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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↗
NASA NTRS1974

Apparatus for microbiological sampling

An automatic apparatus is described for microbiologically sampling surface using a cotton swab which eliminates human error. The apparatus includes a self-powered transport device, such as a motor-driven wheeled cart, which mounts a swabbing motor drive for a crank arm which supports a swab in the free end thereof. The swabbing motor is pivotably mounted and an actuator rod movable responsive to the cart traveling a predetermined distance provides lifting of the swab from the surface being sampled and reversal of the direction of travel of the cart.

Wilkins, J. R.↗
NASA NTRS1972

Microbiological surface sampling cart

Mobile sampling cart automatically swabs surfaces for the recovery of microorganisms. Unit operates without human involvement and provides for control of swabbing speed, rotation of cotton swab, and the pressure and angle applied to swab. Capability of reverse direction is also available. Sampling cart use is limited to flat surfaces.

Wilkins, J. R.↗
NASA NTRS2007

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including (1) the host#s susceptibility, (2) the host#s exposure to the infectious disease agent, and (3) the concentration of the infectious agent, and (4) the characteristics of the infectious agent. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire#s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The first accomplishment of the SWAB experiment was the incorporation of 16S ribosomal DNA sequencing for the identification of bacteria. The use of this molecular technique has increased bacterial speciation of environmental isolates from previous flights three fold compared to conventional methodology. This increased efficiency in bacterial speciation provides a better understanding of the microbial ecology and the potential risk to the crew. Additional SWAB studies focused on the use of molecular-based DNA fingerprinting using repetitive sequencebased polymerase chain reaction (rep-PCR). This technology has allowed contamination tracking of microorganisms between crewmembers and their environment. This study not only demonstrated that ISS has a greater diversity of organisms than originally expected, but also provided insight into possible routes of infection to the crew. Additional ground-based studies used rep-PCR and protein based assays to determine the potential of methicillin resistant Staphylococcus aureus (MRSA) aboard ISS. MRSA has become increasingly common on Earth and pose a treatment problem for infections during flight. While no MRSA have been isolated from ISS to date, the mecA gene product that is responsible for methicillin resistance was isolated in other Staphylococcus species aboard ISS suggesting a potential of MRSA through gene transfer. Using improved sample collection technologies, flight sampling for SWAB was initiated in August 2006 and should continue through spring of 2007. The focus of these flight samples is the collection of DNA for evaluation by Denaturing Gradient Gel Electrophoresis (DGGE). Unlike other techniques, DGGE does not depend on any microbial growth on culture media allowing a more comprehensive assessment of the spacecraft interior. This study should provide insight into the true microbial ecology that is experienced by the crew during flight. This information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Ott, C. M.↗
NASA NTRSDate not supplied

Microbial Monitoring of New Cleanrooms Used to Curate Astrobiologically Relevant Asteroid Samples from Bennu and Ryugu

