基于多种微生物的毒性检测MTOXPlate冻干板制备及优化

Preparation and optimization of lyophilised MTOXPlate for toxicity testing based on eleven different microorganisms

摘要:

【目的】制备基于多种微生物的生物毒性检测MTOXPlate冻干板, 从而简化操作程序, 使其能更灵敏、方便、快速地进行生物毒性测试。【方法】采用真空冷冻干燥法将指示微生物固定在微孔板中。以存活率为指标, 利用荧光分光光度法确定冻干保护剂的组成。通过分析毒性响应灵敏度对检测条件进行优化。【结果】MTOXPlate的冻干保护剂组成为8%海藻糖、2%葡萄糖、2%甘露醇、1%谷氨酸钠。在此组成下, 11株菌株的平均存活率最高, 可达89.41%。复活介质与测试样品同时加入, 在缩短了测试时间的同时也提高了检测灵敏度。该冻干板具有良好的灵敏度和重现性, 能较好地满足污染物生物毒性测试的需要。【结论】MTOXPlate冻干板可作为一种新型的生物毒性测试技术, 在生态毒理检测领域推广应用。

Abstract:

[Objective] In order to simplify the procedure and improve the sensitivity of biological toxicity testing method, the lyophilised MTOXPlate based on 11 genetically diverse microorganisms was prepared. [Methods] The tested microbial strains were fixed on the microplate by the vacuum freeze-drying method. Fluorescence spectrophotometry was used to determine the effects of several cryoprotectants on the freeze dried strains with the survival rate as the selecting index. The test condition was optimized through the sensitivity of toxicity detection. [Results] When the protective agent contains 8% trehalose, 2% glucose, 2% mannitol and 1% sodium glutamate, the mean cell survival rate can reach 89.41% for 11 microbial strains used in this study. The resurrection medium and test sample were added to each well of the pre-prepared 96-well microplate at the same time. So that, short testing time and optimal detection sensitivity could be obtained. This lyophilised MTOXPlate exhibits high sensitivity and good reproducibility, which is suitable for the toxicity assessment. [Conclusion] MTOXPlate could be a new biological toxicity test technology, which can be applied to the field of ecological toxicity testing.