Tetrathionate Brilliant Green Broth Research Articles

Validation of rapid detection methods for Salmonella enterica in green chile.

The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%-25% tests positive and at a level+1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P<.05 were found between the procedures using McNemar's χ2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile.

Determination of best enrichment media for growth of Salmonella injured from cold temperature during process and storage

This purpose of this study was to determine the best enrichment medium for rejuvenating and recovering Salmonella placed in cold temperature prior to the employment of the gold biosensor combined with a light microscopic imaging system. A mixture of nalidixic-resistant Salmonella Typhimurium and Enteritidis were inoculated onto chicken (1,000 CFU/chicken). After cold injury at 4℃ for 24 hr, Salmonella on chicken was enriched for 6 hr with six non-selective media including buffered peptone water broth, lactose broth, brain heart infusion broth (BHI), universal pre-enrichment broth, nutrient broth, and tryptic soy broth, and five selective media including brilliant green broth (BG), rappaport-vassiliadis R10 broth, selenite cystine broth, selenite broth, and tetrathionate brilliant green broth (TBG) for the comparison of Salmonella growth. Various concentrations of Salmonella (10, 50, 100, 500, and 1,000 CFU/chicken) were then enriched for 6 hr in both BHI and BG media to select the best media. BHI was selected as the most effective non-selective enrichment medium, while BG was selected as the most effective selective enrichment medium. Finally, BHI medium was selected as the most efficient enrichment medium for Salmonella growth injured from cold temperature during processing or storage.

Detection of Group D Salmonellae Including Salmonella Enteritidis in Eggs by Polymyxin-Based Enzyme-Linked Immunosorbent Assay

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9–specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

Simple and rapid methods for detecting Salmonella enteritidis in raw eggs

The Centers for Disease Control and Prevention estimates there were 300,000 cases of Salmonella enteritidis (SE) in 1997. Egg products were associated with many of the cases. To address this problem, many producers implemented flock surveillance of the SE situation at their facilities. A rapid and simple method for detecting SE from poultry samples is critical for the effective implementation of such testing strategies. A lateral flow device for the detection of SE utilized in this study was manufactured by Neogen, Lansing, MI. The test panel is a presumptive qualitative test system that detects only members of Group D1 Salmonella species. A series of studies were conducted to optimize the test procedure for raw eggs with different sample preparations. A novel antigen extraction method was developed for use with the test panel kit. The detection limit of the test panel kit was increased approximately tenfold when the extraction method was used. Detection of SE was 100% in raw egg pools inoculated with 10 SE cells per ml of egg and incubated at a 1:10 ratio in buffered peptone water (BPW) or tetrathionate brilliant green broth (TBG) for 24 h at 37 °C. The developed lateral flow test kit could provide a simple, rapid, and inexpensive method for egg producers and processors to test specifically for Salmonella group D1 serovars, such as SE, in egg samples.

Evaluation of culture enrichment procedures for use with Salmonella detection immunoassay

To design efficient culture strategies for use with immunoassays to detect Salmonella in food, the growth of these organisms was investigated according to the Bacteriological Analytical Manual (BAM) and enrichment-immunoassay (EI) culture procedures. The cultures were further evaluated using a commercial enzyme-linked immunosorbent assay (ELISA) kit . The BAM procedure includes pre-enrichment in nutrient broth (NB) for 16 h followed by selective enrichment in either Rappaport–Vassiliadis (RV) or tetrathionate brilliant green (TBG) broth for 16 h. The EI procedure includes pre-enrichment in NB for 4 h, selective enrichment in RV for 16 h and post-enrichment in NB for 4 h. The effects of different incubation times for pre- and post-enrichment, and different culture media for selective enrichment (TBG and RV) and post-enrichment in NB and Brain Heart Infusion broth (BHI) on the growth of the bacteria and ELISA titers in the EI procedure were also investigated. Salmonella enteritidis and S. typhimurium inoculated at different initial concentrations between 0.1 and 35 CFU/ml grew to similar concentrations of 10 7 to 10 8 colony forming unit (CFU)/ml in pure culture and generally 2 to 4 fold lower concentrations ( P<0.05) in mixed culture using spiked chicken rinse. In the BAM procedure, the concentration of Salmonella cultured in RV was higher ( P<0.01) than that in TBG. The cultures in TBG showed positive results for ELISA, but those in RV were generally negative. In the EI procedure, the ELISA titers from cultures post-enriched in NB or BHI were higher ( P<0.01) when TBG, as compared to RV, was used for selective enrichment. Post-enrichment in BHI yielded higher numbers of Salmonella and higher ELISA titers than those in NB ( P<0.05) for post-enrichment. This study demonstrated that in both culture procedures small numbers of Salmonella could be increased to at least 10 7 CFU/ml which is detectable by most ELISAs, and that the type of the culture media used may have a significant impact on ELISA results.

