Swarming Assay for Visualizing Bacterial Surface Motility

Abstract

Source: Morales-Soto, N. et. al., Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays. J. Vis. Exp. (2015)

This video demonstrates a swarming assay using bacteria that express green fluorescent protein to observe surface motility on soft agar. Time-lapse imaging captures coordinated expansion and tendril formation, highlighting bacterial surface motility.

Protocol

1. Swarm Assay Media Preparation and Inoculation

1. Media Preparation
   NOTE: The medium composition described below is applicable to Pseudomonas aeruginosa tendril formation studies. Please see the media specifications in Table 1 for Pseudomonas aeruginosa, Bacillus subtilis, and Myxococcus xanthus surface motility assays.
   1. Mix 200 ml of fluidized aerobic bio-reactor (FAB)-minus ammonium sulfate ((NH4)₂SO₄) swarm medium (Materials Table), 0.9 g of Noble agar, and 0.2 g of Casamino acids (Table 1) by stirring with a magnetic stir bar. Use small volumes (100-300 ml) to improve consistency between experiments.
   2. Autoclave the 200 ml agar/media mixture using an exposure time of 22 min, exposure temperature of 121.1 °C, and a fast vent option. The autoclave settings will allow proper sterilization and agar melting, but prevent agar caramelization.
      NOTE: Noble agar is prone to caramelization; bacterial motility is altered on caramelized agar.
   3. Immediately after the sterilization cycle has finalized, close the cap of the media bottle to prevent water loss by evaporation. However, note that tight capping can cause a “vacuum-sealing”-like effect on the bottle.
   4. Cool the media to 50 °C while stirring at room temperature (RT) and add 2 ml of sterile 1.2 M glucose. Alternatively, place the media in a 60 °C incubator or water bath until ready to use (up to 15 hr later), and proceed as indicated. To prevent the formation of bubbles in the media, mix thoroughly using the magnetic stir bar; bubbles on the surface of the agar will prevent even swarming.
      NOTE: For other assays, add heat-sensitive components that cannot be autoclaved, such as additional nutrients or dyes, at this step, as needed (e.g., 8 µl Invitrogen Syto 63 dye per 100 ml melted agar to image M. xanthus, as shown in Representative Results, below). The addition of some dyes may affect baseline swarming behavior, which should be checked against a non-dye control.
   5. In a laboratory hood, aliquot 7.5 ml of sterile media per 60 mm diameter polystyrene Petri dish and maintain the plates in a single layer (not stacked). For a larger swarming surface, aliquot 25 ml of media per 100 mm diameter Petri dish. It is important to fill the dishes on an even horizontal surface. Use a bull’s-eye level to check if the surface is leveled.
      NOTE: For P. aeruginosa assays, using a specific media volume per plate will improve consistency and reproducibility. For B. subtilis and M. xanthus assays, hand pouring yields results comparable to specific volume aliquots.
2. Plate curing
   1. For small plates (60 mm), allow the melted agar medium to cure (both set to semi-solid and dry excess liquid) in the hood uncovered (i.e., without lids) for 30 min. Larger plates (100 mm) require a longer curing time (see Discussion).
      NOTE: Alternatively, some assays may require plates to cure on the bench top overnight (20-24 hr) covered (i.e., lids on) in a single layer (Table 1). Swarming is sensitive to both excess and inadequate moisture. The humidity, airflow, and temperature of any given lab may necessitate variation in plate curing to promote optimal swarming of your bacterium.
   2. Inoculate plates immediately after the drying period is over. Do not store the plates for further use.
      1. Perform the “ink spread test” by spotting a test plate with a 10 µl mixture of 0.50% (vol/vol) Higgins waterproof black India ink and bacterial inoculum. If the ink/inoculum mixture spreads readily (i.e., does not retain droplet form) on the surface of the media, the media will need additional time to dry.
         NOTE: For species that are particularly sensitive to humidity (e.g., P. aeruginosa), perform a quick “ink spread test” to determine if the plates are dry enough.
3. Swarm Assay Inoculation
   1. Inoculate 6 ml of broth culture media (see Table 1 for details) with an isolated colony from a fresh (< 5 days old if left at room temperature) Lysogeny Broth (LB) plate culture. Incubate broth cultures overnight (≤18 hr) at 30 °C or 37 °C with horizontal shaking (240 rpm).
   2. Inoculate swarm plates by spotting with 1-5 µl of overnight broth culture, or by “poking” the agar with a sterile tooth pick or wire inoculation needle.
      NOTE: We prefer the latter method because it decreases the likelihood of spattering the inoculum and prevents adding additional moisture to the swarm surface area.
4. Swarm Assay Incubation
   1. For a general assay, incubate swarm assay plates at 30 °C or 37 °C (or even 42 °C for B. subtilis; Table 1)—this is bacterium-specific. Invert the plates during incubation so that excess moisture condenses on the lid, not the agar.
      NOTE: Temperature can affect phenotype and kinetics. For P. aeruginosa swarms, incubation at 37 °C leads to faster growth and swarm expansion than incubation at 30 °C; however, the morphology of these swarms often differs with this change in temperature.
   2. For time-lapse imaging, incubate swarm plates at the appropriate temperature before transferring them to the imaging station (see Table 1 for details).
      NOTE: This pre-imaging incubation allows swarms to start their development and become established before being moved to a new environment, which may or may not be optimal for swarming motility.

