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Smart power supply calibration system

This paper details the development of an automated procedure to conduct calibrations of power supplies at Jet Propulsion Laboratory, California Institute of Technology (JPL). The fundamentals of power supply calibrations is given, and discussion on the method by which this custom software handles that calibration. Additionally, this technique provides real time uncertainty quantification of the calibrations. This automated system has demonstrated a time savings over existing automated techniques in use today.

Umansky, Brandon M.↗

Investigating the Effects of Exposure to Blue-Enriched Light or Peppermint Odor on Alertness, Mood, and Performance Upon Awakening from Deep Sleep at Night

Introduction: Sleep inertia refers the transient neurobehavioral impairments experienced immediately after waking from sleep. This period of reduced alertness and performance poses a significant safety risk to on-call workers who may be required to perform a safety-critical task immediately after waking (e.g., emergency services, health care, and military). In these operations, the need for a rapid return to full alertness is critical to mission safety and success. Several factors may exacerbate sleep inertia, resulting in greater impairment upon waking, including: waking from deep sleep, (i.e., slow wave sleep, SWS), waking at night, and waking following prior sleep loss. Awakenings under these conditions are common for on-call and extended shift workers who may need to perform safety-critical tasks soon after waking from unprotected sleep opportunities. Therefore, there is a need for evidence-based reactive countermeasures (i.e., used upon waking) to the cognitive consequences sleep inertia. Specifically, countermeasures that can rapidly restore alertness and performance immediately following sleep. A recent review of the literature on reactive countermeasures highlighted several research gaps and promising candidates for further investigation. The review also emphasized the need for countermeasures that are operationally viable and readily deployed in occupational settings. This study aims to address the identified gaps and limitations by assessing the efficacy of exposure to two known acute alerting stimuli - blue-enriched light and peppermint odor - to improve cognitive performance, alertness, and mood immediately after waking from SWS at night. Materials and Methods: Twelve participants completed a two-week within-subject, randomized, cross-over intervention study including two in-laboratory overnight visits. During each experimental week, the subjects experienced one intervention (light or peppermint) and a control condition upon awakening from SWS at night. The presentation order of the two conditions (intervention or control) at wake-up and the order of intervention (light or peppermint) by week was randomized by sex. Prior to each in-laboratory visit, participants maintained a sleep schedule of 8.5 h for 5 nights and 5 h for one night. Compliance with this sleep schedule was confirmed by actigraphy. In the laboratory, participants went to bed at their habitual bedtime and were monitored by standard polysomnography. After at least five minutes of continuous SWS, participants were awoken and exposed, in a randomized order, to either the control or intervention condition. During the hour after awakening from SWS (at 2, 17, 32, and 47 minutes after waking), participants completed a battery of tasks including a 5-minute psychomotor vigilance task (PVT), a subjective scale of alertness (Karolinska Sleepiness Scale, KSS), and visual analogue scales (VAS) of mood. Following this sleep inertia measurement period, all lights were turned off and participants were allowed to return to sleep. They were then awoken again from their subsequent SWS period and exposed to the alternative condition (control or intervention). Following this second awakening, participants were allowed to sleep until their habitual wake time and were then released from the laboratory. Participants then followed the at-home sleep schedule and returned to the laboratory for the second intervention (light or peppermint) following the procedures described above. The light intervention involved exposure to a blue-enriched light canvas illuminated for 1 hour at a distance of ~56 cm from the participant (~200 lux and ~60 melanopic lux at angle of gaze). For the peppermint intervention, peppermint oil was pipetted onto a mask, and participants inhaled the odor with the mask covering the nose and mouth for 1 minute. The control condition for both weeks involved a dim, red ambient light (<1 lux). An odorless mask, without any oil pipetted onto the mask, was also worn in the peppermint control condition. Results: Compared to the control condition, participants exposed to blue-enriched light had fewer PVT lapses (χ2 = 5.285, p = .022), reported feeling more alert (KSS: F1,77 = 4.955, p = .029; VASalert: F1,77 = 8.226, p = .005), and had improved mood (VAScheerful: F1,77 = 8.615, p = .004; VASdepressed: F1,77 = 4.649, p = .034; VASlethargic: F1,77 = 5.652, p = .020). Exposure to peppermint oil did not improve any outcome measures on any of the tasks compared to control condition (p > .05). Conclusions: We found that participants had fewer lapses of attention upon awakening when exposed to blue-enriched light compared to dim, red light. In addition, participants reported feeling more alert, more cheerful, less depressed, and less lethargic in the blue-enriched light condition. Brief exposure to a peppermint odor, however, did not appear to improve performance, alertness, or mood under the experimental conditions. Our null results in the peppermint condition may have been due to methodological limitations such as the duration and method of administration. Given the need to mitigate the potential impact of sleep inertia on safety-critical tasks in on-call operations, our findings suggest that blue-enriched light exposure upon awakening may help to improve performance and alertness during the sleep inertia period following awakening from deep, nocturnal sleep. We are currently exploring the potential mechanisms for the effect of light on cognitive performance upon awakening as well as investigating its application in real-world settings to explore the translational efficacy of this countermeasure to occupational environments. Continued exploration into light and other reactive countermeasures, and potentially their combination, is needed in order to provide evidence-based guidance on effective sleep inertia countermeasures to improve the alertness and performance of those required to perform safety-critical tasks soon after waking.

