By Stephen J. Hammonds
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Additional info for Diagnostic Bacteriology Protocols
14. , glass beads) (II) and the use of pressure methods such as the French press (12). Cell-breakage methods have been reviewed by Coakley et al. (13). Where studies on specific cellular components such as outer membrane or cell envelope proteins are undertaken, then differential centrlfugatlon techniques may be required for subcellular fractionation after cell breakage (14-16). , Sarkosyl (I 7). In some more delicate bacteria, H. , without washing) into the lysis buffer produces better samples (Z8).
4, in 50-mL centrifuge tubes, and bury in ice. 3. 4, containing 1 mM EDTA and transfer them to the sonication vessel (see Notes 1 and 2). 4. Insert the sonication probe into the bacterial suspension (Fig. 2) and clamp the sonication vessel so that the probe tip is approximately 5 mm from the bottom of the vessel (see Note 3); ensure that the sonication vessel is surrounded with ice. 5. Sonicate (150 W) for 3 min (see Notes 4-7). 6. 4. 7. Pellet any residual whole bacteria by centrifugation at 5OOOgfor 30 min at 4OC, no brake.
During the proteinase K digestion, prepare an SDS-PAGE gel. 2. Analysis of LPS by SDS-PAGE 1. 5% stacking gel (see Chapter 4; see Notes 3 and 4). Lipopolysaccharide Chemotyping 45 2. Remove the bacterial preparations from the 60°C incubatorandcentrifugeat 12,OOOg for 5 s in order to collect condensationfrom the walls of the tubes. 3. L of preparation per lane of the gel. 4. Apply a constant current of 50 mA until the dye-line reaches a point 1 cm from the bottom of the gel. 5. Wearing rubber gloves, remove the gel from the glass plates, place it in a clean plastic box containing approximately 200 mL of fixing solution and mix gently for 2 h to overnight (see Note 5).