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Jan 2018
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Isolation and CryoTEM of Phages Infecting Bacterial Wine Spoilers
感染葡萄酒腐败细菌的噬菌体分离与冷冻电镜观察   

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Abstract

With the objective to isolate phages infecting wine bacterial spoilers, we designed a method for the isolation and purification of phages infecting grape-associated bacteria. The method proved successful to isolate GC1 tectivirus infecting the acetic acid bacterium Gluconobacter cerinus. The isolated phage represents a new genus within the Tectiviridae, named "Gammatectivirus". Using a traditional technique for the concentration of phage particles involving several steps of centrifugation, further insights in the ultrastructure of GC1 could be observed by cryo electron microscopy, saving time and effort. The simple workflow presented may be applied to other viruses infecting bacteria inhabiting other vegetal niches.



[Graphic abstract]



Flowchart illustrating the protocol to isolate, concentrate and observe GC1 under cryo-EM


Keywords: Phage purification (噬菌体纯化), Tectivirus (复层噬菌体属), Phage concentration (噬菌体浓缩), Acid Acetic Bacteria (AAB) (醋酸菌), Oenology (酿酒学), Cryo-EM (冷冻电镜)

Background

Winemaking is a complex and fluctuating environment that is characterized by the temporal succession of distinct communities of microorganisms. The wine-making process starts with the selection of the fruit and the fermentation of sugars into alcohol by yeasts. In most red and dry white wines, this step is followed by a malolactic fermentation, which reduces acidity, increases microbial stability, and creates good-quality grape wine. In contrast to other food fermentations, little knowledge is so far available on the diversity of viruses infecting bacteria, also known as bacteriophages or phages, in the enological ecosystem (de Melo et al., 2018). However, grapes, and more globally the whole fermentation process, are an interesting crossroad between different ecosystems (insect, plant and its rhizosphere, soil and human), and may turn out to represent a valuable source for genome innovation for phages and their bacterial hosts. From the technological point of view, the characterization of phages from the enological niche could also represent an eco-friendly alternative to chemicals to limit bacterial spoilage during wine-making. Among established wine spoilers, acetic acid bacteria (AAB) negatively affect wine quality because of their ability to increase the volatile acidity of wine by the production of acetic acid among other compounds (Du Toit and Pretorius, 2000; De Roos and De Vuyst, 2018). Here, we detail a simple method for the screening and isolation of phages infecting AAB, that can be applied to other fruits (Philippe et al., 2018). Phage particles from small-scale production were concentrated by centrifugation, and time-consuming steps involving PEG precipitation or CsCl gradients were avoided. Cryo electron microscopy (cryo-EM) (Cuervo and Carrascosa, 2018) provided high resolution and distinctive features in the ultratructure of tectiviruses were visible.


Materials and Reagents

Materials

  1. Autoclavable Erlenmeyer flasks (volume: 250 ml) (Dutscher, catalog number: 211907 )

  2. Autoclavable glass tubes (volume: 20 ml) (Dutscher, catalog number: 0 45209 )

  3. Cap-o-test caps (Dutscher, catalog number: 110685B )

  4. Centrifuge tubes (volume: 50 ml) (Fisher Scientific, catalog number: 07-000-983 )

  5. Pasteur pipette

  6. Cryo Grid Box (Electron Microscopy Science, catalog number: 71166-10-W )

  7. Freezer bags (450 ml, VWR, catalog number: 82007-706 )

  8. Lacey Carbon 300 mesh copper grids (Ted Pella, catalog number: 01883)

  9. Polyethersulfone (PES) membrane syringe filters (0.22 μm and 0.45 μm) (Fisher Scientific, catalog numbers: SLGPR33RS ; SLHPR33RS )

  10. Polypropylene microcentrifuge tubes (volume: 1.5 ml) (Fisher Scientific, catalog number: 07-000-244 )

  11. Spatula (Dutscher, catalog number: 442209 )

  12. Spectrophotometer cuvettes (volume: 1.6 ml) (Dutscher, catalog number: 613101 )

  13. Sterile disposable plastic syringes (volume: 50 ml) (Fisher Scientific, catalog number: 13-689-8 )

  14. Sterile 90 x 13 mm Petri dishes (Dutscher, catalog number: 076084B )

  15. Whatmann Filter paper grade 5 (Dutscher, catalog number: 1005055 )


Reagents

  1. Agar-agar (Thermo Fisher Scientific, Fisher ChemicalTM, catalog number: 10548030 )

  2. Calcium chloride dihydrate (CaCl2·2H2O) (Thermo Fisher Scientific, Fisher BioReagents, catalog number: 10306313 )

  3. Ethane (AIR liquide)

  4. Hydrochloric acid, 33.33% (w/v) aqueous solution (Thermo Fisher Scientific, ULTREX, J.T. BakerTM, catalog number: 10782232 )

  5. Liquid nitrogen (cryo-distribution)

  6. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Thermo Fisher Scientific, Fisher BioReagents, catalog number: 10553335 )

  7. Mannitol (Thermo Fisher Scientific, ACROS OrganicsTM, catalog number: 10266523 )

  8. Peptone (Thermo Fisher Scientific, GibcoTM BactoTM Peptone, catalog number: 16299741 )

  9. Sodium chloride (NaCl) (Thermo Fisher Scientific, Technical, catalog number: 10498220 )

  10. Tris-HCl pH 7.5 (Thermo Fisher Scientific, Merck ChemicalsTM, catalog number: 15153685 )

  11. Yeast extracts (Thermo Fisher Scientific, GibcoTM DifcoTM BactoTM, catalog number: 11763553 )

  12. 10x Phage buffer (see Recipes)

  13. CaCl2·2H2O and MgSO4·7H2O 100x stock solutions (see Recipes)

  14. YPM broth medium (see Recipes)

  15. YPMΦ solid (“bottom”) medium (see Recipes)

  16. YPMΦ soft agar (see Recipes)


Bacterial strain and samples of enological origin

  1. Fresh crushed grapes (see Procedure)

  2. Gluconobacter cerinus CRBO11179 (Centre de Resources Biologiques OEnologie, Institut des Sciences de la Vigne et du Vin, ISVV, Villenave-d’Ornon, France)

Equipment

  1. Autoclave

  2. Cryo-electron microscope (Thermo Fisher FEI, model: Tecnai F20 200 kV ) equipped with a CCD camera (Thermo Fisher FEI, model: Eagle 4kx4k)

