Construction of a convenient microbial-growth observation system based on microscopic image analysis applicable to the evaluation of preservative effects

Abstract

The pour-plate method, a conventional approach to the evaluation of food microbial contamination, is costly and time-consuming. We have developed a novel and efficient system to evaluate microbial growth on the surfaces of foods using a membrane filter and microscope followed by image analysis. While Wickerhamomyces anomalus cells directly inoculated onto loin ham were undetectable because of the complex surface structure of the food, the placement of a hydrophilic polytetrafluoroethylene membrane filter on the loin ham enabled us to clearly observe the yeast cells on the filter by microscopy without affecting their growth. Under microscopic observation, cells on the membrane filter as well as those on the agar plate were always observed with surrounding annular dark regions, which were separated from the background by clear boundaries. We defined the areas inside the boundaries, including the cells and the annular dark regions, as the “bounded areas”. In image analysis, the numbers of pixels in the bounded areas increased exponentially in parallel with the viable cell count (CFU/g), suggesting that the pixel numbers were proportional to the viable cell numbers. We applied this system to investigate the adverse effects of sorbic acid preparation (SAP), a common food preservative, on yeast growth. The yeast was incubated on filter-overlaid agar medium in the presence or absence of SAP, resulting in a dose-dependent reduction in the specific pixel increase rate (k [/h]) determined by image analysis as well as in the specific growth rate (μ [/h]) estimated by the agar-plate-based conventional method. Regression analysis revealed a strong correlation between the k and μ values (R2, 0.985) normalized to the corresponding values without SAP. Our system could be applied to estimate the growth rate of microbes on foods and to examine the effects of environmental factors on microbial growth by using the k value as an index. Furthermore, this system would contribute to enhancing food safety, extending shelf life, and solving the important global problem of food loss.