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