Recovery of Bacillus Spore Contaminants from Rough ... · for B. atrophaeus spore recovery from this rough textile, revealing signiﬁcantly different efﬁciencies (0.5 to 15.4%)

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2011, p. 1628–1637 Vol. 77, No. 50099-2240/11/$12.00 doi:10.1128/AEM.02037-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Recovery of Bacillus Spore Contaminants from Rough Surfaces: aChallenge to Space Mission Cleanliness Control�

Alexander Probst,1,2 Rainer Facius,3 Reinhard Wirth,1 Marco Wolf,2 and Christine Moissl-Eichinger1*Institute of Microbiology and Archaea Center, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany1;

Lander Systems and Space Robotics, Astrium Space Transportation, Airbus Allee 1, 28199 Bremen, Germany2; andGerman Aerospace Center, Linder Hoehe, 51147 Cologne, Germany3

Received 29 August 2010/Accepted 19 December 2010

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probableinterference with life detection experiments. Therefore, space agencies measure the cultivable spore load(“bioburden”) of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus sporesfrom Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were comparedfor B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency),was then compared to the current European Space Agency (ESA) standard wipe assay in sampling fourdifferent kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed thestandard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additionalexperiments were performed by sampling Vectran fabric seeded with seven different spore concentrations andfive different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis).Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sternespores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on testsurfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this studyprovide a step forward in understanding the spore distribution on and recovery from rough surfaces. Theresults presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research.

The advent of space travel has raised concerns that biolog-ical cross-contaminations between solar bodies could occurdue to a lack of cleanliness control of space missions. Theconcept of planetary protection includes the avoidance of so-called forward contaminations: spacecraft sent to foreigncelestial bodies may inadvertently serve as a conveyer for bi-omolecules and/or life which originated on Earth. After land-ing or impact on the target, terrestrial contaminants may vio-late the scientific integrity of a mission by causing false-positiveresults in life detection—or even affect the extraterrestrial eco-system.

For cleanliness and contamination control, spacecraft areassembled in clean rooms and must undergo a regular deter-mination of bioload, as required by the United Nations OuterSpace Treaty (2). Therefore, space agencies measure the aer-obic, heterotrophic, and mesophilic spore loads of spacecraftas a proxy indicator to calculate the overall biomatter. Spore-forming bacilli were identified as frequent contaminants onspacecraft-related surfaces (19, 33, 39). Additionally, Bacillusspores are well known to withstand many stresses (28) andeven to survive space conditions (17). Based on these obser-vations, (Bacillus) spores are still considered the major con-tamination source that could affect the integrity of space mis-sions, although the presence of vegetative, extremotolerant

microorganisms in spacecraft assembly clean rooms has beenreported (19, 31, 38).

Assays to sample spacecraft surfaces employ the wipe-rinse technique originally developed by Manheimer in theearly 20th century (23). From that time on, many variants ofthis method have been established, but most previous stud-ies of the method have been related to Bacillus anthracisspore detection due to terrorism incidents in 2001 (8–10, 13,14, 16, 22, 35, 37). On that occasion, the Brentwood MailProcessing and Distribution Center in Washington, DC, washighly contaminated with spores of B. anthracis as a conse-quence of a terroristic attack (1).

Crucial factors for sampling success were attributed to thematerial of the sampling device (35), but the structure of thecontaminated surface was also shown to influence the recoveryof spores significantly (9, 11, 12, 30).

Most studies published so far have been based on analyzingthe spore recovery of a single Bacillus strain, such as B. an-thracis Sterne (6) or B. atrophaeus (26), serving as a surrogatefor B. anthracis. Nevertheless, a significant difference in detec-tion of the two spore types has been reported very recently,resulting in a 2-fold increase of B. atrophaeus spore recoveryefficiency from stainless steel coupons over that of B. anthracisSterne spores (30). In the aforementioned study, we also im-proved and simplified the current National Aeronautics andSpace Administration (NASA) standard swab protocol for thedetection of bacterial spores from various spacecraft surfacesbut found an inefficient detection of spores on textiles. More-over, swab protocols have been designed to sample small areasof spacecraft, but the agencies’ polyester wipe assays are able

* Corresponding author. Mailing address: Institute of Microbiologyand Archaea Center, University of Regensburg, Universitaetsstrasse 31,93053 Regensburg, Germany. Phone: 49 (0) 941 943 4534. Fax: 49 941 9431824. E-mail: [email protected].

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to cover a sample area of 1 m2 (3, 4). Little is known about theefficacy of these wipe assays, since their evaluation in 1979 wasbased on sampling exposed stainless steel and rocket boostermaterials (18).

