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Preventing the Forward Contamination of Mars C Summary of Procedures Currently Used to Assess Bioburden in Spacecraft Assembly Clean Rooms and on Spacecraft NASA NPR 5340.1C is a compilation of procedures for the microbiological examination and assessment of space hardware and associated environments that were developed to meet the requirements of NASA planetary protection and space and life science programs.1 Although discussions are currently under way to develop new assay techniques based on molecular methods, the requirements for currently used procedures are still based on Viking-era culture techniques. Until newer molecular assays and updated procedures are adopted officially, NPR 5340.1C remains in force. This appendix summarizes the current uniform microbiological assay procedures required for use in assessing (1) the degree of microbiological contamination of intramural environments where spacecraft hardware is assembled, tested, and launched; and (2) the level of microbial contamination on spacecraft hardware in relation to the known or anticipated environments of the target planets. Deviation from these procedures is not permitted unless written approval from NASA’s planetary protection officer is granted. All operations summarized below that involve the manipulation of sterile items and sample processing must be performed in laminar flow environments meeting Class-100 air cleanliness requirements of Federal Standard 209B. These procedures are designed primarily for the detection and enumeration of heterotrophic, mesophilic, anaerobic, and anaerobic microorganisms. Procedures for the detection of other microorganisms (e.g., psychrophiles and thermophiles) are also included to meet the needs of specific missions. Since the days of Viking, spore-forming bacteria that remain after heat shocking have been the standard measure for enumerating microbial contamination for planetary protection purposes. Although microbial assays described below are based on culture methods, only those with a heat-shocking regime are used specifically for detection and enumeration of spores on spacecraft hardware as stipulated by planetary protection requirements. ASSAYS TO SAMPLE THE CLEAN-ROOM ENVIRONMENT The clean-room environment at the assembly, test, and launch facilities is assessed in a number of ways in order to test for various categories of contaminants ranging from airborne contaminants accumulating on surfaces, to contaminants on environmental surfaces, and intramural air. 1 The complete version of NASA NPR 5340.1C is available at <www.planetaryprotection.nasa.gov>.
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Preventing the Forward Contamination of Mars Airborne microbial contamination accumulating on surfaces is assessed by methods using fallout strips and ribbons placed throughout a site and collected at intervals. Sample strips are collected, immersed in test tubes, sonicated and heat shocked, and rapidly cooled before portions of the liquid from each tube are transferred onto petri plates for incubation at specified temperatures for specified periods under specified conditions. Plates are aerobically or anaerobically incubated and scanned at 24 and 48 hours to detect any growth. Final colony counts are made at 72 hours. In certain cases, plates are incubated for longer periods under varied conditions to encourage selection of psychrophilic or thermophilic organisms. If required, assay conditions may be modified and directed toward particular groups of microorganisms (e.g., aerobes, aerobic spores, or anaerobic spores). Upon completion of colony counts, results are expressed as number of microorganisms per strip or ribbon, and are reported by general categories of microorganisms (e.g., aerobes, aerobic spores, or anaerobic spores). Microbiological sampling of environmental surfaces is accomplished in several ways. For example, a plate method is used to sample flat, rigid surfaces (e.g., work tables and floors) and nonrigid surfaces (e.g., clothing or packaging materials). The plate method uses preprepared agar plates that are impressed onto the surfaces to be sampled and then incubated under appropriate conditions. Plates are scanned for visible growth at 24 and 48 hours, with final counts taken after 72 hours. If required, plates may be incubated for longer periods under specified conditions to encourage selection of particular groups of microbes. Various swab-rinse methods are also used to assay environmental surfaces, as are wipe-assay methods. As with the aforementioned methods, these involve the use of specific collection materials (cotton swabs or sterile cloths) to sample surfaces, followed by immersion of the sampling material in a solution and then sonication, subsampling, and incubation on petri plates under specified conditions and times. Plates are scanned for growth at appropriate intervals, and results are expressed as microorganisms per square centimeter of surface. Microbiological sampling of intramural air within a facility is conducted by using slit sample agar impaction devices or membrane filter field monitors. In both cases, specified volumes of air are collected on agar plates or membrane filters, and the plates and plated materials are then incubated for specified periods of time. Plates are scanned for growth at appropriate intervals, and results are reported as the number of airborne viable particles per liter of air. SAMPLING OF SPACECRAFT HARDWARE TO DETERMINE CLEANLINESS AND BIOBURDEN Sampling methods for spacecraft hardware use a variety of assays depending on the size, shape, and irregularity of the hardware being sampled. The same assay methods are used to assess bioburden during the assembly, test, and launch operations by the mission team and later by the designee of the Planetary Protection Office to certify and validate required bioburden and cleanliness levels. Surfaces of piece-parts (subcomponent-sized hardware pieces) are sampled by placing the entire part into a flask with sterile rinse solution, sonicating the flask to suspend contaminants in the solution, heat shocking, subsampling the liquid, and plating on petri dishes. Incubation under specified conditions is followed by making counts of microbial colonies at appropriate intervals of time. Results are expressed as the number of microorganisms per piece-part, based on the measured total surface area of the part. Suitable sterility control checks are also required on the entire assay procedure. In addition, as part of the process, an inhibitory test is performed on each type of piece-part after sterilization with dry heat. Spores of Bacillus subtilis var. niger are added to a flask with the part, followed by sonication, removal of the part, and incubation of the flask for 72 hours. A viability check on the spore inoculum is done after incubation. Components, boards, modules, subsystems, systems, and landing capsules are sampled by using swab-rinse or wipe-rinse methods, or both, depending on the area sampled. This is followed by sonication, heat shocking, plating, and culturing for 72 hours. In addition, hardware design provides for the inclusion of detachable strips on the surface of each flight item. A detachable strip assay method is also done as needed by using the method described earlier for assaying airborne contaminants accumulating on strips.
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