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.

1. Bile Solubility
2. Optochin Disk
3. Quellung Reaction

1. Bile Solubility

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.

Method

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.

Results

Positive = disappearance of colony = S. pneumoniae

Negative = no change in colony = viridans streptococci

2. Optochin Disk

The growth of Streptococcus pneumoniae is inhibited by ethylhydrocupreine (optochin). It is a presumptive test for the identification of Streptococcus pneumoniae.

Method

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.

Results

Positive = zone of inhibition ³ mm = Streptococcus pneumoniae

Negative = no zone or < 14 mm = viridans group streptococci

3. Quellung Reaction

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.