1. Animals were sacrificed by cervical dislocation.
2. Samples of the different organs from the tract were aseptically extracted (kidneys, urethers, bladders, and urethra), washed with saline solution, and immediately introduced into fixative.
3. Fixation was with 10% formaldehyde for at least 24 h (see Note 11).
4. The organ must be dehydrated before paraffin embedding, by transfer through increasingly graduated alcohols (see Notes 12 and 13), as follows:
d. Ethyl alcohol 96°, two passages of 1 h each.
e. Ethyl alcohol 100°, two passages of 1 h each.
f. Xylene, two passages of 1 h each.
g. Xylene-paraffin (1:1) in an incubator at 56° for 1 h.
i. Paraffin II in an incubator at 56° for 1 h.
j. Paper boxes were previously prepared to receive the organs in paraffin. The paraffin solidifies when brought to room temperature, and the block is ready. After a wood piece is added, the blocks are ready to be cut in the microtome.
5. The slides must be as narrow as possible; the ideal width is between 2 and 4 m^m. The tissue slides are put into warm water, to be ironed. Later, they are added to glass slides and incubated at 37°C to finish the extension process (see Note 14).
6. The paraffin must be removed; it does not permit staining of the tissue, because it is insoluble in alcohol or aqueous solutions. The slides were immersed in xylene (two successive baths of 1 min each).
7. As the stain used is suspended in aqueous solution, the slides must be progressively hydrated in successive baths, as follows:
f. Distilled water, 1 min.
8. Hematoxyline: 1 min to stain the nucleus. The slides were stained red.
9. Tap water: 20 min. The alkaline salts modify the media and produce a change in color from red to blue (see Note 15).
10. Alcoholic eosin: 0.5% eosin solution in ethyl alcohol 50° to stain the cytoplasm.
11. The slides were transferred successively through different ethyl alcohol gradations (see Note 10).
a. Ethyl alcohol 96°, two passages of 1 min each.
b. Ethyl alcohol 100°, two passages of 1 min each.
c. Xylene, two passages of 1 min each.
12. The slides were covered with 1 drop of Canadian balsam, covered with a glass coverslip, and dried in the air for 24 h (see Note 10).
13. Observation by light microscopy: The nucleus, particularly the chromatin, is stained blue at different intensities and the cytoplasm is stained red.
Immediately after it was obtained, the material was cut into small pieces of less than 1 mm and fixed in 6% glutaraldehyde in cacodylate buffer. The material was later fixed in osmium teroxide. Epoxy resines were used as inclusion media. Ultra-narrow slides 400-600 A wide were obtained by using the ultramicrotome. The slides were mounted on Cupper supports covered with Parlodion and stained with Pb citrate. Tissues were observed under TEM, and the photos were taken with 35-mm Kodak film.
1. For adequate use of microorganisms, the same experimental protocol must always be used. The original culture must be prepared and fractionated in several tubes, which must be kept in freezing conditions. For each experiment, the culture must be obtained from this freezing culture. No more than three subcultures were performed, to avoid changes in adhesion properties.
2. To eliminate excess of Vaseline, we have also tried to wash the beads with a detergent such as sodium deoxycholate (0.125, 0.25, and 0.50% in PBS). We have also studied storage and stability using different temperatures and washing liquids. Different washing liquids such as PBS, peptone water, or a mixture of both, were assayed; better results were obtained with peptone water in terms of more stable beads.
3. The mice used for the experiment must always come from the same inbred, closed colony and be of the same age and weight, to avoid genetic interference. All the control groups must be assayed at the same time. When a certain condition is changed, it must be changed for all the groups.
4. Urine: as the urine volume obtained from each mouse is very low, it is not always possible to perform all the assays.
5. Lactobacillus selection agar is a selective medium for lactobacilli; inhibits the accompanying microbial flora, allowing the growth of colonies with different morphology.
6. Morphological variation in the colonies obtained after growth in plates, isolated from tissue homogenates, was sometimes observed. The colonies were identified by morphological and biochemical tests (not genetic tests). We did not study whether these variations would correspond to rough or smooth variants of the pathogenic microorganisms or whether they were caused by stress on the microorganisms because of host defenses.
7. The slides must be immersed in the fixation liquid for at least 10 min. The time of fixation must not exceed 24 h in any case.
8. The time in which the slides must be kept in each alcohol must be about the indicated time in each step.
9. The slides must be mounted in a very quiet environment to avoid interference of any type before adding the cover slips.
10. The dehydration step is very important, because the transparency of the slide depends on it, as well as the possibility of false-positive reactions when the slides are observed cyto-logically.
11. The amount of fixation liquid must be enough to go into the anatomical piece. The tissues must be fixed as soon as possible so that the slides do not become dry.
12. The dehydration step is very important, because it determines that the tissue and the structures are conserved without modifications, the same as at the time of fixation.
13. Graduation of the different alcohols was determined by measuring the amount of 96° alcohol and adding distilled water until 96° was reached. For example, 80 mL of 96° alcohol plus 16 mL of distilled water results in alcohol 80°.
14. The ironing step is very important, because modifications or folding of the slides can interfere in staining and observation.
15. Differentiation implies the smear changing from red to blue. The differentiation step is very important. The tissues must be in this liquid for at least 20 min to allow the cells to take the appropriate color.
This work was supported by grants from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina), PIP 359, and from the Ministerio de Salud Pública de la República Argentina (Beca Carrillo Oñativia para Investigadores Formados).
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