NASA cleanrooms are certified by particle counts and are humidity-controlled, temperature-regulated, and oligotrophic in nature for assembling spacecraft subsystems. Microorganisms, which are not part of the cleanroom certification metrics, should not be overlooked when assessing the cleanliness of the facility since they can enter through soil or air, shed from humans, adapt to the oligotrophic conditions, and subsequently could contaminate spacecraft. These biogenic particles need to be identified to extend our knowledge of biological contamination for future NASA mission use. This study collected particles from the cleanroom and estimated the distribution of fallout microbial cell and inert dust particles using microscopy and molecular techniques. Aluminum coupon-based polycarbonate filter assemblies were deployed in the spacecraft assembly cleanroom facility to collect fallout particles. Epifluorescence and electron microscopy showed that particles varied in size and structure, and displayed live/dead biological and inert particle signatures from sources that include spores and fungal hyphae. Additionally, correlative epifluorescence and field emission scanning electron microscopy, combined with energy-dispersive X-ray analysis (for elemental compositions) methods, differentiated whether microbes adhering to particles were live/dead cells or inert particles. This visualization approach allowed for the classification of microorganisms as being standalone (free-living) or associated with a particle, as well as its characteristic size. Furthermore, time-course microscopy was used to determine the microbial cell growth and confirm the biological/molecular identification. Routine investigation of cleanroom biological and inert fallout particles will help to determine the biological load of spacecraft components and will also have direct relevance to the pharmaceutical and medical industries. One of the main objectives for NASA’s current and future missions is to prevent forward and back contamination of exploring planets. The goal of this study is to determine the association of microorganisms with the inert, natural cleanroom fallout particles and to ascertain whether microorganisms prefer to adhere to a particle size. A novel microscopy technique was developed, and by utilizing various molecular techniques, particles and associated microbial phylogeny were characterized. An accurate assessment of the microbes associated with cleanroom particles is necessary to protect the health of the people who occupy the room for long duration for aeronautical, medical, and pharmaceutical industries.
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