sleep inertia↗

URANIUM HOLDUP MONITORING WITH COMPTON IMAGING AS FUNCTION OF DEPTH AND MASS

Mass quantification using standoff gamma detection is a logical progression for MC&A applications in uranium processing facilities. While obtaining a ground truth from physical collection and weighing of masses is ideal and routine, it poses a logistical problem from both a standpoint of ALARA programs as well as interruptions to operations. The standard approach is the Generalized Geometry in Holdup (GGH) method that can apply for many cases where geometries of the holdup match simulated models; however, there are many cases where a model may not exist or a feature of the holdup geometry is such that it does not match a model particularly well.1 Gamma imaging at standoff distances and outside process equipment has potential to address these shortcomings and improve mass estimations while reducing personnel exposure; however, algorithms still require a significant degree of benchmarking to build sufficient confidence in algorithms before displacing GGH in some procedures. This paper presents the creation of a set of laboratory standards of depleted U3O8 in fixed size containers covering a range of masses and effective thicknesses up to the infinite thickness for the 185-keV line. These blocks are identically sized aluminum boxes loaded such that a thin, flat face is placed towards the imager, while the back side contains closed cell foam to apply pressure to keep the powder in a fixed position at the front face as the sources are manipulated for measurements. Included is an analysis of the mass estimation using a commercially available Compton imaging instrument where multiple geometries are created showing limitations in current methodologies.

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Outdoor and laboratory testing of photovoltaic modules

An overview of outdoor and laboratory testing being applied to terrestrial photovoltaic modules is presented. Descriptions of the test procedures, examples of results, and discussion of the advantages and shortcomings of each approach are included. The test program consists of real-time outdoor testing of systems, modules and materials, and accelerated outdoor testing and laboratory testing of modules. The test results suggest that the various types of tests complement each other and contribute toward the objective of verifying the environmental suitability of the product. A description of representative photovoltaic systems operational in the U.S. in 1981 is presented, along with an identification, description, and physical inspection summary of JPL test module sites and a summary of degradation and failure data for the modules.

Hoffman, A.↗

January 2023 Semiannual Saltstone Toxicity Characteristic Leaching Procedure (TCLP) Results

