Figure 1. The eclipse phase of pneumococcal bacteraemia.

CD1 mice (n=15) were infected intravenously with 1x10⁶ pneumococci (strain D39). (a) Shows the numbers of bacteria in the blood at 6h, 8h and 24h (filled circles, positive blood cultures; empty circles, negative blood cultures). In the spleen (b), bacteria can be found both at 6h and at 8 h after inoculation. Values are expressed as CFU/ml in blood or CFU/g of spleen [weight 100 to 200 mg]. The limit of detection is shown as a dotted line. Statistical differences (* P < 0.05, Fisher’s exact test, one tailed) are indicated by thin black lines. Panels c to e show representative immunofluorescence microscopy images of on infected spleen sections. Single optical sections (analysis of 5 sections from 3 different spleens for each staining combination) obtained from 6 h infected spleens show the presence of bacterial clusters as small green dots (c, 20x objective and d, 60x objective) which can be seen as clusters of single bacteria at a higher magnification (e, 60x objective). In panel c bacteria are shown in green (α-type2, AF488), bacterial foci are indicated by green circles, metallophilic macrophages in red (Cr-Fc, AF568b) and red pulp macrophages in magenta (α-F4/80, AF647). In panel d, bacterial clusters (green circles, α-type2, AF568) can be observed in the metallophilic macrophages area (Cr-Fc, AF647), in white marginal zone macrophages are also stained (aSIGN-R1b, AF488s). In panel e a big cluster of bacteria is shown in green (α-type2, AF488), nuclei in blue (DAPI) and actin in orange (pAF647 conjugated phalloidin). Antibody details in Supplementary Table 1.