By James Butcher, Alain Stintzi
Campylobacter jejuni (C. jejuni) is usually considered as the only of the commonest factors of bacterial gastroenteritis around the globe. The objective of this quantity is to spotlight key protocols for operating with C. jejuni. specifically, chapters target to focus on contemporary advancements just about in vivo types for C. jejuni pathogenesis, diversified ways to isolate Campylobacter, and a structures biology procedure for learning the impact of all capability Campylobacter gene mutants.Written within the hugely winning Methods in Molecular Biologyseries layout, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and pointers on troubleshooting and averting identified pitfalls.
Authoritative and state-of-the-art, <Campylobacter jejuni: equipment and Protocols encourages current Campylobacter researchers to hire novel tips on how to extra their very own learn and likewise encourages new researchers to incorporate Campylobacter of their destiny study initiatives.
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Additional info for Campylobacter jejuni: Methods and Protocols
1 Accumulation of Ethidium Bromide 1. Harvest an overnight broth culture of C. 5 mL microcentrifuge tube. Prepare triplicate samples for each test strain. To compare a wild-type strain with its cmeABC mutant, the cultures should be prepared concurrently. 2. For each strain, incubate the bacterial suspension at 37 °C for 10 min under normal atmosphere. 3. Add 50 μL of cell suspensions into a well of a black polystyrene 96-well microplate. For each test strain, nine wells are needed, including six test wells and three control wells.
Add vancomycin at a final concentration of 10 μg/mL to the media tempered to 45 °C just before the plates are poured (see Note 3). 3 Solutions and Buffers 1. 8 g NaCl, 2 g MgSO4·7H2O, 5 mL 2 % (w/v) gelatin, 50 mL of 1 M Tris–HCl. Add water to a volume of 900 mL and mix. 5 using HCl. Fill up to 1 L with water and autoclave. 2. 1 M MgSO4 solution. Sterilize by autoclaving. 3. 100 mM CaCl2 solution. Sterilize by autoclaving. 4. Vancomycin, 10–25 mg/mL solution in water. Store at −20 °C. 5. 5 mL microcentrifuge tubes containing 900 μL of SM buffer.
9. 2 μm) them into a sterile tube. 10. Titrate the harvested phage stock on a lawn of propagation strain, as described below, and calculate the pfu/mL. Remember to write the phage name, date of propagation and the achieved pfu/mL on the master stock tubes. Isolation and Purification of C. 6 Titration 25 1. Prepare tenfold serial dilutions of the phage stock in SM buffer up to 10−7 (see Note 18). 2. Divide the bottom of the lawn prepared petri dishes into eight equal segments drawing with a marker and mark the corresponding dilutions from 0 to −7 (see Note 19).