A modified conductance medium for the detection of Salmonella spp

Selenite-cystine/trimethylamine oxide/dulcitol medium has been used in conjunction with conductance instruments to detect the presence of Salmonella spp. in foods and faeces. However, a small but significant number of salmonella strains were missed by this method. The majority of these strains were detected when dulcitol was substituted by mannitol and tested on two separate Malthus conductance instruments. Some strains of Citrobacter freundii and Escherichia coli continued to give false positive results. Attempts are made to explain why the substitution of mannitol for dulcitol gives an improved medium.     

A modified conductance medium for the detection of Salmonella spp.

Selenite-Cystine/trimethylamine oxide/dulcitol medium has been used in conjunction with conductance instruments to detect the presence of Salmonella spp. in foods and faeces. However, a small but significant number of salmonella strains were missed by this method. The majority of these strains were detected when dulcitol was substituted by mannitol and tested on two separate Malthus conductance instruments. Some strains of Citrobacter freundii and Escherichia coli continued to give false positive results. Attempts are made to explain why the substitution of mannitol for dulcitol gives an improved medium.     

Improvements to a lysine medium for detection of salmonellas by elctrical conductance

Improvements in performance of a lysine conductance medium for the detection of salmonellas were achieved from a study of the effects of its various components. When sodium biselenite was included as an inhibitor for non-salmonella organisms conductance signals were depressed. The inclusion of sodium chloride reduced this toxicity and improved conductance responses. Increasing the pH to pH 7.0 prevented the medium becoming too acidic and inhibitory to salmonellas. The new medium detected 70-0% of salmonella-positive animal protein samples.     

Development of a new medium for the culture of agallian leafhopper cells

Summary The optimal composition of a medium for tissue culture of cells from the leafhopperAgallia constricta was estimated from experiments in which the rate of growth of the cells was measured at different concentrations of each component. Wound tumor virus and theconstricta variety of potato yellow dwarf virus multiply, inA. constricta when these viruses are transmitted from one plant to another. Differences weredetected in the suitablility of different batches of fetal bovine serum (after heating at 56°C for 30 min), and histidine as components in the tissue culture medium. Without heat treatment even thebest fetal bovine serum was not suitable. Estimated optimal concentrations of fetal bovine serum, histidine, salts, dextrose, lactalbumin hydrolysate and yeast autolysate were determined. Fungizone (amphotericin B) at 50 or 100 mg per 1 caused harmful effects; effective concentrations of penicillin, neomycin and steptomycin did not. Combinations of histidine-HCl and histidine (free-base) made it possible to prepare buffered medium at my pH between 6.0 and 7.0. Optimal growth ofA. constricta cells occurred at pH 6.43, and ofAceratagallia sanguinolenta at pH 6.30. Osmotic pressures of the new medium between 360 and 405 mOSM were better than lower osmotic pressures. The new medium was still suitable for growth of the cells after, storage for 6 months at 4°C. Portion of a thesis submitted for the Ph.D. degree by the senior author to the Graduate College of the University of Illinois. This research was supported in part by Grant GB 20915 from the National Science Foundation and Grant AI 6392 from the National Institutes of Health.     

Development of a new simplified nutritive medium for the axenic culture of Artemia

A new artificial nutritive medium has been developed for the axenic culture of Artemia enabling the production of adults in less than 1 wk. The techniques for its preparation have been detailed. Its utilization is recommended for the standardized production of experimental animals.     

A new simple medium for the detection of Enterococcus faecalis and Enterococcus faecium by measurement of conductance changes

Streptococcus suis, Streptococcus bovis and the mastitis pathogens Streptococcus dysgalactiae and Streptococcus uberis were the most frequently occurring streptococci in tonsils of cattle. Streptococcus suis dominated in samples from calves between 1 month and 1 year of age, but was much less frequent in calves less than 1 month old. The mastitis pathogen Strep, dysgalactiae was found more often in calves than in older animals. Enterococci were relatively rare, except in the youngest age group. Nearly one third of the strains examined could not be identified to known species.     

Phagomagnetic immunoassay for the rapid detection of Salmonella

This work explores the use of the phage P22 in a phagomagnetic immunoassay for the rapid detection of Salmonella. The covalent attachment of wild-type phages was performed on two different magnetic carriers: carboxyl-activated magnetic nanoparticles (300 nm) and tosyl-activated magnetic microparticles (2.8 μm). The bacteria were captured and preconcentrated by the phage-modified magnetic particles, followed by the detection using specific anti-Salmonella antibodies conjugated to horseradish peroxidase as an optical reporter. Outstanding selectivity and sensitivity was obtained with this approach, achieving detection limits of 19 CFU mL^?1 in 2.5 h without any pre-enrichment, in milk samples. Moreover, if the samples were pre-enriched for 6 h, the method was able to detect as low as 1.4 CFU in 25 mL of milk. Therefore, the proposed strategy based on the combined use of phagomagnetic separation with immunological labeling is promising as a rapid and simple method for food safety.     

Loop-mediated isothermal amplification for the rapid detection of Salmonella

Loop-mediated isothermal amplification (LAMP) assay detected Salmonella within 60 min. The 220 strains of 39 serotypes of Salmonella subsp. enterica and 7 strains of Salmonella enterica subsp. arizonae were amplified, but not 62 strains of 23 bacterial species other than Salmonella. The sensitivity of the LAMP assay was found to be >2.2 cfu/test tube using nine serotypes. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was greater. Both fluorescence and turbidity were able to detect the products in the LAMP assay. S. enteritidis in a liquid egg sample artificially inoculated with the organism was detected by the LAMP assay at 2.8 cfu/test tube, although negative by PCR assay. These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Salmonella.     

