Materials and Reagents

Bacillus subtilis (B. subtilis) 168 bacterial strain (available from ATCC, catalog number: 23857 )
BHI broth/agar (BD, catalog number: 211059/211065)
Amicon Ultra centrifugal filter units Ultra-4 (MWCO 100 kDa) (Millipore, catalog number: UFC910024 )
1x phosphate buffered saline (PBS) (see Recipes)
MSgg medium (see Recipes)

Equipment

Petri dishes (100 x 55 mm) (Corning, catalog number: 351029 ) (dish size can be variable and ultimately depends on the amount of vesicles you wish to purify)
0.22 μm syringe filters (Fisher, catalog number: 09-719C)
Tube (thickwall, polyallomer, 3.5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 349623 )
Optima TL ultracentrifuge (Beckman Coulter)
TLA 100.3 rotor (Beckman Coulter)
Centrifuge capable of 15,000 x g

Procedure

Biofilm growth assay
1. Inoculate a plate of Bacillus subtilis lab strain 168 (from -80 °C) on a BHI agar plate overnight (~18 h) at 37 °C.
2. Inoculate 5 ml BHI broth from overnight agar plate (one colony) and grow for 4 h with shaking (~200 rpm) at 37 °C.
3. Inoculate 1:1,000 of broth culture (12 μl) into 12 ml MSgg media in petri dishes.
4. Let biofilm incubate (covered with dish lid without shaking) at 37 °C for desired time period (usually 3 and 7 days). Ensure that incubator has a water container to ensure the dishes do not dry out.
Purification of vesicles from biofilm
1. After desired time period, remove pellicle and supernatant from plates by pipetting and centrifuge at 15,000 x g for 20 min at 4 °C to remove cells and large debris.
2. To remove remaining cells and debris, filter the spun supernatant with 0.22 μm syringe filters.
3. Centrifuge filtered, cell-free supernatant with centrifugal filter units to concentrate to less than 3 ml in volume.
4. To pellet vesicles from supernatant, ultracentrifuge concentrated supernatant at 195,000 x g for 1 h at 4 °C in polyallomer tubes.
5. Remove supernatant without disturbing the vesicle pellet by pipetting.
6. Repeat spin after washing in 500 μl PBS.
7. Remove PBS wash from tube without disturbing the vesicle pellet.
8. Resuspend pellet in desired volume of PBS.

Notes

Bacillus subtilis grows as a biofilm pellicle on the surface of the media (Figure 1). It may need a few washings to remove all cells for vesicle collection. Figure 2 shows electron micrograph of negatively stained vesicles.
Biofilms can be grown in various plates to achieve desired volume of vesicles. Ideally you want to have a visible pellet after ultracentrifugation. If no pellet is visible, scale up the plate size or number of biofilms for vesicle purification.
Vesicle pellet after ultracentrifugation should be visible, but in cases of small culture volumes the pellet may be difficult to see.

Figure 1. Representation of what biofilm pellicles may look like in a 6-well plate

Figure 2. Negative stain TEM of vesicles isolated from Bacillus subtilis biofilm. Scale bar=100 nm

Recipes

1x phosphate buffered saline (PBS) (1 L, pH 7.4) (filter or autoclave sterilize)
137 mM NaCl
2.7 mM KCl
10 mM Na₂HPO₄
1.8 mM KH₂PO₄
Bring to pH 7.4 with HCl
Dissolve in H₂O up to 1 L
MSgg medium (pH 7) (filter sterilize)
50 μM MnCl₂
5 mM KH₂PO₄
1 μM ZnCl₂
50 μM FeCl₃
2 mM MgCl₂
700 μM CaCl₂
50 μg/ml threonine, tryptophan, and phenylalanine
0.5% glutamate
0.5% glycerol
2 μM thiamine
100 mM morpholinepropanesulfonic acid (MOPS) (pH 7)

Acknowledgments

Funding from NIH Grant Numbers: HL059842, AI033774, AI033142, AI052733 and Center for AIDS Research at Albert Einstein College of Medicine.
Protocol was adapted from Brown et al. (2014) and McLoon et al. (2011).

References

Brown, L., Kessler, A., Cabezas-Sanchez, P., Luque-Garcia, J. L. and Casadevall, A. (2014). Extracellular vesicles produced by the Gram-positive bacterium Bacillus subtilis are disrupted by the lipopeptide surfactin. Mol Microbiol 93(1): 183-198.
Prados-Rosales, R., Brown, L., Casadevall, A., Montalvo-Quirós, S. and Luque-Garcia, J. L. (2014). Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria. MethodsX 1: 124-129.
McLoon, A. L., Guttenplan, S. B., Kearns, D. B., Kolter, R. and Losick, R. (2011). Tracing the domestication of a biofilm-forming bacterium. J Bacteriol 193(8): 2027-2034.