Rappaport-Vassiliadis Medium Research Articles

Relative Effectiveness of Selenite Cystine Broth, Tetrathionate Broth, and Rappaport-Vassiliadis Medium for Recovery of Salmonella spp. from Raw Flesh, Highly Contaminated Foods, and Poultry Feed: Collaborative Study

A collaborative study was performed in 18 laboratories to validate use of Rappaport-Vassiliadis (RV) medium in the standard culture method for recovery of Salmonella spp. from raw, highly contaminated foods and poultry feed. RV medium made from its individual ingredients and incubated at 42 degrees C was compared with selenite cystine (SC) broth incubated at 35 degrees C and tetrathionate (TT) broth incubated at 35 degrees and 43 degrees C for effectiveness in recovery of Salmonella spp. Four artificially contaminated foods (oysters, frog legs, mushrooms, and shrimp) and poultry feed and one naturally contaminated food (chicken) were analyzed. The artificially contaminated foods were inoculated with single serovars of Salmonella at target levels of 0.04 colony-forming units (CFU)/g for the low level and 0.4 CFU/g for the high level. For analysis of 1125 test portions, RV medium (42 degrees C) recovered Salmonella from 409 test portions; TT (43 degrees C), from 368 test portions; TT (35 degrees C), from 310 test portions; and SC (35 degrees C), from 334 test portions. Overall, RV medium was comparable with or better than other selective enrichments for recovery of Salmonella from the foods in this study, except mushrooms. From mushrooms, SC broth (35 degrees C) recovered more positive test portions than did RV medium (42 degrees C) and TT broth (43 degrees C). The method for detection of Salmonella in raw, highly contaminated foods and poultry feed using RV medium has been adopted by AOAC INTERNATIONAL. AOAC Official Method 967.25, Salmonella in Foods, Preparation of Culture Media and Reagents, has been revised to include RV medium, and the applicability of AOAC Official Method 967.26, Salmonella in Foods, Detection, has been restricted to processed foods.

Evaluation of immunomagnetic separation in combination with modified semi-solid Rappaport Vassiliadis medium and Rambach agar for the isolation of Salmonella

The ability of polymagnetic beads coated with Salmonella antibodies to enhance detection of Salmonella using Modified Semi-solid Rappaport Vassiliadis (MSRV) medium and Rambach agar was evaluated. Inoculation of the MSRV medium with pre-enriched buffered peptone water (BPW) resulted in the isolation of Salmonella from 60% of raw poultry samples. Capture of the Salmonella with the beads and inoculation of the beads onto MSRV medium resulted in the isolation of Salmonella from 62% of the samples. If the pre-enriched BPW was centrifuged and the pellet was resuspended in 1 ml of BPW before capture of the Salmonella with the beads and inoculation of the beads onto MSRV medium, Salmonella were isolated from 65% of the samples. Direct plating of the beads to Rambach agar after capture of Salmonella from the pre-enriched BPW, but without selective enrichment in the MSRV medium, resulted in low recovery rates of Salmonella. Plating of pure cultures of Salmonella belonging to subspecies IIIa and IIIb onto Rambach agar resulted in colonies with an atypical appearance.

Comparison between a cultural procedure using Rappaport-Vassiliadis broth and motility enrichments on modified semisolid Rappaport-Vassiliadis medium for Salmonella detection from food and feed

Motility enrichment on modified semisolid Rappaport-Vassiliadis (MSRV) medium was evaluated for a rapid Salmonella detection in naturally contaminated food- and feed samples. The results of this procedure were compared with a conventional cultural procedure using Rappaport-Vassiliadis (RV) broth as selective enrichment and modified brilliant green-, xylose lysine desoxycholate- and mannitol lysine crystal violet brilliant green agar for selective plating. The productivity of motility enrichment was 87% compared to a productivity of 99% for the cultural procedure. Statistical analysis showed that there was a significant difference at the 5% level. The present study shows that a combination of direct motility enrichment with the cultural procedure could be used as a very rapid and highly cost-efficient method for Salmonella detection.

