基于秸秆厌氧干发酵的FISH预处理条件优化

Optimization of Sample Pretreatment for Qualification of the Microbes in Solid State Anaerobic Digestion of Corn Stover by Fluorescence in situ Hybridization

优化了荧光原位杂交技术(FISH)定量测定秸秆厌氧干发酵基质中微生物的样品预处理条件,并在最优条件下做了验证性实验。结果表明:使用无水乙醇作为清洗液,FISH样品的荧光信号强度和丰度比用PBS清洗液(磷酸缓冲盐溶液)时有显著提高;样品预处理过程中加无菌玻璃珠震荡能提高微生物的分散度,从而提高FISH技术对秸秆厌氧发酵基质中微生物定量的准确性;采用离心转速为13 000 r/min时,微生物菌体收集率提高,而且还能保证完好的细胞形态,为后续的FISH杂交奠定基础。

Sample pretreatment method for qualification of the microbes in dry anaerobic digestion by FISH was optimized. Results showed that using absolute ethanol as cleaning fluid, area and abundance of fluorescence signal of the FISH sample was significantly improved than that of PBS. Adding sterilized glass beads in the process of sample stirring can improve the dispersion of microbe cells and then enhance the accuracy of the microbial quantification by the FISH technical for dry anaerobic digestion. When centrifuging with 13 000 r/min for 3 min, the conversion rate is increased, and it will ensure the integrity of cell morphology and lay the foundation for the experiment of subsequent FISH hybridization.