An observation about the relative hardiness of bacterial spores and planetary quarantine

Currently, planetary quarantine spacecraft sterilization requirements are based on the logarithmic model of bacterial death. This model is a single parameter exponential model in which the sole parameter is the decimal reduction time (D-value). The D-value associated with the sterilization of a population of bacteria is only a relative measure of the rate of bacterial inactivation: It is known to depend upon many factors, but generally these dependencies are not thoroughly understood. The D-values chosen for spacecraft sterilization have been selected conservatively relative to defined experimental procedures and bacterial spore stocks. New data indicate that these D-values are not conservative when compared with those of naturally occurring organisms. The possible implications of these new data for planetary quarantine are analyzed here.This work was conducted under Contract W-12,853, Bioscience Division, Office of Space Science and Applications NASA Headquarters, Washington, D.C.     

Release of microbial contamination from fractured solids

Estimation of the probability of release of microbial contamination from the interior of solids upon fracture due to impact is essential to the formulation of planetary quarantine and spacecraft sterilization requirements. A model system was designed in which known concentrations of bacterial spores were incorporated in methyl methacrylate plastic. Pieces of plastic were fractured in a uniform manner exposing interior surface areas of consistent and measurable size. Known surface areas were incubated in sets of 20 culture tubes containing liquid growth medium. The subsequent occurrence of visible growth expressed as percent of tubes positive was interpreted as an estimate of the probability of release of at least one viable micro-organism.From these experiments probability of release as a function of microbial concentration in plastic was estimated for exposed interior surface areas of 30.6, 61.2, 91.8 or 122.4 mm². Good agreement of the empirical results with a theoretical mathematical model of the probability of release of contamination from solids was demonstrated. Analysis of the data using the maximum likelihood procedure provided a means of calculating a proportionality constant representing the effective thickness of the exposed area and the characteristics of the recovery procedure.     

A stochastic bioburden model for spacecraft sterilization

An important factor in the calculation of sterilization cycles for spacecraft is the bioburden on the surface of the spacecraft at the start of the cycle. This bioburden must be predicted by the use of models. This paper presents a stochastic model for the prediction of the bioburden on the surfaces of spacecraft. This model has many desirable properties as well as being consistent with observations.This work was conducted under Contract No. W-12,853, Planetary Programs, Office, of Space Science and Applications, NASA Headquarters, Washington, D.C.     

Microbial contamination associated with the Apollo 6 spacecraft during final assembly and testing

The National Aeronautics and Space Administration (NASA) requires that microorganisms which could contaminate the surface of the moon as the result of lunar missions be enumerated and identified so that life forms in lunar materials returned to earth may be more easily recognized as being of native or terrestrial origin.Assessment of microbial contamination in the intramural environments used for the assembly and test of the manned lunar spacecraft (Apollo) was made using fallout strips and air samplers. Microbial contamination on the surfaces of Apollo Command and Lunar Modules was determined by use of the swab-rinse method.Preliminary results indicate that the levels of microbial contamination which accumulated on exposed stainless steel surfaces, as well as airborne microbial contamination in the high bay assembly areas, were similar to those encountered in the unmanned spacecraft assembly areas. However, higher levels of microbial contamination were detected on the Apollo spacecraft than on the unmanned lunar spacecraft.     

Biological losses and the quarantine policy for mars

An international committee known as COSPAR has agreed that the probability of a single viable organism aboard any unmanned spacecraft intended for planetary landing should be kept less than 10^–3, in agreement with work by Sagan and Coleman. At present, the U.S.A. is committed to remain consistent with this standard. Using a model which includes both expected losses from failures to collect data and from contamination to analyze the quarantine problem, evidence is given which suggests that the current quarantine requirements may be too strict if their implementation forces a program delay. U.S. policy should be re-examined, keeping more fully in mind both the types and the relative sized of the losses which might be encountered.     

Microbiological Profiles of Four Apollo Spacecraft

Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (10(4) per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch(2). A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft.     

Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment

Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5′-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m⁻² of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO₂ (95.54%), N₂ (2.7%), Ar (1.6%), O₂ (0.13%), and H₂O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.     

The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight

The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments.     

