Materials and Reagents

1. S. aureus wild type strain (e.g. the commonly-used NCTC8325 with natural Nal resistance, or its Kan-resistant derivative SH1000, Horsburgh et al., 2002) and/or any mutants of interest.
   Note: S. aureus is a potential human pathogen that is classified as a Biosafety Level (BSL) 2 organism.
   Please see the Center for Disease Control (CDC) resource http://www.cdc.gov/training/QuickLearns/biosafety/ for information on working under BSL2 conditions, including the use of appropriate personal protection equipment, the use of a biological safety cabinet to contain aerosols, and the autoclaving of all trash.
   
2. E. coli strain HT115 expressing cdc-25.1 dsRNA (Ahringer Library, Kamath et al., 2000)
   
3. E. coli strain OP50
   
4. C. elegans wild type strain (most commonly Bristol N2) and/or any mutants of interest (available from the Caenorhabditis Genetics Center at http://www.cbs.umn.edu/cgc)
   
5. Bacto Tryptic Soy Broth (TSB) (BD Biosciences, catalog number: 211825 )
   
6. Difco Tryptic Soy Agar (TSA) (BD Biosciences, catalog number: 236950 )
   
7. Luria Broth (LB) (MP Biomedicals, catalog number: 3002-021 )
   
8. Luria Broth Agar (MP Biomedicals, catalog number: 3002-231 )
   
9. Nalidixic acid sodium salt (Nal) (Sigma-Aldrich, catalog number: N4382 )
   
10. Carbenicillin disodium salt (Carb) (Sigma-Aldrich, catalog number: C1389 )
    
11. 5-fluorodeoxyuridine (FUDR) (Sigma-Aldrich, catalog number: F0503 )
    
12. Nalidixic Acid (Nal) 1,000x stock solution (10 mg/ml) (see Recipes)
    
13. Carbenicillin 1,000x stock solution (100 mg/ml) (see Recipes)
    
14. TSB (see Recipes)
    
15. LB (see Recipes)
    
16. Large TSA + Nal (10 μg/ml) plates (see Recipes)
    
17. Large LB + Carb (100 μg/ml) plates (see Recipes)
    
18. Killing assay plates (see Recipes)
    
19. Nematode growth media (NGM) plates (see Recipes)
    
20. RNAi plates (see Recipes)
    

Equipment

1. Dissection stereo microscope (e.g. Zeiss, model: Stemi 2000 )
   
2. 15 °C and 25 °C C. elegans incubators (e.g. Thermo Fisher Scientific, model: 3940 )
   
3. 37 °C bacterial incubator and shaker
   
4. Platinum wire worm pick* and ethanol lamp (Cole-Parmer, catalog number: EW-48585-84 ) for sterile transfer of worms at dissection scope.
   *Worm picks can either be purchased (e.g. Genesse Scientific, catalog number: 59-AWP ) or made in the lab. To make a pick, insert a 5 cm segment of 90% platinum/10% iridium wire (Tritech Research, catalog number: PT-9010 ) into the narrow end of a glass Pasteur pipet, melt glass over Bunsen burner flame to fasten wire inside, and flatten the tip of the protruding wire (~5 mm) into a flat “spatula”-like structure using a pair of needle-nose pliers. The handle end of the glass pipet can be inserted into foam tubing (Maddak, catalog number: F766900183 ) for more comfortable manipulation (see Figure 1).
   
   
   Figure 1. Example of a worm pick made in the lab. The pick consists of platinum wire inserted into a Pasteur pipet, held in foam tubing for easy handling. See text for details.
   

Software

1. Microsoft Excel or any other spreadsheet software
   
2. GraphPad Prism
   

Procedure