Materials and Reagents

Sterile single-use inoculating loop
200 μl pipette tips (such as: Corning, Axygen^®, catalog number: # T-200-C )
Parafilm (Bemis, catalog number: PM996 )
Syringe-mounted 0.45-µM filter (EMD Millipore, catalog number: SLHV033RS )
Polyethersulfone (PES) filter, 0.45-µM, Ø75-mM membrane diameter, with an attached 500-ml bottle. Operated by a vacuum pump (e.g., Guangzhou Jet Bio-Filtration, catalog number: FPE404500 )
1 ml and 10 ml sterile syringes (without needle)
1 ml syringes (such as: KDL Medical Product, catalog number [as written on the package]: 7290010993604)
10 ml syringes (such as: Medisposables, catalog number [as written on the package]: 9304-10/16933506093041 )
Sterile 15-ml Falcon tube (such as: SPL Life Sciences, catalog number: 50015 )
12 ml polypropylene/polyallomer ultracentrifuge tubes (such as: Beckman Coulter, catalog number: 331372 ) – make sure that they are fit for the ultracentrifuge’s speed
Syringe-mounted 0.22-µM filter (Axiva Sichem Biotech, catalog number: SFPV13 R )
Sterile 1.5-ml tube (1.7-ml clear tube) (Corning, Axygen^®, catalog number: # MCT-175-C )
1 ml pipette tips (such as: Corning, Axygen^®, catalog number: # T-1000-C )
Polystyrene (PS) aseptic Petri dishes, 90 x 15-mm (Miniplast Ein-Shemer, catalog number: 820-090-01-017 )
Xanthomonas strain in glycerol stock stored at -80 °C – for the preparation of this protocol we used X. campestris pv. campestris (Xcc) 33913, which was obtained from American Type Culture Collection (ATCC)
Difco nutrient agar (BD, catalog number: 21300 )
Note: It contains 3 g/L of beef extract, 5 g/L of peptone, and 15 g/L of agar, with a final pH of 6.8 ± 0.2 (as written on the powder’s container).
Cephalexin hydrate (Sigma-Aldrich, catalog number: C4895 ) – stock solution at concentration of 10 mg/ml
Sterile dH₂O
10x phosphate buffered saline (PBS) buffer (optional) (such as: Sigma-Aldrich, catalog number: P5493 )
OptiPrep density gradient medium (Sigma-Aldrich, catalog number: D1556 )
‘Coomassie Plus – The Better Bradford Assay Kit’ (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23236 ) (optional)
Lipid dye FM4-64FX (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: F34653 ) (optional)
Materials and reagents required for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)
Yeast extract powder (EMD Millipore, catalog number: 61931105001730 )
Bacto tryptone (BD, Bacto, catalog number: 211705 )
Sodium chloride (NaCl) (EMD Millipore, catalog number: 7647-14-5 )
Sucrose crystallized (Duchefa Biochemie, catalog number: S0809 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (EMD Millipore, catalog number: 105886 )
NaOH or HCl (in order to adjust pH)
Peptone (EMD Millipore, catalog number: 61930705001730 )
L(+)-glutamic acid (Acros Organics, catalog number: 6106-04-3 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
YEB medium (see Recipes)
PSB medium (see Recipes)
OptiPrep diluent buffer (see Recipes)

Note: (optional) – not necessary for the vesicles’ isolation process.

Equipment

Biological hood
Temperature controlled incubator shaker (28 °C), with a capability to reach 200 RPM (such as: Thermo Fisher Scientific, Thermo Scientific^TM, model: MaxQ^TM 6000 )
Two 2-L Erlenmeyer flasks
Cuvette
Polypropylene Copolymer (PPCO) 250-ml centrifuge bottles (such as: Thermo Fisher Scientific, Thermo Scientific^TM, model: Nalgene^TM PPCO Centrifuge Bottle ) – make sure that they are fit for the centrifuge’s speed
High-speed centrifuge (e.g., Beckman Coulter, model: Avanti J-E , catalog number: 369003; or Thermo Fisher Scientific, model: Sorvall^TM RC-6 , catalog number: 74804^*) and 6 x 250-ml rotor (such as: Beckman Coulter, model: JLA-16.250 , catalog number: 363930, dual-locking lid; or Thermo Fisher Scientific, Sorvall^TM, model: SLA-1500 , catalog number: 46615^* [old model]) at a speed of approx. 32,000 x g
Ultracentrifuge (e.g., Thermo Fisher Scientific, Sorvall^TM, model: Discovery 90SE , catalog number: 40130867^*, Kendro Laboratory Products) and a 6 x 12-ml swinging bucket ultracentrifuge rotor such as: Thermo Fisher Scientific, Sorvall^TM, model: TH-641 , catalog number: 54295^* (old model, Kendro Laboratory Products), or Thermo Fisher Scientific, Thermo Scientific^TM, model: TH-641 , catalog number: 54295 (new model,) at a speed of approx. 175,000 x g
Autoclave
NanoSight device (Malvern) (optional)
Transmission electron microscopy (optional)
Equipment required for SDS-PAGE as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)

Notes

*Catalog number of an old model.
(optional) – not necessary for the vesicles’ isolation process.
Important note: the centrifuge speeds mentioned above, were calculated with Science Gateway's centrifuge rotor speed calculator (http://www.sciencegateway.org/tools/rotor.htm) – if the centrifuge/ultracentrifuge did not give such option.

