Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred. and accepted 22 June 1989     

Effect of Iron on Conversion of Acetic Acid to Methane During Methanogenic Fermentations

A semi-automatic method for the detection of salmonellas in food products is described. This method involves measurement of the fluorescence intensity of preparations stained with fluorescent antibody. An overall agreement of 89·6% was obtained when the method was compared with a standard cultural technique for the detection of salmonellas in foods, feeds and environmental samples, although false negatives were a problem with raw meat products. A saving in time of at least 18 h was achieved.     

A Comparison of the Fluorescent Antibody Method and a Standardized Cultural Method for the Detection of Salmonellas

The results of routine use of the indirect fluorescent antibody (FA) technique using the Spicer-Edwards H antisera set are reported for a range of agricultural and food samples. The FA technique was used on samples after the pre-enrichment incubation period in the proposed ISO method for isolation of salmonellas. The numbers of FA false positive samples ( ca. 5% overall) and FA false negative samples ( ca. 1·3%) were low, but some originally FA false positive results were later shown to be false negative cultural results.     

Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Rapid impedance detection of salmonellas in confectionery using modified LICNR broth

A commercially available broth, with the addition of inhibitors, was used for the rapid impediometric detection of salmonellas in confectionery. Pre-enrichment in skimmed milk was followed by lysine-iron-cystine-neutral red broth in a Bactometer 123 system. Results were obtained 3 d earlier than is possible with conventional microbiological tests. Some false positives were obtained predominantly with Citrobacter freundii, but this problem is also frequently encountered with traditional methods. Organisms responsible for false positives may be isolated and identified more rapidly than is possible by conventional methods.     

Rapid impedance detection of salmonellas in confectionery using modified LICNR broth

A commercially available broth, with the addition of inhibitors, was used for the rapid impediometric detection of salmonellas in confectionery. Pre-enrichment in skimmed milk was followed by lysine-iron-cystine-neutral red broth in a Bactometer 123 system. Results were obtained 3d earlier than is possible with conventional microbiological tests. Some false positives were obtained predominantly with Citrobacter freundii but this problem is also frequently encountered with traditional methods. Organisms responsible for false positives may be isolated and identified more rapidly than is possible by conventional methods.     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods.

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 micrograms of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

Laboratory study of several enrichment broths for the detection of Salmonella spp. particularly in relation to water samples

The selectivity and efficiency of several enrichment broths used for the detection of salmonellas were comparatively evaluated under laboratory and environmental conditions. Media with selenite were less efficient in their inhibition of the growth of Gram-positive micro-organisms. Salmonellas grew slowly in tetrathionate broth and in media containing brilliant green. These media inhibited the growth of Salmonella typhi, which grew only in media containing selenite. The results obtained in the experiments with stressed salmonellas indicate that the media selenite F, selenite F with novobiocin, selenite cystine and Rappaport-Vassiliadis (RV/43), in conjunction with the double agar layer technique, showed an optimal efficiency for the detection of stressed salmonellas. When natural samples (freshwater and seawater) were used to evaluate the media, however, those containing malachite green, whether or not supplemented with sodium novobiocin, enhanced the recovery of salmonellas.     

A complete protocol using conductance for rapid detection of salmonellas in confectionery materials

Increased confidence in conductimetric detection of salmonellas was achieved by combining a bacteriophage-based test with use of a selenite cystine trimethylamine oxide dulcitol medium and a modified lysine decarboxylase broth. All 81 Salmonella isolates tested were detected and few of the 39 non-salmonellas gave false positives. Results from the screening of 43 inoculated product samples further support the use of this simple, rapid method for routine salmonella testing in the food industry.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 μg of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

An improved ELISA method for the detection of Salmonella typhimurium

The applicability of enzyme-linked immunosorbent assay (ELISA) for the detection of salmonellas in foodstuffs was investigated. Several factors affecting the sensitivity of the ELISA, such as the type of protein used for plate post-coating, the method of antibody labelling, and accelerators for antigen-antibody and enzyme-substrate reactions, were studied. Labelling of the antibody with horseradish peroxidase and the use of o -phenylenediamine as substrate in the detection system were demonstrated to be most suitable for the enzyme assay.
Based on these findings, an improved ELISA method was developed for the detection of Salmonella typhimurium. The improved technique was able to detect as few as 5×10⁴-10⁵ cell/ml of salmonellas, and about 24 h were required to enrich the bacteria in food samples and to perform the test. With some modifications, the ELISA assay could reach a very high level of sensitivity and provide excellent repro-ducibility.     

An improved ELISA method for the detection of Salmonella typhimurium

The applicability of enzyme-linked immunosorbent assay (ELISA) for the detection of salmonellas in foodstuffs was investigated. Several factors affecting the sensitivity of the ELISA, such as the type of protein used for plate post-coating, the method of antibody labelling, and accelerators for antigen-antibody and enzyme-substrate reactions, were studied. Labelling of the antibody with horseradish peroxidase and the use of o-phenylenediamine as substrate in the detection system were demonstrated to be most suitable for the enzyme assay. Based on these findings, an improved ELISA method was developed for the detection of Salmonella typhimurium. The improved technique was able to detect as few as 5 x 10(4)-10(5) cell/ml of salmonellas, and about 24 h were required to enrich the bacteria in food samples and to perform the test. With some modifications, the ELISA assay could reach a very high level of sensitivity and provide excellent reproducibility.     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10⁴ cfu/ml (phenyl phosphate system) or 10⁵ cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1–5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS 200 . The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55°C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 μl of 1 mmol 1^-1 AttoPhos^TM (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1–10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR. assay described here can be useful to screen a large number of food samples for contamination by salmonellas.     

Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of Salmonella.

Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars and several related enterobacteria. Those detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media by conventional culture. Of these 58 were detected by the ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. The results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures at an early stage.     

Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control

Polymerase chain reaction (PCR) with real-time detection using two adjacent fluorescent probes in a Lightcycler instrument was applied for detection of the Mycoplasma pneumoniae P1 protein gene. To monitor inhibition in each sample an internal control was constructed that can be amplified by the same primers but detected by different probes and dual color detection. The real-time PCR was applied on 115 respiratory samples from 82 patients and compared to a conventional PCR. There was 100% agreement between the assays, but the real-time PCR proved to be highly superior in speed with a much lower risk of false positives by laboratory contamination.     

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.