|Activity 2: Physiological
The following tests may be performed on alpha-hemolytic streptococci for identification. All results should be recorded on the lab report. The student should draw a flowchart to indicate the identification scheme.
Streptococcus pneumoniae contain autolytic enzymes whose action is accelerated in the presence of bile (sodium deoxycholate). Streptococcus pneumoniae can be distinguished from other alpha-hemolytic streptococci by this property.
Add a drop of 2% sodium deoxycholate to the surface of an isolated colony of alpha-hemolytic streptococcus on sheep blood agar. Incubate at 35 °C for 30 minutes.
Positive = disappearance of colony = S. pneumoniae
Negative = no change in colony = viridans streptococci
The growth of Streptococcus pneumoniae is inhibited by ethylhydrocupreine (optochin). It is a presumptive test for the identification of Streptococcus pneumoniae.
Streak a loop of alpha-hemolytic streptococci for isolation on sheep blood agar. Apply an optochin disk with sterile forceps to the surface of the second quadrant. Incubate at 35°C overnight in 5-10% CO2 atmosphere.
Positive = zone of inhibition ³ mm = Streptococcus pneumoniae
Negative = no zone or < 14 mm = viridans group streptococci
Streptococcus pneumoniae can be identified in body fluids or from culture by antibody enhancement, or "swelling" of the capsule. A small drop of a bacterial suspension or body fluid is placed on a glass slide, then mixed with a loopful of S. pneumoniae-specific antiserum. Aqueous methylene blue suspension is added to give contrast. The slide is allowed to sit with a coverslip in place for about 10 minutes. On microscopic examination the capsule becomes refractile to reduce light and resists the mythelene blue stain if the organism is Streptococcus pneumoniae.