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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   

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mBio
May 2016

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

  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

Definitions
Titer–concentration of infective viruses
MPN–most probable number
Lysate–a solution containing the product released from lysis of cells

  1. Isolation of cyanophages
    1. Collect ca. 50 ml water sample in a clean container (e.g., Falcon tube); pre-rinse container several times with the sample before filling. If possible, keep sample cold and in the dark until the next step.
      Note: Sample volume needed will vary depending on the productivity of the environment, ranging from a few ml for productive environments, to tens of liters for oligotrophic environments. When necessary, viruses can be concentrated from large volumes of water samples to increase the detection limit. For more details on the use of ultrafiltration to isolate viruses, see Suttle et al. (1991).
    2. Remove bacteria and plankton from the sample by filtration; use a 0.45 μm or 0.22 μm pore size syringe filter and a 60 ml plastic syringe. Store filtered sample in the dark, at 4 °C until use.
    3. Prepare exponentially growing target cells.
    4. Transfer ca. 3 ml of exponential growing cultures to 5 sterile glass 13 x 100 mm culture tubes (use a minimum of 3 tubes per water sample and 2 tubes as negative controls).
    5. Add 1 ml of BG-11 media (see Notes for Recipes) to 2 negative control tubes.
    6. Add 1 ml of the filtered water sample to each treatment tube.
    7. Incubate the tubes for a minimum of 7 to 10 days using the specific growth conditions of the target culture. Use a fluorometer (Turner TD700 or equivalent), to measure the in vivo fluorescence (as a proxy for growth), every 1-2 days.
    8. A decrease in relative fluorescence in the treatment compared with the control can be an indication of lysis by a viral agent. Clearance of the culture or distinct change in culture color can also be an indicator.
    9. Select tubes with suspected lytic agents.
    10. Repeat Steps A2 to A7, using these 4 ml samples to confirm that the lytic agent can be propagated. Use the following modifications:
      1. Step A4: Increase volume of cells to 4 ml.
      2. Steps A5 and A6: Reduce volume to 0.1 ml.
      3. Step A7: Expected time for lysis of culture can be shorter.
    11. If lysis is observed, aliquot the lysate into triplicate with a 0.45 μm or 0.22 μm pore size filter to remove most of the bacteria.

  2. Obtaining pure cyanophage stock
    1. Determine the titer of the lysate using the 96-well plate MPN assay (Suttle and Chan, 1993)
      1. Prepare exponentially growing target cells. Add fresh BG-11 media to the exponentially growing culture (1:1). Dispense 150 μl of diluted cells to each well.
      2. Set up a 10-fold dilution series of the lysate. For example, add 1,350 μl of BG-11 media to 8 x 2 ml microcentrifuge tubes. Add 150 μl of lysate to first tube (1/10 dilution). Mix by vortexing. Repeat until the last dilution (1/109 dilution).
      3. Add one dilution per column by adding 50 μl of the proper dilution to each well. Each column consists of 8 wells. One to two columns of the 96-well plate is used as a control which means 50 μl of BG-11 media is added to each of the wells (e.g., columns 1 and 12, Figure 1).


        Figure 1. Diagram showing a 96-wells plate MPN assay. Columns 1 and 12 represent control cultures, where 50 μl of BG-11 media is added into each of the wells. 50 μl of a 10-fold dilution series (10-0 to 10-9) of the lysate is added to the remaining columns. The plate is then monitored after 7 days to record the number and position of clear wells.

