微丝菌(Microthrix parvicella)原位荧光杂交(FISH)定量过程的条件优化

Optimization for Microthrix parvicella Quantitative Processing of Fluorescence in situ Hybridization(FISH)

微丝菌(Microthrix parvicella)是世界范围内诱发活性污泥膨胀现象的主要丝状菌之一,它在活性污泥中准确的原位定量解析对污泥膨胀现象及控制策略研究具有非常重要的意义.由于微丝菌自身的特殊生理生化性质(如表面高疏水性及较厚细胞壁)易导致常规荧光原位杂交(FISH)过程中定量结果偏低.本研究针对FISH过程中存在的探针渗透率低、荧光信号偏弱等现象,从活性污泥样品前处理、杂交过程条件等方面对Microthrix parvicella的FISH定量过程进行了优化.结果表明,在前处理使用溶菌酶(浓度为36 000 U·m L^(-1)),探针浓度为4.5 ng·μL^(-1),杂交时间延长至4 h的条件下,Microthrix parvicella的FISH定量结果可从1.12%提高至96.70%,并与定量PCR(q-PCR)结果和Eikelboom&Jenkins法(镜检观察)定量结果更为趋近一致.

Precise quantification of Microthrix parvicella, which is identified as a dominated filamentous bacterium of bulking sludge in the worldwide, is essential for bulking investigation and related control strategies. However, quantitative processing based on fluorescence in situ hybridization （ FISH） is prone to interference due to the specific characteristics of Microthrix parvicella （hydrophobic surface with thick cell wall）. Our study focused on pretreatment and process optimization to show that the proportion of Microthrix parvicella was increased from 1. 12% to 96. 70% benefited by lysozyme （36 000 U-mL - 1 ）, high probe concentration （4. 5 ng·μL - 1 ） and longer hybridization time （4 h） employed, mapping with the results of q-PCR method and Eikelboom ﹠ Jenkins Observation.