一种分离和纯化噬藻体的液体生物检定方法

Caroline Chénard; Amy M Chan

doi:10.21769/BioProtoc.2691

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Peer-reviewed

Isolation and Purification of Viruses Infecting Cyanobacteria Using a Liquid Bioassay Approach

一种分离和纯化噬藻体的液体生物检定方法

Caroline Chénard

Amy M Chan email

发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2691 浏览次数: 9871

评审: Dennis NürnbergQinglu ZengAnonymous reviewer(s)

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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 (病毒分离)

Virus purification (病毒纯化)

Aquatic microbiology (水生微生物学)

Cyanophage (噬藻体)

Cyanobacteria (蓝藻)

MPN assay (MPN测定)

Liquid bioassay (液体生物检定方法)

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

0.45 μm or 0.22 μm syringe filters (e.g., Millex HV or GV, PVDF) (EMD Millipore)
Sterile disposable plastic syringes, 60 ml (BD, catalog number: 309653 )
Glass 13 x 100 mm culture tubes or 24-well plates (polystyrene, e.g., Corning, catalog number: 3524 )
2 ml microcentrifuge tubes
96-well plates (polystyrene e.g., Corning, catalog number: 3599 )
Parafilm
Sterile disposable plastic syringes, 5 ml (BD, catalog number: 309646 )
Sterile hypodermic needle (18 G 1 ½, BD, catalog number: 305196 )
Aerosol barrier filter tips (SARSTEDT, catalog number: 70.1189.215 )
Centricon Plus-70 (30,000 NMWL, EMD Millipore, catalog number: UFC703008 )
Slide-A-Lyzer^TM Dialysis cassette (20,000 MWCO, Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 66003 )
Target host–clonal cyanobacteria strain, preferably axenic, but not necessary
Freshwater sample (source of viruses)
Optiprep^TM density gradient medium (Axis Shield, catalog number: 1114542 )
MilliQ water
0.1 N HCl
Sodium nitrate (NaNO₃)*
Potassium phosphate dibasic (K₂HPO₄)*
Magnesium sulfate heptahydrate (MgSO₄·7H₂O)*
Calcium chloride dihydrate (CaCl₂·2H₂O)*
Citric acid*
Ferric ammonium citrate*
Disodium EDTA*
Sodium phosphate (Na₂CO₃)*
Boric acid (H₃BO₃)*
Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O)*
Zinc sulfate heptahydrate (ZnSO₄·7H₂O)*
Sodium molybdate dehydrate (Na₂MoO₄·2H₂O)*
Copper (II) sulfate pentahydrate (CuSO₄·5H₂O)*
Cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O)*
Trace metal (TM) stock (see Recipes)
BG-11 media (see Recipes)

*Note: All chemicals used in the media recipe are reagent grade (ACS grade) and were purchased from Thermo Fisher Scientific.

Equipment

P200 pipet (Eppendorf, catalog number: 3123000055 )
Automatic pipet (e.g., P1000)
Fluorometer (e.g., Turner Designs, model: TD-700 with optical kit Turner Designs, catalog number: 10-037R , excitation 350-500 nm, emission > 665 nm)
Vortex (e.g., Scientific Industries, model: Vortex-Genie 2 )
Ultracentrifuge (Beckman Coulter, model: Optima L-90K ) with SW-40Ti rotor (Beckman Coulter, model: SW 40 Ti , catalog number: 331302)
Thin-walled Ultra-Clear^TMopen-top ultracentrifuge tubes (14 ml, 14 x 95 mm) (Beckman Coulter, catalog number: 344060 )
Balance (micro-scale; used to balance centrifuge tubes)
Refrigerated table top centrifuge (e.g., Eppendorf, model: 5810 R with Rotor A-4)
Transmission Electron Microscope (TEM)
Magnetic stir bar
Beaker or Erlenmeyer
Autoclave

Procedure

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Chénard, C. and Chan, A. M. (2018). Isolation and Purification of Viruses Infecting Cyanobacteria Using a Liquid Bioassay Approach. Bio-protocol 8(2): e2691. DOI: 10.21769/BioProtoc.2691.