2. Macroscopic Imaging of Surface Motility Assays

1. For time-lapse imaging, after the pre-imaging incubation period, place the swarm assay plates on a transparent imaging plate inside a commercial in vivo imaging station. Image up to six, 60 mm diameter or four, 100 mm diameter plates at a time. Since the camera captures images from beneath the imaging plane, invert the plates so that the optical path is not obstructed. Alternatively, incubate at 30 °C or 37 °C (Table 1) for the duration of the experiment, and remove the plates to be imaged from the incubator at set time intervals.
2. Place the lids of the Petri dishes upright on top of the plate counterpart that holds the inoculated media. Fill the lids of the Petri dishes with water to prevent excessive drying during imaging, and enclose the entire setup using another clear tray to maintain humidity throughout the experiment.
3. Using Molecular Imaging (MI) software, run assay(s) at room temperature using the imaging settings described in Table 2. For time-lapse imaging, set up a protocol with the necessary steps and specifications.

Table 1: Specifications for Surface Motility Assay Preparation. Includes surface motility assay preparation specifications for P. aeruginosa, B. subtilis, and M. xanthus.

	P. aeruginosa	P. aeruginosa tendril formation studies	B. subtilis	M. xanthus
Overnight broth culture media	FAB plus 30 mM Glucose	FAB plus 30 mM Glucose	LB	CTT (complex media)
Overnight broth culture incubation temperature	37 °C	37 °C	37 °C	30 hr at 30 °C
Swarm media	FAB	FAB minus (NH4)2SO4	2% (wt/vol) LB	CTT
Swarm media: additional components	12 mM Glucosea	10% (wt/vol) Casamino acids (CAA), 12 mM Glucosea	n/a	SYTO® 64a
Agar type	Agar, Noble	Agar, Noble	Granulated agar	Agar, Noble Affymetrix
Agar concentration (wt/vol)	0.45%	0.45%	0.60%	1.50%
Swarm plate size	60 mm	60 mm	100 mm	150 mm
Media volume per plate	7.5 ml	7.5 ml	Hand Poured	Hand Poured
Swarm media setting/drying method	Hood; plates uncovered	Hood; plates uncovered	Benchtop plates covered	Benchtop plates covered
Swarm media setting/drying time	30 min	30 min	Overnight (20 -24 hr)	Overnight (20 -24 hr)
Swarm assay incubation temperature	30 or 37 °C	30 °C	37 °C	30 °C
Incubation for time-lapse imaging	30 °C for at least 4 hr	30 °C for at least 4 hr	37 °C for 2hr	RT for 12 hr
Time-lapse capture length	24 hr	24 hr	10 hr	66 hr
Time-lapse setting	1 frame/10 min	1 frame/10 min	1 frame/6 min	1 frame/10 min
a Added after autoclaving.

Table 2: Imaging Specification. Bruker imaging station specifications for red and green fluorescence and luminescence imaging of bacterial surface growth.

