Growth Experiments

2.1.1. Inoculum Buildup for the Growth Experiments

1. Probiotic microorganisms: Lactobacillus salivarius subsp. salivarius CRL 1328, L. crispatus CRL 1266, and L. paracasei subsp. paracasei CRL 1289.

2. LAPTg broth: 15 g/L peptone, 10 g/L tryptone, 10 g/L glucose, 10 g/L yeast extract, and 1 mL/L Tween-80, pH 6.5. Autoclave at 121°C for 15 min. Store at refrigeration temperature. The chemicals for LAPTg preparation were obtained from Britania (Argentina).

3. MRS broth: Dehydrated MRS was obtained from Biokar Diagnostics (Beauvais, France). Composition: 10 g/L peptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 1.08 g/L Tween-80, 2 g/L dipotassium phosphate, 5 g/L sodium acetate, 2 g/L ammonium citrate, 0.2 g/L magnesium sulfate, and 0.05 g/L manganese sulfate, pH 6.5. Autoclave at 121°C for 15 min. Store at refrigeration temperature.

4. Saline solution (0.85% NaCl). Autoclave at 121°C for 15 min. Store at refrigeration temperature.

5. Spectrophotometer (Spectronic 20, Bausch & Lomb, Miltou Roy). Determinations of optical density (OD) in screw-top glass tubes.

2.1.2. pH Adjustment of Culture Media

1. Solutions for pH adjustment: 1 N HCl and 1 N NaOH. Sterile distilled water, HCl ACS (reagent grade, purity 37%; Anedra, Argentina), NaOH (anhydrous, Anedra). The reagent for the solutions and the 1 N solutions are stored at ambient temperature.

2. Digital pH meter (Digimeter IV, Luftman, Argentina).

3. Standard reference buffers:

a. pH 7.00 (± 0.01 at 25°C): 50 mL 0.1 M KH2PO4 + 29.1 mL 0.1 M NaOH, diluted to 100 mL with distilled water; color-coded yellow (LQT, Argentina). Store at room temperature.

b. pH 4.00 (± 0.01 at 25°C): 50 mL 0.1 M KH phthalate + 0.1 mL 0.1 MHCl, diluted to 100 mL with distilled water; color-coded red (LQT, Argentina). Store at room temperature.

4. Accessories: Distilled water, blotting paper, and plastic bottles to receive the samples.

5. Culture media: LAPTg and MRS broths (described in Subheading 2.1.1.), both at initial pH of 5.0, 6.5, and 8.0.

2.1.3. Incubation Systems

2.1.3.1. Cultures Without Agitation

1. 250-mL Erlenmeyer flasks containing 100 mL of each culture media.

2. Sterile screw-top glass tubes.

3. Water baths (Vicking S.R.L., Masson model, Industria Argentina) at incubation temperatures of 30, 37, or 44°C.

2.1.3.2. Cultures With Agitation

1. 250-mL Erlenmeyer flasks containing 100 mL of each culture media.

2. Water baths (Vicking S.R.L. Shaker, Dubnoff model, Industria Argentina, speed of agitation: 50 opm), incubation temperatures of 30, 37, or 44°C.

2.1.4. Obtaining Samples

1. Automatic pipets.

2. Laminar flow chamber (Clean Room Products).

3. Screw-top glass tubes and Eppendorf tubes.

2.1.5. Optical Density Measurements

1. Digital spectrophotometer (model 250; Gilford Instrument Lab).

2. Glass cuvets with a 10-mm light path.

3. Blanks for the spectrophotometry determinations: Screw-top glass tubes with sterile LAPTg or MRS broths, both to pH 5.0, 6.5, or 8.0 (see item 1 of Subheading 2.1.2.).

2.1.6. Determination of Colony-Forming Units (CFUs)

1. Dilution medium: Peptone water (0.1% meat peptone). Autoclave at 121°C for 15 min. Store at refrigeration temperature.

2. Automatic pipets (for 100 and 1000 ^L), glass tubes containing peptone water (stored at refrigeration temperature), vortex (Vicking, Argentina).

3. Culture media: LAPTg agar or MRS agar (as described in Subheading 2.1.1., but with 15 g/L agar).

4. Incubation system: Incubator, temperature of 37°C (Forma Scientific, model 3185).

2.1.7. Registration of the Experimental Data

Sheets can be obtained using Microsoft Excel for Windows.

2.1.8. Determination of Antagonistic Substances (H2O2 and Bacteriocin)

The experimental details of these determinations are described in Chapter 31 (hydrogen peroxide) and Chapter 32 (bacteriocin).

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