Rappaport Vassiliadis Research Articles

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)

A Comparison of Two Methods for Isolation of Salmonella from Poultry Litter Samples

Two methods were compared to determine their ability and efficiency in detecting Salmonella in poultry litter samples. Method 1 consisted of pre-enrichment in buffered peptone water (BPW), selective motility enrichment in Modified Semisolid Rappaport Vassiliadis (MSRV) agar, and plating onto MacConkey (MC) agar. Method 2 employed tetrathionate brilliant green (TBG) broth and plating on brilliant green agar with novobiocin (BGAN) and on xylose lysine tergitol 4 (XLT4) agar. Method 1 resulted in a significantly higher isolation rate, was cheaper, and was less labor intensive.

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.

Rapid Detection of Salmonellae by Means of a New Impedance-Splitting Method

An Impedance-Splitting method is proposed for the rapid detection of salmonellae in foods. The measuring System, BacTrac™ 4100, permits the registration of changes, caused by bacterial metabolism, not only of the impedance of the culture medium but also of changes in the ionic layers at the measuring electrodes, which has advantages in case of high salt concentrations. These changes are expressed as percentage decreases of the initial values, M-value and E-value, respectively. Food samples were pre-enriched 14 to 16 h at 37°C in peptone water by addition of mannitol, which facilitated the detection of salmonellae on selective culture media. Following this, 0.1 mi of the preenrichment culture was transferred to 9.9 ml of Impedance-Splitting Salmonellae (ISS) medium which consisted of magnesium chloride (hydrated), malachite green oxalate, novobiocin, phosphate buffer, mannitol, peptone and yeast extract. Despite the high magnesium chloride concentration in this medium, salmonellae produced changes of the E-value up to 100%, while the changes in M-values were limited to a few percent. The impedance changes were automatically recorded during incubation in the measuring system for up to 22 h at 40°C, and the time required to exceed a threshold value of 15% (E reaction time) was evaluated. Comparative testing of the ISS method with standard cultural analysis of 250 unknown food samples showed high sensitivity and selectivity in detecting salmonellae. From all of the 122 Salmonella-positive samples, the largest number (119) was obtained by the ISS method, as compared to that obtained by conventional testing with the selenite-cystine (106), Rappaport Vassiliadis soya (95), Rappaport Vassiliadis (92) and tetrathionate brilliant green medium (64). Six samples were false positive by Enterobacter cloaceae. One strain each of Salmonella enteritidis PT8 and Salmonella panama were not recorded. The ISS method is very suitable as a screening test, all the more since a negative investigation result will be obtained within 38 h. In view of the practicability, this method is superior to the enzyme-immunological and molecular-biological procedures.

Evaluation of the toxicity of Salmonella select media for shortening the enrichment period

Five Salmonella serotypes recovering from heat injury exhibited different kinetics of resuscitation and growth. Exponential growth was reached before full resuscitation. Fully resusciated cells and uninjured cells at low cell densities exhibited sensitivity when transferred from non-selective media to selective media, Rappaport-Vassiliadis (RV) and tetrathionate-brillant green (TBG). The miminum munbge of cells required to survive in RV and TBG was determined and ranged from 10 2 to 10 5 CFU depending on the serotype. Salmonella grown in RV produced cell populations which exhibited transferred to fresh RV even at low cell densities. Thus, toxic selective media enriched a resistant population. Selenite cysteine exhibited to toxicity to resuscitated or uninjured Salmonella. The significance of these findings was discussed for the purpose of shortening the Salmonella enrichment period.

Comparison of two enrichment media and three selective media for isolation of salmonellae from fresh chicken carcass rinse fluids and sewer swabs

Slamonella was detected in 214 (45%) of 477 chickens examined. For Salmonella-positive samples of fresh chicken carcass rinse water a sensitivity of 96% and 29% respectively was demonstrated for Rappaport-Vassiliadis (RV) and Selenite broth. A similar superiority of RV was found for the sewer swab samples. Among the three selective agar media investigated a limited sensitivity (below 50%) was found making it imperative to use at least two media, one of which should be deoxycholate citrate agar.

Detection of Salmonella in animal protein by Rappaport-Vassiliadis broth using indirect impediometry

Indirect impedance methodology for the detection of Salmonella was investigated using a rapid automated bacterial impedance technique (RABIT) system. Four commercially available Rappaport-Vassiliadis (RV) enrichment broths were evaluated for their sensitivity and selectivity in detecting Salmonella using this technique. The RV from Lab M and Oxoid (new) gave the shortest detection times and showed good correlation between Salmonella numbers and detection times. Using lab M medium, the indirect impedance technique could distinguish between Salmonella spp. and the closely related genera, Proteus and Citrobacter. The impedance technique showed recoveries of Salmonella from processed animal protein and raw meats equivalent to, or better than, those obtained with RV used in a convensional Salmonella isolation procedure.

Use of Reference Materials (Spray-Dried Milk Artificially Contaminated with Salmonella typhimurium) to Validate Detection Methods for Salmonella

Reference materials consisting of 0.28g of spray-dried milk artificially contaminated with Salmonella typhimurium and contained in gelatin capsules (mean contamination level 4.2 salmonellae per capsule) were used to validate detection of Salmonella species in food. For this, 10 samples of 25 g of mayonnaise, milk powder, minced meat, or poultry meat were spiked with reference materials without repair and with reference materials after repair, respectively. It was found that the physiological condition of Salmonella present in the reference material had no influence on the recovery rate of the organism. The recovery rates of Salmonella were tested by applying various methods [ISO-6579 method, SALMONELLA-TEK™ (Organon Teknika), the modified semisolid Rappaport Vassiliadis agar (MSRV), and the Salmonella rapid test (Oxoid)]. The recovery rates found (averaged on the types of foods tested) were 67% for the Salmonella rapid test, 83% for the MSRV, 86% for the ISO-6579, and 91% for the SALMONELLA-TEK. The use of reference materials is recommended to validate detection of Salmonella species.

