NO ONE has created life in the laboratory. The possibility is vanishingly small that a viable organism can be compounded. Yet that slender chance continues to intrigue some experimenters, and occasionally progress is reported. For example, a particularly dramatic experiment that amateurs can perform was described 17 years ago by Stanley L. Miller, then one of Harold Urey's students at the University of Chicago. Miller circulated a sterile mixture of water vapor, methane, ammonia and hydrogen through an electric spark. At the beginning of the experiment the mixture was crystal clear. Within 24 hours the condensed liquid turned noticeably pink, and after a week it became deep red, turbid and slightly viscid. Chemical analysis disclosed that the fluid contained a number of organic substances, including several amino acids, which are the building blocks of proteins.

Miller had not compounded these substances by controlled chemical synthesis. He merely established a physical environment that favored their spontaneous generation. It was the kind of environment that might have existed on the primitive earth.

Other experimenters have since reported similar achievements. Heat and various forms of electromagnetic radiation have been substituted for Miller's energizing spark to generate various constituents of living organisms, including purines, pyrimidines, protein-like polymers and nucleic acid polymers. In addition techniques have been devised for encouraging some of these materials to unite spontaneously into ordered structures. As such experimentation continues there is a minute but growing probability that a structure will eventually appear that feeds on the nutrients in its surroundings and reproduces itself. As the biologist George Wald has pointed out: "However improbable the even in a single trial, it becomes increasingly probable as the trials are multiplied Eventually the event becomes virtual inevitable" [see "The Origin of Life," by George Wald; SCIENTIFIC AMERICAN August, 1954].

Fascinated by this promise, the experimenters keep trying. Even amateurs are beginning to get in on the fun. On is Carl Fromer of Staten Island, N.Y. who is a premedical student at Columbia University. Fromer explains how to do Miller's experiment and discusses a few others:

"Any experiment that is designed to generate from inorganic substances the constituents of living organisms must provide two basic conditions in the reaction vessel: the vessel must be devoid of life and lacking in free oxygen. The first of these conditions was mentioned more than a century ago in an obscure letter by Charles Darwin: 'It is often said that all of the conditions for the first production of a living organism are not present, which could ever have bee present. But if (And, oh, what a big if!) we could conceive in some warm little pond, with all sorts of ammonia an phosphoric salts, light, heat, electricity etc., present, that a protein compound was chemically formed ready to undergo still more complex changes, at the present day such matter would be instantly devoured or absorbed, which would not have been the case before living creatures were formed!'

"The second basic condition, the absence of free oxygen, was first emphasized by the Russian biologist A. I. Oparin in his classic volume of 1936, The Origin of Life. Essentially Oparin pointed out that free oxygen, a highly reactive gas, would promptly combine with and destroy all organic compounds of interest. The reaction vessel must be sterile and airtight.

"Miller's apparatus consisted of two glass bulbs interconnected by a pair of glass tubes in an O configuration so that it was a closed system. Vapor from boiling water in one bulb rose through the outlet tube and entered the second bulb. The outlet tube of the second bulb contained a spark gap and was cooled just below the spark gap by a surrounding water jacket. Condensation that formed in this region drained through a U trap to the boiler, ready for another trip through the apparatus. Reaction products that formed in the vicinity of the spark gap accumulated in the water. Miller's construction involves some rather tricky glassblowing. The configuration did not seem critical, and so I made a simplified version that works just about as well.

"My apparatus consists of a closed tube of Pyrex glass 3.5 centimeters in diameter and 60 centimeters long fitted with a homemade heating mantle, a cooling jacket and platinum-wire electrodes for the spark gap [see illustration at left]. Access to the interior is provided by a side arm of eight-millimeter Pyrex tubing supplied with a stopcock. The tube operates vertically, with the spark gap at the top. Vapor rises by convection from boiling water at the bottom. Some vapor circulates through the spark gap, where the enclosed gases react, condenses on the chilled wall below and trickles back to the boiler. As in Miller's apparatus, the reaction products accumulate in the water.

"The tube is homemade. I used an ordinary gas-air blowtorch for softening the glass. The air supply was enriched with oxygen to develop the temperature required for working Pyrex. If the experimenter does not have access to glassblowing facilities, arrangements could be made to have the tube assembled by a neon-sign shop.

"One end of the 35-millimeter tubing was collapsed m the flame and blown into a hemisphere. A small hole was then blown in the side, and the access tube was sealed to the hole. After welding a brace of glass rod to support the access tube, I collapsed the remaining open end of the 35-millimeter tubing and expanded it into a hemisphere by blowing into the access tube. The electrodes were sealed to a pair of opposing holes blown near the top of the tube and in the center of the hemispherical bottom. Platinum does not make a vacuum-tight seal with Pyrex, and so I dabbed epoxy cement around each wire at the point where it emerged from the glass.