The aqueous waste from Tank 50 (salt solution) is sampled semiannually for transfers to the Saltstone Production Facility (SPF). Salt solution is treated at SPF and disposed of in the Saltstone Disposal Facility (SDF). Per request of customer, X-TTR-Z-00027, Revision 0, two SDF waste form (saltstone) samples were prepared in the Savannah River National Laboratory (SRNL) from the Tank 50 Waste Acceptance Criteria (WAC) sample and Z-area premix material for January 2023 semiannual Toxicity Characteristic Leaching Procedure (TCLP) samples. One sample contained a Full Premix which included 10:45:45 (by weight) of cement, slag, and fly ash. The second sample contained 60:40 (by weight) of slag and fly ash only referred to as the “Cement-Free grout sample.” Results from the technical report support Task 2: ‘Grout Leaching Analyses’ of the Task Technical Request (TTR) prepared by Savannah River Mission Completion (SRMC). After at least 28 days cured, a sample of each of the SDF waste forms was collected and shipped to a certified laboratory for analysis using the Toxicity Characteristic Leaching Procedure (TCLP). The January 2023 semiannual saltstone (Full Premix) and the Cement-Free grout samples met the South Carolina (SC) Code of Regulations for Hazardous Waste Management Regulations (HWMR) 61-79.261.24 and 61-79.268.48 requirements for a non-hazardous waste form with respect to the Resource Conservation and Recovery Act (RCRA) metals and Underlying Hazardous Constituents (UHCs), and also met the SPF WAC that was in effect at the time of the tank sampling.

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Application of LANDSAT to the surveillance of lake eutrophication in the Great Lakes basin

The author has identified the following significant results. A step-by-step procedure for establishing and monitoring the trophic status of inland lakes with the use of LANDSAT data, surface sampling, laboratory analysis, and aerial observations were demonstrated. The biomass was related to chlorophyll-a concentrations, water clarity, and trophic state. A procedure was developed for using surface sampling, LANDSAT data, and linear regression equations to produce a color-coded image of large lakes showing the distribution and concentrations of water quality parameters, causing eutrophication as well as parameters which indicate its effects. Cover categories readily derived from LANDSAT were those for which loading rates were available and were known to have major effects on the quality and quantity of runoff and lake eutrophication. Urban, barren land, cropland, grassland, forest, wetlands, and water were included.

Rogers, R. H.↗

January 2022 Semiannual Saltstone Toxicity Characteristic Leaching Procedure (TCLP) Results

The aqueous waste from Tank 50 (salt solution) is sampled semiannually for transfers to the Saltstone Production Facility (SPF). Salt solution is treated at SPF and disposed of in the Saltstone Disposal Facility (SDF). Per request of customer, X-TTR-Z-00025, Revision 0, two SDF waste form (saltstone) samples were prepared in the Savannah River National Laboratory (SRNL) from the Tank 50 Waste Acceptance Criteria (WAC) sample and Z-Area premix material for the January 2022 semiannual Toxicity Characteristic Leaching Procedure (TCLP) samples. One sample contained a Full Premix which included 10:45:45 (by weight) of cement, slag and fly ash. The second sample contained 60:40 (by weight) of slag and fly ash only referred to as the “Cement-Free grout sample.” Results from this technical report support Task 2: ‘Grout Leaching Analyses’ of the Task Technical Request (TTR) prepared by Savannah River Mission Completion (SRMC). After at least 28 days cured, a sample of each of the SDF waste forms was collected and shipped to a certified laboratory for analysis using the Toxicity Characteristic Leaching Procedure (TCLP). The January 2022 semiannual saltstone (Full Premix) and the Cement-Free grout samples met the South Carolina (SC) Code of Regulations for Hazardous Waste Management Regulations (HWMR) 61-79.261.24 and 61-79-268.48 requirements for a non-hazardous waste form with respect to Resource Conservation and Recovery Act (RCRA) metals and Underlying Hazardous Constituents (UHCs), and also met the SPF WAC that was in effect at the time of the tank sampling.

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Validation Testing for Molten Chloride Reactor Experiment Equipment Removal and Disposal Techniques