Salmonella Detection in Dried Milk Products by Motility Enrichment on Modified Semisolid Rappaport-Vassiliadis Medium: Collaborative Study

A collaborative study involving 19 laboratories was performed to validate motility enrichment on modified semisolid Rappaport-Vassiliadis (MSRV) medium for rapid detection of motile Salmonella in dried milk products. The MSRV method was compared with the AOAC culture method for detection of Salmonella in nonfat milk powder, whole milk powder, whey powder, casein powder, and butter milk powder. Samples were artificially inoculated with Salmonella at 2 levels of contamination. Uninoculated control samples were included for each type of product. The sensitivity rates were 100% for the MSRV method and 99.0% for the AOAC culture method, while the specificity rate was 100.0% for both methods. Only for the samples of whey powder, which were inoculated with H2S negative S. tennessee, was there a significant difference in the proportion of samples positive by MSRV and the culture procedure. The MSRV method for detection of motile Salmonella in dried milk products has been adopted first action by AOAC INTERNATIONAL.

Comparison of detection of Salmonellaby the Tecra RUnique TMSalmonellatest and the modified Rappaport Vassiliadis medium

One hundred samples consisting of chicken carcass rinses, chicken parts, dust samples from chicken barns and mechanically separated turkey meat were tested forSalmonella by pre-enrichment in modified buffered peptone water (MBPW), and then by either the Tecra ®Unique ™ Salmonella test, or by cultivation for selective migration in modified semisolid Rappaport Vassiliadis (MSRV) medium. Thirty-six per cent of the samples were presumptively positive forSalmonella with the Tecra ®test of which one was a false positive result. Fifty-nine per cent were positive by selective migration in the MSRV medium. The Tecra ®Unique ™ Salmonella test had a sensitivity of 59.3% and a specificity of 97.6% compared with the MSRV method.

Relative Effectiveness of Selective Plating Agars for Recovery of Salmonella Species from Selected High-Moisture Foods

The relative effectiveness of 6 selective plating media were compared for effectiveness in recovery of Salmonella spp. from selected high-moisture foods. Three new plating agars (EF-18, Rambach, and xylose lysine Tergitol-4) and 3 selective plating agars (bismuth sulfite, Hektoen enteric, and xylose lysine desoxycholate) recommended by AOAC INTERNATIONAL and the Bacteriological Analytical Manual (BAM) were compared. The agars were streaked from cultures selectively enriched in selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium. The high-moisture foods studied were naturally contaminated pork sausage, chicken parts, turkey parts, and frog legs and artificially contaminated shrimp, oysters, egg yolks, and lettuce. The relative effectiveness of each selective plating agar was determined by recovery of Salmonella spp. and enumeration of false-positive and false-negative reactions. Although the new selective plating agars compared favorably with the AOAC/BAM-recommended agars, they offered no advantage. Incubation of selective enrichment broths at elevated temperatures decreased the numbers of false-positive and false-negative reactions for all 6 selective plating agars.

Relative Effectiveness of Selenite Cystine Broth, Tetrathionate Broth, and Rappaport-Vassiliadis Medium for the Recovery of Salmonella from Raw Flesh and Other Highly Contaminated Foods: Precollaborative Study

The effectiveness of selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium for recovery of Salmonella spp. from 8 highly contaminated foods was determined. RV medium prepared from individual ingredients and incubated at 42 degrees and 43 degrees C was compared with 2 commercial (Difco and Oxoid) media incubated at 42 degrees C. Naturally and artificially contaminated foods were tested under 2 protocols. For Protocol 1, each food was preenriched in the appropriate medium. After incubation, serial 10 fold dilutions of the preenriched foods were inoculated into selective enrichment media and incubated at 35 degrees, 42 degrees, or 43 degrees C. Effectiveness of these conditions was evaluated by most probable number determination of Salmonella spp. recovered. Productivity of selective enrichments did not differ significantly with this protocol, except that with Oxoid RV medium the number of Salmonella cells recovered from most of the foods was significantly reduced. For Protocol 2, twenty 25 g test portions from artificially inoculated foods were examined qualitatively for Salmonella spp. The effectiveness of the broth/temperature combinations was determined by the number of positive tests under each condition. RV medium prepared from individual ingredients and TT broth incubated at 43 degrees C yielded significantly more Salmonella-positive tests from frog legs and lettuce than did SC and TT broths incubated at 35 degrees C or commercial RV medium incubated at 42 degrees C. With pork sausage and ground beef, significantly fewer Salmonella-positive tests were found with Oxoid RV medium incubated at 42 degrees C and SC incubated at 35 degrees C than from other selective enrichments.(ABSTRACT TRUNCATED AT 250 WORDS)