The Viking Lander Capsule of the first Mars mission wassubjected to extensive heat treatment, sterilizing the spacecraftcomprehensively (33). To this day, spacecraft hardware ismostly manufactured under microbiologically uncontrolledconditions. Consequently, these novel components, which arevery sensitive to dry-heat sterilization, must undergo alterna-tive sterilization methods (e.g., H2O2) prior to assembly. Inte-gration after sterilization poses a danger of recontamination,requiring a regular determination of bioburden during assem-bly, testing, and launching operations. Although Vectran fab-ric, a typical airbag material, can be sterilized via dry-heattreatment prior to clean room entry, the manual integration of,e.g., heat-prone pyrocutters poses a potential contaminationrisk arising from human activity, which is known to be a crucialfactor in microbial contamination of clean room environments(25, 31, 33, 38).

Emerging improvements in space science hardware alsopresent novel surface materials that are integrated into thespacecraft and must be monitored via microbiological sam-pling. Previous studies demonstrated negative effects of highlyporous sample surfaces, resulting in a lower detection of con-taminants (9, 11, 12, 30). In particular, the above-mentionedVectran fabric was proven to retain the vast majority of con-taminants when sampled with the present European SpaceAgency (ESA) swab assay (30). Hence, there is a potentialthreat to life detection missions arising from a biased detectionof spores on rough surfaces. These issues, coupled with theESA’s interest in validating and improving the present stan-dard methods in spore detection, led to setting up this com-prehensive study. Its results will help researchers to understandthe distribution of spores on and possible recovery from roughsurfaces, which are also of great importance for the successfuldetection of, e.g., B. anthracis spores on textiles. The proposedimprovements in sampling and spore recovery will be of highinterest in the fields of planetary protection and public health.

MATERIALS AND METHODS

Experimental design and general background. One of our recent studies,evaluating a nylon-flocked-swab protocol, revealed high aberrations in collectionefficiency depending on the porosity of the test surface (30). The material re-taining most of its spores turned out to be Vectran fabric type A, which wasconsequently the reference test surface in this subsequent study. Ten differentsampling devices and their corresponding sampling protocols were evaluated forB. atrophaeus spore recovery from Vectran fabric type A, the reference surfacein this study. The nominal CFU concentration was set to 4 � 104 per m2

according to observations made in International Organization for Standardiza-tion standard 8 (ISO 8) clean rooms (20). This concentration corresponds to 100CFU per 25 cm2, 400 per 100 cm2, and 1,600 per 400 cm2. This standardconcentration was also used for specificity tests using five different Bacillusstrains. Spore solutions were spotted onto the test surface in a 50% (vol/vol)ethanol (EtOH) solution, if not given otherwise. In addition to efficiency mea-surements, the sampling devices were evaluated with regard to usability, han-dling, and possible disintegration during sampling. Different criteria thereof weredocumented and are listed in Table 1. For linearity studies, different amounts ofB. atrophaeus spores were spotted on Vectran fabric type A in order to gaininformation about the efficiency of the foam-spatula method when varying theconcentrations in inocula (1,600, 1,200, 800, 600, 400, 200, and 100 spore CFUper 400 cm2). Tests to determine the dependence of the recovery on different

inoculation methods (spotting and aerosolization) were also performed at 1,600CFU per 400 cm2 (foam-spatula protocol).

Bacillus species. Five different Bacillus species were used for specificity testswithin this study: B. atrophaeus DSM 675T (Deutsche Sammlung von Mikroor-ganismen und Zellkulturen [DSMZ]) served as the reference test strain. B.anthracis Sterne 34F2 (U. Reischl, Universitaetsklinik, Regensburg, Germany),B. megaterium 2c1 and B. thuringensis E24 (original clean room isolates; P.Rettberg, DLR, Cologne, Germany), and B. safensis DSM 19292T (DSMZ) werethe same strains as described in one of our previous publications (30).

Spore stock solutions. Pure Bacillus cultures were grown on plates until asporulation of �95% was observed. Isolation and purification were performed asdescribed previously with DNase and lysozyme (24). Afterwards, spores werestored in 50% (vol/vol) and absolute (abs.) EtOH, respectively. Dilution serieswere performed to create spore stock solutions with an appropriate number ofCFU determined via positive controls (see below).

Sample surface materials were (i) Vectran fabric type A, (ii) Vectran fabrictype B, (iii) carbon fiber-reinforced plastic (CFRP), and (iv) roughened carbonfiber-reinforced plastic (Fig. 1; Table 1). The sizes of the test surfaces varieddepending on the assay: 5- by 5-cm2 surfaces were used for swab assays, 10- by10-cm2 surfaces were used for direct extraction from the surface, and 20- by20-cm2 surfaces were used for other protocols. Detailed descriptions of theproperties of the materials are provided elsewhere (30).