Procedure

Preparations prior to OMV isolation
1. Preparation of 1 L of YEB (for starters, see Recipes).
2. Preparation of 1 L of PSB (the main medium, see Recipes).
3. Plating the bacteria
  Plate Xcc 33913 (from glycerol stock) on Difco nutrient agar plates, by using a sterile single-use inoculating loop.
  Note: It is recommended to plate the bacteria 2-5 days before making the starters with Xcc.
4. Making Xcc starters (2 sterile tubes with 3 ml of sterile YEB in each)
5. Inoculation of the main medium (PSB)
  1. To every 500 ml of PSB medium in each 2-L Erlenmeyer flask, add 500 µl of cephalexin hydrate 10 mg/ml (final concentration will be 10 µg/ml of medium).
  2. Then, from one of the Erlenmeyer flasks, transfer 1 ml of medium (+ cephalexin) into a clean, new cuvette (will serve as a blank for the spectrophotometer). Cover the cuvette with 2 layers of Parafilm, and keep the cuvette at 4 °C.
  3. Then, to each Erlenmeyer flask, add 300 µl from the same starter (has to be very cloudy).
  4. Put the Erlenmeyer flasks into 28 °C shaker, at the speed of 180-200 rpm, for overnight (Video 1).
  Notes:
6. On the next day – object density measurement
  1. The cuvette stored at 4 °C (with the PSB medium and cephalexin hydrate) serves as a blank. This cuvette should be kept at 4 °C between measurements.
  2. At the ‘normal range’ described in Table 1, the concentration of Xcc 33913 is approximately 10⁸ cfu/ml, and the cells are at around the mid log phase of the growth curve. Just before cell harvest, a small sample of the culture is taken, serially diluted and plated to determine cell number and to assess the amount of purified vesicles per cell.
    
    Table 1. The object density ranges
  Notes:
The isolation process
1. Removal of bacteria
  1. Pour the cultures into 250-ml bottles, and centrifuge them at approx. 13,200 x g for 22 min, at the temperature of 4 °C to pellet bacterial cells. Rotors that can be used: GSA (Sorvall), JLA-16.250 (Beckman-Coulter) (Video 2).
    
    Video 2. Removal of bacteria
  2. Then, filter the supernatant through a 0.45-µM PES filter with Ø75-mM membrane diameter (operated by a vacuum pump).
    Optional: It is possible to filter the entire 1 L of the culture through such filter; thus, you can connect it to a 1-L bottle (if the filter fits). The filtered supernatant can be stored at 4 °C up to 1 day.
2. Pelleting the vesicles
  1. Pour the filtered supernatant into 250-ml bottles, and centrifuge them at approx. 32,000 x g for 2 h, at 4 °C. The vesicles will be pelleted (Figure 1) in the outward direction from the center of the rotor. Rotors that can be used: SLA-1500 (Sorvall), JLA-16.250 (Beckman-Coulter) (Video 3).
  2. Discard the supernatant and resuspend the pelleted vesicles, using 5-8 ml of sterile dH₂O, by pipetting.
    Optional: Filter the resuspended vesicles through 0.45-µM (operated by a 10-ml syringe) into a sterile 15-ml Falcon tube, so you can keep them at 4 °C for a few days. Must be done in a biological hood.
    
    Figure 1. The pelleted OMVs with a translucent yellow color can be seen in the bottom of the tubes (within the marked area of each tube), after centrifugation at approx. 32,000 x g for 2 h
    
    Video 3. Pelleting the vesicles
3. Ultracentrifugation
  1. Pour the resuspended vesicles, from the previous step, into two 12-ml ultracentrifuge tubes, and use sterile dH₂O to fill the remaining volume of the tubes.
  2. Ultracentrifuge the resuspended vesicles, at approx. 175,000 x g, for 1 h and 35 min, at 4 °C. Rotor that can be used: Sorvall TH-641. Ultracentrifuge that can be used: Sorvall Discovery 90SE. The vesicles will be pelleted in the bottom of the tubes (Figure 2, Video 4).
    