Introduction: NASA has constructed two new cleanrooms to house materials from the OSRIS-REx and Hayabusa2 missions to the asteroids Ryugu (162173) and Bennu (101955), respectively. In accordance with standard astromaterials curation practices, these cleanrooms will be monitored for particulate contamination and maintained to ISO 5 equivalent standards1. Since the samples in these collections are expected to contain prebiotic organic compounds that may help explain the origin of life on Earth, these labs will also be monitored for organic and biological contamination2. Samples from Ryugu arrived on Earth in December, 2020. After basic characterization in Japan, NASA received a subset of these samples at the astromaterials curation facility in Houston in December of 2021. OSIRIS-REx is expected to return samples in September, 2023. Here we present preliminary microbial monitoring results from monthly monitoring of these new labs and the connected microtomy and staging areas that support them, as they are being commissioned. We also compare these results to baseline values for other astromaterials curation labs. We will also briefly describe additional cleaning efforts employed to reduce the bioburden in these new cleanrooms. Methods: Microbial samples were collected from surfaces using a dry macrofoam swab (Puritan Brand 2518051PFRNDFD). Swabs were also opened in the lab but not touched to any surfaces to function as negative controls. Samples and controls were processed inside a class II biosafety cabinet to avoid inadvertent cross contamination. The swabs were suspended in 15 ml of PBS (Phosphate Buffered Saline) and vortexed for 20 seconds to remove cells from the swab surface. The PBS was used to inoculate Petri dishes filled with TSA (Tryptic Soy Agar), Blood Agar, or Reasoners 2 agar to check for microbial growth. Each plate was inoculated with 0.1 ml of PBS. The TSA and blood agar plates were incubated at 35˚C and the Reasoners 2 agar plates were incubated at 25˚C for seven days. Petri dishes filled with Potato dextrose agar, Saboraud dextrose agar, or Saboraud dextrose agar with 0.1 mg/ml of chloramphenicol, an antibiotic, were used to check for fungal growth. These plates were inoculated with 0.3 ml of PBS and incubated at 30˚C. The remaining PBS was frozen at -80 ˚C for DNA sequencing. After incubation, isolates were counted and reisolated for identification. Isolates were identified using the VITEK23 system or by sequencing a portion of the 16S rRNA gene for bacteria or the ribosomal internal transcribed spacer (ITS) for fungi. Sequencing was performed with an ABI 3500 Sanger sequencer. Results: During our initial sampling, six of the seven sites sampled (86%) displayed bacterial or fungal growth. Samples collected from the staging areas and microtomy labs are not included in this calculation since those areas are maintained at a lower ISO 7 equivalent cleanliness standard. A month later, only three of the seven sites (43%) displayed bacterial growth. No fungal growth was detected in the second sampling. Since new equipment had been introduced to the Hayabusa2 lab since the first round of sampling, an additional three sampling sites were included in the second round of sampling. None of these sites displayed microbial growth. These sites will be included in all future sampling efforts. Bacterial isolates have been identified from the following genera at multiple time points: Micrococcus, Staphylococcus, and Bacillus. Isolates from the genera: Microbacterium, Nocardioides, Methylocystis, and Microvirga were identified in the initial sampling, but were not present at later time points. Identification of fungal isolates is in progress. Results are summarized in Table 1. Discussion: The recovery rate or percentage of positive samples4 was initially 86%, which is higher than the median recovery rate for comparable ISO 5 equivalent curation labs like Stardust (33%), Hayabusa (33%), and Cosmic Dust (50%). However, after a month of operation, the recovery rate for these same sites decreased to 43%, which is similar to what we observe in comparable curation cleanrooms with no microbial control requirements. Adding in the new sampling sites further decreases the recovery rate to 30%. With the reduction in recovery rate, we also observed a decrease in microbial diversity. At the first time point, we observed at least 10 different bacterial species and at least two different fungi. This is a higher diversity than the median values for comparable ISO 5 equivalent labs (2-4 isolates per sampling event). After the second sampling, we observed at least 4 bacterial species and no fungi, which is more consistent with comparable labs. We expect the recovery rate and diversity in both labs to continue to decrease as routine operation continues. We will use ultrapure hydrogen peroxide to disinfect equipment and work areas prior to opening any sample containers. Most of the bacterial and fungal isolates were detected on samples from the cleanroom floors. This is consistent with baseline results from other curation labs. Organisms from the genera Bacillus, Staphylococcus, and Micrococcus that were repeatedly detected are common in cleanrooms and on human skin5,6. These organisms are generally thought to be introduced when people enter the cleanroom. Microbacterium, Nocardioides, and Microvirga have also previously been identified in astromaterials cleanrooms, but not as frequently as Bacillus, Staphylococcus, and Micrococcus. Methylocystis is a novel genus in the astromaterials cleanrooms, but it was identified with low accuracy (93% match in the sequenced region of the 16S rRNA gene) and further work is needed to confirm this identification. Microbacterium is a diverse genus with isolates identified from terrestrial and aquatic sediments. Some species of Microbacterium are capable of degrading complex organic compounds found in crude oil. The presence of these bacteria in the OSIRIS REx and Hayabusa2 cleanrooms should be closely monitored. Methylocystis is a genus of methanotrophic bacteria capable of oxidizing methane. If this identification proves to be correct and it is detected again, it should be closely monitored as well. Under nominal operating conditions, samples should not ever encounter the cleanroom floor or other high traffic areas. If we observe an increase in the bioburden in sensitive work areas that appears to be influenced by organism transfer from high traffic areas like the floors, we can employ additional hydrogen peroxide treatments to disinfect high traffic areas. Routine microbial monitoring of these labs will ensure that NASA’s astromaterials collections remain pristine and useful for scientific study. Table 1. Sampling Locations and Colony Counts Bacterial CFUa Fungal CFU Bacterial CFU Fungal CFU Lab - Location 11/2/2021 11/2/2021 12/13/2021 12/13/2021 H2b-Floor 4 8 1 0 H2-staging pass through 3 0 0 0 H2-microtomy pass through TNTCc 0 0 0 H2 Microscope 1 NA NA 0 0 H2 Microscope 2 NA NA 0 0 H2-Table NA NA 0 0 OREXd- microtomy pass through 0 0 6 0 OREX – Anteroom pass through 0 0 0 0 OREX – Floor 1 2 0 0 OREX Witness Foil Table 3 0 1 0 Staging-Floor 16 0 15 0 Microtomy-Floor 3 0 2 0 a: CFU = Colony Forming Unit b: H2 = Hayabusa2 Lab c: TNTC = too numerous to count d: OREX = OSIRIS-REx Lab References: 1. ISO 14644-1:2015 - Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration. 37 (2015). 2. McCubbin, F. M. et al. Space Sci Rev 215, (2019). 3. Pincus, D. H. Encyclopedia of Rapid Microbiological Methods (2005). 4. The United States Pharmacopeial Convention. USP General Chapter <1116> 17, 784–794 (2013). 5. Sheraba, N. S., Yassin, A. S. & Amin, M. BMC Research Notes 3, 278 (2010). 6. Utescher, C. L. de A., Franzolin, M. R., Trabulsi, L. R. & Gambale, V. Brazilian Journal of Microbiology 38, 710–716 (2007).

