A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters
A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters
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计数和鉴定吸附在硝酸纤维素膜过滤器上的可培养病毒的简单方法
DOI：
10.1128/aem.66.1.194-198.2000
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发表时间：
2000
影响因子：
4.4
通讯作者：
J. Jofre
中科院分区：
文献类型：
--
作者：
Georgios T. Papageorgiou;L. Mocé;C. Christodoulou;F. Lucena;D. Akkelidou;E. Ioannou;J. Jofre
摘要我们确定了水牛绿猴细胞在硝酸纤维素膜过滤器表面生长的条件，使其覆盖每个过滤器的整个表面并穿透毛孔。当使用这种条件时，以前吸附在细胞膜上的脊髓灰质炎病毒感染细胞并复制。采用空斑分析方法和定量方法(最可能的细胞病变单位数)检测和计数吸附在滤膜上的病毒。水悬浮液中的脊髓灰质炎病毒然后通过吸附到纤维素膜过滤器上进行浓缩，随后无需洗脱即可计数，这一步骤在使用常用方法时是必要的。针对自来水、海水和0.25M甘氨酸缓冲溶液，对膜过滤器的孔径、样品含量和样品体积进行了优化。在优化的条件下，回收的病毒数比标准空斑计数多50%以上。在常用抗生素的基础上加入头孢他啶，可用于研究微生物污染程度较低和中等的天然样品，无需对样品进行去污处理。这种方法还允许在转移制剂后直接在硝酸纤维素膜上或在Hyond N+膜上进行斑块杂交。
ABSTRACT We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.