    Figure 2. The pelleted OMVs seen in the bottom of the 12-ml tubes, after ultracentrifugation at approx. 175,000 x g for 1 h and 35 min
    
    Video 4. Ultracentrifugation
4. Final resuspension and storage
5. Density gradient centrifugation: In order to purify the OMV further, for analyses that require highly-purified samples (e.g., protein analysis in LC-MS/MS), a density gradient centrifugation step is required (Chutkan et al., 2013).
OMV preparation quality and quantity checks
1. Checking if the preparation is sterile
  1. Pipette 10 µl of OMV preparation on a plate with Difco nutrient agar, and leave open until the 10 µl drop dries out. Must be done in a biological hood.
  2. Then, place the plate in a 28 °C incubator for 2-5 days, and check if colonies are formed where the drop was placed.
2. Quantification of the OMVs
3. Transmission electron microscopy (TEM)
  TEM can be used to view OMV (Figure 4) and estimate size range and concentration as well as examine the purity of the preparation.
  
  Figure 4. TEM image of an OMV preparation, diluted in sterile dH₂O by a ratio of 1:20. OMV diameter may range from 20-250. A scale bar is at the bottom left.
4. SDS-PAGE
  In order to get the profile of the proteinaceous cargo of OMV, run an OMV sample, which was denatured in a sample buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT)/β-mercaptoethanol, on a protein gel. If you are searching for a specific protein associated with OMV, you can also run a Western blot or a dot blot assays. SDS-PAGE is also a way to compare the proteinaceous profiles of OMV isolated from different bacteria.

Data analysis

Perhaps one of the main challenges in purifying OMVs from bacterial culture is to prove its purity. Since OMVs are purified from culture supernatants the risk of contamination by surface, extracellular bacterial appendages, such as flagella, pili, fimbriae, must be taken into consideration. Carefully examining multiple TEM grid samples and determining whether such appendages had co-purified with the OMVs or not is highly recommended to assure purity. Another possible contaminant of OMV preparations are broken cell debris and large protein complexes. To minimize the chances of broken cell contamination cultures should be harvested during the logarithmic growth phase when cell death and breakage is less likely to occur. Additionally, we highly recommend that the density gradient centrifugation step will be carried out for any down-stream studies, especially for analytical studies such as proteomics, to avoid co-purification of large protein or protein complexes from the supernatant.

Recipes

YEB (1 L, for starters)

Add:
5 g of yeast extract powder
10 g of Bacto tryptone
5 g of NaCl
5 g of sucrose crystallized
5 g of MgSO₄·7H₂O
to 900 ml of dH₂O
Adjust the pH to 7.3 using NaOH or HCl
Note: Do not heat while measuring pH.
Bring to 1 L by adding more dH₂O, and autoclave at 121 °C for 20 min

PSB (1 L, the main medium)

Add:
10 g of peptone
10 g sucrose crystallized
1 g of L(+)-glutamic acid
to 900 ml of dH₂O
A slight heating while stirring might assist in dissolving the components, yet it’s not compulsory. Turn off the heat, and transfer the beaker to another, colder, stirrer
Adjust the pH to 7.3 using NaOH or HCl
Note: Do not heat while checking pH.
Add more dH₂O in order to reach 1 L
Mix by passing the medium between the beaker and a measuring cylinder
Divide the medium between two 2-L Erlenmeyer flasks (500 ml in each), and autoclave at 121 °C for 20 min

OptiPrep diluent buffer (1 L)

Add:
8.5 g of NaCl
2.38 g of HEPES
to approx. 750 ml of dH₂O in a beaker
Adjust the pH to 7.4 with NaOH
Note: Do not heat while checking pH.
Bring to 1 L with dH₂O and filter-sterilize with 0.45-μM filter (operated by a vacuum pump)
Suggestion: keep the buffer at 4 °C

Acknowledgments

Work at O. Bahar lab was supported by the German-Israeli Foundation for Scientific Research and Development (GIF), grant No. I-2392-203.13/2015 and by the Israel Science Foundation, grant No. 2025/16. We would like also to thank our lab members for every help given, S. Burdman’s lab, M. Levy’s lab, and N. Sela, for collaboration in experiments, and also M. Mawassi’s lab, V. Gaba’s lab, A. Dombrovsky’s lab, and S. Manulis-Sasson’s lab, for some of the equipment needed for the experiments.

References

Chutkan, H., Macdonald, I., Manning, A. and Kuehn, M. J. (2013). Quantitative and qualitative preparations of bacterial outer membrane vesicles. Methods Mol Biol 966: 259-272.
Goel, A. K., Rajagopal, L. and Sonti, R. V. (2001). Pigment and virulence deficiencies associated with mutations in the aroE gene of Xanthomonas oryzae pv. oryzae. Appl Environ Microbiol 67(1): 245-250.
Munhoz, C. F., Weiss, B., Hanai, L. R., Zucchi, M. I., Fungaro, M. H., Oliveira, A. L., Monteiro-Vitorello, C. B. and Vieira, M. L. (2011). Genetic diversity and a PCR-based method for Xanthomonas axonopodis detection in passion fruit. Phytopathology 101(4): 416-424.