Growth of Salmonella and competing flora in five commercial Rappaport-Vassiliadis (RV)-media

Growth of 18 Salmonella strains belonging to 11 serotypes and the bacterial flora of deep-frozen broiler carcasses was examined in five commercial Rappaport-Vassiliadis (RV) media. Growth was measured by an automated turbidometer (Bioscreen R). Significant differences in performance between the media investigated were observed. The unequal performances were ascribed to differences in the concentration of magnesium chloride (MgCl 2) and the type of peptone used.

Techniques for the isolation of salmonellas from eggs.

1. When the contents of 4 or more naturally-contaminated intact eggs were combined, the isolation rate of Salmonella enteritidis was improved by extending incubation at 37 degrees C from 24 to 48 h before sub-culture. 2. The isolation rate of salmonellas from raw homogenised whole egg was significantly increased by the inclusion of Novobiocin and Cefsulodin in the primary culture media. 3. Rappaport Vassiliadis broth was found to be superior to Selenite as a selective enrichment medium.

Comparison between semisolid Rappaport and modified semisolid Rappaport-Vassiliadis media for the isolation of Salmonella spp. from foods and feeds

Two semisolid media poured in petri dishes, semisolid Rappaport and modified semisolid Rappaport Vassiliadis were compared for the recovery of Salmonella spp. from 154 high- and low-moisture foods and feeds. Semisolid media were inoculated after pre-enrichment in buffered peptone water and after enrichment in Müller-Kauffmann tetrathionate broth and selenite cystine broth. Also a conventional procedure was used, where the enrichment broths were streaked on Brilliant Green and bismuth sulphite agars. Of 62 samples found positive for Salmonella by one or more methods, semisolid Rappaport detected all of them and 60 when inoculated directly from the pre-enrichment broth. Modified semisolid Rappaport-Vassiliadis gave only 20 positive samples. The difference in productivity between semisolid Rappaport and the other media poured in plates (modified semisolid Rappaport-Vassiliadis, Brilliant Green and bismuth sulphite agar) was statistically significant ( P < 0.001). The incubation temperature recomended for modified semisolid Rappaport-Vassiliadis (42 °C) was found to be too high. By comparison to the other media, semisolid Rappaport gave a 500% increase in the detection of the enteritidis serotype of Salmonella.

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.

Comparison of the TECRA Salmonella Immunoassay with the conventional culture method

The TECRA Salmonella Enzyme Immunoassay was compared with a conventional culture method for the detection of salmonella in naturally contaminated foods and animal feeds. Rappaport Vassiliadis enrichment prior to ELISA and photometric reading of the results gave 95% agreement with the conventional method. Presumptive positive results can be reported within 48 h.

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.

Rapid identification of Salmonella from poultry meat products by using ‘Mucap test’

A study was made in order to improve a new Salmonella identification test (Mucap Test) in which umbelliferone is released, giving a blue fluorescent light under a Wood lamp, after contact with Salmonella colonies. The study concerned 354 colonies, previously isolated from 55 poultry meat samples. Two enrichment media [Tetrathionate Bile Broth (TBB) and Rappaport Vassiliadis (RV)] and two isolation media [Brilliant Green Agar (BGA) and Desoxycholate Agar (DA)] were used, and the results of the test obtained respectively with each association were compared. The sensitivity was consistently good, but the specificity of the test was generally poor. The best association seemed to be RV/DA which gave 85% specificity, against 39% for TBB/BGA, 58% for TBB/DA, and 77% for RV/BGA. The predominant genera responsible for false-positive results were Pseudomonas and Proteus Providencia.

Salmonella isolation using RVS broth and MLCB agar

Experiments were carried out to determine sources of variation which might influence the reliability of Rappaport Vassiliadis (RV) broth and Mannitol lysine crystal violet brilliant green agar (MLCB). The amount of MgCl 2.6H 2O in the RV broth formula is critical and should be approximately 29 gl −1 of the ready to use medium. When soya peptone is used instead of tryptone, the medium should be buffered when stored for longer periods of time before use. This buffered soya peptone medium is called RVS broth in order to prevent confusion with the ‘classical’ RV broth. MLCB agar proved in our hands to be an excellent medium for the isolation of H 2S positive Salmonella when used after enrichment in RVS broth. Care should be taken however that when ‘home-made’ the agar should contain sufficient Mg.

Evaluation of reference samples for the detection of Salmonella

Reference samples consisting of spray-dried milk artificially contaminated with Salmonella typhimurium II 505 contained in a gelatin capsule have been developed to evaluate the standard method for the detection of Salmonellae in foods and feeding stuffs. The present study was carried out to evaluate these reference samples. Salmonella isolations from reference samples in the absence of food were satisfactory in 4 out of 6 laboratories when Müller-Kauffmann tetrathionate broth prepared according to ISO-6579 (TBB-ISO) was used and in all laboratories when the secondary enrichment medium was the magnesium chloride-malachite green broth of Rappaport-Vassiliadis (RV). The proportion of Salmonella isolations from reference samples decreased from 80% to 47% with TBB-ISO and from 95% to 63% with RV in the presence of food. Most negative results were obtained with foods such as minced pork and minced beef, which had high aerobic and Enterobacteriaceae plate counts. It is concluded that the reference samples can be used to test the performance of media and the reproducibility of methods when it is used without the addition of food. The results obtained for Salmonella isolations in the presence of food questioned the role of numbers and types of competitive micro-organisms. In particular these latter results indicated that more research is needed to gain a better understanding of the Salmonella isolation procedure.