The Molten Chloride Reactor Experiment (MCRE) will be the first reactor featuring a fast-spectrum molten chloride circulating nuclear fuel system in the world. Planning for equipment removal and disposal (ERD) of MCRE has identified several technology gaps due to the unique environment of this nuclear experiment. Some of the gaps arise from the application of existing disassembly and/or sizing methods to novel material forms or in novel configurations. Others arise from unknown material behavior. This paper summarizes proposed test plans for ERD validation experiments to address these complicated or unknown equipment removal procedures. At the Waste Management Symposia in 2024, the Idaho National Laboratory (INL) MCRE ERD team presented the challenges associated with hosting multiple nuclear experiments in series with only brief transition periods between systems. Such difficulties include higher dose rates, the presence of radioisotopes infrequently encountered in reactor decommissioning and radioactive waste management, lack of intrinsic remote-operations infrastructure in the test bed, space constraints in the test bed, and contamination minimization requirements. To address these challenges, remote or semi-remote technologies are planned to be implemented in a non-hot cell environment with limited space availability. The team also discussed how a systems engineering approach is being used for conceptual development and design of equipment removal systems to address these challenges. For example, to reduce constraints for the removal of more difficult components, non-activated, noncontaminated elements are planned to be taken out first where possible. Still, there are complexities associated with the remaining components. In this work, the operational framework for MCRE ERD was reviewed for technical gaps and open questions, and test plans were drafted to address these areas. The tests plans were written for the following categories: vision systems, pipe cutting, drill/grout/filler, flush salt, and miscellaneous, with the miscellaneous group consisting of tests like techniques for removing bearings and reflector bricks. The test plans explore material, infrastructure, and staffing requirements needed for test execution. The test plans additionally focus on the evaluation of success. Determining the outcome of a test is imperative - as these explorative actions have the potential to rearrange or re-scope planned ERD activities. Success criteria identified thus far include required tool output, required area(s), debris production and mitigation, and repeatability. Test plans are an essential aspect of the systems engineering approach to MCRE ERD. They are used as the beginning steps in defining use cases for the ERD system. Performance of the validation tests is expected to begin in the summer of 2025 and will take approximately 9 to 12 months to complete. Execution of these plans will be expedited by specifying test needs ahead of time, facilitating efficient interactions with any subcontractors tasked with running the requested tests. Evaluating the outcomes of these tests will inform MCRE ERD procedures and timing and will also identify additional technical constraints for the MCRE ERD System. This upfront process optimization effort will help the project save time and resources at the end of the experiment.

21 - SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLAN↗

Radial coordinate maps, radial vectors, and binormal vectors for 5/6, 5/5 and 5/4 edge island domains in W7-X

Abstract The edge island domain in Wendelstein 7-X consists of divertor islands whose position coincide with the location of rational values of the rotational transform - ι ≈ ( 5 / 6 , 5 / 5 , 5 / 4 ) and surround the main confinement volume (the ‘main plasma’), providing the magnetic topology for an island divertor. The 5/5 edge consists of five individual islands that are unconnected. In contrast, a single island connects onto itself after 6 or 4 toroidal transits in the 5/6 or 5/4 edge, respectively. Many interesting phenomena are related to these islands and diagnostic analyses require a mapping from ‘laboratory’ or real space coordinates to the island coordinate system (ICS). A procedure is described here to calculate several scalar and vector quantities for closed island structures which can be utilized in fast interpolation schemes for inverse maps. For the 5/5 edge, a fixed-boundary vacuum (zero beta) magneto-hydrodynamic solution of the 5/5 island is found with the Variational Moments Equilibrium Code, VMEC. The solution is compatible with already existing routines which determine the radial and binormal vectors of VMEC solutions at arbitrary laboratory coordinates. VMEC does not support solutions for the 5/4 and 5/6 islands, but the radial and binormal vectors are available in a local 2D Fourier ICS.

Physics↗

Planning for Space Station Freedom laboratory payload integration

Space Station Freedom is being developed to support extensive missions involving microgravity research and applications. Requirements for on-orbit payload integration and the simultaneous payload integration of multiple mission increments will provide the stimulus to develop new streamlined integration procedures in order to take advantage of the increased capabilities offered by Freedom. The United States Laboratory and its user accommodations are described. The process of integrating users' experiments and equipment into the United States Laboratory and the Pressurized Logistics Modules is described. This process includes the strategic and tactical phases of Space Station utilization planning. The support that the Work Package 01 Utilization office will provide to the users and hardware developers, in the form of Experiment Integration Engineers, early accommodation assessments, and physical integration of experiment equipment, is described. Plans for integrated payload analytical integration are also described.