      4. Seal the 96-well plates with Parafilm, and incubate the plates using the specific conditions of the cyanobacteria cultures.
      5. Monitor plates for lysis every few days, recording the number and position of clear wells on the plate. When new clear or nearly clear wells no longer appear for 1 week, record the final ‘score’ for each dilution level. (Figure 1)
      6. Determine the concentration of infective virus, using a Most Probable Number (MPN) calculation program* (Jarvis et al., 2010).
        *Note: The program can be downloaded from: http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich/index.html.
    2. Purification of a clonal virus isolate (see Middelboe et al., 2010 In: Manual of Aquatic Viral Ecology. The American Society of Limnology and Oceanography p118-133 for more details)
      1. Prepare exponentially growing target cells, dispense ca. 4 ml into 13 x 100 mm culture tubes.
      2. Use BG-11 media to dilute lysate of known concentration to 1 infective virus/ml.
      3. Add 0.2 ml (0.2 infectious units) to each of 20 tubes of susceptible host cells.
      4. Monitor tubes (by eye or by in vivo fluorescence) for 2 to 3 weeks. Cultures in which lysis occurs are assumed to be the result of a single-virus infection.
        Notes:
        1. If lysis occurs in ≤ 4 tubes out of 20, it is assumed that lysis in each tube was caused by one infectious unit, therefore each tube would contain a separate phage clone. Transfer a small aliquot from all the tubes to fresh target cells to confirm the results. Proceed to Step B2e.
        2. If lysis occurs in more than 4 tubes, repeat Steps B2a-B2d, but reduce the volume of diluted lysate added in Step B2c accordingly in order to achieve lysis in ≤ 4 tubes out of 20 (e.g., add 0.1 ml instead of 0.2 ml).
      5. Scale up each lysed culture (clonal isolate) to make primary phage stocks; e.g., add 5 μl of 0.45 μm filtered lysate to 40 ml of exponentially growing cells.
      6. Incubate the tubes for 7 to 10 days using the specific growth conditions of the culture. Once the culture has lysed, centrifuge the lysate (5,000 x g, 15 min) to remove most of the cellular debris. Carefully transfer the supernatant to a fresh tube. Afterwards, filter the supernatant with a 0.22 μm pore size syringe filter (PVDF, Millipore), and store at 4 °C in the dark until further use. This is the primary phage stock solution.
      7. The phage stock can be further concentrated using a centrifugal UF device such as the Centricon Plus-70 (follow the manufacturer’s instructions).
        1. Pre-rinse the Centricon Plus-70 with 50 ml of MilliQ water.
        2. Centrifuge at 3,500 x g for 10 min. Pour out the filtrate.
        3. Add 40 ml of sample into the centricon.
        4. Centrifuge at 3,500 x g for 15 min. To collect the viral concentrate, connect the Centricon to the concentration collection cup, invert the Centricon, and remove the filtrate collection cup. Centrifuge at 1,000 x g for 2 min. The final volume should be less than 1 ml. Store concentrate at 4 °C until use.

  3. Purification of the cyanophage stock using an OptiprepTM gradient and the SW40 rotor (see Lawrence and Steward, 2010 for more details)
    Note: The viral concentrate is further purified on a continuous, isopycnic density gradient.
    1. Prepare OptiprepTM solutions using culture media as the diluent. We used 20%, 30% and 40% (v/v) final-OptiprepTM concentration solutions.
    2. Using a syringe and needle, prepare 3-step gradients with thin-walled open-top ultracentrifuge tubes (e.g., Beckman Ultra-ClearTM tube, 14 ml, 14 x 95 mm), beginning with the least dense solution. Be sure to leave enough room at the top of the centrifuge tube to layer the sample.
    3. Allow the gradient to equilibrate for at least 2 h at room temperature (or overnight at 4 °C).
    4. Carefully overlay ~0.2 to 1 ml of the viral concentrate on top of the OptiprepTM gradient using an adjustable pipet fitted with an aerosol barrier tip. Balance the underweight tubes by adding culture media one drop at a time, being careful not to disturb the gradient (use a fine-tipped transfer pipet).
    5. Carefully load the tubes into rotor and ultracentrifuge. For a 12 ml gradient with 1 ml virus concentrate sample in a Beckman Coulter SW-40Ti, centrifuge for 8 h at 207,570 x g (34,100 rpm) and 20 °C.
    6. Once the run is finished, carefully remove the tube from the rotor and clamp it vertically on a stand. Look for the presence of band(s) in the OptiprepTM gradient and remove the band(s) by side puncturing the tube with a needle. In a darkened room, shine a light from the top of the tube to help to visualize the bands. Gentle extract the band with the syringe and transfer the contents to a small tube (see Lawrence and Stewart, 2010 for more details).
      Note: OptiprepTM should not affect the infectivity of the viral isolates, however, proper controls should be performed in order to eliminate any possible negative effects that OptiprepTM may have on the growth of specific viral-hosts.
    7. To remove any leftover OptiprepTM material from the sample, a Slide-A-LyzerTM Dialysis cassette can be used. Once the band sample is loaded into the cassette according to the manufacturer’s instructions, the cassette is stored overnight at 4 °C into a beaker of fresh and sterile BG-11 media. Afterward, the dialysis sample is removed from the dialysis cassette according to the manufacturer’s instructions.

