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Methods and Implementation

NASA uses a variety of methods to measure, control and reduce spacecraft microbial contamination for planetary protection purposes. Assembly of spacecraft hardware is carefully controlled and often takes place in clean-room facilities using, aseptic techniques in order to meet planetary protection requirements. Dry heat microbial reduction techniques first used on the Viking spacecraft are still used today. Measurement techniques are cultivation-based microbial assays using well characterized biological methods.

Clean Rooms and Microbial Barriers

NASA requires that planetary protection procedures involving sterile items and sample processing must be conducted in Class 100 clean rooms, as defined by federal standard (equivalent to ISO Class 5). Such clean rooms feature laminar-air-flow systems to filter out contaminants; these systems work by keeping the air within a space moving in one direction along parallel flow lines at a uniform velocity through very fine filters. Planetary protection procedures specify the types of devices that may be used for air sampling in these environments.


Clean room assembly of a Viking component

Clean room assembly of a Viking component

Individuals executing planetary protection procedures during assembly, testing and launch operations must wear protective clothing to ensure that they do not bring contamination into the procedures. Clean-room garmenting requirements include the use of hoods, masks, surgical gloves, booties and the protective suits known as bunny suits. Garmenting requirements may be slightly less stringent for planetary protection work during non-critical operations – for instance, before cleaning and microbial-assay activities.

Planetary protection plans may call for the use of active or passive microbial barriers to protect against the recontamination of spacecraft after microbial reduction processing. For planetary protection purposes, microbial barriers may operate under high pressure or ambient pressure. If pressurized, a barrier must be maintained at a specific, continuous static pressure level above ambient air pressure to prevent microbiological recontamination. Air must flow from inside a pressurized microbial barrier toward the outside. A barrier operating at ambient air pressure employs filters to protect against microbiological recontamination. These ambient filter systems must be capable of retaining 99.97 percent of all particles or organisms greater than 3 × 10^ -7 ^ meter in size.

Passive detection methods include the use of sterile stainless steel fallout strips and Teflon fallout ribbon to collect samples of airborne microbial contamination accumulating on surfaces. Periodically, these strips or ribbons are assayed using one of the methods discussed below.

Sterilization


Clean room technicians prepare a Viking Lander for dry heat sterlization

Preparing a Viking Lander for dry heat sterlization

NASA currently has only one approved method of spacecraft sterilization – dry heat microbial reduction. This technique was used on the Viking Mars landers, which were built and launched in the 1970s. Before launching the Viking landers, NASA cleaned them to reduce their total surface biological burden to a specific level, then packaged them in a fully enclosed bioshield (resembling a large casserole dish) and baked them in an oven at 111.7 degrees Celsius (233.1 degrees Fahrenheit) for 30 hours. Advanced materials, electronics, and other heat-sensitive equipment being used on spacecraft today could be damaged by such high-temperature treatment, however.

Consequently, NASA researchers are developing an alternative sterilization method, a low-temperature, vapor-phase, hydrogen-peroxide-based sterilization process. The hydrogen-peroxide-based process has been used in the pharmaceutical industry for some time without degrading component materials or devices. The process being developed for planetary protection applications uses an off-the-shelf hydrogen peroxide sterilizer and involves repeated sterilization cycles. A single cycle features evacuation of the sterilization chamber to form a vacuum and injection of hydrogen peroxide into the chamber to establish a specified vapor concentration. Researchers have tested the efficacy of this process using chemical indicators to ensure that peroxide has been adequately dispersed for sterilization and biological indicators such as microbial spores to verify that sterilization has occurred. Researchers have also tested a number of different metals for compatibility with the hydrogen peroxide process, exposing samples to the process and then examining them for material property changes. Other types of spacecraft materials tested for compatibility include tapes and encapsulating materials, thermal blankets, solder, composite materials, gaskets, lubricants, adhesives and plastics. So far it appears that the hydrogen peroxide sterilization process appears to be an effective alternative to dry-heat sterilization.

Current Assay Methods

The current standard assay method is derived from the bioassay work performed on the Mars Viking spacecraft prior to their launch in 1975. The Viking project conducted more than 6000 assays in total on the entire mission and averaged a minimum of 1000 assays on each of the landed spacecraft. This research established requirements for Category IV-A missions, demonstrated the feasibility of Category IV-B missions (terminal sterilization), and refined analytical procedures currently used today.

