Materials and Reagents

General use

Standard pipette filter tips (TipOne filter tips) (StarLab, catalog numbers: S1120-3810 , S1120-1810 , S1120-8810 and S1112-1720 or equivalent)
Pipettes (10 ml serological pipette) (Sarstedt, catalog number: 86.1254.001 )
Ultrapure water

Cell culture

175 cm² flasks (Greiner Bio-One, catalog number: 660175 , or equivalent)
Filtropur S 0.2 (Sarstedt, catalog number: 83.1826.001 )
Stericup Quick Release Express Plus 0.22 µm PES (Merck Millipore, catalog number: S2GPU11RE )
Acanthamoeba castellanii (Douglas) Page (ATCC, catalog number: 30010 )
D-(+)-glucose (Sigma, catalog number: G8270 )
Sodium citrate tribasic trihydrate (Sigma, catalogue number: C7254 )
Proteose-Peptone (Sigma, catalog number: 82450 )
Yeast extract (Fisher BioReagent, catalog number: BP1422 )
Ampicillin Sodium Salt, Cell Culture/Molecular Biology Grade (Euromedex, catalog number: EU0400 )
Kanamycin Sulfate, Cell Culture Grade (Euromedex, catalog number: UK0010 )
D-(+)-Glucose (Sigma, catalog number: G8270 )
Proteose Peptone Yeast Extract medium (PPYG; see Recipes)
Base medium
Complete medium
36% Glucose solution (see Recipes)
Ampicillin stock solution (100 mg/ml) (see Recipes)
Kanamycin stock solution (25 mg/ml) (see Recipes)

Gradient preparation and recovery

Plastic Syringes (5 ml syringe) (Terumo, catalog number: SS*05SE1 )
21G x 1^1/2” needle (Terumo, catalog number: AN*2138R1 or equivalent)
Sterile 50 ml conical tubes (Sarstedt, catalog number: 62.547.254 , or equivalent)
Polyallomer Tubes, 38.5 ml (Beckman, catalog number: 326823 )
UltraPure Cesium chloride Optical Grade (Invitrogen, catalog number: 15507-023 )
Cesium chloride solutions (1.2 g/cm³, 1.3 g/cm³, 1.4 g/cm³ and 1.5 g/cm³; see Recipes)
K36 buffer (see Recipes)

Purification quality control

Glass microscope slide (Biosigma, catalog number: VBS653/A )
Coverslip (Biosigma, catalog number: VBS636 )
UVette 220-1600 nm (Eppendorf, catalog number: 952010051 )
Formvar and carbon coated 200 mesh copper/rhodium grids (Electron Microscopy Sciences, catalog number: FCF200-Cu )
Uranyl acetate 1% in water
Note: Uranyl acetate is a radioactive chemical. It is provided by electron microscopy facilities as we are not allowed to use it in the lab.

Equipment

Microbiological safety hood (ADS laminaire, model: Optimal 12 , or equivalent)
Incubator Bio Performance (Froilabo, catalog number: BP240 , or equivalent)
Centrifuge (Beckman Coulter, model: JXN-30 )
Swinging-Bucket Rotor (Beckman Coulter, model: JS24-38 )
Spectrophotometer (Eppendorf, model: Biophotometer )
Inverted microscope (Zeiss, model: Axio Observer.Z1 )
Transmission Electron Microscope (FEI, model: Tecnai G2 )
Autoclave (SHP Steriltechnik SG, model: Laboklav 135 )
Peristaltic pump (KNF Lab, model: Laboport )

Procedure

Seed 20 175 cm² flasks with 1,000 Acanthamoeba castellanii cells per cm² in 20 ml of PPYG medium. Incubate at 32 °C for two days.
Note: You can add ampicillin and kanamycin at final concentrations of 0.1 mg/ml and 0.025 mg/ml, respectively, in each flask to avoid bacterial contamination.
After two days, the cell confluence should be between 130,000 and 180,000 cells/cm². Infect the cells with a Mimivirus stock at a multiplicity of infection of 0.2.
Incubate at 32 °C for two days or until cell lysis is complete.
Note: There is no need to change the medium before infection or to remove the virus from the flasks.
Harvest the cell debris-containing supernatants from the flasks and pool them into 50 ml conical tubes.
Note: For 20 175 cm² flasks containing 20 ml of medium you will need 7 tubes.
Centrifuge at 500 x g for 10 min at room temperature (~25 °C, RT) to remove the cell debris.
Decant the supernatant into new 50 conical tubes.
Centrifuge at 10,000 x g for 25 min at RT to pellet the virus.
Discard the supernatant.
Resuspend the resulting virus pellets in 10 ml K36 buffer by pipeting and pool in two new 50 ml conical tubes (35 ml per tube). Complete the volume to 50 ml with K36 buffer.
Centrifuge at 10,000 x g for 25 min at RT to pellet the virus.
During the centrifugation, prepare four CsCl gradients in Beckman polyallomer tubes. Add 7.5 ml of CsCl solution density 1.5 g/cm³ at the bottom of each tube, then carefully overlay with 9 ml of 1.4 g/cm³ CsCl solution and 9 ml of 1.3 g/cm³ CsCl solution, dropwise. Hold for the final virus-containing 1.2 g/cm³ density layer.
Discard the supernatant from the centrifugation in Step 9.
Resuspend the two viral pellets with 16 ml of 1.2 g/cm³ CsCl solution for each pellet by pipeting.
Overlay 8 ml of resuspended virus pellets on top of each of the four gradients, dropwise (Figure1A).
Centrifuge overnight (between 16 and 18 h) at 100,000 x g at 20 °C in a Beckman Coulter JS24-38 rotor.
After centrifugation, harvest the white ring corresponding to the virus fraction (Figure 1B) by carefully pipetting through the gradient or by puncturing the polyallomer tube and aspirating with a needle and a syringe (Figure 2). Transfer the virus fraction to two new 50 ml conical tubes, add K36 buffer up to 50 ml in each tube and mix by inverting the tubes.

