GreenLight® System for Determination of Microbial Load An assessment of Bacterial Load in Raw Milk using GreenLight® Technology

The GreenLight® system was designed for applications across the food industry with further uses in environmental measurements. The core technology is a novel oxygen-depletion sensor that can detect very small changes in oxygen content making the system ideal for use in enumerating aerobic microbes. Here we describe a small comparative study conducted by Luxcel Biosciences, assessing correlation between the GreenLight system and standard plate count (SPC) for local raw milk. GreenLight demonstrated strong correlation to SPC with improvements in time-to-result, limit of detection and preparation costs over SPC. GreenLight has been AOAC and Microval certified for raw meats and poultry and further certifications in dairy products is ongoing. Introduction GreenLight Model 930 is designed to address the increasing demand for faster, simpler methods of determining bacterial load in food samples. The industry standard for TVC determination (ISO 4833:2003), also known as Standard Plate Count (SPC), is widely used but presents users with some very significant drawbacks. The method is both material and labor intensive, requiring the preparation and analysis of multiple agar plates per sample. More importantly, the method is slow, with 48-72 hours typically required for a definitive result [1-3] and determinations are inherently subjective. Application Note F13002 GreenLight® System for Determination of Microbial Load An assessment of Bacterial Load in Raw Milk using GreenLight® Technology Minneapolis, MN 55428 USA Phone 763.493.6370 E-Mail [email protected] www.microbialdetection.com Figure 1: GreenLight Model 930 The GreenLight series of instruments address these limitations, with GreenLight Model 930 providing a rapid high-throughput method for the assessment of bacterial load through analysis of microbial oxygen consumption [4,5]. The test sample is prepared in the usual manner and then simply added to a APCheckTM test vial. An oxygen sensor at the base of each vial is then interrogated kinetically by the GreenLight 930 instrument, producing oxygen profiles which reflect microbial growth (Fig. 2). As bacteria replicate, oxygen consumption rate increases. At a critical point, oxygen consumption exceeds back-diffusion and the sensor signal increases significantly. It then passes a pre-set threshold (Fig. 2A). The higher the initial load, the earlier this threshold level is reached, with instrument software automatically determining the timeto-result (Onset time) for each sample and subsequent bacterial load values using a pre-determined calibration (Fig. 2B). This simple procedure allows the rapid determination microbial contamination levels with results available in 1-12 hours depending on microbial load. GreenLight provides the capacity to handle high sample volumes and facilitates rapid turn-around times in critical limited shelflife applications. Figure 2: A) Oxygen-based growth curves from a serial dilution of E.coli across a range of contamination levels. B) The correlation between measured GreenLight time to result and bacterial load as determined by agar plate counting (ISO 4833:2003). To assess the feasibility of determining bacterial load in raw milk samples, a study was performed examining discrete samples over multiple days. A calibration curve was generated relating GreenLight time-to-result to SPC. Discrete samples were then measured using GreenLight 930 and the SPC reference method in parallel in order to assess assay performance and method correlation. Materials & Methods Standard Plate Count Prior to sampling, the raw milk samples were mixed thoroughly by inverting. A serial 10-fold dilution of each raw milk sample was prepared in PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 KH2PO4 g/L, pH 7.4) with 5 dilution steps per sample. 1mL of each dilution was added to a sterile agar plate in duplicate. 15mL of plate count agar, pre-cooled to 45°C, was poured onto each plate and swirled gently to mix. After agar solidification the plates were inverted and incubated for 72h at 30°C. Plates containing less than 300CFU/mL were counted and used to calculate the concentration of cells in the corresponding GreenLight 930 sample. GreenLight 930 Measurement Prior to sampling, the raw milk samples were mixed thoroughly by inverting. For standard curve generation, 1:10 and 1:100 dilutions were then prepared in a sterile nutrient diluent (10g/L granulated yeast extract, 3.5 g/L Na2HPO4, 1.5 g/L KH2PO4, 5g/L NaCl). For enumeration, only 1:10 dilutions were performed. 2mL of each dilution was added to an APCheck vial in duplicate. The samples were loaded as per manufacturer’s instructions and measured kinetically at 5 minute intervals on the GreenLight 930 instrument at 30°C. Maximum run time was set for 24 hours in order to accommodate negative control readings. Results & Discussions Preliminary Calibration Generation Samples were measured as outlined above at multiple dilutions to generate data points at both high and low levels of bacterial contamination. Samples were diluted in a nutrient broth to ensure optimum and uniform growth conditions, thereby circumventing any potential difficulties associated with milk sample variability and also facilitating the enumeration of samples with a bacterial load of greater than ~2x107CFU/mL. This approach generated a dataset in excess of 70 data points between 102 and 106 CFU/mL which provided a calibration function to convert measured time-to-result into microbial load values in units of LogCFU/mL. Method Evaluation & Comparison Using the calibration function generated as outlined above, a series of discrete samples were measured at 1:10 dilution and the predetermined calibration used to convert the measured time-to-result into a calculated microbial load in units of Log CFU/mL. These values