[Microbiological determination of netilmicin using a thin-layer chromatography scanner]

We presented a new colorimetric bioassay of aminoglycoside antibiotics, which were represented by netilmicin (NTL) in this study, based on the discoloration of thymolphthalein (TP) in paper (indicator-disc) by carbon dioxide produced by Bacillus subtilis. To evaluate the amount of the carbon dioxide, the following experiment was carried out. One milliliter of B. subtilis suspension containing 4.5 x 10(7) colony forming units/ml, 1 ml of nutrient broth, 0.9 ml of 0.1 M phosphate buffer (pH 8.0) and 0.1 ml NTL sample solution were added to an incubation container, which was then placed in a water-bath (37 degrees C) for 3 hours. The oxygen concentration in the head space of flask was determined using gas-chromatograph. The dose-response curves showed good correlation between amounts of NTL and carbon dioxide produced by B. subtilis. The indicator-disc containing TP and sodium hydroxide was placed into the Reacti-flask and then incubated in the same manner as described above. After incubation, concentration of blue colored TP was determined using a TLC scanner. The discoloration of blue color to white showed the proportionality between NTL concentrations and the degrees of discoloration of TP. The method can accurately measure NTL levels down to 2.5 micrograms/ml in water using 0.1 ml samples, and should be adequate for rapid bioassay.