3.3.1. Preparation of Bacterial Suspension
1. Inoculate 5 mL of MRS broth with an active culture of propionibacteria and incubate statically at 37°C up to the late exponential phase (42 h).
2. Harvest cells by centrifugation (10,000g, 10 min), wash twice with 50 mM PBS, pH 7.2, and finally resuspend in the same buffer.
3. Adjust the bacterial concentration in a spectrophotometer to approx 5 x 108 colony-forming units (CFU)/mL (OD560 = 0.8; see Note 6).
3.3.2. Preparation of Intestinal Epithelial Cells Suspension
1. The mice, maintained in metal cages with free access to food and water, must be fasted for 24 h before the performance of adhesion experiments.
2. Sacrifice mice by cervical dislocation and immobilize the body on the dissection board by fixing the extremities with metallic pins.
3. Cut the skin with scissors and open the abdominal cavity in aseptic conditions.
4. Remove the small bowel and place it in a sterile Petri dish.
5. Discard the intestinal contents by flushing PBS, pH 7.2, through the intestine with a glass syringe.
6. Cut the intestine longitudinally and open it in the Petri dish. Wash gut walls repeatedly with sterile PBS, pH 7.2.
7. Scrap the mucosa gently with the edge of a microscope slide and suspend the exfoliated cells in chilled PBS, pH 7.2.
8. Keep the suspended scrapings on ice for 15 min to allow the large debris to settle. Remove the fluid supernatant containing the intestinal epithelial cells (IECs) carefully and transfer to another sterile recipient.
9. Wash the IEC suspension twice with PBS by centrifuging at low speed (120g, 10 min) to avoid cell damage.
10. Resupend the IECs in NCTC 135 medium supplemented with 2% fetal bovine serum and adjust the suspension to a concentration of 5 x 105 cells/mL by counting cells in a Neubauer chamber (see Note 7).
11. Keep the IEC suspension on ice and use within 2 h for binding assay.
1. Mix suspensions of propionibacteria and IECs at a 1:4 proportion and incubate at 37°C for 1 h in a 5% CO2/95% air atmosphere. Make mixtures in duplicate.
2. After incubation, wash the mixtures once with PBS (120g, 5 min) and resuspend to the original volume in cell culture medium. Keep the mixtures on ice and determine adhesion indexes within 1 h.
3. Adhesion of propionibacteria to IECs is determined by examining 30 epithelial cells selected at random using phase-contrast microscopy (17). Two adhesion parameters can be determined: (1) the adhesion percentage, which indicates the number of intestinal cells containing attached bacteria per 100 IECs; and (2) the adhesion index, which gives the mean number of adherent bacteria per IEC. They are expressed as follows:
a. Adhesion percentage: Number of IECs with adherent bacteria extrapolated to 100 IECs.
b. Adhesion index: Average number of bacteria attached per IEC, only including IECs with bacteria.
4. To illustrate and identify adhesion patterns clearly, wash the duplicate reaction mixtures (120g, 5 min), resuspend in PBS, and filter through an 8-^m-pore membrane. Transfer filters retaining cells with adherent bacteria to albumin-coated microscope slides, fix with methanol, and Gram stain. Observe under the light microscope.
3.4. Adhesion to Intestinal Epithelial Cells After Digestion In Vitro
1. Repeat steps 1 and 2 of Subheading 3.3.1. but resuspend the cells in the 10th part of the original volume.
2. Add the cell suspension to 5 mL of artificial gastric juice at pH 3. Mix with a vortex and incubate each bacterial suspension at 37°C with agitation at 200 opm (Dubnoff Metabolic Shaking Incubator, Precision CGA Corp.).
3. After 60 min of gastric digestion, centrifuge the bacterial suspension at 20,000g for 10 min. Suspend the pellet to the original volume in intestinal fluid and incubate as before.
4. After 60 min of intestinal digestion, harvest cells by centrifugation (10,000g, 10 min), wash twice with 50 mM PBS, pH 7.2, and finally resuspend in the same buffer.
5. Adjust the bacterial concentration in a spectrophotometer to approx 5 x 108 CFU/mL (OD560 = 0.8; see Note 7).
6. Carry out the assay as in Subheading 3.3.3.
3.5. Intestinal Transit Rate
1. Incubate one ampoule of B. stearothermophilus in a stove at 65 °C for 24 h.
2. Open the ampoule and inoculate 100 ^L of the grown culture in 5 mL of BHI broth. Incubate at 65°C for 24 h. Subculture twice in BHI (see Note 8).
3. Make a third subculture in duplicate and incubate at 65 °C for 24 h. Let cells from one culture sporulate at room temperature for 7 d. With the other grown culture, make a calibration curve of absorbance against viable cells per mL by plating serial 10-fold dilutions of known OD560 into PCA with incubation at 65°C for 24 h.
4. Wash the sporulated stored culture three times with saline solution (2000g, 10 min) and suspend the cells in the appropriate volume of sterile deionized water in order to obtain 5 x 107 CFU/mL.