Miniaturized Most Probable Number and Enrichment Serology Technique for the Enumeration of Salmonella spp. on Poultry Carcasses

The ability of two 8-tube most probable number (MPN) techniques to quantitatively recover Salmonella spp. from 26 fresh, naturally contaminated chicken skin samples was compared. Individual macerated skin samples were tested in parallel using a traditional (tMPN) and a miniaturized (mMPN) analytical procedure. In the tMPN assay, replicate aqueous portions from each macerated sample were preenriched individually in buffered peptone water, selectively enriched in Müller-Kauffmann tetrathionate brilliant green broth (MKTBG), and plated on Rambach agar. Each MKTBG was also postenriched in M Broth, and the resulting postenrichment culture screened for the presence of Salmonella cells by enrichment serology (ES). Although a similar analytical approach was used in the mMPN assay, it differed from the tMPN in the use of smaller test volumes dispensed in microplates, and on a sedimented portion of skin macerate as test material. Of the 26 Salmonella-contaminated samples examined in this study, the tMPN coupled to Rambach agar or ES identified 23 and 24 positive samples, respectively. Under homologous conditions, the mMPN detected all 26 positive Salmonella contaminated samples. The most probable numbers in 100-g skin samples analyzed by the tMPN ranged from 18/100 g to 9,530,000/100 g with a median value of 570/100 g. Levels of contamination by the mMPN procedure ranged from 90/100 g to 556,000/100 g with a median value of 1,200/100 g. Statistical analysis of experimental data underlined the equivalence of the tMPN and the mMPN procedures and nonequivalence of the Rambach plating and ES conditions. It is suggested that the microplate mMPN coupled to ES offers a reliable and more cost-effective analytical approach for the quantitative recovery of Salmonella on broiler carcasses.

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal.

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37 degrees C, Selenite Broth with Brilliant Green and Sulphapyridine at 37 degrees C and 43 degrees C, and Rappaport-Vassiliadis Broth (RV 10) at 42 degrees C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar. Enrichment in RV/42 degrees C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37 degrees C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment. A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow, and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81-92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.

Comparison of Modified Immunodiffusion and Bacteriological Analytical Manual (BAM) Methods for Detection of Salmonella in Raw Flesh and Highly Contaminated Food Types

A wide variety of naturally contaminated and inoculated raw flesh and highly contaminated food types was analyzed by a modified immunodiffusion enrichment protocol and the Bacteriological Analytical Manual (BAM) method to determine the equivalence of these methods. This modification was developed by Agriculture Canada to allow addition of a high-temperature selective enrichment step in tetrathionate brilliant green broth at 42 degrees C while maintaining a 2-day total test time. Foods representing red meat, white meat, frog, and seafoods and one type of animal meal were evaluated. A total of 320 samples was tested, resulting in false negative rates of 5.2 and 3.5%, respectively, for the modified immunodiffusion and the BAM culture methods. The overall agreement rate was 96.9%.

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.

Development of an enzyme-linked antibody hydrophobic grid membrane filter method for the detection of Salmonella in foods

An enzyme-linked antibody hydrophobic grid membrane filter (ELA-HGMF) method was developed using M105 monoclonal antibody and EF-18 as a selective agar. Food was inoculated into nutrient broth and incubated overnight followed by enrichment in tetrathionate brilliant green broth incubated at 35°C (TBG 35) for 18-24 h. One ml of the 10 -3, 10 -4 and 10 -5 dilutions of the TBG 35 were filtered through HGMFs which were placed on EF-18 selective agar. Growth on the EF-18 medium was stained with the ELA, whether it was suspected to be Salmonella or not. Growth on a replicated filter that corresponded to the stained grid cells was then picked and confirmed biochemically and serologically. One hundred and twenty-one foods, feeds, animal by-products and feces were assayed using the ELA-HGMF method with the Health Protection Branch (HPB) standard cultural method as a reference procedure. Fifty-three samples (31 naturally contaminated and 22 artificially inoculated) were positive for Salmonella by both procedures, and strains were isolated from two samples by the ELA-HGMF method alone. An attempt to shorten the time for enrichment from 24 to 6 h decreased the sensitivity of the method and this modification was not pursued any further. The main advantage of the ELA-HGMF method is the relative ease of selection of Salmonella growth from the HGMFs. This, in turn, meant that few strains with atypical reactions needed to be further tested for determination as Salmonella or non-Salmonella. The use of ELA stain to detect the organisms on HGMFs enabled detection of a positive sample in five days, whereas, with few exceptions, a negative sample was detected within four days.

