Recovery of bacillus spore contaminants from rough surfaces: a challenge to space mission cleanliness control

Abstract

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference 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 spores from 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 compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). 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 four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five 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 Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research.

FIG. 1.

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

FIG. 2.

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

FIG. 3.

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

FIG. 4.

Scanning electron micrograph of Vectran fabric type A showing four fibers and B. atrophaeus spores spotted onto the surface (concentration, 1.3 × 10⁴ per mm²). Bar, 10 μm. A, spores free on fibers; B, spores between fibers; C, spores attached to free, thin fibers; D, spores in furrows of the fibers.

FIG. 5.

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