噻唑蓝比色法检测细菌生长和存活状况的研究

建立了细菌生长和存活状况检测的噻唑蓝 (MTT)比色分析法。将S .aureus和E .coli菌以平板菌落计数法计数活菌数量 ,将一定数量细菌或经煮沸处理细菌加入微孔板 ,同时加入MTT ,于 36～ 37℃培养 ,加入DMSO溶解 ,于 51 0nm波长处测定A值 ;将一定数量活菌 ,加入不同数量MTT ,孵育不同时间 ,用不同量DMSO溶解 ,于不同波长处测定A值 ,以优化细菌MTT比色分析法条件。结果表明 :(1 )MTT能被活细菌利用 ,形成的fomazan数量与细菌浓度成线性关系 ;(2 )细菌经煮沸后MTT显色 ,A值明显降低 ,与煮沸时间成正比例 ;(3)MTT加入后在 0～ 2h内A值与作用时间成正相关 ;(4)MTT浓度增加 ,A值有一定程度增大 ;(5)以DMSO为Formazan溶解剂 ,其最大吸收波长范围为 51 0～ 530nm ;(6)以菌液体积的 2、3、4倍量加入DMSO均可较好地溶解Formazan颗粒。MTT比色分析法能反映活细菌的生长 ,存活状况 ;其较适宜的参数 :MTT终浓度0 3mg ml,作用时间 2h ,溶解剂DMSO量为菌液量 2倍 ,最大吸收波长范围为 51 0～ 530nm。

Study on tetrazolium salt colorimetric assay for growth and survival of bacteria

To establish a tetrazolium salt(MTT) colorimetric assay for bacteria growth and survival, living or boiling killed bacteria S. aureus and E. coli were incubated with MTT in microdilution plate for 36-37 degrees C at different time. DMSO was added as formazan dissolvent and the mixtures were measured for optical density(A) or absorbence at 510 nm wave length. Living bacteria were incubated with different amount of MTT for different time. The formazan were dissolved with different amount of DMSO and the A value were measured at different wave length. The results showed that yellow MTT could be reduced into blue formazan by living bacteria S. aureus and E. coli and the A value was directly proportional to concentration of the bacteria. A values of bacteria decreased with increasing of boiling time. To some extents, absorbence of living bacteria was direct proportion to MTT concentration and incubation time in the range of 0-2 hours. DMSO was good dissolvent for bacteria formazan and the highest absorbence could be reached in wave length range of 510-530 nm. It was suggested that MTT colorimetric assay could be used for measurement of bacteria growth and survival, and its optimal parameters were as follows: MTT concentration is 0.3 mg/ml. MTT incubation time was 2 hours. DMSO amount as dissoventis was 2-fold volume of bacteria suspension. Measurement wave length was 510-530 nm.