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Four decades after NASA’s Viking landers acquired humanity’s first view of the surface of Mars, a planet whose reality lived in the imaginations of skywatchers for millennia, a definitive smoking gun for microbial life there still eludes scientists.

Since the two Viking missions turned up no concrete evidence for life on Mars, at least according to the interpretations of most astrobiologists, all landers dispatched to the red planet have carried no similar instrumentation for a direct detection of living microbes.

Mars missions since Viking have followed the trail of water, seeking evidence that Mars was once habitable. The latest results obtained by NASA’s Curiosity rover point to the answer to that question being yes.

NASA last week celebrated the 40th anniversary of the Viking 1 probe’s touchdown on Mars on July 20, 1976, becoming the first lander to reach the Martian surface and return images and scientific data.

The twin Viking 2 lander reached Mars a few weeks later on Sept. 3.

“We ended up with more questions than we had when we formulated that mission,” said Ellen Stofan, NASA’s chief scientist, and daughter of a member of the Viking team.

Viking was NASA’s boldest robotic mission to date, with two pairs of landers and orbiters each launched aboard Titan-Centaur rockets from Cape Canaveral three weeks apart in August and September 1975.

Each spacecraft included two parts, with the descent probe designed to detach once the carrier module slipped into orbit around the red planet. Viking 1 was originally due to land July 4, 1976, on the 200th anniversary of U.S. Declaration of Independence.

But engineers looking over images from the Viking 1 orbiter deemed the planned landing site unsafe, prompting quick thinking to find another target for the lander.

Using a heat shield, parachutes and throttled rocket thrusters, Viking 1 reached the Martian volcanic plain at Chryse Planitia, then beamed to Earth the first images from the planet.

Equipped with eyes on the ground, scientists immediately began piecing together Mars’ geologic puzzle, a thrilling, but sometimes frustrating, process that continues to this day.

“To the geologists that worked on this mission, right away they could start understanding that that hill on the distant horizon was actually the rim of an impact crater, (and) that those rocks across the surface were probably excavated and dropped onto the surface when that crater formed,” Stofan said.

Carrying a complex suite of instruments to look for microbial life, measure seismic activity and weather, and study the properties of Martian rocks and soils, the Viking landers tried to do it all.

“Viking is one the greatest missions that NASA has ever undertaken,” said Roger Lanius, a former NASA chief historian who is now a senior official at the Smithsonian Institution.

Originally dubbed Voyager — not the famed outer solar system probes — the tandem Mars mission was initially slated to launch on a huge Saturn 5 rocket. The hefty payload consisted of an orbiter and a lander that would pave the way for follow-on human missions, and the mission would have cost $2 billion in 1967 dollars, according to Erik Conway, the resident historian at NASA’s Jet Propulsion Laboratory.

That is more than $14 billion at today’s dollar value, a cost deemed unaffordable in the wake of the expensive Apollo moon landing program.

The downsized Viking program cost NASA about $1 billion in the 1970s, equivalent to around $4 billion today.

Each lander launched piggyback on the Viking orbiters, then separated to streak through the Martian atmosphere protected by an ablative cork-like heat shield. A 53-foot (16-meter) diameter polyester parachute and three hydrazine-fueled terminal descent thrusters helped slow the three-legged lander’s speed through the plant’s rarefied atmosphere, and a radar guided the plutonium-powered craft to its landing site.

Each lander had two general purpose computer channels, each with storage capacity equivalent to 18,000 words. The spacecraft recorded science data to a tape recorder for long-term storage before eventual transmission back to Earth

The stationary probes made groundbreaking discoveries about the history of Mars, including clues indicating the planet’s atmosphere may have leaked into space, driving climate change. The landers also reported on Martian weather and seasons.

But Viking’s most ambitious experiment was to search for life, and the results deflated hopes that the mission would find iron-clad evidence that microbes inhabit the alien world.

“Viking, of course, had a very public role in trying to determine whether or not there was some biological material they might encounter on the red planet,” Lanius said last week at a Viking 40th anniversary celebration at NASA’s Langley Research Center in Virginia. “That was a major part of the mission, not the only part by any means … but from the public’s perspective, I think the life story is really important.”

Lanius thinks scientists may have over-sold the potential discovery of active life in the run-up to Viking.

Carl Sagan, the famous celebrity-astronomer, was a member of the Viking science team, and the idea for the highly-rated Cosmos television series was hatched during his time on the Viking mission.

Nevertheless, Lanius argues the Viking life experiments’ inconclusive results dampened the spirits of researchers and the public, and was one of several reasons NASA did not send another mission to Mars for 17 years.

“It was played up celebrity scientists like Carl Sagan, who thought they were going to find something and ballyhooed it on a regular basis including on Johnny Carson (The Tonight Show) at night,” Lanius said last week.