Inoculation. Prior to inoculation, surface materials were rinsed with 70%(vol/vol) EtOH, dried, and sterilized via UV irradiation (360 s; GS Gene Linker,UV Chamber; Bio-Rad) and autoclaving (121°C, 200 kPa, 1 h). Spore stocksolutions were sonicated (3 min, 120 W, 35 kHz; Sonorex super DK 102P;Bandelin, Germany) prior to usage in order to ensure seeding of single spores.For wet inoculation, spores were spotted in 50% (vol/vol) EtOH solutions ontotest surfaces and dried for 24 h in a laminar flow hood. For aerosolization, sporestock solutions (abs. EtOH) were sprayed into a settling chamber (dimensions,28 by 28 by 25 cm; zinc sheet) as an alternate inoculation procedure. Twocontrasting types of spraying methods were used: aerosolization via a spray gun(Sogolee HP-200 [0.2 mm]; Taiwan Airbrushes, Taoyuan, Taiwan) and via aspray diffuser (Roth GmbH & Co. KG, Karlsruhe, Germany).

In the case of the spray diffuser, the chamber, including the sample surfacematerial, was preheated to 60°C for 15 min prior to each seeding in order toincrease the aerosolization effect. Here, three pump strokes were necessary tomeet CFU requirements. After the seeding process, spores were allowed to settlein the chamber for 15 min before the test surface was removed.

General handling and procedures. A biosafety cabinet (Microflow biologicalsafety cabinet; Nunc, Wiesbaden, Germany) ensured the cleanliness and sterilityof the sampling assays described herein. Spacecraft bioburden assays usuallyfocus on enumerating the number of germinable spores; vegetative cells arekilled by implementing a heat step (15 min, 80°C) during the spore extractionprocedure (3, 4). Therefore, this step was omitted for the recovery efficiencystudies.

Sampling methods. The procedures described herein were based on the wipe-rinse technique (23) if not stated otherwise. An overview of the methodologies,the sampling devices (including manufacturers), and the corresponding samplesurface size is provided in Table 1. In general, sampling was performed by wipingthe surface three times with a premoistened sampling device, rotating the direc-tion of motion 0°, 90°, and 135°. For extraction of spores from the samplingmaterial, phosphate-buffered saline including Tween 80 (0.02% [vol/vol]; PBST)was added to the removed head of the sampling tool; this suspension wasincubated at room temperature (RT; 22°C � 2°C) for 1 h prior to extraction inorder to simulate transport from the sampling site to the laboratory. Cultivationwas performed according to ESA standard assays (3): the cultivation medium forgrowing the extracted spores was R2A (BD, Heidelberg, Germany), and colonycounts of the spread/pour plates were performed after 24, 48, and 72 h ofincubation at 32°C.

The sampling protocols of the study were as follows.(i) Swab assays. Swab assays were carried out according to protocol A in the

work of Probst et al. (3, 30). The sterile swabs were removed from their con-tainer, and the head of the swab was premoistened with water. Swabbing wasperformed by moving the swab in one direction while rotating the head andapplying steady pressure. This was repeated twice by changing the linear direc-tion of the swabbing process by 90° and 135°, as explained above. Afterwards, theswab head was broken into a tube containing 2.5 ml sterile PBST. For sampleprocessing, the tube was vortexed at full speed for 5 to 6 s before four 0.5-mlsamples of the spore suspensions were plated. Modifications of this protocolwere accomplished when and where necessary, depending on the type of swabused: Whatman FTA card applicators were processed in a 50-ml Falcon tube(VWR, Darmstadt, Germany) for extraction due to their size; for Puritan foam-

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tipped applicators, only three instead of four spread plates were achieved due toPuritan foam’s high absorption of the extraction solution.

(ii) Foam-spatula protocol. The foam spatula (22) had a 17-cm-long and1.5-cm-wide handle. The bilateral broadening at the forefront was covered by a4- by 4-cm large piece of polyurethane sponge on both sides. For premoistening,the sponge was slightly swirled in a wide-necked bottle containing 30 ml of sterilewater. During sampling, the sampling device was held at a 30° angle toward the400-cm2 test surface and the side of the sponge was changed for each of the threesampling steps (0°, 90°, and 135°). Then the sponge was broken into a wide-necked bottle (VWR; 100-ml total volume, with screw cap) by using its frontbreaking point, and 40 ml of PBST was added. For extraction, the capped bottlewas shaken for 20 s horizontally and sonicated for 120 s (120 W, 35 kHz).Twenty-four milliliters of the extracted solution was portioned into six petridishes of 4 ml each, and pour plates were achieved by adding molten (50°C),sterile R2A agar.

(iii) Nylon-flocked-spatula protocol. This sampling device had the same basicstructure as that described for the foam spatula, but its forefront was flocked withnylon fibers as described previously (30). The sampling protocol was identical tothe foam-spatula protocol, with the exception of using 10 ml of PBST forextraction to result in two pour plates only.

(iv) SpongeSicle protocol. The SpongeSicle sampling device was 20 cm longand had a 2-cm-wide handle. The two-sided forefront was covered by a cellulosesponge having a size of 4 by 4 cm. For moistening the sponge, 10 ml of sterilewater was added into the sterile bag of the sampling tool. The rest of the protocolwas accomplished according to the foam-spatula protocol, except that 20 ml ofPBST was used for extraction and three pour plates were achieved.

