Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Abstract

Source: Smith, S., et al., Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging. J. Vis. Exp. (2021).

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

1. 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.
2. Transform strains with stable plasmids encoding genes for different fluorescent proteins (e.g., GFP or RFP) to visually distinguish strain types on the microscope. Here, the inhibitor strain is tagged with a GFP-encoding plasmid (pVSV102), and the target strain is tagged with a dsRed-e....

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 mar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	Fisher	12-518-100B
Thermo Scientific Richard-Allan Scientific Cover Glass	Fisher	22-050-235	#1.5 cover glass, 25 mm2
Type F Immersion Oil	Fisher	NC0297589
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
<strong>LBS media</strong>
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
<strong>mPBS (marine PBS)</strong>			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
Sterile Vacuum Filter Units	Fisher	SCGVU01RE	Used to filter-sterilize mPBS
Vacuum pump			Used to filter-sterilize mPBS