Willenberg, Harvey J.↗

Army Reactor Program Regulatory Bases: Supporting Army Regulation 50–7

This report describes the regulatory bases for the 2025 revision of Army Regulation (AR) 50–7 - Nuclear and Chemical Weapons and Materiel: Army Reactor Program and the new associated Department of the Army pamphlet (DA PAM) Army Reactor Program Procedures.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

A study of TRIGLYCINE SULFATE (TGS) crystals from the International Microgravity Laboratory Mission (IML-1)

Preliminary evaluation of the data was made during the hologram processing procedure. A few representative holograms were selected and reconstructed in the HGS; photographs of sample particle images were made to illustrate the resolution of all three particle sizes. Based on these evaluations slight modifications were requested in the hologram processing procedure to optimize the hologram exposure in the vicinity of the crystal. Preliminary looks at the data showed that we are able to see and track all three sizes of particles throughout the chamber. Because of the vast amount of data available in the holograms, it was recommended that we produce a detailed data reduction plan with prioritization on the different types of data which can be extracted from the holograms.

Lal, R. B.↗

Curating NASA's Past, Present, and Future Extraterrestrial Sample Collections

As codified in NASA Policy Directive 7100.10F, the Astromaterials Acquisition and Curation Office at NASA Johnson Space Center (hereafter JSC Curation) is charged with curation of all extraterrestrial material under NASA control, including future NASA missions. JSC Curation curates all or part of nine astromaterial collections in seven clean room suites: (1) Apollo Samples (1969; ISO 6-7), (2) Luna Samples (from USSR; 1972; ISO 7), (3) Antarctic Meteorites (1976; ISO 7), (4) Cosmic Dust (1981; ISO 5), (5) Microparticle Impact Collection (formerly called Space Exposed Hardware; 1985; ISO 5), (6) Genesis Solar Wind Atoms (2004; ISO 4); (7) Stardust Comet Particles (2006; ISO 5), (8) Stardust Interstellar Particles (2006; ISO 5), (9) Hayabusa Asteroid Particles (from JAXA; 2010; ISO 5). In addition to the labs that house the samples, we have installed and maintained a wide variety of facilities and infrastructure required to support the clean-rooms: more than 10 different HEPA-filtered air-handling systems, ultrapure dry gaseous nitrogen systems, an ultrapure water system (UPW) and cleaning facilities to provide clean tools and equipment for the labs. We also have sample preparation facilities for making thin sections, microtome sections, and even focused ion-beam (FIB) sections to meet the research requirements of scientists. To ensure that we are keeping the samples as pristine as possible, we routinely monitor the cleanliness of our clean rooms and infrastructure systems. This monitoring includes: daily monitoring of the quality of our UPW, weekly airborne particle counts in the labs, monthly monitoring of the stable isotope composition of the gaseous N2 system, and annual measurements of inorganic or organic contamination in processing cabinets. We track within our databases the current and ever-changing characteristics of more than 250,000 individual samples across our various collections (including the 19,141 samples on loan to 433 Principal Investigators in 24 countries). The next sample return missions that NASA will participate in are Hayabusa2 and OSIRIS-REx (Origins Spectral Interpretation Resource Identification Security - Regolith Explorer). The designs for a new state-of-the-art suite of clean rooms to house these samples at JSC have been finalized. This includes separate ISO class 5 clean rooms to house each collection, a common ISO class 7 area for general use, an ISO class 7 microtome laboratory, and a separate thin section lab. Additionally, a new cleaning facility is being designed and procedures developed that will allow for enhanced cleaning of cabinets and tools in an inorganically, organically, and biologically clean manner. We are also designing a large multi-purpose Advanced Curation laboratory that will allow us to develop the techniques necessary to fully support the Hayabusa2 and OSIRIS-REx missions, as well as future possible sample return missions (e.g., Lunar Polar Volatiles, Mars, Comet Surface). A micro-CT (micro Computed Tomography) laboratory dedicated to the study of astromaterials has come online within JSC Curation, and we plan to add additional facilities that will enable non-destructive (or minimally-destructive) analyses of astromaterials in the near future (e.g., micro-XRF (micro X-Ray Fluorescence), confocal imaging Raman Spectroscopy). These facilities will be available to: (1) develop sample handling and storage techniques for future sample return missions, (2) be utilized by PET (Positron Emission Tomography) for future sample return missions, (3) for retroactive PET-style analyses of our existing collections, and (4) for periodic assessments of the existing sample collections.