  4. Confirmation of virus isolate
    1. To confirm that the lytic agent of the cyanobacteria was purified, a subsample of the purified band (from Step C7), is diluted and added back to an exponentially growing culture of cyanobacteria.
    2. Once the culture is lysed, filter the lysate through a 0.45 μm or 0.22 μm pore size filter (PVDF), titer (as in Step B1) and store the stock at 4 °C in the dark.
    3. A subsample of the purified virus can also be used for examination on a Transmission Electron Microscope (TEM) to confirm that a virus is the lytic agent and to examine its morphology.
      Samples might need to be concentrated using the centricon or by ultracentrifugation. In general, a minimum of 1010 virus particles/ml are need for TEM examination using the negative staining procedure. More details on the TEM examination can be found in Suttle and Chan, 1993.

Recipes

Notes:

  1. Stock solutions can be conveniently prepared at 1,000x for each ingredient. Add 1 ml of each 1,000x stock to prepare 1 L of media. Store stock solutions at 4 °C.
  2. When preparing the trace metal stock mix, dissolve each ingredient separately prior to adding the next one from the list.


  1. Trace metal (TM) stock
    2.86 g/L H3BO3
    10 mg/L CuSO4·5H2O
    10 mg/L Co(NO3)2·6H2O
    6 mg/L Na2MoO4·2H2O
    22 mg/L ZnSO4·7H2O
    180 mg/L MnCl2·4H2O
  2. BG-11 media
    Notes:
    1. A freshwater media for the culture of cyanobacteria; ref Ripka et al. (1979), modified as described below
    2. We use the trace metal stock (TM) recipe for F/2 media, more as a matter of convenience. The composition is very similar to the TM stock for BG-11.
    1. To prepare 1 L of media, carefully slide a clean magnetic stir bar into a clean acid-washed beaker or Erlenmeyer. Add 1 L of Milli-Q water, and place the container onto a magnetic stir plate
    2. With gentle stirring, add the above nutrients in the order specified using either an automatic pipet (e.g., P1000) fitted with sterile tips or sterile serological pipets
    3. Adjust pH to ca. 8.0 (initial pH is ca. 8.5) using 0.1 N HCl (a couple of drops)
    4. Once thoroughly mixed, dispense the media into 500 ml portions and autoclave for 20 min at 121 °C

