Institute of Microbial Technology, Sector 39 A, Chandigarh, 160036, India,
Cytotechnology. 2006 Jun;51(2):111-7. doi: 10.1007/s10616-006-9027-2. Epub 2006 Nov 3.
Preparation of isolated cells and microorganisms for ultrastructural examination always provides a challenge in terms of adequate immobilization of the cells and prevention of subsequent sample loss and damage during various steps of sample processing. Using a positively charged nylon membrane substrate we demonstrate that it is possible to easily immobilize and retain a sample of isolated cells in culture for a wide variety of microscopy-based techniques. Radiolabelled E. coli cells when immobilized on the charged membrane were seen to be highly resistant to detachment when subjected to the normal sample processing procedures associated with microscopy. In contrast cells on regular millipore membranes were rapidly lost during sample preparation. We demonstrate the utility of charged nylon membranes for a wide variety of microscopy based analysis including scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), TEM based immunogold labelling, laser confocal microscopy and SEM based elemental analysis.
为了进行超微结构检查,分离细胞和微生物的制备工作总是面临着挑战,需要找到合适的固定方法,防止细胞在处理过程中的各个步骤中丢失或受损。我们使用带正电荷的尼龙膜作为基质,证明了对于各种基于显微镜的技术来说,使用这种带正电荷的尼龙膜非常便于固定和保留培养中的分离细胞样本。当放射性标记的大肠杆菌细胞被固定在带电荷的膜上时,与显微镜相关的正常样本处理程序所导致的细胞脱离情况几乎可以忽略不计。相比之下,普通的 Millipore 膜上的细胞在样本制备过程中迅速丢失。我们证明了带电荷的尼龙膜在各种基于显微镜的分析中的应用价值,包括扫描电子显微镜(SEM)和透射电子显微镜(TEM)、原子力显微镜(AFM)、基于 TEM 的免疫金标记、激光共聚焦显微镜和基于 SEM 的元素分析。