The Effect of BioBurden on In-Depth Disinfection of Denture Base Acrylic Resin

Timothy R. Saunders, DDS; Villa L. Guillory, DDS; Stephen T. Gregoire, MS; Meade Pimsler, PhD; and Mary S. Mitchell, MHS

Copyright 1998 Journal of the California Dental Association.

This study evaluated the effectiveness of three different disinfectant solutions against denture bioburden absorbed within the depth of acrylic resin. Specimens were taken from dentures that had been worn by the patients for 15 to 20 years and were scheduled for replacement.

Previous studies have demonstrated that dental prostheses brought into the dental office for repair or adjustment are contaminated with bacteria, viruses, and fungi.1-3 Several authors have discussed effective means of surface disinfection of dental prostheses, however, the vast majority or research on acrylic resin disinfection has been specific to surface contamination and not subsurface disinfection.4-16 Chau, et. al., contaminated and then disinfected acrylic resin specimens in various disinfectant solutions.17 These specimens were then fractured and cultured to determine whether the disinfectant solutions penetrated the depth of the acrylic resin. They observed that a 10-minute immersion in 5.25 percent sodium hypochlorite disinfects dental acrylic resin more effectively than either Biocide (Iodophor) or Alcide LD (Chlorine dioxide) disinfectant solutions when used in accordance with manufacturer's recommendations. Prior to this, no studies have been performed sampling existing patient prostheses that have been worn for extended periods of time nor evaluating the effectiveness of subsurface disinfection.

The purpose of this study was to evaluate in-depth disinfection of complete denture prostheses that had been worn for periods of 15 to 20 years.

Materials and Methods

Complete Dentures

Four maxillary/mandibular complete dentures that had been worn by patients for 15 to 20 years and were scheduled for replacement were used as the source of samples. Eight denture acrylic resin specimens, 8 mm x 8 mm, were taken from each maxillary and mandibular denture flange areas (distal buccal flange of the maxillary denture and the distal lingual flange of the mandibular denture using sterile discs).

Pre-disinfection Culture and ID

The 64 denture acrylic resin specimens were briefly rinsed in sterile saline and dropped into individual test tubes of sterile Mueller-Hinton broth. The specimens were separated into labeled tubes identifying their site of origin. These tubes were incubated at 37C with 5 percent CO2 for 18 to 24 hours. An uninoculated Mueller-Hinton broth tube of the same lot number was also incubated. Any Mueller-Hinton broth tubes showing no growth at 24 hours were reincubated for an additional 24 hours.

Disinfection Procedure

The specimens were removed from the culture medium, rinsed briefly in sterile saline, then immersed in one of the three different freshly mixed disinfectant solutions using the following disinfection protocols:

1. Biocide (Biotrol International, Louiseville, Colorado): Immerse for 10 minutes in a 0.48 percent solution of Biocide in warm (>20 C) deionized water (2 ml of Biocide in 14 oz of sterile water).

2. Alcide LD (Alcide Corp., Norwalk, Connecticut): Immerse for three minutes in solution that consists of 10 parts per deionized water, one part LD base LD activator.

3. Sodium Hypochlorite (James Austin Co., Mars, Pennsylvania): Immerse for 10 minutes using 5.25 percent (undiluted) sodium hypochlorite and deionized water in the following concentrations: 10 percent solution, 20 percent solution, 30 percent solution, 40 percent solution, 50 percent solution, and 100 percent solution (or undiluted).

4. Sterile 0.9 N saline: Immerse for 10 minutes.

The specimens from all four dentures were disinfected in the three different solutions and 0.9 N sterile saline. The contaminated specimens were placed in sterile specimen cups when immersed in the disinfectants. The specimens were removed from the solution at specified intervals and harvested for bacterial growth.

Microbiological Testing

Isolation Procedure

Following disinfection, the specimens were rinsed briefly with a sterile 0.9 N saline solution and then placed between two sterile metal blocks, wrapped in several layers of sterile gauze and pulverized with a manual impact device. The impact device was also wrapped in new sterile gauze for each plug pulverizing action. The pulverized specimens were dropped into test tubes of sterile Muller-Hinton broth and cultured at 37C for 48 hours. If growth was detected, gram stains were performed and the organisms were subcultured to blood agar, eosin methylene blue agar, and chocolate agar plates.

Identification Procedure

Gram positive organisms were presumptively identified by catalase and coagulase reactions. For catalase testing, suspect colonies from blood agar plates were transferred, using a sterile loop, to a clean, sterile glass slide and tested with one to two drops of 3 percent hydrogen peroxide. Coagulase activity was detected using the Remel Staph Latex Kit. Additional testing included: sensitivity to bacitracin and optochin, reactivity in the CAMP test, the ability to hydrolyze esculin in the presence of bile, and the organisms' ability to grow in Infusion Medium (Remel) containing 6.5 percent NaCl. Confirmatory testing was performed using the Vitek Identification System, GPS-SA and GPI cards.

Gram negative organisms were presumptively identified by their ability to ferment lactose on eosin methylene blue agar, reactivity with indole, and oxidase activity. Confirmatory testing was performed using the AFI 20E system.

Results

All specimens were shown to be colonized by bacteria following incubation in sterile Muller-Hinton broth medium prior to disinfection. Samples were disinfected in two commercial disinfectant solutions (Alcide and Biocide), or in dilutions of 5.25 percent sodium hypochlorite. Growth was observed on all saline disinfection controls. The most common organisms recovered were S. aureus, P. aeruginosa, and E. coli. Gram positive cocci were also recovered from some of the saline control specimens.