A B Regberg↗
NASA NTRSDate not supplied

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↗
NASA NTRS1974

Automatic inoculating apparatus

An automatic inoculating apparatus for agar trays is described and using a simple inoculating element, such as a cotton swab or inoculating loop. The apparatus includes a movable carriage for supporting the tray to be inoculated, a drive motor for moving the tray along a trackway, and a swabbing motor for automatically swabbing the tray during the movement. An actuator motor controls lowering of the inoculating element onto the tray and lifting of the inoculating element. An electrical control system, including limit microswitches, enables automatic control of the actuator motor and return of the carriage to the initial position after inoculating is completed.

Wilkins, J. R.↗
NASA NTRS1993

Microbiological test results of the environmental control and life support systems vapors compression distillation subsystem recycle tank components following various pretreatment protocols

Microbiological samples were collected from the recycle tank of the vapor compression distillation (VCD) subsystem of the water recovery test at NASA MSFC following a 68-day run. The recycle tank collects rejected urine brine that was pretreated with a commercially available oxidant (Oxone) and sulfuric acid and pumps it back to the processing component of the VCD. Samples collected included a water sample and two swab samples, one from the particulate filter surface and a second from material floating on the surface of the water. No bacteria were recovered from the water sample. Both swab samples contained a spore-forming bacterium, Bacillus insolitus. A filamentous fungus was isolated from the floating material. Approximately 1 month after the pretreatment chemicals were changed to sodium hypochlorite and sulfuric acid, a swab of the particulate filter was again analyzed for microbial content. One fungus was isolated, and spore-forming bacteria were observed. These results indicate the inability of these pretreatments to inhibit surface attachment. The implications of the presence of these organisms are discussed.

Huff, Tim↗
NASA NTRS2009

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including the concentration and the characteristics of the infectious agent. Thus, a thorough knowledge of the microorganisms aboard spacecraft is essential in mitigating infectious disease risk to the crew. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The translation of 16S ribosomal DNA sequencing for the identification of bacteria from the SWAB experiment to nominal operations has increased bacterial speciation of environmental isolates from previous flights three fold compared to previous conventional methodology. The incorporation of molecular-based DNA fingerprinting using repetitive sequence-based polymerase chain reaction (rep-PCR) into the capabilities of the laboratory has provided a methodology to track microorganisms between crewmembers and their environment. Both 16S ribosomal DNA identification and bacterial fingerprinting have improved NASA s capability to better understand spacecraft environments and determine the source of contamination events. Preflight sampling has been completed for air, surface, and water samples. In-flight sample collection has been completed for a total of 8 air and surface sample collection sessions. In-flight hardware has performed well and the surface sampling device received positive feedback from the crew for its ease of use. While processing and analysis continue for these samples, early results have begun to provide information on the spacecraft environment. Using a method called Denaturing Gradient Gel Electrophoresis (DGGE), several air and samples were evaluated to determine the types of organisms that were present. Using only molecular techniques, DGGE does not depend on any microbial growth on culture media, allowing a more comprehensive assessment of the spacecraft interior. Preliminary results have identified several microorganisms that would not have been isolated using current technology, though none of these organisms would be considered medically significant. Interestingly, the isolation of Gram negative organisms is greater using DGGE than conventional media based isolation. The cause of this finding is unclear, though it may be the result of the technique s ability to isolate both viable and non-viable bacteria. The next phase of the SWAB sample analysis is the use of quantitative polymerase chain reaction (QPCR) to look for specific medically significant organisms. While not as broad as DGGE, QPCR is much more sensitive and may reveal findings that were not seen during the initial evaluation. Together, this information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Castro, V.A.↗
NASA NTRSDate not supplied

