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All sourcesNASA NTRSDOE OSTI
NASA NTRS2007

Early Results and Spaceflight Implications of the SWAB Flight Experiment

Microbial monitoring of spacecraft environments provides key information in the assessment of infectious disease risk to the crew. Monitoring aboard the Mir space station and International Space Station (ISS) has provided a tremendous informational baseline to aid in determining the types and concentrations of microorganisms during a mission. Still, current microbial monitoring hardware utilizes culture-based methodology which may not detect many medically significant organisms, such as Legionella pneumophila. We hypothesize that evaluation of the ISS environment using non-culture-based technologies would reveal microorganisms not previously reported in spacecraft, allowing for a more complete health assessment. To achieve this goal, a spaceflight experiment, operationally designated as SWAB, was designed to evaluate the DNA from environmental samples collected from ISS and vehicles destined for ISS. Results from initial samples indicate that the sample collection and return procedures were successful. Analysis of these samples using denaturing gradient gel electrophoresis and targeted PCR primers for fungal contaminants is underway. The current results of SWAB and their implication for in-flight molecular analysis of environmental samples will be discussed.

Ott, C. Mark↗
NASA NTRS2015

Extravehicular Activity (EVA) Microbial Swab Tool

When we send humans to search for life on Mars, we'll need to know what we brought with us versus what may already be there. To ensure our crewed spacecraft meet planetary protection requirements--and to protect our science from human contamination--we'll need to know whether micro-organisms are leaking/venting from our ships and spacesuits. This is easily done by swabbing external vents and surfaces for analysis, but there was no US EVA tool for that job. NASA engineers developed an EVA-compatible swab tool that can be used to collect data on current hardware, which will influence eventual Mars life support and EVA hardware designs.

Rucker, Michelle↗
DOE OSTI2020

Success Using A Granular-Activated Carbon Filtration Swab to Rehabilitate Westbay{sup R} Multi-level Groundwater Monitoring Wells at the INL - 20544

In November 2015, tetrachloroethylene (PCE) was discovered in groundwater samples collected from a Westbay{sup R} multi-level groundwater monitoring well at the Idaho National Laboratory (INL).a Subsequent investigation determined that PCE was not present in the groundwater. The detected PCE was instead due to inadvertent contamination of the internal tubing fluid in the Westbay well-monitoring system, which is isolated from the surrounding groundwater. Further investigation showed that three of the 11 Westbay wells at the INL contained PCE-contaminated tubing fluid at concentrations above the U.S. Environmental Protection Agency's maximum contaminant level for drinking water (5 μg/L). Loose sediment at the bottom of some Westbay wells was also found to contain PCE; the three wells were then identified for rehabilitation as part of the INL well maintenance program. The unique construction of the Westbay wells - i.e., the small diameter (5.72 cm [2.25 in.]) and the depths of the tubing fluid ranging from 144.8 to 419 m (475 to 1,376 ft) below ground surface - posed unique challenges to the rehabilitation. Because the tubing fluid provides structural integrity, the fluid cannot be removed and then replaced without damaging the integrity of the Westbay well. Additionally, the small diameter of the systems and the associated depths limit the availability of equipment that can be used for the rehabilitation. After an evaluation of potential methods, it was determined that the use of an in-well, custom-fabricated, granular-activated-carbon filtration swab deployed via a wire line would provide the safest and most cost-effective method for rehabilitation. Westbay well rehabilitation started at well MIDDLE-2051 in late June 2018 and paused in December 2018 for the season. The PCE concentration in the tubing fluid at MIDDLE-2051 was initially reduced to a maximum concentration of 3.81 μg/L from a maximum of 642 μg/L, while the concentration of PCE in the sediment had been reduced from 5,360 to 2,270 μg/kg. Sampling was performed at MIDDLE-2051 again in August 2019, and the tubing fluid increased to a maximum concentration of 45.4 μg/L, while the concentration in the sediment decreased further to 1,890 μg/kg due to desorption of PCE from the sediment to the tubing fluid. Rehabilitation resumed at well MIDDLE-2050A in late August 2019, and the PCE concentration in the tubing fluid had been reduced to a maximum concentration of 19.0 μg/L. Although the method has been shown to be time-consuming, it has proven to be effective and safe for personnel performing the work and to the integrity of the well. Future sample data results at all wells will help determine an estimated completion of the rehabilitation. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2022