“What we found was apparently nobody home,” said Penny Boston, director of NASA’s Astrobiology Institute. “That was a big shock, at least to me, as then a member of the public who was sort of expecting — naively, more or less — instant results. I wanted to hear that year stuff was living. It was giving us evidence of that. So this was a really big bummer.

“I was sort of set aback,” Boston said. “I was thinking, ‘Gosh, I want to work in exobiology, as we called it at the time, and now it seems like it’s just a pile of rocks, and there’s no life there at all.'”

The Viking results “really put a dark cloud over mission thinking for a long time,” Boston said.

The biological package on each lander included three instruments, each tackling the life question differently. One experiment sought signs of carbon-based organisms, and another tried to detect metabolic processes. Both returned a negative finding.

A third instrument, called Labeled Release, injected nutrients into a radioactive solution with Martian soil, creating a culture that could be monitored for growth. The objective was to look for carbon dioxide or methane gas bubbles released by microbes, a process similar to the way inspectors on Earth check the quality of drinking water.

The results of the that bio-investigation are still open to interpretation, said Gilbert Levin, a former sewage engineer who designed the Labeled Release experiment.

In fact, to Levin, the outcome of his experiment is crystal clear.

Take Martian soil, put it in a sealed test chamber, douse it with a radioactive fluid and organic nutrients, then eureka?

“Here is gas streaming out immediately,” Levin recalled. “We were so excited. We sent out and got champagne and cigars.”

Each lander then carried out the same experiment with a lifeless control sample irradiated and heated to scorching temperatures. And?

“Much greater than a 3-sigma difference,” the 92-year-old Levin said last week, meaning that something was very different about the freshly-sourced Martian soil. “We clearly had now satisfied the pre-mission requirements for the detection of life.”

Levin said there was a “modest” difference in the experiment’s signature on Mars and the result of a similar measurement made on soil collected from California, which he said adds another check to the scorecard favoring an affirmative result for life.

While most scientists acknowledge the results of Viking’s Labeled Release experiment were consistent with active microbial life, the lack of supporting data from the landers’ other instruments threw a shade of doubt over the findings. For example, outgassing of oxygen from the Martian soil detected by Viking’s gas chromatograph is most easily explained by a chemical reaction, not microorganisms, scientists concluded.

“It showed us really how little we did understand about biology at the time,” Boston said. “This was not the fault of the community. The best science of the time was put into that mission. Of course, it (also) showed us how little we knew about Mars.”

Viking found no clear yes/no answer to the life question, Boston said.

“The conclusion by the principal experimenters (was that) a biological interpretation of the results was unlikely,” said Joel Levine, a professor of applied science at the College of William and Mary, and a former member of the Viking science team at NASA’s Langley Research Center.

“One of the criticisms that one could level at the biological package is that, in retrospect, there were clear logical gaps that were left hanging,” Boston said.

NASA did not send another robot toward Mars until 1992, when the Mars Observer orbiter blasted off from Florida. But that spacecraft was lost three days before reaching Mars, and the next successful red planet probe was Mars Pathfinder, the first in a series of NASA rovers that touched down July 4, 1997, 21 years after Viking 1’s original landing date.

Many scientists blame the long gap between Mars missions on the sour taste left by Viking’s discovery of an apparently barren world, blowing up fantastical expectations that microbes would be easy to unroot and analyze on another planet.

“Of course, there were other reasons why we didn’t go back with a successful mission to Mars for 20 years, but this failure of instant gratification, where maybe we thought that it was easier to find life than it appears to have been, was a definite component,” Boston said.

Conway, the JPL historian, said the space agency’s science budget was under pressure in the late 1970s to help pay for development of the space shuttle. In the 1980s, the Reagan administration backed large astrophysics programs — particularly the Hubble Space Telescope — at the expense of planetary science, he said.

“The gap would not have been as bad as it turned out to be had Mars Observer worked, but there are very specific political reasons that there was almost no funding for planetary science, except for Galileo (the Jupiter orbiter), during that timeframe,” Conway said last week.

Since Mars Pathfinder, NASA’s explorers at the red planet have been on the hunt for water, not life.

NASA’s Spirit and Opportunity rovers launched in 2003 and found evidence of ancient hot springs and a coastline with the discovery of fine-grained silica soil and layered rock patterns, environments that could support hardy microorganisms known to live on Earth. Opportunity also returned data suggesting water on Mars may have been drinkable, before an acidic period in the planet’s ancient past, according to mission scientists.

NASA’s Phoenix lander arrived at Mars in May 2008, descending to Mars’ arctic plains for a five-month mission that scooped up the uppermost layer of soil to find ice deposits lying just inches deep. Phoenix also detected light snowfall from high-altitude cirrus clouds, adding to scientists’ understanding of the Martian water cycle.

Phoenix discovered perchlorate, an unexpected chemical in Martian soil that is toxic to some Earth-based life forms and serves as food for others. Perchlorate also has implications for future human expeditions to Mars because it can be refined into rocket fuel.

