laborious technology remains ineffective in assessing the total bioburden of spacecraft, since some microorganisms may still elude cultivation.

The current NASA approach (described in Chapter 2) of using the cultivation of spore-forming bacteria does not provide an accurate estimate of total bioburden. Moreover, it is possible that some of the organisms of potentially greatest concern for the forward contamination of Mars, such as psychrophiles (see Chapter 5), may not be well correlated with the counts of spore-forming bacteria on spacecraft. New rapid and less expensive methods that provide more accurate estimates of total viable bioburden, and that do not require growth of microorganisms in the laboratory, are described below.

Cytological Methods

Epifluorescent Microscopy

The use of epifluorescent microscopy3 and flow cytometry4 to directly count cells labeled with high-quantumyield fluorescent nucleic acids stains such as acridine orange, DAPI, and Sybr-Gold has increased estimates of microbial biomass in nature several-fold and has revised understanding of the importance of microorganisms in the natural environment. However, the detection of nucleic acids is not an indicator of viability. Fluorescent stains must be used in concert with other assays that enable the detection of essential metabolites or enzymes, if accurate estimates of total viable biomass are to be obtained.

Detection of Membrane Integrity

Membrane integrity is an indication of cell viability that can be detected using commercial fluorescence stains such as the BacLight Live/Dead kit.5 This kit uses two nucleic acid stains, SYTO 9 and propidium iodide, which differ in their ability to penetrate healthy cells. SYTO 9 can penetrate both live and dead cells, whereas propidium iodide can only penetrate cells with damaged membranes (dead or damaged cells). When those stains are used together, live cells fluoresce green and dead or damaged cells are red when viewed with an epifluorescent microscopic. Epifluorescent microscopy techniques will require either witness coupons (replicate coupons made of the same material as the spacecraft that can follow the spacecraft through the entire assembly and cleaning process and can be mounted under a microscope for viewing) or microscopic procedures in the clean-room and launch facilities to examine surfaces directly.6 Irrespective of how cells are stained, the sensitivity of all light microscopy techniques is constrained by the volume of material that can be viewed in a field. At a magnification of 1000×, the fluid volume represented in a viewing field will be ~0.05 µl. There is a 50 percent probability of finding a single cell in a field if the concentration of cells in the original sample is 2 × 103 cells per milliliter.

Flow Cytometry and Cell Sorting

Flow cytometers equipped with a fluorescently activated cell sorter (FACS) analyze particles in a laminar flow by their light-scattering and fluorescent properties.7 Flow cytometers are used to enumerate and characterize

3  

Epifluorescent microscopy relies on the excitation of susceptible molecules in a sample with short-wavelength, high-energy light and observation of the emitted lower-energy light (fluorescence). Susceptible molecules include autofluorescent compounds such as chlorophyll and F420 (an enzyme cofactor found in methanogens) or fluorophores that bind with specific cell structures. Epifluorescent microscopy enables researchers to observe and detect cellular components that were not visible with conventional contrast microscopy and is sensitive enough to detect a single molecule.

4  

Flow cytometry is the analysis of cells that have been labeled with fluorophores as they pass in a narrow stream through a laser beam.

5  

BacLight is just one of many commercially available kits that may be used for detecting viable biomass.

6  

The suggested technique would be performed on witness coupons and would leave no residue. Should microscopy techniques be developed that could be used to examine the spacecraft directly, any residues would presumably be cleaned before launch.

7  

This technology was originally developed to analyze cell suspensions combined with fluorescently labeled antibodies by conjugated



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