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

  1. Chénard, C., Chan, A. M., Vincent, W. F. and Suttle, C. A. (2015). Polar freshwater cyanophage S-EIV1 represents a new widespread evolutionary lineage of phages. ISME J 9(9): 2046-2058.
  2. Jarvis, B., Wilrich, C. and Wilrich, P. T. (2010). Reconsideration of the derivation of Most Probable Numbers, their standard deviations, confidence bounds and rarity values. J Appl Microbiol 109(5): 1660-1667.
  3. Lawrence, J. E. and Steward, G. F. (2010). Purification of viruses by centrifugation. In: Wilhelm, S. W., Weinbauer, M. G. and Suttle, C. A. [Eds.]. Manual of Aquatic Viral Ecology. ASLO pp: 116-181.
  4. Middelboe, M., Chan, A. M., and Bertelsen, S. K. (2010). Isolation and life cycle characterization of lytic viruses infecting heterotrophic bacteria and cyanobacteria. In: Wilhelm, S. W., Weinbauer, M. G. and Suttle, C. A. [Eds.]. Manual of Aquatic Viral Ecology. ASLO pp: 118-133.
  5. Ripka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stainer, R. Y. (1979). Generic assignment, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1-61.
  6. Suttle, C. A. (2000). The ecological, evolutionary and geochemical consequences of viral infection of cyanobacteria and eukaryotic algae. In: Hurst, C. J. (Ed.). Chapter 6 (248-296) in Viral Ecology. Academic Press pp: 639.
  7. Suttle C. A. and Chan, A. M. (1993). Marine cyanophages infecting oceanic and coastal strains of Synechococcus: abundance, morphology, cross-infectivity and growth characteristics. Mar Ecol Prog Ser 92: 99-109.
  8. Suttle C. A., Chan, A. M. and Cottrell, M. T. (1991). Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine phytoplankton. Appl Environ Microbiol 57 (3): 721-726.
  9. Waterbury, J. B. and Valois, F. W. (1993). Resistance to co-occuring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater. Appl Environ Microbiol 59: 3393-3399.

简介

以下方案描述了使用液体生物测定方法分离和纯化感染蓝细菌的病毒。 感染蓝细菌的病毒也被称为噬藻体。 本协议是专门为分离感染淡水蓝藻的蓝藻,特别是不能在固体培养基上培养的蓝细菌而编写的。 推荐使用克隆蓝藻培养物来分离病毒。 生长条件(即,介质,光周期和温度)应根据感兴趣的主体进行修改。

【背景】蓝藻是海洋和淡水系统中重要的营养生物。作为其他的水生微生物,蓝藻受到病毒感染(Suttle,2000)。例如,在海洋沿海地区,感染聚球蓝细菌的病毒滴度。 (Suttle和Chan,1993; Waterbury和Valois,1993),可以达到10-5 ml-1,并且基于温度,盐度和宿主丰度而不同。尽管蓝细菌及其病毒(也称为“蓝藻”)具有生态重要性,但只有少数病毒已经从有限的蓝藻菌株中分离出来。因此,通过筛选新的蓝藻菌株来分离新病毒是非常有意义的。以下议定书对于海洋和淡水系统都是相关的,但下面的例子将重点讨论分离和纯化感染淡水蓝藻的病毒(Chénardet al。,2015)。液体生物测定方法优于使用固体基质的公开方案的优点是可以靶向无法耐受噬菌斑测定方法经常使用的较高温度或不能在固体培养基上生长的蓝细菌。