Zeigler, Ryan A.↗

October 2024 Semiannual Composite Salt Waste Processing Facility (SWPF) Decontaminated Salt Solution (DSS) Toxicity Characteristic Leaching Procedure (TCLP) Results

The aqueous waste from the Salt Waste Processing Facility (SWPF) is sampled semiannually for transfers to the Saltstone Production Facility (SPF). Salt solution is treated at SPF and disposed of in the Saltstone Disposal Facility (SDF). Per request of customer, X-TTR-Z-00027, Revision 0, one SDF waste form (saltstone) sample was prepared in the Savannah River National Laboratory (SRNL) from the SWPF Decontaminated Salt Solution (DSS) Waste Acceptance Criteria (WAC) sample and Z-area premix material for the October 2024 semiannual Toxicity Characteristic Leaching Procedure (TCLP) sample. The sample contained 60:40 (by weight) of slag and fly ash (referred to as the “Cement-Free grout sample”). Results from the technical report support Task 2: ‘Grout Leaching Analyses’ of the Task Technical Request (TTR) prepared by Savannah River Mission Completion (SRMC). At 64 days cured, a sample of the SDF waste form was collected and shipped to a certified laboratory for analysis using the Toxicity Characteristic Leaching Procedure (TCLP). The October 2024 semiannual Cement-Free grout sample met the South Carolina (SC) Code of Regulations for Hazardous Waste Management Regulations (HWMR) 61-79.261.24 and 61-79.268.48 requirements for a non-hazardous waste form with respect to the Resource Conservation and Recovery Act (RCRA) metals and Underlying Hazardous Constituents (UHCs), and met the SPF WAC that was in effect at the time of the sample collection at SWPF.

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April 2024 Semiannual Composite Salt Waste Processing Facility (SWPF) Decontaminated Salt Solution (DSS) Toxicity Characteristic Leaching Procedure (TCLP) Results

The aqueous waste from the Salt Waste Processing Facility (SWPF) is sampled semiannually for transfers to the Saltstone Production Facility (SPF). Salt solution is treated at SPF and disposed of in the Saltstone Disposal Facility (SDF). Per request of customer, X-TTR-Z-00027, Revision 0, one SDF waste form (saltstone) sample was prepared in the Savannah River National Laboratory (SRNL) from the SWPF Decontaminated Salt Solution (DSS) Waste Acceptance Criteria (WAC) sample and Z-area premix material for the April 2024 semiannual Toxicity Characteristic Leaching Procedure (TCLP) sample. The sample contained 60:40 (by weight) of slag and fly ash (referred to as the “Cement-Free grout sample”). Results from the technical report support Task 2: ‘Grout Leaching Analyses’ of the Task Technical Request (TTR) prepared by Savannah River Mission Completion (SRMC). After at least 28 days cured, a sample of the SDF waste form was collected and shipped to a certified laboratory for analysis using the Toxicity Characteristic Leaching Procedure (TCLP). The April 2024 semiannual Cement-Free grout sample met the South Carolina (SC) Code of Regulations for Hazardous Waste Management Regulations (HWMR) 61-79.261.24 and 61-79.268.48 requirements for a non-hazardous waste form with respect to the Resource Conservation and Recovery Act (RCRA) metals and Underlying Hazardous Constituents (UHCs), and met the SPF WAC that was in effect at the time of the tank sampling.

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Techniques for Solution- Assisted Optical Contacting

A document discusses a solution-assisted contacting technique for optical contacting. An optic of surface flatness Lambda/20 was successfully contacted with one of moderate surface quality, or Lambda/4. Optics used were both ultra-low expansion (ULE) glass (Lambda/4 and Lambda/20) and fused silica (Lambda/20). A stainless steel template of the intended interferometer layout was designed and constructed with three contact points per optic. The contact points were all on a common side of the template. The entire contacting jig was tilted at about 30 . Thus, when the isopropanol was applied, each optic slid due to gravity, resting on the contact points. All of the contacting was performed in a relatively dusty laboratory. A number of successful contacts were achieved where up to two or three visible pieces of dust could be seen. These were clearly visible due to refraction patterns between the optic and bench. On a number of optics, the final step of dropping isopropyl between the surfaces was repeated until a successful contact was achieved. The new procedures realized in this work represent a simplification for optical contacting in the laboratory. They will both save time and money spent during the contacting process, and research and development phases. The techniques outlined are suitable for laboratory experiments, research, and initial development stages.