Tissue replication and mucosal swab detection of Sosuga virus in Syrian hamsters in the absence of overt tissue pathology and clinical disease

Human infection with Sosuga virus (SOSV), a recently discovered pathogenic paramyxovirus, has been reported in one individual to date. No animal models of disease are currently available for SOSV. Here, we describe initial characterization of experimental infection in Syrian hamsters, including kinetics of virus dissemination and replication, and the corresponding clinical parameters, immunological responses, and histopathology. Here, we demonstrate susceptibility of hamsters to infection in the absence of clinical signs or significant histopathologic findings in tissues.

60 APPLIED LIFE SCIENCES↗
NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗
NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗
NASA NTRS2008

Apparatus for Sampling Surface Contamination

An apparatus denoted a swab device has been developed as a convenient means of acquiring samples of contaminants from surfaces and suspending the samples in liquids. (Thereafter, the liquids can be dispensed, in controlled volumes, into scientific instruments for analysis of the contaminants.) The swab device is designed so as not to introduce additional contamination and to facilitate, simplify, and systematize the dispensing of controlled volumes of liquid into analytical instruments. The swab device is a single apparatus into which are combined all the equipment and materials needed for sampling surface contamination. The swab device contains disposable components stacked together on a nondisposable dispensing head. One of the disposable components is a supply cartridge holding a sufficient volume of liquid for one complete set of samples. (The liquid could be clean water or another suitable solvent, depending on the application.) This supply of liquid is sealed by Luer valves. At the beginning of a sampling process, the user tears open a sealed bag containing the supply cartridge. A tip on the nondisposable dispensing head is engaged with a Luer valve on one end of the supply cartridge and rotated, locking the supply cartridge on the dispensing head and opening the valve. The swab tip includes a fabric swab that is wiped across the surface of interest to acquire a sample. A sealed bag containing a disposable dispensing tip is then opened, and the swab tip is pushed into the dispensing tip until seated. The dispensing head contains a piston that passes through a spring-loaded lip seal. The air volume displaced by this piston forces the liquid out of the supply cartridge, over the swab, and into the dispensing tip. The piston is manually cycled to enforce oscillation of the air volume and thereby to cause water to flow to wash contaminants from the swab and cause the resulting liquid suspension of contaminants to flow into the dispensing tip. After several cycles to ensure adequate mixing, liquid containing the suspended contaminant sample is dispensed. The disposable components are then removed from the dispensing head, which may then be reused with a fresh set of disposable components.

Wells, Mark↗
NASA NTRS2024

ISS External Microorganisms: A Planetary Protection Experiment to Inform Requirements for Crewed Missions to Mars

