Respirometric sensor vials for rapid and selective detection of coliforms in raw milk

Abstract

Traditional microbiological methods for coliforms detection, such as the Most Probable Number (MPN) assays, are as essential for food hygiene and safety as they are time-consuming and labor-intensive. We propose a new methodology for the rapid and sensitive detection and enumeration of coliforms in raw milk, which uses disposable 30 mL respirometric vials with built-in oxygen sensors and a handheld autonomous reader. Milk samples are added in sensor vials containing selective media and incubated at 37 °C with hourly readings of the sensor signals with FirestingO2 (Pyroscience) until the preset sensor signal threshold is reached, thus indicating cell growth and viability. The resulting time to threshold (TT, h) is then calculated and used to determine coliforms load in the milk sample by applying pre-determined calibration equation(s). Three versions of the sensor vial based coliform test were assessed: one-step assay in M-lauryl sulphate Broth (LSB), one-step assay in Brilliant Green Bile Lactose Broth (BGB) and the two-step sequential assay in LSB followed by BGB broths. The results of the sensor vials tests were benchmarked against the standard MPN ISO4831:2006 method for coliform detection, showing strong statistical correlation (demonstrated by correlation analysis), and time to result reduced more than 2-fold (≤11 h vs 24–72 h). Microbial isolates from the sensor vial tests were also analyzed by 16S rRNA sequencing, confirming the selectivity and accuracy of the respirometry technique. Thus, offering rapid results, ease of use, real-time monitoring, on-site deployment, cost and labor-saving capabilities, the sensor vial test provides an attractive alternative to traditional methods of coliform testing in milk and dairy products.