Rapid Salmonella Detection in Foods by Motility Enrichment on a Modified Semi-Solid Rappaport-Vassiliadis Medium

Abstract

Modification of Rappaport-Vassiliadis enrichment broth into a semisolid motility medium (MSRV) provided a sensitive means for detecting Salmonella in contaminated foods. The type of peptone, the concentration of magnesium chloride, the presence of novobiocin and the temperature of incubation were determinants in medium performance. The analytical procedure consists of preenrichment for 20 h, followed by motility enrichment on MSRV for 24 h and, if there is migration, serological tests with the motile culture. The test result is obtained within 48 h from the start of preenrichment. This approach gave 39% more Salmonella-positiwe samples than enrichment in tetrathion-ate brilliant green broth with subsequent plating. Many methods are available for isolating salmonellae from food (3), including: cultural methods (6), the fluorescent antibody technique (6), enrichment serology (75), enzyme immunoassay (72) and molecular DNA-DNA hybridization (5). An important step in all of these methods is preenrichment of samples in a nonselective broth to allow resuscitation and growth of the often few and injured Salmonella cells (2). Subsequently, enrichment in a selective broth is done to enhance the ratio of salmonellae to the competitive flora. Different selective enrichment broths are used for cultural procedures (2), including: tetrathionate broth, sele-nite broth and Rappaport broth based on magnesium chloride and malachite green oxalate. Recent studies have shown that Rappaport-Vassiliadis (RV) medium (20), a modification of the Rappaport broth, is superior to other enrichment media (7,77,79). Several investigators (1,9,16) reported successful isolation of salmonellae from feces on the basis of motility in semisolid enrichment media in U-rubes. Fecal suspen-sions were inoculated on one side of the tube. During incubation, motile bacteria migrated through the medium and were selected from the other side for further testing. After migration, the salmonellae were isolated in a pure or nearly pure culture state. The procedure was simple, effective and even superior to the standard isolation method, and capable of screening rapidly a large number of fecal specimens. However, the method is not useful for isolating Salmonella typhi and nonmotile serotypes. Motility enrichment is not widely used in Salmonella methodology for food products (10,11). Goossens et al. (8) recently reported a procedure involving motility enrichment on a semisolid Rappaport medium in petri dishes for detecting Salmonella in fecal specimens. This procedure yielded a 22.3% increase in sensitivity compared to standard cultural methods. The present study investigates the use of a semisolid RV medium in petri dishes for Salmonella isolation from food products. Several modifications were tested to identify a medium on which Salmonella strains migrate into a pure culture state so that rapid detection is possible. MATERIALS AND METHODS Preparation of semisolid media Rappaport-Vassiliadis (RV) broth. This broth (20) was prepared by dissolving 5 g of tryptone (Oxoid L 42), 8 g of NaCl and 1.6 g of KH 2 P0 4 in 1 L of water (solution A) and adding 100 ml of solution B (400 g of MgCl 2 «6H 2 0 in 1 L of water) and 10 ml of solution C (4 g of malachite green oxalate in 1 L of water). The pH of RV broth was 5.2. 60% Semisolid RV (60% SRV). The semisolid medium was prepared by mixing six volumes of RV broth with four volumes of 0.8% agar solution in distilled water. Both solutions were autoclaved (115°C, 15 min) and cooled to 50°C. After mixing, the medium was poured into petri dishes. When agar is added directly to the RV broth, the medium does not form a stable gel after sterilizing and pouring into petri dishes because the gel strength of agar is diminished by heating at pH lower than 6.0. Semisolid RV (SRV). The full concentration of RV broth was used in this medium. The components of solution A were dissolved in 600 ml of water to which was added 100 ml of solution B and 10 ml of solution C. This solution was sterilized and, after cooling to 50°C, was mixed with 400 ml of sterile 0.8% agar solution and poured into petri dishes. Semisolid RV medium with novobiocin (SRVN). This medium was composed of filter-sterilized novobiocin solution (20 mg/