Science is hard. That’s not exactly a newsflash to any of the readers of this blog, but it’s a point that Science has been reminding me of recently. This has been reminding me of an earlier Science Is Hard episode that my lab went through. I’ve been reminding myself that we got through that one (and that one was definitely worse), and we’ll get through this one, too.
Both of these Science Is Hard episodes have involved culturing problems, and, in both cases, I feel like we did really good science to figure out the cause of the problem. But it’s the sort of science that goes completely unreported. In many ways, it fits as the story behind the paper – really, for a whole number of papers, because none of them would have existed if we hadn’t figured out the problem.
The first major culture problem occurred when I was at Georgia Tech, after I’d been there for about a year. For the first semester or so, I was mainly ordering things and setting up the lab. And then, in my second semester, we started doing research. There were definitely stumbling blocks (it took us a long time to get our algae chemostats really going, for example), but things were moving along.
Until they weren’t. At some point, we started having lots of problems with animals dying. It was so frustrating, because we had felt like we were about to be going full steam, only to find ourselves unable to do any experiments. So, of course, we started trying to figure out why.
My grad student Rachel was in her first year in the lab, and had recently started doing some experiments. She was really worried that maybe she was doing something wrong in the lab that was causing the problems. And, frankly, the timing was a little suspicious, as the problems had started right around when she really started to do work in the lab. So, she and I tried to set up an experiment side-by-side, doing everything at the same time. We both had tons of animals die. That ruled out the Rachel hypothesis (which was a relief to Rachel and to me!), but didn’t tell get us much further to figuring out what was going on.
Rachel ended up having the key insight that got us moving on the right track: she was the one who first noticed that the deaths were a beaker-level phenomenon. Either all of the animals died in a beaker or none died. Based on that observation, we put some beakers in the acid bath, rinsed them well with DI water, and then set up animals in them. None died. Breakthrough!
So, it was something on the beakers. But what? And when was it getting on there? To get at that, we first did an experiment where we acid washed a bunch of beakers, and then rinsed half with DI water and put the other half through our normal dishwashing process (which involves scrubbing with soapy water, then rinsing with tap water, then putting them in the dishwasher for a tap rinse followed by DI rinses. We are serious about getting our beakers clean.) The animals in the beakers that had only been rinsed with DI water all lived. The ones that had gone through the regular dishwashing process died. More progress!
So, then we needed to figure out which part of the dishwashing process was a problem. We had the people who ran our water system come and put in a way for us to draw from the DI tanks that fed the dishwasher in between the tanks and the dishwasher. We then used the DI water from that new feed to rinse the dishes (after washing them with soapy water and rinsing with tap water), and compared those to beakers that were rinsed on a DI cycle in the dishwasher. Again, the animals in beakers we’d rinsed by hand did great; the ones in beakers that had gone in the dishwasher died.
By this point we’d been troubleshooting for months, but we had at least made lots of progress. At this point, Al Dove from the Georgia Aquarium heard about our problems and very kindly offered to run some water samples for us. We put a beaker in the dishwasher upright to collect water and sent it over. The copper in the water was 74 ug/L. As my colleague Terry Snell pointed out, the LC50 for copper for the rotifer Brachionus is 30 ug/L.
At that point, I was so ready to buy a new dishwasher and put the problem behind us! I had been using a dishwasher purchased at Lowe’s, because that’s what the lab I’d been in as a grad student had done, and there weren’t any problems. But I decided that, given that the problem was with the dishwasher, I needed to get a fancy lab-grade dishwasher. So, I did. When the new dishwasher came and they took the old one out to replace it, they found that the DI line had been attached with a copper fitting. This is a huge no-no, since DI water is very pure and leaches the copper from the pipe into the water. But, it explained why we had so much copper in the water!
At that point, our problem was identified, but not solved. We have a LOT of glassware in my lab (thousands of beakers), and we had no way of knowing which had gone through the dishwasher while the copper contamination was occurring. So, we concluded that we had to acid wash every piece of glassware in the lab. That took weeks of work, mostly done by my excellent technician, Jessie.
In the end, we lost about a semester of work due to the copper problem. We haven’t figured out the source of our current problem yet. One thing that I find interesting is that, when I run into a problem like this, the first person I contact is my PhD advisor, Alan Tessier. I’d like to think I’m a grown up scientist now, but I still really value advice from Alan!
For now, we’re going through all the trouble-shooting. Acid washing beakers didn’t help, nor did using brand new beakers. So, it doesn’t seem to be a glassware problem. Now we’re on to testing whether it’s an issue with the water. One possibility is that something about the water has changed as we stored it over the winter. (We culture our Daphnia in filtered lake water.) Perhaps some compound the Daphnia really like has broken down over the winter. So, we’ll go out and get new water and see if that solves the problem. I was dragging my heels on going out and breaking through the ice, since it seems like a major pain, but my lab is really excited about our upcoming winter limnology expedition. And we’re all really excited about the prospect of getting this problem solved soon!
We’ll get through it. But, boy, science is hard.