The fluorescence spectrum of bacterially bound acridine orange (AO) was investigated to evaluate its use for the rapid enumeration of bacteria. Escherichia coli ATCC 25922 samples were stained with 2 × 10-2, 2 × 10-3 or 2 × 10-4% w/v AO, followed by 3, 2 or 0 washing cycles, respectively, and fluorescence spectra were recorded using a fibre-based spectroscopic system. Independent component analysis was used to analyse the spectral datasets for each staining method. Bacterial concentration order of magnitude classification models were calculated using independent component weights. The relationship between fluorescence intensity of bound AO and bacterial concentration was not linear. However, the spectral signals collected for AO stain concentration-bacterial concentration pairs were reproducible and unique enough to enable classification of samples. When above 105 CFU ml-1, it was possible to rapidly determine what the order of magnitude of bacterial concentration of a sample was using a combination of two of the sample preparation methods. A relatively inexpensive (around US$10 per test) rapid method (within 25 min of sampling) for enumeration of bacteria by order of magnitude will reduce the time and cost of microbiological tests requiring gross concentration information. Graphical Abstract Fluorescence spectra of bacterially bound acridine orange (AO) were used for the rapid enumeration of bacteria. Order of magnitude bacterial concentration classification models were calculated using independent components analysis of these fluorescence spectra. When above 105 CFU ml-1, it was possible to rapidly determine the order of magnitude of bacterial concentration of a sample using a combination of two sample preparation methods.
Keywords: Acridine orange; Bacteria; Enumeration; Fluorescence spectroscopy; Microbial contamination.