By Jenny Howard, David M. Whitcombe
Diagnostic Bacteriology Protocols offers a extensive diversity of presently used recommendations for detecting, settling on, and differentiating bacterial mobile elements, together with established proteins, nucleic acids, enzyme actions, lipopolysaccharides, and metabolites. It describes every one approach in basic easy-to-follow steps that warrantly reproducible effects for rookies and senior researchers alike. Troubleshooting information, alternative routes of acting systems, and informative motives approximately why definite steps are necessary-aids now not frequently present in commonplace magazine recipes aid even hugely expert researchers to acquire the consequences they wish.
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Extra resources for Diagnostic Bacteriology Protocols
Silver stain fixing solution: 40% (v/v) methanol, 5% (v/v) acetic acid. 5. 7% (w/v) periodic acid m fixing solution. 6. Silver nitrate: 20% (w/v) In deionized water. 7. Ammonium solution (concentrated). 8. lM NaOH. 44 Chart 9. lM sodmm hydroxide mixed with 2 mL of ammonium hydroxide followed by 5 mL of 20% silver nitrate solution. Make the volume up to 150 mL with deionized water, mix, and use wrthin 5 min of preparation. 10. 5 mL formalin in 1 L deionized water (prepared freshly). 11. Gel storage solutron: 10% (v/v) acetic acid.
Coli are described. This involves the culture of E. coli under inducing conditions (in the presence of an iron chelator), isolation of outer membrane proteins, protein quantitation, and, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, described in Chapter 4), analysis of the protein profile. 2. Materials 1. Culture media: For the examination of constitutive outer membrane proteins, bacteria should be grown m a medium suitable for the species under mvestigation; for example, strains of E.
Results Examples of LPS chemotypes are shown in Fig. 2. Lane 1 shows longchain LPS, expressed by S. enteritidis, with chains composed of equal numbers of sugar units separating together to give a banded profile resembling the rungs of a ladder (see Note 9). Lipid A-core LPS migrates very near the dye-front and stains very intensely, appearing as an almost black region. The profile in lane 2 shows the LPS from a strain of S. enteritidis that does not express long-chain LPS; although LPS “rungs” cannot be detected, lipid A-core can be seen as a black band on the dyeline.
Diagnostic Bacteriology Protocols by Jenny Howard, David M. Whitcombe