Visualization of Single-Cell Bacterial Interactions

Abstract

Source: Yarringto, K. D., et al. Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions. J. Vis. Exp. (2020)

This video demonstrates the preparation of a coculture system using motile and non-motile bacteria under an agarose pad for live-cell imaging. It shows how motile bacteria detect chemical attractants and invade non-motile colonies, revealing bacterial interactions.

Protocol

1. Preparation of Bacterial Cells and Inoculating Pads

1. Measure the optical density, OD₆₀₀, of each subculture and dilute Pseudomonas aeruginosa to an OD₆₀₀ = 0.03 and Staphylococcus aureus to an OD₆₀₀ = 0.10 in M8T (Minimal Medium 8 + tryptone) prewarmed to 37 °C. Mix P. aeruginosa and S. aureus in a 1:1 ratio and vortex.
   NOTE: If strains require antibiotics, they can be added to the overnight and subculture, but they should not be added when mixing species for imaging if they affect the other species in the coculture. Plasmid stability and the effects of antibiotics on all species in the coculture will need to be determined for each organism/plasmid being used.
2. Pipette 1 µL of coculture evenly across the bottom of a pre-warmed, sterile 35 mm glass coverslip dish.
3. Remove the silicone cutout from the mold using sterile tweezers.
4. Remove the pad from the dish using sterile spatulas.
   1. Slip the slightly bent spatula under the edge of the pad while holding the mold upside down to drop it out onto a sterile Petri plate lid.
      NOTE: Do not force the pad out, or it will rip. Keep track of which side of the pad is the bottom.
5. Transfer the pad to the dish with the bacterial cells, bottom-side-down, by sliding the 90° angled spatula under the pad and placing it on top of the inoculated coverslip. Use the 90° angled spatula to make the pad flush against the coverslip and gently press out any air bubbles.
6. Remove excess moisture from moist wipes and place them around the edge of the dish, making sure they do not touch the pad. The sample is now ready for imaging.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose pads
35 mm glass bottom dish with 20 mm micro-well with 1.5 cover glass	Cellvis	D35-20-1.5-N	One for agarose pad molds, one for experiment
Round-Tapered Spatulas	VWR	82027-530
Silicon isolator, press-to-seal, 1 well, 2.0 mm diameter, 20 mm, silicone/adhesive	Sigma-Aldrich	S6685-25EA	For agarose pad molds
Tweezers	VWR	89259-944
M8T Minimal Media
D (+) Glucose	RPI	G32045
KH₂PO₄	RPI	P250500
MgSO₄	Sigma-Aldrich	208094
NaCl	RPI	S23025
Na₂HPO₄·7H₂O	Sigma-Aldrich	230391
Tryptone	BD Biosciences	DF0123173
Bacterial Strains
Pseudomonas aeruginosa PA14 (WT) pSMC21 (Ptac-GFP)	PMID: 9361441
Staphylococcus aureus USA300 LAC (WT)	PMID: 23404398		USA300 CA-Methicillin resistant strain LAC without plasmids