Colonization Capability of Lactobacilli and Pathogens in the Respiratory Tract of Mice

Microbiological, Cytological, Structural, and Ultrastructural Studies

Rosa Cangemi de Gutierrez, Viviana M. Santos, and María Elena Nader-Macías

1. Introduction

Respiratory tract infections are among the bacterial infections that affect humans with higher frequency. Those produced by Streptococcus pneumoniae are reported to have the highest incidence in the world, affecting both children and old people. As a 2001 report from the World Health Organization (I) expressed it, the basic fight of children under 5 yr old is to survive. Five different conditions (acute respiratory infections, diarrhea, measles, palludism, and undernutrition) directly produce more than 50% of the deaths in this age group. Respiratory tract infections in the developing countries in the Americas are among the first three causes of death in children under 1 yr and between the first and second cause in children between 1 and 4 yr old. Pneumonia is responsible for 85 and 90% of deaths in children under 5 yr old (approx 150,000 annually), 95% of them occurring in the developing countries in the Americas.

There is an increased worldwide tendency to use preventive measures and to consume products that help to maintain the health status of the individual. Thus the use of probiotics has increased systematically during the last decade, and the scientific literature trying to demonstrate the positive effect of such preparations has also increased. The term probiotic (2) has been applied to products that (1) contain live microorganisms, freeze-dried or included in fermented products or (2) improve the health status of humans and animals, exerting effects in the mouth or gastrointestinal tract (included in foods or capsules), in the respiratory tract (as aerosols), or in the urogenital tract (by local application)

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

Having in mind the high incidence of respiratory tract infections, and looking for preventive measures as well as the possible applications of probiotics, the aim of this chapter was to use mice as experimental models to determine whether members of the genus Lactobacillus were able to colonize and give protection from infections after inoculation by the intranasal route. To this end, the following procedures were carried out:

1. Screening of the predominant bacterial species in respiratory organs (3).

2. Study of the kinetics of colonization of the different groups of microorganisms from 15 d up to adult (2 mo) (3).

3. Screening of the probiotic characteristics of all the isolated strains.

4. Determination of the colonization ability of the selected Lactobacillus strains after intranasal inoculation, as well as the optimal dose and amount of microorganisms (4).

5. Experiments to evaluate whether intranasal administration of Lactobacillus fermentum produces some type of modification at the structural or ultrastructural level in the different organs of the respiratory tract (5).

6. Determination of whether L. fermentum could prevent an infection with Streptococcus pneumoniae in the respiratory tract when given by the intranasal route (6).

The first procedures were developed by applying in vitro techniques described in previously published papers (3,4), and also in some other chapters of this book. In this chapter, a detailed description of the methodology applied to animals is given.

2. Materials

2.1. Microorganisms

The microorganisms used in this study were L. fermentum and S. pneumoniae. Lactobacilli were isolated from the respiratory tract of mice, as described previously (3,4). S. pneumoniae was isolated from a human source and characterized as described in a previous paper (6).

2.2. Culture Media

1. Storage: Milk-yeast extract: 10% skim milk, 1% glucose, 0.5% yeast extract, pH 6.5. Sterilize by autoclaving and store at refrigeration temperature. L. fermentum was stored at -70°C in milk-yeast extract.

2. Supplemented Brain-Heart Infusion (BHI): BHI: 5 mg/mL hemin, 0.005% cystein chlorhydrate, 0.5% yeast extract, 100 mg/mL kanamycin, 1 mg/mL vitamin K, final pH 7.4 ± 0.2. Sterilize by autoclaving prior to the addition of antibiotics and vitamins and store at refrigeration temperature. At the time of use, the sterilized basal media is melted and the antibiotic and vitamins added before pouring onto the plates. The vitamins and antibiotics were sterilized by filtration and stored at freezing conditions. S. pneumoniae was stored in glycerol-BHI broth at -70°C.

