Establishment of a Single Temperature Incubation Approach for Environmental Monitoring Samples with Focus on Mold Recoveries

Abstract

A robust environmental monitoring program is essential to properly estimate and identify microorganisms in cleanrooms, ensuring that microbial contamination remains acceptably low, and a good state of control is maintained in the manufacturing areas. The incubation conditions are important to support optimal microbial recoveries, considering that there is no single culture medium, temperature and incubation time that can recover all microorganisms. Particularly molds are quite sensitive microorganisms, and some species may have very specific nutritional and environmental needs. In this study, a two-phase approach was used to identify a single incubation-temperature approach that could recover most of the cleanroom microbial flora with focus on molds. Phase 1 included a growth promotion study performed in the laboratory using pharmacopeial and in-house strains, comparing different media (SDA and TSA) at single or dual incubation-temperature approaches for 5 or 6 days. Phase 2 was based on an in situ study where sampling was performed in different areas of a pharmaceutical facility, comparing the recoveries at different incubation conditions. In addition, extension studies of phase 1 and phase 2 were performed to get a better understanding of growth requirements for in-house molds. The results show that an incubation on Tryptone Soy Agar (TSA) at 25-30° for 3-4 days was able to recover most tested microorganisms in phase 1 and a large variety of microorganisms in phase 2, indicating that the single incubation-temperature is an optimal approach for the recovery of microorganisms in cleanrooms. Exceptions were noted for one strain of the species Cutibacterium acnes, a microaerophilic bacterium for which anaerobiosis and higher temperatures were needed, and two mold strains (Sistotrema brinkmannii and Stereum hirsutum), indicating that those molds required a specific media (Sabouraud Dextrose Agar, SDA) for their proliferation. The results showed that TSA incubated at single or dual incubation-temperature approach cannot compensate for the absence of SDA for some environmental molds that may be atypical in cleanrooms. Therefore, in addition to TSA, certain monitoring with SDA at e.g., cleanroom entrance points may be beneficial to recover molds with very specific nutritional requirements.