For isolating Salmonella (other than S. typhi) from water and associated materials, such as sewage liquor, sewage sludge, digested sludge and pressed sludge cake:

1. Concentrate the sample by filtering it through a plug of sterile absorbent cottonwool inserted in the neck of a large sterile funnel or through a Whatman No. 17 absorbent pad. Pre-enrichment

2. Using aseptic technique, transfer the cottonwool plug or the pad to 100 mL of a suitable pre-enrichment medium such as Buffered Peptone Water.

3. Incubate at 37 ± 0.5°C for 18-24 hours. Selective Enrichment

4. Inoculate 10 mL of Rappaport Vassiliadis R10 Broth with 0.1 mL of the pre-enrichment culture. Inoculate 10 mL of Muller-Kauffman Tetrathionate Broth with 1 mL of the pre-enrichment culture.

5. Incubate Rappaport Vassiliadis R10 Broth at 41.5 ± 0.5°C. Incubate Muller- Kauffman Tetrathionate Broth at 42 ± 1°C for 48 hours. Expected Results

6. After incubation, subculture both selective enrichment broths to Brilliant Green Agar and XLD Agar. Incubate at 35 ± 2°C for 18-24 hours.

7. Examine for typical Salmonella colonies. Confirm identification of isolates by biochemical and serologic tests.

Milk and Foods

For isolating Salmonella (other than S. typhi) from milk and milk products, raw flesh foods, highly contaminated foods and animal feeds:

Pre-enrichment

1. Add 25 g or a 25 mL sample of the specimen to 225 mL of pre-enrichment medium. Consult appropriate references for the type of product being tested.

2. Incubate at 35°C for 24 ± 2 hours5 or at 37°C for 16-20 hours,4 depending on the referenced procedure being followed.

Selective Enrichment

3. Inoculate 10 mL of Rappaport Vassiliadis R10 Broth with 0.1 mL of pre-enrichment culture. Inoculate 10 mL of another selective enrichment medium such as Tetrathionate Broth or Selenite Cystine Broth with 1 mL of the pre-enrichment culture.

4. Incubate Rappaport Vassiliadis R10 Broth at 41.5 ± 0.5°C4 for 24 ± 2 hours. Incubate the other selective enrichment broths appropriately. Expected Results

5. After incubation, subculture Rappaport Vassiliadis R10 Broth and the other selective enrichment broths to selective agar media and incubate at 35 ± 2°C for 24 ± 2 hours.4 6. Examine for typical Salmonella colonies. Confirm identification of isolates by biochemical and serologic tests.

2. Warm slightly to completely dissolve the powder.

3. Dispense 10 mL amounts into suitable containers.

4. Autoclave at 116°C (10 psi pressure) for 15 minutes.

5. Test samples of the finished product for performance using stable, typical control cultures.

2. Vassiliadis, Trichopoulos, Kalandidi and Xirouchaki. 1978. J. Appl. Bacteriol. 44:233.

3. Peterz, Wiberg and Norberg. 1989. J. Appl. Bacteriol. 66:523.

4. International Dairy Federation. 1995. Milk and milk products: detection of Salmonella. IDF Standard 93B:1005. Brussels, Belgium.

5. Horwitz (ed.). 2000. Official methods of analysis of AOAC International, 17th ed. AOAC International, Gaithersburg, Md.