The applied dilution of the antipneumococcal antiserum effortlessly stained encapsulated phenotypes, as established by immunofluorescence microscopy. For morphological examination of the structure, products were set by the LRR fixation technique. Samples were then dehydrated using a graded series of ethanol on ice for 30 min for each step. Products were penetrated with the acrylic resin LRWhite by using 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 natural resin was added, and samples were incubated pifithrin for 8 h on ice, improved, and left immediately. Finally, samples were put in gelatin capsules, which were filled up with genuine LRWhite resin at room temperature. The LRWhite resin was polymerized for 48 h at 60 C. Ultrathin sections were cut with a diamond blade, and sections were acquired with Formvar coated copper grids. Counterstaining of the areas was performed with four to five 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. Lymphatic system and 2 l of each pellet was put on a steel sample holder and straight away frozen in melting nitrogen, for cryo FESEM samples were centrifuged. Frozen samples were then moved into a cryo model, 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 cryo stage in a very Zeiss DSM982 Gemini discipline emission scanning microscope and examined at 135 C at an acceleration voltage of 2 kV. Quelling reactions were conducted using supplement type 3 specific antiserum. Cell related production and cell released polysaccharides were determined using the Stains all assay for detecting acidic polysaccharides. Pneumococci were cultured in semisynthetic ATP-competitive ALK inhibitor method to a cell density of 4 108 cells/ml, and the microorganisms and culture supernatant were separated by centrifugation. Germs were washed twice with 2, and PBS. 5 109 pneumococci were resuspended in 0. 5 ml water. This content of bacteriumassociated polysaccharides or even the amount of polysaccharides in 0. 5 ml of culture supernatant was determined by 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 m 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. Feminine inbred mice were challenged once they were 10 weeks 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 blend of ketamine and xylazine. pneumoniae administered in the nostrils. Get a handle on mice received 20 m of sterile PBS without bacteria.