Making Solutions by Jonathan Knight
Science - precise, meticulous, quantifiable study of the world, life and everything. Who would ever doubt the rigor of science or the exactness of its measure? Certainly not an eager young graduate student, as I was in 1986. To my mind, good science depended on accuracy. Bad measurement could lead to false understanding, rendering the foundation of scientific knowledge porous and weak, like old mortar between the ivy-covered bricks of great research institutions.
Erected on a hillside overlooking San Francisco Bay, the science buildings at the University of California are composed mainly of concrete, steel, and glass. They suggest solidity and precision, neatness and care. Near the entrance of the building I worked in as a graduate student hangs a 1950s portrait of Wendell M. Stanley, a pioneer in the molecular biology of viruses. Beside him are enlarged black-and-white photographs of what appear to be six-legged Martian landing craft and towers made of candy corn. These are the viruses that Stanley saw in the electron microscope - ultraminuscule invaders of plant cells and bacteria, consisting entirely of the two most basic molecules of life, nucleic acid (DNA or RNA) and protein.
I still marvel at the geometry of these simple parasites. The twenty-sided head, an icosahedron, of one bacteria-killing virus sits atop a long cylindrical neck, which leads to a cone pointing down for spiking into a bacterial cell. Through this protein needle the virus injects its DNA to start an infection. Stanley was the first person to see some of these creatures. By studying them he helped launch the field of molecular biology.
Stanley's portrait inspired me daily as I began my career as a molecular biologist. He too was once a new graduate student, and he too once had to perform the task I then had before me as a new member of a lab - making chemical solutions. Experiments in molecular biology depend on good solutions, so everyone has their own personal set of 20 or 30 bottles crowding the shelves above their work bench. People are reluctant to lend solutions out, for fear that the borrower might unwittingly spill or contaminate them.
Most solutions are clear liquids in glass containers the size of a lunch soda, distinguishable only by arcane markings on a piece of colored tape -5M NaCl, a certain concentration of salt; 3M NaOH, a strong solution of lye. Mixed in the right proportions, solutions can become a buffered ion soup that resembles the inside of a cell. There, the biologist hopes that proteins, DNA, and RNA feel at home so they will perform in a test tube the way they do in an animal.
Since I couldn't do any real experiments until I had made my solutions, I set about the task with Boy Scout zeal. I began lining my shelf with bottles of zinc chloride and sodium hydroxide and silver sulfate and lithium acetate, all dissolved in water to exactly the right concentration. I assumed that any solution even slightly out of whack would jeopardize my experiments, rendering them uninterpretable or at least unreliable. If I got the solutions wrong, I could spend meaningless hours doing useless work.
Today, I am a bit embarrassed to admit the precautions I took to make sure I got the concentrations right. For days I measured and weighed, carefully spooning out white powders, some as coarse as granulated sugar, some as fine as confectioner's, to quantities within one hundredth of a gram. I measured the volume of double-distilled water I needed for each solution in a glass graduated cylinder, adding the last bit of water dropwise with a pipette for ultimate precision. I mixed the water and powder in a beaker, rinsing the last few grains off the weighing paper with some of the liquid. After all the powder had dissolved, I poured the solution back and fourth from the beaker to the cylinder to the beaker to incorporate the last drops of water still clinging to the sides of the containers. I fantasized about shrinking myself down and counting the molecules to make sure the ratio was right: one molecule of zinc to every five molecules of H2O. I wanted the most accurate solutions in the lab, in the whole university if possible, so that I could have the most accurate, most reliable experiments of anyone.
"What are you doing?" an older graduate student said to me one day when he saw how much care and time I was taking at the expensive Mettler balance, generally used for only the most sensitive experiments. The Mettler was so sensitive you had to hold your breath and stand perfectly still to get the final reading of the weight, as any motion of the air would send the digital readout spinning like a one-armed bandit. "Just making solutions,"I said. And I thought silently, "The best solutions in the lab."
After four days I was ready to begin my experiments. I had 25 glistening bottles of pristine reagents, lined up like sodas at a deli counter. Each one sported a little yellow label, which I knew with confidence stated the exact contents. My 2M LiAc was exactly two molar lithium acetate - not 1.99999999, but 2.00000000.
The hard work began. Every morning I lined up 48 little plastic tubes in a rack on my bench. I numbered each one with a Sharpie pen and began adding solutions. Using pipettes specialized for minuscule quantities of liquid, I added tiny amounts of various solutions to the tubes. I varied the proportion from one tube to the next, so I could test the function of the protein I was studying in different liquid environments. Then I took from the freezer a portion of the lab's precious protein solution, one which a postdoc had spent two months making.
