Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed ß-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the ß-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.

The effect of anode potential on bioelectrochemical and electrochemical tetrathionate degradation.

The effect of poised anode potential on electricity production and tetrathionate degradation was studied in two-chamber flow-through electrochemical (ES) and bioelectrochemical systems (BES). The minimum anode potential (vs. Ag/AgCl) for positive current generation was 0.3V in BES and 0.5V in the abiotic ES. The anode potential required to obtain average current density above 70mAm-2 was 0.4V in BES and above 0.7V in ES. ES provided higher coulombic efficiency, but the average tetrathionate degradation rate remained significantly higher in BES (above 110mgL-1d-1) than in the abiotic ES (below 35mgL-1d-1). This study shows that at anode potentials below 0.7V, the electrochemical tetrathionate degradation is only efficient with microbial catalyst and that significantly higher tetrathionate degradation rates can be obtained with bioelectrochemical systems than with electrochemical systems at the tested anode potentials.

Long-term stability of bioelectricity generation coupled with tetrathionate disproportionation.

To prevent uncontrolled acidification of the environment, reduced inorganic sulfur compounds (RISCs) can be bioelectrochemically removed from water streams. The long-term stability of bioelectricity production from tetrathionate (S4O6(2-)) was studied in highly acidic conditions (pH<2.5) in two-chamber fed-batch microbial fuel cells (MFCs). The maximum current density was improved from previously reported 80mAm(-2) to 225mAm(-2) by optimizing the external resistance. The observed reaction products of tetrathionate disproportionation were sulfate and elemental sulfur. In long-term run, stable electricity production was obtained for over 700days with the average current density of 150mAm(-2). The internal resistance of the MFC decreased over time and no biofouling was observed. This study shows that tetrathionate is an efficient substrate also for long-term bioelectricity production.

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.

Comprehensive simultaneous kinetic study of sulfitolysis and thiosulfatolysis of tetrathionate ion: unravelling the unique pH dependence of thiosulfatolysis.

The kinetics of the reactions of tetrathionate with S(IV) species and with thiosulfate in slightly acidic and neutral media were studied concurrently at 25.0 ± 0.1 °C by simultaneous high-performance liquid chromatography monitoring of the concentrations of polythionates (including trithionate, tetrathionate, and pentathionate), thiosulfate, and sulfite. The tetrathionate-sulfite and tetrathionate-thiosulfate reactions were found to be first-order with respect to both reactants. The tetrathionate-sulfite reaction was found to be pH-dependent under the conditions studied. In contrast, the tetrathionate-thiosulfate reaction was experimentally demonstrated to be pH-independent at neutral medium, where the pKa2 value of sulfurous acid plays a key role, whereas under slightly acidic conditions, between pH 4 and 5 the consumption of tetrathionate during the course of reaction was found to become pH-dependent. We show that the pH dependencies in both systems can be readily explained by the reactivity difference between sulfite and bisulfite toward the ß-sulfur of the tetrathionate. A simple two-step kinetic model incorporating the protonation equilibrium of sulfite is proposed on the basis of the simultaneous evaluation of the kinetic curves of the two systems, which allowed us to determine reliable rate coefficients for both the forward and backward reactions. Furthermore, the powerful ability of simultaneously evaluating the two chemical systems to yield reliable rate coefficients of the kinetic model is demonstrated.

Electricity generation from tetrathionate in microbial fuel cells by acidophiles.

Inorganic sulfur compounds, such as tetrathionate, are often present in mining process and waste waters. The biodegradation of tetrathionate was studied under acidic conditions in aerobic batch cultivations and in anaerobic anodes of two-chamber flow-through microbial fuel cells (MFCs). All four cultures originating from biohydrometallurgical process waters from multimetal ore heap bioleaching oxidized tetrathionate aerobically at pH below 3 with sulfate as the main soluble metabolite. In addition, all cultures generated electricity from tetrathionate in MFCs at pH below 2.5 with ferric iron as the terminal cathodic electron acceptor. The maximum current and power densities during MFC operation and in the performance analysis were 79.6 mA m(-2) and 13.9 mW m(-2) and 433 mA m(-2) and 17.6 mW m(-2), respectively. However, the low coulombic efficiency (below 5%) indicates that most of the electrons were directed to other processes, such as aerobic oxidation of tetrathionate and unmeasured intermediates. The microbial community analysis revealed that the dominant species both in the anolyte and on the anode electrode surface of the MFCs were Acidithiobacillus spp. and Ferroplasma spp. This study provides a proof of concept that tetrathionate serves as electron donor for biological electricity production in the pH range of 1.2-2.5.