2.3. Preparation of Microorganisms for Inoculation Into Mice

1. LAPTg broth (7): 15 g/L meat peptone, 10 g tryptone, 10 g yeast extract, 10 g glucose, 1 mL Tween-80, final pH 6.5. Sterilize by autoclaving at 121°C for 15 min). Store at refrigeration temperature. All the media ingredients were obtained from Britania Laboratories (Buenos Aires, Argentina).

2. LAPTg + rifampicin: 15 g meat peptone, 10 g tryptone, 10 g yeast extract, 10 g glucose, 1 mL Tween-80, 1.5% agar, final pH 6.5, prepared as described in step 1. The antibiotic rifampicin was prepared by dissolving the powder in ethyl alcohol and sterilizing by filtration. It was stored for 1 wk at freezing temperature. At the time of use, the antibiotic was defrozen and added aseptically to the melted media for a concentration of 1 mg/mL.

3. Blood-agar: Columbia agar (Britania Laboratories) with 5% human blood. The agar base was sterilized by autoclaving and stored at refrigeration temperature. At the time of use, sterile blood was added for a concentration of 5%.

4. Columbia agar with 5% heated blood: The agar base was sterilized by autoclaving. At the time of use it was melted, the human blood was added for a concentration of 5%, and it was later heated for 15 min at 70°C. This media was prepared prior to use.

5. de Man-Rogosa-Sharpe (MRS) agar (8): 10 g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 0.8 g/L Tween-80, 2 g/L dipotassium phosphate, 5 g/L sodium acetate, 2 g/L ammonium citrate, 0.2g/L magnesium sulfate, 0.05 g/L manganese sulfate, 15 g/L bacteriological agar, pH 6.5.

2.4. Animal Maintenance Conditions

a. Adult male 2 V2-mo-old Balb/c mice weighing 25-30 g were obtained from the inbred colony maintained in our laboratory at the Microbiology Institute of the National University of Tucuman.

b. They were housed in plastic cages, fed ad libitum with a conventional balanced diet for rodents, given water ad libitum, and maintained under constant environmental conditions.

c. The experimental protocol was previously approved by the Ethical Committee of Animal Care at CERELA.

d. Each experimental assay was carried out with groups of 25-30 mice.

e. A group of control animals of the same size was similarly inoculated with a solution without microorganisms (placebo).

f. Mice were randomly assigned to the different experimental groups.

2. Keeping standard conditions in the mice colony.

a. The ideal room temperature is around 18°C with small fluctuations.

b. The cages were cleaned daily, and the wood-chip bed was changed.

3. Other materials.

a.

Spatulas.

b.

Pipets.

c.

Petri dishes.

d.

Glass tubes.

e.

Calibrated loops.

f.

Tuberculin syringes

g.

Sterile plastic tips.

h.

Gloves.

4. Mice sacrifice.

a. Dissection boards.

b. Scissors.

c. Material for dissection.

2.5. Histological Technique

1. Baker phormol-calcium reagent: 10% anhydrous sodium chloride (Cicarelli), 100 mL distilled water, pH 6.5 (Biopack), 10 mL formaldehyde 40% (stabilized in methanol; Biopur). Store at 15-25°C and prepare a new set of reactive every 30 d (9).

2. Activated hematoxylin for histological purposes (Biopur-Argentina), ready for use; does not require the addition of acid. Store at 15-25 °C.

3. Alcoholic yellow eosin (Biopur-Argentina), ready to use. Store at 15-25°C (9).

4. Ziehl fucsin diluted with distilled water (1:5) (Biopack); prepared at the time of use. Store at 15-25 °C.

5. Indigo picrocarmin (Biopack).

6. Watery acuose solution of acetic acid (0.2%) (Biopur) (9).

7. Graduated ethyl alcohols (50°, 70°, 80°, 96°, and 100°; Cicarelli). Store at 15-25°C; prepared from pure ethanol (B6) with demineralized water added, pH 6.5 (Biopack).

9. Other materials:

a. Coplin flasks.

b. Glass bottles for organ fixation.

c. Glass cuvets for staining slides.

d. Stove for impregnation (54-58°C).

e. Glass slides.

f. Glass cover slides.

g. Canadian natural balsam (Biopack).

h. Paraffin microtome, Minot type.

i. Thermostatized iron for the paraffin slides. j. Light microscope.

