Investigations into the detection of injured Salmonella typhimurium in foodstuffs

A thesis submitted to the Department of Biology and Health Science, University of Luton, in partial fulfilment of the requirements for the degree of Doctor of Philosophy DepartmentA chemically defined medium for Salmonella growth was developed and optimised using supplements of amino acids, nucleosides, vitamins and different carbon sources. The medium developed was compared to commercially available pre-enrichment media BPW and Salmosyst. Growth of Salmonella was significantly higher in BPW and Salmosyst than the medium developed. The amino acid and nucleoside supplement was directly compared with peptone. The results show peptone to be nutritionally superior and promoting better growth of Salmonella. Three different ELISA assays were used to detect Salmonella growing in four different media. The ELISA assay sensitivity was determined and a degree of media interference with the immunoassays was established. Salmonella culture viability was investigated using three different procedures: differential culturing on selective and non-selective media; fluorescence microscopy with BACLIGHT stained cells and flow cytometry analysis of BACLIGHT and BEP stained cells. Flow cytometry was found to be the most consistent, sensitive and rapid procedure for cell viability measurement. Clusters of viable cells unable to grow on solid media and therefore remaining undetectable by cultural methods were identified using flow cytometry. Severely heat injured Salmonella was used to determine media recoverability. The results indicate that media which contain peptone recover injured Salmonella better than chemically defined or other media. Detection of Salmonella was performed using PCR assay after sample pre-enrichment. The amplification of Salmonella DNA extracted using a crude method resulted in an assay sensitivity of 20 Salmonella cells in pure cultures. The specificity of the oligonucleotide primers employed in the PCR assay was confirmed. Non-salmonella organisms present in high numbers interfered with PCR detection of Salmonella. Food components also interfered with PCR amplification and reduced the assay sensitivity. Interference by food components and non-salmonella DNA was eliminated by the use of a 24 hour pre-enrichment followed by a 3 hour secondary enrichment, a rapid DNA extraction and template preparation. Using this system it was possible to detect 3 Salmonella cells per gram of food in the presence of 106 non-salmonella cells within 28 hours