Studies on a colorimetric bioassay for netilmicin using an indicator disc

Abstract

A new colorimetric bioassay of an aminoglycoside antibiotic, netilmicin (NTL), was investigated. Discoloration of the color-indicator on the paper disc (indicator disc) is due to the presence of CO₂ produced by Bacillus subtilis. Discoloration of the indicator disc by CO₂ was studied as follows: A mixture of B. subtilis suspension, nutrient broth, 0.1 M phosphate buffer (pH 8.0) and NTL solution was transfered to a 5 ml Reacti-Vial^®. An indicator disc containing thymolphthalein (TP), methyl red (MR) and NaOH was attached to the inside of the Reacti-Vial septum. The vials were then placed in a shaking water-bath (37±1°C) for 3 h. The blue indicator disc was discolored, the degree of discoloration depending on the growth of B. subtilis in the medium. When the concentration of NTL was high and the level of CO₂ low, no change was observed. The color of the indicator disc was determined spectrophtometically after 3 h of incubation, using complementary tristimulus colorimetry, which is useful in determining pH-dependent chemical species like TP and MR. Our proposed method was found to save more than 13 h of working time when compared with the conventional bioassay method, so it is a useful and convenient method for determining NTL.