Quantitative validation of media for transportation and storage of Streptococcus pneumoniae

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 degrees 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 degrees 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 degrees C (P < 0.0001) and 30 degrees C (P < 0.0001). Samples can be stored for 4 and 2.5 days at 6 to 8 degrees C, 28 and 17 h at 21 degrees C, and 15 and 7 h at 30 degrees 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.

FIG. 1.

The viability of two different isolates of each serotype (ST) stored in STGG medium at different temperatures. Pneumococcal cells from overnight cultures were transferred to 4.0 ml of STGG medium, and 1-ml aliquots were stored frozen at −70°C (A) and refrigerated at 6 to 8°C (B), 21°C (C), and 30°C (D). Frozen samples were thawed at each sampling point and frozen immediately afterward. Two different isolates (Royal Free numbering) were tested for each capsular ST: ST 6 no. 10 (♦) and 156 (⋄), ST 18 no. 9 (▪) and 107 (□), ST 19 no. 5 (•) and 25 (○), and ST 23 no. 6 (▴) and 13 (▵). Data are shown as the means of results from three independent experiments, and error bars indicate standard errors of the means. Each experiment was performed in triplicate.

FIG. 1.

The viability of two different isolates of each serotype (ST) stored in STGG medium at different temperatures. Pneumococcal cells from overnight cultures were transferred to 4.0 ml of STGG medium, and 1-ml aliquots were stored frozen at −70°C (A) and refrigerated at 6 to 8°C (B), 21°C (C), and 30°C (D). Frozen samples were thawed at each sampling point and frozen immediately afterward. Two different isolates (Royal Free numbering) were tested for each capsular ST: ST 6 no. 10 (♦) and 156 (⋄), ST 18 no. 9 (▪) and 107 (□), ST 19 no. 5 (•) and 25 (○), and ST 23 no. 6 (▴) and 13 (▵). Data are shown as the means of results from three independent experiments, and error bars indicate standard errors of the means. Each experiment was performed in triplicate.

FIG. 1.

The viability of two different isolates of each serotype (ST) stored in STGG medium at different temperatures. Pneumococcal cells from overnight cultures were transferred to 4.0 ml of STGG medium, and 1-ml aliquots were stored frozen at −70°C (A) and refrigerated at 6 to 8°C (B), 21°C (C), and 30°C (D). Frozen samples were thawed at each sampling point and frozen immediately afterward. Two different isolates (Royal Free numbering) were tested for each capsular ST: ST 6 no. 10 (♦) and 156 (⋄), ST 18 no. 9 (▪) and 107 (□), ST 19 no. 5 (•) and 25 (○), and ST 23 no. 6 (▴) and 13 (▵). Data are shown as the means of results from three independent experiments, and error bars indicate standard errors of the means. Each experiment was performed in triplicate.

FIG. 1.

The viability of two different isolates of each serotype (ST) stored in STGG medium at different temperatures. Pneumococcal cells from overnight cultures were transferred to 4.0 ml of STGG medium, and 1-ml aliquots were stored frozen at −70°C (A) and refrigerated at 6 to 8°C (B), 21°C (C), and 30°C (D). Frozen samples were thawed at each sampling point and frozen immediately afterward. Two different isolates (Royal Free numbering) were tested for each capsular ST: ST 6 no. 10 (♦) and 156 (⋄), ST 18 no. 9 (▪) and 107 (□), ST 19 no. 5 (•) and 25 (○), and ST 23 no. 6 (▴) and 13 (▵). Data are shown as the means of results from three independent experiments, and error bars indicate standard errors of the means. Each experiment was performed in triplicate.

FIG. 2.

Pneumococcal cell viability of serotypes 6, 18, 19, and 23 stored in either STGG medium or SGG medium at various temperatures. Pneumococcal cells from overnight cultures were transferred to 4.0 ml of STGG medium (upper panel) or 4.0 ml of SGG medium (lower panel), and 1-ml aliquots were stored refrigerated at 6 to 8°C, at 21°C, or at 30°C. Although t_1/2 values were determined from nontransformed data, the log₁₀ of viable cell counts is plotted against storage time for each temperature for clarity, as indicated. Each data point is the mean value for all of the isolates studied (n = 12).