The immunoreactivity of very encapsulated bacteria was four-fold less than that of a nonencapsulated pneumococcal alternative or nonencapsulated pneumococci. The infected cells were washed 3 times with PBS, and extracellular germs were incubated with a fluorescein Hedgehog inhibitor isothiocyanate labeled goat antirabbit immunoglobulin. After permeabilization with 0. 10 percent Triton X 100 for 5 min, the extracellular and intracellular pneumococci were stained using antipneumococcal antiserum and tetramethyl rhodamine isocyanatelabeled goat anti rabbit immunoglobulin. Extracellular pneumococci were yellow, and intracellular pneumococci were red. Bacterial adherence and invasion were scored for a minimum of 50 cells per glass coverslip by fluorescence microscopy. Each test in this study was repeated at least five times, and the mean standard deviation was calculated. For the traditional fixation process, contaminated Gene expression monolayers grown on coverslips were fixed with a fixation solution containing five full minutes formaldehyde and 14 days glutaraldehyde in cacodylate buffer for 1 h on ice and subsequently washed several times with cacodylate buffer. For the chemical glutaraldehyde ruthenium red osmium fixation approach, pneumococci were mounted in a fixation solution containing three times glutaraldehyde and 0. 150-200 ruthenium red in cacodylate buffer for 1 h on ice. After washing in cacodylate buffer containing 0. 15.4-inch ruthenium red, samples were fixed in hands down the osmium in cacodylate buffer containing 0. 15% ruthenium red for 1 h at room temperature and washed with cacodylate buffer with 0. Fifteen minutes ruthenium red. For that acetate based formaldehyde glutaraldehyde ruthenium red osmium fixation treatment, contaminated monolayers were first fixed with 2 and the next day formaldehyde. Five minutes glutaraldehyde in cacodylate buffer containing 0. 075% ruthenium red and 0. 075 M lysine acetate for 20 min on ice. After washing with cacodylate buffer containing 0. 075% ruthenium red, products were set another time with 2% formaldehyde and 2. Five hundred glutaraldehyde in cacodylate buffer with 0. 075% ruthenium red for 3 h, cleaned with cacodylate buffer contact us containing 0. 075% ruthenium red, and then fixed with 1% osmium in ruthenium red containing cacodylate buffer for 1 h at room temperature. Eventually, samples were washed repeatedly with ruthenium red cacodylate buffer. All samples were then dehydrated using a graded series of acetone on ice for 15 min for each stage. Examples in the 100% acetone stage were allowed to reach room temperature before yet another change of 100% acetone. Samples were then subjected to critical point drying with liquid CO2. The dry samples were covered with an approximately 10 nm thick silver film by sputter coating before assessment with a field emission scanning electron microscope utilizing an Everhart Thornley SE detector and an in lens detector at a 50 rate at an acceleration voltage of 5 kV.