(v) Wipe assays. Wipe assays were performed as described for the ESAstandard assay for microbial examination of flight hardware (3), except that thesample surface area was 400 cm2. In brief, a sterile 15- by 15-cm polyester wipewas premoistened with 3 ml sterile water and placed flat onto the test surface.After each step of wiping (0°, 90°, and 135°), the wipe was folded, so that the usedsurface was wrapped inside. Afterwards, it was placed into a 50-ml Falcon tube(VWR, Germany) and 40 ml PBST was added, as given in the ESA standardprocedure (3). Spores were extracted from the wipe by shaking the tube for 15 s

followed by sonication (120 s, 120 W, 35 kHz). Subsequently, 32 ml of theextraction solution was utilized for eight pour plates.

(vi) Direct extraction. The direct-extraction bulk method was not based on thewipe-rinse technique; instead, spores were directly extracted from the surfacematerial. Therefore, a 10- by 10-cm piece of (contaminated) Vectran fabric wasrolled and placed into a 50-ml Falcon tube followed by addition of 40 ml ofextraction solution. For extraction, the tube was shaken for 15 s and sonicated for120 s (120 W, 35 kHz).

Controls. For positive controls, spore stock solutions that were used for spot-ting spores onto test surfaces were directly plated on R2A agar plates. Foraerosolization controls, sterile bioassay dishes (24 by 24 cm) with R2A mediumwere placed into the settling chamber and seeding was performed as describedabove. Colony counting was done after 24, 48, and 72 h of incubation (32°C) inorder to determine the number of CFU.

For each test series, one negative control was carried along; that is, sterile 50%(vol/vol) EtOH and abs. EtOH were spotted or sprayed, respectively, onto thetest surface as mentioned above. Then the corresponding sampling method wasapplied, and samples were processed as described above.

Direct inoculation. For determination of the extraction efficiency of the foam-spatula protocol, 1,600 CFU of B. atrophaeus spores was directly spotted ontotwo-thirds of the sponge, 1 ml of the spore solution (50% EtOH [vol/vol]) foreach side. Afterwards, the sponge was broken into a wide-necked bottle followedby extraction and CFU enumeration as already described above. For statisticalanalysis (see below), the volume was adjusted to 42 ml (40-ml storage and 2-mlspore stock solution).

Statistical analysis. In adherence to the stipulation of USP (United StatesPharmacopeia) 1223 (5), the statistical processing of the data makes proper useof the underlying probability distribution of the raw data, i.e., the Poisson dis-tribution, instead of approximations to the normal distribution. In particular, twofeatures of Poisson distributed data are exploited in the analysis (29, 32, 36).Given count data, x and y, from two Poisson populations with expectation values(means) � and �, i.e., X�Poisson(�) and Y�Poisson(�), then the sum, s � x � y,is again a Poisson variable, S�Poisson(), with mean � � � �. This can begeneralized to more than two samples. The test of equality of two means or the

FIG. 1. Scanning electron micrographs of different surfaces for recovery tests. White boxes indicate enlarged areas shown in the next pictureof a series. Bars, 200 m (row 1), 50 m (row 2), and 20 m (row 3). (A) Vectran fabric type A; (B) Vectran fabric type B; (C) carbonfiber-reinforced plastic (CFRP); (D) roughened CFRP.



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construction of confidence intervals for an observed ratio r � x/y is based on asecond property of the Poisson distribution. Given the above observed sum, s �x � y, the distribution of Y or X conditional on S � s is a binomial distribution.

One-sided type I error probabilities, �(1), for the null hypothesis H0: � � � areobtained by summing the probabilities of this binomial distribution pertaining tothose events which belong to the alternative hypothesis H1 for an envisageddeviation from H0 (H1: less than and/or H1: greater than).

Confidence intervals for ratios, R, estimating � � �/� from observations ofcounts u and v, U�Poisson(a�) and V�Poisson(b�) with u � v � w and knowna and b, again are derived from the binomial distribution.

Raw data were processed by a program script for the statistical programmingenvironment R (http://www.R-project.org). Further details are given in the workof Probst et al. (30). Confidence levels were chosen as 0.95 (95%). Nonoverlap-ping confidence intervals of two assortments of sampling methods were evidencefor a significant difference of their recovery efficiencies.

For different recovery methods, average recoveries across the test species oracross the test inocula were determined as weighted averages (for the procedure,see reference 30).

