Jul 07, 2020
- 1Cardiff University
External link: https://doi.org/10.1371/journal.pone.0237748
Protocol Citation: Laura Espina 2020. Preparation of soil bacteria for FCM. protocols.io https://dx.doi.org/10.17504/protocols.io.biazkaf6
Manuscript citation:
Espina L (2020) An approach to increase the success rate of cultivation of soil bacteria based on fluorescence-activated cell sorting. PLoS ONE 15(8): e0237748. doi: 10.1371/journal.pone.0237748
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 07, 2020
Last Modified: July 07, 2020
Protocol Integer ID: 38969
Extraction of soil bacteria
Extraction of soil bacteria
Measure the conductivity of the soil sample with a conductivimeter.
Prepare solution N: Add the needed amount of NaCl to 100 mL of distilled water to prepare a saline solution with the same conductivity that of the soil (see figure below). Autoclave.
Correlation between the concentration of NaCl in NaCl dilutions and their conductivity values
Prepare the soil slurry: Weight 20 g of soil and add 40 mL of sterile solution N.
Vortex the soil slurry for 00:20:00 .
Let the soil slurry settle for 00:05:00 .
Remove big particles from soil slurry: Prepare a sterile vacuum filtration system with a hydrophilic membrane of 12 μm of pore size. Filter the soil slurry and retain the pass-through.
Wash the soil particles: Prepare a sterile vacuum filtration system with a hydrophilic membrane of 0.1 μm of pore size. Filter the pass-through from step 6. Vortex the filter in a sterile standard tube with 5 mL of solution N for 00:02:00 .
Concentration of the soil particles: Centrifuge the suspension from step 7 (6000 x g, 20°C, 00:05:00 ). Remove supernatant.
Expected result
Pellet of approximately 109 particles between the sizes of 0.1 and 12 μm.
Resuspend in 2 mL of solution N.
Concentration of soil bacteria by density centrifugation: Prepare solutions of 60 Mass Percent w/v (1.3 g/mL) Histodenz (Sigma-Aldrich). Autoclave. Take 2 mL from step 8 and carefully pour onto a Histodenz cushion of 2 mL.
Centrifuge (7155 x g, 20°C, 00:30:00 ).
Carefully recover the microbial fraction (shown in picture below inside the red circle).
Centrifuge the recovered fraction (6000 x g, 20°C, 00:05:00 ).
Expected result
Pellet of approximately 109 microorganisms (mostly bacteria).
Extraction of soil bacteria after cultivation on agar
Extraction of soil bacteria after cultivation on agar
Resuspend the pellet from step 9 in solution N. Prepare serial dilutions in solution N and plate onto R2A agar plates.
Incubate the plates at 17 °C for 96:00:00 .
Resuspend the grown biomass on agar plates from the previous step in solution N in several microcentrifuge tubes. Centrifuge them (6000 x g, 20°C, 00:05:00 ) and remove supernatant.
Expected result
Pellet of approximately 109 readily culturable microorganisms (mostly bacteria).
Staining procedure
Staining procedure
Prepare staining solution:
To a solution of Potassium phosphate buffer 7.0 at 0.1 Molarity (M) , add: Propidium iodide (80 micromolar (µM) in water ), 5(6)-carboxyfluorescein diacetate (10 micromolar (µM) in water ) and EDTA (60 micromolar (µM) in water ).
Add 1 mL of staining solution to each pellet from steps 9 and/or 12. Mix by vortexing.
Incubate samples from step 14 in the dark at 30 °C for 00:30:00 .
Prepare FCM tubes with 1 mL of PBS added with bovine serum albumin (0.8 Mass / % volume ) and refrigerate them for 01:00:00 .
Add 10 µL of a sample from step 15 to a tube from step 16 for FCM analysis. Filter all the content of the tube through a hydrophilic membrane of 0.2 μm of pore size. Proceed to FCM analysis.