Growth Experiments

3.1.1. Inoculum Buildup for the Growth Experiments

1. Probiotic microorganisms: Lactobacilli were isolated from vaginal swabs of women from Tucuman, Argentina. The microorganisms were identified by the methods described in Bergey's Manual of Determinative Bacteriology (16), standard techniques used in the laboratory, and API 50 CHL (Biomerieux-France) (4). Some of the selected strains have good surface properties and probiotic characteristic, as follows: L. salivarius subsp. salivarius CRL (Centro de Referencia para Lactobacilos Culture Collection) 1328, producer of a bacteriocin (5) and L. crispatus CRL 1266 and L. paracasei subsp. paracasei CRL 1289, producers of H2O2 (6,7).

2. Before the growth experiments, 5 mL of either MRS or LAPTg broths (tubes) are inoculated with 30 ^L of lactobacilli stored at -20°C in milk-yeast extract and incubated at 37°C for 24 h. A 150-^L aliquot of these precultures is cultivated twice at 37°C for 12 h in 5-mL of the same media (see Note 1).

3. The third culture is centrifuged (10 min, 2000g), and the spent supernatant is discarded. The pellet is washed with 3 mL saline solution and then resuspended in the same solution to reach a final OD of 1.4 at 540 nm. This bacterial cell suspension is used as the inoculum for the growth experiments (see Note 2).

3.1.2. pH Adjustment of Culture Media

1. 1 N HCl and 1 N NaOH solutions were aseptically prepared with sterile distilled water (see Note 3).

2. Before the pH determinations of culture media, the pH meter must be calibrated with the reference standard buffers. The electrode is washed with distilled water and dried with blotting paper; 2 mL of each buffer is placed in small plastic bottles, and the pH meter is calibrated first at pH 7.00 and then at pH 4.00. When the pH meter is not used, the electrode should be submerged in 3 M KCl solution (see Note 4).

3. Before inoculation, the initial pH of the LAPTg and MRS broths is aseptically adjusted to 5.0 with 1 N HCl, or to 6.5 and 8.0 with 1 N NaOH, determining the pH with the pH meter as described above in step 2 (see Note 5).

3.1.3. Incubation Systems Cultures Without Agitation

1. 100 mL of each medium (in 250-mL Erlenmeyer flasks) is inoculated (2% v/v) with precultures of each Lactobacillus strain, prepared as described above in Subheading 3.1.1. (see Note 6).

2. The flasks are agitated and later distributed (in 5 mL samples) into glass tubes for growth determinations (see Note 7).

3. The tubes are incubated in a water bath in aerobic conditions, without agitation, at a constant temperature of 30, 37, or 44°C (see Note 8).

4. This incubation system is used for all the growth conditions corresponding to the complete factorial experimental design used to study growth and bacteriocin production of L. salivarius CRL 1328, and for the nonaerated conditions tested in the fractional factorial experimental design for growth and H2O2-production of L. paracasei CRL 1289 and L. crispatus CRL 1266. Cultures With Agitation

1. The lactobacilli are inoculated (2% v/v) into 100 mL of each medium (250-mL Erlenmeyer flasks).

2. The cultures are incubated in a water bath with agitation (see Note 9).

3. This incubation system is used for evaluation of the effect of aerated conditions tested in the fractional factorial experimental design used for both growth and H2O2-production of L. paracasei CRL 1289 and L. crispatus CRL 1266.

3.1.4. Obtaining Samples

1. For determination of antagonistic substances, pH, and CFU/mL, the samples are withdrawn every 3 h; the OD measurements are performed every one hour.

2. For nonagitated cultures, at each time point a tube is removed from the incubator; for agitated cultures, the samples are aseptically withdrawn from the flasks (4 mL), placed in sterile tubes, and labeled (see Note 10). In both cases, different aliquot are taken, as follows:

a. First, 500 ^L are placed in sterile tubes, and the supernatants are separated by cen-trifugation (5 min at 6000g and 20°C) and stored at -20°C (up to 1 mo) or at 4°C (23 h) for the bacteriocin and H2O2 determinations, respectively.

b. Then 100 ^L are diluted with peptone water in glass tubes and stored at 4°C for the viable count. They cannot be kept under these conditions for more than 1 h (see Subheading 3.1.7.).

c. The remaining volume is used for the OD and pH determinations (see Subheadings 3.1.5. and 3.1.6., respectively).

3.1.5. Optical Density Measurements (see Note 11)

1. The selected wavelength of the spectrophotometer is 540 nm (see Note 12).

2. The initial turbidity of the growth media must be corrected to absorbance value 0 (zero) with the corresponding blanks of culture media placed in glass cuvets (see Note 12).

3.1.6. pH Determination of Culture Samples

The pH of the culture media during the lactobacilli growth is determined as described in Subheading 3.1.2.

3.1.7. Determination of Colony-Forming Units

1. The samples are serially diluted (10-fold) in peptone water, from 1:10 to 1:1,000,000, using glass tubes with peptone water and automatic pipets; each dilution tube was vigorously mixed with a vortex.

2. A 0.5-mL aliquot of each sample dilution is placed on a plate, and 12-15 mL of melted culture medium (LAPTg or MRS agar, depending on whether the previous lactobacilli growth was in LAPTg or MRS broth) are added and mixed carefully while the plate is rotated for an even distribution of the bacteria. The agar media should be melted and maintained at a temperature of 45-50°C before use.

3. The plates are incubated at 37°C for 48 h. Then the number of CFUs was determined by selecting data for the plates that contained between 30 and 300 CFU. The results are expressed as CFU/mL.

3.1.8. Registration of the Experimental Data

Registration of the experimental data and some transformations of these original data were performed using Excel sheets.

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