Evolutionary Processes Transpiring in the Stages of Lithopanspermia

Lithopanspermia is a theory proposing a natural exchange of organisms between solar system bodies as a result of asteroidal or cometary impactors. Research has examined not only the physics of the stages themselves but also the survival probabilities for life in each stage. However, although life is the primary factor of interest in lithopanspermia, this life is mainly treated as a passive cargo. Life, however, does not merely passively receive an onslaught of stress from surroundings; instead, it reacts. Thus, planetary ejection, interplanetary transport, and planetary entry are only the first three factors in the equation. The other factors are the quality, quantity, and evolutionary strategy of the transported organisms. Thus, a reduction in organism quantity in stage 1 might increase organism quality towards a second stress challenge in stage 3. Thus, robustness towards a stressor might in fact be higher in the bacterial population surviving after transport in stage 3 than at the beginning in stage 1. Therefore, the stages of lithopanspermia can themselves facilitate evolutionary processes that enhance the ability of the collected organisms to survive stresses such as pressure and heat shock. Thus, the multiple abiotic pressures that the population encounters through the three stages can potentially lead to very robust bacteria with survival capacities considerably higher than might otherwise be expected. This analysis details an outcome that is possible but probably rare. However, in addition to lithopanspermia, spacecraft mediated panspermia may also exist. The analogous stages in a spacecraft would result in a greater likelihood of increasing the stress tolerance of hitchhiking organisms. Furthermore, missions seeking life elsewhere will frequently be sent to places where the possibility of life as we know it is assumed to exist. Thus, we not only can transport terrestrial organisms to places where they are potentially more likely to survive but also may increase their invasive potential along the way. This analysis highlights further requirements that planetary protection protocols must implement and also provides a framework for analyses of ecological scenarios regarding the transmission of life, natural or artificial, between worlds in a solar system.

     

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars

Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.     

A multi-stage decision model for mission non-contamination requirements

There is apt to be much uncertainty in any program of planetary exploration. This uncertainty naturally leads to possible uncertainty in the time period in which planetary quarantine is desirable, to possible uncertainty in the total number of missions to be launched in the vicinity of any given planet, and indeed, to possible uncertainty in the meaning of the word contamination.A model is developed in this paper which makes possible the derivation of mission non-contamination requirements withouta priori knowledge of either the time period in which planetary quarantine is to be observed or the total number of missions to be used in exploring the planet being quarantined. On the basis of this model, some general observations are made about the need for carefully defining contamination.A rather liberal interpretation of CETEX reports (CETEX, 1958) and (CETEX II, 1959).     

Estimating the number of terrestrial organisms on the moon

NASA policy requires that the number and spatial distribution of terrestrial organisms deposited on the Moon be estimated. This paper describes the way in which such an ‘inventory’ is being maintained, and some of the conclusions that may be drawn from it. This work was conducted under Contract W-12,853, Planetary Programs, Office of Space Science and Applications, NASA Headquarters, Washington, D.C.     

Importance of ultrasonication conditions in recovery of microbial contamination from material surfaces

The conditions of ultrasonication to recover micro-organisms from material surfaces are not standardized and often not validated. This leads to discrepancies in experimental results which can affect the sterilization dose setting, cycle development and process validation, and can have an impact on sterilization processing economics and product sterile quality. The sample loading pattern, species of micro-organism, materials and configurations of sample containers were examined under defined conditions. All of these were found to be important factors contributing to the efficiency of microbial recovery from material surfaces by ultrasonication. Validation of sonication conditions in routine laboratory operations is therefore important for an accurate estimate of the surface contamination to assure the medical device processing and the product sterile quality.     

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.     

Mathematical Estimation of the Level of Microbial Contamination on Spacecraft Surfaces by Volumetric Air Sampling

Microbiological sampling methods presently used for enumeration of microorganisms on spacecraft surfaces require contact with easily damaged components. Estimation of viable particles on surfaces using air sampling methods in conjunction with a mathematical model would be desirable. Parameters necessary for the mathematical model are the effect of angled surfaces on viable particle collection and the number of viable cells per viable particle. Deposition of viable particles on angled surfaces closely followed a cosine function, and the number of viable cells per viable particle was consistent with a Poisson distribution. Other parameters considered by the mathematical model included deposition rate and fractional removal per unit time. A close nonlinear correlation between volumetric air sampling and airborne fallout on surfaces was established with all fallout data points falling within the 95% confidence limits as determined by the mathematical model.     

