ASTROBIOLOGY Volume 4, Number 3, 2004 © Mary Ann Liebert, Inc. Research Paper Evaluation of Various Cleaning Methods to Remove Bacillus Spores from Spacecraft Hardware Materials KASTHURI VENKATESWARAN, 1 SHIRLEY CHUNG, 1 JUDITH ALLTON, 2 and ROGER KERN 1 ABSTRACT A detailed study was made of the biological cleaning effectiveness, defined in terms of the ability to remove bacterial spores, of a number of methods used to clean hardware surfaces. Aluminum (Al 6061) and titanium (Ti 6Al-4V) were chosen for the study as they were deemed the two materials most likely to be used in spacecraft extraterrestrial sampler construction. Metal coupons (1 cm  2.5 cm) were precleaned and inoculated with 5.8  10 3 cultivable Bacil- lus subtilis spores, which are commonly found on spacecraft surfaces and in the assembly en- vironments. The inoculated coupons were subsequently cleaned using: (1) 70% isopropyl al- cohol wipe; (2) water wipe; (3) multiple-solvent flight-hardware cleaning procedures used at the Jet Propulsion Laboratory (JPL); (4) Johnson Space Center-developed ultrapure water rinse; and (5) a commercial, semi-aqueous, multiple-solvent (SAMS) cleaning process. The biolog- ical cleaning effectiveness was measured by agar plate assay, sterility test (growing in liquid media), and epifluorescent microscopy. None of the cleaning protocols tested completely re- moved viable spores from the surface of the aluminum. In contrast, titanium was capable of being cleaned to sterility by two methods, the JPL standard and the commercial SAMS clean- ing process. Further investigation showed that the passivation step employed in the JPL stan- dard method is an effective surface sterilant on both metals but not compatible with alu- minum. It is recommended that titanium (Ti 6Al-4V) be considered superior to aluminum (Al 6061) for use in spacecraft sampling hardware, both for its potential to be cleaned to steril- ization and for its ability to withstand the most effective cleaning protocols. Key Words: Space- craft materials—Cleaning—Spores—Disinfection. Astrobiology 4, 377–390. 377 INTRODUCTION T HE SEARCH FOR LIFE on other planets (in situ or sample-return missions) will likely involve ultrasensitive detection of biosignatures. Biolog- ical contaminants on spacecraft outbound from Earth to other planetary bodies could contami- nate those pristine environments and compro- mise both in situ life-detection experiments and returned extraterrestrial samples by yielding false positives. Protecting other solar system bodies from biological contamination originating from 1 Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California. 2 Johnson Space Center, Houston, Texas.