5. Give an oral dose of 1 x 106 spores of B. stearothermophilus in 50 ^L of water to each of 39 mice fasted overnight.
6. Sacrifice three mice immediately and at 30-min intervals up to 360 min.
7. Obtain the stomach, small bowel, and cecum contents. Weigh the organs before and after removing the contents.
8. Heat the contents at 80°C for 10 min and prepare 10% homogenates with saline solution (see Note 9).
9. Make serial 10-fold dilutions in 0.1% peptone water. Pour different dilutions into PCA and incubate at 65°C for 24 h.
10. Calculate percentage of recovery in each section as: (no. of spores counted in each GI section/no. of spores orally delivered) x 100.
3.6. Adhesion to Intestinal Epithelial Cells In Vivo
1. Put mice at random into two treatment groups of five animals each.
2. Develop the selected Propionibacterium strain in MRS broth up to the late exponential phase (42 h). Wash by centrifugation (10,000g, 10 min) with sterile PBS, and suspend the cells in 10% nonfat milk to a concentration of 1 x 1010 CFU/mL.
3. Orally administer 50 ^L of the bacterial suspension (5 x 108 CFU) to each mouse of each test group with a plastic-tipped pipet. Give an equal volume of sterile milk to control mice.
4. At the optimum time of sampling for maximal recovery of propionibacteria from the small bowel (determined from the intestinal transit rate experiment), sacrifice the animals and obtain IECs as described in steps 1-10 of Subheading 3.3.2.
5. Determine the bacterial adhesion as in steps 3 and 4 of Subheading 3.3.3. and compare results with controls obtained from animals receiving 50 ^L of 10% nonfat milk administered orally.
6. Adhesion in vivo of propionibacteria can be also expressed as CFU/g of IECs by counting the bacteria in selective media (11) (see Note 10).
3.7. Enzyme Activity (fi-Galactosidase Assay)
3.7.1. Determination of fi-Galactosidase Activity of Bacterial Suspensions
1. Harvest cells by centrifugation (10,000g, 10 min, 4°C), wash twice with 0.05 M KH2PO4/ Na2HPO4, pH 7.0 containing 0.1% 2-mercaptoethanol (phosphate buffer), suspend in the same buffer to adjust the OD560 to 0.5, and finally concentrate 20 times (approx 1 x 1010 cells/mL, or 3.85 ± 0.16 mg of dry weight).
2. Carry out the enzymatic reaction by mixing: 200 ^L of cell suspension (see Note 11), 250 ^L o-nitrophenyl |3-D-galactopyranoside (1 mM final concentration of ONPG), and 550 ^L phosphate buffer, pH 7.0 (see Note 12).
3. Prepare substrate and sample blanks by excluding sample and substrate, respectively, in the reaction mixtures.
4. Incubate all mixtures at 37°C for 15 min and stop with 800 ^L of 0.5 M Na2CO3.
5. Remove bacterial cells by centrifugation (5000g, 10 min) and determine the absorbance at 440 nm of supernatants.
3.7.2. Determination of fi-Galactosidase Activity of Intestinal Contents
1. Obtain the intestinal contents in a glass tube by flushing saline solution through the intestine with a glass syringe and a catheter.
2. Wash the contents with saline solution (1000g, 5 min) to eliminate residues of lactose that could compete with ONPG for |3-galactosidase enzyme (see Note 9).
3. Make 10% homogenates with 0.05 M phosphate buffer, pH 7.0 (step 1 of Subheading 3.2.).
4. Carry out the enzymatic reaction as in steps 2 and 3 of Subheading 3.7.1. using the homogenates instead cell suspensions in the reaction mixtures. Use 200 ^L of small bowel and 30 mL of large bowel contents (see Note 9).
5. Centrifuge at 10,000g, 10 min, 4°C and read the OD440 of supernatants (see Note 9).
1. The P-galactosidase activity is expressed as the rate of hydrolysis of o-nitrophenyl |3-d-galactopyranoside. The enzyme activity is defined as nanomoles of ONP released from ONPG per milliliter of cell suspension or grams of intestinal contents and per minute of reaction under the assayed conditions.
2. Calculate activity with the following formula:
(OD440 sample - OD440 higher blank) x F = nanomoles OPN/min/mL/cell suspension reaction time (min) x sample volume (mL)
(OD440 sample - OD440 higher blank) x F = nanomoles ONP/min/g/content reaction time (min) x sample volume x 0.1 g*
*For 10% homogenates ( 0.1 g of content in 1 mL of sample.
3 Factor F is the slope of a calibration curve of nanomoles of o-nitrophenyl (ONP) in function of absorbance at 440 nm. To obtain the standard curve include different concentrations of ONP, between 0 and 2 mM, in a final volume of 1.8 mL (which corresponds to the volume of stopped reaction mixtures), and read the OD440 of the different dilutions.
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