  3. Cryo-holder and cryo-transfer station (Gatan, model: 626 )

  4. Glow Discharge unit (Cordouan Technologies, model: ELMO )

  5. Incubator set to 25 °C

  6. High-speed refrigerated centrifuge (Hitachi, model: CR 22N ) and R15A-0688 rotor

  7. Microwave (Thermo Fisher Scientific, ManutanTM MW20G, catalog number: 15540739 )

  8. Orbital shakers (Stuart, model: SSL1 )

  9. pH meter (Hanna, model: pH211 )

  10. Spectrophotometer (Shimadzu, model: UV-1280 )

  11. Vitrification Robot (Leica Microsystem, model: EM-GP 2 )

  12. Water bath (colume: 4 L) (Benchmark Scientific, MyBath model)

Software

  1. ImageJ (NIH)

  2. Tecnai version 4.3 (ThermoFisher FEI)

  3. TEM Imaging & Analysis version 4.4 (ThermoFisher FEI)

Procedure

  1. Collect grapes and recover juices

    1. Collect fresh grapes into freezer bags (~200 x g) from selected vineyards/wineries.

    2. Store at 4 °C during transport and process samples as quickly as possible.

    3. Crush grapes manually and centrifuge the samples (5,000 x g, 10 min).

    4. Collect the supernants (juices).

    5. Filter the juices using successively 0.45 µm then 0.22 µm PES membrane filters under sterile conditions.

    6. Store filtered juices in a 4 °C fridge.


  2. Isolation of viral plaques, purification and production of a primary stock (Stock I)

    1. Gluconobacter cerinus culture preparation

      1. Prepare a sterile 250 ml Erlenmeyer flask with 15 ml of YPM broth.

      2. Inoculate with 10 µl of a ready-to-use frozen bacterial strain in glycerol stock and incubate over night with shaking (200 rpm) at 25°C.

      3. The following morning, back dilute the culture 1/10 into 15 ml of fresh YPMΦ broth and incubate at 25 °C in a shaking incubator until OD600nm 0.2 is obtained (4 to 5 h).

    2. Preparation of the bottom top YPMΦ plates

      1. Melt carefully the bottom top YPMΦ agar by heating in a microwave oven.

      2. Cool down to 55 °C in a water bath.

      3. Pour plates, using ~20 ml melted medium (prepare 3 plates per juice sample to analyze).

      4. Allow the agar based media to solidify for 1 h at room temperature.

    3. Double agar overlay assays

      1. For each juice sample to analyze, melt 3 YPMΦ soft agar tubes in a boiling water bath.

      2. Maintain the tubes in a 55 °C water bath prior to use.

      3. Just before use, take the filtered juices out from the 4 °C fridge (Step A6) and prepare serial dilutions in phage buffer 1x (10-1, 10-2).

      4. Remove a YPMΦ soft agar tube from the water bath and place it on a rack for 1-2 min at room temperature (RT).

      5. Add 200 μl of bacterial culture at OD600nm 0.2 (Step B1) and 100 μl of undiluted filtered juice in the YPMΦ soft agar tube.

      6. Vortex 3 times and spread evenly on top of a solid YPMΦ plate.

      7. Repeat Steps B3d-f with each dilution (10-1, 10-2) of the juice samples (Step B3c) using the two remaining YPMΦ agar plates.

      8. Incubate the plates at 25 °C for 24 h.

    4. Isolation of plaques

      1. Select the plate with individual plaques.

      2. Visually identify the different morphotypes of plaques (large, small, clear, turbid) on the YPMΦ plate (if relevant).

      3. Use a marker to draw a circle on the bottom of the plate around each selected plaque.

      4. Punch each plaque out using a sterile Pasteur pipette with an aspiration bulb.

      5. Place each recovered agar plug containing a single plaque into a micro-tube containing 500 μl of YPMΦ broth.

      6. Vortex gently.

      7. Allow the phages to diffuse in the broth medium for 20 min at RT.

      8. Write R1 (first round of purification) on this microtube with a permanent marker.

      9. Store at 4 °C or proceed immediately to Step B5.

    5. Perform two additional rounds of purification

      1. Dilute the R1 sample in phage buffer 1x (10-1 to 10-4) in micro-tubes.

      2. Perform double layer agar plating for the 4 diluted samples (repeat Steps B1 to B3 for each diluted sample).

      3. After incubation for 24 h, observe and select the plate which contains individual plaques.

      4. Punch a single plaque into a micro-tube containing 500 µl of YPMΦ.

      5. Write R2 (second round of purification) on your microtube with a permanent marker.

      6. Repeat a third and last round of phage infection on the same sensitive bacterial strain to ensure purity. All steps need to be carried out under sterile conditions.

      7. The 500 µl R3 sample is your primary pure phage stock (Stock I). Store it at 4 °C.


  3. Preparation of a secondary phage stock (stock II) by using the confluent plate lysate method

    This step is necessary to obtain an appropriate volume of phage lysate.

    1. Double agar overlay assays

      1. Prepare a 15 ml culture of G. cerinus CRBO 11179 culture (OD600nm 0.2) in YPMΦ broth (see Step B1).

      2. Fill 3 sterile 1.5 ml microtubes with 900 µl of 1x sterile phage buffer and label the tubes with the dilution numbers (10-1 to 10-3).

      3. Add 100 µl of stock I in the 10-1 tube and perform serial dilutions.

      4. Prepare 24 YPMΦ agar plates.

      5. Melt 24 YPMΦ soft agar tubes and place them in a water bath at 55 °C.

      6. Remove 8 YPMΦ soft agar tubes from the water bath and place them on a rack for 1-2 min at room temperature (RT).

      7. In each tube, mix 100 µl of the ten-fold phage stock I with 200 µl of the sensitive bacterium as shown in Steps B1 to B3.

      8. Pour each tube on an YPMΦ agar plate.

      9. Repeat Steps C1f to C1h with 8 additional soft agar tubes and YPMΦ agar plates and use 100 µl of stock I diluted to 10-2.

      10. Repeat Steps C1f to C1h with the remaining 8 soft agar tubes and YPMΦ agar plates and use 100 µl of stock I diluted to 10-3.

      11. Incubate the 24 plates 24 h at 25 °C.

    2. Recovery of stock II

      1. For each dilution plated on YPMΦ plates (10-1, 10-2, 10-3), observe the density of plaques.

      2. Select the highest dilution giving confluent lysis (see Figure 1). Only the 8 plates corresponding to this dilution will be processed.