Keeping the tube cold on ice, I added a bit of the clear, protein-charged liquid to each mix. Finally, I added a small quantity of DNA to each tube. Later in the day I evaluated how much of the protein had bound to the DNA with a laborious, solution-intensive procedure that kept me in lab till the evening. I thought about the results on the way home at midnight so that I would know how to set up the next day's experiments when I got in the next morning at ten.
One day, three months later, I happened to be watching our Swedish postdoc make a solution. He did everything just as I had done it before, except that he chose not to use the Mettler balance. But when it came time to measure the water, he didn't bother with a graduated cylinder. He just estimated, adding too little water, it seemed to me. Amazed, I kept watching. He poured in the powder, rinsed off the weigh paper and stirred until the chemical dissolved. Only then did he transfer the solution to a graduated cylinder and bring the volume up to a precise level.
Suddenly I realized what he was doing. I staggered for a moment and had to catch myself on the edge of a sink. Of course! What an idiot I had been! You have to measure the volume after the powder is added, not before as I had done. I had forgotten that even powder takes up space.
As any freshman chemistry student knows, to make half a liter of solution, you weigh out the chemical, dissolve it in water, and then bring the volume up to half a liter. By adding the chemicals last, I had changed the final volume of my solutions, just as a glass of iced tea filled to the brim overflows when you add sugar. My solutions were all more than half a liter, sometimes much more. Every solution in the lot was 10 or 20 percent less concentrated than I thought. Rather than using 2M LiAc in my reactions, I had been adding 1.8 M. Three months of experiments would have to be redone.
"What's the matter?" asked Arne, the Swedish postdoc, in his musical, flawless English. I told him. "So," he said, "you screwed up. Well, it probably won't make any difference."
He was just trying to be nice. Of course it would make a difference. I moped back to my bench and began pouring my solutions down the drain. To dispose of the toxic acrylamide solutions, I had to add a catalyst that causes the acrylamide to solidify and become less toxic. Normally, one dilutes acrylamide before adding catalyst, but in my haste to remove the offending solution from my life, I didn't bother. The liquid started to get hot, very hot. Then it boiled. And as the acrylamide began to harden, the bottle cracked and shattered, sending smoke and steam in all directions. "Holy shit, what was that," someone yelled from across the lab. She came over to my bench and we both watched the little volcano for a few minutes until it cooled off and stopped sputtering. It was the most fun I had had all day. I felt like a kid playing with a chemistry set. Having thus relieved some tension, I set about making all my solutions from scratch.
I began repeating my experiments from the past three months with the new solutions, doing the most important ones first. I did 48 tubes a day, just as before. I was quite sure that the new solutions would give me new and different results, that I would see things I hadn't seen before, that riddles would be solved and new windows to the miniature world of proteins and DNA would open.
Instead, from one experiment to the next, I saw exactly the same things I had seen the first time around. The same reactions took place in the same way at the same rate. I didn't imagine that everything would change, but surely something had to come out different.
Then I remembered Arne's words, "it probably won't make any difference." So I went to him and asked what he meant by that. He told me that my protein-DNA reactions are a bit like flies and flypaper. If you hang a strip of flypaper in a room full of flies, some flies will stick, and it doesn't matter if the air in the room has a little extra oxygen or nitrogen in it, or if the temperature is warm or cool. The flies seek out the paper and land, usually for good. Many proteins find their targets and stick regardless of the soup they are floating in. While boiling tar might not work well as a medium for biological reactions, a few salt molecules more or less often won't make a difference.
It makes sense when you think about it. These dauntless proteins have weathered the storms of evolution, carrying out their functions faithfully as species have transformed over eons from fish to lungfish, from dinosaur to bird, from trilobite to fruit fly. Proteins can't always count on finding themselves in precisely the same chemical soup generation after generation as organisms evolve. But those that perform regardless of small changes in the concentration of sodium have made it through millions of years of natural selection and are still encoded in the genes of living things today.
Biology has been called a soft science, partly because it deals with soft things like flesh and fat and sinew. Even bone is soft compared to steel rods machined to sub-micron tolerances for a physics experiment. Biology experiments are also soft in that they sometimes to work under a wide range of conditions, and the important parameters can be hard to nail down. And then sometimes experiments stop working, even though you are following the protocol as carefully as possible and you are convinced you have thought of everything. No one knows why, but clearly you haven't thought of everything. Some variable is not under control.
Biology is also part art. We speak of people's hands. "She has good hands, all her stuff works." "This procedure never works in my hands." I can't imagine any self-respecting physicist talking that way. In physics, the lines are sharp and clear like the metal casing of a particle accelerator. But in biology, sometimes you can smack a fly and it still gets away.
I didn't let Arne's revelation about the nature of proteins drive me to carelessness. I still tried to be consistent in my experiments from day to day, to keep control of as many variables as possible. But I spent a lot less time making solutions, and I never went back to the Mettler balance. And sometimes, when my experiments weren't working and I was sure no one was looking, I threw in a little extra salt, just for good measure.