Doctoral Degrees (Food Science)

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Browsing Doctoral Degrees (Food Science) by Author "Brand, Amanda Salome"

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    Critical evaluation of the accuracy of the enumeration methodology of Coliforms and E. Coli in water from rivers used for the irrigation of fresh produce
    (Stellenbosch : Stellenbosch University, 2012-12) Brand, Amanda Salome; Britz, T. R.; Sigge, G. O.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: The accuracy of methods for the enumeration of coliforms and Escherichia coli present in river water intended for the irrigation of fresh produce has been critically evaluated to determine whether the results of the traditional method were reliable in indicating faecal pollution. The potential of rapid alternative methods were also explored. Baseline monitoring of the Berg River showed the presence of potential pathogens such as Salmonella, and also that E. coli levels exceeded international guidelines for the safe irrigation of minimally processed foods (MPFs) in 20.5% of cases, which indicated faecal intrusion. An exploratory study into the use of microbiological and physico-chemical parameters in predicting E. coli numbers, as a rapid alternative to direct enumeration, was conducted. These measurements, neither individually nor in combination, could accurately predict the E. coli numbers. The rapid method Colilert-18 was compared against multiple tube fermentation (MTF) for the enumeration of coliforms and E. coli. Spearman rank correlation coefficients showed that Colilert-18 had acceptable (r2=0.69) and fair (r2=0.74) correlations with MTF for coliform and E. coli enumeration, respectively. Bland and Altman statistics were used to determine pollution influence, and Colilert-18 showed increasing disagreement with MTF at very high concentrations of coliforms and E. coli. Bacterial isolates obtained from MTF reactions were identified using biochemical and mass spectrometry methods. These identifications revealed that the greatest contributors to inaccurate coliform enumeration by MTF were false negative coliforms which fail to produce gas from lactose. Numerical biochemical data suggested that these isolates may be able to use other carbohydrates preferentially over lactose. Inaccurate E. coli enumeration was caused by E. coli strains which could not utilise lactose or 4-methylumbelliferyl- -D-glucuronide (MUG), as well as non-E. coli isolates which were able to hydrolyse MUG. The method of transfer of bacteria between MTF media was also identified as problematic for accuracy. Monoplex polymerase chain reaction (PCR) differentiation of MTF isolates showed that detection of the uidA gene showed the greatest accuracy in the detection of E. coli, while the multiplex PCR protocol for detecting diarrheagenic E. coli pathotypes identified one strain of enteroaggregative E. coli (EAEC). A qualitative methodological risk classification was used, in combination with the individual reactions of MTF isolates, to elucidate their contribution to enumeration inaccuracy and to evaluate the effect of MUG and Levine-eosin methylene blue (L-EMB) agar. The classification indicated that inaccurate enumeration of E. coli was more problematic than that of coliforms, but revealed that the exclusion of MUG from MTF may increase the accuracy of E. coli enumeration. The omission of L-EMB would have very little effect on E. coli enumeration accuracy. This work confirmed that MTF is fairly reliable in the enumeration of coliforms and E. coli. Inaccuracies are primarily attributable to atypical organisms which are considered to make up a small proportion of the total bacterial population. Colilert-18 was shown to be an acceptably accurate alternative, and its rapid production of results can be highly advantageous in the monitoring of irrigation water used for MPFs.