Metabolism of Di- and Mono-n-Butyl Phthalate by Soil Bacteria

Samples for mycological analysis were collected from surfaces in the Skylab spacecraft before launch and during flight for each manned mission. Fungal contamination levels were low during the first two flights; however, the species recovered were different for each mission. On the third mission, widespread contamination of the Skylab spacecraft with Aspergillus and Pencillium spp. was detected. This contamination was traced to several contaminated space suit undergarments.     

Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.Planetary protection efforts work toward protecting (i) solar system bodies from contamination by terrestrial biological material (forward contamination), thus preserving opportunities for future scientific investigation, and (ii) the Earth from harmful contamination by materials returned from outer space (back contamination) (5). These approaches apply directly to the control and eradication of microorganisms present on the surfaces of spacecraft intended to land, orbit, fly by, or be in the vicinity of extraterrestrial bodies. Consequently, current planetary protection policies require that spacecraft be assembled and readied for launch in controlled clean room environments. To achieve these conditions and maintain compliance with good manufacturing practice regulations, robotic spacecraft components are assembled in ultraclean facilities. Much like facilities in the medical, pharmaceutical, and semiconductor sectors, National Aeronautics and Space Administration (NASA) spacecraft assembly clean rooms (SAC) are kept extremely clean and are maintained to the highest of industry standards (17). Filtered air circulation, controlled temperature and humidity, routine exposure to disinfectants and surfactants, and nutrient-limiting, oligotrophic conditions make it very challenging for microorganisms to persist in such environments, but these measures by no means eradicate biological contaminants entirely (18). Several investigations, both culture based and culture independent, have demonstrated that a variety of bacterial taxa are repeatedly isolated under clean room conditions (18, 24, 26; P. Vaishampayan, S. Osman, G. Andersen, and K. Venkateswaran, submitted for publication). However, despite a growing understanding of the diverse microbial populations present in SAC, predicting the true risk of any such microbes’ compromising the findings of extraterrestrial life detection efforts remains a significant challenge (30). A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination (33).The purification of nucleic acids, subsequent PCR amplification, and shuttling of 16S ribosomal “fingerprint” genes from noncultivable microorganisms into genetically amenable lab strains of Escherichia coli have evolved into a gold standard of molecular means to elucidate the microbial diversity in a given sample. In theory, the cloning and sequencing of 16S ribosomal genes from each and every cell present, regardless of cultivability and inclusive of novel taxa, would result in a comprehensive survey of microbial communities on the surfaces of SAC and colocated spacecraft (24, 26). Unfortunately, the full-length sequencing of all 16S rRNA genes from environmental samples would be prohibitively expensive, making the approach unfeasible for generating comprehensive phylogenetic profiles of complex microbial communities.Attempting to infer population membership from clone libraries limited to hundreds or thousands of sequences has proven to be insufficient for detecting extremely low-abundance organisms. Recent analyses of phylogenetic DNA extracted from soil, water, and air revealed that laboriously derived clone libraries severely under-represent complex bacterial communities compared to very rapid (i.e., requiring only hours) DNA microarray approaches (1, 6, 11, 23, 36). One of the reasons for this is the high sensitivity of PhyloChip methodologies, which are able to detect organisms present in amounts below 10⁻⁴ abundance of the total sample (12). Numerous validation experiments using sequence-specific PCR have confirmed that taxa identified by the microarray were indeed present in the original environmental samples, despite their absence in corresponding clone libraries (3). This highlights the utility of the method compared to classical cloning. Although the analysis of each sample by the PhyloChip provides detailed information on microbial composition, the highly parallel and reproducible nature of this array allows tracking community dynamics over time and treatment. Even without prior sequence information, PhyloChip can identify specific microbial interactions that are key to particular changing environments.A comprehensive census of the microbial communities on the surfaces in three NASA SAC supporting two distinct missions was conducted. To ensure that the maximum diversity of resident microbiota was uncovered, subsamples from each clean room surface sampling were subjected to both DNA microarray protocols and conventional cloning and sequencing of 16S rRNA genes. This study, to our knowledge the first of its kind, focused on comparing the microbial diversity profiles resulting from DNA microarray analyses and conventional cloning and sequencing of 16S rRNA genes arising from a variety of low-biomass surfaces.     