        Figure 1. Ilustration of a confluent lysis

      3. Under sterile conditions, add 1 ml of sterile YPMΦ broth on the surface of each of the 8 double layer agar plate selected.

      4. Incubate 20 min at RT. Alternately, plates can be stored overnight at 4 °C at this step.

      5. Use a sterile spatula to carefully collect the soft agars from each plate and the liquid medium in a centrifugation tube.

      6. Centrifuge the sample at 10,000 x g for 10 min at 15 °C.

      7. Filter the supernatant (6-8 ml) using 0.45 µm PES membrane filters.

      8. Store stock II at 4 °C.

    3. Determine the titer of stock II as follows (Figure 2)

      1. Prepare a 15 ml culture of G. cerinus CRBO 11179 culture (OD600nm 0.2) in YPMΦ broth (see B1).

      2. Prepare 8 sterile 1.5 ml micro-tubes containing 900 µl of 1x sterile phage buffe and serially dilute stock II 10-1 through 10-8.

      3. Take an YPMΦ agar plate and draw a grid of 8-10 spaces on the plate bottom.

      4. Remove a 5 ml YPMΦ soft agar tube from the water bath at 55 °C and inoculate with 0.2 ml of the culture prepared in Step C3a.

      5. Vortex briefly to homogenize, pour on the YPMΦ agar plate and let the agar solidify at RT (30 min).

      6. Spot a 10 µl drop of each dilution (pure to 10-8) of stock II in the available spaces of the grid; put the lid and let the Petri dish on the bench until the drops are adsorbed (1 h).

      7. Incubate overnight at 25 °C.

      8. Determine the dilution giving an optimal range to count between 2 to 20 plaques per space. The titer (PFU/ml) may be calculated by the following formula: PFU/ml (of secondary stock) = 1/dilution factor x number of plaques x 1/(0.01).



      Figure 2. Rapid titration of phage stocks (II and III)

  4. Preparation of highly concentrated GC1 phage suspension (stock III) by the liquid method

    1. Introduce aseptically 15 ml YPMΦ broth in a sterile 250 ml Erlenmeyer flask.

    2. Inoculate 10 µl of a frozen culture of G. cerinus CRBO11179.

    3. Incubate overnight under 25 °C in a shaking incubator at 200 rpm.

    4. Measure the OD600nm of the culture and transfer a volume in new flask containing 60 ml YPMΦ broth to obtain an OD600nm of 0.1, corresponding to approximately 108 CFU/ml.

    5. Divide into three separate sterile 250 ml erlenmeyer flasks (20 ml/flask) (see Figure 3).



      Figure 3. Summary of the preparation of phage stocks I, II and III

    6. Infect two flasks with phage GC1 with a multiplicity of infection (MOI) of approximately 1 phage for 200 bacteria. The third culture is used as a non-infected negative control.

    7. Allow the phages to adsorb to bacterial cells 30 min at RT without skaking.

    8. Incubate at 25 °C in a shaking incubator at 200 rpm.

    9. Measure the OD600nm periodically (mean values of 1 and 2.5 are observed for the infected assays and the control, respectively, after 14-16 h).

      Note: Don’t extend incubation beyond 14-16 h as a 2-Log reduction in final phage titer is usually observed.

    10. Centrifuge the lysed cultures at 10,000 x g for 10 min at 15 °C.

    11. Filter the supernatant through a 0.22 μm PES filter.

    12. Measure the titer of the lysate as described in Step C3.

      Note: A titer of ~1010 PFU/ml is routinely obtained in the laboratory. The phage titer should exceed 107 PFU/ml to be observed under cryo-EM.

    13. Transfer the filtered supernatant in a 50 ml centrifuge tube (ca. ~38 ml) and centrifuge 2 h at 20,000 x g at 4 °C (ensure all sample tubes are evenly filled to properly balance the centrifuge).

    14. Carefully remove the supernatant and air dry the pellet.

    15. Resuspend gently the phage pellet in 20 to 50 µl of sterile phage buffer 1x

    16. Store at 4 °C.

      Note: Sample should not be opaque for cryo-EM observation, if needed increase the dilution with phage buffer 1x.


  5. Observation of GC1 phage particles by cryo-EM

    We used the following standard protocol.

    1. Vitrification: freezing of the preparation was made using the EM-GP apparatus (the procedure has been summarized Figure 4).

      1. Fill the humidifier with distilled water and change the blotting paper using a Whatman grade 5.

      2. Set the chamber conditions: temperature of 25 °C and 80% humidity and ethane temperature at -184 °C.

      3. Set the forceps conditions: “blotter settings” must be adjusted to see the filter paper touching the grid without pulling it; “grid blot position” must be adjusted to place the grid 1 mm above the edge of the paper and “Grid Tf position” is adjusted to totally immersed the grid into the ethane container.

      4. Adjust your freezing’s settings: Unselect the rotations and sensor. Select “A-plunge” to automatically plunge the grid after blotting. Set the time(s) settings as follows: Delay at 0.0; Blot at 2.0 s and Hold at 0.0.

      5. Fill the container with liquid nitrogen until 100% after placing the ethane container and the grid box support.

      6. Around -120 °C, condense the ethane at a pressure of 0.1 mBar.

      7. Glow discharge the grids. Place the Lacey grids, carbon facing upwards, on a piece of glass slide covered with a Parafilm. Use the following glow discharge condition: vacuum at 0.3 mBar, intensity of 3 mA and timer of 40 s.

      8. Apply 4 µl of the sample on the carbon side of the grid and activate the blotting and the plunge freezing by pressing “blot/A plunge”. Blotting is done on the opposite side of the sample drop.

      9. Transfer the grid in the cryo-grid box. When all frozen grids are inside the grid box, screw the lid and transfer the grid box to a pre-cooled pierced 50 ml Falcon Tube under liquid nitrogen. Store the grid box container in a Dewar with liquid nitrogen until observation.



      Figure 4. Graphical summary of the vitrification step

    2. Cryo-EM observation: acquisitions were made using a Tecnai F20 operating at 200 kV (ThermoFisher FEI) equipped with a CCD camera (Eagle 4kx4k, ThermoFisher FEI)

      1. Pump the GATAN 655 cryo-holder until the vacuum is better than 10-4 Torr.

      2. Mount the grid on the cooled Cryo-holder.

      3. Rotate the goniometer of the microscope to -55° to insert horizontally the holder to prevent all the liquid nitrogen going out. Carefully introduce the cryo-holder in the microscope and wait until the end of the count-down in the “vacuum overview”. Once the count-down in the “vacuum overview” has ended, introduce the cryo-holder completely into the microscope and rotate the goniometer to 0°.

