Assessment of bioburden on human and animal tissues: Part 1--results of method development and validation studies

Recovered human and animal tissues are used extensively in surgery for wound repair and reconstruction. In preparation for the validation of chemical disinfection and radiation sterilization processes, studies were performed on the development and validation of quantitative bioburden recovery methods for human bone and soft tissue and also for porcine dermis. The use of a swab-based method was not considered due to the known poor efficiency of recovery for this technique. The "exhaustive extraction" and "inoculated product" approaches to validation of a bioburden recovery efficiency factor have inherent strengths and weaknesses; in this study, tissues were inoculated and also subjected to a series of extractions to determine if/when "exhaustion" occurred. Femoral and tibial shaft rings, iliac crest wedges, sections of Achilles tendon, a soft tissue composite sample, and porcine dermis, were inoculated at several sites with Bacillus atrophaeus spores, and then subjected to either shaking by hand, mechanical shaking, or sonication plus mechanical shaking. Each of these methods of agitation were performed in combination with three rinse (extraction) fluids: phosphate buffer (Butterfield's buffer), phosphate buffer with 0.2% polysorbate 80 (a surfactant), and water with 1% peptone and 1% polysorbate 80 (Fluid D). The highest recovery efficiencies were observed with sonication plus mechanical shaking; of the three extraction media, Fluid D gave the highest first-rinse recovery efficiency (65%) and Butterfield's buffer gave the lowest (39%). Each of the three recovery methods, however appeared to reach "exhaustion", a subsequent rinse giving less than 10% of the recovery found in the first rinse. The results demonstrated the importance of performing bioburden method development and validation studies. The method validation strategy described here, using a combination of tissue inoculation and repetitive extraction, showed the superiority of sonication plus mechanical shaking using Fluid D as the rinse medium. In addition, the use of only the exhaustive extraction approach could have resulted in the development of a methodology that consistently underestimated the bioburden present on/in recovered tissue.