Selenite Cystine Research Articles

Millipore Filtration and Use of RV Medium for Isolation of Salmonella from Preenrichment Broths

A filtration procedure for isolation of Salmonella from preen-richment broth is described. Five ml of broth are passed through a Millipore AP15 pre-filter above a .45μm Millipore filter. The filter is transferred to 25 ml of Rappaport-Vassiliadis broth and incubated at 42°C. Of 76 naturally and 55 artificially contaminated meat and poultry samples, 102 were positive for Salmonella. Of these, 99 were isolated using the filtration technique, 93% of which were obtained following a 6-h incubation period. Isolations from tetrathionate brilliant green and selenite cystine broth were 93 and 84, respectively.

Comparison of Salmonella Bio-EnzaBead™ Immunoassay Method and Conventional Culture Procedure for Detection of Salmonella in Crustaceans

The sensitivity of the Bio-EnzaBead™ enzyme immunoassay (EIA) for recovery of Salmonella in crustaceans was compared to a standard cultural method. Presumptive positive samples by the EIA procedure were confirmed culturally by plating their respective selective enrichments and post-enrichment M-broth cultures on bismuth sulfite, xylose desoxycholate, and Hektoen enteric agars. Use of tetrathionate, selenite cystine, and M-broth cultures was necessary for reliable confirmation of positive EIA reactions. Of 287 samples tested, 129 were positive by culture, while 130 were EIA-positive and were confirmed from the M-broth and/or the selective enrichments. When M-broth alone was used for confirmation of positive EIA readings, only 112 samples were shown to be positive, while 127 were confirmed from tetrathionate and selenite cystine. The importance of using all three media to confirm positive EIA readings was clearly demonstrated. There was a large number (76) of positive EIA assays which could not be confirmed culturally. These uncon-firmed positives were probably due to a cross-reacting organism present in the lot of material tested.

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.

Rapid Detection of Salmonellae in Foods Using Immunoassay Systems

A standard cultural method, radioimmunometric (RIMA) and enzyme immunometric (EIMA) assays were compared for detection of salmonellae in 235 food samples. The immunoassays used titanous hydroxide as the solid-phase, commercial Spicer-Edwards salmonella polyvalent H antisera (SEA) or pooled antisera produced against 10 salmonella flagellins (PFA). Nineteen food samples were positive for Salmonella by the standard cultural method. These as well as one additional sample were also positive for Salmonella by RIMA and EIMA. No false-negative results were obtained from the immunoassays using PFA, whereas two false-negative results were observed when SEA was used. The incidence of false-positive results when SEA and PFA were used were, respectively, 3.0 and 0.9% with RIMA and 2.6 and 0.9% with EIMA. The immunoassays were also able to detect 77 Salmonella serotypes when grown alone or in association with other species of Enterobacteriaceae, in mannitol selenite cystine broth. Both immunoassays performed reliably on enrichment cultures stored under refrigeration for up to 9 d. Also, of 6 non-motile salmonellae, 5 were detectable by the immunoassays. The immunoassays were simple, rapid and cost-efficient.

Salmonella typhimurium Phage-Type 10 from Cheddar Cheese Implicated in a Major Canadian Foodborne Outbreak

Levels of Salmonella typhimurium phage-type 10 in Cheddar cheese implicated in a major Canadian foodborne outbreak ranged from 0.36 to 9.3 salmonellae/100 g. Such a low level contamination likely accounted for the uneven distribution of the organism among subsamples of individual lots. Coliform and Escherichia coli counts were within acceptable limits, whereas three of the 11 lots tested contained ≥105Staphylococcus aureus per gram but no staphylococcal enterotoxins. Campylobacter and Yersiniaspp. were not detected in any of the 12 lots examined. Ability of S. typhimurium to survive up to 8 months in Cheddar cheese stored at refrigerator temperature (5°C) underlines the inadequacy of current regulations requiring a 60-d storage of cheese manufactured from heat-treated (unpasteurized) milk before sale. Results underlined the greater sensitivity of selective enrichment in tetrathionate brilliant green (43°C) than in selenite cystine (35°C) for detection of Salmonella in cheese.

Rapid and automated detection of salmonella by electrical measurements.

A rapid method for determining the presence of salmonella in food is described. It consists of pre-enrichment in buffered peptone water modified by the addition of dulcitol and trimethylamine oxide, followed by selective enrichment in a selenite-cystine broth with similar modifications. Changes in the conductance of the selective enrichment broth are monitored continuously using a suitable impediometric instrument. Most of the Salmonella spp. tested gave a fast (approximately 100 microS/h) and large (greater than 600 microS) change in conductance, other enteric bacteria much less or no change. The assay is usually complete within 24 h. Samples of foodstuffs, naturally and artificially contaminated with Salmonella spp., were all correctly classified. Some strains of Citrobacter freundii produced a false positive conductance response, and they could not be selectively eliminated using antibiotics or cyanide. The conductance method is simple and easy to use, gives rapid results and involves less media and subculturing than is required for traditional methods.

