Barry DiGregorio:

You actually started your career looking for microbes in municipal water systems correct?

Dr. Levin:

My professional career started as a 'sanitary engineer.' During my senior year in high school I met a sanitary engineer who was a commissioned officer in the U.S. public health service. He told me of the many facets to this profession and the multiple scientific and engineering disciplines involved in protecting the public health. Career possibilities included water supply, wastewater, drainage, air pollution, foods, and all aspects of the environmental protection, and the development of relevant processes and products. I applied to the Johns Hopkins University to enroll in its sanitary engineering program and was accepted. The first step was to obtain a bachelor's degree in civil engineering, and then take a master's degree in sanitary engineering and public health. Upon completion of both degrees, I went to work for the Maryland state health department as a junior sanitary engineer. My assigned responsibilities dealt with municipal water supplies, wastewater disposal, industrial waste disposal, shellfish sanitation, and swimming pools. Water quality analysis, especially microbial, was involved in all these activities. Early on I became especially interested in the microbiology-concerned projects I was assigned.

Barry DiGregorio:

You worked with a microbial detection technique called radiorespirometry in the late 1950's that was extremely sensitive for the detection microbes in water and in blood. Are you the inventor of this method and how does it work?

Dr. Levin:

I am the inventor. It is a very simple test, patterned after the long-used, classic method for detecting bacteria. That method placed a sample of the material suspected of bacterial contamination into a test tube containing a liquid broth designed to culture the bacteria. If bacteria were present, they would eat the nutrient and reproduce. At the same time they were exhaling gas as part of their metabolism of the food. Eventually enough gas would be expired to create small, visible bubbles. The bubbles were proof that bacteria were present. Some tests were designed to detect any bacteria. Others were designed to detect specific species. The types of nutrient used determined which bacteria would respond. Varied depending on the specific test, the length of time required to detect the bacteria ranges from one to several days, even up to a week. My invention was simply to add tiny amounts of radioactive nutrient into the nutrient(s) used in the test. Chemically there was no difference between the radioactive molecules and the nonradioactive ones. The bacteria could not tell the difference between them and metabolized them both. However, when radioactive molecules were metabolized the gas produced was radioactive. Methods to detect radioactivity are so sensitive that the gas can be detected within minutes, providing answers almost immediately compared to the length of time required by the classic method. In the standard test, bacteria have to reproduce to about a million per milliliter of culture broth to produce visible bubbles. The radioactive method is so sensitive that as few as ten bacterial cells in the sample can be detected in about half an hour, before any growth occurs. Growth is not needed. I developed the method to detect total bacteria and to detect coliform organisms (of sewage origin) for use in detecting contamination of drinking water and swimming water. This was adopted by several states as an emergency water supply public method. I then developed the method and associated instrumentation to be able to detect and identify specific pathogenic microorganisms of public health interest. The method is now used in hospitals and clinics worldwide to detect human blood infection very quickly.

Barry DiGregorio:

Didn't you have a problem selling the invention initially?

Dr. Levin:

My carbon-labeled microbial respirometry technique worked very well, both to detect and to identify microorganisms. However, potential user agencies feared the public relations aspect of using radioactive material. Of course, hospitals were using increasing amounts of isotopes and X-rays, but even they resisted (until sometime later) expanding that use into microbiological testing. This was frustrating.

Barry DiGregorio:

How did you get involved with NASA?

Dr. Levin:

In 1958, I accompanied my wife, then a reporter for Newsweek magazine, to a Christmas party at the home of the Washington bureau chief, Ernest Lindley. There I met the first NASA administrator, Keith Glennan and we had a nice talk about space research. I had long been interested in the possibility of life beyond the earth. When I was 9 years old, my cousin, pointing out Mars to me, told me about an astronomy course she was taking at college where the possibility of life on mars and elsewhere was discussed. An idea dawned on me at the party. Putting down my martini, I asked, only half-jokingly, whether NASA might ever look for life on Mars. Glennan surprised me by saying he was planning to do so, and that he had just hired an M.D., Clark Randt, to head up a new NASA biology program. Glennan suggested I go see Randt and tell him about my test. I made an appointment very soon after. Randt was most receptive and told me to submit my idea as a proposal for possible funding for me to do the research. This was very exciting, and I promptly went to work crafting a proposal explaining what needed to be done to develop my microbial radiorespirometry experiment and an instrument to perform it on Mars. He said NASA intended to fund several such experiments and to choose a number of them for a Mars lander.