      4. Quickly refill the Dewar of the cryo-holder with liquid nitrogen.

      5. Connect the cable of the control station to the cryo-holder and check the temperature around -175 °C.

      6. Record images in low dose conditions and spot size 5. Search mode at x5,000 and exposure mode at x50,000 and 20 e-2s.

    3. Data analysis

      Reliable measurements of the phages morphology and sizes were obtained from cryo-TEM images and performed with ImageJ software.

Data analyses

Plunge freezing of GC1 particles allows us to observe their exact shape with reduced shrinkage of the capsid (Figures 5A-5D). GC1 is a tailless phage of about 60 nm in diameter, with the presence of an inner lipid membrane (Figure 5D, white arrow). The virion presents eleven vertices on its surface where spike complexes are anchored. A unique vertex ensures the viral DNA packaging and injection through the formation of a tubular structure from the membrane of about 10 nm in diameter and 60 nm in length (Figure 5D, black arrow). When DNA injection is not triggered, viral particles are fully filled with genetic material (Figure 5C) while it is partially filed when the tubular structure occurs (Figure 5D) (Saren et al., 2005; Peralta et al., 2013). We refer the readers to the original paper (Philippe et al., 2018).


Figure 5. Comparative images of negatively stained and cryo-fixed GC1 phage particles. A-B: GC1 phage prepared by negative staining and observed by TEM (Philippe et al., 2018). C-D: GC1 phage prepared by plunge freezing and observed by cryo-EM. Scale bar = 50 nm.

Notes

  1. Acetic acid bacteria (AAB) are generally recognized as safe bacteria and all the experiments can be performed in a biosafety level 1 laboratory. AAB can be easily recovered from mature/overippe grapes, and isolated on YPM agar supplemented with 0.1 ml of a 0.25% solution of penicillin (to inhibit the growth of Lactic acid bacteria) and 0.2 ml of a 0.25% alcoholic solution of pimaricin (to suppress the growth of yeasts and moulds). 5

  2. Alternatively, musts can be collected from wineries (instead of grapes).

  3. We analyzed several musts from red and white grapes. A single sample from white grapes yielded plaques on G. cerinus CRBO11179. All had the same morphotype. Two individual plaques were isolated and both corresponded to phage GC1. 5

  4. During preparation of stock I (Steps B4 and B5), intermediate R1, R2 and R3 lysates can be stored at 4 °C for a few weeks.

  5. Don’t use chloroform to avoid bacterial contamination of your phage stocks as it leads to Tectiviridae phage particles inactivation.

  6. Phage titer of stock II is 1,000 to 10,000-fold higher than that of stock I at this step.

  7. Step C produces a sufficient volume of fresh crude phage stock (stock II) to prepare a more concentrated lysate (stock III) by the Liquid Method (Procedure D). Stock II is not suitable for visual analysis by cryo EM.

Recipes

  1. Phage Buffer 10x (for 1 L)

    1. Dissolve 58 g of NaCl and 20 g of MgSO4·7H2O in 500 ml of 1 M Tris-HCl pH 7.5

    2. Complete to 1 L with distilled water

    3. Sterilize by autoclaving

    4. Store at room temperature

    5. To prepare 1x phage buffer, dilute 10 ml of 10x phage buffer with 90 mL distilled water and sterilize it by autoclaving (1 bar, 20 min)

  2. CaCl2·2H2O and MgSO4·7H2O 100x stock solutions

    1. Dissolve 35.5 g of CaCl2·2H2O in 100 ml of distilled water

    2. Dissolve 19.3 g of MgSO4·7H2O in 100 ml of distilled water

    3. Sterilize both stock solutions at 1 bar for 15 min

  3. Yeast-Peptone-Mannitol (YPM) broth (for 1 L)

    1. Weight 5 g of yeast extract, 3 g of peptone and 25 g of mannitol

    2. Add 800 ml distilled water

    3. Adjust pH to 5 with hydrochloric acid aqueous solution

    4. Complete to 1 L with distilled water

    5. Sterilize by autoclaving (1 bar, 20 min)

  4. Modified Yeast-Peptone-Mannitol solid medium (YPMΦ “bottom” agar) (for 1 L)

    1. Prepare 1 L of YPM broth (3a. to c.)

    2. Add 10 ml of CaCl2·2H2O and MgSO4·7H2O 100x stock solutions

    3. Add 20 g of granulated agar

    4. Complete to 1 L with distilled water

    5. Sterilize by autoclaving (1 bar, 20 min)

    6. Store at room temperature

  5. Modified Yeast-Peptone-Mannitol soft agar medium (YPMΦ) (for 1 L)

    1. Prepare 1 L of YPMΦ broth (4a. to c.)

    2. Add 6 g of granulated agar

    3. Dissolve agar by heating in a microwave until complete melting of the agar

    4. Distribute in 5 ml aliquots in glass tubes (20 ml) and close with cap-o-test caps

    5. Sterilize by autoclaving

    6. Store at 4 °C

References

  1. Cuervo, A. and Carrascosa, J. L. (2018). Observation of bacteriophage ultrastructure by cryo-electron microscopy. Methods Mol Biol 1693: 43-55.
  2. de Melo, A. G., Levesque, S. and Moineau, S. (2018). Phages as friends and enemies in food processing. Curr Opinion Biotechnol 49: 185-190.
  3. De Roos, J. and De Vuyst, L. (2018). Acetic acid bacteria in fermented foods and beverages. Curr Opinion Biotechnol 49: 115-119.
  4. Du Toit, M. and Pretorius, I. (2000). Microbial spoilage and preservation of wine: Using weapons from Nature's own arsenal-A review. S Afr J Enol Vitic 21: 74-96.
  5. Peralta, B., Gil-Carton, D., Castaño-Díez, D., Bertin, A., Boulogne, C., Oksanen, H. M., Bamford, D. H. and Abrescia, N. G. (2013). Mechanism of membranous tunnelling nanotube formation in viral genome delivery. PLoS biology 11(9): e1001667.
  6. Philippe, C., Krupovic, M., Jaomanjaka, F., Claisse, O., Petrel, M. and le Marrec, C. (2018). Bacteriophage GC1, a Novel Tectivirus Infecting Gluconobacter cerinus, an Acetic Acid Bacterium Associated with Wine-Making. Viruses 10(1).
  7. Saren, A. M., Ravantti, J. J., Benson, S. D., Burnett, R. M., Paulin, L., Bamford, D. H. and Bamford, J. K. (2005). A snapshot of viral evolution from genome analysis of the tectiviridae family. J Mol Biol 350(3): 427-440.

