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

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

1. 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.
2. 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 (10 x 10 cm). Dry the plates and store them at 4 °C.
3. Modified LB medium: Add 10 g/L of tryptone and 5 g/L of yeast extract in DI water.....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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