"The water jacket consists of a 22 centimeter length of Pyrex tubing five centimeters in diameter equipped with eight-millimeter inlet and outlet tubes. The ends of the jacket were closed with rings of plastic tape wrapped around the 35-millimeter tube. The rings make a snug fit with the jacket. I sealed the rings to the jacket with a thick coating of epoxy cement.

"The heating mantle was made by winding a coil of Nichrome wire, along with asbestos insulation, around the lower end of the tube. A strip of asbestos paper 11 centimeters wide was cemented around the glass with sodium silicate (water glass). A flexible copper lead, insulated with asbestos, was crimped to one end of the Nichrome wire and lashed to the paper with twine near one edge. A layer of wire was wound over the paper at a turn spacing of five millimeters. Four layers of wire and paper were applied. After a flexible lead was attached to the outer end of the wire and lashed to the end of the final layer the coil was completed with a cover of asbestos paper lagged in place with twine and cemented.

"The finished tube was exhausted to the limit of a mechanical air pump. I used the compressor from a discarded electric refrigerator for an air pump, connecting the tube to the inlet of the compressor. The tube was then sterilized by electron bombardment. One terminal of a 15,000-volt, 60-milliampere transformer from a neon sign was connected to both spark-gap electrodes; the other terminal was attached to the electrode in the bottom of the tube. Increasing potential was gradually applied to the input terminals of the high-voltage transformer by a variable transformer until the tube filled with glow discharge. The tips of the electrodes became red-hot. The bombardment was continued for five hours, after which the stopcock was closed.

"The apparatus was transferred to a sterile chamber for filling, along with a pressure cooker containing about a liter of distilled water that had been boiled at a pressure of 15 pounds per square inch for 30 minutes. Some 500 milliliters of the cooled water was transferred to a sterile beaker. The access tube was sterilized briefly in a flame and, when it had cooled, was dipped into the water. Water rushed into the tube when the stopcock was opened. I filled the tube to the upper edge of the heating mantle, using about 100 milliliters of water.

"The experiments were made with gases of reagent grade that come compressed in lecture bottles. The gases and the other required materials, including the glass, are available from distributors of scientific supplies. The gases are mixed in the proportion of one part hydrogen and two parts each of anhydrous ammonia and methane (by volume). The proportions need not be exact. I had no precise gauges, and so I measured the gases with three toy rubber balloons that expand into approximate spheres.

"After the balloons had been sterilized in the pressure cooker one was coupled to the outlet of the hydrogen cylinder and filled to a diameter of six inches. The remaining two balloons were similarly filled to a diameter of 7.5 inches with ammonia and methane respectively. The gases were combined in one of the balloons by coupling, with a short glass tube, the balloon containing hydrogen to the one containing ammonia. The balloon containing hydrogen was squeezed to force the gas into the ammonia. The methane was combined with the ammonia in the same way.

"The balloon now containing all three gases was coupled to the access tube of the apparatus and the stopcock was opened to admit the mixture. After the stopcock was closed and the balloon (still containing a substantial amount of gas) had been removed, an empty balloon w as coupled to the access tube. When the stopcock was opened the trapped gas expanded to atmospheric pressure. A more elegant technique for metering the gas can doubtless be devised, but this one works and is inexpensive.

"The filled tube was supported upright by an apparatus stand. After power was applied to the heater, the water came to a gentle boil in about two hours. In the meantime a Tesla coil was connected to one of the spark-gap electrodes and the companion electrode of the spark gap was grounded. When the water reached the boiling point, the coil was turned on and adjusted to an output potential just sufficient to jump the spark gap. Coils of this type are designed for locating leaks in vacuum systems made of glass and are available commercially.

"Within 24 hours the water assumed the color of weak tea, and in seven days it became dark brown. A brown waxlike deposit formed in the upper portion of the tube. After the power was turned off and the tube had cooled, I transferred the fluid under vacuum to a sterile flask. The flask had been fitted with a sterile rubber stopper containing two holes, one of which made a snug fit with the access tube of the apparatus. A hose attached the air pump to the other hole.

"The apparatus was placed in the horizontal position so that the fluid would dram into the flask when the air was pumped out. The fluid was concentrated by evaporation under vacuum in the flask to a volume of approximately 20 milliliters and transferred to a sterile test tube of known weight in which it was evaporated to dryness under vacuum. The net weight of the solid reaction products was 816 milligrams.

"The material was analyzed for amino acids by thin-layer chromatography and by an ion-exchange column. To make the chromatographic analysis I dissolved 25 milligrams of the dried material in 100 milliliters of distilled water to which 364 milligrams of hydrochloric acid was added. Similar solutions, each containing one of 20 known amino acids, were prepared by the same procedure and stored in separate, labeled containers.