2.6. Cytological Technique

1. Pure methyl alcohol. Store at 15-25°C (Cicarelli).

2. Giemsa stain: Azur II eosin, methylene blue (Merck, ready to use). Store up to the date recommended on the label (10).

3. Graduated ethyl alcohols (50°, 70°, 80°, 96°, and 100°; Cicarelli). Store at 15-25°C. They were prepared from pure ethanol with demineralized water, pH 6.5 (Biopack).

5. Other materials:

a. Glass bottles.

b. Cover slides.

c. Glass slides.

d. Glass cuvets for staining.

e. Natural Canadian balsam (Biopack).

f. Ethyl alcohols (Cicarelli).

g. Light microscope.

2.7. Electron Microscopy

1. 3.5% Glutaraldehyde solution buffered with 0.1 M phosphate buffer, pH 7.4 (Biopack). Store at 5-10°C in a cold room.

2. 1% Osmium tetroxide.

3. Aqueous solution of 2% uranyl acetate.

4. Ethyl alcohols of increasing strength (Cicarelli).

5. Spurr resin.

6. Uranyl acetate (Pelco).

7. Lead citrate (Pelco).

8. Zeiss EM 109 transmission electron microscope at 50 kV.

2.8. Microbiological Techniques

1. Glass tubes for samples.

2. Peptone water.

3. Sterile plastic syringes.

4. Culture media for lavage.

5. Homogeneizor (Omni-Mixer 17106, Sorvall).

6. Glass tubes for diluting samples.

7. MRS and MRS agar with 100 ^g/mL rifampin (Sigma). Incubator.

8. For Gram stain: Standard reactants or kit.

9. For microbiological identification in enriched, selective, and differential culture media: MRS, LBS, BHI, blood agar, and chocolate agar.

10. Biochemical tests to identify the microorganisms:

a. Standard biochemical tests described in the Bergey's Manual of Bacteriological Procedures (11) and API Strepto.

b. API Staph and API 50 CHL (Biomerieux, France).

c. Supplementary test to identify Lactobacilli (Bergey's Manual [11])

d. Additional tests for streptococci: production of urease, growth in media with sodium azide and 6.5% NaCl, esculin bilis reaction.

11. For the optoquine test: 5 ^g optoquin (Difco; Bacto-differentiation disks, Optoquin BBL, or Taxo P disks), blood agar plates, surface spreaders.

12. For oxgall solubility: 10% Sodium deoxycholate (BBL or Difco).

13. For the Quellung test: Glass slides, specific antisera, methylene blue.

2.9. Antibody Determinations

1. Suspension of L. fermentum as antigen: L. fermentum was killed by heating at 100°C for 1 h.

2. Suspension of S. pneumoniae: By tyndalization (heating at 60°C for 1 h for three consecutive days).

3. For the agglutination reaction: Glass tubes, saline solution, pipets.

3. Methods

3.1. Microorganisms

1. Isolation of Lactobacilli: L. fermentum was isolated from the respiratory tract (pharynx) of adult Balb/c mice. Lactobacilli were identified by the methods described in Bergey's Manual of Determinative Bacteriology (11), standard techniques used in the laboratory, and API 50 CHL (Biomerieux-France).

2. Rifampicin-resistant L. fermentum.

a. L. fermentum mutants resistant to antibiotics were obtained after determining their sensitivity to different antibiotics (by the monodisc diffusion test and minimal inhibitory concentration [MIC]).

b. Mutants were grown on MRS-R. L. fermentum RR (resistant to rifampyn) showed the same phenotypical properties as the parental strain (tested by the API System).

c. To obtain the suspensions for inoculation, strains were subcultured in LAPTg broth (7) at 37°C three times, alternatively in media with and without antibiotic.

d. They were centrifuged, washed with saline solution (SS), and resuspended to the desired concentration.