简介

[摘要]以分离噬菌体感染葡萄酒细菌的目的为目标,设计了一种分离纯化葡萄相关细菌噬菌体的方法。该方法证明成功地分离到了感染醋酸细菌葡糖杆菌(Gluconobacter cerinus)的GC1病毒。分离的噬菌体代表了大肠病毒科中的一个新属,名为“丙种病毒”。使用叔raditional技术为噬菌体颗粒的浓度,涉及离心几个步骤,在超微结构进一步的见解GC1可以通过观察到低温 电子显微镜,节省时间和精力。提出的简单工作流程可以应用于感染其他植物生态位的细菌的其他病毒。


[图形摘要]





流程图说明了在低温EM下分离,浓缩和观察GC1的方案



[背景]酿酒是一个复杂而多变的环境,其特征是不同微生物群落的时间顺序。葡萄酒酿造过程开始于水果的选择以及酵母将糖类发酵为酒精的过程。在大多数红葡萄酒和干白葡萄酒中,此步骤之后是苹果酸乳酸发酵,降低了酸度,增加了微生物的稳定性,并生产出优质的葡萄酒。与其他食品发酵相比,到目前为止,关于生态系统中感染细菌(也称为噬菌体或噬菌体)的病毒的多样性知之甚少(de Melo等人,2018)。但是,葡萄,乃至整个发酵过程中的全球,是不同生态系统(昆虫,植物及其根际,土壤和人类)之间的有趣交汇处,并且可能证明它是噬菌体及其细菌宿主基因组创新的宝贵来源。 。从技术的角度来看,从生态位上表征噬菌体也可以代表一种生态友好的替代品,以限制酿酒过程中细菌的腐败变质。在成熟的葡萄酒破坏剂中,乙酸细菌(AAB)会对葡萄酒质量产生负面影响,因为它们能够通过产生乙酸等其他化合物来增加葡萄酒的挥发性酸度(Du Toit和Pretorius,2000年; De Roos和De Vuyst ,2018年)。在这里,我们详细介绍了一种简单的方法来筛选和分离感染AAB的噬菌体,该方法可以应用于其他水果(Philippe et al 。,2018)。噬菌体颗粒从小-规模的生产,通过离心浓缩,并涉及PEG沉淀或耗时的步骤的CsCl被避免梯度。低温电子显微镜(cryo-EM)(Cuervo和Carrascosa ,2018)提供了高分辨率,并且在tectivirus病毒的超微结构中具有明显的特征。

关键字:噬菌体纯化, 复层噬菌体属, 噬菌体浓缩, 醋酸菌, 酿酒学, 冷冻电镜

材料和试剂

用料

1 耐高压加热的ë rlenmeyer瓶(体积:250米升)(Dutscher ,目录号:211907)
2 耐高压加热的玻璃管(体积:20米升)(Dutscher ,目录号:045209)
3 测帽测试盖(Dutscher ,目录号:110685B)
4 离心管(容积:50 ml )(Fisher Scientific,目录号:07-000-983)
5 巴斯德移液器
6 低温栅格盒(电子显微镜科学,目录号:71166-10-W)
7 冷冻袋(450毫升,VWR,目录号:82007-706)
8 莱西碳纤维300目铜网格(Ted Pella,目录号:01883)
9 聚醚砜(PES)膜注射过滤器(0.22微米和0.45微米)(Fisher Scientific公司,产品目录号:SLGPR33RS; SLHPR33RS)
10 聚丙烯微量离心管(体积:1.5米升)(Fisher Scientific公司,目录号:07-000-244)
11 锅铲(Dutscher ,目录号442209)
12 分光光度计的比色皿(体积:1.6米升)(Dutscher ,目录号:613101)
13 无菌一次性塑料注射器(容量:50 ml)(Fisher Scientific,目录号:13-689-8)
14 90 x 13毫米无菌培养皿(Dutscher ,目录号:076084B)
15 5级Whatmann滤纸(Dutscher ,目录号:1005055)
 


试剂种类


1 琼脂(Thermo Fisher Scientific,Fisher Chemical TM ,目录号10548030)
2 二水合氯化钙(CaCl 2 ·2H 2 O)(Thermo Fisher Scientific,Fisher BioReagents ,目录号:10306313)
3 乙烷(液化空气)
4 盐酸,33.33%(w / v)水溶液(Thermo Fisher Scientific,ULTREX,JT Baker TM ,目录号:10782232)
5 液氮(低温分布)
6 七水合硫酸镁(MgSO 4 ·7H 2 O)(Thermo Fisher Scientific,Fisher BioReagents ,目录号:10553335)
7 甘露醇(Thermo Fisher Scientific,ACROS Organics TM ,目录号:10266523)
8 蛋白ept(Thermo Fisher Scientific,Gibco TM Bacto TM蛋白ept,目录号:16299741)
9 氯化钠(NaCl)(Thermo Fisher Scientific,Technical,目录号:10498220)
10 Tris-HCl pH为7.5(赛默飞世尔科技,默克化学TM ,目录号:15153685)
11 酵母提取物(Thermo Fisher Scientific,Gibco TM Difco TM Bacto TM ,目录号:11763553)
12 10x噬菌体缓冲液(请参阅食谱)
13 CaCl 2 ·2H 2 O和MgSO 4 ·7H 2 O 100x储备溶液(请参阅配方)
14 YPM肉汤培养基(请参见食谱)
15 YPM Φ固体(“底部”)培养基(见配方)
16 YPM Φ软琼脂(见配方)
 


细菌菌株和病原学样本


1 新鲜碎葡萄(请参阅步骤)
2 cerucus cerinus cerbous cernous cerinus cernous cerinus cernous cerinus CRBO11179(法国生物资源科学研究院,ISVV,法国维拉纳夫-德奥农)
 


