Adhesion to Intestinal Epithelial Cells In Vitro

2.4.1. Preparation of Bacterial Suspension

1. Tubes with 5 mL of MRS broth.

2. Phosphate-buffered saline (PBS): 50 mM KH2PO4/Na2HPO4, containing 8 g/L NaCl and 2 g/L KCl, pH 7.2. Autoclave at 121°C for 15 min and store at room temperature.

3. Refrigerated centrifuge.

4. Spectrophotometer for glass tubes (Spectronic 20, Bausch & Lomb, Miltou Roy Company, USA).

2.4.2. Preparation of Intestinal Epithelial Cell Suspension

1. Three Balb/c mice.

2. Dissection board, pins, scissors, and dissection pliers.

3. Sterile microscope slides.

5. NCTC 135 medium (Sigma) supplemented with 2% fetal bovine serum, pH 7.5. Prepare according to the manufacturer's instructions (see Note 3).

6. Neubauer chamber for adjustment of the number of epithelial cells.

7. Refrigerated centrifuge

8. Phase-contrast microscope.

2.4.3. Adhesion Assay

1. Stove at 37°C with gas supply to provide a 5% CO2/95% air atmosphere.

3. Membrane filters of 8 ^m pore size (filter membranes, nitrocellulose, cat. no. N-4021, Sigma).

4. Kit for Gram staining.

5. Phase-contrast microscope.

2.5. Intestinal Transit Rate

1. Spore suspension of B. stearothermophilus (Britania Laboratories, Argentina). Store at refrigeration temperature.

4. Dissection board, pins, scissors, and dissection pliers.

5. Sterile saline solution.

6. Diluent medium: 0.1% peptone water.

8. Sterile Petri dishes and screw-top glass tubes.

9. Precision digital balance.

2.6. Adhesion to Intestinal Epithelial Cells In Vivo

1. Five Balb/c mice per treatment group.

3. Dissection board, pins, scissors, and dissection pliers.

5. NCTC 135 medium.

6. Membrane filters of 8 ^m pore size.

7. Kit for Gram staining.

8. Phase-contrast microscope.

2.7. Enzyme Activity (fi-Galactosidase Assay)

1. Phosphate buffer: 50 mM KH2PO4/Na2HPO4, pH 7.0 containing 0.1% 2-mercaptoethanol (cat. no. M 6250, Sigma).

2. Substrate solution: 4.16 mM o-nitophenyl-|3-D-galactopyranoside in water (ONPG; cat. no. N-1127, Sigma). Store prepared solution in a dark flask at 4°C for 1 mo or at -20°C for several months.

3. Stop solution: 0.5 M Na2CO3. Store at room temperature.

4. Calibration curve: 5 mMo-nitrophenol (ONP; cat. no. N-9256, Sigma) in phosphate buffer (step 1), as the product released by enzyme activity.

5. Spectrophotometer.

3. Methods

3.1. Propionibacteria Storage and Growth Conditions

1. For storage, harvest by centrifugation the cells from 10 mL of a late exponential phase culture (36-42 h). Wash once with saline solution and resuspend the pellet in 5 mL of milk-yeast extract. Transfer the suspension to 1-mL screw-top sterile cryovials and store at -20°C. Propionibacteria remain viable for several months.

2. Activate propionibacteria strains from stock cultures at -20°C in milk-yeast extract, by inoculating 100 ^L in the selected medium and incubate statically at 37°C for 48 h. Make at least three successive transfers every 24 h before experimental use.

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