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.     

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.     

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.     

Detection of salmonellas in animal feeds by electrical conductance

A comparison was made between standard culture methods and electrical conductance using a Malthus AT Microbiological Analyser for the examination of animal feeds for salmonella. Conductance testing with a selenite cystine/trimethylamine-N-oxide/dulcitol medium resulted in the detection of salmonellas in 49 of 55 known positive animal feeds, 13 of 19 spiked feed samples and 36 of 47 salmonella cultures. Testing with a lysine decarboxylase/glucose medium gave significantly better results (P less than 0.05) than with selenite cystine medium but five lysine decarboxylase negative strains of salmonella were undetected. When both media were used in parallel all salmonella positive samples were detected. No difference was found between preenrichment in buffered peptone water containing trimethylamine/mannitol and that containing lysine/glucose. Positive detection criteria for selenite medium of conductance peak at greater than or equal to 500 microsiemens (microS) with a rate of change of greater than or equal to 60 microS/h or 400-499 microS with a rate of change of greater than or equal to 40 microS/h and for lysine medium with a peak of greater than or equal to 100 microS have been established. The method offers savings in media and operating costs over conventional standard culture methods, provides results within 48 h and is recommended for statutory feed monitoring purposes.     

Development of a new culture medium for the rapid detection of Salmonella by indirect conductance measurements

The main difficulties in conductance medium development are to allow Salmonella to grow and produce a conductance signal while impeding growth of related species such as Escherichia coli and Citrobacter freundii . Various selective agents were screened for these capacities and a new medium was derived, named KIMAN (Whitley Impedance Broth basal medium supplemented with three selective components: novobiocin, malachite green and potassium iodide). This medium supported the growth of Salmonella serotypes and inhibited non-salmonella strains in pure cultures.     

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.     

Use of a commercially available ELISA kit for detection of salmonellas in water

The accuracy and specificity of a commercially available ELISA kit (Locate, Rhone-Poulenc Diagnostics) were assessed when applied to enrichment cultures of naturally contaminated water and sewage. The kit showed 66/180 samples positive by both culture and ELISA. The ELISA showed six positives which could not be confirmed by culture.     

The effect of air on the response of salmonellas in conductance media

The effect of air on the conductance response of salmonellas in three selective media was investigated. When assays were carried out aerobically, the time to observe a presumptive positive in all media was reduced and the conductance change was larger than in assays done under microaerophilic conditions. Low (10²/ml) numbers of pre-enriched salmonellas were detected only under aerobic conditions.     

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).     

Evaluation of lysine-iron-cystine-neutral red for the detection of salmonellas in the Bactometer

Ninety-five salmonellas and 40 non-salmonellas were screened in the Bactometer using lysine-iron-cystine-neutral red (LICNR) in order to evaluate the selectivity of the medium for the detection of salmonellas. Results for blackening of the medium in the well (indicating hydrogen sulphide, H₂S, production) and step size are presented. Five out of 95 salmonellas tested failed to produce blackening in the well, four of these five are known to be non-H₂S producers. Although salmonellas generally gave a larger step value than non-salmonellas, this criterion could not be used to distinguish reliably the two groups of organisms.     

Improvements to enhanced horseradish peroxidase detection sensitivity

At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13-fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.     

An ion-exchange based extraction method for the detection of salmonellas in soil

A method that uses a cation-exchange resin (Chelex 100) and differential centrifugation for the extraction and detection of salmonellas in soil was developed. The extraction efficiencies of a range of materials were examined and Chelex plus polyethylene glycol was identified as the best combination. Shake speeds, shake times and differential centrifugation speeds were selected to give an optimum salmonella recovery. The Chelex method accurately enumerated 1 cell per 10 g of nonsterile soil within 24 h. Addition of glycerol to soil samples enabled storage at — 70°C for 85 d without significant decreases in salmonella numbers. The Oxoid Salmonella Rapid Test (SRT) could be used to pre-screen large numbers of soil samples for the presence of salmonellas, prior to analysis by the Chelex method. The SRT method detected Salmonella typhimurium at levels as low as 2·5 cells per 10 g of nonsterile soil.     

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.     

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     

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).     

Use of conductance measurements to detect growth of Clostridium botulinum in a selective medium

Growth of Clostridium botulinum in a selective medium (SBM) was monitored using conductance measurements. The correlation of log ₁₀ counts of spores and vegetative cells with detection times was highly significant ( r = 0.96, 0.97; P < 0.001). Log₁₀ counts of Cl. botulinum growing in pasteurized pork slurries also showed a clear linear relationship with detection times ( r = 0.77) but the confidence limits (± log₁₀ 2.3) of the regression line were too wide to estimate numbers of Cl. botulinum. For growth studies of Cl. botulinum in pork slurries detection times were useful in determining whether or not a visually unspoiled sample contained growing cells. Knowledge that a sample contained growing cells allowed a count to be made within 24 h, whereas 48 h would elapse before results from a traditional plate count were available.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas

D.CHOI, R.S.W. TSANG AND M.H. NG. 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10⁶/ml of a smooth wild strain of Salm. typhimurium , and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.