Two Methods to Evaluate the Antimicrobial Action of Calcium Hydroxide Paste

Abstract

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.