Putting Water Throughtap In Vagina

2 doses

2 doses

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H, inoculation of 0.5 mg of estradiol valeratel; C, control mice inoculated with peptone water without lactobacilli; L, inoculation of lactobacilli in peptone water; S, days of mice killing and sample processing.

H, inoculation of 0.5 mg of estradiol valeratel; C, control mice inoculated with peptone water without lactobacilli; L, inoculation of lactobacilli in peptone water; S, days of mice killing and sample processing.

9. Immunofluorescence assays were carried out to evaluate whether local administration of lactobacilli produces stimulation of cells involved in the immune response at the local level, as detailed in Subheading 3.7.

3.4. Microbiological Assays

1. To determine the number of microorganisms in the vagina of mice, samples were obtained by using automatic pipets with tips loaded with 100 ^L of sterile saline solution. Aliquots (50 ^L) of the washing samples were added to the surface of MRS agar with antibiotics and later incubated at 37°C for 48-72 h to determine the number of viable microorganisms. The samples were also spread on glass slides for different staining techniques, including direct Gram stain.

2. The organs were opened longitudinally in aseptic conditions with a sterile scissors, and transferred to the homogenizor tube containing 1 mL of peptone water. The Teflon pestle connected to the homogenizor was used, applying a speed of 1400 rpm. Different aliquots (100 ^L and 200 ^L) of the organ homogenates were added to Petri dishes, where melted MRS agar plus antibiotic was added. The method of the successive dilutions was also performed by diluting 0.5 mL of the organ homogenate in successive dilutions of peptone water, plating later aliquots of the respective dilutions in agar plates. The plates were incubated in microaerophilic conditions at 37° C for 48-72 h. Then, the number of microorganisms was determined by counting plates showing between 30 and 300 colonies. Some colonies were picked and Gram-stained to confirm the lactobacilli characteristics.

3. Vaginal washings were Gram-stained, and the characteristics of the microflora were observed, looking for the characteristic shape of the lactobacilli, which is sometimes very different from the indigenous Gram-positive bacilli. This observation gave us an idea of the direct microbiological results before the results of the plates were available.

3.5. Histological Technique

1. After the organs were extracted, they were transferred to 96° ethyl alcohol at 4°C for 15 h to perform fixation of the material and to maintain the cellular structure without modifications (see Note 5).

2. Dehydration was performed by passing the tissues successively three times through alcohol 100° at 4°C for 2 h, to dehydrate the samples.

3. As the next step was the addition to paraffin, a substance nonsoluble in alcohol, the method required the alternative passage of the tissues in an intermediate reactive sub stance able to solubilize both alcohol and paraffin. With this objective, the tissues were passed through three different glass bottles containing xylol, the two first cold (4°C) and the last one at room temperature.

4. Three paraffin passages were performed, each one for 2 h at 60°C, to obtain material with uniform consistency.

5. Microtome slides were performed in a 2-^m-wide Minot-type microtome. The slides/cuts were stretched in a thermostatized water bath maintained at 37°C. The tissue slides were put into glass slides to be later stained and evaluated by microscopy.

3.6. Staining: H&E Stain

1. The paraffin was taken out of the histological slides by two successive passages in xylol for 2 min each.

2. The material was hydrated by submerging the tissues in flasks containing different decreas-ingly gradated alcohols (two transfers from absolute alcohol and one in 96°, 80°, 70°, and 50° ethyl alcohols) for 2 min each at room temperature.

3. The staining procedure applied was a double staining that give color to both the nucleus and the cell cytoplasm. The slides were submerged for 10 min in Mayer's hematoxylin and then washed three times with water (distilled watter: 2 min; tap water: 15 min; distilled water: 1 min).The passage through tap water was an important step because it helped to develop the color of the stain. Then the slides were submerged in eosin (0.5% aqueous solution w/v) for 5 min and immediately put in a distilled water bath.

4. The object of dehydration was to make the material ready to be included in Canada balsam, by passing successively through ethyl alcohols of increasing concentrations (80°, 96°, 100°, and 100°) for 2 min each. Then two xylol passages were performed, for 2 min each.

5. Canada balsam (50 ^L) was added to the material on the surface of the glass slides, covering the tissues impregnated in xylol.

6. The number of cells and their characteristics, from epithelial layer and lamina propria were evaluated by using an optical microscope at 100 and 40X.

3.7. Immunofluorescence

This technique was applied to identify and localize the antigens by using fluorescence-labeled antibodies such as FITC with ultraviolet wavelengths. The specificity of this technique allows recognition of all cells with surface inmunoglobulins expressed on their surface (17) (sees Note 6).

1. The paraffin was removed from the histological slides by three successive passages in xylol for 5 min each one at 4°C. Then three alcohol baths were performed (96°, 70°, and 40°) for 2 min each at 4°C. The material was then transferred to PBS, pH 7.2 (two transfers for 10 min each).

2. FITC-conjugated antibody (0.1 mL) diluted with PBS was added to each slide, and the mixture was incubated at 37°C for 30 min in a humid chamber. Then the slides were washed for 10 min with PBS at 4°C. One drop of mounting liquid was then added to the slides, which were covered with a cover slip. The number of cells that were IgA+ or IgM+ in the lamina propia was determined.

4. Notes

1. Before carrying out the inoculation of the microorganisms, the purity of the lactobacilli under study must be checked carefully, as for any other microbiological assay. Also, in terms of the process used to obtain the microorganisms they must be derived from stock kept in freezing conditions, with the same number of subcultures, the same culture media, and all other requirements kept the same.

2. The strain of mice used under all the experimental conditions must be the same. They must also come from an inbred colony to ensure the same genetics. If some of the experimental conditions must be modified, all the controls must be set up again.

3. When intramuscular inoculation is performed, it is convenient to use the internal part of the mouse paw, because correct inoculation of the hormone can be easily observed.

4. Intravaginal inoculation of lactobacilli must be performed as fast as possible, because the agarized peptone water cools down quickly.

5. The paraffin must be filtered every time the technique is performed. The material must be kept in the alcohol bath room for the time indicated. Do not keep the material longer than indicated, because it can harden, making it very difficult to cut later in the appropriate way. Paraffin must be added carefully when the blocks are prepared to avoid the formation of bubbles, which can hinder cutting.

6. Before the fluorescent labeled antisera is diluted, the room must be prepared for darkness; dilution and all subsequent steps must be performed in the dark to avoid inactiva-tion of the fluorescence.

Acknowledgements

This work was supported by grants from the Ministerio of Salud Pública de la República Argentina (Beca Ramón Carrillo) and CONICET (PIP 359).

References

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9. Ocaña, V. S., Ruiz Holgado, A. P., and Nader-Macías, M. E. (1999) Selection of vaginal H2O2-generating Lactobacilli species for probiotic use. Curr. Microbiol. 38, 279-284.

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