Study by Microbiological Methods Virginia S. Ocaña and María Elena Nader-Macías
Adhesion of lactobacilli to the epithelium has been described as the first step in the formation of a barrier to prevent undesirable microbial colonization (1); consequently, it has been defined as a characteristic of interest for selecting probiotic strains (2-4).
Several methods have been described to predict the adhesion ability of Lactobacillus. Early studies were phenomenological: it was useful to determine whether a particular bacterium could hemagglutinate, or bind to coated particles. Later, studies based on the adhesion of bacteria to epithelial cells in vitro were developed (1,5-10). These last assays were based on the numbers of bacteria attached to epithelial cells, which were determined by counting stained microorganisms under light microscopy or by measuring the radioactivity of previously radiolabeled bacteria. Because the microscope technique, had some disadvantages, a modification was developed in our laboratory. The technique described in this chapter is a modification of the Mardh and Westron method (5,11). The method allows the study of the adhesion ability of bacteria, even if they are aggregating or if they are high-adherent bacteria covering a large area of the epithelial cell surface. Determination of the number of adherent bacteria by counting colonies grown in a selective media avoids the time-consuming, tedious, and hazardous counting under the light microscope and the use of radioactive methods.
The microorganisms employed for this study were previously selected because of their ability to produce antimicrobial substances or because of their self-aggregating pattern, as shown by the salt aggregation test (12-15). They were: L. crispatus CRL 1266, L. salivarius subsp. salivarius CRL 1328, L. acidophilus CRL 1259, and L. acido-
philus CRL 1294. L. salivarius S256 isolated from human saliva and Lactobacillus sp.
CC18 isolated from chicken crop (kindly donated by María Ahumada and Carina
Audisio, CERELA, Tucumán, Argentina) were also employed to obtain comparative results.
1. LAPTg broth: 1.5% peptone, 1% tryptone, 1% glucose, 1% yeast extract, 0.1% Tween-80, pH 6.5. Sterilize at 121°C for 15 min (by autoclaving) and store under refrigeration.
2. LAPTg agar: 1.5% peptone, 1% tryptone, 1% glucose, 1% yeast extract, 0.1% Tween-80, 1.5% agar-agar, pH 6.5. Sterilize at 121°C for 15 min and store under refrigeration.
3. Milk-yeast extract (MYE): 10% low-fat milk, 0.5% yeast extract, 1% glucose. Sterilize at 121°C for 15 min and store at room temperature.
4. LBS agar: 1% peptone from casein, 0.5% yeast extract, 2% glucose, 0.6% potassium dihydrogen phosphate, 0.2% ammonium citrate, 0.1% Tween-80, 1.5% sodium acetate, 0.0575% magnesium sulfate, 0.0034% iron (II) sulfate, 0.012% manganese sulfate, 1.5% agar-agar, pH 5.5. Sterilize at 121°C for 15 min and store under refrigeration.
5. Eagle's minimal essential medium (EMEM), pH 7.0 (Gibco), used as prepared by the manufacturer. Store at 0-4°C until the expiration date (see Note 1).
2.3. Adherence Assay
1. Epithelial cells.
2. Cytobrush (Cytosoft, Medical Packaging) and Neubauer chamber.
3. Dulbecco's modified Eagle's medium (Gibco, cat. no. 11995-065). Store at 2-8°C and protect from light.
7. pH strips
8. Filters with 8-^m pore membranes (Millipore)
9. Shaker (for agitation at 37°C and 100 opm/min)
10. Peptone water: 1% peptone in distilled water. Store at room temperature.
1. Microorganisms isolated in LAPTg or LBS agar are grown in 5 mL of LAPTg for 12 h at 37°C, subcultured twice in the same media under identical conditions, and then separated by centrifugation at 2000g for 15 min.
2. The pellet is resuspended in 5 mL of MYE and stored at -20°C.
1. Frozen lactobacilli (stored in MYE at -20°C) are subcultured three times in LAPTg broth (16) for 12-14 h at 37°C prior to adhesion studies. An inocula of 2% is used.