A METHOD TO REDUCE BIOBURDEN IN ASTROMATERIALS CURATION FACILITIES WITHOUT INTRODUCING UNWANTED CONTAMINATION

Introduction: NASA curates its Astromaterials collections in cleanrooms that are carefully monitored for particulate, inorganic and trace metal contamination. Current sample collections are not particularly susceptible to organic contamination or biological alteration. However, new collections like those from the OSIRIS-REx and Hayabusa2 missions will have organic contamination requirements and are susceptible to biodegradation. It will be necessary sterilize or at least disinfect curation labs, as well as tools and equipment in a manner that does not introduce additional contamination and does not affect the samples 1. Current curation cleaning procedures utilize isopropyl alcohol which offers some bioburden reduction, but is not effective against spore-forming bacteria or fungal spores 2. We present a modified disinfection method that uses ultrapure hydrogen peroxide to reduce bioburden inside curation labs and glove boxes without introducing contamination or damaging curation equipment. We tested this method in the meteorite processing lab as well as on a glovebox being cleaned for use in processing ANGSA (Apollo Next Generation Sample Analysis) samples and present the results of those tests. We discuss the limitations of this method and describe potential situations in which it will not be applicable. The CDC guidelines for disinfection andsterilization in healthcare facilities discusses over 15different methods for reducing bioburden in hospitalsettings 3. The most common method, steamsterilization, is well suited to sterilizing curationprocessing tools but cannot easily be used to sterilizecleanroom surfaces or large equipment likegloveboxes. Chemical sterilization with bleach(NaOCl) is also a common strategy in healthcare andpharmaceutical settings that presents materialcompatibility issues as well as serious inorganiccontamination concerns for curation facilities.Introducing a new source of Na and Cl into curationlabs is not acceptable. Other chemical methods likeethylene oxide, formaldehyde, iodophors andquaternary ammonium compounds could introduceorganic and inorganic contamination. We chose tofocus on hydrogen peroxide because it is generallycompatible with commonly used curation materialslike stainless steel, aluminum and Teflon and becauseit decomposes to oxygen and water. The CDCguidelines for hydrogen peroxide specify using a 7.5wt% solution at 25 ̊C with a contact time of 30 minutesfor high level disinfection and 6 hours for sterilization.High level disinfection is defined as a technique thatwill kill all microorganisms except large numbers ofbacterial spores 3. Methods: We prepared a solution of 7.5 wt%hydrogen peroxide from a stock solution of ultrapure30 wt% peroxide (JT Baker) and curation gradeultrapure water. This ultrapure water is already used incuration cleaning procedures and thus is not consideredand additional source of contamination. We conducteda materials compatibility test by exposing unanodizedand anodized 6061 T6 Al alloy to the peroxide solutionfor up to six hours and periodically inspecting thesurfaces for visible defects. We used this peroxide todisinfect the floor of the meteorite processing lab andthe interior of a curation glovebox by exposing thesesurfaces to the peroxide solution for 30 min. Thesurfaces were swabbed with a dry macrofoam swabbefore (Puritan Brand 2518051PFRNDFD) and afterperoxide treatment to collect microbes present on thesurfaces. Microbes were extracted by sonication fromthe swab into 15 ml of PBS (phosphate buffered saline)and inoculated onto the following media: TSA (trypticsoy agar) BA (blood agar), R2A (Reasoners 2 agar),Potato Dextrose Agar, Saboraud Dextrose Agar andSaboraud Dextrose Agar with 0.1 mg/ mlchloramphenicol. Four TSA plates and two BA plateswere inoculated with 0.1 ml of PBS each andincubated at 35 and 37 for 48 hours. Two R2A°C°Cplates (0.1 ml of PBS each) were incubated at 25 .°CThe remaining plates were inoculated with 0.2ml ofPBS and incubated at 30 ̊C for seven days. Afterincubation bacterial and fungal isolates were countedand transferred to new plates for identification usingthe VITEK24 automated system or by sequencing aportion of the barcode gene (16S rRNA for bacteria,small subunit gene for fungi) on an ABI 3500 Sangersequencer. Negative controls consisted of swabs thatwere opened in the sampling environment andanalyzed alongside the experimental samples.Results: A 6 hour exposure to hydrogen peroxideresulted in visible pitting on un-anodized 6061 Al, butnot on anodized surfaces. No visible pitting occurredafter a 30 minute exposure. Therefore, we decided tolimit our experimental tests to 30 min. exposures. 17bacterial CFU (colony forming units) representing 4distinct organisms were isolated from the meteorite processing lab floor prior to hydrogen peroxidetreatment. We were unable culture any organisms afterperoxide treatment. In the glovebox we were able toculture three bacterial CFU representing three distinctspecies, including a spore forming bacterium prior todisinfection with peroxide. After the peroxidetreatment we were unable to culture any organisms.Routine monitoring of the meteorite processing lab andthe glovebox did not indicate any increase in unwantedinorganic contamination after these peroxidetreatments. Discussion: A 30 minute treatment with 7.5 wt%peroxide appears to be an effective method forreducing bioburden on typical cleanroom surfaces. Themethod does not introduce unwanted organic orinorganic contamination and is compatible withcommonly used curation materials like stainless steel,Teflon and anodized aluminum alloys. Special careshould be taken with un-anodized aluminum.Prolonged exposure to hydrogen peroxide can causepitting on this material. We recommend using thismethod to disinfect curation labs and equipment whenbiological alteration is a concern. This method iseffective at room temperature and cannot be used todisinfect labs and equipment where the ambienttemperature is < 0 ̊C. Astromaterials samples shouldbe removed from the area where disinfection is tooccur. Hydrogen peroxide is a powerful oxidizingagent and will react with any organic carbon present inthe sample. References: [1.] Mccubbin, F. M. et al.Sp. Sci Rev(2019) doi:10.1007/s11214-019-0615-9. [2.] Mogul, R.et al.Astrobiology 18, ast.2017.1814 (2018). [3.]Rutala, W. A. & Weber, D. J. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. [4.] Pincus, D. H. in Encyclopedia of Rapid Microbiological Methods (2005).