"The specimens were applied, as small spots with a micropipette, to an Eastman Chromagram sheet coated with cellulose. To minimize the area covered by the spots and thus improve the resolution of the chromatogram I applied a third of a microliter of each specimen to the cellulose, dried the spot thoroughly and then made similar applications on top of the first one until one microgram had been deposited.

"The unknown material was placed near one corner of a Chromagram sheet at a distance of three centimeters from - the edges. The known specimens were spotted in a row on another sheet, two centimeters apart and three centimeters from one edge of the sheet. Both sheets were then placed for development in a rectangular, closed glass dish that contained wash solution.

"The wash solution was prepared by shaking together 100 milliliters each of distilled water, acetic acid and butanol. After standing for a few minutes this mixture separates. Fifty milliliters of the top layer was transferred to the rectangular container. The container was lined with a sheet of filter paper saturated with wash solution. The Chromagram sheets were inserted in the rectangular container with the specimens at the bottom.

"Wash solution migrates upward by capillary attraction and carries the acids along at characteristic rates that vary with the affinity of the acids for cellulose. Development was continued until the wash solution migrated to within five millimeters of the top edge. Both developed sheets were dried.

"The sheet containing the unknown substances was then redeveloped by turning it 90 degrees and again inserting it into the wash solution with the specimen at the bottom. The specimen substances migrated at right angles with respect to their first transit and separated still more. The dried chromatograms appeared blank to the eye, but the amino acids became visible as purple spots after the sheets were sprayed with ninhydrin solution and baked at 100 degrees Celsius in a kitchen oven for five minutes. I made up the solution by dissolving 200 milligrams of ninhydrin in 100 milliliters of acetone to which one drop of collidine was added immediately before use.

"Five spots of color appeared on the sheet of the unknown substances. The distance through which each spot migrated matched the migration distance of one acid of the sheet that had been spotted with known acids. A third sheet was spotted with these five known substances, developed and compared with the unknown substances. The unknown substances were identified as aspartic acid, glutamic acid, serine, glycine and alanine, in the approximate proportions of one aspartic acid, two glutamic acid, 15 glycine, 12 serine and 13 alanine [see illustration above right].

"At about the time the chromatograms were made a biochemist volunteered to run a sample of the material on an automatic ion-exchange analyzer. In this instrument compounds are washed through a vertical tube of glass packed with granular ion-exchange resins. The compounds migrate at characteristic rates and emerge from the column separately into a stream of clear fluid that reacts with the fractions to become colored.

"For the analysis of amino acids the clear fluid is ninhydrin solution. Color changes are monitored by a photoelectric cell that actuates a pen recorder. The emergence of each amino acid appears as a peak in the graph that is plotted automatically by the recorder. The machine includes several columns that can be operated simultaneously for plotting slightly displaced graphs of two or more mixtures, for example a known and an unknown mixture of amino. A graph of the amino acid mixture that had been synthesized by my apparatus, combined with a superposed graph of known amino acids, confirmed the chromatographic analysis and also indicated the proportionate yields mentioned above [see illustration above].

"The synthesized amino acids can be used to perform a fascinating experiment. About 10 years ago Sidney W. Fox of the Institute for Space Biosciences at Florida State University developed a simple technique that encourages the amino acids to unite spontaneously in the form of well-organized structures that he refers to as microspherules. Fox shared the opinion of some experimenters that amino acid molecules, which formed in the vaporous atmosphere of the primitive earth, tended to be carried into the oceans by rain. These compounds did not break down readily, and the oceans became an increasingly rich mixture of organic compounds. Fox reasoned that some of the compounds would occasionally be washed onto hot lava, where they would dry, cook and subsequently be washed again into the sea. How would the compounds respond to this sequence of events? Fox put the question to nature by an experiment. The answer turned out to be that the amino acids would organize themselves into microspherules!

"His experiment sounded interesting, and so I decided to try it. Hot lava is scarce on Staten Island, but I own a flat plate of ceramic that contains a number of hemispherical depressions about a centimeter in diameter and half as deep. I placed about 50 milligrams of the dried reaction product from my apparatus in each of four depressions and the same amount of a mixture (in one-to-one proportions) of commercially prepared aspartic and glutamic acids in four other depressions to serve as a control. The ceramic plate was put in an electric oven at a temperature of 85 degrees C. At the end of about an hour the powdered materials became a highly viscous mass resembling dark brown tar.

"I then filled each depression with a 1 percent solution of salt water to simulate the concentration of sodium chloride in the primordial sea and thereafter, for six hours, replaced the evaporating fluid from time to time with distilled water. Finally, with a pipette I transferred the end products (the synthesized amino acids and the control) to separate centrifuge tubes and rotated them for 25 minutes at 2,600 revolutions per minute. Smears of the dense fraction of both specimens that settled to the bottom of the tubes were examined with a microscope. Both contained microspherules that were identical in appearance, but the population in the control specimen was substantially larger. The accompanying photomicrograph [right] depicts microspherules made with the synthesized amino acids.

