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

Microb Pathog. 1995 Feb;18(2):141-52. doi: 10.1016/s0882-4010(95)90125-6.


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.

MeSH terms

  • Animals
  • Bacteriological Techniques
  • Colony Count, Microbial
  • Cryopreservation / standards*
  • Culture Media
  • Humans
  • Immunization, Passive
  • Lethal Dose 50
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Pneumococcal Infections / genetics*
  • Pneumococcal Infections / immunology
  • Pneumococcal Infections / microbiology
  • Streptococcus pneumoniae / genetics
  • Streptococcus pneumoniae / growth & development
  • Streptococcus pneumoniae / pathogenicity*
  • Time Factors
  • Vaccination
  • Virulence


  • Culture Media