Improved Enrichment for Recovery of Shigella sonnei from Foods

Shigella species were recovered from foods by the procedure described in the Bacteriological Analytical Manual, 5th Ed. The method is effective if Shigella species are present at about 10(6) cells/g. A 25 g food portion was incubated in Gram-negative (GN) and selenite cystine broths for 16 h at 35 degrees C and streaked onto MacConkey, Levine's eosin methylene blue, desoxycholate citrate, and xylose lysine desoxycholate agars. S. sonnei cells were recovered quantitatively at 44.5 degrees C, and along with other Shigella species, were grown with Escherichia coli in a tryptone broth under anaerobic conditions. Shigella species were also grown in a mixed microflora from foods. S. sonnei cells were inoculated into an enrichment broth containing 20 g tryptone, 2 g K2HPO4, 2 g KH2PO4, 1 g glucose, 5 g NaCl, 1.5 mL Tween 80, and 0.5 mg novobiocin/L (pH 7.0) and incubated for 20 h at 44 degrees C. Enrichments were streaked onto MacConkey agar and the plates were incubated 20 h at 35 degrees C. Suspect Shigella colonies were screened in glucose, tryptone, and lysine broths and in triple sugar iron and motility agars. The sensitivity varied from 0.3 to 1000 bacteria/g. The method has been examined with artificially inoculated lettuce, celery, brussels sprouts, mushrooms, and hamburger. It is also applicable to S. flexneri if incubation is conducted at 42 degrees C.

Effective Enrichment-Plating Conditions for Detection of Salmonella in Foods

The performance of tetrathionate brilliant green (TBG) and selenite cystine (SC) enrichments, and bismuth sulfite (BSA) and brilliant green sulfa (BGS) plating media was assessed on the basis of data on 2085 Salmonella-contaminated low and high moisture foods that were collected during a 6-year (1977 to 1983) study involving 22 laboratories. None of the eight enrichment- plating combinations considered identified all positive samples. TBG was markedly more sensitive than SC for detection of salmonellae in high moisture foods, where enrichment in TBG and plating on both BSA and BGS identified 92% of contaminated samples. Plating of SC enrichment cultures on the two agar media was substantially less productive (63%). With low moisture foods, TBG and SC rates of isolation varied by less than 10% under homologous conditions. Plating media did not affect method sensitivity. Identification of numerous contaminated samples by TBG or SC enrichment alone underlines the value of using multiple enrichment and plating media in standard methods.

Recovery of Salmonella from Artificially Contaminated Poultry Feeds in Non-Selective and Selective Broth Media

The efficacy of four preenrichment media (lactose broth, lactose broth with Tergitol, buffered peptone and M-9) and of direct enrichment in selenite cystine and modified tetrathionate broths for recovering two Salmonella serotypes from dry poultry feed was determined. The salmonellae, artificially inoculated and stored for 7 wk in the dry feed (1.5 to 2.2 cells of salmonellae/g of feed analyzed), were recovered from significantly more samples after preenrichment with M-9 or buffered peptone than with any of the other procedures tested.

Evaluation of Direct Enrichment at Elevated Temperature for Recovery of Salmonellae from Oysters

A comparison of the recovery of salmonellae from naturally contaminated oysters was made by using either Lactose broth preenrichment at 35°C followed by selective enrichment in Tetrathionate and Selenite Cystine broths at 35, 41 and 43°C, or direct enrichment in Tetrathionate and Selenite Cystine broths with incubation at 35, 41 and 43°C. Direct enrichment in Selenite Cystine broth at both elevated temperatures produced the greatest number of positive samples. Similar results were obtained with artificially contaminated oyster samples (1 Salmonella/5 g of meat). Statistical analysis of the data showed that significantly higher recovery of salmonellae was obtained by using direct enrichment of samples in Selenite Cystine broth at the elevated temperature.

Isolation of Salmonella from Raw Chicken in Venezuela

Forty-five samples of raw chicken carcasses obtained from three poultry processing plants were examined for presence of Salmonella by the whole carcass rinse and skin maceration methods. Sensitivity of the selenite cystine (SC), tetrathionate brilliant green (TBG) and Rappaport (RAP) enrichment broths at 37°C and 43°C was compared following preenrichment in nutrient broth incubated at 37°C for 24 h. The rinse method and skin maceration resulted in isolation of Salmonella from 41 and 22 of the 45 samples, respectively. RAP incubated at 43°C resulted in higher rates of isolation of Salmonella than TBG and SC incubated at the same temperature. Incubation at 37°C was less productive. The sensitivity and selectivity of bismuth sulfite agar (BSA) exceeded that obtained with desoxycholate citrate agar (DCA) and brilliant green agar (BGA). With the methods and media we compared, sampling by the whole carcass rinse method and enrichment in RAP (43°C) and plating on BSA provide optimal conditions for detection of Salmonella in raw chicken. Eleven serotypes were identified with Salmonella anatum showing the highest frequency of isolation.

