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Experimental infections of either cells or animals require the preparation of good quality inocula. Unfortunately, the important pulmonary pathogen Streptococcus pneumoniae is a fastidious microorganism that suffers an autolysis process when cultured in vitro. Supplementation of Todd-Hewitt broth with a biological buffer (20 mM Tris-HCl, pH = 7.8) promotes a six hours delay in the beginning of the autolysis process. Additional improvements include washing bacteria before freezing, avoiding manipulations after thawing, and the use of glycerol (&lt;18%) as a cryoprotectant, instead of reagents like skimmed milk that may affect cell cultures. With the proposed protocol &gt;70% of the frozen bacteria was viable after 28 weeks at -80 °C, and aliquots were highly homogeneous. We have tested their utility in a whole blood infection model and have found that human plasma exhibits a higher microbicidal activity than whole blood, a result that we have not found previously reported. Additionally, we have also observed significant variations in the antimicrobial activity against different strains, which might be related to their virulence.•Media culture buffering extends S. pneumoniae viability for 6 h.•Washing before freezing of single use aliquots minimizes manipulation after thawing.•Experimental infection with the frozen inocula has shown that plasma has higher bactericidal activity than blood.

Preparation of inocula for experimental infection of blood with Streptococcus pneumoniae