Alonso M C, Rodríguez J, Borrego J J
Department of Microbiology, Faculty of Sciences, University of Málaga, Spain.
Int Microbiol. 1999 Dec;2(4):227-32.
A method for concentrating, enumerating and isolating viral particles from marine water samples was developed and evaluated. The method consists of a concentration step by a tangential flow filtration (TFF) system, ultrafiltration by centrifugal concentrator, and visualization by transmission electron microscopy (TEM). This procedure allows to reduce volumes of ca. 21 of seawater to 10-20 microliters, which can be dispensed on electron microscopy grids to count total viral particles. This method allows the recovery of small numbers of viral particles from oligotrophic seawater samples, in which viral numbers ranged from 10(5) to 10(6) viral particles/ml. The tangential flow filtration system was evaluated as quantitative technique using suspensions of two different bacteriophages (T6 and phi X174) in autoclaved seawater. Recovery rates varied depending on both the viral morphology and flow rate; recovery percentages reached 117.4% for T6 and 60.6% for phi X174 using low flow rate.
开发并评估了一种从海水样本中浓缩、计数和分离病毒颗粒的方法。该方法包括通过切向流过滤（TFF）系统进行浓缩步骤、通过离心浓缩器进行超滤以及通过透射电子显微镜（TEM）进行可视化。此程序可将约2升海水的体积减少至10 - 20微升，这些体积可分配到电子显微镜网格上以计数总病毒颗粒。该方法能够从贫营养海水样本中回收少量病毒颗粒，其中病毒数量范围为每毫升10⁵至10⁶个病毒颗粒。使用两种不同噬菌体（T6和phi X174）在高压灭菌海水中的悬浮液，对切向流过滤系统作为定量技术进行了评估。回收率因病毒形态和流速而异；使用低流速时，T6的回收率达到117.4%，phi X174的回收率达到60.6%。