关键字:病毒分离, 病毒纯化, 水生微生物学, 噬藻体, 蓝藻, MPN测定, 液体生物检定方法

材料和试剂

  1. 0.45μm或0.22μm注射器过滤器(例如Millex HV或GV,PVDF)(EMD Millipore)
  2. 无菌一次性塑料注射器,60毫升(BD,目录号:309653)
  3. 玻璃13×100mm培养管或24孔板(聚苯乙烯,例如Corning,目录号3524)
  4. 2毫升微量离心管
  5. 96孔板(聚苯乙烯,如Corning,目录号:3599)
  6. Parafilm
  7. 无菌一次性塑料注射器,5毫升(BD,目录号:309646)
  8. 无菌皮下注射针(18 G1½,BD,目录号:305196)
  9. 气溶胶屏障过滤嘴(SARSTEDT,目录号:70.1189.215)
  10. Centricon Plus-70(30,000 NMWL,EMD Millipore,目录号:UFC703008)
  11. Slide-A-Lyzer TM透析盒(20,000MWCO,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:66003)
  12. 靶向宿主 - 克隆蓝细菌菌株,优选无菌,但不是必需的
  13. 淡水样本(病毒来源)
  14. Optiprep TM密度梯度培养基(Axis Shield,目录号:1114542)
  15. MilliQ水
  16. 0.1 N HCl
  17. 硝酸钠(NaNO 3)*
  18. 磷酸二氢钾(K 2 HPO 4)*
  19. 七水合硫酸镁(MgSO 4•7H 2 O)*
  20. 氯化钙二水合物(CaCl 2•2H 2 O)*
  21. 柠檬酸*
  22. 柠檬酸铁铵*
  23. EDTA二钠*
  24. 磷酸钠(Na 2 CO 3)*
  25. 硼酸(H 3 BO 3)*
  26. 锰(II)四水合物(MnCl 2•4H 2 O)*
  27. 七水硫酸锌(ZnSO 4•7H 2 O)*
  28. 脱水钼酸钠(Na 2 MoO 4•2H 2 O)*
  29. 硫酸铜(II)五水合物(CuSO 4•5H 2 O)*
  30. 硝酸钴(II)六水合物(Co(NO 3)2•6H 2 O)*
  31. 痕量金属(TM)库存(见食谱)
  32. BG-11媒体(见食谱)

注意:介质配方中使用的所有化学试剂均为试剂级(ACS级),均购自Thermo Fisher Scientific。

设备

  1. P200移液器(Eppendorf,目录号:3123000055)
  2. 自动移液器(,例如,P1000)
  3. 荧光计(例如,Turner Designs,型号:TD-700,带有光学试剂盒Turner Designs,目录号:10-037R,激发350-500nm,发射> 665nm)。
  4. 涡流(例如,Scientific Industries,型号:Vortex-Genie 2)
  5. 带有SW-40Ti转子的超速离心机(Beckman Coulter,型号:Optima L-90K)(Beckman Coulter,型号:SW 40 Ti,目录号:331302)
  6. 薄壁Ultra-Clear TM敞开式超速离心管(14ml,14×95mm)(Beckman Coulter,目录号:344060)
  7. 平衡(微型;用于平衡离心管)
  8. 冷冻台式离心机(例如,Eppendorf,型号:5810R,带转子A-4)
  9. 透射电子显微镜(TEM)
  10. 磁力搅拌棒
  11. 烧杯或锥形瓶
  12. 高压灭菌器

程序

定义
效价浓度的感染性病毒
MPN最可能的数字
裂解液 - 含有细胞溶解释放的产物的溶液

  1. 分离的cyanophages
    1. 将50毫升水样收集在一个干净的容器中(例如,Falcon管)。在充装之前用样品预冲洗容器数次。如果可能的话,保持样品冷,并在黑暗中,直到下一步。
      注意:所需的样品量取决于环境的生产力,从生产环境的几毫升到寡营养环境的几十升。必要时,可以从大量的水样中浓缩病毒以提高检测限。有关使用超滤分离病毒的更多细节,请参见Suttle et al。 (1991)。
    2. 过滤除去样品中的细菌和浮游生物;使用0.45μm或0.22μm孔径的注射器过滤器和一个60 ml塑料注射器。将过滤的样品在黑暗中存放在4°C直到使用。
    3. 准备成倍增长的靶细胞。
    4. 转移 ca。将3ml指数生长培养物加入到5个无菌玻璃13×100mm培养管中(每个水样品使用最少3个试管,2个试管作为阴性对照)。

