Analysis of Total Aerobic Viable Counts in Raw Fish by High-Throughput Optical Oxygen Respirometry

Abstract

A simple, miniaturized, and automated screening assay for the determination of total aerobic viable counts in fish samples is presented here. Fish tissue homogenates were prepared in peptone buffered water medium, according to standard method, and aliquots were dispensed into wells of a 96-well plate with the phosphorescent, oxygen-sensing probe GreenLight. Sample wells were covered with mineral oil (barrier for ambient oxygen), and the plate was monitored on a standard fluorescent reader at 30°C. The samples produced characteristic profiles, with a sharp increase in fluorescence above the baseline level at a certain threshold time, which could be correlated with initial microbial load. Five different fish species were analyzed: salmon, cod, plaice, mackerel, and whiting. Using a conventional agar plating method, the relationship between the threshold time and total aerobic viable counts load (in CFU per gram) was established, calibration curve generated, and the test was validated with 169 unknown fish samples. It showed a dynamic range of 10(4) to 10(7) CFU/g, accuracy of ± 1 log(CFU/g), assay time of 2 to 12 h (depending on the level of contamination), ruggedness with respect to the key assay parameters, simplicity (three pipetting steps, no serial dilutions), real-time data output, high sample throughput, and automation. With this test, quality of fish samples, CFU-per-gram levels, and their respective time profiles were determined.

Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation

“…In addition to the generation of a more complete picture of capture capability, the Greenlight TM system is automated, hands-on time is minimal, a simple 'mix and measure' protocol is used, it is high-throughput, producing 96 simultaneous results and in real-time with results available within 12-24 h. These attributes have also been reported by Hempel et al (2011) and Borchert et al (2012).…”

High throughput quality and safety assessment of packaged green produce using two optical oxygen sensor based systems

“…The new rapid TVC test for green produce was also compared to the similar tests described previously for raw meat (O'Mahony et al, 2009) and fish (Hempel et al, 2011) samples. TVC calibrations for these three tests reproduced from the corresponding analytical functions are very similar.…”

“…Respirometric TVC assays were performed as described previously (Hempel et al, 2011). Briefly, a vial of the O 2 -sensitive probe GreenLight was reconstituted in 1 ml of PBW and 0.1 ml of this solution was dispensed in each assay well of the 96-well plate.…”

“…A number of rapid microbiological methods have been developed in recent years. In particular, optical oxygen respirometry represents a simple, high-throughput, automated micro-assay platform which allows rapid TVC testing and enumeration of bacteria in complex food samples such as raw meat and fish (Hempel et al, 2011;O'Mahony, Green, Baylis, Fernandes, & Papkovsky, 2009). It has recently become available commercially as the GreenLight Ò system (http://www.mocon.com).…”

Rapid detection and respirometric profiling of aerobic bacteria on panels of selective media

“…This methodology is also very efficient in the detection of aerobic bacteria. In particular, it has allowed simple and rapid determination of total viable counts (TVC) in complex food samples (crude homogenates of raw meat and fish), with the use of standard 96-well plates and commercial fluorescence reader (Hempel et al 2011). The assay combines high degree of automation, instrumental readout, and minimal labour and space requirements.…”