In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

Published: April 30, 2023

Abstract

Source: Karki, P. et al. High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence. J. Vis. Exp. (2021)

In this video, we demonstrate a method to evaluate the effect of osmolytes on the formation of bacterial persisters. The bacterial cells are first exposed to osmolytes to induce osmotic stress, triggering persister formation, then treated with antibiotics to enumerate the persisters.

Protocol

1. Preparation of growth medium, ofloxacin solution and E. coli cell stocks Regular Luria-Bertani (LB) medium: Add 10 g/L of tryptone, 10 g/L of sodium chloride (NaCl), and 5 g/L of yeast extract in deionized (DI) water. Sterilize the medium by autoclaving. LB agar plates: Add 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract, and 15 g/L agar in DI water and sterilize the medium by autoclaving. At the desired temperature (~55 °C), pour ~30 mL of agar medium into square plates …

開示

The authors have nothing to disclose.

Materials

14-ml test tube Fisher Scientific 14-959-1B
Flat-bottom 96-well plate USA Scientific 5665-5161
Gas permeable sealing membrane VWR 102097-058 Sterilized by gamma irradiation and free of cytotoxins
Half-area flat-bottom 96-well plate VWR 82050-062
LB agar Fisher Scientific BP1425-2 Molecular genetics grade
Ofloxacin salt VWR 103466-232 HPLC ≥97.5
Phenotype microarray (PM-9 and PM-10) Biolog N/A PM-9 and PM-10 plates contained various osmolytes and buffers respectively
Round-bottom 96-well plate USA Scientific 5665-0161
Sodium chloride Fisher Scientific S271-500 Certified ACS grade
Sodium nitrate Fisher Scientific AC424345000 ACS reagent grade
Sodium nitrite Fisher Scientific AAA186680B 98% purity
Square petri dish Fisher Scientific FB0875711A
Tryptone Fisher Scientific BP1421-500 Molecular genetics grade
Varioskan lux multi mode microplate reader Thermo Fisher Scientific VLBL00D0 Used for optical density measurement at 600 nm
Yeast extract Fisher Scientific BP1422-100 Molecular genetics grade

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記事を引用
In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence. J. Vis. Exp. (Pending Publication), e21228, doi: (2023).

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