Tail or artefact? Illustration of the impact that uncertainty of the serial dilution and cell enumeration methods has on microbial inactivation.

The estimation of the concentration of microorganisms in a sample is crucial for food microbiology. For instance, it is essential for prevalence studies, challenge tests (growth and/or inactivation studies) or microbial risk assessment. The application of serial dilutions followed by viable counts in Petri dishes is probably the most extended experimental methodology for this purpose. However, this enumeration technique is also a source of uncertainty. In this article, the uncertainty of the serial dilution and viable count methodology related to the sampling error is analyzed, as well as the approximation of the microbial concentration by the number of colonies in a Petri dish. We analyze from a theoretical point of view (statistical analysis) the application of the binomial and Poisson models, demonstrating that the Poisson distribution increases the variance when used to model individual serial dilutions. On the other hand, the binomial model produces unbiased results. Therefore, the Poisson distribution is only applicable when it is a good approximation of the binomial distribution, so the use of the latter is recommended. The relevance of this uncertainty is demonstrated by Monte Carlo simulations of a generic microbial inactivation experiment, where the only source of uncertainty/variability considered is the one generated by serial plating and viable cell enumeration. Due to both the uncertainty of the methodology and the omission of zero-count plates because of the log-transformation, the simulated survival curve can have a tail. Therefore, this phenomenon, which is usually attributed to biological variability, can be to some extent an artefact of the experimental design and/or methodology.