[Oxidation of sulfur-containing substrates by aboriginal and experimentally designed microbial communities].

Aboriginal and experimental (constructed of pure microbial cultures) communities of acidophilic chemolithotrophs have been studied. The oxidation of elemental sulfur, sodium thiosulfate, and potassium tetrathionate as sole sources of energy has been monitored. The oxidation rate of the experimental community is higher as compared to the aboriginal community isolated from a flotation concentrate of pyrrhotine-containing pyrite-arsenopyrite gold-arsenic sulfide ore. The degree of oxidation of the mentioned S substrates amounts to 17.91, 68.30, and 93.94% for the experimental microbial community and to 10.71, 56.03, and 79.50% for the aboriginal community, respectively. The degree of oxidation of sulfur sulfide forms in the ore flotation concentrate is 59.15% by the aboriginal microbial community and 49.40% by the experimental microbial community. Despite a higher rate of oxidation of S substrates as a sole source of energy by the experimental microbial community, the aboriginal community oxidizes S substrates at a higher rate in the flotation concentrate of pyrrhotine-containing pyrite-arsenopyrite gold-arsenic sulfide ore, from which it was isolated. Bacterial-chemical oxidation of the flotation concentrate by the aboriginal microbial community allows for the extraction of an additional 32.3% of gold from sulfide minerals, which is by 5.7% larger compared to the yield obtained by the experimental microbial community.

Preparation of molecularly imprinted polymer by surface imprinting technique and its performance for adsorption of dibenzothiophene.

The novel surface imprinted polymer composites (MIP/K(2)Ti(4)O(9)) were prepared using dibenzothiophene (DBT) as the template, 4-vinylpyridine as the functional monomer and potassium tetratitanate whisker (K(2)Ti(4)O(9)) as the carrier. The synthetic product was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Parameters influencing DBT adsorption such as contact time, temperature and DBT initial concentration were investigated. The adsorption kinetics were evaluated with the pseudo-first-order and pseudo-second-order models, and the adsorption isotherms were fitted by Langmuir and Freundlich models. Selectivity experiments showed that MIP/K(2)Ti(4)O(9) exhibited excellent recognition capacity and binding affinity to DBT compared with the comparative substrates. MIP/K(2)Ti(4)O(9) could also be easily regenerated and reused ten times with only about 20% loss of adsorption capacity.

Sodium tetrathionate effect on papain purification from different Carica papaya latex crude extracts.

Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.

An improved chemical model for the quantitative description of the front propagation in the tetrathionate-chlorite reaction.

It is experimentally proven that the stoichiometry of the tetrathionate-chlorite reaction is 2S4O(2-)6 + 8(1/2)ClO-(2) + 6H2O = 8SO(2-)4 + ClO-(3) + 7(1/2)Cl- + 12H+ near 1:4 molar ratio of the reactants. Re-evaluation of the previously measured front velocity--concentration curves also shows that this stoichiometry along with both the rate equation r = (1.6 x 10(5) M(-3) s(-1) [H+]2 + 3.6 x 10(7) M(-4) s(-1) [H+]3)[S4O(2-)6][ClO-(2)] and the protonation processes existing in the present system allow us to describe the front velocity as a function of the initial concentration of the reactants quantitatively. Some consequences detailed in the conclusions may concern not only uniquely the tetrathionate-chlorite reaction but any front propagation study including H+ as an autocatalyst.

A spectroscopic investigation into the reaction of sodium tetrathionate with cysteine.

A spectroscopic investigation into the reaction of sodium tetrathionate with cysteine at pH 5 both at the boil and at room temperature has been carried out. The Raman and infrared spectra of the model compounds cysteine, cysteine-S-sulfonate, cysteine-S-thiosulfonate, sodium thiosulfate and sodium sulfite were also obtained and vibrations involving the sulfur atoms were analyzed in detail. These results were utilized in the interpretation of the spectra obtained from tetrathionate-cysteine reaction mixtures. The reaction supernatants were analyzed by high performance thin layer chromatography while the precipitates were analyzed gravimetrically. It was found that during the reaction, the thiol groups of cysteine are oxidised to give predominantly cysteine-S-sulfonate. Cystine was also detected but was determined gravimetrically to be a minor reaction product. No significant amounts of cysteine-S-thiosulfonate were detected. The reaction is accompanied by the formation of elemental sulfur and a small amount of sulfite. Major reaction pathways are put forth that are consistent with the experimental data.

