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Sensitivity of detection of bacteria with fluorescent and luminescent phenotypes using different instruments

The problem of bacterial enumeration in different samples is of great importance in many fields of research. Construction of recombinant fluorescent and luminescent bacteria that can be easily detected by nondestructive instrumental methods proves us with an opportunity to monitor bacteria in a wide variety of clinical, environmental and food samples in real time. Three different labels were employed: Green Fluorescent Protein (GFP), Bacterial luciferase (BL) and Firefly Luciferase (FFL). Both plasmid and chromosomal transformants of different strains of E. coli, P. putida and S. enteritidis were used. For the detection of the in vivo GFP the Shimadzu RF 540 spectrofluorimeter, Labsystems FL- 500 plate fluorimeter and Night Owl LB 98 CCD-camera from EG and G Berthold supplied with excitation light source and proper spectral filters both in macroscopic and microscopic mode were used. For the detection of in vivo luminescence of BL and FFL, tube luminometer BG-P from GEM Biomedical Inc., luminometric plate reader from BioOrbit, BIQ Bioview CCD camera from Cambridge Imaging Ltd and Night Owl LB 98 CCD camera both in macroscopic and microscopic mode were used. The expression levels of the labels, their stability, stability of the signal and detection limits of tagged bacteria were investigated. The detection limits for GFP tagged bacteria were 5 X 104 - 6 X 106, for BL tagged bacteria 5 X 102 - 2 X 105, and for FFL tagged bacteria - 4 X 103 - 106 CFU/ml, depending on the instrument used. Single bacteria could be detected with the help of the Night Owl in the microscopic mode.