3. S. pneumoniae.

a. S. pneumoniae was isolated from human pneumonia-suffering subjects and identified by standard techniques.

b. The virulence of this microorganisms was tested in mice.

c. Pathogenicity in mice was increased by inoculating S. pneumoniae intraperitoneally.

d. The following day, mice were bled by heart puncture, and capsulated S. pneumoniae was isolated from blood agar plates.

e. The pathogen was stored in glycerol-BHI broth at -70°C.

f. Prior to challenge, S. pneumoniae was plated onto blood agar plates, and a suspension prepared from the microorganisms was grown on the surface.

g. Mice inoculated with S. pneumoniae without passing through animals as described in Subheading 3.2., item 1a, were not susceptible to the pathogen and its subsequent colonization and infection.

h. The S. pneumoniae strain belongs to the 6A serotype. Serotyping was performed in the Servicio de Bacteriología, Instituto Nacional de Enfermedades Infeccionsas-ANLIS (Dr. Carlos Malbrán, Buenos Aires, Argentina).

i. The technique applied was the Quellung techique using antisera produced by the Statens Serum Institute (Copenhagen, Denmark).

4. The number of lactobacilli was determined by the serial dilution method; the aliquots were plated in LAPTG agar plates and incubated at 37°C for 48 h.

5. The number of S. pneumoniae was determined by the serial dilution method; the aliquots were plated in blood agar plates, and incubated microaerophilically at 37°C for 48 h.

3.2. Inoculation Procedure

1. Intranasal inoculation of mice with L. fermentum.

a. Mice were intranasally inoculated with 50 ^L of L. fermentum in SS (1 x 109 CFU/ mL) every 12 h.

b. The exposure time was approx 1 min per animal, with one intranasal inoculation of 10 ^L each time.

c. Inoculation was performed by using an automatic micropipetor equipped with a nose tip, and the suspension was sniffed in by the animals with normal tidal breathing.

d. To facilitate downward migration of the inoculum, mice were held in a vertical position for 2 min.

e. Different groups of mice (three to four animals each) were inoculated with one, two, or four doses of 1 x 109 CFU/mL (50 ^L each), administered every 12 h.

f. Other groups of mice were reinoculated 10 d after the first inoculation with four doses (50 ^L) of the same L. fermentum suspension in SS (1 x 109 CFU/mL).

2. Intranasal inoculation with S. pneumoniae.

a. Inoculation with S. pneumoniae was performed with 50 ^L of a suspension containing 1 x 109 CFU.

b. The suspension of the pathogenic microorganisms was obtained as described in Subheading 3.3.

Fig. 1. Scheme of the inoculation and killing steps. Black arrows indicate the intranasal inoculation of lactobacilli, performed every 12 h. Open arrows indicate the days of mice sacrifice. The upper scheme indicates the regular protocol, while the lower figure indicates the reinoculatio procedure.

3. Intranasal inoculation with L. fermentum and S. pneumoniae.

a. Mice were intranasally inoculated with L. fermentum, receiving four doses of 107 CFU every 12 h, as described in step 1.

b. The next day, S. pneumoniae (109 CFU/mouse) was also intranasally inoculated. This was the experimental group for studing the preventive effect of treatment with L. fermentum prior to S. pneumoniae challenge.

c. Control animals were inoculated with sterile 0.1% peptone water intranasally.

4. The inoculation and sacrifice protocol is shown in Fig. 1.

3.3. Experimental Procedure to Obtain Samples From Mice

1. Mice were sacrificed by cervical dislocation on d 2, 4, and 7 after inoculation depending on the experimental group under study.

2. Nasally instilled pharynx, trachea, bronchia, and lungs were extracted aseptically and transferred to 0.1% peptone water.

3. The organs were homogenized with a Teflon pestle in a tissue homogenizor.

4. Aliquots of the samples, or their dilutions, were added to selective media to determine the numbers of viable microorganisms.

5. Plates containing between 30 and 300 bacteria were counted.

6. Identification of isolated microorganisms was checked either by Gram stain or by biochemical tests of single colonies.