A review on microbiological decontamination of fresh produce with nonthermal plasma

Food safety is a critical public health issue for consumers and the food industry because microbiological contamination of food causes considerable social and economic burdens on health care. Most foodborne illness comes from animal production, but as of the mid‐1990s in the United States and more recently in the European Union, the contribution of fresh produce to foodborne outbreaks has rapidly increased. Recent studies have suggested that sterilization with nonthermal plasma could be a viable alternative to the traditional methods for the decontamination of heat‐sensitive materials or food because this technique proves capable of eliminating micro‐organisms on surfaces without altering the substrate. In the last 10 years, researchers have used nonthermal plasma in a variety of food inoculated with many bacterial species. All of these experiments were conducted exclusively in a laboratory and, to our knowledge, this technique has not been used in an industrial setting. Thus, the purpose of this review is to understand whether this technology could be used at the industrial level. The latest researches using nonthermal plasma on fresh produce were analysed. These evaluations have focused on the log reduction of micro‐organisms and the treatment time.     

Validation of a Nylon-Flocked-Swab Protocol for Efficient Recovery of Bacterial Spores from Smooth and Rough Surfaces

In order to meet planetary-protection requirements, culturable bacterial spore loads are measured representatively for the total microbial contamination of spacecraft. However, the National Aeronautics and Space Administration''s (NASA''s) cotton swab protocols for spore load determination have not changed for decades. To determine whether a more efficient alternative was available, a novel swab was evaluated for recovery of different Bacillus atrophaeus spore concentrations on stainless steel and other surfaces. Two protocols for the nylon-flocked swab (NFS) were validated and compared to the present NASA standard protocol. The results indicate that the novel swab protocols recover 3- to 4-fold more (45.4% and 49.0% recovery efficiency) B. atrophaeus spores than the NASA standard method (13.2%). Moreover, the nylon-flocked-swab protocols were superior in recovery efficiency for spores of seven different Bacillus species, including Bacillus anthracis Sterne (recovery efficiency, 20%). The recovery efficiencies for B. atrophaeus spores from different surfaces showed a variation from 5.9 to 62.0%, depending on the roughness of the surface analyzed. Direct inoculation of the swab resulted in a recovery rate of about 80%, consistent with the results of scanning electron micrographs that allowed detailed comparisons of the two swab types. The results of this investigation will significantly contribute to the cleanliness control of future life detection missions and will provide significant improvement in detection of B. anthracis contamination for law enforcement and security efforts.The recent discovery of liquid water on Mars has sparked debate about the possibility of extraterrestrial life (37). Consequently, highly sensitive biosensors will be deployed onboard spacecraft like the Mars Science Laboratory (MSL), using technologies such as gas chromatographical analysis to search for the smallest traces of life (http://mars.jpl.nasa.gov/msl/mission/). Contamination of equipment by terrestrial microorganisms resulting from a lack of spacecraft cleanliness could significantly compromise the integrity of life detection missions and result in falsely positive extraterrestrial life signals. The prevention of this so-called “forward contamination” is one major goal of American and European space agencies'' planetary-protection efforts. Regular determination of a spacecraft''s bioload and the mission components throughout assembly are mandatory for detecting unacceptably high contamination that exceeds levels set by the United Nations treaty (Outer Space Treaty [11]).Modern spacecraft hardware is very susceptible to standard heat sterilization protocols, so baking the entire spacecraft, such as the Viking Lander Capsule at 111.7°C ± 1.7°C for 23 to 30 h is no longer feasible (30). Alternative cleaning and sterilization methodologies for spacecraft components prior to assembly (i.e., nonthermal plasma technologies) have been discussed (36). However, after integration, sterile hardware is exposed to a significant risk of contamination during assembly, testing, and launching operations. Because of limited access to integrated spacecraft components, the microbial cleanliness of a spacecraft and its surroundings is meticulously maintained through frequent cleaning and sterilization routines. Therefore, the regular and frequent detection of possible contaminants in the assembly environment is more important than ever.To estimate the severity of microbial contamination, the National Aeronautics and Space Administration''s (NASA''s) standard procedure focuses on aerobic, mesophilic spores (26). Briefly, surface samples are taken from spacecraft using moist cotton swabs or wipes. After an extraction procedure, the samples are subjected to a short heat shock (15 min; 80°C) to kill vegetative cells and then pour plated in Trypticase soy agar (TSA) for the enumeration of CFU. This protocol was originally developed for the Viking mission more