We have developed, tested, and flown a caddy capable of collecting aseptic samples from external surfaces of the ISS (International Space Station). The sampling caddy is certified for use during US EVA (extra vehicular activity) and was launched to ISS in the summer of 2023. We are scheduled to collect samples from 6 locations outside ISS during an EVA in May of 2024. We will freeze these samples at -80°C on orbit and return them to Earth. We will then extract and sequence any DNA collected during the EVA using next generation sequencing technologies to characterize the community composition and function of each sample. Measuring the type and quantity of microbes present on the exterior of ISS will allow us to address knowledge gap 2B, “Acceptable levels of microbial/organic releases from humans and support systems” described in a 2019 COSPAR report. Collecting data about microbial release from current crewed vehicles will inform requirements for acceptable leak rates for future crewed missions to Mars. The sampling kit consists of eight commercially available, sterile, DNA free, macrofoam swabs ( 23 mm. diameter ) installed in custom aluminum end effectors. Each end effector is housed in and individual aluminum canister. Each canister contains a 0.2 μm Teflon filter to allow the interior volume to accommodate pressure changes without permitting microbial contaminants to enter the sterile interior volume. A handle repurposed from the space shuttle tile repair kit is used to remove the end effector from the sample canister, collect a sample by swabbing a surface and then replace the end effector in its canister. The canisters and end effectors were cleaned and assembled on Earth. Prior to installing the sterile swab the canisters and end effectors were sterilized in an autoclave at 134°C, 215 kPA, for 7 min.. The final assembly occurred in a sterilized class II biosafety cabinet. We will collect six samples from the 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the CDRA (Carbon Dioxide Removal Assembly) vent, and 6) the VES (Vacuum Exhaust System) vent. The remaining two swabs will be reserved as controls. One swab will be exposed during the EVA without touching any surfaces to act as a blank. The final swab will remain sealed until the entire sampling kit is returned to earth. Based on previously published results from the Russian segment, we hypothesize that there will be detectable microbes at some or all of these locations. Ground-based testing of this sampling caddy confirms that the swabs remain sterile as the canisters transition in and out of vacuum. We were able to retrieve, viable bacterial and fungal cells as well as DNA from samples collected from US space suits during vacuum chamber tests lasting as long as seven hours. Based on these results and feedback from the test subjects the sampling caddy was modified to improve ergonomics and meet US EVA safety requirements. Bayonet probes were added to the sides of the sample kit as alternate mounting points. Additional locking features were added to the end effector and the filter stack to prevent inadvertent release during use. The opening mechanism was changed from one where the end effector was rocked laterally to defeat a ball detent to a twist-to-open threaded closure for similar reasons. Demonstrating, this sampling caddy’s effectiveness during a US EVA will allow us to address knowledge gaps identified in COSPAR reports and begin to define planetary protection requirements for life support systems on crewed missions to mars. This kit could also be used to collect contamination control samples during Artemis missions to verify requirements and could be easily modified for robotic sample collection.

Planetary Protection↗
NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

We have developed, tested, and flown a caddy capable of collecting aseptic samples from external surfaces of the ISS (International Space Station). The sampling caddy is certified for use during US EVA (extra vehicular activity) and was launched to ISS in the summer of 2023. We are scheduled to collect samples from 6 locations outside ISS during an EVA in May of 2024. We will freeze these samples at -80°C on orbit and return them to Earth. We will then extract and sequence any DNA collected during the EVA using next generation sequencing technologies to characterize the community composition and function of each sample. Measuring the type and quantity of microbes present on the exterior of ISS will allow us to address knowledge gap 2B, “Acceptable levels of microbial/organic releases from humans and support systems” described in a 2019 COSPAR report. Collecting data about microbial release from current crewed vehicles will inform requirements for acceptable leak rates for future crewed missions to Mars. The sampling kit consists of eight commercially available, sterile, DNA free, macrofoam swabs ( 23 mm. diameter ) installed in custom aluminum end effectors. Each end effector is housed in and individual aluminum canister. Each canister contains a 0.2 μm Teflon filter to allow the interior volume to accommodate pressure changes without permitting microbial contaminants to enter the sterile interior volume. A handle repurposed from the space shuttle tile repair kit is used to remove the end effector from the sample canister, collect a sample by swabbing a surface and then replace the end effector in its canister. The canisters and end effectors were cleaned and assembled on Earth. Prior to installing the sterile swab the canisters and end effectors were sterilized in an autoclave at 134°C, 215 kPA, for 7 min.. The final assembly occurred in a sterilized class II biosafety cabinet. We will collect six samples from the 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the CDRA (Carbon Dioxide Removal Assembly) vent, and 6) the VES (Vacuum Exhaust System) vent. The remaining two swabs will be reserved as controls. One swab will be exposed during the EVA without touching any surfaces to act as a blank. The final swab will remain sealed until the entire sampling kit is returned to earth. Based on previously published results from the Russian segment, we hypothesize that there will be detectable microbes at some or all of these locations. Ground-based testing of this sampling caddy confirms that the swabs remain sterile as the canisters transition in and out of vacuum. We were able to retrieve, viable bacterial and fungal cells as well as DNA from samples collected from US space suits during vacuum chamber tests lasting as long as seven hours. Based on these results and feedback from the test subjects the sampling caddy was modified to improve ergonomics and meet US EVA safety requirements. Bayonet probes were added to the sides of the sample kit as alternate mounting points. Additional locking features were added to the end effector and the filter stack to prevent inadvertent release during use. The opening mechanism was changed from one where the end effector was rocked laterally to defeat a ball detent to a twist-to-open threaded closure for similar reasons. Demonstrating, this sampling caddy’s effectiveness during a US EVA will allow us to address knowledge gaps identified in COSPAR reports and begin to define planetary protection requirements for life support systems on crewed missions to mars. This kit could also be used to collect contamination control samples during Artemis missions to verify requirements and could be easily modified for robotic sample collection.