设备


 
1 高压釜
2 配备CCD照相机的低温电子显微镜(Thermo F isher FEI,型号:Tecnai F20 200 kV)(Thermo F isher FEI,型号:Eagle 4kx4k)
3 低温容器和低温转移站(加坦,型号:626)
4 辉光放电装置(Cordouan Technologies,型号:ELMO)
5 保温箱设置为25°C
6 高速冷冻离心机(日立,型号:CR 22N)和R15A-0688转子
7 微波(Thermo Fisher Scientific,Manutan TM MW20G,目录号:15540739)
8 轨道振动器(Stuart,型号:SSL1)
9 pH计(Hanna,型号:pH211)
10 分光光度计(岛津制作所,型号:UV-1280)
11 玻璃化机器人(Leica Microsystem,型号:EM-GP 2)
12 水浴(colume :4 L)(基准科学,MyBath模型)
 


软件


 
1 ImageJ(NIH)
2 Tecnai版本4.3(Thermo F isher FEI)
3 TEM成像与分析4.4版(赛˚F isher FEI)
 


程序




A 收集葡萄并回收果汁
1 从选定的葡萄园/酿酒厂将新鲜的葡萄收集到冷冻袋中(约200 xg)。
2 在运输过程中应将其储存在4°C,并尽快处理样品。
3 手动粉碎葡萄并离心样品(5,000 xg ,10分钟)。
4 收集上级(果汁)。
5 在无菌条件下依次用0.45 µm和0.22 µm PES膜过滤器过滤果汁。
6 将过滤后的果汁储存在4°C的冰箱中。
 


B 分离病毒斑块,纯化和生产一级原种(原种I)
1 葡萄糖酸蜡状文化准备
a 制备无菌250米升ë与15米rlenmeyer烧瓶升YPM肉汤。
b 接种用10μ升一个的准备使用的冷冻在细菌菌株甘油小号滴答孵育过夜,在25°振荡(200rpm)下进行。
c 第二天早晨,回稀释培养1/1015米升新鲜YPM的Φ肉汤并孵育在25℃下在振荡培养箱中直至OD 600nm处获得0.2(4〜5小时)。
2 底部顶部YPM的制备Φ板
a 熔融仔细底部顶部YPM Φ通过在加热琼脂烤箱微波。
b 在水浴中冷却至55 °C。
c 倾注平板,使用〜20 m升熔化介质(准备3块板每汁样品分析)
d 让琼脂基培养基在室温下固化1小时。
3 双琼脂覆盖试验
a 对于每个样品汁来分析,熔液3 YPM Φ软琼脂管在沸水浴中。
b 使用前,将试管保持在55°C的水浴中。
c 刚好在使用之前,采取过滤果汁出从4 ℃的冰箱(步骤A6) ,并准备连续稀释在缓冲液噬菌体1×(10 -1 ,10 -2 )。
d 删除YPM Φ从水浴软琼脂管,并将其放置在支架上,在室温下(RT)1-2分钟。
e 加入200 μ升细菌培养物在OD 600nm处0.2(步骤B1)和100 μ升未稀释滤汁在YPM Φ软琼脂管。
f 涡旋3次,并均匀地散布在固体YPM的顶部Φ板。
g 重复步骤B3D-F与每个稀释(10 -1 ,10 -2 )的果汁样品的(步骤乙使用两个剩余3C)YPM Φ琼脂平板上。
h 将板在25°C下孵育24小时。
4 斑块的隔离
a 选择带有单个噬菌斑的板。
b 视觉上识别斑块在YPM不同形态(大,小,清晰,混浊)Φ板(如果相关)。
c 用记号笔在板的底部每个选定的噬菌斑周围画一个圆圈。
d 冲床各斑块出使用无菌巴斯德吸管与抽吸灯泡。
e 将每个回收含单个噬菌斑到含有500微管琼脂塞μ升YPM的Φ肉汤。
f 轻轻涡旋
g 在室温下,使噬菌体在肉汤培养基中扩散20分钟。
h 用永久性标记在该微管上书写R1(第一轮纯化)。
i 储存于4 ℃或立即进行小号TEP B5 。
5 再进行两轮纯化
a 将R1样品在微管中的噬菌体缓冲液1x(10 -1至10 -4 )中稀释
b 对4个稀释样品进行双层琼脂平板接种(每个稀释样品重复S teps B1至B3)。
c 孵育24小时后,观察并选择包含单个噬菌斑的板。
d 冲一个单个噬菌斑到含有500μ的微管升YPM的Φ 。
e 用永久性标记在微管上写R2(第二轮纯化)
f 在同一敏感细菌菌株上重复第三轮也是最后一轮噬菌体感染,以确保纯度。所有步骤都必须在无菌条件下进行。
g 500 µl R3样品是您的主要纯噬菌体原种(原种I)。储存在4℃。
 


C 使用融合板裂解液方法制备二级噬菌体原液(原液II)
该步骤对于获得适当体积的噬菌体裂解物是必需的。


1 双琼脂覆盖试验
a 制备15微米升的培养G.蜡状CRBO 11179培养物(OD 600nm的在YPM 0.2)Φ肉汤(见步骤B1) 。
b 填3个无菌1.5米升微管与900微升1个的X无菌噬菌体缓冲液,并用稀释的数字(10标注管-1 〜10 -3 )。
c 在10 -1试管中加入100 µl储备液I并进行系列稀释。
d 制备24个YPM Φ琼脂平板上。
e 熔体24 YPM Φ在55软琼脂管,并放置在一水浴℃下。
f 除去8 YPM Φ从水浴软琼脂试管并将其放置在支架上,在室温下(RT)1-2分钟。
g 在每个试管中,将100 µl的十倍噬菌体原液I与200 µl的敏感细菌混合,如S teps B1至B3所示。
h 倾上的YPM每个管Φ琼脂平板。
i 重复小号TEPS C1F到C1H与8个额外的软琼脂管和YPM Φ琼脂板上,并使用100μ升的股票我稀释至10 -2 。
j 重复小号TEPS C1F到C1H与剩余的8支软琼脂管和YPM Φ琼脂板上,并使用100μ升的股票我稀释至10 -3 。
k 在25°C下将24个板孵育24小时。
2 回收存货II
a 对于涂布在YPM每个稀释Φ板(10 -1 ,10 -2 ,10 -3 ),观察噬斑的密度。
b 选择最高的稀释度以进行融合裂解(见图1 )。仅处理与该稀释液相对应的8个板。
 






