Control of microorganisms in vitro by calcium hydroxide pastes.

AIM: The aim of this study was to determine the influence of vehicles on the antimicrobial efficiency of calcium hydroxide. METHODOLOGY: A total of 588 size 50 sterile absorbent paper points, were immersed in various microbial suspensions for 3 min. The points were then placed on Petri dishes and covered with intracanal dressings containing calcium hydroxide: Ca(OH)2 + saline; Ca(OH)2 + camphorated paramonochlorophenol; Ca(OH)2 + 1% chlorhexidine solution: Ca(OH)2 + 3% sodium lauryl sulphate; Ca(OH)2 + Otosporin. After 1 min, 48 and 72 h and 7 days, 147 absorbent paper cones were removed from contact with the intracanal dressings and individually transported and immersed in 5 mL of Letheen Broth, followed by incubation at 37 degrees C for 48 h. Microbial growth was evaluated by turbidity of the culture medium. A 0.1-mL inoculum obtained from the Letheen Broth was transferred to 5 mL of BHI, and incubated at 37 degrees C for 48 h. Bacterial growth was again evaluated by turbidity of the culture medium. Positive BHI tubes were selected and inocula were spread on the surface of BHI agar and incubated at 37 degrees C for 48 h. Gram staining of the BHI growth and from colonies growing on BHI agar was carried out. RESULTS: An antimicrobial effect occurred after 48 h on the cultures of S. mutans, E. faecalis, S. aureus, P aeruginosa, B. subtilis, C. albicans and a mixed culture, irrespective of the intracanal dressing. CONCLUSIONS: Under the conditions of this study, the various vehicles associated with calcium hydroxide pastes did not influence the time required for microbial inactivation.

Two methods to evaluate the antimicrobial action of calcium hydroxide paste.

The objective of this study was to analyze two methods for determining the antimicrobial effectiveness of (i) calcium hydroxide plus saline, (ii) calcium hydroxide plus polyethylene glycol, and (iii) calcium hydroxide plus camphorated paramonochlorophenol. Four microorganisms (Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 6633)), one yeast (Candida albicans (ICB/USP-562)), and one mixture of these organisms were used. The strains were inoculated in Brain Heart Infusion (BHI) and incubated at 37 degrees C for 24 h. Two methods, the direct exposure test and the agar diffusion test were used to evaluate antimicrobial effects. For the direct exposure test (DET) 288 paper points were contaminated with the standard microbial suspensions and exposed to the intracanal dressings for 1, 24, 48, and 72 h. The points were immersed in Letheen Broth, followed by incubation at 37 degrees C for 48 h. An inoculum of 0.1 ml obtained from Letheen Broth was then transferred to 7 ml of BHI under identical incubation conditions, and microbial growth was evaluated. Pastes showed activity between 1 and 72 h, depending on the microorganism/mixture tested. For the agar diffusion test 36 Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the same microbial suspension used for the DET, using sterile swabs that were spread on the medium. Five cavities were made in each of two agar plates (total = 10) and completely filled with one of the calcium hydroxide pastes. The plates were preincubated for 1 h at environmental temperature and then incubated at 37 degrees C for 24 to 48 h. The inhibition zone around each well was recorded in millimeters, and the results were submitted to ANOVA and Tukey test (alpha = 0.05). All intracanal dressings induced inhibition zones (range 5.0-10.0 mm). Data obtained showed that both the DET and agar diffusion test are useful in establishing the calcium hydroxide antimicrobial spectrum, thus improving infection control protocols. The direct exposure method is independent of other variables and is a practical laboratory procedure. A complete antimicrobial effect was observed after 48 h on indicator microorganisms, in both tests, irrespective of the calcium hydroxide paste vehicle.

Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealapex and Dycal.

The objective of this study was to investigate the antimicrobial action of mineral trioxide aggregate (MTA), Portland cement, calcium hydroxide paste (CHP), Sealapex and Dycal. The chemical elements of MTA and two Portland cements were also analyzed. Four standard bacterial strains: Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633), one wild fungus, Candida albicans (ICB/USP-562), and one mixture of these were used. Thirty Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the experimental suspensions. Three cavities, each one measuring 4 mm in depth and 4 mm in diameter, were made in each agar plate using a copper coil and then completely filled with the product to be tested. The plates were pre-incubated for 1 h at environmental temperature followed by incubation at 37 degrees C for 48 h. The diameters of the zones of microbial inhibition were then measured. Samples from diffusion and inhibition halos were extracted from each plate and immersed in 7 ml BHI broth and incubated at 37 degrees C for 48 h. Analyses of chemical elements present in MTA and in two samples of Portland cement were performed with a fluorescence spectrometer Rx. The results showed that the antimicrobial activity of CHP was superior to those of MTA, Portland cement, Sealapex and Dycal, for all microorganisms tested, presenting inhibition zones of 6-9.5 mm and diffusion zones of 10-18 mm. MTA, Portland cement, and Sealapex presented only diffusion zones and among these, Sealapex produced the largest zone. Dycal did not show inhibition or diffusion zones. Portland cements contain the same chemical elements as MTA except that MTA also contains bismuth.