Some modification to the media for rapid automated detection of salmonellas by conductance measurement

A selenite medium for the automated detection of salmonellas by conductance measurements has been modified to eliminate the negative results given by some dulcitol-negative strains. The dulcitol is replaced with mannitol and pre-enrichment is best done in buffered peptone water containing mannitol and dimethylsulphoxide. It is suggested that both versions of the selenite medium be used initially.     

Development,validation, and applications of a new laboratory-scale indirect impedancemeter for rapid microbial control

We introduce a new laboratory-scale impedance-meter which is specially intended for indirect technique. It consists of a software system enabling data acquisition via a connected bus which is wired to the measuring cells. These measuring cells are individual impedance-meters that can be activated independently of one another. In the current configuration, the device is slightly affected by temperature, but it can register as little as 10.9 micromol of CO(2). With Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae cultures, the conductance responses were highly replicable and repeatable for inocula concentrations of 1-10(8) colony-forming units (CFU) ml(-1). The main use for such devices could be the detection of contamination in foodstuffs. Several of these foodstuffs, when incubated at 37 degrees C, spontaneously release quite large amounts of CO(2). Our impedance meter, however, was able to detect an E. coli presence in canned French beans at 2.35 x 10(-2) CFU ml(-1) and a S. cerevisiae contamination of apple purée in glass jars at 6.1 x 10(-3) CFU ml(-1). The conductance response and the detection time (the time needed for a significant change in conductance) were correlated to the concentration of ampicillin (an antibiotic added to E. coli cultures). The device is thus able to detect the presence of inhibitory compounds in milk or other foodstuffs. Some industrial assays are in process to complement these laboratory tests. Compared with other available techniques for CO(2) measurement (manometry, infrared, radioactive labeling), the technique put forward here appears to be the best compromise between sensitivity, technical constraints, and cost. A commercial version of the impedance meter would enable routine measurements in the quality control of foodstuffs, pharmaceuticals, cosmetics and in R&D laboratories.     

Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of Salmonella

AIMS: Evaluation of iQ-Check PCR Salmonella for Salmonella detection in artificially and naturally contaminated food and environmental field samples. METHODS AND RESULTS: Artificially contaminated samples (poultry meat and ground red meat) subjected to cold- and freeze-stress, and 120 naturally contaminated samples (swabs and meat) were tested for Salmonella using the diagnostic semi-solid Salmonella medium (DIASALM) method, the Vidas assay and the iQ-Check PCR assay after 24 h enrichment in buffered peptone water. CONCLUSIONS: Both the iQ-Check PCR and the Vidas assay provide a rapid and user friendly screening method for detection of Salmonella. False negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed. In total 45 of 120 naturally contaminated field samples showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was respectively 92% for the iQ-Check PCR and 95% for the Vidas method. SIGNIFICANCE AND IMPACT OF THE STUDY: False-negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed, e.g. freezing at -18 degrees C for 7 days. Of the 120 naturally contaminated field samples 45 showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was 92% for the iQ-Check PCR and 95% for the Vidas method respectively.     

Development of a rapid method for the detection of prostate-specific antigen by immunochromatography

A single-step qualitative rapid test for the determination of prostate-specific antigen (PSA) in samples of human blood serum by immunochromatography using a complex of colloidal gold with monoclonal antibodies to PSA as the detection agent was developed. The determination limit for PSA in serum blood samples is 10 ng/ml; the analysis time, 15-25 min; the sensitivity of the method, 100%; and its specificity, 92.5%.     

Development of a rapid method for the detection of prostate-specific antigen by immunochromatography

A single-step qualitative rapid test for the determination of prostate-specific antigen (PSA) in samples of human blood serum by immunochromatography using a complex of colloidal gold with monoclonal antibodies to PSA as the detection agent was developed. The determination limit for PSA in serum blood samples is 10 ng/ml; the analysis time, 15–25 min; the sensitivity of the method, 100%; and its specificity, 92.5%.     

A rapid method to improve protein detection by indirect ELISA

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.     

A medium for the rapid growth and detection of moulds

A method is described for encouraging rapid germination and growth of mould propagules and enhancement of their detection. This inovles the design of a new growth medium Apple Pulp Gelatin (APG) which contains an optical brightener.     

Development of a surface adhesion immunofluorescent technique for the rapid detection of Salmonella spp. from meat and poultry

A rapid method based on bacterial adhesion was developed for the detection of Salmonella in an enriched meat system. Minced beef samples inoculated with Salm. enteritidis (10 cfu g-1) were incubated overnight (18 h) at 37 degrees C in buffered peptone water. Salmonella enteritidis cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane attached to a glass slide. The organisms attached to this polycarbonate membrane were subsequently visualized using immunofluorescent microscopy. The technique had a detection level of log10 3.5 Salmonella ml-1. The surface adhesion immunofluorescent technique correlated well with Salmonella plate counts (r2 = 0.99). Application of the rapid method to retail beef and poultry samples (n = 100) confirmed the correlation between this technique and traditional microbiological procedures. Thirty-one retail samples were reported positive for Salmonella species. No false positives or negatives were recorded for the rapid method.