Gliding Motility in Phormidium lacuna Filaments

Abstract

Source: Weber, N., et al., Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna. J. Vis. Exp. (2022)

This video demonstrates the procedure to assess gliding motility in Phormidium lacuna by homogenizing filament cultures to create a uniform suspension, preparing a Petri plate assay setup over a microscope slide, and visualizing filament movement.

Protocol

1. Assay for movement in liquid medium
   1. For direct observation of motility, transfer 8 mL of medium containing P. lacuna into a 6 cm Petri dish. Wait a few minutes until the sample reaches room temperature, then cover the Petri dish with cellophane foil.
   2. Place a microscope slide on the x-y table of a standard microscope with a camera. Switch on the microscope light. Ideally, always use the same electrical and optical settings for the lighting. Move a 4x or 10x objective into the light's path.
   3. Place the Petri dish on top of the slide. Adjust single filaments or filament bundles by x, y, and z movements of the table.
      NOTE: Due to the three-dimensional arrangement, only a part of the relevant section can be in focus. The cellophane foil allows adjusting the focus without restriction.
   4. Observe movements of single filaments or bundles. Ensure that the objective lens does not touch the liquid. Record the movements of filaments with a standard microscope camera.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glassware, different
Microscope DM750	Zeiss
Ocular camera MikrOkular Full HD	Bresser		for time-lapse recordings, coupled to Raspberry Pi minicomputer
Petri dishes polystyrole, 60 mm x 15 mm	Merck	P5481-500EA
Software, URL
ImageJ		software for immage processing (pixel intensities, circle diameter)
Culture media
Artificial seawater	0.41 M NaCl, 53 mM MgCl₂, 28 mM Na₂SO₄, 10 mM CaCl₂, 9 mM KCl , 2.4 mM NaHCO₃ ,0.84 mM KBr, 0.49 mM H3BO₃, 90 µM SrCl₂, 72 µM NaF
f/2 -liquid medium	artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution, 0.88 mM NaNO₃, 36 µM NaH₂PO₄
Trace element solution	0.36 mM NaH₂PO₄, 12 µM Na₂EDTA, 39 nM CuSO₄, 26 nM Na₂MoO₄ , 77 nM ZnSO₄, 42 nM CoCl₂, 0.91 µM MnCl₂
Vitamin solution	0.3 µM thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin