As confirmed by immunofluorescence microscopy, the applied dilution of the antipneumococcal antiserum effortlessly stained encapsulated phenotypes. For morphological examination of the capsule structure, samples were fixed from the LRR fixation procedure. Samples were then dehydrated with a graded series of ethanol on ice for 30 min for each step. Products were penetrated with the acrylic resin LRWhite by applying 1 part 100% ethanol and 1 part LRWhite for 2 h on ice, followed by 2 parts and 1 part ethanol LRWhite and overnight incubation on ice. A day later genuine resin was added, and samples were incubated ALK inhibitor for 8 h on ice, changed, and left overnight. Finally, samples were placed in gelatin capsules, which were filled up with natural LRWhite resin at room temperature. The LRWhite glue was polymerized for 48 h at 60 C. Ultra-thin sections were cut with a stone blade, and sections were picked up with Formvar coated copper grids. Counterstaining of the sections was performed with 4% aqueous uranyl acetate for 5 min. After air drying, samples were analyzed using a Zeiss EM 910 transmission electron microscope at an acceleration voltage of 80 kV. For cryo FESEM samples were centrifuged, Plastid and 2 m of each pellet was put on a brass sample holder and straight away frozen in melting nitrogen. Frozen samples were then transferred in to a cryo system, freeze fractured at 110 C, and freeze etched for 30 s at 110 C. After sputter coating with a thin layer of gold palladium, samples were transferred onto a phase in the Zeiss DSM982 Gemini area emission scanning microscope and examined at 135 C at an acceleration voltage of 2 kV. Quelling reactions were conducted using capsule type 3 specific antiserum. Cell connected capsule production and cell released polysaccharides were determined using the Stains all assay for detecting acidic polysaccharides. Pneumococci were cultured in semisynthetic conjugating enzyme channel to a cell density of 4 108 cells/ml, and the bacteria and culture supernatant were separated by centrifugation. Germs were washed twice with 2, and PBS. 5 109 pneumococci were re-suspended in 0. 5 ml water. The content of bacteriumassociated polysaccharides or the level of polysaccharides in 0. 5 ml of culture supernatant was based on measuring the absorbance at 640 nm after addition of 2 ml of an answer containing 20 mg of 1 ethyl 2 naphtho thiazolium bromide and 60 l of glacial acetic acid in 100 ml of 50% formamide. Values were normalized by subtraction of values calculated for culture medium or water. Virus free C57BL/6 mice were obtained from Charles River. Female inbred mice were challenged if they were 10 months old and weighed 19 h. Mice were challenged with 20 l of sterile PBS containing 5 106 CFU of serotype 3 S and were anesthetized by intraperitoneal injection of 40 l of a 5:2 mixture of ketamine and xylazine. pneumoniae administered in the nostrils. Control rats received 20 l of sterile PBS without bacteria.