Barry DiGregorio:

When did NASA officially fund you for this?

Dr. Levin:

In 1959, NASA funded my proposal to develop my radiosrespirometry experiment to go to Mars. I named it 'Gulliver,' because it was to seek Lilliputian life forms on a far away land, and I hired a small team to help me in the laboratory. The development went exceedingly well. Within the first year we had developed a suitable nutrient for detection of a broad array of microorganisms, selected and incorporated the radioactive carbon label, and demonstrated the sensitivity and quickness of the technique. Later, NASA changed the name to 'Labeled Release' to indicate the seriousness of its purpose. Before the end of the year we had a working instrument that a subcontractor manufactured to meet our concepts. We tested the instrument on a nearby playground and it promptly detected microorganisms.

Barry DiGregorio:

Can you describe how the Gulliver worked?

Dr. Levin:

Barry DiGregorio:

How did you eventually get a place as a Principal Investigator for biology on the Viking mission to Mars?

Dr. Levin:

Ten years after I started my work for NASA, the agency announced the creation of the Viking mission to Mars: twin spacecraft to be launched in 1975 to rendezvous with the red planet in 1976. Each spacecraft consisted of an orbiter that was to circle mars, and a lander that was to be gently deposited on the surface. The first lander was scheduled to land July 4, 1976. NASA then asked for proposals for experiments for the mission, the chief announced objective of which was to search for life. All of us developing life detection experiments under NASA's science program submitted proposals applying for the Viking mission. The selection process was rigorous, with each candidate being examined by 4 separate review panels, one of NASA scientists and the others consisting of renowned university researchers. Four experiments were selected to be flown aboard each Viking lander. The excellent success in rapidly detecting the broadest possible array of microorganisms in soils from around the world and also in laboratory cultures, together with its small and efficient instrumentation, won a spot for my labeled release experiment. The other experiments selected were the "Wolf Trap," that monitored a vial of water for increasing turbidity after a soil sample was placed in it; the 'Gas Exchange' experiment that measured for changes in the composition of the atmosphere above a vial of "chicken soup" nutrient into which the soil sample was placed; and the 'Pyrolytic Release' experiment that was a modification of my dark release experiment which looked for the photosynthetic incorporation of radioactive carbon dioxide and/or carbon monoxide by organisms in the soil sample.

Barry DiGregorio:

But only three of the biology experiments were actually flown. What happened to the fourth biology experiment?

Dr. Levin:

Intense development of the experiments and the Viking spacecraft went on in parallel, all under the direction of Jim Martin, Sr., the Viking project manager. Frequent meetings were held to assess progress against the rigid schedules set for each component over the ensuing 10 years until launch. When coming down to the wire for instrument delivery, some 2 years before launch since the instruments had to be 'buttoned up' and placed within the spacecraft by then, it became apparent that the landers had space, weight and power problems, and could take only 3 life detection instruments. A frantic selection process ensued. NASA constructed a selection rationale based on the Mars environment. The concept was to select experiments that tested for life under Martian conditions. The principal environmental condition was water. It was, therefore, decided to select experiments that covered the entire spectrum of possible water abundances. The Pyrolytic Release experiment was based on the presumption that mars was bone dry and thus added no water. The Labeled Release experiment added only one drop of water, placed at the center of the soil sample so that, as it migrated to the edges, a continuum of wetness would be supplied, declining with distance from the center. The Gas Exchange experiment added enough nutrient solution such that the entire sample was wetted. The Wolf Trap, however, placed a small sample of soil into a relatively large volume of water, inundating any microorganisms in the sample. Accordingly, NASA eliminated the Wolf Trap. Development of the other three experiments was completed and they were, indeed, flown to mars aboard each of the Viking spacecraft.

Barry DiGregorio:

What kinds of terrestrial environments did you test your instruments in?

Dr. Levin:

We tested the LR experiment in the laboratory on pure cultures, mixed cultures and wild cultures, all including the widest array of microbial genera we could get. In addition, we obtained soil samples from widely differing geographic regions including the Antarctic, the Gobi Desert and Alaska. Test instruments were constructed so that the samples could be tested under anaerobic conditions and simulated Martian conditions as well as their normal environment. Field tests with 4 generations of LR instruments that obtained their own samples were made locally, on deserts, such as Death Valley, on mountains, such as White Mountain, CA, the Rocky Mountains, CO, the Salton Sea flats, CA, and on a wide variety of other locations.

