin test.


The addition of bile salts, such as sodium deoxycholate, accelerates the natural lytic reaction observed in pneumococcal cultures by increasing the activation of autolytic enzymes produced by Streptococcus pneumoniae.

The bile solubility test is performed by adding a bile-salt solution to an established broth or blood-agar culture of the organism in question. A positive result in broth culture is obtained by noting visible clearing of the culture's turbidity, as compared to a control tube, after addition of the bile salt solution and re-incubation for up to 3 hours. On blood-agar plates, bile-soluble pneumococcal colonies "disappear" leaving behind their green zone of α-hemolysis, after placing a drop or two of the bile-salt solution on the colony and re-incubating the plate for 30 minutes.

Additional testing, by serology, may occasionally be necessary as only 86% of pneumococcal strains lyse completely with the addition of bile salts.



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