The use of fluorescent optical respirometry for microbiological development and evaluation of pharmaceutical preparations

The determination of the number of microorganisms is very important in the biotechnology, pharmacy and food industries. Monitoring the quality of pharmaceuticals and food products requires fast, sensitive and selective methods to detect a small number of viable bacterial cells. Isolation of the natural compounds presented in the food with antibacterial properties requires testing of many samples, against many bacteria in a short time.

Counting bacteria on the agar plates, membrane filters, and using the “most probable number" are basic methods used to determine of the living bacteria. The methods require a long incubation time, colonies may be formed by several related species of bacteria, and full identification takes up to seven days. The serial dilution method in broth, used in clinical microbiology allows determination of the minimal inhibitory concentration. The length of assay time and the impact of the physical properties of the sample affect the results.

We used a fluorescence oxygen-sensitive sensor, ruthenium-tris(4,7-diphenyl-1,10-phenanthroline) dichloride (Ru(DPP)₃Cl₂), the fluorescence of which depends on the amount of oxygen in the tested sample, applied in the fluorescent optical respirometry (FOR) method. Molecular oxygen is a fluorescence quencher. Growing microorganisms consume oxygen, thus influencing the intensity of fluorescence in the sample. The FOR method was performed to evaluate the effect of chemical and environmental factors, plant extracts on aerobic bacteria. The FOR method allows to detect bacteria in sterile and non-sterile pharmaceutical products. This method allows also for a rapid, unequivocal detection and counting of living bacterial cells.