7. The organs were also added to 10% formaldehyde to be processed for histological purposes.

3.4. Weight Determinations

Mice were weighed every day throughout the experiment to determine whether S. pneumoniae challenge produces some type of clinical condition that makes the animals lose weight. Weight was determined before sacrifice by weighing the animals in an OHAUS balance, Precision Standard Model T.S 400 D.

3.5. Histological Studies

1. From each experimental group, two mice were randomly selected for histological examination.

2. Trachea, bronchia, and lungs were separated for further studies after cervical dislocation.

3. The areas studied were:

a. The high trachea located in the neck base near the thyroid gland.

b. The bronchia, where they divide.

c. The lungs (left and right lobule): terminal bronchiole and alveolar wall with their capillary and connective tissue to which the alveolar macrophages are associated.

4. The samples were fixed with 10% paraformaldehyde in glass flasks with 10-20 vol more than the sample for 24 h at room temperature.

5. The samples ere passaged in alcohol 70° (1 h each), and then embedded in paraffin for 24 h according to the method standardized at our laboratory.

6. The samples were cut into 5-^m-thick sections.

7. On the histological slides, different techniques were used to allow analysis of the modifications produced in the lamina propria of the trachea by the increase in the number of lymphocytes.

8. Slides were stained with H&E and by the Ramón y Cajal technique and then processed for light microscopy (40x objective).

9. Soon after the organs were obtained, they were fixed in Baker's fixative (sample size 23 cm and volume of fixation liquid approx twice the sample size).

10. Dehydration was performed with graduated alcohols: 50°, 70°, 80°, 96°, and 100°, keeping the samples in each one for 2-3 min.

11. The organs were embedded in paraffin at 56-58°C.

12. Slides (5-6 ^m wide) were obtained with a sliding microtome.

13. The staining technique used (either H&E or Ramón y Cajal technique [9]) depends on the type of cell of interest.

3.5.1. H&E Technique

1. The slides underwent the following steps:

a. Xylene: two passages of 3-5 min.

b. Ethyl alcohol 100° to take out the excess xylene: 1 min.

c. Ethyl alcohols 96°, 70°, and 50°: 1 min each.

d. Distilled water: 1 min.

2. Staining was performed with Erlich hematoxylin for 2-5 min.

3. The slides were washed with distilled water and transferred for the differentiation step (top water with concentrated HCl).

4. Slides were allowed to change color until they were violet and stored for 10 min.

5. Alcoholic eosin was applied for 3 min.

6. Slides were washed in distilled water.

7. For dehydration and mounting, the slides were passed very quickly through:

c. 100° Ethyl alcohol: two different passages at 1 min each.

d. Xylene: two passages of 1-2 min each.

e. The slides can be mounted in Canadian balsam.

9. The results were as follows:

a. Hematoxylin stains the nuclear components blue or purple, the intensity depending on the concentration of nucleic acids (DNA and RNA). The cytoplasm can show an aggregation of substances stained blue with hematoxylin; this basophilic reaction also depends on the presence of nucleic acids in the cytoplasm.

b. Eosin stains the cytoplasm red or pink, the degree depending mainly on the amount of structural proteins.

This method can be used after any previous method of fixation. It is useful for the differentiation of cellular categories.

1. Soon after the organs were obtained, they were fixed in Baker's fixative (sample size of 2-3 cm, fixation liquid volume approx twice the sample size).

2. Dehydration was performed with graduated alcohols: 50°, 70°, 80°, 96°, and 100°, keeping the samples in each for 2-3 min.