    5. 加入1 ml BG-11培养基(见食谱注释)到2个阴性对照管

    6. 每个处理管中加入1毫升过滤水样
    7. 使用目标培养物的特定生长条件将培养物培养至少7至10天。使用荧光计(Turner TD700或同等产品),每1-2天测量一下体内荧光(作为生长代理)。
    8. 与对照相比,治疗中相对荧光的减少可以是病毒剂溶解的指示。文化的清除或文化色彩的明显变化也可以作为一个指标。
    9. 选择疑似溶解剂的试管。
    10. 重复步骤A2到A7,使用这4毫升的样本,以确认裂解剂可以传播。使用以下修改:
      1. 步骤A4:增加细胞的体积为4毫升。
      2. 步骤A5和A6:减少体积到0.1毫升。
      3. 步骤A7:培养裂解的预期时间可以缩短。
    11. 如果观察到裂解,用0.45μm或0.22μm孔径的过滤器将裂解物等分成三份以去除大部分细菌。

  2. 获得纯粹的cyanophage股票
    1. 使用96孔板MPN测定(Suttle和Chan,1993)确定裂解物的效价,
      1. 准备指数增长的目标细胞。将新鲜的BG-11培养基添加到指数生长的培养物(1:1)中。
        分配150μl稀释的细胞到每个孔
      2. 建立10倍稀释系列的裂解物。例如,向8 x 2 ml微量离心管中加入1,350μlBG-11培养基。向第一管中加入150μl裂解物(1/10稀释)。通过涡旋混合。重复,直到最后一次稀释(1/10稀释)。
      3. 每个柱加入一个稀释液,每孔加入50μl适当的稀释液。每个柱子由8个井组成。使用96孔板的1至2列作为对照,这意味着将50μl的BG-11培养基加入每个孔(例如,图1的第1栏和第12栏)中。


        图1.显示96孔板MPN测定的图。第1列和第12列代表对照培养物,其中每孔加入50μlBG-11培养基。向剩余的柱中加入50μl的10倍稀释系列(10→-0→10→-9)裂解物。然后在7天后监测平板,记录透明井的数量和位置。

      4. 用Parafilm密封96孔板,并使用蓝细菌培养物的特定条件培养板。
      5. 每隔几天监测一次裂解板,记录板上清孔的数量和位置。当新的清澈或几乎清澈的孔在1周内不再出现时,记录每个稀释水平的最终“分数”。 (图1)
      6. 使用最大可能数(MPN)计算程序*(Jarvis等,,2010)确定感染性病毒的浓度。
        *注意:该程序可以从以下网站下载: http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich/index.html
    2. 克隆病毒分离物的纯化(参见Middelboe等人,2010年:水生病毒生态学手册,美国湖沼学和海洋学协会p118-133,以获得更多细节)
      1. 准备指数增长的目标细胞,分配4毫升到13×100毫米的培养管。
      2. 使用BG-11培养基稀释已知浓度的裂解液至1感染性病毒/ ml。

      3. 加入0.2毫升(0.2感染单位)到20管易感宿主细胞
      4. 监测管(通过眼睛或通过体内荧光)2至3周。发生裂解的文化被认为是单一病毒感染的结果。
        注意:
        1. 如果裂解发生在20个以内的≤4个管中,则认为每个管中的裂解是由一个感染单位引起的,因此每个管将包含单独的噬菌体克隆。将所有试管中的一小份转移到新鲜的靶细胞中以确认结果。继续步骤B2e。
        2. 如果在超过4个试管中发生裂解,重复步骤B2a-B2d,但相应地减少步骤B2c中加入的稀释裂解物的体积,以便在≤4个试管中实现裂解(例如,添加0.1ml而不是0.2毫升)。
      5. 放大每个裂解培养物(克隆分离物)以制备初级噬菌体库;例如,将5μl的0.45μm过滤的溶解产物加入到40ml指数生长的细胞中。
      6. 使用培养物的特定生长条件将培养管温育7至10天。一旦培养物裂解,离心裂解物(5,000×g,15分钟)除去大部分细胞碎片。小心地将上清转移到新鲜的试管中。之后,用0.22μm孔径的注射器过滤器(PVDF,Millipore)过滤上清液,并在4℃下在黑暗中储存直至进一步使用。这是主要的噬菌体原液。
      7. 可以使用Centricon Plus-70等离心UF装置(遵照制造商的说明书)进一步浓缩噬菌体原液。
        1. 用50毫升MilliQ水预冲洗Centricon Plus-70。
        2. 在3,500×g下离心10分钟。倒出滤液。