Antimicrobial evaluation of calcium hydroxide in infected dentinal tubules.

The objective of this study was to evaluate the antimicrobial activity of calcium hydroxide in infected dentinal tubules. Four microorganisms, strains of ATCC (Streptococcus faecalis (ATCC-29212), Staphylococcus aureus (ATCC-6538), Bacillus subtilis (ATCC-6633), and Pseudomonas aeruginosa (ATCC-27853)) and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. Sixty-three human maxillary central incisors were prepared and sterilized by autoclaving. Five groups of 12 teeth each were contaminated for 28 days using new 24-h cultures every 72 h, prepared and adjusted to tube 2 of the MacFarland scale (6 x 10(8) cells/ml). Root canals were then irrigated with 5 ml of saline, dried, and completely filled with calcium hydroxide paste. At intervals of 0, 48, and 72 h, and 7 days, dressings were removed and teeth were immersed in 5 ml of BHI and incubated at 37 degrees C for 48 h to observe the growth and multiplication of the microorganisms. Three uninoculated teeth were maintained in a humid environment as an aseptic control. These teeth were immersed in BHI and maintained at 37 degrees C for 7 days to determine microbial growth. Bacterial growth was shown by turbidity of the culture medium and confirmed by seeding these broths on BHI agar at 37 degrees C for 24 h. The positive BHI tubes were selected, and inoculum was spread on the surface of BHI agar, followed by the same incubation conditions. Gram stain was conducted from BHI growth and from colonies growing on solid medium. Calcium hydroxide in infected dentinal tubules showed no antimicrobial effect on S. faecalis, S. aureus, B. subtilis, P. aeruginosa, or on the bacterial mixture used throughout the experiment.

Effect of vehicle on antimicrobial properties of calcium hydroxide pastes.

The current discussion about the importance of intracanal dressings and the effect of vehicles on calcium hydroxide pastes is justified by controversy concerning the achievement of complete disinfection after preparation of infected root canals and the real antimicrobial effect of these vehicles. The aim of this study is to investigate the role of vehicles in the antimicrobial effect of calcium hydroxide pastes. Well-conducted research about the characteristics of calcium hydroxide, such as antimicrobial potential, physico-chemical aspects and histocompatibility, gives credibility to the choice of this medication in several clinical situations. Different vehicles have been added to calcium hydroxide in an attempt to enhance its properties. Scientific reasoning indicates the use of hydrosoluble vehicles (distilled water, saline) associated with calcium hydroxide because of their chemical characteristics of dissociation, diffusibility and filling capability which are decisive for the biological behavior, i.e., antimicrobial qualities and induction of tissue repair.

In vitro determination of direct antimicrobial effect of calcium hydroxide.

The objective of this study was to determine in vitro the time required for calcium hydroxide in direct contact with microorganisms to express its antimicrobial effect. The microorganisms used were: Micrococcus luteus (ATCC-9341), Staphylococcus aureus (ATCC-6538), Fusobacterium nucleatum (ATCC-25586), Pseudomonas aeruginosa (ATCC-27853), Escherichia coli, and Streptococcus sp. The strains were cultivated in Brain Heart Infusion (BHI), with the exception of F. nucleatum (BHI-PRAS). Pure and mixed suspensions of the microorganisms were prepared. Paper cones immersed in these substances were covered with calcium hydroxide paste, and after 0, 1, 2, 6, 12, 24, 48, and 72 h and 7 days they were transferred to an appropriate medium to observe the growth and multiplication of the microorganisms. Incubation was conducted at 37 degrees C for 48 h, according to the requirements of oxygen of each microorganism. The antimicrobial effect of calcium hydroxide was shown to occur after 12 h on M. luteus and F. nucleatum, 24 h on Streptococcus sp, 48 h on E. coli, and 72 h on S. aureus and P. aeruginosa. Mixture II (M. luteus + Streptococcus sp + S. aureus) was sensitive to calcium hydroxide antimicrobial potential after 48 h, whereas mixture I (M. luteus + E. coli + P. aeruginosa), mixture III (E. coli + P. aeruginosa), and mixture IV (S. aureus + P. aeruginosa) were inactivated after 72 h of exposure.

Mechanism of action of calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria.

The biological and bacteriological action of calcium hydroxide confer to its current success as an intracanal dressing. For this reason the mechanism of action of calcium and hydroxyl ions on tissue and bacteria deserves further study. The objective of the present paper is to analyze and discuss the mechanism of action of calcium and hydroxyl ions on anaerobic bacteria, starting from the isolated study of the influence of pH on these bacteria , as well as the mechanism of action of calcium hydroxide on tissue.

Immunoglobulin A antibodies reactive with Streptococcus mutans in saliva of adults, children, and predentate infants.

