BLT Stages

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This bachelor’s thesis has two subjects: measurement uncertainty tests for different parameters in microbiology food testing and the method validation of Pseudomonas cetrimide naladixic acid (PCN) agar and Rapid Pseudomonas aeruginosa (RPSA) agar for quantitative analysis of Pseudomonas aeruginosa in water samples.

The measurement uncertainty is one of four validation parameters for a microbiological quantitative method validation. It is a parameter that quantifies the dispersion of results. These results are results of analyses in double with several quantitative methods, used in Eurofins Food Testing Belgium. The dispersion of these results is caused by varying the in double analyses on the basis of black box parameters. These black box parameters include a different analyst, different sampling, different matrices and others.  For the measurement uncertainty tests, four matrix categories must be tested. Category 1 are liquids and powders, category 2 are mixed solid food samples, category 3 are small solid food samples and category 4 are other solid food samples. The target is to test 10 samples for each matrix category for each quantitative method.

For the measurement uncertainty tests, previously analysed samples are used. These samples are naturally contaminated, which cause that these samples contain realistic concentrations of microorganisms. Each of these samples are registered in double and analysed in with other food samples in the flow of the routine.

A lot of widely tested methods in Eurofins Food Testing Belgium mostly achieved the target of ten samples for each matrix category. For a lot of less tested methods, the target isn’t achieved. There are various reasons. First, category 1 products aren’t widely tested in Eurofins Food Testing Belgium. Second, for some parameters food companies take precautions to avoid these micro-organisms in their products (ex. Listeria monocytogenes). Third, a lot of parameters (like anaerobic parameters or coliforms) experience difficulties to survive in the storing conditions (-20°C and aerobically).

Pseudomonas cetrimide naladixic acid (PCN) agar and Rapid Pseudomonas aeruginosa (RPSA) are both growth media for Pseudomonas aeruginosa. Pseudomonas aeruginosa is quantitative analysed by a membrane filtration. For this analysis method two media can be used. PCN agar is routinely used in Eurofins Food Testing Belgium for the quantitative analysis of Pseudomonas aeruginosa in water samples. This medium has a major drawback. The confirmation tests are complex and intensive. As an alternative, RPSA agar can be used. This medium needs no confirmation. In the method validation, both methods must be compared, to decide if RPSA agar can be used as an alternative for PCN agar.

A method validation for a quantitative method has four validation parameters: trueness, repeatability, reproducibility and measurement uncertainty. Both media are compared based on these four parameters.

Two separate validations were performed. For trueness, no data was acquired. In the first validation, samples of drinking water, groundwater and surface water were spiked with Pseudomonas aeruginosa. The samples were tested on both media and evaluated on the parameter repeatability. Both media have comparable results, but the variability is high, because there is a low colony count on the plates.

In the second validation, samples of drinking water and water in bottles for consummation were spiked with a higher concentration of Pseudomonas aeruginosa and were evaluated on repeatability, reproducibility and measurement uncertainty for both media. The results for both media were comparable and have a low variability.

Based on the results of both validations can be concluded that RPSA agar is a possible alternative to PCN agar.