Treatment of Bacteria with Plant Root Exudates to Emulate In Vitro Plant-Microbe Interactions

Abstract

Source: Yi, Y., et al. Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates. J. Vis. Exp. (2018)

This video demonstrates the preparation and treatment of Bacillus mycoides cultures to study plant-microbe interactions. It outlines the steps from streaking a stock culture and incubating it in liquid medium to exposing the bacteria to plant root exudates under agitation. The process culminates in harvesting and flash-freezing the bacterial cells for subsequent analysis of gene expression changes induced by the exudates.

Protocol

1. Growing Bacteria

1. Streak a B. mycoides strain from a -80 °C glycerol stock onto an Luria-Bertani (LB) agar plate, and incubate the plate at 30 °C overnight.

2. Inoculate an LB liquid medium with a single colony from the plate, and grow the culture in a shaking incubator at 200 rpm overnight at
30 °C.

2. Treatment and Sampling of Bacteria

1. To compare the growth curve of the bacteria treated with root exudates to a negative control, prepare a series of flasks containing 50 mL of LB liquid medium. Add 0.5 mL of the overnight bacterial culture to all flasks, and add either 0%, 5%, 10%, or 15% (v/v) root exudate or sterile deionized water to the medium, respectively.

2. Use a culture with deionized water instead of root exudates as a control. Measure the optical density at 600 nm (OD₆₀₀) every 1 h afterward. Generate a growth curve by plotting the OD₆₀₀ values versus time.

3. Dilute an overnight B. mycoides culture with 90 mL of pre-warmed LB medium to an initial OD₆₀₀ of ~ 0.05 in a 300 mL flask. Add 10 mL of root exudates to the culture and incubate it at 30 °C for 1 h.

4. Use a 10 mL sterile deionized water treatment as a control. Collect cells from the culture by centrifugation at 9,000 x g for 2 min at 4 °C. Discard the supernatant, and immediately freeze the pellet in liquid nitrogen, then store it at -80 °C until use.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium hypochlorite	Sigma	CAS: 7681-52-9	10-15% active chlorine
Luria-Bertani (LB) broth
Incubater	New Brunswick Scientific	Innova 4000
Spectrophotometer	Thermo Fisher Scientific	Genesys 20
Liquid nitrogen
Glass beads	Sigma	G8893	0.5 µm
2.0 ml tube with screw cap			RNase free
1.5 ml and 2.0 ml eppendorf tube			RNase free
Centrifuge	Eppendorf	5430