Science is hard: culturing problems edition

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.


Related posts:
1. System envy and experiment failures
2. Tractable != easy

13 thoughts on “Science is hard: culturing problems edition

  1. Preach on Meg! Culturing problems are the worst. I had some in spring 2005, our experiments kept dying. The summer undergrads eventually figured it out–we think the floods in the city changed the chemistry of the city’s water. We switched to using water from a spring in a provincial park; I now drive out there periodically to fill carboys and get funny looks from anyone who’s picnicking in the park.

  2. While this is obviously a post about road blocks in the research process, I wonder if its not indicative more generally about the nature of ecology and the high levels of contingency in ecology (any of dozens of factors could dominate a system and which one does can seem close to random).

  3. ‘Burps’ like this can be, and usually are frustrating. But in my 30 years in the labs, I learned they happen at very inconvenient times, and to treat them as a Sherlock Holmes ‘game’. Such as the alkaline phosphatase in our water purification system affecting our ELISAs, fittings on the CO2 tanks interfering with colony growth in the incubators, etc. I found such anomalies a valuable contribution to lab meetings under the heading of ‘SH Deduction’.😀

    • Yes! It’s definitely a mystery that needs to be solved, and treating it that way makes it seem less frustrating. But I still want to solve this current mystery ASAP.🙂

  4. I have a question about how “muddy boots field ecologists” react to stories about the technical challenges of culturing things in the lab.

    One reaction would be to sympathize–to recognize that, even in highly controlled lab environments, s**t happens, just as it does in the field. It’s different s**t–you don’t get freak storms or ground squirrels gnawing on your flagsticks in the lab, just as you don’t get copper contamination of your glassware in the field–but s**t happens all the same. Like Meg’s old post said, “tractable” does not equal “easy”. The natural variability that you see in the field still happens in the lab, it just has different proximate sources.

    But another reaction would be to see all the fiddling needed to culture organisms in the lab as just part of “rigging” the experiments so that they come out as you anticipated. To see all the s**t that happens in the lab as somehow fundamentally different from the s**t that happens in the field.

    I’m curious which reaction is more common. Perhaps Meg can answer this one herself since she’s a muddy boots field ecologist as well as a lab ecologist.🙂

    • Hmmm. I don’t know which is more common! I think the most common reaction I get when I talk about culture problems (or things along those lines) is simply surprise. As I said in that earlier post, it’s easy to think that tractable and easy are the same thing.

      That said, I have discussed with others the idea of whether it’s a problem when animals seem stressed in the lab. On the one hand, if the most offspring any of your life table animals produce is 3, then you probably don’t have enough variation to see differences between genotypes, for example. And figuring out which of my treatments died a bit more slowly than the others (e.g., due to a temperature fluke) doesn’t seem very satisfying. On the other hand, conditions in the field are probably pretty stressful (but maybe in a different way). So, is it better to measure things in cushy lab conditions or stressful ones? I don’t think there’s a right answer to that.

  5. Pingback: Links 2/24/15 | Mike the Mad Biologist

  6. Reminds me of when we bought our first PCR machine, somewhere around 1990 (or later, or earlier?). PCR’s run with the reaction tubes that came with the machine never worked, whereas they did using another batch of tubes. We thought to test this because we had already run the PCR in question using tubes and machine belonging to someone else. I hate to think of the frustration people just starting out with this machine and these tubes would have had–they would obviously have concluded that they were doing something wrong and that the fault was with themselves.

    • Oh my goodness, I can only imagine how long it would take to figure out that the tubes were the problem for people with no experience!

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