"Microspherules appear to consist of an inner core enclosed by a transparent membrane. A careful study of the structures would require the use of a phasecontrast microscope. I do not have one, and so I stained a smear of the specimen with bromophenol blue and examined it with a conventional microscope at a magnification of 1,000 diameters. For some minutes following their preparation microspherules appear to be jellylike in consistency and somewhat tacky. Later they solidify and retain their spherical shape for weeks.

"Fox demonstrated that freshly made microspherules tend to unite into long chainlike bodies that resemble a string of microscopic beads. To encourage the formation of these structures Fox applied slight pressure to the freshly cooked mixture. I made them by first depositing a thin ring of a cement known as gold size on a microscope slide. In the resulting shallow cavity I placed a drop of freshly made microspherule solution. The cavity was closed with a cover slip. When pressure was applied to the cover slip by the point of a needle, the microspherules linked into chains [see illustration at left].

"Fox has also demonstrated that various organic compounds, including amino acids, can be generated by exposing a mixture of the same gases to heat. His apparatus resembles Miller's in that it consists of a closed loop containing a cooling jacket and a pair of bulbs, one of which serves as a boiler. For the spark gap Fox substituted a reaction vessel of quartz that contains hot sand. Unreacted gases flow through a Pyrex tube to the bottom of the vessel and migrate upward through the sand to an outlet tube for another transit through the apparatus. The sand is maintained at a temperature of 900 to 1,100 degrees C. by an electric furnace that surrounds the quartz reaction vessel [see illustration below].

"The gases must be forced through the sand by pressure, which is developed by an aspirator that Fox inserted in the system immediately above the boiler. Condensed fluid is admitted to the boiler periodically by opening a stopcock in the return line. So far Fox has produced with quartz sand 13 amino acids, including aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, alloisoleucine, isoleucine, leucine, tyrosine and phenylalanine. Twelve of the substances have also been produced by packing the reaction vessel with silica gel. On the other hand, b-alanine, sarcosine and N-methylalanine have not been synthesized by heat although they appear in Miller's apparatus.

"In a typical experiment Fox passes methane gas through concentrated aqueous ammonia and transfers the resulting mixture to the apparatus. After the gases have circulated through the heated sand for various periods of time that may range from minutes to hours, they are transferred to a closed flask and absorbed by a cold solution of 3 N aqueous ammonia. The cold solution is then heat to 75 degrees C. for various lengths o time in a closed flask and finally evaporated under vacuum. A solution of 4 N hydrochloric acid is circulated through the dried residue (refluxed) for five hours.

"The resulting hydrolysate is evaporated to dryness under vacuum and dissolved in a citrate buffer at pH 2.2. The mixture is then ready for analysis and use. Fox found that the yield of different amino acids varies with the materials in the reaction vessel and with the temperature of the vessel. For example, when the reaction vessel is packed with quartz sand maintained at a temperature of 950 degrees C., aspartic acid constitutes 3.4 percent of the final product, but when silica gel is used at the same temperature, the production falls to 2.5 percent. When the temperature of the silica gel is raised to 1,050 degrees, the production of aspartic acid rises to 15.2 percent but the production of glycine falls from 68.S percent to 24.4 percent. "In undertaking these experiments I made a point of keeping the hazards in mind. Hydrogen and methane form explosive mixtures with air. Anhydrous ammonia is toxic and can burn the skin. Ideally the gases should be handled in a fume hood. I filled the balloons and transferred the gases to the apparatus in my backyard. Ammonia migrates slowly through rubber. If you squeeze the balloon with your bare hands, the gas may burn your palms. I wore neoprene gloves. The cooling jacket prevents explosive steam pressure from building up inside the apparatus, but only if a continuous stream of cold water circulates through the jacket.

"As time permits I intend to continue the experimentation by including phosphoric acids in the mixture, in the hope of synthesizing some of the nucleic acid polymers and perhaps inducing them to combine with the protein-like bodies."

IN discussing the liquefaction of gases in this column last November, I stated correctly that methyl chloride is a harmless gas. A number of readers subsequently pointed out that methyl chloride combines explosively with aluminum and should not be admitted to the sealed compressors of home refrigerators. Most of these machines contain parts made of aluminum to which the gas is exposed.

Bibliography

THE ORIGIN OF LIFE. Alexander Ivanovich Oparin. Dover Publications, 1953.

THE ORIGINS OF PREBIOLOGICAL SYSTEMS AND OF THEIR MOLECULAR MATRICES Edited by Sidney W. Fox. Academic Press, 1965.

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