Enumeration of salmonellae in crocodile pond water by direct plate counts and by the mpn technique

The use of Rappaport-Vassiliadis (RV) selective enrichment medium for the enumeration of salmonellae in pond water from four crocodile farms by the most probable number (MPN) method was compared to direct plating on xylose Lysine Desoxycholate agar. Direct plate counts (DPC) were consistently higher than MPN estimations (means of DPC 4.6 log 10 viable bacteria (v.b.) per 100 ml, as opposed to MPN 2.6 log 10 v.b. per 100 ml). It is suggested that the difference may be caused by differing viability of Salmonella subspecies or serovars in the RV medium, as four laboratory strains of S. enterica subspp. enterica and salamae were enumerated equally well by both DPC and MPN determinations, whereas the MPN failed to detect two strains of S. enterica subsp. diarizona at a concentration level of approximately 2 log 10 v.b. per 100 ml. It is concluded that the MPN determination method may not be sufficient and reliable when applied to samples containing Salmonella subspecies and serovars of reptilian origin.

Evaluation of motility enrichment on Modified Semisolid Rappaport-Vassiliadis medium (MSRV) and automated conductance in combination with Rambach agar for Salmonella detection in environmental samples of a milk powder factory

The efficacy of motility enrichment on modified semisolid Rappaport-Vassiliadis medium (MSRV) and an automated conductance method for the detection of Salmonella in environmental samples was evaluated. Two hundred and ten environmental samples from unrestricted areas of a milk powder factory, 49 of which were artificially contaminated with Salmonella infantis, were examined. From exactly 100 samples Salmonella could be isolated. With the conventional (ISO-DIS 6579) method a 100% score was obtained, whereas the MSRV method gave 82 positive results. The conductance method permitted the detection of Salmonella in only 66 samples. The use of Rambach agar improved isolation efficiency of Salmonella from enrichment broths.

Salmonella Detection in Cocoa and Chocolate by Motility Enrichment on Modified Semi-Solid Rappaport-Vassiliadis Medium: Collaborative Study

A collaborative study was performed in 13 laboratories (including the authors' laboratories) to validate motility enrichment on modified semi-solid Rappaport-Vassiliadis (MSRV) medium for rapid detection of motile Salmonella in cocoa powder and chocolate. The MSRV method was compared with the AOAC standard culture method for detection of Salmonella in cocoa powder, milk chocolate, sweet chocolate, and dark chocolate. Chocolate samples were artificially inoculated with Salmonella at 2 levels of contamination. Cocoa powder was inoculated at 2 levels with naturally contaminated dust from cocoa beans. Uninoculated control samples were also included for each type of product. The sensitivity rate was 98.1% for the MSRV method and 94.9% for the AOAC culture method. The specificity rate was 100.0% for both methods. There was no significant difference in the proportion of samples positive by MSRV and the AOAC culture methods for any of the food types. The MSRV method has been adopted first action by AOAC INTERNATIONAL.

Collaborative study of the international office of cocoa, chocolate and sugar confectionery on Salmonella detection from cocoa and chocolate processing environmental samples

A comparative collaborative study was performed in 13 laboratories to evaluate the use of motility enrichment on Modified Semisolid Rappaport-Vassiliadis medium for rapid Salmonella detection from food-processing environmental samples. Artificially contaminated chocolate scrapings and naturally contaminated cocoa bean dust samples were used in the study. Pre-enrichment was performed in buffered peptone water with added casein and malachite green oxalate. Motility enrichment was compared with a conventional cultural procedure using Rappaport-Vassiliadis broth and selenite cystine broth as selective enrichment. The productivity of motility enrichment was 93.5% compared to a productivity of the cultural procedure of 92%. Statistical analysis showed that there was no significant difference between the two procedures. Modified Semisolid Rappaport-Vassiliadis medium is a sensitive and simple diagnostic tool for the microbiological safety evaluation of food-processing environments.

