By Patrik M. Bavoil Virginia L. Clark
This quantity of Methods in Enzymology includes contributions overlaying the vast spectrum of interactions among bacterial pathogens and their eukaryotic hosts. It enhances quantity 235, during which tools for the isolation and id of bacterial pathogens and linked virulence determinants are defined. because the learn of bacterial pathogenesis borrows from an outstanding number of applied sciences and disciplines, we goal to supply the reader with a consultant pattern of the main complex pathogenesis study
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Extra resources for Bacterial Pathogenesis Part B: Interaction of Pathogenic Bacteria with Host Cells
Optimal growth conditions are at 37° in a humidified atmosphere of 5% CO2 in air. The cells do best if the culture medium is changed every 3 days and if the L-929 cells are subcultured at the point where they have grown to form a confluent monolayer on the bottom of the flask (confluence usually occurs 5 to 6 days following inoculation of flasks with L-929 cells). Cell suspensions for subculture are made using an inoculum which contains I x 105 cells/ml (as assayed by a hemocytometer chamber). A cell scraper (S/P T4206-I Baxter Scientific Products, McGaw Park, 1L) may be used to remove the adherent cells from the inner surface of the tissue culture flasks.
54, 603 (1975). zt Q. N. Myrvik, E. S. Leake, and B. Fariss, J. lmmunol. 86, 128 (1961).  ENDOTOXIN INDUCTION OF PROSTAGLANDIN RELEASE 37 The lung, heart, and trachea are taken out as a block. They are washed with phosphate-buffered saline (PBS) and the heart is taken out carefully, avoiding any injury to the lung. The lung and the trachea are washed well with saline to remove the adhering blood. An oral cannula is inserted into the trachea and fixed to it by binding with a string. About 30 to 40 ml of sterile saline is injected into the lung with a syringe connected to the oral cannula and the lung is massaged gently.
The marrow cells are counted and added to the tempered soft agar medium at a final concentration of 5 × 104 nucleated cells/ml of medium. 7. One milliliter of soft agar/cell suspension is added to each of the assay plates. Each plate is gently swirled immediately after the addition of medium. 8. The plates are left at room temperature for 15 min or until agar mixture has gelled. 9. The assay is incubated for 5 to 7 days at 37 ° in a humidified, 7% CO2 incubator. 10. Colony formation is scored by observing the plates under 35 × magnification with an inverted microscope.
Bacterial Pathogenesis Part B: Interaction of Pathogenic Bacteria with Host Cells by Patrik M. Bavoil Virginia L. Clark