María Claudia Otero, Virginia S. Ocaña, and María Elena Nader-Macías
For the study of probiotic microorganisms, the in vitro selection tests need to be based on a solid scientific foundation (1). Surface characteristics, one of the in vitro properties are used to evaluate the potentially probiotic strains of lactobacilli (2).
Bacterial surface properties have been associated with attachment to a variety of substrata. Bacterial adhesion to tissues is considered the first step, and such adhesion can also determine the colonization capability of a microorganism (3). Through adhesion ability and colonization of tissues, probiotic microorganisms can prevent pathogen access by steric interactions or specific blockage on cell receptors (4).
One of the main characteristics studied is the hydrophobic nature of the bacterial cell surface. To test this property, Rosenberg and Doyle (3) divided microbial cell hydrophobicity assays into two categories. The first includes contact angle measurements (CAMs), partitioning of cells into one or another liquid phase (TTP), and adsorption of individual hydrophobic molecular probes at the cell surface. The second category includes microbial adhesion to hydrocarbons (MATH), hydrophobic interaction chromatography (HIC), and adhesion to polystyrene and other hydro-phobic solid surfaces. The tests included in the first category measure hydrophobic properties of the outer cell surface as a whole; those in the second measure hydropho-bicity in terms of adhesion (3). Finally, those bacterium classified as hydrophobic can be considered as able to mediate adhesion (5,6).
The objective of this chapter is to describe three different methods applied in our laboratory for the study of bacterial surface properties. They can be used to screen characteristics of lactobacillus strains for probiotic purposes. They are:
• Microbial adhesion to hydrocarbons (MATH).
• Hemagglutination (HA) reaction.
MATH was assayed by mixing washed cell suspensions with the test hydrocarbons (n-hexadecane, n-octane, and p-xylene) for a given time and measuring adhesion simply as the decrease in turbidity in the aqueous phase (7). This method was first described for bacteria isolated from the oral environment, more specifically, from dental plaque (8).
The SAT was developed, among other reasons, to study the hydrophobicity of the bacterial cell surface (9). Is based on the fact that highly hydrophobic bacteria are able to aggregate with characteristic patterns in the presence of different ammonium sulfate concentrations.
The HA reaction can be an important tool for monitoring bacterial adhesion, because the erythrocyte surface shares the ontogenetic origin of the tissue cells normally colonized by the organisms (10). In particular, epithelial cells express outer molecules similar to the red blood cell membrane (11).
2.1. Preparation of Microorganisms
1. LAPTg broth: 15 g/L peptone, 10 g/L tryptone, 10 g/L glucose, 10 g/L yeast extract, 1 mL/L Tween-80, pH 6.5, autoclaved at 121°C for 15 min, and stored at refrigeration temperature. The chemicals for LAPTg preparation were obtained from Britania Laboratories (Argentina).
2. Milk-yeast extract: 10% skim milk, 1% glucose, 0.5% yeast extract, pH 6.0, autoclaved for 20 min at 115°C (3/4 atm) and stored at refrigeration temperature.
3. Accessories: sterile screw-capped cryovials.
2.1.2. Culture Buildup for the Assay
2. de man-Ragosa-Sharpe (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, 0.05 g/L manganese sulfate, pH 6.5, autoclaved at 121°C for 15 min and stored at refrigeration temperature.
2.2. Hydrophobic Partition Assay: MATH
1. Saline solution (0.85% NaCl), autoclaved at 121°C for 15 min and stored at refrigeration temperature.
2. Spectrophotometer (Spectronic 20, Bausch & Lomb, Miltou Roy, USA) for determinations of optical density (OD) in tubes.
3. Microorganisms: obtained as described in Subheadings 3.1. and 3.2.1.
4. Hydrophobic solvents (all from Merck, Darmstadt): n-hexadecane, pro-analysis; p-xylene, pro-analysis; and toluene, pro-analysis. (see Note 1).
5. Accessories: Vortex (Vicking, Argentina), pro-pipets, screw-top glass tubes.
6. Determination of the degree of hydrophobicity.
ODb = optical density before, obtained as described in Subheading 3.2.1. ODa = optical density after, obtained as described in Subheading 3.2.3.
2.3. Salt Aggregation Test
1. Microorganisms: obtained as described in Subheading 3.1.3.
2. Phosphate buffer: 0.02 M, pH 6.8, stored at room temperature.
3. Bacterial suspension: obtained as described in Subheading 3.3.1.
4. Ammonium sulphate ([NH4]2SO4) solutions: 0.2-4 M in distilled water. The different concentration solutions are prepared by diluting the 4 M solution with distilled water. The solutions are stored at room temperature.
2.4. HA Characteristics
1. Microorganisms: obtained as described in Subheading 3.1.3.
2. Saline solution (see Subheading 2.2., step 1).
3. Spectrophotometer (see Subheading 2.2., step 2) and tubes.
4. Blood samples: from human or bovine sources, obtained by venipuncture.
5. Sodium citrate solution (3.8%), autoclaved at 121°C for 15 min.
6. Sterile syringes and screw-top tubes.
7. Capillary test tubes used in hematology to determine red cell concentrations.
8. Accessories: round-bottomed microplates, automatic pipets, and laminar flow chamber (Clean Room Products, USA).
3.1. Preparation of Microorganisms
Lactobacilli were isolated from vaginal swabs of women and cows under aseptical conditions. The microorganisms were identified by the methods described in Bergey's
Manual of Determinative Bacteriology (12), standard techniques used in the laboratory, and API 50 CHL (Biomerieux-France).
1. Lactobacilli subcultivation: Screw-top glass tubes containing 5 mL LAPTg broth were inoculated with colonies of each of the isolated lactobacilli, and incubated at 37°C for 24 h. A 150-^L aliquot of these cultures was subcultured twice at 37°C for 12 h in 5 mL of the same medium.
2. Storage in milk-yeast extract: The third lactobacilli culture was centrifuged (10 min, 2000g), and the spent supernatant was discarded. The pellet was resuspended in 2 mL milk-yeast extract. This bacterial suspension was distributed in cryovials (1 mL) and stored at -20°C.
1. Lactobacilli subcultivation: Stored lactobacilli were subcultured three times in LAPTg broth (5 mL were inoculated with 30 ^L of lactobacilli stored at -20°C in milk-yeast extract), incubating the cultures between 12 and 14 h at 37°C. The third culture was used to prepare the bacterial cell suspension for each experiment.
3.2. Hydrophobic Partition Assay (MATH)
1. Bacterial-cell suspension preparation. The third culture was centrifuged (10 min, 2000g), and the spent supernatant was discarded. The pellet was washed three times with 3 mL
saline solution and then resuspended in the same solution to an OD600 from 0.4 to 0.6. The exact OD values were registered as ODb values (OD before).
2. Hydrophobic partition assay. Aliquots of 3.6 mL of the bacterial suspensions were distributed in glass tubes. The solvents (0.6 mL) were added to each tube. The tubes were closed and vortexed vigorously for 1 min. They were kept still to allow the immiscible solvent and aqueous phase to separate.
3. Optical density measurements. After separation of the two phases, the aqueous layer was removed by using Pasteur pipets, and transferred to clean tubes. Absorbance was measured in this aqueous phase: OD values were registered as ODa (OD after).
4. Calculation. The percent of hydrophobicity was obtained from the application of the following formula:
The percentage of hydrophobicity was calculated for each strain with the three solvents. Each test was performed in triplicate, and the means of the data were calculated.
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