All of the biology instruments looked for evidence of active metabolism. The LR sought catabolism of organic substrates and respiration of gases produced. The PR sought evidence of active photosynthetic fixation of CO and CO₂. The GEX looked for metabolically caused changes in the headspace atmosphere above its sample of soil. The Wolf Trap (if flown) would have looked for increased opacity in a suspension of the soil as evidence of growth and metabolism. All agreed, as had the various selection teams, that the observation metabolism would provide the surest evidence for life.

Barry DiGregorio:

What measures were taken by NASA to insure the Viking biology experiments would not simply detect any surviving Earth microbes carried to Mars within the spacecraft?

Dr. Levin:

From the start of the exploration of Mars, NASA and COSPAR were concerned with the possibility that Mars might be infected with terrestrial life brought on spacecraft, and that experiments looking for Martian life might detect terrestrial microorganisms brought by the spacecraft and reach a false conclusion. The Viking project was planned to preclude any chance of contamination of Mars to one in a million. This number was achieved through a series of calculations and estimates on the probabilities assigned to each step required for the delivery of a viable microorganism from earth to Mars. Accordingly, NASA established strict procurement rules in accordance with recommendations from Cospar. All manufacturers of Viking components were required to fabricate and assemble their products in clean rooms using aseptic technology. The rooms were monitored with "coupons," similar to microscope slides, distributed around the room. These were periodically cultured in microbiological media to assess microbial populations, if any, and appropriate measures taken, if indicated. Chemical cleansing was also performed on all product surfaces. The components were then aseptically shipped to the spacecraft assembly building. There they entered a clean room where the Viking spacecraft were assembled. The components were integrated into the spacecraft under clean room technology. When the entire Viking spacecraft were assembled, they were heated to a temperature and for a period of time to sterilized the spacecraft and all components. The spacecraft were then placed in shields that maintained their sterility and transported to the launch platform for attachment with the Titan booster rocket. The shields remained in place until after the spacecraft had been launched and had exited the Earth's atmosphere in order to preclude contamination by air-borne microorganisms. After the spacecraft departed the earth's atmosphere, the shields were explosively ejected.

Barry DiGregorio:

Why did NASA include the same set of biology instruments on Viking Lander 1 and 2?

Dr. Levin:

The whole idea of sending two spacecraft was to have a backup in the quite likely event that one was lost on takeoff, space travel or landing. Thus, the very best selection of instruments was made, and the same array was placed on each spacecraft.

Barry DiGregorio:

How long did the actual Viking biological sampling testing period on Mars last?

Dr. Levin:

Each instrument went through its own cycle of testing. An 8-sol cycle (sol=one Martian day), beginning about 2 sols after the samples were acquired, was used for each of the 9 completed experiments of the LR instrument. However, inasmuch as two of the samples tested had been held in the soil hopper for 2 and 3 months, respectively, those periods might be added to the tests. The GEX ran 5 tests ranging in length from 0.1 to 103 sols. The PR ran 9 experiments, each soil sample being incubated for 120 hours. As most of the biology samples were shared, storage times for the PR also ranged from the approximate 2-day pre-test period to 139 sols before testing.

Barry DiGregorio:

In 1997 you made the claim that you discovered microbial life on Mars with Viking in 1976. Why did it take so long for you to reach this conclusion?

Dr. Levin:

Barry DiGregorio:

Why have all the NASA Mars missions since Viking ignored placing updated life sciences experiments aboard their spacecraft?

Dr. Levin:

The failure to pursue NASA's highest priority (the search for life in the solar system), and the goal NASA once described as "probably the greatest experiment in the history of science," cannot be logically explained. It results from NASA's fear of finding out that its original conclusion about Viking was wrong, supplemented by philosophical and religious elements who insist, for non-scientific reasons, there can be no life elsewhere but Earth.

Barry DiGregorio:

What kind of robotic life detection experiment do you think we could send to Mars that would unambiguously demonstrate once and for that life does or doesn't exist?