3. The organs were embedded in paraffin 56-58°C.

4. Slides (5-6 ^m wide) were obtained with a sliding microtome.

5. Slides were stained with Ziehl's fuscin for 10 min.

6. Slides were washed with running water.

7. Slides were washed with acetic water to eliminate fucsin excess.

8. Slides were stained with indigo picrocarmine in a bottle for 10 min.

9. Slides were washed with acetic water for 10 min and dehydrated with absolute alcohol to eliminate the red (excess).

10. Dehydration was performed in alcohols of increasing gradation for 2-3 min.

11. Slides were washed with pure xylene for 3-4 min.

12. Slides were mounted in Canadian balsam for longer storage (see Note 2).

13. To quantify the lymphocyte population, an area of the epithelium corresponding to 50 epithelial cells was taken as a reference. This area correlates with the number of lymphocytes present in the lamina propria area (see Note 3).

3.6. Cytological Studies

1. The left lobule was used to perform lung cytological slides. Before light micrography, the organ was immersed in 9% saline solution. Macerated cellular material was spread on the glass slides, which had been previously immersed in 96° ethyl alcohol and air-dried.

2. The glass slides with the lung impress were fixed with methyl alcohol for 3 min.

3. Later they were placed into the Giemsa stain (diluted 1:10 with tap water), pH 5.0 for 10 min.

4. Then the slides were washed several times to prevent contamination by the stains.

5. The slides were dried in air.

6. Slides were observed with a light microscope (Leitz, Germany).

7. Numbers of cells on the lung slides were counted based on the method used for bone marrow counts, according to Grignaschi et al. (10). A total of 500 cells was counted in different areas of the slide and according to the proportion of cells, the percentage was later determined.

3.7. Electron Microscopy Techniques

1. Higher trachea and lung samples (bronchia, bronchioli, and alveoli) were obtained after sacrificing mice by cervical dislocation.

2. Samples were fixed in 3.5% glutaraldehyde solution buffered with 0.1 Mphosphate buffer (pH 7.4) for 3 h and postfixed in 1% osmium tetroxide with the same buffer overnight.

3. Samples were treated with an aqueous solution of 2% uranyl acetate for 40 min.

4. After fixation, tissues were gradually dehydrated in a series of alcohols of increasing strength, passed through acetone, and embedded in Spurr resin.

5. Ultrathin sections were stained with uranyl acetate and lead citrate.

6. Observation was with a Zeiss EM 109 transmission electron microscope at 50 kV (see Note 4).

3.8. Microbiological Techniques

1. Determination of the number of Microorganisms in the respiratory tract organs.

a. Nasal and pharynx instillations were obtained by washing each open cavity with l mL of peptone water and later scraping them gently with mini-sterile cotton tips.

b. Trachea, bronchia, and lung were aseptically removed and homogenized in 1 mL peptone water with a Teflon pestle.

c. The number of microorganisms was determined by the serial dilution method.

d. The organs were diluted in peptone water (1% peptone) and inoculated on MRS-rifampin (15 ^g/mL) agar plates (Biokar Diagnostics, Beauvais, France).

e. Plates were incubated at 37°C for 48-72 h in a microaerobic environment.

f. Lactobacilli resistant to rifampin were counted after that period.

g. The number of microorganisms was determined, taking into account those plates showing between 30 and 300 CFU/plate.

2. Identification of microorganisms was performed by Gram staining and biochemical tests. The streptococci were also identified by the test described next, in step 3.

3. Direct examination: Gram stain. Direct observation of the samples was performed after Gram staining the slides. The technique and reactants are those standards set up in the laboratory for routine Gram stain.

4. Microbiological identification in enriched, selective, and differential culture media. The microorganisms present in the respiratory tract of mice were isolated by standard techniques used in the laboratory. The culture media used were BHI, blood agar, and chocolate agar.

5. Biochemical tests to identify the microorganisms.

a. Identification was performed by standard morphological and biochemical tests used for clinical diagnosis. The criteria applied for identification were (1) observation of B-hemolytic colonies grown in blood agar with typical morphology and (2) variable predominance.

b. Identification of the following microorganisms was performed: a-hemolytic Streptococcus, Staphylococcus, Micrococcus, Corynebacterium, Enterobacteriaceae,

Peptocococcus, Veillonella, Clostridium, Bacteroides, Citrobacter, Escherichia, Klebsiella, and Aeromonas. c. The phenotypic properties studied were (1) standard biochemical tests as described in the Manual of Bacteriological Procedures and (2) API Strepto, API Staph, and API 50 CHL (Biomereux, France).