Signal	Green Fluorescence	Red Fluorescence I	Red Fluorescence II	Luminescence
Protein or dye	Green Fluorescent Protein (GFP)	mCherry protein or Nile Red rhamnolipid stain	SYTO® 64	Luciferase from the lux operon
Excitation wavelength (nm)	480 ± 10	540 ± 10	590 ± 10	Off
Emission wavelength (nm)	535 ± 17.5	600 ± 17.5	670 ± 17.5	No filter
Exposure time (sec)	30	60	60	240
f-stop	4.0	4.0	2.5	1.1
FOV (mm)	190	190	140	120
Focal plane (mm)	27.5	27.5	12.2	4
Binning (pixels)	None	2 x 2	None	8 x 8

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagentsa:
FAB minimal media:				Prepare every ~4 weeks. Top to 1 L with nanopure H2O.
(NH4)2SO4	Sigma	A4418	2 g	Not used in P. aeruginosa tendril formation studies.
Na2HPO4 x 7H2O	Sigma-Aldrich	S9390	9 g
KH2PO4	Sigma	P5655	3 g
NaCl	BDH	BDH8014	3 g
MgCl2 x 6H2O solution (198 g/L)	Fisher Scientific	M33	1 ml
CaCl2 x 2H2O solution (14 g/L)	Fisher Scientific	C79	1 ml
Trace metal solution (see below)	n/a	n/a	1 ml
Trace metal solution:				Top to 1 L with nanopure H2O. Maintain in a glass bottle, stirring and covered with foil.
CaSO4 x 2H2O	Sigma-Aldrich	255548	200 mg
MnSO4 x H2O	Sigma-Aldrich	M7634	20 mg
CuSO4 x 5H2O	Fisher Scientific	C493	20 mg
ZnSO4 x 7H2O	Sigma-Aldrich	Z4750	20 mg
CoSO4 x 7H2O	Sigma-Aldrich	C6768	10 mg
NaMoO4 x 2H2O	Sigma	S6646	10 mg
H3BO3	Fisher Scientific	A74	5 mg
FeSO4 x 7H2O	Sigma-Aldrich	F7002	200 mg
CTT media:				Prepare as needed. Top to 100 ml with nanopure H2O.
Tris-HCl, 1 M solution (adjust to pH 8.0)	Amresco	234	1 ml	Prepare a 1 M stock solution in nano pure H2O. Adjust pH to 8.0 and filter sterilize (0.2 μm pore).
K2HPO4, 1 M solution (adjust to pH 7.6)	Sigma-Aldrich	P3786	0.1 ml	Prepare a 1 M stock solution in nano pure H2O. Adjust pH to 7.6 and filter sterilize (0.2 μm pore).
MgSO4 solution	Fisher Scientific	M65	0.8 ml	Prepare a 1 M stock solution in nano pure H2O. Filter sterilize (0.2 μm pore).
Casitone	BD Diagnostics	225930	1 g
Additional reagents:
LB Broth, Lennox	BD Diagnostics	240230	2 % (wt/vol)
D-(+)-Glucose	Sigma-Aldrich	G5767	30 mM for overnight broth cultures; 12 mM for swarm media	Prepare a 1.2 M filter sterilized stock solution in nano pure H2O. Add to media after autoclaving.
Casamino acids (CAA)	Amresco	J851	0.10 % (wt/vol)	Recommended for P. aeruginosa tendril formation studies. Add to media prior to autoclaving.
Agar, Noble	Sigma-Aldrich	A5431	0.45 % (wt/vol)	Preferred Noble agar for P. aeruginosa surface motility studies. Add to media prior to autoclaving.
Agar, Noble	Affymetrix	10907	1.50 % (wt/vol)	Used in M. xanthus surface motility studies. Not recommended for P. aeruginosa motility studies. Add to media prior to autoclaving.
Agar, Granulated	Fisher Scientific	BP1423	0.60 % (wt/vol)
Higgins waterproof black India ink	Higgins	HIG44201	0.50 % (vol/vol)	Mix ink with inoculum to test swarm media surface moisture.
SYTO® 64 red fluorescent nucleic acid stain	Invitrogen	S-11346		Use 4 μl (for P. aeruginosa) or 8 µl (for M. xanthus) of SYTO® 64 per 100 ml of molten agar (added after autoclaving).
Relevant materials and equipment:
Petri dish, sterile, 150 mm x 15 mm (Dia.x H)	VWR	25384-326
Petri dish, sterile, 100 mm x 15 mm (Dia.x H)	VWR	25384-342
Petri dish, sterile, 60 mm x 15 mm (Dia.x H)	VWR	25384-092
In-Vivo Xtream	Bruker			Use for the macroscopic imaging of surface motility studies. http://www.bruker.com/products/preclinical-imaging/opticalx-ray-imaging/in-vivo-xtreme/overview.html