Modified Semisolid RV Medium Base

A semisolid medium for detection of motile Salmonella species from food and environmental specimens

Composition**

Final pH ( at 25°C): 5.5±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 32.95 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 115°C for 15 minutes. Cool to 45-50°C and aseptically add rehydrated contents of 1 vial of IMRV/ RV Selective Supplement (FD193). Mix well and dispense as desired (butts or Petri plates)

Note: The motility of Salmonellas can be drastically reduced when the agar surface becomes too dry. Hence the plates should be well dried before use. If visible moisture occurs on the lid of the plates or the surface of agar, it must be removed. While incubation, incubate the plates aerobically in an upright position for no longer than 24 hours at respective temperatures.

Principle And Interpretation

Salmonella species have been isolated from humans and almost all animals throughout the world. They cause many types of infections from mild, self-limiting gastroenteritis to life threatening typhoid fever (1).

Successful isolation of Salmonella is often both time-consuming and complex. As over 90% of all Salmonella species are motile, a number of isolation procedures have been based on motility. Use of Modified Semisolid RV Medium Base provides a method for isolating motile Salmonella species from clinical, food and environmental samples (2,3,4,5).

Peptone special provide the nitrogenous and carbonaceous substances and other essential growth nutrients. Yeast extract serves as a source of B complex vitamins. Phosphate gives good buffering capacity to the medium. Malachite green serves as a selective agent. Addition of novobiocin as a supplement (FD193) in the medium selectively inhibits most gram-positive organisms. Sodium chloride maintains the osmotic equilibrium of the media. Salmonella generally survives a little high osmotic pressure (due to MgCl2 in the medium), grows at slightly low pH and are resistant to malachite green compared to other bacteria. All these factors make the medium selective for the isolation of Salmonella. These medium enrich Salmonella and the semi solid nature of the medium helps to differentiate the motile Salmonella from non-motile ones.

The working of these media is based on the ability of Salmonella species to migrate in the selective medium competing other motile organisms, thus producing opaque halos of growth. The motile bacteria will show a halo or zone of growth originating from inoculation spot. These media can be used in combination with direct culture. Selenite F Broth (M052) can be used for enrichment, while for isolation of Salmonella species, XLD Agar(M031) can be used. Subculturing on XLD Agar (M031) or Mannitol Lysine Agar (M1071) results in higher recovery rates (6). These medium is not suitable for the detection of non-motile strains of Salmonella (7).

Inoculate 3 drops (0.1 ml) of pre-enrichment culture (16-20 hours old) in separate spots on the air-dried medium surface. Incubate the plates in an upright position at 42°C for upto 24 hours. The motile bacteria will show a halo or zone of growth originating from inoculation spot. Salmonella species show straw-coloured colonies. Sub-cultures can be carried out from the outside edge of the halo to confirm purity and for further biochemical and serological tests.

Quality Control

Appearance Light yellow to light blue homogeneous free flowing powder

Gelling Semisolid, comparable with 0.26% Agar gel.

Colour and Clarity of prepared medium Blue coloured clear to slightly opalescent gel forms in Petri plates or in tubes as butts.

Reaction Reaction of 3.29% w/v aqueous solution at 25°C. pH : 5.5±0.2

pH 5.30-5.70

Cultural Response M1482: Cultural characteristics observed after an incubation at different temperatures for 18-24 hours with added IMRV/RV Selective Supplement (FD193).

Storage and Shelf Life

Store below 30°C and the prepared medium at 2-8°C. Use before expiry date on the label.

Reference

1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
2. DeSmedt J. M., Bolderjik R. F., Rappold H., Lautenschleager D., 1996, J. Food. Prot., 49: 510-514.
3. DeSmedt J. M., Bolderjik R. F., 1987, J. Food Prot.; 50: 658-661.
4. Chau P. Y., Huang C. T., 1974, J. Clin. Pathol.; 27: 405 - 407.
5. Goossens H., Wauters G., De Boeck M., Janssens Butzler J. P., 1984, J. Clin. Microbiol., 19 : 940-941.
6. Aspinall S. T., Hindle M. A. and Hutchinson D. N., 1992, Europ. J. Clin. Microbiol. Inf. Dis., 11:936.
7. Holbrook R., Anderson J. M., Baird-Parker A. C., Dodds L. M., Sawhney D., Struchbury S. H. and Swaine D., 1989, Lett. Appl. Microbiol., 8:139.