Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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 Caroline Chénard
Amy M Chan Amy M Chan

发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2691 浏览次数: 10423

评审: Dennis NürnbergQinglu ZengAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of mBio, featuring study using the protocol.

May 2016

Advertisement

Technologies for RNA Detection
“科研可重复性与创新”系列网络研讨会
Special Issue on Protocols for Organoid Research
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

原代人单核细胞的体外HIV-1侵染

原代人单核细胞的体外HIV-1侵染

Patrick O’Connell [...] Yasser A. Aldhamen

2019年07月05日 6177 阅读

固有层聚集培养模式中HIV-1介导的Gut CD4+ T细胞凋亡的量化分析

固有层聚集培养模式中HIV-1介导的Gut CD4+ T细胞凋亡的量化分析

Stephanie M. Dillon [...] Cara C. Wilson

2020年01月20日 5285 阅读

水稻条纹病毒纯化

水稻条纹病毒纯化

Gang Lu [...] Xiaorong Tao

2020年03月20日 4864 阅读

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) search Virus purification (病毒纯化) search Aquatic microbiology (水生微生物学) search Cyanophage (噬藻体) search Cyanobacteria (蓝藻) search MPN assay (MPN测定) search Liquid bioassay (液体生物检定方法) search

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

  1. 0.45 μm or 0.22 μm syringe filters (e.g., Millex HV or GV, PVDF) (EMD Millipore)
  2. Sterile disposable plastic syringes, 60 ml (BD, catalog number: 309653 )
  3. Glass 13 x 100 mm culture tubes or 24-well plates (polystyrene, e.g., Corning, catalog number: 3524 )
  4. 2 ml microcentrifuge tubes
  5. 96-well plates (polystyrene e.g., Corning, catalog number: 3599 )
  6. Parafilm
  7. Sterile disposable plastic syringes, 5 ml (BD, catalog number: 309646 )
  8. Sterile hypodermic needle (18 G 1 ½, BD, catalog number: 305196 )
  9. Aerosol barrier filter tips (SARSTEDT, catalog number: 70.1189.215 )
  10. Centricon Plus-70 (30,000 NMWL, EMD Millipore, catalog number: UFC703008 )
  11. Slide-A-LyzerTM Dialysis cassette (20,000 MWCO, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 66003 )
  12. Target host–clonal cyanobacteria strain, preferably axenic, but not necessary
  13. Freshwater sample (source of viruses)
  14. OptiprepTM density gradient medium (Axis Shield, catalog number: 1114542 )
  15. MilliQ water
  16. 0.1 N HCl
  17. Sodium nitrate (NaNO3)*
  18. Potassium phosphate dibasic (K2HPO4)*
  19. Magnesium sulfate heptahydrate (MgSO4·7H2O)*
  20. Calcium chloride dihydrate (CaCl2·2H2O)*
  21. Citric acid*
  22. Ferric ammonium citrate*
  23. Disodium EDTA*
  24. Sodium phosphate (Na2CO3)*
  25. Boric acid (H3BO3)*
  26. Manganese(II) chloride tetrahydrate (MnCl2·4H2O)*
  27. Zinc sulfate heptahydrate (ZnSO4·7H2O)*
  28. Sodium molybdate dehydrate (Na2MoO4·2H2O)*
  29. Copper (II) sulfate pentahydrate (CuSO4·5H2O)*
  30. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O)*
  31. Trace metal (TM) stock (see Recipes)
  32. 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

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

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

ris ris Download Citation in RIS Format

分类

微生物学 > 微生物细胞生物学 > 细胞分离和培养

细胞生物学 > 细胞分离和培养 > 细胞分离

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件