A. B. Regberg↗
NASA NTRSDate not supplied

ISS External Microorganisms: A Payload to Close Planetary Protection Knowledge Gaps for Crewed Missions

Before NASA or COSPAR is able to set planetary protection requirements for crewed missions to locations like Mars there are a number of critical knowledge gaps that must be addressed (1). One of the most important knowledge gaps is an understanding of microbial leakage from crewed habitats and space suits. Current ECLSS (Environmental Control and Life Support System) and PLSS (Portable Life Support System) requirements do not include any provisions to control microbes that may escape along with vented or leaked gasses. The current generation of NASA space suits can leak at rates as high as 100 cm2 /min. during nominal operation (2). ISS (International Space Station) intentionally vents atmospheric gases like CO2 to maintain habitable conditions for the crew. Furthermore, every time an airlock is used for EVA (extravehicular activity)there is an accompanying release of internal atmosphere. Since it is not possible to sterilize a crewed mission, it is important that we understand what if any microbes are entrained in these vented and leaked products. It is also important to understand if these microbes can survive on exterior surfaces. Recent sampling of the Russian segments of ISS suggest that bacteria and fungi from inside ISS may be capable of surviving on external surfaces(3). NASA is developing an aseptic sampling tool for use during EVA and plans to collect samples from vents on ISS to build on these results. The results of this work will be used to develop planetary protection requirements for vented and leaked gasses from crewed volumes. NASA has developed and tested a tool kit for collecting microbiological samples during EVA(4). This tool kit contains eight commercially available, 23 mm. diameter, foam swabs that can be used to aseptically collect samples while at vacuum. The swabs are individually housed in aluminum canisters that are equipped with 0.2 μm Teflon filters. These filters allow the canisters to equilibrate to pressure changes while preventing microbiological contamination. The canisters will be cleaned and sterilized before flight. Results from ground-based testing indicate that this tool kit is capable of aseptically collecting microbes while at vacuum without becoming contaminated during pressure changes(5). Based on the results of this ground testing we have modified the tool kit to meet NASA safety requirements and improve the ergonomics. We added additional mounting points to the tool kit to give astronauts more options for securing it during use. We also changed the opening mechanism to improve the precision with which swabs can be extracted from the tool kit. We plan to use this kit on an upcoming EVA to collect samples from non-propulsive vents and areas near the U.S. airlock on ISS. These samples will be frozen at -80 ̊C and stored on station until they can be returned to Earth. We will analyze these returned samples using next generation DNA sequencing to determine the community composition and function of external ISS environments. The results of this study will close planetary protection knowledge gaps for crewed missions and will help NASA determine appropriate planetary protection requirements for life support systems. The tool kit will also be useful for collecting aseptic samples on upcoming crewed or robotic missions and could easily be modified to collect samples with organic contamination control requirements as well.

