Quantitative Validation of Media for Transportation and Storage of Streptococcus pneumoniae

Abstract

ABSTRACT The need to design effective Streptococcus pneumoniae vaccines and to monitor resistance means that it is essential to have efficient methods to determine carriage rates. Two liquid media, consisting of skim milk, glycerol, glucose, and tryptone soya broth (STGG) or skim milk, glycerol, and glucose (SGG) alone, were evaluated for their ability to maintain pneumococcal viability. Optimal recovery of S. pneumoniae was achieved when swabs were transferred to STGG medium prior to plating onto blood agar-gentamicin selective plates (22%) compared to 7% when plated out directly ( P < 0.0001 by Fisher's exact test). Both STGG and SGG media are appropriate for the long-term storage of pneumococci and primary swab samples at −70°C, with no decrease in viable count observed following repeated freeze-thaw cycles. Samples could be stored refrigerated for up to 3 days in either STGG or SGG medium with no significant loss of viability. Viability decreased progressively in storage at 20 to 30°C, with greater losses of viability occurring at the higher temperatures. There were no significant differences in viability between isolates in the two media. STGG preserved pneumococci significantly better (about twofold) than SGG medium at 21°C ( P < 0.0001) and 30°C ( P < 0.0001). Samples can be stored for 4 and 2.5 days at 6 to 8°C, 28 and 17 h at 21°C, and 15 and 7 h at 30°C in STGG and SGG media, respectively. For field studies undertaken in resource-limited environments, SGG medium can be prepared by using locally available materials. The quantitative data reported in this study will enable researchers to plan appropriate transport and storage protocols.