DeVine, Glenn↗

Enumeration and Fluorescence In Situ Hybridization of Microbial Bioburden on Cleanroom Surfaces

Introduction: Microorganisms are everywhere on Earth, even in the cleanest of places. Spacecraft assembly cleanrooms can harbor low levels of living and dead microbial cells (e.g., [1,2]), and cleanroom bioburden can also include organic molecules from industrial sources and in situ biomass. Life detection missions require careful attention to avoid contaminants that can be easily convoluted with analytical targets. We are evaluating epifluorescent microscopy and fluorescence in situ hybridization (FISH) as methods to complement organic contamination detection techniques. Epifluorescent cell counting offers an accurate and cost-effective way to quantify low levels of surface biomass. FISH could allow for the identification of residual organisms, and can be targeted to detect active populations of specific organisms such as bacteria known to resist cleaning procedures. This effort is part of a larger study that is concentrated on characterizing the surface and airborne molecular organic contamination background in Johnson Space Center (JSC) Astromaterials curation laboratories and Goddard Space Flight Center (GSFC) spacecraft assembly rooms, and understanding contaminants in the context of cleaning procedures and residual bioburden. Methods: Samples were collected by swabbing surfaces in ISO 5 and ISO 7 equivalent cleanrooms at JSC. Swabs for FISH were fixed in 4% paraformaldehyde (PFA) for 3 hours and then stored in 1:1 ethanol:PBS, while swabs for cell counting were stored in 4% PFA until analysis to avoid any cell loss during centrifugation that could impact quantification of very low biomass samples. Cell counting was performed with SYBR Gold as in [3], but adapted for very low biomass. FISH was performed as in [4], using DAPI as a counterstain for all DNA-containing cells. Negative controls included wells with no probe applied, to test for natural fluorescence, as well as the nonsense probe NONEUB (reverse complement of EUB338) to evaluate non-specific probe binding. Results and Discussion: Cleanroom surfaces had 102-103 cells cm-2. The extremely low biomass of these samples was challenging for enumeration, and required careful and routine use of “field” and laboratory blanks. FISH was performed with the general archaeal and bacterial probes ARCH915 and EUB338 (EUBMIX, [4]), probe GAMBET ([4]), and PSE227, which targets the genus Pseudomonas [5]). The latter two probes were selected because Pseudomonas spp. and other Gammaproteobacteria have not been isolated from cleanroom surfaces but do appear frequently in rRNA gene libraries from these surfaces. While some active bacteria were identified (Fig. 1c), most cells detectable by DAPI did not have a strong or any fluorescent signal (e.g., Fig. 1d), indicating that the vast majority of cells are dead or inactive. This suggests that cleaning protocols are effective at inactivating microbial contaminants, but that dead or inactive cells can remain on surfaces. Cells were often clumped in a weakly autofluorescent matrix, possibly biofilm material (Fig. 1c,d). We also observed other particulate material that was collected by the swabs, including apparent textile fibers (Fig. 1b). Our results are consistent with other studies that show that the bioburden present in clean rooms includes active, dormant, and dead cells. We will discuss how FISH and epifluorescent cell counting could be applied in planetary protection protocols, including the advantages and disadvantages of FISH and cell counting for routine use, as well as different possible applications for more specialized FISH procedures. References: [1] Moissl-Eichinger et al. (2015) Sci Rep, 5, 9156 [2] Hendrickson et al. (2021) Microbiome, 9, 238 [3] Jones et al. (2017) Appl Environ Microbiol, 83, e00909-17 [4] Jones et al. (2015) Appl Environ Microbiol, 81, 1242-1250. [5] Watt et al. (2006) Environ Microbiol, 8, 871-884

C J Huff↗