Appendix I.

Bacterial isolation, culturing and identification:

Insect and manure/litter samples will be prepared for serial dilutions. Serially diluted aliquots will be plated directly onto selective media (Campy-Cefex; Stern et al., 1992). Plates will be incubated at 42_C utilizing the Campy-Pac microaerobic system. Following 24 hours incubation bacterial colonies suggestive of Campylobacter will be gram stained and wet mounted to determine motility and cell morphology characteristic of Campylobacter. Campylobacter colony forming units (CFUs) in the original sample will be enumerated, and colonies will be subcultured at 42_C microaerobically. After 36 hours of growth, the subcultures will be catalogued, and the cell mass will be swabbed off the plates and preserved in freezing medium (Brucella broth with 15% sterile glycerol) at B70_C for further study.

Stern, N. J., B. Wojton, and K. Kwiatek. 1992. A differential selective medium and dry-ice generated atmosphere for recovery of Campylobacter jejuni. J. Food Prot. 55:514-517.

Salmonella species will be cultured from insect and manure/litter treatments using methods for bacterial isolation developed by Bager et al. (1991). One gram of manure/litter, or bacteria from insects will be pre-enriched in 10 ml buffer peptone water and incubated over night at 371C. Then, 100 _l of this will be enriched overnight in Rappaport Vassilliadas medium at a 1:100 ratio (Andrew et al. 1998). Samples will be plated on xylose lysine tryptone (XLT4) agar or Hektoen enteric agar and identified as Salmonellae. Identification will be confirmed using triple sugar iron (TSI) and Urea media.

Harein et al. (1970) isolated 26 serotypes of E. coli known as human pathogens from darkling beetles collected from Minnesota brooder houses. Isolated cultures can be grown anaerobically in 10 ml LST broth for 24 hr at 37 °C. The population is verified through serial dilutions in 4.5 ml LST and plated on CHROMagar O157 (Chromagar Microbiology, Paris, France). Other attaching efacing E. coli strains may be important in poultry related studies (McAllister et al. 1996).

Samples for Listeria analysis (insect and manure/litter samples) will be held on ice until processed. Samples will be suspended in 25 ml of enrichment broth (EB) and placed in a blender or stomacher for thorough mixing. The blended culture is then transferred to a 500 ml Erlenmeyer flask and incubated 4 h at 301C. After initial incubation a selective agent (acriflavin, nalidixic acid, and cycloheximide) is added to the culture and continued incubating another 44 h, for a total of 2 d, at 301C. At 48 h, streak the EB culture onto both OXA (Curtis et al., 1989) and incubate OXA plates at 351C for 24-48 h. Listeria colonies have a black halo. Purified isolates can be rapidly identified by conventional tests commercial kits: API Listeria (BioMerieux sa Marcy-l'Etoile, France).