Identification and Characterization of Streptococcus pneumonia : Bile Solubility Test
The bile (sodium deoxycholate) solubility test distinguishes S. pneumoniae from all other alphahemolytic
streptococci. S. pneumoniae is bile soluble
whereas all other alpha-hemolytic streptococci are bile resistant. Sodium
deoxycholate (2% in water) will lyse the pneumococcal
cell wall.
A. Preparation of 2% sodium deoxycholate (bile salt) solution
1. Dissolve 2 g of sodium deoxycholate into 100 ml sterile distilled water.
B. Performing the bile solubility test
1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C
with ~5% CO2 (or in a candle-jar).
2. Add bacterial growth from the overnight BAP to 1.0 ml of 0.85% saline to
achieve turbidity in the range of a 0.5-1.0 McFarland standard.
3. Divide the cell suspension equally into 2 tubes (0.5 ml per tube).
4. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube. Add 0.5
ml of 0.85% saline to the other tube. Mix each tube well.
5. Incubate the tubes at 35-37°C in CO2.
6. Vortex the tubes.
7. Observe the tubes for any clearing of turbidity after 10 minutes.
Continue to incubate the tubes for up to 2 hours at 35-37C in CO2 if negative after 10 minutes.
Observe again for clearing.
Read Also :Identification and Characterization of Streptococcus Pneumoniae
C. Reading the bile solubility test results
A clearing of the turbidity in the bile tube but not in the saline control
tube indicates a positive test.
D. Troubleshooting
Partial clearing (partial solubility) is not considered positive for
pneumococcal identification. Partially soluble strains that have optochin
zones of inhibition of less than 14 mm are not considered pneumococci.
E. Quality control
Each new lot of sodium deoxycholate should be tested with positive and
negative QC strains. S. pneumoniae strain ATCC
49619 can be used as a positive control and S. mitis strain ATCC
49456 can be used as a negative control.
IV. Commercial test kits for identification
Several commercial identification systems that use slide agglutination tests are available for identification of colony growth from a BAP as S. pneumoniae. These identification tests use suspensions of latex beads with rabbit antibody specific for S. pneumoniae capsular antigens.
Visible agglutination occurs when the S. pneumoniae capsular antigen reacts with the antibodycoated latex beads. The manufacturer’s instructions should be followed precisely when using these kits.
These kits should be regularly subjected to QC using a non-pneumococcal streptococcal species, since they can become cross-reactive with prolonged storage.
V. Determining S. pneumoniae capsular serotypes using serologic methods
Although serotyping of pneumococci is not usually necessary for a clinical response, capsular serotype determination is a critical component of successful pneumococcal disease surveillance
efforts. Effective current multivalent vaccines target combinations of key serotypes.
Determination of serotype distributions associated with disease in certain regions provides information regarding the potential usefulness of applying existing vaccines and is also critical for assessing vaccine impact.
Serotype distribution can be determined by culture of the organism followed by serological determination of the capsular type by latex agglutination and the quellung reaction. Many
laboratories have opted to use simpler and less expensive methods of deducing capsular
serotypes through the use of specific PCR reactions.
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