Optical time-lapse microscopy for rapid assessment of microbial quality in hygroscopic food samples

In the food industry, time-to-result is crucial for faster release of products, minimising recalls, mitigation of microbial contamination problems and, ultimately, food safety. Carrageenan is isolated from red seaweed (Rhodophyta) and applied in various foods and beverages as a gelling, thickening, texturing, or stabilising agent due to its hygroscopic properties. Currently, the standard industry plate count method entails a one-hundred-fold dilution of the sample before mixing with molten agar for assessing the level of microbial contamination in carrageenan samples prior to business-to-business shipment. However, even at this dilution, carrageenan swells, forms clumps, clogs pipettes, and leaves thick gel structures, bubbles, and debris in agar plates causing microbial enumeration to be challenging and subject to human error. Here, we report, for the first time, the application of mini agar plates monitored by the automated time-lapse microscopy IntuGrow solution to assess the microbiological quality in the challenging food ingredient. Without dilution of the food sample, the carrageenan powder is scattered between two layers of Plate Count Agar to enumerate bacteria within 12-20 h, while enumeration by traditional plate counts requires 72 h. A DELAY algorithm for optical time-lapse microscopy was developed and added to IntuGrow analysis software to suppress the effects of swelling and enhance detection of the presence of growing microbial colonies by normalising the background using previous images. Time-lapse microscopy image-based monitoring made it possible to obtain results from carrageenan samples that could not be obtained by traditional plate counts due to swarming bacteria. Comparison between the two methods showed a nearly perfect Demings slope of 0.96, while an observed bias of -0.33 log CFU/g indicated that IntuGrow counts were lower than traditional plate counts. This is likely due to carrageenan artefacts being counted as colonies in the latter plates. The ability of IntuGrow to enumerate bacteria in challenging food ingredients such as carrageenan implies that the technology should be easy to apply for easy-to-dilute samples or non-hydrocolloid powders. Further testing in an industrial setting by different operators should be used to validate the reproducibility of the method.