Dr. Levin:

I have proposed my chiral LR experiment to NASA several times since 1995, and to ESA and the Canadian Space Agency. It has been rejected (1995) or ignored every time. The only chance to investigate life on Mars came when I was a member of the JPL Mars Oxidant team, the MOx was an experiment designed to seek the putative oxidant on Mars and was placed aboard the Russian Mars 96 mission. I managed to include a chiral life detection test by modifying a small portion of the MOx experiment. Unfortunately, that spacecraft failed to reach Mars. The detection of active chiral metabolism in a soil sample would be uncontrovertibly evidence for life. Everyone I have spoken to about that experiment has agreed that it could give unambiguous results, including Dr. Wesley Huntress, former NASA Deputy Administrator for Science, and even Chris McKay, who worked with me on MOx.

Barry DiGregorio:

Is there any possibility that all three Viking biology experiments gave indications for life on Mars? For example, the GEX samples demonstrated that CO₂ was being absorbed while giving off oxygen. Couldn't this be interpreted as photosynthesis? If not, why?

Dr. Levin:

No. The GEX reported an abrupt (about 2 hr) outpouring of O₂. This occurred in the dark, and even before any liquid nutrient was added to the soil. Merely exposing the soil to the humidity supplied by the aqueous nutrient solution caused the brief response. This is not an indication of biology, but of chemistry - if the response were indeed correct - the raw data obtained by GEX were never published. What was published was the result of applying various correction factors to that data. Those factors themselves are suspect in that they presume knowledge about the Martian soil that we still do not have. Moreover, upon heating to the control temperature, the GEX still gave a positive response.

Barry DiGregorio:

Seven out of nine PR experiments on Mars showed minute quantities of organic material had formed from the soil samples in contrast to the negative GCMS findings. Two of these PR samples tested positive for organic material even though they were run totally in the dark without the solar simulating Xenon lamp on. How can this be explained if not by organisms?

Dr. Levin:

Prior to the mission, the PR experimenters stated that, because of the variable first peak results obtained in testing, a positive result would require a response in the amount of about 10,000 cpm. The PR Mars responses were only about 100 cpm, well within the noise level. Moreover, both before and after the mission, Jerry Hubbard reported PR experiments in which, even fitted with the UV filter, the instrument produced results from sterilized glass beads and soils above the "active" responses obtained on Mars. The explanation is that, both on Earth and Mars, the PR produced small amounts of organic matter. Moreover, this organic matter remained and even accumulated in the PR over time. However, the survival of the organics in the PR proved that there were no oxidants in the soil capable of destroying the organics.

Barry DiGregorio:

Finally a report in the November 7^TH 2003 issue of Science "Mars-Like Soils in the Atacama Desert, Chile, and the Dry Limit of Microbial Life" casts heavy doubt on whether Viking and in particular your LR instrument detected living microbes on Mars. Have you read this report and do you have a rebuttal?

Dr. Levin:

The authors, three of whom I personally know to be fully aware of my numerous and recent publications on the subject of the presumptive oxidants on Mars, problems with the results of the Viking GCMS, and the chiral LR experiment I have published and proposed to NASA several times, mislead the reader by omitting references that contradict their approach and conclusions. Particularly egregious is their presentation of the very experiment I have reviewed with each of them, the use of chiral compounds to distinguish biological from chemical reactions, as their own. Chris McKay has told me he deliberately omits references to the papers I presented to the preeminent Astrobiology Division meetings of SPIE (The International Society for Optical Engineering) because their publications are not "peer reviewed." This conveniently allows the authors to ignore addressing the specific points I made in my 1997 publication that first announced that the LR had detected living microorganisms, and in my many follow-up papers. However, even this protective device does not shield the authors from papers that I did publish in peer reviewed journals that:

1. Rule out strong oxidants as the cause of the LR response.
2. Rule out UV as the source of the LR response.
3. Shows that the extended control regimens run on Mars strongly mitigate against chemical or physical cause of LR reaction.
4. Shows a metabolic response from Antarctic soil in which GCMS detected no organics.
5. Rule out specific oxidants recently proposed by others, and rule out oxidants in general.

Not to mention any of the above is indicative of an extreme determination to mislead the reading public.

With respect to the article itself, there are a number of points I will comment on to Science. They show a somewhat amateurish, but still strongly biased approach with respect to microbiological technique, the use of appropriate controls, the interpretation of results, and the use of long-dead straw men arguments in a vain attempt to cover up, rather than to discuss objectively, the results of the Mars Viking LR experiment.