阿利新兰刚果红染色法在烟曲霉菌生物膜胞外多糖染色中的应用研究  

Application of alcian blue congo red staining in dyeing of extracellular polysaccharide in Aspergillus fumigatus biofilm

作　　者：黄宏[1] 陈一强[1] 孔晋亮[1] 温汉春[1] 侯长春[1] 王可[1] 

机构地区：[1]广西医科大学第一附属医院呼吸疾病研究所,广西南宁530021

出　　处：《中华医院感染学杂志》2015年第9期1934-1936,共3页Chinese Journal of Nosocomiology

基　　金：国家自然科学基金资助项目(81260002)

摘　　要：目的研究烟曲霉生物膜中胞外多糖染色的新方法 ,评价阿利新兰刚果红染色对烟曲霉胞外多糖染色的可靠性。方法将烟曲霉受试菌株的浓度为1×105孢子/ml的孢子悬液1ml,加入到24孔的组织培养板中的无菌玻璃细胞爬片上,37℃孵育,分别在孵育的不同时间用阿利新兰刚果红复合染色染胞外多糖,在光镜下观察曲霉生物膜胞外多糖的变化,与电镜下观察到的生物膜胞外基质相比较。结果阿利新蓝刚果复合染色后光镜下观察发现,菌丝被染成蓝色,胞外多糖为深红色;第8小时孢子萌芽,第12小时菌丝延长,第16小时菌丝的周围开始出现深红色的胞外多糖,第20小时深红色的菌丝之间有深红色的胞外多糖,第24小时深红色的胞外多糖布满载体表面,第48小时菌丝基本完全被胞外多糖包裹;整个生长过程与扫描电镜观察生物膜变化的结果完全相符。结论用阿利新兰刚果红染色观察烟曲霉生物膜胞外多糖简单、可靠、重复性好,适用于临床检验和实验室研究。OBJECTIVE To seek a new method to dye the extracellular polysaccharide in the A spergillus f umigatus biofilm and evaluate the reliability of alcian blue congo red staining used for dyeing extracellular polysaccharide in A . f umigatus .METHODS In vitro biofilm of A .f umigatus formed on the surface of sterile glass cell culture cov‐ered slipes in 24‐well tissue culture plates by adding 1 ml of 1×10^5 spores·ml cell suspension to each well .After incubation at 37 ℃ for various time periods ,the extracellular polysaccharide in biofilms were stained with alcian blue congo red stains .The changes of the extracellular polysaccharide were observed under light microscopy and were compared with the results of the extracellular matrix that were observed under scanning electronic microscope （SEM ） .RESULTS After being stained by alcian blue congo red and observed under light microscopy ,the strains were dyed blue ,while the exopolysaccharides were deep red under an optical microscope .As the culturing time in‐creased ,spore germination developed into mycelial grow th;part of the spores had germinated at hours;mycelial extension was observed at 12 hours ;mycelia continued to grow and the blue mycelia were surrounded by a few deep red exopolysaccharides at 16 hours;the exopolysaccharides appeared among the mycelia and connected the mycelia at 20 hours ;the carrier was covered by exopolysaccharides at 24 hours ,except for a few blue mycelia un‐covered by exopolysaccharides on the top;the mycelia were completely covered by the unevenly distributed exopo‐lysaccharides at 48 hours .The whole process was in accordance with the results that were observed under SEM . CONCLUSION It is convenient and reliable to observe the A .f umigatus biofilms by using alcian blue congo red staining ,which is suitable for the clinical laboratory examination .

关 键 词：生物膜 烟曲霉菌 胞外多糖 阿利新蓝刚果红染色 

分 类 号：R379[医药卫生—病原生物学]