Oscillatory reactions involving hydrogen peroxide and thiosulfate-kinetics of the oxidation of tetrathionate by hydrogen peroxide.

The reaction between tetrathionate and hydrogen peroxide forms an important part of several pH oscillators based on the oxidation of thiosulfate. The kinetics of this reaction were examined in a batch reactor by measurement of the initial pH values in the range from 8 to 10.5. Experimental data were evaluated by the method of initial reaction rates combined with the assumption of instantaneously equilibrated acid-base reactions. The rate-determining step was found to be of the first order with respect to tetrathionate, hydrogen peroxide, and OH- ions with the value of rate constant k = (1.50 +/- 0.03) x 10(2) (M2 s)(-1) at 25 degrees C. In the alkaline solution, no distinct catalytic effect of Cu2+ was observed in contrast to earlier assumptions. The waveform of measured pH over the course of the reaction indicates that thiosulfate is probably an intermediate because characteristics of the curves are very similar to those for the oxidation of thiosulfate. We also measured the time evolution of concentrations of major components by the attenuated total internal reflectance infrared spectroscopy to further elucidate the underlying reaction mechanism. These measurements confirm the suspected role of thiosulfate as an intermediate in the studied reaction and provide valuable detailed information on the time evolution of thiosulfate, sulfite, sulfate, tetrathionate, and trithionate. These experimental observations are included in a simple mechanism that accurately simulates the reaction course in an alkaline solution. The results provide considerable new insights into the nature of autocatalysis in the hydrogen peroxide-thiosulfate-Cu2+ reaction and suggest that a new role for Cu2+ in the oscillatory dynamics observed in a flow-through reactor needs to be found.

Post-translational regulation of mercaptopyruvate sulfurtransferase via a low redox potential cysteine-sulfenate in the maintenance of redox homeostasis.

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S(4)O(6)(2-)) inhibited rat MST (k(i) = 3.3 min(-1), K(i) = 120.5 microM and k(i) = 2.5 min(-1), K(i) = 178.6 microM, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys(247), suggesting that Cys(247) is a target of the oxidants. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S(2)O(3)(-)), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO(-)) at Cys(247), which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.

[Oxidation of inorganic sulfur compounds by obligatory organotrophic bacteria]. / Okislenie neorganicheskikh sernykh soedinenii obligatno khemogeterotrofnymi bakteriiami.

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some of denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.

Determination of salmonellae from municipal wastewaters.

This study compared the efficiency of culture methods for salmonellae detection in wastewaters collected from three Finnish municipal treatment plants and from one laboratory-scale plant. The performance of one-step enrichment in Preuss tetrathionate broth was better than that of two-step enrichment (buffered peptone water pre-enrichment (BPW) and selective enrichment in Rappaport-Vassiliadis (RV) medium. The best combinations for Salmonella isolation were xylose-lysine-deoxycholate (XLD) and Rambach (RB) agars after Preuss enrichment and did not differ when brilliant green-magnesium chloride (BM) or brilliant green phenol red (BP) agars were used. The two-step enrichment inhibited the growth of both salmonellae and interfering accompanying flora. Salmonella-positive plates were generally easier to read when inoculated from RV than from Preuss medium because of less growth of competing flora. XLD and BM agars supported growth of salmonellae and inhibited growth of competing flora better than BP and Rambach agars. XLD and BM agars gave the highest numbers of salmonellae isolations but XLD and Rambach agars gave the best differentiation. Salmonella levels were < 3- > 1100 MPN/100 mL.

Sulfur speciation and tetrathionate sulfitolysis monitoring by capillary electrophoresis.

Capillary electrophoresis (CE) with indirect detection was used to analyse mixtures of sulfur-containing compounds. An optimised electrolyte, composed of 2 mM sulfosalicylic acid-0.5 mM Waters osmotic flow modifier OFM-OH, pH adjusted to 7.00 with Bis-Tris, allowed the analysis of sulfide (H2S/HS-), thiosulfate (S2O(3)2-), tetrathionate (S4O(6)2-), trithionate (S3O(6)2-), sulfite (HSO3-/SO(3)2-), sulfate (SO(4)2-), and peroxodisulfate (S2O(8)2-). Only sulfate showed a single-peak electropherogram that gives evidence of its stability in aqueous solutions. Thiosulfate, tetrathionate and peroxodisulfate showed an additional minor peak of sulfate that did not seem to be time-dependent and was supposed to be due to salt impurities. Sulfide and sulfite showed rapid conversion into their oxidation products in solutions exposed to air. Linear calibration curves were obtained for all these species, taking the oxidation process into account for sulfide and sulfite. Limits of detection were in the low- to mid-microM range, except for sulfide, for which the limit of detection was 10(-5) M. The identity of trithionate was not determined via standard solutions (not commercially available) but after the rearrangement of tetrathionate with sulfite that yields thiosulfate and trithionate. The time-dependence of the concentrations of both the reagents and products could be followed simultaneously by the proposed CE method and the reaction kinetics were found to be second order in the pH range 7 to 11. A calibration curve for trithionate could be deduced from this study.