Immunoglobulin A (IgA) antibodies reactive with Streptococcus mutans MT3 cells (serotype c) were sought, using an indirect enzyme-linked immunosorbent assay, in the saliva of humans who either harbored or did not harbor detectable levels of this organism. Samples of unstimulated whole saliva from three adults and one child who were infected with S. mutans contained IgA which bound to MT3 cells. Saliva samples of two adults studied also contained IgA which reacted with S. mutans strains of serotypes e, g, a, and b, the latter two of which are rarely isolated from humans. The saliva of three children who did not harbor detectable levels of S. mutans and of three of five predentate infants also contained IgA reactive with MT3 cells. The latter observation is of special interest since S. mutans does not colonize the mouth before eruption of teeth. Thus, the presence of salivary IgA reactive with S. mutans cells is not necessarily related to present or past infection by this organism. Absorption with MT3 cells markedly reduced the reactivity of adult saliva without greatly altering the total concentration of IgA present; this suggests that the IgA was not binding to S. mutans MT3 cells via Fc receptors. The possibility that the antibodies which reacted with S. mutans MT3 may have been induced to other bacteria with cross-reactive antigens was supported by the finding that absorption of saliva with mixed bacterial growth derived from common dairy products significantly reduced its reactivity. Absorption experiments further suggested that a significant portion of the salivary IgA antibodies was binding to glucans on the cell surface.

Impaired colonization of gnotobiotic and conventional rats by streptomycin-resistant strains of Streptococcus mutans.

Colonization of streptomycin-resistant mutants derived from Streptococcus mutans strain LB1, a human isolate, and strain FA-1, a rodent isolate, was studied in gnotobiotic and conventional rats. Mutants resistent to 2.0 mg of streptomycin per ml were isolated by using both stepwise (suffix "R"M) and one-step (suffix "R"1) selections. Rats were infected with mixtures of parental and streptomycin-resistant strains, and the proportions of each strain present in samples from the intestinal canal, tongue dorsum, teeth, and fissure plaque were determined. Combinations of strains investigated were LB1 and FA-1"R"M; FA-1 and LB1"R"M; LB1 and LB1"R"1; FA-1 and FA-1"R"1. In gnotobiotic rats, nonresistant strains predominated in every oral sample studied at 7 and 21 days after infection. Similarly, when conventional exgermfree rats were infected with FA-1 and FA-1"R"1, FA-1 dominated in all samples. Streptomycin-sensitive revertants were not detected in rats monoinfected with strains LB1"R"1 and FA-1"R"1 for 21 days. No antagonistic interactions were observed between the strains in in vitro experiments. Streptomycin-resistent mutants attached to hydroxyapatite treated with rat or human saliva in equal or higher numbers than did parental strains. However, parental strains appeared to grow faster in Trypticase soy broth then streptomycin-resistant mutants. These observations indicate that induction of streptomycin resistance frequently impairs the colonization properties of S. mutans strains, possibly by altering their rate of growth.

Ability of Streptococcus mutans and a glucosyltransferase-defective mutant to colonize rodents and attach to hydroxyapatite surfaces.

A mutant of Streptococcus mutans forming little or no cell-associated glucosyltransferase attached to the teeth of rats and to saliva-treated hydroxyapatite comparably to its parent.

Comparative estimates of bacterial affinities and adsorption sites on hydroxyapatite surfaces.

The adsorption of strains of prominent oral bacteria to hydroxyapatite (HA) surfaces was studied by use of an adsorption model based on the Langmuir adsorption isotherm; this permitted comparative estimates of the number of adsorption sites and the strength of the adsorption bonds on untreated and salivatreated HA surfaces for strain of Streptococcus mutans, S. salivarius, S. sanguis, S. mitis, Actinomyces viscosus, and A. naeslundii. The experimental data closely followed the adsorption model as judged by the high correlation coefficients obtained for all strains studied. Adsorption to untreated HA was similar for strains of the six species studied, suggesting that a common adsorption mechanism, possibly Ca(2+) bridging, may exist for attachment to HA. More complex interactions appeared to be involved in bacterial adsorption to saliva-treated HA since adsorption of the strains tested at unsaturating cell concentrations varied more than 30-fold. This indicates that adsorbed salivary components on HA surfaces impart a higher order of specificity for subsequent bacterial adsorption. Fewer cells of strains of S. mutans, S. salivarius, and A. naeslundii adsorbed to saliva-treated HA than to untreated HA because adsorbed salivary components presented fewer adsorption sites. Substantially higher numbers of cells of strains of S. sanguis, S. mitis, and A. viscosus adsorbed to saliva-treated HA because the film of adsorbed salivary components increased the number of adsorption sites for these strains. The affinity constants for all but one strain studied were lower on saliva-treated HA than on untreated HA. The number of bacterial cells which adsorbed to saliva-treated HA more closely related to the number of available binding sites than to the strength of their adsorption bonds when tested at an initial concentration of 2 x 10(7) organisms/ml. Although some differences were observed in the adsorption of strains of S. mutans representative of five serological groups, the numbers which attached to saliva-treated HA did not vary widely; this suggests that factors other than their ability to attach to a pellicle-covered HA surface may be responsible for their varying geographic distribution in human populations.