The standard assay procedure targets spore-forming organisms using a cultivation-based technique. In general, this procedure requires that a sterile cotton swab wetted with sterile water be used for sampling. A sample area of no more than 25 square centimeters per swab is taken. After sampling, the swab is inserted aseptically into a test tube containing sterile water and processed in the laboratory within a 24 hour period. Processing the sample includes extracting it via vortex and sonication, followed by a heat shock step of 80 degrees Celsius (176 degrees Fahrenheit). The resulting solution is plated into nutrient agar and incubated for 72 hours. Contamination load estimates are calculated from the number of microbial colonies grown and counted. A variation on this procedure is the wipe method, using a cleanroom cloth instead of a swab, which allows sampling of a larger area. The images below illustrate some of the steps involved in the assay method.

A clean room technician collects the samples.

Collecting the samples

These samples are bottled, labeled, and ready to be processed.

Collected samples ready to be processed

Spore colonies are visible in this petri dish following incubation.

Spore colonies after incubation

Research In New Assay Techniques

Future solar system exploration missions may require a more precise way of accounting for viable and non viable bacteria at the time of launch. Even more stringent analytical methods may be required for samples that will be returned to Earth. The current requirement does not provide data on thermally intolerant viable organisms, viable but non-culturable organisms, non-viable organisms, and molecular fragments of organisms. Therefore, the development of new, quick, more sensitive assay techniques to precisely detect both culturable and non-culturable microorganisms and fragments of organisms on spacecraft are being developed.

The focus of the current research is to advance the state of the art and augment the current method, first used on Viking, which is limited to spore analysis. The ultimate goal of the research is not to increase the cleanliness requirement currently levied on various missions, but rather to better understand the nature of the bioburden through the use of well-characterized standard methods. Consequently, an array of standard techniques is needed to provide various analytical methodologies that can be used to assess bioburden, depending upon mission specifications.

Preliminary research has been conducted on a number of methods that may be suitable for use on spacecraft. Methods under consideration include Polymerase Chain Reaction (PCR), Epifluorescence Air Monitoring and Analysis of Live/Dead Cells, Limulus Amebocyte Lysate, Adenosine Triphosphate, Mass Spectrometry of Proteins and Lipids, and Capillary Electrophoresis. The ultimate goal of the research and development of each method is to provide a comprehensive and statistically valid data package which can provide the means by which a NASA specification can be proposed. The certification process to support this goal is lengthy and requires substantial fundamental research and method standardization. Two methods being considered for near-term submission to NASA for use on spacecraft are Limulus Amebocyte Lysate assay, and Adenosine Triphosphate assay.

The Limulus Amebocyte Lysate (LAL) assay method currently under development tests for the presence of microbial cell wall materials. The method is based upon an enzyme cascade, isolated from the blood cells, or amebocytes, of the primitive horseshoe crab, Limulus polyphemus, which is part of its anti-microbial defense mechanisms. The method detects lipopolysaccharide (endotoxins) and beta glucan from Gram negative bacteria, yeast and mold, which is a general indicator of the bioburden present on space-bound hardware. The LAL method was originally developed for use in the pharmaceutical industry, where the presence of endotoxin in injectable drugs and medical devices is known to cause fever in patients. Its application under development for planetary protection has been adapted to the use of ultra-clean polyester swabs to sample spacecraft surfaces. Microbial material is extracted from the swab with water and quantified using either a portable instrument or laboratory-based microplate reader. The method has several strong points; it is exquisitely sensitive (10-12 g lipopolysaccharide), rapid (15 – 30 minutes), and reactive with both live organisms as well as organic material from dead or non-cultivable organisms. Research is continuing that would further increase the breadth of microbes reactive to the assay. This would further improve its usefulness to assess microbial bioburden and organic cleanliness of spacecraft.

Adenosine Triphosphate (ATP) is generated by all living cells, including microorganisms. Used as an essential energy source, ATP may be found in both live and dead cells in concentrations that directly relate to the size of the cellular body. As a result, the presence of ATP is a good indication of biological contamination. The method was originally developed by the Kikkoman Corporation and is widely used in the food industry to determine gross bacterial contamination. The standard Kikkoman method uses a bioluminescence regent containing firefly luciferin and luciferase. ATP transfers its energy to the luciferen molecule. The reaction is catalyzed by the presence of the luciferase enzyme and magnesium ions. The resulting bioluminescence is directly proportional to the amount of ATP in the total cell mass present in the sample. The analysis is performed using a portable ultra-violet spectrophotometer, and can be completed in about 1 hour. Because the amount of ATP per cell varies (ranging from trace amounts for spores to high amounts for yeast), a quantitative calibration between the amount of ATP and the total cell number will need to be established. However, the ATP assay is a good indicator of cleanliness if correlated with the other methods based on cell mass.