        3. 添加40毫升样品到centricon
        4. 在3,500×g 离心15分钟。要收集病毒浓缩液,将Centricon连接到浓缩杯,倒置Centricon,并取出滤液收集杯。 1000×g离心2分钟。最终体积应该小于1毫升。
          在4°C储存浓缩液直到使用
  3. 使用Optiprep TM梯度和SW40转子纯化cyanophage原液(详见Lawrence and Steward,2010)
    注意:病毒浓缩物在连续的等密度梯度下进一步纯化
    1. 使用培养基作为稀释液准备Optiprep™解决方案。我们使用了20%,30%和40%(v / v)的最终Optiprep TM浓度溶液。
    2. 使用注射器和针头,用薄壁敞开式超速离心管(例如Beckman Ultra-Clear TM管,14ml,14×95)准备3步梯度毫米),从最低密度解决方案开始。确保在离心管顶部留出足够的空间以层叠样品。

    3. 在室温下(或在4°C过夜)使梯度平衡至少2小时
    4. 使用装有气雾剂阻挡端的可调移液管,在Optiprep TM梯度顶部小心覆盖〜0.2至1ml的病毒浓缩物。通过一滴一滴添加培养基来平衡体重不足的管子,注意不要打扰梯度(使用细尖的移液管)。
    5. 小心地将管装入转子和超速离心机。对于在Beckman Coulter SW-40Ti中含有1ml病毒浓缩物样品的12ml梯度,在207,570xg(34,100rpm)和20℃下离心8小时。
    6. 一旦运行完成,小心地从转子上取下管子,并将其垂直夹在支架上。在Optiprep TM 梯度中查找带的存在,并通过用针刺穿管来移除带。在黑暗的房间里,从灯管顶部照射一束光,以帮助观察乐队。温和地用注射器提取带子,并将内容物转移到一个小管中(详见Lawrence和Stewart,2010)。
      注意:Optiprep TM不应该影响病毒分离株的感染性,但是应该进行适当的对照以消除Optiprep可能会对特定病毒宿主的生长产生负面影响。
    7. 为了从样品中除去剩余的Optiprep TM材料,可以使用Slide-A-Lyzer TM透析盒。一旦条带样品根据制造商的说明书装入盒中,将盒在4℃下过夜储存在新鲜无菌BG-11培养基的烧杯中。之后,根据制造商的说明将透析样品从透析盒中取出。

  4. 确认病毒分离
    1. 为了确认蓝细菌的溶解剂被纯化,将纯化条带(来自步骤C7)的子样品稀释并加回到指数增长的蓝细菌培养物中。
    2. 一旦培养物被裂解,通过0.45μm或0.22μm孔径的过滤器(PVDF),效价(如步骤B1)过滤裂解物并在黑暗中将该物质储存在4℃。
    3. 纯化病毒的子样本也可用于透射电子显微镜(TEM)检查,以确认病毒是裂解剂并检查其形态。
      样品可能需要使用centricon浓缩或超速离心。通常,使用负染色程序需要最少10 10病毒颗粒/ ml进行TEM检查。有关TEM检查的更多细节可以在Suttle和Chan,1993中找到。

食谱

注意:

  1. 库存解决方案可以方便地准备在每种成分1,000倍。每1,000x库存添加1毫升,以制备1升培养基。将原液储存在4°C。
  2. 制备痕量金属原料混合物时,先将各成分分开溶解,然后再添加下一个成分。