Reversible protection of disulfide bonds followed by oxidative folding render recombinant hCGbeta highly immunogenic.

Active immunization of women against human chorionic gonadotropin (hCG) has been considered as a promising option for contraception. However, prototype hCG vaccines based on natural sources of antigen are expected to be costlier for use by common people. In the present report, a functionally active, cost-effective antigen of bacterial origin has been described. Sulfonation of thiol groups of the protein, anion-exchange purification, refolding with concomitant formation of disulfide bonds in the presence of cysteamine-cystamine redox buffer, and slow removal of denaturant resulted in 95% homogeneous, monomeric form of the antigen. The recombinant processed antigen [CGbeta(p)] obtained this way was highly immunopotent. Cellular DNA and endotoxin contaminants were appreciably low in the final product. The immunogenic response was drastically reduced with the unprocessed antigen. This finding envisages better prospect of a cost-effective hCG vaccine for birth control.

Isolation of Salmonella organisms from the mesenteric lymph nodes of horses at necropsy.

OBJECTIVES: To determine the prevalence of Salmonella infections in horses at necropsy. DESIGN: Cross-sectional prevalence survey. ANIMALS: 102 horses. PROCEDURE: Mesenteric lymph nodes were collected from horses that were necropsied. Horses had died or were euthanatized because of severe disease or at the request of the owner. Twenty-eight of the horses were racehorses euthantized following acute catastrophic injuries on the racetrack. Mesenteric lymph nodes were submitted for Salmonella culture via direct plating of tissue specimens on MacConkey agar and by use of 4 enrichment culture techniques that used tetrathionate and selenite enrichment broth and brilliant green and Salmonella-Shigella selective plating media. RESULTS: Salmonella typhimurium was isolated from the mesenteric lymph nodes of 2 foals (2/102, 1.96% of the horses). Salmonella organisms were not isolated from the mesenteric lymph nodes of adult horses. CONCLUSIONS AND CLINICAL RELEVANCE: Prevalence of Salmonella infections in horses of our study (1.96%) suggests that the results of cross-sectional surveys, using bacteriologic culture to determine prevalence of Salmonella infection, should be interpreted with caution. Prevalence of Salmonella infections determined in a single facility may not reflect the prevalence of Salmonella-infected horses in the general population; furthermore, obtaining a Salmonella isolate from a horse does not establish that the horse is a chronic Salmonella carrier.

Purification and partial characterization of kininogenase activity from Schistosoma mansoni adult worms.

An enzyme presenting kallikrein-like activity (designated sK1) was purified from the supernatant of Schistosoma mansoni adult worm homogenate. The enzyme cleaves bradykinin from purified rat plasma kininogen. Activity was optimal at pH 9.0 and the enzyme showed amidolytic activity, since it hydrolysed the kallikrein synthetic substrate D-Pro-Phe-Arg-p-nitroanilide. The activity of sK1 upon rat plasma kininogen was strongly inhibited by the serine proteinase inhibitors phenylmethanesulfonyl fluoride, aprotinin or soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid or sodium tetrathionate. The molecular mass of sK1, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 66 kDa and the pI value, estimated by analytical chromatofocusing, was 4.2. Physical and chemical properties suggest that sK1 is a serine proteinase of the kallikrein family. Evidence is presented which suggests that sK1 is a component of the tegumental surface of the parasite and the levels of its activity in the male adult worm are approximately 21 times higher than those in the female adult worm. The intravenous injection of 3 micrograms of sK1 into an anaesthetized rat induced a drastic reduction in the arterial blood pressure of the animal. This effect lasted for about 1 min, and was followed by a progressive recovery of the arterial pressure. Neither bradycardia nor cardiac arrhythmias were noticed, suggesting a peripheral vasodilation effect. The presence of sK1 on the surface of adult male worms could play an important role in the wandering capacity of coupled worms into the visceral vasculature of the host.

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.