Neither Alcide nor Biocide, used in accordance with the manufacturer instructions, was effective in disinfecting the denture acrylic resin. Growth was obtained from seven of eight denture samples disinfected in either solution (effective kill rate 12.5 percent). Dilutions of sodium hypochlorite, ranging from 10 percent to 50 percent of a 5.25 percent solution in distilled water, proved to be three to five times more effective than either Alcide or Biocide. 10, 20, 30, 40, and 50 percent dilutions of 5.25 percent sodium hypochlorite in saline effectively disinfected between three and five of eight denture samples (effective kill rates of 37.5 -- 62.5 percent, Table 1).

In a final set of experiments, denture base material was disinfected in undiluted (100 percent) 5.25 percent sodium hypochlorite (Table 2). No growth was obtained from six to eight samples in this series. There was a 75 percent effective kill rate, which was six times more effective than Biocide or Alcide. Normal oral flora were isolated from two of the samples which yielded growth. Control samples cut from the same dentures and incubated in saline instead of sodium hypochlorite all produced growth.

Discussion

In our previous study we observed that 10 minutes immersion in 5.25 percent sodium hypochlorite more effectively disinfected both the interiors and exteriors of purposely contaminated acrylic resin specimens than did either Alcide or Biocide.17 Specimens were prepared from three different acrylic resins (Lucitone 199, Ortho, and Repair), and were incubated in broth cultures containing a mixture of bacteria. Biocide failed to disinfect two of five Lucitone 199 specimens and four of five Repair specimens. In the same study, Alcide did not disinfect two of five Lucitone 199 specimens, and one of five Repair specimens. In contrast, all specimens were disinfected by immersion in undiluted 5.25 percent sodium hypochlorite.

Similar results were obtained in this study, which employed resin-based material derived from dentures worn by patients for 15-20 years. One striking observation was the effect of microbial bioburden on disinfection. In addition to bacteria, resin-based denture material is coated and infiltrated with protein, polysaccharides, and other materials from the oral environment. This material creates a protective microenvironment for microbes living on and in denture material which must be overcome for effective disinfection in the laboratory. Our results show that bioburden reduces the effectiveness of disinfection by all test disinfectants in this study. Alcide and Biocide were both relatively ineffective, killing all cultivable bacteria and fungi in one of eight denture specimens tested. The effectiveness of disinfection of sodium hypochlorite proved to be three to five times more effective than Alcide or Biocide, however, the protective nature of bioburden was evident (i.e., a ten minute immersion in undiluted 5.25 percent sodium hypochlorite produced an effective kill rate of only 75 percent).

Furthermore, organisms from specimens exposed to disinfection were recovered that were not recovered from the saline control specimens. This may be reflective of heterogeneous colonization of denture material by oral flora which may result from: (1) establishment of microenvironments within the denture material, (2) competition between organisms, or (3) changing oral microflora and oral environment over the years. We believe this does not obscure the results of the study, rather it demonstrates the complexity of the oral microenvironment and the necessity for effective disinfection of a broad range of microorganisms.

Summary

The studies described in this report clearly indicated that 5.25 percent sodium hypochlorite is more effective in disinfecting complete dentures submitted by patients for repair or replacement than either of two commercially available disinfection solutions. Sodium hypochlorite is relatively inexpensive and easily available. Further, material studies have shown common bleach has no discernible structural or cosmetic effect on resin-based denture material. These results indicate sodium hypochlorite is the best available disinfectant for use in dental offices and laboratories.

The data obtained in this study support the hypothesis that acrylic resin complete dentures worn by patients for 15 to 20 years may be contaminated with bacteria both superficially and within the body of the protheses. The data also suggests that a ten-minute immersion in 5.25 percent sodium hypochlorite more effectively disinfects acrylic resin than do either Alcide or Biocide disinfectant solutions used in accordance with the manufacturer's recommendation.

Conclusion

Disinfection of the dental prostheses is the minimum standard of care protocol prior to sending them to the laboratory. This study shows a dental prosthesis must be disinfected on the exterior as well as the interior surface because of contaminating microorganisms. Data from this study suggests that a ten-minute immersion in full strength sodium hypochlorite (5.25 percent) more effectively disinfects dental acrylic resin than either Biocide or Alcide disinfectant solutions.

Future studies are recommended which would evaluate the reaction of varing concentrations and time of disinfection in sodium hypochlorite and partial denture prostheses (eg. Chromium-cobalt).

Authors / Timothy R. Saunders, DDS, is Associate Professor of Clinical Dentistry at the University of Southern California School of Dentistry. Villa L. Guillory, DDS, is a Captain in the United States Air Force Dental Corps and is a Prosthdontic Resident. Stephen T. Gregoire, MS, is a Major in the United Stateds Air Force Biomedical Science Corps at the Office of the Surgeon General. Meade Pimsler, PhD, is a Lieutenant Colonel in the United Stateds Air Force Biomedical Science Corps at the Office of the Surgeon General. Mary S. Mitchell, MHS, is a Medical Technologist.

The views expressed herein are those of the authors and do not necessarily reflect the views of the United States Air Force or the Department of Defense. This paper was originally presented before the American Academy of Maxillofacial Prosthetics, Kansas City, MO, October, 1996.

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