图1 。Ilustration一个融合的裂解


 


c 在无菌条件下,添加1米升的无菌YPM Φ肉汤每个所选琼脂平板8双层的表面上。
d 在室温下孵育20分钟。或者,在此步骤中,板可以在4 °C下保存过夜。
e 用无菌刮刀小心地将每块板中的软琼脂和液体培养基收集在离心管中。
f 在15°C下以10,000 xg离心10分钟。
g 使用0.45 µm PES膜滤器过滤上清液(6-8 ml )。
h 将储存液II储存在4°C下。
 
3 按以下方法确定原料II的效价(图2 )
a 制备15微米升的培养G.蜡状CRBO 11179培养物(OD 600nm的在0.2)YPM Φ肉汤(参见B1) 。
b 制备8个无菌1.5毫升微管含有900μ升1X无菌的噬菌体buffe和š erially稀股票II 10 -1至10 -8 。
c 采取YPM Φ琼脂平板和绘制的8-10位的网格在板底部。
d 除去5μm的升YPM Φ在55从水浴软琼脂管℃,并用0.2M接种升中制备的培养的步骤C 3a中。
e 短暂涡旋均化,倒在YPM Φ琼脂板,并让在室温琼脂固化(30分钟)。
f 在网格的可用空间中滴入10 µl稀释的每份储备液II (纯至10 -8 )。盖上盖子,将培养皿放在工作台上,直到液滴被吸附(1小时)。
g 在25°C下孵育过夜。
h 确定稀释液的最佳范围,以每个空间计算2至20个噬菌斑。滴度(PFU /米升)可以通过下面的公式来计算:PFU /米升(二次库存的)=斑块X 1的1 /稀释因子x个/(0.01)。
 






FIGURE  2 。快速滴定噬菌体原液(II和III)


 


D 液相法制备高浓度GC1噬菌体悬浮液(原液III)
1 引入无菌15米升YPM Φ肉汤在无菌250米升ë rlenmeyer烧瓶中。
2 接种10 µl的G. cerinus CRBO11179冷冻培养物。
3 在25°C下于200 rpm的振荡培养箱中孵育过夜。
4  测量OD 600nm的培养物并在含有60米新烧瓶转移的体积升YPM Φ肉汤以获得OD 600nm处的0.1,对应于大约10 8 CFU /米升。
5 划分成三个独立的无菌250米升锥形烧瓶(20米升/烧瓶)(见图URE 3 )。
 






图3 。噬菌体储备I,II和III的制备摘要


 


6 用噬菌体GC1感染两个烧瓶,其中200个细菌的感染复数(MOI)约为1个噬菌体。第三种培养物用作未感染的阴性对照。
7 允许噬菌体在RT下30分钟吸附到细菌细胞上,而不会产生斑点。
8 在200 rpm的振荡培养箱中于25°C孵育。
9 定期测量OD 600nm (在14-16小时后,被感染的检测物和对照物的平均值分别为1和2.5)。
注意:不要È XTEND孵育14-16以外H作为在最终的噬菌体效价的2数值的减少通常被观察到。                           


10 将裂解的培养物在10,000 xg下于15°C离心10分钟。
11 通过0.22过滤上清液微米PES过滤器。
12 如步骤C3所述,测量裂解物的效价。
注:〜10的滴度10 PFU /米升在实验室中常规获得。噬菌体滴度应超过10 7 PFU / ml ,才能在冷冻电镜下观察到。


13 将过滤后的上清液转移到50 ml的离心管(约38 ml )中,并在4°C下以20,000 xg离心2 h (确保所有样品管均已装满以适当平衡离心机)。
14 小心除去上清液,然后风干沉淀。
15 在20至50 µl无菌噬菌体缓冲液1x中轻轻重悬噬菌体沉淀
16 储存在4℃。
注意:用于冷冻-EM观察的样品不应不透明,如果需要,可将噬菌体缓冲液稀释1倍。


 


E冷冻EM观察GC1噬菌体颗粒
我们使用以下标准协议。


1玻璃化:使用EM-GP设备冷冻制剂(该过程已总结在图4中)。
a 向加湿器中注入蒸馏水,并使用Whatman 5级更换吸墨纸。       


b 设置腔室条件:温度为25°C,湿度为80%,乙烷温度为-184°C。      


c 设置镊子条件:必须调整“吸墨设置”,以使滤纸在不拉动的情况下接触网格。必须调整“栅格印迹位置”以将栅格放置在纸张边缘上方1毫米处,并调整“栅格Tf位置”以将栅格完全浸入乙烷容器中。       


d 调整冻结设置:取消选择旋转和传感器。选择“ A插入”以在印迹后自动插入网格。如下设置时间设置:延迟为0.0;以2.0 s吸干并保持0.0。      


e 放置乙烷容器和栅格箱支架后,用液氮填充容器,直到100%。       


f 在-120°C附近,在0.1 mBar的压力下冷凝乙烷。        


g 辉光放电网格。将Lacey网格(碳朝上)放在一块覆盖有Parafilm的载玻片上。使用以下辉光放电条件:真空度为0.3 mBar ,强度为3 mA,计时器为40 s。      


h 在网格的碳侧上涂抹4 µl的足够量的水,然后通过按“印迹/ A浸入”激活吸水和浸入冻结。在样品滴的另一侧进行印迹。      


i 将栅格转移到低温栅格盒中。当所有冷冻的油脂都在格栅盒内时,拧紧盖子,将格栅盒转移至在液氮下预先冷却的,穿孔的50 ml Falco n管中。将格栅盒容器存放在带有液氮的杜瓦瓶中,直到观察。        


 






图4 。玻璃化步骤的图形摘要


 


2 Cryo-EM观测:使用配备CCD摄像机(Eagle 4kx4k,Thermo F垫圈FEI),运行在200 kV的Tecnai F20(Thermo F垫圈FEI)进行采集。
a 抽气GATAN 655低温保持器,直到真空度超过10-4托。       


b 将栅格安装在冷却的低温支架上。      


C 将显微镜的测角仪旋转到-55°,以水平插入支架,以防止所有液氮流出。小心地将低温保持器引入显微镜,并等待直到“真空概览”中的倒数计时结束。一旦“真空概览”中的倒数结束,就将冷冻保持器完全引入显微镜,并将测角仪旋转至0°。       


d 快速用液氮重新填充冷冻柜的杜瓦瓶。      


e 将控制站的电缆连接到低温保持器,并检查-175°C附近的温度。       


f 在低剂量条件和光斑尺寸5.搜索模式记录的图像在X5 ,000和曝光模式在X50 ,000和20e中- / A 2秒。        


3 数据分析
噬菌体形态和大小的可靠测量是从冷冻TEM图像获得的,并使用ImageJ软件进行。


 