6. Lactobacilli identification.

a. Additional tests performed for the identification of lactobacilli were the following: catalase test, Gibson's test, fermentation of calcium gluconate, growth at different temperatures (15° and 45°C), and growth in skim milk.

b. Carbohydrate fermentation was evaluated from lactose, mannitol, raffinose, sorbitol, and inulina.

c. Hydrolysis was used for arginine, esculine, and hippurate.

7. Streptococci identification: Streptococci were evaluated by production of urease, growth in media with sodium azide and 6.5% NaCl, and the esculin bilis reaction.

8. S. pneumoniae identification: The optoquin and oxgall test was performed as follows:

a. The colonies were spread into the surface of blood agar, where the optoquin discs were added aseptically. The S. pneumoniae colonies are inhibited by the optoquin.

b. Colonies morphologically suspected to be S. pneumoniae must also be tested for oxgall solubility.

c. Oxgall discs were also used.

9. Oxgall solubility: This test is based on the characteristics of S. pneumoniae, because the colonies are lysed when incubated in 10% sodium deoxycholate (BBL or Difco ).

10. Quellung test.

a. Serological identification of S. pneumoniae is based on the reaction of specific antisera against capsular polysaccharides using the Quellung reaction. It can be applied directly to the clinical samples (sputum, pleural exudates, cerebrospinal fluid, or other exudates) for fast identification of pneumococci. It is also valuable as a confirmatory procedure for the identification of S. pneumoniae from cultures.

b. One small drop of the clinical material or the young culture in broth is added to the glass slide.

c. Three drops of the antiserum are added, which are mixed with the bacterial suspension.

d. Then, one drop of saturated solution of methylene blue is added, and the slide is covered with a glass cover slip.

e. After 15 min, the slides are observed under an immersion microscope.

f. S. pneumoniae stains dark blue, surrounded by a very strong transparent halo.

3.9. Serum Antibodies

1. Mice were bled from the retro-orbital venous plexus at different days post S. pneumoniae challenge.

2. Serum was kept frozen until determination of antiserum level.

3. Antibody titration was performed using an agglutination reaction with L. fermentum and S. pneumoniae suspensions (1 x 109 microorganisms/mL each).

4. The agglutination reaction was performed with serial dilutions of the sera and microorganism suspensions.

5. Tubes were incubated for 1 h at 37°C and then kept at 4°C for 12 h.

6. Antibody titers were defined as the inverse of the highest dilution presenting a positive agglutination reaction.

3.10. Statistical Analysis

Experiments were performed at least three times, and the results obtained were used to calculate the mean and SD. Results are shown in the tables and graphs. Student's t-test was used to determine the statistical significance of the differences between data.

4. Notes

1. Screening of the surface characteristics of the microorganisms allowed selection of some lactobacilli strains from the respiratory tract of mice.

2. Inoculation of S. pneumoniae by the intranasal route in mice was hard to assess. No respiratory infections were produced in mice. Only after animalization of the microorganisms (which means ip inoculation of young mice and bleeding after 24 h by a heart puncture) was respiratory tract infection produced in the experimental mice model.

3. Inoculation of the microorganisms by the intranasal route must be performed very carefully to allow the small drop to be sniffed into the nasal cavity. The mice must be kept in a vertical position for some minutes to allow the microorganisms to enter the nasal area.

4. When performing the histological technique, care must be taken before immersing the trachea and lung into the fixation step. All blood must be extracted from the tissues by washing twice with buffered solution.

5. The macerated lung is soaked in sterile saline solution. The impress must be performed very soon after extraction of the organ, always using a wet fixation to avoid cell retraction.

6. For trachea samples for electronic microscopy purposes, it is recommended to perform a glutaraldehyde (fixate) passage with a tuberculin syringe in the central area of the trachea, and later to put the trachea into the fixative.

Acknowledgments

This work was supported by grants from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas).

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