Materials and Reagents

1. 50 ml plastic tubes for handling harvested cultures (VWR International, catalog number: 62406-200 )
   
2. 0.45 μm syringe filters (VWR International, catalog number: 28145-477 )
   
3. 0.22 μm syringe filters (VWR International, catalog number: 28144-050 )
   
4. 30 ml syringes (VWR International, catalog number: 66064-760 )
   
5. 1.5 ml Eppendorf tubes (VWR International, catalog number: 89213-152 )
   
6. 200 mesh formvar coated TEM grids (Electron Microscopy Sciences, catalog number: EMS200-Cu )
   
7. Nunc^TM MicroWell^TM 96-Well Optical-Bottom Plates with Coverglass Base (Thermo Fisher Scientific)
   
8. Myxococcus xanthus wild type strain DZ2 (UC Regents, Berkeley)
   
9. Phosphate buffered saline (PBS) (VWR International, catalog number: 97064-158 )
   
10. Fluorescent lipid dye FM 4-64 (Life Technologies, Molecular Probes^®, catalog number: T-13320 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: T-13320”.
    
11. Uranyl acetate (Electron Microscopy Sciences)
    
12. 10 mM MOPS (pH 7.6)
    
13. 2 mM MgSO₄
    
14. 10% (w/w) Bacto casitone
    
15. 5% (w/w) Bacto yeast extract
    
16. CYE Media (see Recipes)
    

Equipment

1. Sterile, side arm 250 ml Erlenmeyer Flasks
   
2. Shaking incubator (e.g., Thermo Fisher Scientific, model: MaxQ4000 )
   
3. Vortexer
   
4. Centrifuge with capacity for 25 ml cultures, 5,000 x g (Refrigeration not required)
   
5. Ultra-centrifuge with capacity for 1 ml samples, 140, 000 x g required and refrigeration required. (e.g., GMI, Beckman Coulter, model: L8-70M )
   
6. Ultra-centrifuge tubes (fit to machine specifications)
   
7. -80 °C freezer
   
8. Fluorescence plate reader (e.g., Tecan Trading AG, model: Vission-100 )
   
9. Transmission Electron Microscope (Philips/FEI, model: 5350 NE Dawson Creek Drive ), capable of imaging negatively-stained samples at voltages ranging between 80 kv and 200 kV equipped with a 2 k x 2 k CCD camera
   
10. Autoclave for sterilizing media and flasks (or access to sterile media and growth vessels).
    

Software

1. Digital Micrograph software and 2 k x 2 k CCD camera (Gatan Inc.)

Procedure