The Curiosity rover has traversed across Gale Crater since August 2012, and measurements collected at multiple stops near the mission’s landing site led scientists to surmise the 96-mile (154-kilometer) impact basin once had flowing rivers and a large lake, or system of lakes.

One of the most eye-catching discoveries in recent years comes from aerial imagery taken by a high-resolution camera aboard NASA’s Mars Reconnaissance Orbiter. Scientists analyzing the images, and spectral data to go with it, found intermittent seasonal briny water flows on steep hillsides and crater walls, an environment that could be ripe for microbial life today.

The flows could be triggered by the warmth of the sun causing shallow ice to melt and flow down the slopes, or the recurring features, which appear as dark streaks to MRO’s camera, could stem from water bubbling up from shallow aquifers.

“That means we have the potential to access that subsurface water on Mars quite easily,” Stofan said. “So, again, it goes back to this question (of life). If there’s life on Mars, that’s probably the environment in which we would find it potentially in the future, so these are extremely exciting sites for potential future exploration.”

But finding a recognizable organism near one of the water flows will be difficult. NASA has designated the flow sites as “special regions” that should be avoided by humans or any spacecraft not sterilized with extreme care, and many scientists don’t agree on what a new space instrument designed to find life should measure.

One dream scenario proposed by NASA officials planning human missions to Mars might involve an astronaut base in orbit around the planet, or on the surface miles away from the sensitive water flows. In such a case, crew members could direct ultra-clean rovers via real-time remote control radio links to explore for life without putting astronauts, or the potential alien microbes, in jeopardy from contamination.

Rover drivers on Earth can only send the Curiosity and Opportunity craft pre-programmed commands due to the minutes-long communications lag. At closer distances, astronauts could drive rover scouts as they would fly a drone or play a video game.

Two rovers set for launch in 2020 will come closer to resolving the Martian life quandary than any mission since the Viking landers.

Europe’s ExoMars rover has a drill to bore up to 6 feet (2 meters) into the Martian crust and pull out core samples. Due to arrive at Mars in early 2021, the craft will dump the material into an on-board mini-laboratory jointly developed by U.S. and European scientists charged with sniffing out low concentrations of organics, then determining whether the molecules are from extinct or extant life.

It’s a big question, and one that hasn’t been directly posed by a Mars mission since Viking.

The ExoMars rover’s focus is on underground samples, where any existing life or remains of extinct organisms could be better preserved, shielded from ionizing radiation at the planet’s surface.

An immobile science station going to the red planet with the ExoMars rover in 2020 may also be able to conduct a follow-up Labeled Release measurement similar to the Viking experiment, Levin said. The jury is still out on whether that objective will be part of the ExoMars flight plan, he said.

The ExoMars rover will be systematically cleansed before its launch, and it will be the first interplanetary probe sterilized to the same life-seeking standard as the Viking landers.

NASA’s next Mars rover is also set for launch in 2020, and part of its mission is to gather and tag rock and soil samples for retrieval by another mission later in the 2020s. Future spacecraft will recover the specimens and return to them to Earth, or perhaps to a human-tended lab near Earth, for inspection.

“This is a very important mission,” said Jim Green, director of NASA’s planetary science division. “It actually, in many ways, is a follow-up to the Vikings.

“It enables us to bring the right environment back, put it in our laboratories to study Mars, and resolve the question of whether there is life on Mars or not,” Green said. “In addition to the rock samples, we’ll also have regolith and soil samples. We’ll be able to perform the Labeled Release experiment in our own laboratories, and understand really what the physics is behind the observations that were made by the Labeled Release experiment while it sat on Mars and performed its job (on Viking 1 and 2).”

Nevertheless, Levin still stands by the initial results from the Labeled Release investigation on the Viking landers.

A group of biologists in 2012 took another look at the Viking data, plugged the measurements into a numerical model, and determined the experiment likely scooped up respirating microbes. The team was led by Joseph Miller, a neurobiologist at the University of Southern California, who previously led a study that found the gas output from the Viking Labeled Release samples fluctuated on cycles of nearly 24.7 hours, roughly the duration of a Martian day.

Miller said that could be an indication of a circadian rhythm, the day-night cycle observed in most living things.

“In addition to the 55,000 pictures of the surface of Mars taken from orbit, in addition to the 5,500 pictures of the Mars surface (from the landers), in addition to the first measurements of atmospheric composition, pressure and density,” Levine said in a NASA science meeting last year, “in addition to the discovery that the surface of Mars is unlike any other surface in the solar system, it just may be that in 1976 the people sitting in this room actually discovered the presence of life on the surface of Mars.”

Speaking at last week’s anniversary gathering in Hampton, Virginia, Levin harkened back to his early career as a municipal wastewater expert and offered this advice to future Mars colonists: “When you do get to Mars, do not drink the water.”

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Follow Stephen Clark on Twitter: @StephenClark1.