Recovery of Salmonella from High-Moisture Foods by Abbreviated Selective Enrichment

Five high-moisture foods were used to evaluate both the effect of a 6 h, rather than the standard 24 h, selective enrichment incubation period, and the efficiency of Rappaport-Vassiliadis (RV) medium relative to the use of selenite cystine (SC) and tetrathionate (TT) broths for the recovery of Salmonella. Cheese and lettuce were artificially inoculated with a pool of two serotypes, whereas the other foods were naturally contaminated. Significantly higher numbers of Salmonella-positive test portions were obtained at 24 h with the following food and media combinations: cheese (TT and RV media), lettuce (SC, TT, and RV media), raw chicken (RV medium), and pork sausage (SC, TT, and RV media). There were no significant differences between the two incubation periods in recovery of Salmonella from turkey. Overall, more Salmonella-positive test portions were obtained from samples of lettuce, chicken, and pork sausage selectively enriched in RV medium than in SC or TT broths. The results of this study indicate that not all high-moisture foods can be selectively enriched for 6 h without a significant loss in recovery of Salmonella. RV medium was superior to SC and TT broths for recovery of Salmonella from some meats and was at least as productive in its recovery from the other high-moisture foods tested.

Isolation of Salmonella from Fluid Milk with the Use of Rappaport-Vassiliadis Medium

The performances of Rappaport-Vassiliadis (RV) medium and tetrathionate brilliant green broth (TT) for the detection of salmonellae in pasteurized fluid whole milk, artificially contaminated, were compared. The RV medium was found to be more sensitive and more selective than the TT medium, as far as Salmonella typhimurium was concerned. From the 100 samples contaminated with S. typhimurium only, 97 were found positive witb RV medium, while 86 were found positive with TT medium (P<0.001). From the 100 samples contaminated with S. typhimurium plus gram-negative competing organisms, 79 were found positive with RV medium, while only 41 with TT medium (P<10−6). The mean values of growth of competing organisms in RV medium after 24 and 48 h of incubation at 43°C were 0.81 and 0.92, respectively, while the corresponding values in TT medium were 1.81 and 1.87.

Collaborative study on the use of motility enrichment on modified semisolid Rappaport-Vassiliadis medium for the detection of Salmonella from foods

A collaborative study was performed in 15 laboratories to evaluate the use of motility enrichment on modified semisolid Rappaport-Vassiliadis (MSRV) medium for rapid Salmonella detection in a variety of food products. The results of this procedure were compared with those obtained by the cultural procedure using Rappaport-Vassiliadis (RV) broth as selective enrichment and modified brilliant green agar for selective plating. The tests were performed with Salmonella reference samples (SRS) as well as with naturally contaminated food products. When SRS were used without added food the productivity of both MSRV and RV was 96%. When SRS were combined with reference samples containing competitive bacteria the productivity was 98% for MSRV and 95% for RV. In the tests with food samples the productivity of MSRV was 92% with SRS added to food and 96% with naturally contaminated samples, while the productivity of RV was 88% and 90%, respectively. Statistical analysis showed that there was no significant difference between the procedures.

Recovery of Salmonella from Frozen Shrimp: Evaluation of Short-Term Selective Enrichment, Selective Media, Postenrichment, and a Rapid Immunodiffusion Method

Frozen shrimp was used as a high-moisture food matrix to evaluate the effect of the following conditions and media on the recovery of Salmonella: comparative efficiency of 6 and 24 h selective enrichment incubation periods; efficiency of Rappaport-Vassiliadis (RV) medium relative to selenite cystine (SC) and tetrathionate (TT) selective enrichment broths; need for postenrichment; and reliability of the immunodiffusion method (Salmonella 1–2 TEST) as a rapid screening procedure. From a total of 244 Salmonella-positive, samples, recoveries at 6 h for selective enrichments SC, TT, RV(1) receiving 1 ml of inoculum, and RV(2) receiving 0.1 ml of inoculum, were 147, 149, 200, and 169, respectively; at 24 h, recoveries were 148, 142, 193, and 205, respectively. As a selective enrichment, RV medium was generally more productive than either SC or TT broths. Postenrichment reduced method sensitivity. Test kit reactions were read independently by three analysts to evaluate the immunodiffusion method. Examination of 200 shrimp samples by standard cultural and 1–2 TEST methods detected 52.5–57% and 56.5–60.5% positive samples, respectively.