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.

Evaluation of Enrichment Protocols for the 1–2Test™ for Salmonella Detection in Naturally Contaminated Foods & Feeds

The 1–2 test™ system was used to detect Salmonella in naturally contaminated foods and feeds. The results were compared with the official Canadian Wet Culture Method. In Phase I, the analytical procedure used for the 1–2 test™ was that suggested by the manufacturer. For both methods, 196 food and feed samples were analyzed. Thirty-four samples were found positive by the Wet Culture Method but only 26 were found positive by the 1–2 test™. In Phase II, the procedure for the 1–2 test™ was modified to include an enrichment stage using tetrathionate brilliant green broth. Of the 314 samples analyzed, 84 samples were found to be positive. There were 79 samples found positive by both methods. In the remaining positives, 3 tested positive only by the 1–2 test™, and 2 tested positive only by the Wet Culture Method. The majority of positive samples in Phase II were animal feeds. The selective enrichment modification to the 1–2 Test™ procedure has enhanced its reliability for Salmonella detection in foods and feeds. The manufacturer's current procedure specifies a selective enrichment step.

Dynamics of Salmonella Isolation with Modified Semi-Solid Rappaport-Vassiliadis Medium

The effect of low Salmonella cell concentrations in combination with high numbers of competitive flora after pre-enrichment on subsequent isolation with motility enrichment in modified semisolid Rappaport-Vassiliadis (MSRV) was studied. Salmonella was always detected on MSRV if the cell concentration in the preenrichment culture was at least 60 per ml, even if they were outnumbered by competitive bacteria by a factor of 107. In a total of 52 experiments, Salmonella detection on MSRV directly after preenrichment was successful in 46 tests. Combination of direct motility enrichment with additional motility enrichment after 8 h in tetrathionate brilliant green (TBG) broth resulted in 49 positive tests, compared to only 33 positive results after enrichment in TBG for 24 h with subsequent plating on selective agars.

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

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-positive samples than enrichment in tetrathionate brilliant green broth with subsequent plating.

Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis.

A major outbreak of gastroenteritis was traced to Cheddar cheese contaminated with Salmonella typhimurium. There were no significant differences in pH values of the contaminated (mean pH 5.31) and non-contaminated (mean pH 5.39) cheese. The isolation rates of Salm. typhimurium were about the same when cheese samples were homogenized in lactose broth, lactose broth containing 1% Tween 80, or in aqueous 2% sodium citrate. Salmonella typhimurium was isolated regardless of preenrichment in lactose broth, but required selective enrichment in selenite cystine or tetrathionate brilliant green broth. There were no marked differences in the isolation rates obtained with different selective enrichment media, or after incubation at 36 degrees and 43 degrees C for 24 or 48 h. Contaminated samples of cheese failed to yield Salm. typhimurium consistently despite large and multiple samplings; samples from the interior of cheese blocks yielded positive results more frequently than the samples from the exterior. The number of Salm. typhimurium in factory sealed blocks as well as in samples obtained from the homes of known cases of salmonellosis was found to range from less than 3/100 g to 9/100 g of cheese. The infective dose of Salm. typhimurium in contaminated cheese was probably no greater than 10(4) organisms, and a rapid decline in numbers of Salm. typhimurium must have occurred subsequent to the outbreak.

Comparison of Rappaport-Vassiliadis Enrichment Medium and Tetrathionate Brilliant Green Broth for Isolation of Salmonellae from Meat Products

The effectiveness of Rappaport-Vassiliadis enrichment medium (RV medium) and Difco's tetrathionate brilliant green broth (TBG) for detection of Salmonella in 553 samples of meat products was compared. All samples were preenriched for 20 h in buffered peptone water. Then 0.1 ml of the preenrichment was inoculated into 10 ml of RV medium, 1 ml was added to 9 ml of TBG broth, and 1 ml was inoculated into 10 ml of Muller-Kauffman (MK) tetrathionate broth. All enrichments were incubated at 43°C for 24 h, except for MK broth which was incubated for 48 h, and all were subcultured onto brilliant green deoxycholate agar and bismuth sulfite agar. The Rappaport-Vassiliadis medium was superior to Difco's tetrathionate brilliant green broth, being considerably more sensitive and more specific. The superiority of RV medium concerned the number of positive samples (36% and 28%, respectively), and also the number of Salmonella serotypes and strains. The RV medium inhibited the lactose- and sucrose-negative competing organisms much more than the Difco's tetrathionate broth. The performance of Difco and Muller-Kauffman tetrathionate brilliant green broths was similar. Addition of the brilliant green solution after boiling the tetrathionate broth slightly increased its efficacy. The effectiveness of brilliant green deoxycholate agar and bismuth sulfite agar was similar, whether after enrichment in RV medium or in any of the studied tetrathionate brilliant green broths.

