Abstract
The following protocol describes the isolation and purification of viruses infecting cyanobacteria using a liquid bioassay approach. Viruses infecting cyanobacteria are also known as cyanophages. This protocol was written specifically for the isolation of cyanophages infecting freshwater cyanobacteria particularly, cyanobacteria that cannot be cultured on solid media. The use of a clonal cyanobacterial culture is recommended for the isolation of viruses. Growth conditions (i.e., media, light cycle and temperature) should be modified based on the host of interest.
Keywords: Virus isolation, Virus purification, Aquatic microbiology, Cyanophage, Cyanobacteria, MPN assay, Liquid bioassay
Background
Cyanobacteria are important phototrophs in both marine and freshwater systems. As other aquatic microorganisms, cyanobacteria are subject to viral infection (Suttle, 2000). For instance, in marine coastal regions, titers of viruses infecting Synechococcus spp. can reach up to 105 ml-1 and differ based on temperature, salinity and host abundance (Suttle and Chan, 1993; Waterbury and Valois, 1993). Despite the ecological importance of cyanobacteria and their viruses (also known as cyanophages), only a small number of viruses have been isolated from only a limited number of cyanobacterial strains. Consequently, it is of great interest to isolate new viruses by screening with new strains of cyanobacteria. The following protocol is relevant for both marine and freshwater systems but the example given below will focus on the isolation and purification of viruses infecting freshwater cyanobacteria (Chénard et al., 2015). The advantage of the liquid bioassay method over published protocols using solid substrate, is the opportunity to target cyanobacteria that cannot tolerate the higher temperatures often employed with the plaque assay method or cannot grow on solid media.
Materials and Reagents
*Note: All chemicals used in the media recipe are reagent grade (ACS grade) and were purchased from Thermo Fisher Scientific.
Equipment
Procedure
Definitions Titer–concentration of infective viruses MPN–most probable number Lysate–a solution containing the product released from lysis of cells
Recipes
Notes:
Acknowledgments
CC is supported by the Singapore’s National Research Foundation under its Marine Science Research and Development Programme (Award No. MSRDP-P13). Development of these protocols is supported in part by the Canadian Foundation for Innovation, Leaders of Opportunity Fund (Project # 25412) and National Sciences and Engineering Research Council of Canada (EQPEQ 375995-09). This protocol was inspired and adapted from previous works: Middelboe et al. (2010); Suttle and Chan (1993); Chénard et al. (2015). The authors do not have any conflicts of interest or competing interests to declare.
References
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