Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level
Published: April 30, 2023
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 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…
Disclosures
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 | 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 | ||
| 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 |
| Sterile Vacuum Filter Units | Fisher | SCGVU01RE | Used to filter-sterilize mPBS |
| Vacuum pump | Used to filter-sterilize mPBS |
Tags
Cite This Article
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).