Enumeration and isolation of viral particles from oligotrophic marine environments by tangential flow filtration.

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.