Virulence of Streptococcus pneumoniae in mice: a standardized method for preparation and frozen storage of the experimental bacterial inoculum.

Animal models of pneumococcal infection are important to evaluate the protective capacity of new vaccine candidates. We have established a method to prepare and store the experimental inoculum without loss of virulence or number of bacteria. This allows a standardized inoculum from the same culture batch to be used in several experiments. Pneumococci were cultured to mid-logarithmic growth phase in Todd-Hewitt broth with 17% fetal calf serum. The bacterial broth was distributed into smaller volumes and immediately frozen on liquid nitrogen and stored at -70 degrees C. We have tested the virulence of five different pneumococcal serotypes in BALB/c, C57BL/6, and NIHS mice using inocula prepared by this method and stored without loss of virulence for up to 4 years. Serotypes 1, 4, 5 and 8 were highly virulent for the strains of mice tested whereas type 6B showed lower virulence and a peculiar, protracted course of infection. There were no clear differences in virulence between the different strains of mice with the exception of serotype 6B, which showed higher virulence in BALB/c and NIHS mice than in C57BL/6 mice.