Figure 1. Centrifuge tubes before (A) and after (B) separation of Mimivirus particles on CsCl gradient. The white ring indicated by the black arrow corresponds to Mimivirus particles.

Figure 2. Collection of the viral fraction by side puncture

Centrifuge at 10,000 x g for 25 min at RT to pellet the virus.
Resuspend the two pellets with 10 ml of K36 buffer each, complete the volume to 50 ml with K36 and centrifuge them at 10,000 x g for 25 min at RT to wash the virus.
Repeat Steps 16 and 17 twice. For the last wash, pool the resuspended virus in a single 50 ml conical tube.
Resuspend the final pellet in 10 ml of K36 buffer.
The virus is ready to be titrated or stored at -80 °C until further use.

Notes:

Typically, when purifying Acanthamoeba polyphaga Mimivirus using that protocol, we recover 10 ml of viral solution containing 1 x 10¹⁰ to 4 x 10¹⁰ particles/ml.
The purified particles are infectious and can be used to performed new infections or any other experiments.

Data analysis

Light microscopy
Drop 4.5 µl of the purified virus suspension on a microscope slide and overlay with a coverslip. Invert for a few hours in order to let the virus settle onto the coverslip for easier focusing. Observe with a light microscope using the 63x objective to confirm the purified virus does not contain any obvious contaminants (Figure 3).

Figure 3. Observation of Mimivirus purified particles with a light microscope (63x objective, 1.6x optovar)

Transmission Electron Microscopy (TEM)
For TEM, dilute the purified Mimivirus sample 1:2 (v/v) in K36 buffer and incubate a 10 µl droplet on a formvar- and carbon-coated 200 mesh copper/rhodium grid for 1 min at RT. Wash the grid with three successive 1% uranyl acetate droplets. Leave the residual uranyl acetate on the grid for 1 min and remove by gently touching the edge of the grid with a filter paper. After drying, examine the grid using an electron microscope (Figure 4).

Figure 4. Observations of Mimivirus purified particles by negative staining

Recipes

Glucose solution
Dissolve 36 g of glucose and 2 g of sodium citrate in 100 ml of slightly warm water.
When the solution is clear, use it to prepare the complete PPYG medium.
Proteose Peptone Yeast Extract (PPYG) medium
Base medium:
Autoclave 40 g of proteose peptone and 2 g of yeast extract diluted in 1.8 L of ultrapure water in a 2 L glass bottle.
Let it cool at room temperature.
Complete medium:
1. Add the following to the basal medium
  20 ml of MgSO₄ 400 mM
  16 ml of CaCl₂ 50 mM
  20 ml of Fe(NH₄₎₂(SO₄₎₂ 5 mM
  20 ml of Na₂HPO₄ 250 mM
  20 ml of KH₂PO₄ 250 mM
  and 100 ml of glucose solution.
2. Filter through 0.22 µm Stericups using a peristaltic pump and store at room temperature
Ampicillin stock solution (100 mg/ml)
1. Dissolve 1 g of ampicillin in 10 ml of ultrapure water
2. Filter through a 0.22 µm filter plugged on a syringe
3. Aliquot in 1.5 ml tubes and store at -20 °C
Kanamycin stock solution (25 mg/ml)
1. Dissolve 250 mg of kanamycin in 10 ml of ultrapure water
2. Filter through a 0.22 µm filter plugged on a syringe
3. Aliquot in 1.5 ml tubes and store at -20 °C
K36 buffer
1. Dissolve 2.25 g of KH₂PO₄, 5.8 g of K₂HPO₄·3H₂O, 7.4 g of KCl and 0.9 g of NaCl in 1 L of ultrapure water
2. Filter through a 1 L 0.22 µm Stericup linked to a peristaltic pump and store at room temperature
Cesium chloride solutions
Density 1.5 g/cm³: dissolve 22.7 g of CsCl in 27.3 ml of ultra pure water
Density 1.4 g/cm³: dissolve 19.4 g of CsCl in 30.6 ml of ultra pure water
Density 1.3 g/cm³: dissolve 15.62 g of CsCl in 34.38 ml of ultra pure water
Density 1.2 g/cm³: dissolve 11.2 g of CsCl in 38.8 ml of ultra pure water
Once the solutions appear clear, filter through a 0.22 µm filter plugged on a syringe

Acknowledgments

This work was supported by the French National Center for Scientic Research and the “coup de chapeau” of the Mediterranneen Institute of Microbiology.

Competing interests

There are no conflicts of interest or competing interest.

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