  1. 痕量金属(TM)库存
    2.86g / L H 3 BO 3 10mg / L CuSO 4•5H 2 O 10mg / L Co(NO3)2•6H2O 2
    6mg / L Na 2 MoO 4•2H 2 O
  2. BG-11媒体
    注意:
    1. 用于培养蓝细菌的淡水培养基;参考Ripka等。 (1979),如下所述进行修改
    2. 为方便起见,我们使用F / 2介质的痕量金属原料(TM)配方。该组合物与BG-11的TM原料非常相似。
      “”src
    1. 要准备1 L的介质,请小心地将干净的磁力搅拌棒放入清洁的酸洗烧杯或锥形瓶中。加入1升Milli-Q水,将容器置于磁力搅拌盘上。
    2. 在温和搅拌下,按照规定的顺序加入上述营养素,使用装有无菌吸头或无菌血清吸管的自动移液管(例如,P1000)。
    3. 使用0.1N HCl(几滴)将pH调节至8.0(初始pH为8.5)。
    4. 一旦彻底混合,分配媒体成500毫升的份量和121℃高压灭菌20分钟

致谢

CC由新加坡国家研究基金会根据其海洋科学研究与发展计划(MSRDP-P13号奖)提供支持。加拿大创新基金会,机会基金领导者(项目#25412)和加拿大国家科学与工程研究委员会(EQPEQ 375995-09)支持了这些协议的制定。这个协议是从以前的作品启发和改编的:Middelboe 等。(2010); Suttle和Chan(1993); Chénard等(2015)。作者没有任何利益冲突或竞争利益的申报。

参考

  1. Chénard,C.,Chan,A. M.,Vincent,W. F.和Suttle,C. A.(2015年)。 极性淡水cyanophage S-EIV1代表了一种新的广泛的噬菌体进化谱系。 ISME J 9/9(9):2046-2058。
  2. Jarvis,B.,Wilrich,C。和Wilrich,P.T。(2010)。 重新考虑推导最可能数字,它们的标准偏差,置信区间和稀有值。 J Appl Microbiol 109(5):1660-1667。
  3. Lawrence,J. E.和Steward,G. F.(2010)。 通过离心纯化病毒在:Wilhelm,SW,Weinbauer,MG和Suttle,CA [编辑]。水生病毒生态学手册。 ASLO pp:116-181。
  4. Middelboe,M.,Chan,A. M.和Bertelsen,S. K.(2010)。 感染异养细菌和蓝细菌的裂解病毒的分离和生命周期表征 :Wilhelm,SW,Weinbauer,MG和Suttle,CA [编辑]。水生病毒生态学手册。 ASLO pp:118-133。
  5. Ripka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M.和Stainer,R.Y。(1979)。 蓝藻纯培养物的一般分配,应变历史和特性。 J Gen Microbiol 111:1-61。
  6. Suttle,C.A。(2000)。蓝藻和真核藻类的病毒感染的生态,进化和地球化学后果。作者:赫斯特,CJ(赫斯特,CJ),“生物地球化学”,“生物地球化学”,“生物地球化学”,“生物地球化学” (编)。第六章(248-296)病毒生态学。学术出版社 pp:639.
  7. Suttle C.A.和Chan,A.M。(1993)。 海洋蓝藻感染海洋和沿海球菌菌株:丰度,形态,交叉感染性和生长特性。 Mar Ecol Prog Ser 92:99-109。
  8. Suttle C.A.,Chan,A.M。和Cottrell,M.T。(1991)。 利用超滤技术从海洋浮游植物的病原体海水中分离出病毒。 Appl Environ Microbiol 57(3):721-726。
  9. Waterbury,J.B。和Valois,F.W。(1993)。 耐受同时发生的噬菌体使海洋聚球藻群落与丰富的海水中的碳水化合物共存。 Appl Environ Microbiol 59:3393-3399。
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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.
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