Sampling Methods for Evaluating Microbial Contamination of Oral Devices

Objectives: Due to the absence of existing protocols, the purpose of this study was to develop laboratory procedures for the isolation and identification of the microorganisms found on the surfaces and in the depths of oral devices such as dentures and protective athletic mouth-guards (PAM). Methods: Sterile end-cutting nippers were used to section dentures and PAM. Surfaces and depths of the sectioned material were touched three times to blood agar plates (BAP), chocolate agar (CA), and Sabaroud dextrose agar (Sab). All media were incubated at 37°C for 24 hours. Colony forming units (CFU) were enumerated by serial dilution of previously weighed oral device material, minced by either sterile nippers (dentures) or sterile razor blades (PAM) and vortexed in known volumes of sterile water. Media demonstrating microbial growth were scored by three independent investigators using a previously validated microbial intensity scale (0 to 4). Results: Microorganisms were isolated and identified by using pure culture techniques, selective and deferential media, physiological tests, and molecular biochemical techniques. Standard laboratory procedures and sterile techniques were observed for all procedures throughout these studies. The potential danger of fracturing oral devices required strict observation of safety precautions for all procedures. Conclusion: These techniques have been used successfully to isolate and quantify a wide spectrum of aerobic and facultative bacteria, yeasts, molds, mycoplasma, and amoebae from a variety of oral devices and can be used in a glove box for the isolation of strict anaerobes. Companion studies of the surfaces, porosities, and biofilms of these oral devices by electron microscopy were consistent with the biological findings.