This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.
Protocol
1. Coincubate bacterial strains Choose two bacterial strains for single-cell bacterial competition assays. Here, two strains of V. fischeri are used: a target strain (ES11424) and an inhibitor strain (MJ1125) that is known to kill the target strain using the type VI secretion system on chromosome II (T6SS2)1, which is a contact-dependent killing mechanism. Transform strains with stable plasmids encoding genes for different fluorescent proteins (e.g., GFP or RFP) to visually distinguish stra…
Representative Results
Figure 1: Agarose pad preparation and slide setup for coincubation assays. (A) Setup for making 2% agarose pads. Five layers of lab tape (green) are wrapped around a cover slip at two points approximately 20 mm apart. Next, warm 2% agarose in marine Phosphate-Buffered Saline (yellow) is pipetted between the pieces of tape and immediately covered with a 25 mm2 cover sli…
Divulgations
The authors have nothing to disclose.
Materials
1.5 mL Microcentrifuge tube
Fisher
05-408-129
10 uL single channel pipette
1000 uL single channel pipette
20 uL single channel pipette
200 uL single channel pipette
Agarose
Fisher
BP165-25
Low melting agarose
Cellvis 35 mm Dish
Fisher
NC0409658
#1.5 cover glass bottom
Chloramphenicol
Sigma
C0378
stock (20 mg/mL in Ethanol); final concentration in media (2 μg /mL LBS)
FIJI image analysis sofware
ImageJ
https://imagej.net/Fiji/Downloads
open-source software
Fisherbrand Cover Glasses: Circles
Fisher
12-545-81P
#1.5 cover glass; 12 mm diameter
Kanamycin Sulfate
Fisher
BP906-5
stock (100 mg/mL in water, filter sterilize); final concentration in media (1 μg/mL LBS)
Lens Cleaning Tissue Paper
Fisher
S24530
Petri Plates
Fisher
FB0875713
sterile with lid
Razor Blades
Fisher
S65921
Semi-micro Cuvettes
VWR
97000-586
Spectrophotometer
Thermo Scientific Gold Seal Plain Microscope Slides
Upright or inverted fluorescence microscope with camera and imaging software
Images in this article were acquired on a Nikon TI-2 inverted fluorescent microscope outfitted with an ORCA-Fusion Digital CMOS camera using NIS-Elements software.
Vortex
Water bath
Used to keep agarose warm prior to pipetting
LBS media
1M Tris Buffer (pH ~7.5)
50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base)
Agar Technical
Fisher
DF0812-17-9
15 g (Add only for plates)
DI water
950 mL
Sodium Chloride
Fisher
S640-3
20 g
Tryptone
Fisher
BP97265
10 g
Yeast Extract
Fisher
BP9727-2
5 g
mPBS (marine PBS)
Phosphate buffered saline with marine salts added; used for making agarose pad
10X PBS
Fisher
ICN1960454
Instant Ocean Sea Salt
Instant Ocean
SS1-160P
Adjust concentration to appropriate salinity; 20 psu used here
Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level. J. Vis. Exp. (Pending Publication), e21230, doi: (2023).