Sample preparation and isolation of microbes from the Mars Odyssey spacecraft.
Components of the Mars Odyssey spacecraft were manufactured in various geographical locations. The spacecraft underwent several months of assembly in the Spacecraft Assembly Facility at the Jet Propulsion Laboratory, Pasadena, CA, USA, before it was transported to the Kennedy Space Center, FL, USA. Samples were taken from 25 different surface areas (25 cm2) of the spacecraft using sterile, water-moistened polyester swabs (Texwipe) during final assembly and encapsulation in the Spacecraft Assembly and Encapsulation Facility II, Kennedy Space Center (February 2001). Upon collection of a surface sample, each swab was placed individually into sterile water (final volume 10 ml). The 25 samples were pooled into one sterile container and processed immediately. All samples were analysed for both spore-formers and total cultivable heterotrophs.
Samples were sonicated for 2 min and heat-shocked at 80 °C for 15 min, at which time appropriate aliquots were placed into Petri dishes and total aerobic spores were enumerated by pour-plate techniques using tryptic soy agar (TSA; Difco) as the growth medium (32 °C for 2 days) (Anonymous, 1980). Samples that were not heat-shocked were enumerated for total aerobic cultivable heterotrophs on TSA. C.f.u. were counted after incubation at 32 °C for up to 7 days. Isolates were selected, purified and stored in glycerol at -80 °C. Identification of purified strains was accomplished by rDNA sequencing (see below). Type strains of various Bacillus species for use as controls were either procured from the American Type Culture Collection or received as gifts from the USDA Research Centre (National Center for Agricultural Utilization Research) collection.
A nutrient sporulation medium (NSM) was used to produce spores (Schaeffer et al., 1965; Nicholson & Setlow, 1990). A single purified colony of the strain to be sporulated was inoculated into NSM liquid medium. After 2–3 days growth at 32 °C, cultures were examined in wet mounts to ascertain the level of sporulation. Once the number of free spores was greater than the number of vegetative cells, the culture was harvested and spores were purified. Spores were purified by treating with lysozyme and washing with salts and detergents (Nicholson & Setlow, 1990). These chemical treatments did not remove the exosporium surrounding the spore coat. Purified spores were resuspended in sterile deionized water, heat-shocked (80 °C for 15 min) to ensure inactivation of the vegetative population and stored at 4 °C in glass tubes.
The refractile nature of the spores was examined by phase-contrast microscopy using an Olympus microscope (BX-60). Non-destructive examination of spores and vegetative cells was also exploited using a field-emission environmental SEM (Phillips XL30). In addition, standard SEM and TEM were utilized to examine surface details and cross-sections, respectively, according to established methods (Cole & Popkin, 1981).
Characterization of spores for various physical and chemical conditions.
Radiation dosimetry at the Co60 source was performed using an ion chamber with accuracy to the USA Bureau of Standards (Coss, 1999). All irradiations were carried out in glass vials using spore samples in water. Spores (108 spores ml-1) were exposed to both 1 Mrad (50 rad s-1 for 330 min) and 0·5 Mrad (25 rad s-1 for 330 min) and survival was quantitatively verified by growing the gamma radiation-treated samples on TSA at 32 °C.
Purified spores (106 spores ml-1) were diluted in PBS (pH 7·2), placed in an uncovered Petri dish and exposed to UV radiation (254 nm; UV Products). At appropriate intervals, samples of spores were removed, diluted serially tenfold in PBS and plated onto NSM agar medium. Plates were incubated at 37 °C for up to 5 days and colonies were counted.
A liquid H2O2 protocol, developed by Riesenman & Nicholson (2000), was modified and used to examine H2O2 resistance in spores. Known concentrations of spore suspensions prepared in PBS (108 spores ml-1) were treated with H2O2 (5 % final concentration) and incubated at room temperature (25 °C) with gentle mixing. After 60 min incubation, 100 µl was removed and diluted in bovine catalase (100 µg ml-1 in PBS). Serial 1 : 10 dilutions of the catalase-treated suspension were prepared in tryptic soy broth (TSB; Difco) to check viability and spread onto TSA for quantitative measurement of H2O2-resistant spores.
To test desiccation resistance, spore suspensions (20 µl) were dispensed onto pre-sterilized metal and glass-fibre discs (103 spores per disc; Millipore). The spore-inoculated discs were incubated in a glass desiccation chamber with a relative humidity of 15 % for 1 or 2 days before c.f.u. were counted on TSA medium. Briefly, the desiccated sample was placed in sterile PBS, vortexed thoroughly and placed in a sonicating water bath (Branson Ultrasonics) for 2 min at room temperature before plating onto TSA medium. Plates were incubated at 32 °C for 2 days and the number of spores that survived was counted. Untreated aliquots of purified spores at equivalent concentrations were included and worked up alongside test aliquots for all treatments as a means of determining relative percentage survivability.
(i) Phenotypic characterization.
The ability to grow in NaCl concentrations of 1–10 % was determined in T1N1 liquid medium (1 % Bacto tryptone containing the appropriate amount of NaCl) and the ability to grow without NaCl was determined in 1 % sterile tryptone water (Colwell & Grimes, 2000). The commercially available Biolog identification system was used, according to the manufacturer's specifications, to characterize utilization of various carbon substrates. In addition, API NE test strips (bioMérieux) were used to characterize the strain further.
(ii) 16S rDNA sequencing.
Approximately 10 ng purified DNA (Johnson, 1981) from liquid cultures was used as a template for PCR amplification. Universal primers (Bact 11 and 1492) were used to amplify the 1·5 kb PCR fragment according to protocols established by Ruimy et al. (1994). Purified amplicons were sequenced (MWG Biotech) and the identity of a given PCR product was verified by bi-directional sequencing analysis. The phylogenetic relationships of organisms covered in this study were determined by comparison of individual 16S rDNA sequences to existing sequences in public databases (http://www.ncbi.nlm.nih.gov/). Evolutionary trees based on parsimony and maximum-likelihood analyses were constructed with PAUP software (Swofford, 1990).
(iii) DNA–DNA hybridization.
Cells were suspended in 0·1 M EDTA (pH 8·0) and cell walls were digested by lysozyme treatment (final concentration, 2 mg ml-1). DNA was isolated by standard procedures (Johnson, 1981). DNA–DNA relatedness was studied by microplate hybridization methods (Ezaki et al., 1989) with photobiotin labelling and colorimetric detection, using 1,2-phenylenediamine (Sigma) as the substrate and streptavidin–peroxidase conjugate (Boehringer Mannheim) as the colorimetric enzyme (Satomi et al., 1997).