SEM and spore preparation. Scanning electron microscopy (SEM) (30) wascarried out with a digital scanning microscope (DSM 950; Zeiss, Oberkochen,Germany). Prior to the analysis, preparations were coated with a gold-palladiumtarget, creating a layer of 1.4 nm (Polaron SC 515, SEM coating system). Toanalyze the polyurethane material of the foam spatula, a 1- by 1-cm-large piecewas cut out of its sponge and glued onto a stab by using nonconductive adhesiontabs (Plano GmbH, Wetzlar, Germany). For analysis of the sample surfacematerial, pieces of 1 cm in diameter were prepared and placed on SEM stabs alsoby using nonconductive adhesion tabs. For further purification of spores, ultra-centrifugation was performed using a CsCl gradient (40% [wt/wt], 16 h; 50,000rpm [336,239 � g]) and a swing-out rotor (SW 60 Ti; Beckman Optima LE-80K;Beckman Coulter Inc.) (15, 30). In order to gain insights into the distribution ofspores on the sample surface material, highly purified spore solutions (50%[vol/vol] EtOH) were sonicated (3 min, 120 W, 35 kHz) and spotted ontoprepared test surfaces. For aerosolization, the sample surface was preheated to60°C and highly purified spores were sprayed (abs. EtOH) onto the test surfaceby using a spray diffuser (Roth GmbH & Co. KG, Karlsruhe, Germany).

RESULTS

Comparison of sampling methods. In general, means of theCFU recovery efficiencies from Vectran fabric type A rangedfrom 0.5% (wipe protocol) to 15.4% (direct extraction) de-pending on the sampling method applied (Table 1). Consider-ing the swab protocol, Whatman FTA card applicators col-lected the highest number of CFU from the textile (6.7%recovery efficiency) but showed no significant difference fromPuritan 3655 swabs (5.3%). Fewer CFU were recovered byfoam-tipped applicators and Puritan 3600 swabs (1.4% and2.4% recovery efficiencies, respectively). The wipe methods

based on Spec wipe 7 wipers (ESA standard method [3]) andVectra Alphasorb turned out to be insignificantly different indetection, whereas both resulted in a very poor recovery effi-ciency of 0.5%. The nylon-flocked spatula and SpongeSicleprotocols revealed efficiencies of 4.2% and 3.4% recovery,respectively. Moreover, no significant differences were de-tected between nylon-flocked spatulas and Puritan 3655 swabsor SpongeSicles and Puritan 3600 swabs. Direct extraction(bulk method; 15.4%) and the foam-spatula protocol (13.2%)revealed the highest recovery efficiencies, both being signifi-cantly different from all other methods. For the bulk method,however, the surface sample material itself was used for ex-traction; hence, this procedure was studied for comparison butnot as a method applicable to spacecraft surfaces. The foam-spatula protocol consequently out-performed all other meth-ods in spore detection on Vectran fabric type A.

Sampling surfaces of different degrees of roughness. Thepresent ESA standard procedure for large surface samplingwas compared to the foam-spatula protocol in CFU recoveryof B. atrophaeus spores from four different spacecraft surfaces(Fig. 1). An overview of the results of this section is providedin Table 2, whereas Fig. 2 additionally shows results from thenylon-flocked-swab protocol of a previous study for compari-son (30). The wipe method was most efficient when samplingroughened CFRP (19.6% recovery efficiency) and detectedfewer CFU on smooth CFRP (9.0%). Sampling of Vectranfabric type B resulted in a significantly higher recovery of CFU(6.5%) than that of Vectran fabric type A (0.5%). Consideringthe foam-spatula protocol, it revealed the highest recoveryefficiencies when sampling smooth CFRP (57.1%) and rough-ened CFRP (38.1%), with significant differences. In the case ofVectran fabric, more CFU were detected on type B than ontype A (24.4% and 13.2% recovery efficiencies, respectively),which correlates with results from the wipe assay. In summary,at a concentration of 1,600 CFU per 400 cm2, the foam-spatulaprotocol was superior to the present ESA standard wipe assayregardless of the porosity of the surface analyzed. The averagerecovery efficiency for B. atrophaeus spores (CFU) for thefoam-spatula protocol was 41.1%, approximately 3 timeshigher than that for the standard wipe assay (13.9%).

TABLE 2. Comparison of B. atrophaeus spore recoveries from surfaces with different degrees of roughnessd

Sampling method/tool Sample surface materialSampling

area(cm2)

No. ofreplicates

Recovered CFU Recoveryefficiency (%) 95% CIa Avg recovery (%)b

Mean Range

Foam-spatula protocol CFRPc 400 10 886.4 680.0–1,111.7 57.1 55.4–58.6 41.1 � 9.7 (19.5)Roughened CFRP 400 10 591.0 461.7–711.7 38.1 36.7–39.5Vectran fabric type A 400 13 204.4 136.7–246.7 13.2 12.5–13.9Vectran fabric type B 400 10 378.0 280.0–458.3 24.4 23.3–25.5