2. Overnight cultures grown in LAPTg broth are centrifuged at 2000 rpm for 15 min, and the supernatant was discarded.
3. Lactobacilli were washed twice with saline and once with EMEM, pH 7.0. They were resuspended in EMEM to obtain a final concentration of 107 bacteria/mL. An OD600 of 0.11 was determined previously by a calibration curve.
1. Vaginal epithelial cells were collected by scraping the vaginal wall of healthy volunteers with a cytobrush and resuspended immediately in EMEM, pH 7.0.
2. Indigenous bacteria from epithelial cells were removed by washing three times with 10 mL of EMEM and performing a final filtration through an 8-^m-pore filter (Millipore) of the prewashed cells resuspended in 10 mL of EMEM. This filtration step allowed bacteria to pass through but retained epithelial cells (see Note 2).
3. Vaginal cells were resuspended in EMEM and adjusted to 105 cells/mL as determined in a Neubauer chamber under light microscopy. The OD600 of 105 cells/mL was approx 0.15. The OD of the cell suspension can be adjusted with EMEM to 0.15 before counting in the Neubauer chamber, which is done to confirm the epithelial cell concentration.
3.3. Adherence Assay
1. 1 mL of vaginal cell suspension is mixed with 1 mL of the bacterial suspension and incubated at 37°C for 1 h on a platform mixer (100 opm).
2. A control was prepared by substituting EMEM for the bacterial suspension.
3. Assays can be performed at pH 7.0 and pH 4.0, adjusting the pH with 2 NNaOH and 10% lactic acid, respectively.
4. Suspensions are centrifuged for 10 min at 500 rpm, the supernatant was discarded, and the pellet was resuspended in 10 mL of EMEM.
5. The suspension was passed again through an 8-^m pore membrane to remove nonadherent bacteria.
6. Membranes are washed with manual agitation for 1min in Petri dishes containing 5 mL of saline.
7. The remaining washing solution, containing the epithelial cells with the attached bacteria, was employed for counting viable adherent bacteria.
1. The number of viable bacteria is determined by the plate dilution method, using peptone water as diluent and LBS agar plates as growth media. The dilutions used are 1:10, 1:100, and 1:1000. Pure washing cell suspension (100- and 200-^L aliquots) were also added to LBS plates to detect poorly adherent bacteria (see Note 3).
2. LBS plates were incubated in a microaerophylic environment for 48 h at 37°C, and colonies were counted to calculate adhesion.
4. Adhesion can be expressed as adhesion percentage calculated as follows (see Note 4):
% Adhesion = Log UFC (adhered bacteria) x 100 Log of total number of bacteria and as mean percentage of adhesion per epithelial cell:
% Adhesion/cell = Log UFC (adhered bacteria) x 100 Log epithelial cell number
1. Do not use if the pH indicator turns yellow or another color, or if there is any sign of contamination (turbidity).
2. Washing of the epithelial cells obtained from the vagina (see Subheading 3.2., step 1 is a very important step. By this procedure, indigenous bacteria are removed, as is the mucus. The presence of mucus can block the membrane pores and affect the later filtration steps.
3. To verify that all t he bacteria and cells are removed form the filters after the washing step, the filter can be transferred to a new Petri dish and LBS agar can be added on top. The plate can be incubated in a microaerophilic environment for 48 h at 37°C, and the absence of colonies grown can be tested.
4. In the original Mardh and Westron technique, the procedure applied to determine the number of attached bacteria is different. After the last filtration, which allows the separation of epithelial cells with the attached bacteria, cells retained on filters are transferred to slides by gently pressing the membranes against slides (that had previously been coated with a thin layer of albumin, as indicated in Subheading 3.3., item 6. The cells transferred to slides are fixed with 96° ethanol and Gram-stained. Then bacteria are counted under the light microscope (40X and 100X). With this method, self-aggregating bacteria cannot be individually counted. With the plate dilution method described in this chapter, it is possible to count all the bacteria by using the agitation procedure.
This work was supported by grants from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), PID-BID 395, and Beca Carrillo Oñativia from the Ministerio de Salud Pública de la República Argentina.
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