RAPPAPORT-VASSILIADIS (RV) ENRICHMENT BROTH

Code: CM0669

A selective enrichment broth for the isolation of salmonellae.

THIS MEDIUM IS VERY HYGROSCOPIC AND MUST BE PROTECTED FROM MOISTURE.
The quantities given for the formula as classically described made 1110ml of medium. They have been published this way in the Oxoid literature to coincide with the scientific literature.

The directions for reconstituting Oxoid Rappaport-Vassiliadis (RV) Enrichment Broth follow usual Oxoid practice and specify the weight needed for 1 litre of medium.

Directions
Add 30g (the equivalent weight of dehydrated medium per litre) to 1 litre of distilled water. Heat gently until dissolved completely. Dispense 10ml volumes into screw-capped bottles or tubes and sterilise by autoclaving at 115°C for 15 minutes.

Description
Rappaport-Vassiliadis (RV) Enrichment Broth is based on the formulation described by van Schothorst and Renaud¹ and is recommended as the selective enrichment medium when isolating Salmonella species from food and environmental specimens. It can also be used to isolate Salmonella from human faeces without pre-enrichment but the inoculum must be small. The original formulation described by Rappaport et al² was specifically developed to exploit the four characteristics of Salmonella species when compared with other Enterobacteriaceae.
1. The ability to survive at relatively high osmotic pressures.
2. To multiply at relatively low pH values.
3. To be relatively more resistant to malachite green.
4. To have relatively less demanding nutritional requirements.

Oxoid’s Rappaport-Vassiliadis (RV) Enrichment Broth is similar to that described by Vassiliadis et al.³ except that the peptone used is soya peptone, which has been reported to enhance the growth of salmonellae^1,11.

Rappaport Broth was found to be superior to Selenite Enrichment Broth and Tetrathionate Broth for enrichment of salmonellae with the exception of Salmonella typhi². Vassiliadis et al.³ modified Rappaport Broth by lowering the concentration of malachite green and raising the incubation temperature to 43°C. This modified Rappaport Enrichment Broth is RV or Rappaport-Vassiliadis Medium and has been found to be superior to other salmonella selective enrichment media, especially when small inocula of pre-enrichment broth are used^4,5,6,7,8.

In an evaluation of different enrichment media for isolation of Salmonella from seawater, Rappaport-Vassiliadis (RV) Broth and the same broth supplemented with novobiocin were the best for detection and enumeration of salmonellae in samples with low and moderate pollution levels⁹.
In another study¹⁰, Rappaport-Vassiliadis (RV) Broth was found to be superior to tetrathionate-brilliant green broth for the detection of salmonellae in artificially contaminated fluid whole milk.

It is important that the inoculum size used for enrichment culture in RV Broth is sufficiently small not to interfere with its selectivity. Inoculum/broth ratios 1:100 to 1:2000 have been suggested¹².

Technique
Food and Environmental Specimens
1. Prepare Buffered Peptone Water CM0509 or Buffered Peptone Water (ISO) CM1049 as directed in containers containing 225ml of the medium.
2. Prepare Rappaport-Vassiliadis (RV) Enrichment Broth CM0669 as directed.
3. Add 25g of the test specimen to 225ml of Buffered Peptone Water and incubate at 35°C for 16-20 hours.
4. Inoculate 0.1ml of the pre-enrichment peptone water culture to 10ml of Rappaport-Vassiliadis (RV) Enrichment Broth and incubate at 42 ± 1°C for 24-48 hours.‡
5. Subculture the broth by streaking on to plates of X.L.D. Agar CM0469 plus one other agar. Incubate at 35°C for 18-24 hours.
6. Colonies showing typical Salmonella colonial morphology should be confirmed by biochemical or serological methods.

‡ The recommended incubation temperature is 43°C but this is a critical upper limit. To allow for incubator temperature fluctuation 42 ± 1°C is a preferred recommendation with 42 ± 0.1°C for water baths.
Refer to standard methods for exact techniques.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Pale green coloured, free-flowing powder
Prepared medium: Blue coloured solution

Quality control

* This organism is available as a Culti-Loop®

Precautions
RV Broth should not be used if Salmonella typhi is suspected.
Note the difference in weight between the classical formula on the label and the reduced weight per litre, using anhydrous magnesium chloride.

References
1. van Schothorst M. and Renaud A.M. (1983) J. Appl. Bact. 54. 209-215.
2. Rappaport F., Konforti N. and Navon B. (1956) J. Clin. Path. 9. 261-266.
3. Vassiliadis P., Pateraki E., Papaiconomou N., Papadakis J.A. and Trichopoulos D. (1976a) Annales de Microbiologie (Institut Pasteur) 127B. 195-200.
4. Vassiliadis P., Trichopoulos D., Kalapothaki V. and Serie C. (1981) J. Hyg. Camb. 87. 35-39.
5. Harvey R.W.S., Price T.H. and Xirouchaki E. (1979) J. Hyg. Camb. 82. 451-460.
6. Vassiliadis P. (1983) J. Appl. Bact. 54. 69-75.
7. Vassiliadis P., Kalapothaki V., Trichopoulos D., Mavromatte C. and Serie C. (1981) Appl. & Environ. Microbiol. 42. 615-618.
8. Vassiliadis P. (1983) J. Appl. Bact. 56. 69-76.
9. Moriñigo M.A., Muñoz M.A., Cornax R., Castro D. and Borrego H.J. (1990) J. Microbiol. Methods 11. 43-49.
10. Vassiliadis P., Kalapothaki V. and Trichopoulos D. (1991) J. Food Prot. 54. 421-423.
11. McGibbon L., Quail E. and Fricker C.R. (1984) Inter. J. Food Microbiol. 1. 171-177.
12. Fricker C.R. (1987) J. Appl. Bact. 63. 99-116.