By Jan Michiels, Maarten Fauvart
This quantity offers a finished choice of equipment which were instrumental to the present realizing of bacterial persisters. Chapters within the ebook disguise subject matters starting from normal tools for measuring persister degrees in Escherichia coli cultures, protocols for the choice of the persister subpopulation in Candida albicans, quantitative measurements of style I and sort II persisters utilizing ScanLag, to in vitro and in vivo models for the learn of the intracellular job of antibiotics. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and state of the art, Bacterial endurance: equipment and Protocols brings jointly the main revered researchers in bacterial patience whose reports will stay very important to figuring out this box for a few years to come.
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Extra info for Bacterial Persistence: Methods and Protocols
High-persistence (hip) mutants were isolated from the original screens for E. coli mutants with altered persister frequency . Later, it was found that although the screens were made with ampicillin, increased persister frequency was observed also against fluoroquinolone antibiotics and vice versa . This suggested that persisters are equally tolerant to many or all antibiotics. Current results suggest that the mechanism of antibiotic action considerably influences the persister levels and different (but probably overlapping) populations survive different antibiotics [24, 41, 42].
3. 45 g Na2HPO4 (see Note 4) in 800 mL water, and fill up to 1000 mL. Dilute 10Â in water to prepare 1Â PBS. Autoclave and store at room temperature. 4. Stock solutions of antibiotics (see Note 5). (a) Ampicillin: Dissolve ampicillin sodium salt in water at a concentration of 100 mg/mL. Do not freeze and melt. Store at 4 C for up to 1 week (see Note 6). 1 M NaOH. Dissolve, dilute with water to a concentration of 5 mg/mL, aliquot, and freeze. Store at À20 C for up to 3 months. 5. Dimethyl sulfoxide (DMSO).
Remove supernatant and resuspend bacteria in 1 mL LB medium. Repeat centrifugation, remove supernatant, and resuspend cells in 1 mL of fresh PBS. Continue with serial dilutions and plating as in the case of the non-treated and ampicillin-treated samples (see Note 16). Example results of the persister measurement are shown in Fig. 1. Alternatively, the results of antibiotic treatment and growth resumption can be analyzed by flow cytometry (see Note 17). 4 Notes 1. Let the powdered medium hydrate by pouring it slowly on the surface of the water.
Bacterial Persistence: Methods and Protocols by Jan Michiels, Maarten Fauvart