Wipe assay CFRP 400 10 138.9 61.3–235.0 9.0 8.4–9.5 13.9 � 3.8 (7.68)Roughened CFRP 400 10 304.5 236.3–426.3 19.6 18.8–20.5Vectran fabric type A 400 10 8.1 3.8–17.5 0.5 0.4–0.7Vectran fabric type B 400 10 101.5 48.8–143.8 6.5 6.1–7.0

a CI, confidence interval of recovery efficiency.b Mean � standard error of the mean (standard deviation) of the recovery efficiencies of the different test series of a single method.c CFRP, carbon fiber-reinforced plastic.d The spore concentration (nominal) was 4 � 104 CFU per m2. Inoculation was performed via spotting. The spore inocula were 1,600 (nominal) and 1,551.8 (actual)

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Operational range of the foam-spatula protocol. A summaryof the results of these test series is provided in Table 3. Themethod’s recovery efficiency declined from 13.2% to 7.0% forinocula of 1,600 and 1,200 CFU per 400 cm2, respectively. Thiswas also observed in additionally performed experiments. Atand below 1,200 CFU per sample area, the recovery efficiencystayed constant with an average of 6.7%.

The specificity of the foam-spatula protocol was tested byvarying the Bacillus species used for the recovery test; numer-ical values of those recovery efficiency experiments are pro-vided in Table 4. Spores of B. atrophaeus were recovered withthe highest efficiency (13.2%), and those of B. anthracis wererecovered with the lowest efficiency (0.3%); the two were sig-nificantly different from one another. Spores of clean roomisolates, B. megaterium 2c1 and B. thuringiensis E24, were de-tected with an insignificant difference (5.1% and 5.4% recoveryefficiencies, respectively). B. safensis spores were recovered

with an efficiency of 0.5%. The average recovery efficiencybased on all species was determined to be 4.9%.

Extraction efficiency of the foam-spatula protocol. Themean of the extraction efficiency was determined to be 84.2%with a confidence interval (95%) ranging from 81.9% to 86.6%.Moreover, the mean of recovered CFU was 1,306.4 per testwith a maximum of 1,442.0 and a minimum of 1,209.3 spores.CFU that were consequently lost during sample processing orretained in the sponge were calculated to add up to 15.8% ofthe inoculum.

SEM analysis of the foam-spatula sponge. The tip of thesampling device was a spongy network of polyurethane withbubble-like cavities (Fig. 3). The sponge comprised severallayers of this network, whereas only the uppermost layer was indirect contact with the sample surface material. The cavities ofthe top surface of the sponge had a diameter of approximately60 m, and the polyurethane junctions varied from 10 to 30m in width. Moreover, these connections had gaps and holesof different sizes ranging from approximately 2 to 10 m.

SEM analysis of the sample surface material. Vectran fabrictype A was made of bundles of single fibers, which were inter-woven into a network in a 90° angle. These cylindrical fibershad a diameter of approximately 20 m. An oblique view ofthe bundles (Fig. 1A) showed that there were also gaps be-tween the fibers of a single bundle (Fig. 1A3). Additionally,there were free thin fibers found to be detached from theactual fibers and protruding in a disorderly way from the sur-face. When these free, thin fibers, which were variable in width(2 to 15 m) and length, came apart, furrows were left on themain fiber, thus increasing the porosity of the surface.

The surface structure of Vectran fabric type B was almostidentical to that of type A (Fig. 1B). However, the fibers had adiameter of 25 m and were flattened cylinders in shape.Consequently no gaps occurred between the fibers within abundle.

CFRP was shown to be a very smooth surface with sporadic,low rises (Fig. 1C). In comparison, roughened CFRP consistedof blocks of 20 grooves that alternated at 90° angles (Fig. 1D).These grooves were approximately 20 m wide and 20 mdeep. Although the structure of the surface was very regular, itrevealed many particulates of different sizes and diffuse struc-tures protruding from its surface.

Distribution analysis of spores on sample surfaces. Whenspotted onto the test surfaces, B. atrophaeus spores (1.3 � 104

spores per mm2) turned out to be well distributed on CFRP

FIG. 2. Histogram of recovery efficiencies of B. atrophaeus spores(1,600 CFU per 400 cm2 or 100 CFU per 25 cm2) from differentsurfaces using the foam-spatula protocol, the ESA standard wipe assay,and the nylon-flocked-swab protocol. Error bars indicate the confi-dence interval (95%). CFRP, carbon fiber-reinforced plastic. The as-terisk indicates that data are from the work of Probst et al. (30).

TABLE 3. Recovery efficiency of the foam-spatula protocol at different spore concentrationsb

Nominalinoculum(CFU)/m2

Nominalinoculum

(CFU)/400 cm2

Actualinoculum

(CFU)/400 cm2

No. ofreplicates

Recovered CFU Recoveryefficiency (%) 95% CIa

Mean Range

2,500 100 104.8 11 6.8 1.7–15.0 6.5 4.7–8.75,000 200 209.6 10 12.5 8.3–18.3 6.0 4.7–7.510,000 400 387.5 11 26.8 15.0–50.0 6.9 5.9–8.015,000 600 632.5 10 47.5 36.7–60.0 7.5 6.7–8.520,000 800 775.9 10 52.3 50.0–76.7 6.7 6.0–7.530,000 1,200 1,265.0 13 87.9 41.7–135.0 7.0 6.4–7.540,000 1,600 1,551.8 13 204.4 136.7–246.7 13.2 12.5–13.9

a CI, confidence interval, in percentage of recovered spores.b The sample surface was Vectran fabric type A, 400 cm2. The organism was B. atrophaeus spores; inoculation was performed via spotting.



