TITLE: Preparation of soil bacteria for FCM
AUTHORS: Laura Espina

[STEPS]
?. [Extraction of soil bacteria]
Measure the conductivity of the soil sample with a conductivimeter.
?. [Extraction of soil bacteria]
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.
?. [Extraction of soil bacteria]
Prepare the soil slurry: Weight  of soil and add  of sterile solution N.
20 g
40 mL
?. [Extraction of soil bacteria]
Vortex the soil slurry for  .
?. [Extraction of soil bacteria]
Let the soil slurry settle for  .
?. [Extraction of soil bacteria]
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.
?. [Extraction of soil bacteria]
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   of solution N for .
5 mL
?. [Extraction of soil bacteria]
Concentration of the soil particles: Centrifuge the suspension from step 7 (). Remove supernatant. Resuspend in 2 mL of solution N.
Centrifuge: 6000 34, 5 min, 20 10
Pellet of approximately 109 particles between the sizes of 0.1 and 12 μm.
?. [Extraction of soil bacteria]
Concentration of soil bacteria by density centrifugation: Prepare solutions of  Histodenz (Sigma-Aldrich). Autoclave. Take 2 mL from step 8 and carefully pour onto a Histodenz cushion of 2 mL.Centrifuge ().Carefully recover the microbial fraction (shown in picture below inside the red circle).Centrifuge the recovered fraction ().
[w/v (1.3 g/mL)]
Centrifuge: 7155 34, 30 min, 20 10
Centrifuge: 6000 34, 5 min, 20 10
Pellet of approximately 109 microorganisms (mostly bacteria).
?. [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.
?. [Extraction of soil bacteria after cultivation on agar]
Incubate the plates at   for .
17 °C
?. [Extraction of soil bacteria after cultivation on agar]
Resuspend the grown biomass on agar plates from the previous step in solution N in several microcentrifuge tubes. Centrifuge them () and remove supernatant.
Centrifuge: 6000 34, 5 min, 20 10
Pellet of approximately 109 readily culturable microorganisms (mostly bacteria).
?. [Staining procedure]
Prepare staining solution: To a solution of Potassium phosphate buffer  at ,  add: Propidium iodide (), 5(6)-carboxyfluorescein diacetate () and EDTA ().
[in water]
[in water]
[in water]
?. [Staining procedure]
Add 1 mL of staining solution to each pellet from steps 9 and/or 12. Mix by vortexing.
?. [Staining procedure]
Incubate samples from step 14 in the dark at  for .
30 °C
?. [Staining procedure]
Prepare FCM tubes with  of PBS added with bovine serum albumin (  and refrigerate them for  .
1 mL
?. [Staining procedure]
Add  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.
10 µl