Estimation of most probable number of Salmonella in retail samples of minced pork

The number of salmonellae in 174 samples of minced pork was determined by the Most Probable Number (MPN) method in two separate laboratories. The material examined was taken from a collection of samples which were Salmonella positive in an earlier study. The MPN estimation was carried out using portions of original samples which had been divided (2 × 100 g) before the initial examination and which had been deep frozen and stored for 1 to 14 weeks at −18°C until re-examination. Of the 174 samples initially positive for Salmonella, 131 (75.3%) were positive on re-examination using pre-enrichment in buffered peptone water (BPW) and selective enrichment in Rappaport-Vassiliadis medium (RV) and in tetrathionate medium according to Muller-Kauffmann (MK). The majority of the samples gave Salmonella counts below 1000/100 g (96.7% at lab. A and 97.3% at lab. B). Comparison of the results from both laboratories showed good agreement in the distribution pattern of the frequencies within the MPN classes, but agreement between the same sample pair was poor (r = 0.23). RV medium proved to be superior to the MK medium.

Collaborative Study of the International Office of Cocoa, Chocolate, and Sugar Confectionery on the Use of Motility Enrichment for Salmonella Detection in Cocoa and Chocolate

A comparative collaborative study was performed in 15 laboratories to validate the use of motility enrichment on Modified Semisolid Rappaport-Vassiliadis (MSRV) medium for Salmonella detection in cocoa and chocolate products. The use of MSRV was compared with a cultural procedure using Rappaport-Vassiliadis broth and selenite cystine broth as selective enrichments. Artificially contaminated milk chocolate samples as well as Salmonella reference capsules added to Salmonella-free cocoa and milk chocolate were used as test samples. Motility enrichment produced 347 positive test results compared to 320 for the cultural procedure. For samples containing a lactose positive Salmonella strain, motility enrichment was far more productive than the cultural procedure, while for the other samples no significant statistical difference in the productivity of both procedures was observed at the 5% level.

An improved method for detection of Salmonella in foodstuffs using anti- Salmonella antibody

A rapid and reliable procedure was developed for detection of Salmonella spp. in foodstuffs. After pre-enrichment in nutrient broth for 6 h and an 18-h selective enrichment in Rappaport-Vassiliadis medium, cells were collected on a membrane filter. The presence of Salmonella cells was detected with enzyme-labeled polyclonal antibody to Salmonella common structural antigen (CSA-1). The detection method did not appear to be influenced by the presence of Escherichia coli, a commonly found interfering organisms in contaminated foodstuffs.

Abbreviated Selective Enrichment, Post Enrichment and a Rapid Immunodiffusion Method for Recovery of Salmonella from Instant Nonfat Dry Milk

Recovery of Salmonella from instant nonfat dry milk was studied by determining the efficiency of 6 and 24 h selective enrichment incubation periods, the productivity of Rappaport-Vassiliadis (RV) medium relative to selenite cystine and tetrathionate broths, and the potential enhancement of post enrichment with GN and M broths. Efficiency of the immunodiffusion method recently approved by the Association of Official Analytical Chemists (AOAC) for recovering Salmonella was also determined. Samples of artificially contaminated instant nonfat dry milk were selectively enriched for 6 h without compromising recovery. There was no advantage in using RV medium, and post enrichment did not enhance recovery. For the evaluation of the immunodiffusion method, three analysts independently read the test kit reactions. Of the 220 samples examined, 42.7–45.5% were positive by the immunodiffusion and the AOAC/Bacteriological Analytical Manual (BAM) culture methods, 42.3–43.2% were negative by both methods, 11.4–14.1% were negative by the immunodiffusion test and positive by the AOAC/BAM culture method, and 0–0.9% were negative by the AOAC/BAM method and positive by the immunodiffusion method.

An Assessment of Rappaport-Vassiliadis Medium Based on Growth Kinetics

The growth of eleven Salmonella serotypes cultured in modified Rappaport-Vassiliadis medium was compared with the growth in selenite broth and in nutrient broth. The growth of other enterobacteria in the three media was also studied. The mean generation times of all the serotypes were calculated by fitting the experimental data to the exponential growth law. The results showed that Rappaport-Vassiliadis medium sustained the growth of nine Salmonella serotypes and proved more successful in limiting the growth of other enterobacteria than did the selenite or nutrient broths.