A B Regberg↗
NASA NTRSDate not supplied

ISS External Microorganisms: A Payload to Close Planetary Protection Knowledge Gaps for Crewed Missions

Before NASA or COSPAR is able to set planetary protection requirements for crewed missions to locations like Mars there are a number of critical knowledge gaps that must be addressed (1). One of the most important knowledge gaps is an understanding of microbial leakage from crewed habitats and space suits. Current ECLSS (Environmental Control and Life Support System) and PLSS (Portable Life Support System) requirements do not include any provisions to control microbes that may escape along with vented or leaked gasses. The current generation of NASA space suits can leak at rates as high as 100 cm2 /min. during nominal operation (2). ISS (International Space Station) intentionally vents atmospheric gases like CO2 to maintain habitable conditions for the crew. Furthermore, every time an airlock is used for EVA (extravehicular activity)there is an accompanying release of internal atmosphere. Since it is not possible to sterilize a crewed mission, it is important that we understand what if any microbes are entrained in these vented and leaked products. It is also important to understand if these microbes can survive on exterior surfaces. Recent sampling of the Russian segments of ISS suggest that bacteria and fungi from inside ISS may be capable of surviving on external surfaces(3). NASA is developing an aseptic sampling tool for use during EVA and plans to collect samples from vents on ISS to build on these results. The results of this work will be used to develop planetary protection requirements for vented and leaked gasses from crewed volumes. NASA has developed and tested a tool kit for collecting microbiological samples during EVA(4). This tool kit contains eight commercially available, 23 mm. diameter, foam swabs that can be used to aseptically collect samples while at vacuum. The swabs are individually housed in aluminum canisters that are equipped with 0.2 μm Teflon filters. These filters allow the canisters to equilibrate to pressure changes while preventing microbiological contamination. The canisters will be cleaned and sterilized before flight. Results from ground-based testing indicate that this tool kit is capable of aseptically collecting microbes while at vacuum without becoming contaminated during pressure changes(5). Based on the results of this ground testing we have modified the tool kit to meet NASA safety requirements and improve the ergonomics. We added additional mounting points to the tool kit to give astronauts more options for securing it during use. We also changed the opening mechanism to improve the precision with which swabs can be extracted from the tool kit. We plan to use this kit on an upcoming EVA to collect samples from non-propulsive vents and areas near the U.S. airlock on ISS. These samples will be frozen at -80 ̊C and stored on station until they can be returned to Earth. We will analyze these returned samples using next generation DNA sequencing to determine the community composition and function of external ISS environments. The results of this study will close planetary protection knowledge gaps for crewed missions and will help NASA determine appropriate planetary protection requirements for life support systems. The tool kit will also be useful for collecting aseptic samples on upcoming crewed or robotic missions and could easily be modified to collect samples with organic contamination control requirements as well.

A B Regberg↗
NASA NTRS1979

Wipe-rinse technique for quantitating microbial contamination on large surfaces

The evaluation of an improved wipe-rinse technique for the bioassay of large areas was undertaken due to inherent inadequacies in the cotton swab-rinse technique to which assay of spacecraft is currently restricted. Four types of contamination control cloths were initially tested. A polyester-bonded cloth (PBC) was selected for further evaluation because of its superior efficiency and handling characteristics. Results from comparative tests with PBC and cotton swabs on simulated spacecraft surfaces indicated a significantly higher recovery efficiency for the PBC than for the cotton (90.4 versus 75.2%). Of the sampling area sites studied, PBC was found to be most effective on surface areas not exceeding 0.74 sq m (8.0 sq ft).