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and roughened CFRP, occurring individually or in pairs (notshown). Only about 24% of 150 spores enumerated were at-tached to diffuse structures or to large particles in the case ofroughened CFRP (not shown). On Vectran fabric type B, ahighly accumulative effect, resulting in single layers of cluster-ing spores, was observed (not shown). Analysis of spore distri-bution on Vectran fabric type A was carried out by enumerat-ing more than 1,700 spores via SEM (Fig. 4). Spores of B.atrophaeus and other species (B. megaterium 2c1, B. thuringien-sis E24, and B. safensis), which were spotted on Vectran fabrictype A, were observed to cluster between the single fibers ofthe textile (56%) (Fig. 5). Others attached to free, thin fibers

protruding from the surface or were located in furrows, butonly 13% of the spores were lying on the fibers. In contrast,when aerosolized/sprayed onto Vectran fabric type A, sporesof B. atrophaeus were observed to be mainly free on the fibers(62%), and only 7% were located in between.

Variation of inoculation method. Based on the differentdistributions of B. atrophaeus spores when sprayed and spottedonto the test surface (see above), different inoculation meth-ods were applied to seed B. atrophaeus spores onto Vectranfabric type A; the results are summarized in Table 5. Using thespray diffuser and a preheated settling chamber for inocula-tion, the foam-spatula protocol resulted in low recovery effi-ciency (3.0%) compared to previous results from spottingspores (50% EtOH; 13.2% recovery efficiency). In addition,the spray gun method for inoculation allowed a recovery ofspores from the surface of 18.7%. The comparison of all threeinoculation methods revealed significantly different results forthe foam-spatula protocol based on confidence interval analy-sis (95%).

TABLE 4. Specificity of the foam-spatula protocolb

OrganismActual CFU in

inoculum(nominal, 1,600)

No. ofreplicates


Mean Range

Bacillus anthracis Sterne 1,566.6 10 5.3 1.7–15.0 0.3 0.2–0.5Bacillus atrophaeus 1,551.8 13 204.4 136.7–246.7 13.2 12.5–13.9Bacillus megaterium 2c1 1,509.4 10 76.8 40.0–123.3 5.1 4.6–5.6Bacillus safensis 1,640.2 11 8.5 5.0–15.0 0.5 0.4–0.7Bacillus thuringiensis E24 1,519.6 10 81.7 51.7–108.3 5.4 4.9–5.9

a CI, confidence interval, in percentage of recovered spores.b The sample surface was Vectran fabric type A, 400 cm2. The nominal spore concentration was 4 � 104 CFU per m2; inoculation was performed via spotting.

FIG. 3. SEM images of the sponge material of the foam spatula(polyurethane, macrofoam). (A) Top view; bar, 500 m. (B) Enlarge-ment of the white box in panel A; bar, 50 m. (C) Oblique view (85°);bar, 100 m. (D) Enlargement of the white box in panel C; bar, 10 m.

FIG. 4. Scanning electron micrograph of Vectran fabric type Ashowing four fibers and B. atrophaeus spores spotted onto the surface(concentration, 1.3 � 104 per mm2). Bar, 10 m. A, spores free onfibers; B, spores between fibers; C, spores attached to free, thin fibers;D, spores in furrows of the fibers.



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DISCUSSION

In a recent study evaluating a nylon-flocked-swab protocol,we reported an unsatisfactory recovery of B. atrophaeus sporesfrom Vectran fabric, an airbag material used for space travel(30). As a matter of fact, the present study was subsequentlyundertaken to further investigate this problematic surface, elu-cidate the recovery efficiency of ESA’s current wipe assay, andfind a possible alternative.

Many of the current spore detection protocols have beenevaluated on stainless steel (16, 18, 35), some studies haveincluded alternative test surfaces like bare concrete coupons(8–10, 12, 22), and only a few investigations have been per-formed on rough surfaces, wherein carpeting has been of pri-mary interest (11, 14). Direct extraction of spores from theVectran fabric type A (bulk method) showed low recoveryefficiency compared to previous data based on carpeting (11),indicating that this fabric has a higher potential to retainspores. Interestingly, the wipe-rinse protocol that out-per-formed other sampling strategies—including ESA’s wipe as-say—was based on a foam spatula. This sampling device ismade of polyurethane (macrofoam [16]) and has already beenidentified as a sampling material with high recovery efficiencies(12, 13, 16, 22, 35). The evaluation of this protocol according tointernational standards (5) showed a decrease of the sporerecovery at lower concentrations of B. atrophaeus spores

(13.2% to 6.7%). This is in contrast to a previous report in theliterature (22) in which the same sampling device was used.Hence, the rough surface (Vectran fabric) rather than thefoam spatula may be responsible for the nonlinearity, which,however, has been observed for other protocols before (7–10,14, 16).