Recovery of Salmonella from refrigerated preenrichment and refrigerated enrichment media

The productivity of the standard cultural procedure for the isolation of Salmonella using preenrichment in buffered peptone water (BPW) followed by inoculation into Rappaport-Vassiliadis (RV), tetrathionate (TBG) (Difco) and selenite-cystine (SC) (Difco) enrichment broths, was compared with that using preenrichment and enrichment cultures which had been held under refrigeration (72 h at 5–10°C). Seventy-seven of 251 samples of food products were found to contain Salmonella. Refrigerated preenrichment cultures inoculated into RV medium (43°C), TBG broth (43°C) and SC broth (37°C) yielded salmonellae from 93.5, 85.7 and 54.5% contaminated samples, respectively. Refrigerated cultures in RV, TBG and SC broths, inoculated onto three selective plating media, identified 100, 87.0 and 41.6% of the contaminated samples, respectively. The selective plating medium brilliant green deoxycholate agar was at least as productive as brilliant green sulpha agar and bismuth sulphite agar, when streaked from RV and TBG broths, but was less effective when streaked from SC broth.

Efficacy of an International Method for Detection of Salmonella in Chocolate and Cocoa Products

The methods of the International Office of Cocoa and Chocolate and International Sugar Confectionery Manufacturers' Association (IOCC/ISCMA), and of the Health Protection Branch (HPB) were compared for their ability to detect Salmonella in chocolate and cocoa products. Of 152 samples tested, 13 contaminated samples were identified, 10 by the HPB and 8 by the IOCC/ISCMA method. Prolonged (48 h) incubation of enrichment media produced two false-negative results each with the Muller-Kauffman tetrathionate and the selenite cystine broths, exerted no effect on tetrathionate brilliant green, and identified one additional positive sample with the selenite brilliant green broth. More samples were found positive on bismuth sulfite than on brilliant green and brilliant green sulfa agar media. The present study underlines the limited sensitivity of both standard methods and questions the determinant role of casein in the neutralization of toxic agents in cocoa products.

Rapid Cultural Methods for Detection of Salmonella in Feeds and Feed Ingredients

Efficacy of standard, 6-h standard and direct enrichment methods for detection of Salmonella in naturally contaminated feeds and feed ingredients was compared. Analysis by the standard method involved preenrichment of feed slurries in nutrient broth, selective enrichment in tetrathionate brilliant green (43°C) and selenite cystine (35°C), and isolation of presumptive isolates on bismuth sulfite and brilliant green sulfa agar media. Sample analysis by the 6-h standard method was identical to the above except that incubation of enrichment broths was reduced to 6 h; for direct enrichment. preenrichment in nutrient broth was omitted. Of 287 samples tested, 75 were found to contain salmonellae by the three methods combined. Ability of the standard and 6-h standard methods to identify the same 58 contaminated samples underlines the reliability of the 6-h standard method for the more rapid detection of Salmonella in animal feeds. Identification of 68 positive samples by direct enrichment presumably resulted from equilibration (3 to 4 h) of feed slurries at reduced water activity before analysis. Addition of novobiocin (40 μg/ml) to selective enrichment broths did not facilitate isolation of Salmonella through repression of competitive flora. Productivity of the six enrichment-plating combinations used in this study was comparable, and no single medium played a determinant role in recovery.

Comparison of Preenrichment to Direct Enrichment and the Effect of Pyruvate in Media for Recovery of Salmonellae in Feed

Nineteen mash and 13 pelleted samples of poultry feed were collected from 10 commercial feed mills and analyzed for Salmonellae with six different media as the initial medium in the analysis: 1) lactose broth (L), 2) L plus 1% sodium pyruvate (LP), 3) selenite cystine broth (SC), 4) SC plus 1% sodium pyruvate (SCP), 5) tetrathionate (TT) broth, and 6) TT plus 1% sodium pyruvate (TTP). Thirteen of the 19 (68%) mash samples and 3 of the 13 (23%) pelleted samples were found to contain Salmonellae. When samples were preenriched in L, Salmonellae were recovered from only 7 of the 16 positive samples, whereas direct enrichment in SC and TT recovered Salmonellae from 10 and 11 of the 16 positive samples, respectively. When the combination of SC and TT were used, direct enrichment resulted in recovery of Salmonellae from 14 of the 16 positive samples. One percent sodium pyruvate was found to be highly inhibitory, resulting in identification of only 1 of 16 known positive samples.