资料分析


 


彻底冻结GC1颗粒使我们能够观察到衣壳收缩减少的确切形状(图5 A- 5 D)。GC1是直径约60 nm的无尾噬菌体,带有内部脂质膜(图5 D,白色箭头)。病毒体在其表面上固定了钉状复合物的表面上具有11个顶点。独特的顶点可通过从直径约10 nm,长度约60 nm的膜形成管状结构来确保病毒DNA的包装和注射(图5 D,黑色箭头)。当不触发DNA注射时,病毒颗粒被遗传物质完全填充(图5 C),而当管状结构出现时病毒颗粒被部分填充(图5 D)(Saren等人,2005 ;Peralta等人,2013)。 。我们向读者推荐原始论文(Philippe等,2018)。


 






图5 。负染色和冷冻固定的GC1噬菌体颗粒的比较图像。AB:通过阴性染色制备的GC1噬菌体,并由TE M观察到(Philippe等,2018)。C- D:通过骤冷制备的GC1噬菌体,并通过冷冻-EM观察。比例尺= 50 nm。


 


笔记


 


1. 乙酸细菌(AAB)通常被认为是安全细菌,所有实验都可以在生物安全1级实验室中进行。可以很容易地从成熟/过量葡萄中回收AAB ,并在YPM琼脂上分离,补充0.1 ml的0.25%青霉素溶液(抑制乳酸菌的生长)和0.2 ml的0.25%pimaricin乙醇溶液(抑制酵母菌和霉菌的生长)。5
2. 另外,也可以从酿酒厂(而不是葡萄)收集芥末。
3. 我们分析了红葡萄和白葡萄的几种葡萄汁。从白葡萄单一样本产生上斑块G.蜡状CRBO11179。全部具有相同的形态型。分离出两个单独的噬菌斑,并且都对应于噬菌体GC1。5
4. 在制备过程中的库存I(小号TEPS B4和B5),中间R1,R2和R3的裂解物可以储存在4 ℃下几个星期。
5. 不要使用氯仿来避免细菌污染您的噬菌体原种,因为它会导致Tectiviridae噬菌体颗粒失活。
6.股票II的噬菌体效价为1 ,000至10 ,在该步骤比的库存000倍更高的I。
7. 步骤C产生足够量的新鲜噬菌体原液(原液II),以通过液体方法制备更浓缩的裂解液(原液III)(步骤D)。Stock II不适合通过cryo EM进行视觉分析。
 


菜谱


 
1. 噬菌体缓冲液10 x (1 L)
将58 g NaCl和20 g MgSO 4 ·7H 2 O溶于500 ml 1 M Tris-HCl pH 7.5中
用蒸馏水补足至1 L
高压灭菌
室温保存
为了制备1 X噬菌体缓冲液,D ilute10米升10的X噬菌体缓冲液用90毫升蒸馏水和通过高压灭菌进行灭菌(1巴,20分钟)
2. CaCl 2 ·2H 2 O和MgSO 4 ·7H 2 O 100 x储备溶液
将35.5 g CaCl 2 ·2H 2 O溶解在100 ml蒸馏水中
将19.3 g的MgSO 4 ·7H 2 O溶于100 ml的蒸馏水中
将两种储备溶液在1巴下灭菌15分钟
3. 酵母蛋白ept甘露醇(YPM)汤(1升)
重量5克酵母提取物,3克蛋白ept和25克甘露醇
添加800米升蒸馏水
用盐酸水溶液调节pH至5
用蒸馏水补足至1 L
高压灭菌(1 bar,20分钟)
4 改性酵母蛋白胨-甘露醇固体培养基(YPM Φ “底部”琼脂) (1 L)
准备1 L的YPM汤(3a 。至c 。)
添加10米升的CaCl 2 ·2H 2 O和硫酸镁4 ·7H 2个ö100 X原液
加入20克颗粒琼脂
用蒸馏水补足至1 L
高压灭菌(1 bar,20分钟)
室温保存
5 修饰的酵母-蛋白胨-甘露醇软琼脂培养基(YPM Φ )(1 L)
制备1升的YPM Φ肉汤(4a上。至c。)
加入6克颗粒琼脂
通过在微波炉中加热直至琼脂完全融化来溶解琼脂
分布在5米升在玻璃管中的等分试样(20米升与帽邻测试帽)和密切
高压灭菌
储存在4°C
 


参考文献

1 Cuervo,A.和Carrascosa,J.L .(2018年)。冷冻电子显微镜观察噬菌体的超微结构。方法分子生物学1693:43-55。
d2 e Melo,AG,Levesque,S.和Moineau ,S.(2018年)。噬菌体在食品加工中是朋友和敌人。Curr O pinion Biotechnol 49:185-190。
3 De Roos ,J.和De Vuyst ,L.(2018)。发酵食品和饮料中的乙酸细菌。Curr O pinion Biotechnol 49:115-119。
4 Du Toit,M。和Pretorius,I。(2000)。微生物变质和葡萄酒的保存:使用自然界自己的武器库中的武器-A评论。S Afr J Enol Vitic 21:74-96。              
5 佩拉尔塔,B.,吉尔-纸箱,D.,卡斯塔NO-DI EZ ,D.,贝尔坦,A.,布洛涅,C.,奥克萨宁,HM,班福德,DH和Abrescia ,NG(2013)。病毒基因组传递中膜隧穿纳米管形成的机制。PLoS生物学11(9):e1001667。
6 菲利普,C.,Krupovic ,M.,Jaomanjaka ,F.,CLAISSE,O.,海燕,M。和乐Marrec ,C。(2018)。噬菌体GC1,新型复层病毒侵染葡糖Ç erinus ,醋酸菌与关联酿酒。病毒10(1)。
7 Saren ,AM,Ravantti ,JJ,Benson,SD,Burnett,RM,Paulin,L.,Bamford,DH和Bamford,JK(2005)。病毒进化的快照,来自tectiviridae家族的基因组分析。分子生物学杂志350(3):427-440。              
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引用:Chaïb, A., Decossas, M., Philippe, C., Claisse, O., Lambert, O. and Le Marrec, C. (2020). Isolation and CryoTEM of Phages Infecting Bacterial Wine Spoilers. Bio-protocol 10(21): e3801. DOI: 10.21769/BioProtoc.3801.
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