Comparison of the Milliflex RMDS Method with Conventional Cultivation Assays for Bioburden Determination on Spacecraft and in Cleanrooms

Kurzfassung

The COSPAR planetary protection guidelines define bioburden limits for interplanetary missions that aim to prevent biological contamination of both the target celestial body and the Earth. This principle arises from the scientific need to preserve planetary conditions for future biological and organic constituent exploration—especially astrobiology. All bioburden constraints are defined with respect to the number of aerobic microorganisms that survive a heat shock of 80°C for 15 min (“spores”) and are cultured on TSA at 32°C for 72 h. For the assembly and integration of space hardware under bioburden-controlled conditions a method giving results in a shorter period of time would be highly preferable. We have compared the Milliflex RMDS (Rapid Microbial Detection System) with conventional cultivation assays using the same biological endpoint, i.e. the colony forming capability of microorganisms. With the Milliflex RMDS aqueous samples are filtered with membrane filters. These filters are placed on top of nutrient agar plates and incubated under appropriate conditions so that viable microorganisms grow into microcolonies. The cells are lysed directly on the filter and the number of microcolonies is determined by the detection of ATP with a luciferin-luciferase reaction. The resulting in bioluminescence is measured with a CCD camera. For method comparison we have used pure cultures of spores from Bacillus atrophaeus (DSM 675), Bacillus safensis (DSM 19292 T), Bacillus thuringensis (E24 and DSM 2046 T), Bacillus megaterium (2c1 and ATCC 14581) as well as environmental samples from cleanrooms. Bioburden assays with the RMDS are fast and sensitive. With this technique the bioburden detection time is reduced from over 72 h to less than 8 h. Both methods show equivalent sensitivity and are able to detect one CFU. Therefore this Milliflex RMDS method can be used as an alternative to the standard assays used for bioburden determination on spacecraft and in associated environments.