Ein methodenvergleich zur anreicherung von salmonellen aus menschlichen faeces

Abstract 154 samples of faeces contaminated with salmonellae belonging to 21 species were investigated each in 23 enrichment broths for compare the efficiency. The best results were obtained with selenite cystine broth (no. 5) followed by Rappaport broth (no. 17) and tetrathionate brillant green bile broth — TBG — (no. 12); see also Fig. 1–4 and Tables 3 and 4. S. typhi is recovered the best by selenite broths. Rappaport broth seems to be the optimal enrichment broth for the most salmonellae with exception of S. typhi, S. bovismorbificans , and some other species. Preuss's modification of tetrathionate enrichment broth was significantly more efficient than Muller-Kauffmann 's tetrathionate. Brillant green bile broths are not very selective. Its efficiency is comparable with unselective broths without any inhibiting substances. The unspecific broths show a litlle enrichment effect during short incubation times. Therefore, they are used for preenrichment. However, incubation during 24 h is recommanded for optimal recovery in selective enrichment broths, i.e. Rappaport , selenite, and tetrathionate media. The selenite broth should be used unconditionally for detecting salmonellae in human faeces. It should be combined additionally with Rappaport or tetrathionate broth in the modification of Preuss but not of Muller-Kauffmann .

Behavior of Salmonellae During Manufacture and Ripening of Manchego Cheese

Six Salmonella strains were inoculated into 12 vats (2 vats/strain) of pasteurized sheep milk at a level of 104 cells/ml, and Manchego cheese was manufactured by usual procedures, with 1% of a Streptococcus lactis culture as starter. Growth of Salmonella occurred during the first 6–9 h, with mean increases in log counts of 1.67, 1.49 and 1.71 respectively for Salmonella enteritidis, S. typhi and S. typhimurium; data inversely correlated to pH decrease. Mean numbers of Salmonella declined during the first week by 4.43, 1.18 and 3.97 log cycles for the three serotypes, respectively, with a significant correlation between decreases in pH and in Salmonella log counts. Salmonella survived for 4 weeks in 9 vats, for 6 weeks in 3 vats and was absent from all lots of 8-week Manchego cheese. Brilliant green agar yielded the highest productivity among five selective agars used for the enumeration of Salmonella by direct-plating procedures, while enrichment in selenite cystine broth followed by streaking to bismuth sulfite agar gave the highest Salmonella recovery of all eight broth-agar combinations tested.

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.

Surfactants for the Effective Recovery of Salmonella in Fatty Foods

Inhibitory concentrations of 8 surfactants were determined for Salmonella typhimurium and Salmonella enteritidis. Pure culture work resulted in the exclusion of Tween 20, Teepol 610 and Brij 35 and retention of Tergitol-7 (T-7), Tween 80 (TW 80), Triton X-100 (TX), Myrj 52S (M), and Arlacel 80 + Tween 60 (AT) for a study on the quantitative recovery of Salmonella in 45 naturally contaminated fatty foods. Replicate food samples (100 g) were preenriched overnight at 35 C in nutrient broth supplemented with 3%(w/v) surfactant except AT (10%). Serial dilutions of preenrichment cultures were selectively enriched overnight in tetrathionate brilliant green (43 C) and selenite cystine (35 C) broths and streaked on bismuth sulfite and brilliant green sulfa agar media. Recovery with all test surfactants was comparable to that obtained with nutrient broth controls; of 270 preenrichment cultures tested, only 7 false-negative results attributable to TX (3), AT (2), M (1), and nutrient broth control (1) were obtained. None of the surfactants consistently yielded greater populations of Salmonella for given foods or food categories; median counts for preenrichment cultures were 104–105 salmonellae/ml for low and high moisture foods and 106–107 salmonellae/ml for animal feeds. These results suggest that use of surfactants to facilitate detection of Salmonella in fatty foods is not warranted.

Recovery of Salmonellae from Foods using a Combined Enrichment Technique

ABSTRACTThe separate steps of preenrichment in lactose broth and selective enrichment in tetrathionate (TET) or selenite‐cystine (SC) broths were combined into single procedures for the detection of both injured and uninjured salmonellae in foods. Following 4 hr of incubation, iodine or selenite were added to combined lactose‐tetrathionate (CTET) or selenite basal broths, forming selective environments. CTET broth was as efficient as conventional TET and SC broths but the combined selenite media were not. Salmonellae were detected in foods in 30 hr using direct fluorescent antibody and combined enrichment techniques. The combined procedure resulted in savings of assay time, labor and materials.