Planetary Protection↗
DOE OSTI2022

Recovery of DNA from fired and unfired cartridge casings: comparison of two DNA collection methods

For over 10 years, various studies have attempted to increase the recovery of DNA from ammunition by modifying the DNA collection, extraction, purification, and amplification procedures, with varying levels of success. This study focused on the “soaking” method of Montpetit & O’Donnell [1] and the “rinse-and-swab" method of Bille et al. [2]. First, testing for the presence of exogenous DNA, 210 boxed cartridges (brass, steel, and nickel-plated) from nine manufacturers were swabbed and DNA was extracted, concentrated, and quantified. Extracts that quantified > 0 ng/µL (44 of 210) were amplified and genotyped with GlobalFiler™. Of those, only one extract yielded two alleles indicating that the manufacturing and packaging of ammunition was virtually DNA free. Next, to obtain a baseline comparison of two DNA collection methods on a non-metallic substrate and identify a suitable number of cells to spot on cartridges, different DNA input amounts of primary human adult epidermal keratinocytes (HEKa) were tested. Thereafter, 300 brass and 300 nickel-plated, cartridges were spotted with HEKa cells containing ~5 ng of DNA, fired or unfired, and processed with either method. Finally, five methods representing hybrids of the soaking and rinse-and-swab methods were tested to determine if variations of those methods could be used to increase DNA yield and recovery. The results show that the soaking method consistently yielded more DNA than the rinse-and-swab method from a non-metallic substrate. However, the comparison study demonstrated that both methods performed comparably for cartridges. On average, the soaking method recovered 0.25 ng of DNA (5.1% recovery) and the rinse-and-swab method recovered 0.28 ng (5.8% recovery). However, average recoveries were significantly different among three analysts and considerable variation in yields were observed, possibly due to storage time. Furthermore, consistent with prior reports, the DNA recovered from brass casings was only 16% of that recovered from nickel-plated casings and the average yield of DNA from fired casings was reduced to 67% of unfired casings. Moreover, DNA extracts from brass or nickel-plated casings did not appear to contain amplification inhibitors and only 30/596 appeared severely degraded. Finally, both the published rinse-and-swab and soaking methods yielded more DNA than all modifications of the two methods. Overall, both methods yielded equivalent DNA quantities. Additionally, recovery of DNA from any given cartridge casing may be dependent on storage time as well as the skill, proficiency, and experience of the analyst and may reflect stochastic effects, particularly for casings containing low copy and/or degraded DNA.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2021

Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station using Nanopore Sequencing

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies’ MinION™ sequencer and miniPCR bio’s miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

microbiology↗
NASA NTRS2026

Does the International Space Station Leak DNA? Preliminary Results from the ISS External Microorganisms Payload