Sampling Methods for Detection of Salmonella in Raw Chicken Carcasses

ABSTRACTDetection of Salmonella in thaw water, pericloacal skin and whole carcass rinse samples from naturally contaminated broiler chickens was compared. Of 100 birds tested, 70 were found to contain salmonellae by the three sampling techniques combined; the median level of contamination was 0.27 salmonellae/100g eviscerated carcass weight. Recovery with the whole carcass rinse technique (93%) was significantly greater than that obtained with thaw water (63%) and skin (74%) methods; performance of the latter two methods was not statistically different. Thaw water does not provide a reliable index of Salmonella contamination and therefore is not a suitable method for the nondestructive analysis of poultry carcasses. Selective enrichment in tetrathionate brilliant green broth and plating on bismuth sulfite agar identified the greatest number of contaminated carcasses under all sampling conditions.

Effect of Various Media and Incubation Conditions on Recovery of Inoculated Salmonella from Poultry Feed

Samples of commercially pelleted poultry feed (30 g) were inoculated with nalidixic acid-resistant marker strains of Salmonella heidelberg or S. montevideo at levels of 1 (low), 20 (medium), or 40 (high) cells/g of feed, then 100 ml of either a nonselective preenrichment medium (lactose broth) or a selective enrichment medium [selenite cystine (SC) or tetrathionate brilliant green (TT) broth] was added and incubated at either 37 or 43 C for 24 or 48 hr in different experiments. Four selective plating media [MacConkey with 100 ppm nalidixic acid, brilliant green (BG) sulfa, modified BG, bismuth sulfite] were then streaked. MacConkey agar with nalidixic acid served as a control to which the other three plating media were compared. After 24 hr incubation at 37 C, colonies with characteristics typical of Salmonella were selected from the plates and examined biochemically and serologically to determine if they were the marker organism.In this study, preenrichment was counterproductive, because the marker organisms were recovered much more often with direct enrichment in SC or TT broth than with preenrichment in lactose broth. The TT broth produced 135 positive recoveries, whereas only 88 positive recoveries were made with SC. No differences were observed between 37 and 43 C or 24 and 48 hr incubation of the selective enrichment media. Overall efficiency of the plating media for Salmonella recovery from feed, when compared to the control (MacConkey), was BG sulfa, 65%; modified BG, 64%; and bismuth sulfite, 40%. The most effective enrichment broth-plating medium combination was TT-BG sulfa, yielding 86% positive recoveries when compared to the control.

Update on Preenrichment and Selective Enrichment Conditions for Detection of Salmonella in Foods

The search for short and sensitive cultural methods for detection of Salmonella in foods has met with limited success. Short (3–8 h) incubation of non-selective enrichment media do not provide conditions for effective resuscitation of stressed or injured salmonellae and result in unacceptably high numbers of false-negative results. Isolation of Salmonella is not dependent on the nutritional value of preenrichment media; simple media such as lactose and nutrient broths are equally reliable as highly nutritive sterility testing media. The need for detergents in non-selective enrichment of fatty foods and use of preenrichment transfer volumes greater than 1 ml is not indicated. Although selective enrichment in tetrathionate brilliant green (TBG) broth at an elevated temperature (43 C) increases method sensitivity, use of Mueller-Kauffman TBG under similar analytical conditions may be inhibitory to Salmonella. Refrigeration of preenrichment and selective enrichment broth cultures has been used successfully to provide greater analytical flexibility by increasing the number of days on which analyses can be initiated without engendering weekend work.

Limitations of lysine-iron-cystine-neutral red broth in the presumptive identification of Salmonella

Lysine-iron-cystine-neutral red broth performed satisfactorily in the presumptive identification of Salmonella in preenriched food and animal feed samples enriched in tetrathionate-brilliant green broth. Homologous results from selenite-cystine enrichment broths yielded unacceptably high numbers of false negative reactions.