My wife, infant son, and I moved to Amherst the first weekend of April 1982. A beautiful snow fell on Sunday. Then, early on Monday morning, my new boss Bruce Levin cross-country skied by the old house we were renting, knocked on the door, and asked me when I’d be coming to the lab!
I had much to learn, of course. I remember learning how to use a pipettor from a technician in Bruce’s lab, and how exciting it was to estimate the number of cells in a flask (typically many millions or even billions). That estimation is done not by counting the cells directly, but instead involves precisely diluting small amounts through a series of test tubes, each tube containing a large, known volume of a sterile solution. At the end of the dilution series, one takes a tiny amount from the final tube and spreads it across an agar plate. The plate is then incubated for a day or so, during which time each of the few hundred cells that survived the dilutions grows into a separate colony. A colony is a clump of millions of cells that can be seen with the naked eye, unlike the individual cells that can be seen only by using a microscope. One counts the colonies on the plate and, using that number and the dilutions that one made, one can then back-calculate the density of cells in the original flask.
In my first effort at this most basic procedure, I did three replicates from the same flask. I was thrilled when I counted the colonies on the first two plates, and the numbers differed by only a few percent. The third plate, however, differed by perhaps a factor of two, which meant I had done something wrong—maybe I’d let an air bubble into the pipettor’s tip, displacing some of the liquid—and I realized the importance of attention to details.
A little later, while I was still learning the ropes, Bruce had me perform a more complicated experiment to measure the rate at which a certain virus, called T6, adsorbs to and infects E. coli cells. The experiment required a lot of repetitive dilutions and plating of samples that I had to process quickly and accurately. The basic idea is that free viruses should decline in number over time as more and more of them enter cells. (This decline continues only until the first viruses to infect cells have had enough time to produce the next generation of viruses, hence the need to process the samples quickly.) Alas, my experiment was a total failure. What was I doing wrong? I think Bruce had me repeat the experiment, with the same lousy outcome. Though he never said it, perhaps he would regret hiring me. After all, given my lack of experience, Bruce had also taken a leap of faith.
After my second failure, Bruce checked his notes about the particular strain that we were using. As it turned out, he had given me a strain of E. coli that was resistant to T6! Hence, there were no infections, and that explained my failed experiments. Later on, I was able to use the same protocol to measure the rate at which a different virus, T2, adsorbed to and infected E. coli.
Oh, and what about my experiment to look for evolutionary changes that compensated for the cost of bacterial resistance to infection by viruses? That’s what I had proposed in my letter to Bruce asking about a postdoc. I never got to that experiment while I was in Bruce’s lab. However, it provided the seed for a project that I eventually conducted as an early-career faculty member at the University of California, Irvine.
[Bacterial colonies growing on agar plates. Photo credit: Brian Baer, MSU.]
3 responses to “A Leap of Faith, Part 2”
I think all of us have learned over the course of our careers how important it is to be able to quickly figure out if what’s written on the vial you’ve been handed is what’s actually inside – chemicals, strains, plasmids, etcetera. It’s good to learn this very early on.
for my PhD, I was given a cryo vial with a mutant E. coli strain by my supervisor – It took me ~3 years to find out that the mutation was ~30 min away (ftsI) from the initially suspected locus (rplW). ha! but almost everything I know about E. coli genetics I have learned hands-on in this three-year search. never regretted it.
Yes, eternal vigilance!
vigilance: yes / paranoia: no.
except for *very* rare cases, all tube/vial mix-ups I ran into turned out to be sloppiness, not malice.