Materials and Reagents

1. Worm pick made with platinum wire (Tritech Research, catalog number: PT-9901 )
   
2. Filter paper (110 mm diameter) (GE Healthcare, Whatman, catalog number: 1001-110 )
   
3. Petri dish 140 mm diameter (VWR, catalog number: 391-1500 )
   
4. 90 mm Petri dishes (Techno Plas, catalog number: S9014UV20 )
   
5. C. elegans strains
   The following protocol applies to strains derived from wild type animals (N2, Bristol strain). Suggested controls (available from the Caenorhabditis Genetics Center (CGC)):
   1. N2 (wild-type): positive control
      
   2. gcy-31(ok296): unable to respond to downshifts of O₂ levels
      
   3. gcy-35(ok769): unable to respond to upshifts of O₂ levels
      
   4. gcy-9(n4470): unable to respond to changes in CO₂ concentration
      
   5. tax-4(p678): deficient in O₂ and CO₂ sensing
   Note: All strains should be grown and maintained under standard conditions (Brenner, 1974). All strains should be crosses a minimum of 4 times with wild type animals. Changes in temperature or starvation can affect the results of the assay. Therefore, strains should be maintained at a constant temperature (routinely 20 °C) and well-fed with OP50 Escherichia coli for at least two generations prior to the assay.
   
6. Copper(II) chloride dihydrate (CuCl₂·2H₂O) (Sigma-Aldrich, catalog number: 221783-100G )
   
7. NGM maintenance plates seeded with OP50 E. coli (see Recipes)
   
8. KPO₄ buffer (1 M, see Recipes)
   1. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662-500G )
      
   2. Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: 3786-500G )
      
9. Assay NGM plates (see Recipes)
   1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-250G )
      
   2. Agar (SERVA high-gel strength agar) (SERVA Electrophoresis, catalog number: 11396.03 )
      
   3. Cholesterol (Sigma-Aldrich, catalog number: C8667-25G )
      
   4. Calcium chloride (CaCl₂) (Merck, catalog number: 1.02382.0500 )
      
   5. KPO₄ buffer (1 M)
      
10. Starvation plates (see Recipes)
    

Equipment

1. Computer compatible with the software
   Intel core i7, 32GB RAM, Windows 7, 64 bit. Needs one additional gigabit Ethernet card for connecting the GigE camera (Figure 1)
   
2. Camera
   We use a 4-megapixel CCD camera (JAI, model: BM-500GE ), 35 mm objective: QIOPTIQ MEVIS 3516. Extension rings: PENT EXTENSION RING 1 (1 mm C-Mount) (Figure 1)
   
   
   Figure 1. Computer and camera setup
   
   
3. Flow controllers
   Vögtlin Instruments, CO₂: GSC-A9TA-BB21 50 mln/min, N₂: GSC-A9TA-BB22 200 mln/min, O₂: GSC-A9TA-BB21 50 mln/min (Figure 2)
   
   
   Figure 2. Gas and flow controller setup. A. Overview of mains gas supply connected to the flow controller. B. Close-up of the flow controller.
   
   
4. LED illumination (CCS, catalog number: TH-211/200RD ), cable (CCS, catalog number: FCB-1 ), power supply (CCS, catalog number: PD2-5024 )
   
5. Individual gas bottles for O₂, CO₂ and N₂
   Note: Alternatively, premixed bottles may be used, depending on the flow controllers that are used. Nevertheless, we recommend individual gas bottles as they provide flexibility on the gas parameters that can be tested.
   
6. Assay chamber: custom-made transparent Plexiglas device with a flow arena of 60 x 60 x 0.7 mm (Figure 3)
   
   
   Figure 3. Custom-designed assay chamber. A. Top diagram: view from the top. Bottom diagram: lateral view. Note that the diagram is not scaled. B. Photograph of the assay chamber taken from above.
   
   
7. Autoclave
   
8. Dryer (Binder, catalog number: 9010-0102 )
   
9. 500 ml autoclaved bottle
   

Software

1. Labview (to program and regulate the flow controllers)
   
2. MATLAB (R13) (MathWorks) and Image Acquisition and Image Processing Toolbox (Figure 4)
   
3. Parallel WormTracker (freely available: https://sourceforge.net/projects/wormtracker/)
   
4. Streampix software (Norpix)
   
   
   Figure 4. Screenshots of WormTracker analysis. A. Tracks of individual worms (red lines) detected by the software. B. MATLAB analysis software screen showing tracking frames.
   

Procedure