than 3 decades ago (30) and has remained, for the most part, unchanged.Recent studies have shown that cotton swabs have acceptable recovery efficiencies for Bacillus spores (41.7%) (32) but, due to their organic nature, may raise residue problems on surfaces. Furthermore, their comparatively high DNA content could lead to false positives or inhibition should NASA one day incorporate molecular technologies into their microbial-detection protocols (7).Based on these observations, researchers are beginning to move away from cotton in favor of alternative swabs made from rayon or macrofoam (6, 18). A recent study reported high recovery efficiencies for various vegetative cells from stainless steel surfaces by applying a novel swab with a bulb-shaped head flocked with nylon fibers (12). Patented in 2004, this design facilitates the release of particulates and microbes, resulting in a significantly higher detection rate. The broad applicability of these nylon-flocked swabs (NFS) has been demonstrated by their use in various clinical studies isolating pathogens from medical environments (1, 10, 20).General studies on surface-sampling tools have clearly shown that the swab material and the extraction method are the dominant factors in spore recovery efficiencies (32). Additionally, the properties of the surface to be sampled affect sample recovery (8). For planetary-protection applications, the broad variety of novel materials used in spacecraft construction must be considered. The Mars Exploration Rover mission craft, for example, was composed of at least five kinds of surface materials (http://marsrovers.jpl.nasa.gov/overview). While the cruise stage was constructed primarily of aluminum and the aeroshell consisted of aluminum honeycomb structures, the lander itself was made of titanium and graphite composite (carbon fiber-reinforced plastic [CFRP]). The airbag and the parachutes were made of Vectran and polyester/nylon fabrics. These different materials are quite challenging for sampling tools. Accurate sampling of materials with various surface textures will require planetary-protection programs to introduce novel swab materials.To our knowledge, no investigations have been performed to compare the recovery of spores from different spacecraft surfaces. Previous studies have compared cotton and synthetic sampling materials, but only on stainless steel surfaces (19), and no studies have compared sampling methods on actual spacecraft materials (7).Recently published protocols for spore detection have been based on one specific Bacillus species and/or on one type of surface. Unfortunately, these protocols provide no insight into the effects of varying these factors (4-6, 8, 9, 14, 18), as requested by USP (United States Pharmacopeia) 1223 for validation of alternative microbial methods (3). Some of the aforementioned studies were conducted in response to B. anthracis terrorism incidents in 2001 and used B. atrophaeus as a surrogate. Consequently, information about the actual sampling efficiency of B. anthracis spores is quite limited and may vary significantly from the B. atrophaeus data.In this comprehensive study, we evaluated the novel nylon-flocked swab and a corresponding protocol to recover Bacillus spores from five different spacecraft-related surfaces. It should be noted that although stainless steel served as the standard test surface, it is not a predominant material in spacecraft; however, since the majority of previous (sampling) studies were performed on stainless steel, it represents a universally recognized carrier and also serves as a conservative proxy for the average roughness of the materials used in space science.Our nylon-flocked-swab protocol was validated with respect to accuracy, precision, limit of detection, linearity, and robustness (3). Moreover, its specificity was determined by applying spores of seven different Bacillus species, including the avirulent, attenuated strain Bacillus anthracis Sterne, and by comparing the resulting recovery efficiencies. The results in this communication will significantly contribute to planetary-protection protocols and could also be of high interest for public health issues.     

Microbiological profiles of the Viking spacecraft.

Planetary quarantine requirements associated with the launch of two Viking spacecraft necessitated microbiological assessment during assembly and testing at Cape Canaveral and the Kennedy Space Center. Samples were collected from selected surface of the Viking Lander Capsules (VLC), Orbiters, (VO), and Shrouds at predetermined intervals during assembly and testing. Approximately 7,000 samples were assayed. Levels of bacterial spores per square meter on the VLC-1 and VLC-2 were 1.6 x 10(2) and 9.7 x 10(1), respectively, prior to dry-heat sterilization. The ranges of aerobic mesophilic microorganisms detected on the VO-1 and VO-2 at various sampling events were 4.2 x 10(2) to 4.3 x 10(3) and 2.3 x 10(2) to 8.9 x 10(3)/m2, respectively. Approximately 1,300 colonies were picked from culture plates, identified, lypholipized, and stored for future reference. About 75% of all isolates were microorganisms considered indigenous to humans; the remaining isolates were associated with soil and dust in the environment. The percentage of microorganisms of human origin was consistent with results obtained with previous automated spacecraft but slightly lower than those observed for manned (Apollo) spacecraft.