Kirschner, L. E.↗
NASA NTRS1988

Passive damping concepts for free and forced member and grillage vibration

The performance of potential passive damping concepts is investigted for a long tubular aluminum alloy member, and a two-bar grillage structure. The members are restrained partially at the ends and are of the type being considered by NASA for possible use in the construction of a future space station. Four different passive damping concepts are studied and include nylon brush, wool swab, copper brush, and silly putty in chamber dampers. Both free and forced vibration tests are conducted. It is found that the silly putty in chamber damper concept provides considerably greater passive damping as compared to that of the other three concepts. For the grillage natural vibration, a five wool swab damper configuration provides greater damping than the five silly putty dampers in chamber configuration. Due to the constrained motion imposed by the vibrator used in the tests, the effectiveness of the passive dampers could not be adequately evaluated for the individual member. However, it is found that for the grillage under forced vibration, the five silly putty dampers in chamber damper configuration provides very effective passive damping although only at and around the resonant frequency. At resonance, these dampers provide a 51 percent reduction in the dynamic magnification factor for this case.

Razzaq, Zia↗
NASA NTRS1998

Contamination sampling device

A contamination sample collection device has a wooden dowel with a cotton swab at one end, the cotton being covered by a nylon cloth and the wooden dowel being encapsulated by plastic tubing which is heat shrunk onto the dowel and onto a portion of the cotton swab to secure the cotton in place. Another plastic tube is heat shrunk onto the plastic that encapsulates the dowel and a portion of the nylon cloth to secure the nylon cloth in place. The device may thereafter be covered with aluminum foil protector. The device may be used for obtaining samples of contamination in clean room environments.

Delgado, Felix A.↗
NASA NTRS2008

Rapid Biochemical Analysis on the International Space Station (ISS): Preparing for Human Exploration of the Moon and Mars

The Lab-on-a-Chip Application Development - Portable Test System, known as LOCAD-PTS, was launched to the International Space Station (ISS) aboard Space Shuttle Discovery (STS-116) on December 9th,2006. Since that time, it has remained onboard ISS and has been operated by the crew on 10 separate occasions LOCAD-PTS is a handheld device for rapid biochemical analysis; it consists of a spectrophotometer, a series of interchangeable cartridges, a pipette and several clean/sterilized swabbing kits to obtain samples from ISS surfaces. Sampling, quantitative analysis and data retrieval is performed onboard, therefore reducing the need to return samples to Earth. Less than 20 minutes are required from sampling to data, significantly faster than existing culture-based methods on ISS, which require 3-5 days. Different cartridges are available for the detection of different target molecules (simply by changing the formulation within each cartridge), thereby maximizing the benefit and applications addressed by a single instrument. Initial tests on ISS have focused on the detection of the bact.erial macromolecule endotoxin, a component of bacterial cell walls. LOCAD-PTS detects endotoxin with a cartridge that contains a formulation known as Limulus Amebocyte Lysate (LAL) assay. LAL is derived from blood of the horseshoe crab, Limulus polyphemus, and detects enodotoxin with an enzyme cascade that triggers generation Of a yellow colored dye, p-nitroanaline. The more p-nitroanaline product, the more endotoxin is in the original sample. To enable quantitative analysis, the absorbance of this color is measured by LOCAD-PTS through a 395 nm filter and compared with an internal calibration curve, to provide a reading on the LED display that ranges from 0.05 Endotoxin Units (EU)/ml to 5 EU/ml. Several surface sites were analyzed within ISS between March 2007 and February 2008, including multiple locations in the US Laboratory Destiny, Node 1 Unity, AMock, and Service Module Zvezda. The goals of this initial study were to i) test the cleanliness of reagents/supplies on orbit, ii) test the crew's ability to collect and process a sample in microgravity without contamination, iii) demonstrate nominal function of the LOCAD-PTS, and iv) provide a general survey of endotoxin within the ISS. The surface sites varied greatly in terms of their frequency-of-use and material texture/composition; from relatively smooth aluminum, to fabric, to the room temperature vulcanizing (RTV) rubber of a Extravehicular Mobility Unit (EMU) spacesuit. Results showed that: i) the swabbing kits and reagents remained clean on orbit, ii) the crew could collect and process a sample without contamination, and iii) the LOCAD-PTS functioned nominally in > 99% of the 55 tests completed. We will present detailed results of the survey of endotoxin on ISS surfaces. These results and technology are important in the near-term - by providing an extra tool in the toolbox for ISS microbial monitoring. They are also important in the longer term as valuable preparation for human exploration of the Moon and Mars. One of the proposed science goals for the human exploration of Mars will be to detect and characterize any indigenous biological molecules that may exist on the Martian surface. To achieve that goal, the crew must have the technology available onboard to differentiate indigenous biology from any terrestrial biological material brought to Mars by the spacecraft and crew (termed 'forward contamination'). The LAL assay is already one of the official methods used by NASA's planetary protection program to certify cleanliness of interplanetary robotic spacecraft prior to launch; and therefore endotoxin is a good marker of forward contamination (as well as other microbial molecules detectable with LOCAD-PTS e.g. box-1, 3-glucan and lipoteichoic acid). Furthermore, the distribution and abundance of these molecules on the ISS provides a good indicator of what to expect on the Crew Exploratioehicle Orion, the lunar lander Antares, and future crewed spacecraft destined for Mars. In addition, technology such as LOCAD-PTS has been proposed to help evaluate forward contamination during lunar surface operations by the crew, as preparation for the human exploration of Mars.