Existing crewed spacecraft like the ISS (International Space Station) leak by design. The ISS routinely releases gas to maintain life support systems and when astronauts exit the station to perform space walks. The chemical component of this leakage is well characterized, but the biological components are not. The ISS is not subject to planetary protection requirements, but planned missions to Mars will use similar systems and will be subject to planetary protection requirements. If detectable microorganisms are escaping through vents and or airlocks we may need to redesign our crewed habitats to minimize this type of contamination. To test the hypothesis that microorganisms from inside ISS are detectable on exterior surfaces an astronaut used the ISS External Microorganisms sampling kit (Rucker et al. 2018) to sample exterior surfaces of the ISS during an EVA (Extra Vehicular Activity) in January of 2025. These samples were returned to Earth for DNA extraction and sequencing. We successfully, extracted and sequenced bacterial, fungal and viral DNA from these samples that was not present in the negative controls. These results should help NASA refine the planetary protection requirements for crewed missions. Methods: The samples were collected using sterile, DNA free, buccal swabs (23 mm. diameter) housed in custom canisters. Each canister uses a 0.2 μm Teflon filter to maintain sterility as the caddy, holding 8 swabs moves in and out of vacuum. The astronaut sampled the: 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the Carbon Dioxide Removal Assembly vent, and 6) the Vacuum Exhaust System vent. The seventh swab was exposed to vacuum during the EVA without touching it to a surface. The eighth swab, a negative control, was not opened until the caddy returned to Earth. DNA was extracted from the swabs using a QIamp UCP Pathogen kit and prepared for sequencing on an Aviti (Element Biosciences) sequencer (Arslan et al. 2024). The resulting sequences were analyzed using the EDGE Bioinformatics platform (Li et al. 2017). The sequences were analyzed individually using tools like BLAST, GOTTCHA2, Kraken2, and PanGIA. The data were also assembled into metagenome assembled genomes) using tools like CONCOCT, MaxBin2 and MetaBAT2. Results: We successfully extracted and sequenced bacterial, archaeal, fungal and viral DNA from all seven samples. The handrail swab had the lowest number of reads (768,651) and the airlock thermal cover had the highest number of reads (8,819,230). These samples contain DNA from human associated bacteria (e.g. Crynebacterium riegelii ), fungi (.e.g. Penicillium rubens ), and viruses (e.g Alphapapillomavirus ). Conclusion: Preliminary interpretation suggest that the airlock and the space suits themselves are the largest sources of contaminant DNA. Most if not all of the DNA is from organisms known to be present inside the ISS. Vents attached to life support systems may be a lesser source of biological contamination. Further analysis should help NASA address planetary protection knowledge gaps for crewed missions.

Aaron B Regberg↗
DOE OSTI2025

Effects of sampling techniques on short-term survival and genotyping success of salmonid fry

ABSTRACT Objective Genetics tools have become an integral part of managing and understanding fish populations. Generally, a small tissue sample, such as a fin clip, is taken and then genotyped, with little effect on survival of the fish. However, tissue sampling may have a larger effect on juvenile fish survival compared to their adult counterparts. We evaluated survival and genotyping success of various genetic sampling techniques for Chinook Salmon Oncorhynchus tshawytscha and Rainbow Trout Oncorhynchus mykiss fry. Methods Three sampling treatments were evaluated including control (anesthetized and handled), fin clipping (partial caudal fin clip), and swabbing (OmniSwab was used to collect external mucus). Survival was monitored for 12 d posttreatment, and genotyping success was evaluated. Results Survival was high in all treatment groups (i.e., 0.93–1.00) but, on average, was lower in the swab treatment group. Genotyping was successful in 100% of the fin clip samples and 11–50% of the swab samples. Conclusions Results of this study suggest that sampling caudal-fin tissue does not negatively affect fry short-term survival and the small tissue samples yield highly successful genotyping results. Swabbing did not produce successful genotyping results, and fish sampled with swabs experienced higher mortality than those that received fin clips. Results indicate that fin clips should be used for collection of genetic samples from fry.

McCarrick, Darcy K.↗