Rappaport Vassiliadis Broth EP/USP

Enrichment of Salmonella

Collect, dilute and prepare samples and volumes as required according to specifications, directives, official standard regulations and/or expected results.

Inoculate aseptically the tubes/bottles with the prepared sample or its dilution in a proportion 1/10 (V/V).

Incubate the bottles tightly closed aerobically at 41.5 ± 1 °C for 24 ± 3 h (Pharmacopoeia recommended 30-35 °C).

(Incubation times, temperature and sample volumes may vary depending on the sample, on the specifications, etc.)

Read the turbidity (growth indicator) and inoculate any confirmatory, secondary medium by streaking methodology or by spiral method, like XLD, BPLS, etc., to confirm results. After proper incubation, enumerate all the colonies that have appeared onto the surface of the secondary agar.

Presumptive isolation or recovery of Salmonella must be confirmed by further microbiological and biochemical tests.

Each laboratory must evaluate the results according to their specifications.

Calculate total microbial count per mL of sample by multiplying the average number of colonies per plate by the inverse dilution factor if a diluted sample was streaked. Report results as colony forming units (CFU) per mL or g along with enrichment and secondary media used, incubation time and temperature.

Observations

• The refrigerated medium may present precipitates, but they will not affect the performance.
• The pH may increase slightly during the useful life of the culture medium, but it does not affect the performance.

LC-5187, bottles

This culture medium is a modification of the Rappaport Vassiliadis Broth (ISO) with the composition adjusted to the formulation proposed by the European Pharmacopoeia in the Harmonised Methodology, and also to the requirements of the Japanese and United States Pharmacopoeia.

Faecal specimens and water can be enriched directly on this medium. For pharmaceutical products, food and environmental specimens, a pre-enrichment step in TSB or BPW is recommended. Refer to suitable methodology (Pharmacopoeia or ISO Standard) for the incubation time and temperatures and confirmation subcultures and tests.

Physical/chemical control

Microbiological control

Inoculate: 50-100 CFU (productivity) according to harmonized Eur. Pharmacopoeia.

Inoculate separately each strain in BPW as pre-enrichment for 18 h.

Subculture 1:100 (V/V) after 24 h of incubation in pre-enrichment broths.

LC-5187, bottles: microbiological control

Prepare tubes - inoculate: practical range 10-100 CFU (productivity) according to harmonized Eur. Pharmacopoeia.

Aerobiosis. Incubation at 30-35 °C. Reading at 18-24 h.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiology Media. CRC Press Inc. London.
• EUROPEAN PHARMACOPOEIA 8.0 (2014) 8th ed. § 2.6.13. Microbiological examination of non-sterile products: Test for specified microorganisms. Harmonised Method. EDQM. Council of Europe. Strasbourg.
• ISO Standard 6340 (1995) Water Quality. Detection of Salmonella species.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• PETERZ, M., C. WIBERG & P. NORBERG (1989) The effect of incubation temperature and magnesium chloride concentration on growth of Salmonella in home-made and commercially available dehydrated Rappaport Vassiliadis broths. J. Appl. Bact. 66:523-528.
• USP 33 - NF 28 (2011) <62> Microbiological examination of non-sterile products: Test for specified microorganisms. Harmonised Method. USP Corp. Inc. Rockville. MD. USA.
• VASSILIADIS, P., C.H. MAVROMMATI, M. EFSTRATIOU & G. CHROMAS (1985) A note on the stability of Rappaport Vassiliadis enrichment medium. J. Appl. Bact. 59:143-145.