Maule, J.↗
NASA NTRS2009

Surface, Water and Air Biocharacterization - A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft (SWAB) will use advanced molecular techniques to comprehensively evaluate microbes on board the space station, including pathogens (organisms that may cause disease). It also will track changes in the microbial community as spacecraft visit the station and new station modules are added. This study will allow an assessment of the risk of microbes to the crew and the spacecraft. Research Summary: Previous microbial analysis of spacecraft only identify microorganisms that will grow in culture, omitting greater than 90% of all microorganisms including pathogens such as Legionella (the bacterium which causes Legionnaires' disease) and Cryptosporidium (a parasite common in contaminated water) The incidence of potent allergens, such as dust mites, has never been systematically studied in spacecraft environments and microbial toxins have not been previously monitored. This study will use modern molecular techniques to identify microorganisms and allergens. Direct sampling of the ISS allows identification of the microbial communities present, and determination of whether these change or mutate over time. SWAB complements the nominal ISS environmental monitoring by providing a comparison of analyses from current media-based and advanced molecular-based technologies.

Pierson, Duane L.↗
NASA NTRS2013

Microbiological Sampling Methods and Sanitation of Edible Plants Grown on ISS

Pathogenic microbes on the surfaces of salad crops and growth chambers pose a threat to the health of crew on International Space Station. For astronauts to safely consume spacegrown vegetables produced in NASA's new vegetable production unit, VEGGIE, three technical challenges must be overcome: real-time sampling, microbiological analysis, and sanitation. Raphanus sativus cultivar Cherry Bomb II and Latuca sativa cultivar Outredgeous, two saled crops to be grown in VEGGIE, were inoculated with Salmonella enterica serovar Typhimurium (S. Typhimurium), a bacterium known to cause food-borne illness~ Tape- and swab-based sampling techniques were optimized for use in microgravity and assessed for effectiveness in recovery of bacteria from crop surfaces: Rapid pathogen detection and molecular analyses were performed via quantitative real-time polymerase chain reactiop using LightCycler® 480 and RAZOR® EX, a scaled-down instrument that is undergoing evaluation and testing for future flight hardware. These methods were compared with conventional, culture-based methods for the recovery of S. Typhimurium colonies. A sterile wipe saturated with a citric acid-based, food-grade sanitizer was applied to two different surface materials used in VEGGIE flight hardware that had been contaminated with the bacterium Pseudomonas aeruginosa,. another known human pathogen. To sanitize surfaces, wipes were saturated with either the sanitizer or sterile deionized water and applied to each surface. Colony forming units of P. aeruginosa grown on tryptic soy agar plates were enumerated from surface samples after sanitization treatments. Depending on the VEGGIE hardware material, 2- to 4.5-log10 reductions in colony-forming units were observed after sanitization. The difference in recovery of S. Typhimurium between tape- and swab- based sampling techniques was insignificant. RAZOR® EX rapidly detected S. Typhimurium present in both raw culture and extracted DNA samples.

Parrish, Charles H. II↗