Quantitative determination of pneumococcal capsular polysaccharide serotype 14 using a modification of phenol–sulfuric acid method

• Published in: Journal of microbiological methods 2003, Vol.52 (1), p.69-73
• Main Authors: Cuesta, Gabriela, Suarez, Norma, Bessio, Maria I, Ferreira, Fernando, Massaldi, Hugo
• Format: Article
• Language: English
• Subjects: Bacterial Capsules - analysis; Bacterial polysaccharide; Bacterial Vaccines; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Colorimetric determination; Fundamental and applied biological sciences. Psychology; Microbiology; Morphology, structure, chemical composition; Phenol - chemistry; Reproducibility of Results; Streptococcus pneumoniae - chemistry; Streptococcus pneumoniae - classification; Streptococcus pneumoniae - isolation & purification; Sulfuric Acids - chemistry; Temperature; Vaccines
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(02)00151-3

The capsular polysaccharide of Streptococcus pneumoniae, serotype 14, is part of every pneumococcal vaccine presently in the market or under development. A strategy for the quantitative determination of this polysaccharide by the phenol–sulfuric acid method is described. The modality of acid addition is shown to be the critical step for obtaining reproducible test results between different technicians. Raising the incubation temperature above 80 °C increased the consistency of the method by more than 60% regardless of the acid addition modality, but at the expense of some loss of sensitivity. Incubation at 110 °C was found necessary to obtain reproducible results within 3% for this technique, which was used to follow the enrichment of the polysaccharide during the last steps of purification. A model mixture of the component polysaccharide sugars provided an adequate and economic standard to construct the calibration curve for this assay, with absorbance reading either in the reaction tubes or in a microplate. A similar procedure may be applied to the determination of other bacterial polysaccharides as well.