Based on the constant recovery of �6.7% at low sporeconcentrations, the limit of detection was calculated to beapproximately 15 CFU per 400 cm2 and 368 CFU per m2 forthe foam-spatula protocol. In contrast, planetary protectionrequirements demand a spore load equal to or lower than300 spores (bioburden) per m2 of spacecraft surface (2)—avalue that can hardly be proven with present sampling meth-ods. Similar to ESA’s nylon-flocked-swab protocol (30),sampling methods applied in this study are unable to meetthis requirement for Vectran fabric type A. These methodsalso included the agencies’ wipe protocol, which has beenused in several microbial community analyses and screeningsurveys in the last decade (19–21, 25, 27, 31). Furthermore,Vectran fabric has been used for previous Mars landingmissions (Mars Exploration Rover, http://marsrover.nasa.gov/mission/spacecraft_edl_airvbags.html; Mars Pathfinder,http://marsprogram.jpl.nasa.gov/MPF/mpf/mpfairbags.html), yetdata about the performance of space agencies’ spore detectionmethods on this surface have not been reported in the litera-

FIG. 5. Histogram showing the statistical distribution of Bacillus spores on Vectran fabric type A. Defined localizations are according toFig. 4. Spores were spotted onto the test surface if not stated otherwise. Numbers in parentheses give the numbers of preparations analyzed.Sp, spores.

TABLE 5. Recovery efficiency of the foam-spatula protocol when sampling Vectran fabric type A inoculated with B. atrophaeus spores bydifferent methodsb

Inoculationmethod

Actual CFU ininoculum (nominal, 1,600)

No. ofreplicates


Mean Range

Spray diffuser 1,600.5 7 48.1 20.0–85.0 3.0 2.6–3.5Spray gun 1,601.9 10 298.8 200.0–328.3 18.7 17.8–19.6Spotting 1,551.8 13 204.4 136.7–246.7 13.2 12.5–13.9

a CI, confidence interval, in percentage of recovered spores.b The nominal spore concentration was 4 � 104 CFU per m2.



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ture up to now. As a conclusive statement, positive CFU de-tection on Vectran fabric is indicative of a high contaminationlevel, whereas negative detections on the fabric do not consti-tute fulfillment of planetary protection requirements. More-over, we assert that rough surfaces should be reduced to aminimum on spacecraft in order to ensure proper cleanlinesscontrol and meet planetary protection requirements.

For the purpose of evaluating the specificity of the foam-spatula protocol, spores of different Bacillus species wererecovered from Vectran fabric type A. The causes of aber-rations in the recovery of different types of Bacillus spores,based on, e.g., hydrophobicity of the spore sheath (34), havealready been discussed previously (30). In addition, datafrom SEM analysis in this study clearly support this assump-tion, as all spores were distributed similarly on Vectranfabric, although they were recovered with different efficien-cies. However, the more-than-40-fold-higher recovery effi-ciencies of B. atrophaeus than of B. anthracis Sterne sporesreemphasize our previous statement that B. atrophaeus can-not be used as a surrogate for B. anthracis contaminants(30), and investigations based on this model organism (8–13,22) should be interpreted with high skepticism concerningtheir transferability in the case of a B. anthracis terrorismincident.

Although this communication clearly showed the limitationsof present spore detection methods with regard to fabrics, ourfoam-spatula protocol represents a strong advantage in sporeload detection on large (spacecraft-related) surfaces: thismethod was superior in that it detected an up-to-26-fold in-crease of CFU over that in the present ESA and NASA wipeassay, whereas the applicability of the foam-spatula method interms of particle loss and usability still has to be approved.Clearly, the results presented here have shed more light ontothe distribution of spore contaminants on spacecraft surfacesand have a striking significance for anthrax spore-related stud-ies: those should no longer be based on B. atrophaeus as asurrogate and should also consider textiles and cloths as con-taminated materials.

ACKNOWLEDGMENTS

Gerhard Kminek, Jorg Bolz, Ina Denecke, and Wayne Schubert aredeeply acknowledged for critical inputs and helpful discussion. Re-views by Moogega Cooper, Mary Singer, and Annett Bellack are muchappreciated. We thank Angelika Kuhn for maintenance of the scan-ning electron microscope and Petra Rettberg for providing Bacillusspecies.

We acknowledge funding and research and logistical support fromEADS Astrium, Bremen, Germany. Alexander Probst was supportedby the National German Academic Foundation (Studienstiftung desdeutschen Volkes).

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