Protocol creep

In many respects, our lab is like a farm. We grow things. We grow social amoebae to be precise. Our social amoebae are predators. They hunt down bacteria and engulf them. They are chatty predators, chemically talking to each other all the time, and particularly sharing or eavesdropping on information about the standing crop of bacteria. This is why we like them. They are social. But the stage we like the best happens when they starve, because then they aggregate, form a multicellular body, crawl around, and ultimately form a fruiting body in which about 20% of them die to build a stalk that the others flow up. At the top, the lucky winners transform themselves into hardy spores, and await dispersal, aided by the death of their pals that lifted them up.

This makes it sociobiology, and it is particularly amenable to study in this simple organism. But this post is about protocols, the most basic protocols of our group. We grow our social amoebae, mostly Dictyostelium discoideum, on yeast, sugars, and salts dissolved into agar in a Petri plate. That medium does not feed the amoebae; it feeds the bacteria that the amoebae eat. I suppose for us it is like eating those special Spanish hams from pigs that ate only acorns, compared to hams from pigs that ate slop. What they ate matters. Which bacterium they eat also matters.

But how much do these things matter? Perhaps not very much, because there are lots of protocols out there for growing dicty, and lots of bacteria, mostly gram negative, that they find tasty. I said gram negative because I love it when an old test proves so  informative, but that is a story for another time.  The truth of the matter is we can grow dicty in lots of ways.

But for consistency, a lab group should use one recipe across all users, unless there is a good reason not to. We can drive  people new to the group crazy if they get told there are lots of protocols for the same thing, and it doesn’t matter which they use, but they must use the same one across an experiment.

So, now is the time for the protocol gods to act. We have come through a group bottleneck of 6 people in the lab (not counting me or Dave), down from double that in Houston. Still, it wasn’t a single cell bottleneck, so variation is still there. Hence the need for decisions. What we decide will be put on our web page, and in a big blue notebook of plastic protected pages, for those that like things concrete.

We have chosen a single standard medium. It is the same SM that Formedium makes. Unfortunately, they only supply it with glucose, and there are good reasons to autoclave the sugars separate from the salts to avoid carmelizing. The magnesium should be added later also, since it so easily goes out of solution. So we’ll use that standard recipe, but not necessarily provided by Formedium, unless we can talk them into leaving out the glucose. There, that is a decision.

Why do they put the sugars right in? Because that is how all the dicty people do it, even though the bacteria people do the separate autoclaving. After all, it is bacteria we are growing, so I’m inclined to respect the bacteria people. We have bacteria people in our group to educate us. We also have dicty people.

We will also always measure how much medium goes in a plate. Pouring thin or thick changes numbers and condition of bacteria, we all agree.

Another important standard is which bacterium we use, and how we grow them. We will use the form of Klebsiella aerogenes provided by Dictybase, and we will not do any evolution on the bench. We are an evolutionary lab, so we know it doesn’t take much for bacteria to evolve. We’ll streak out the bacteria, and start a flask of LB with a single clone from a colony that looks normal. We’ll use the bacteria from the still, benchtop flask after a day, and continue to use it only a few days.

OK, that’s enough standardizing for now, but we’re going to keep at it. That way the new people can learn the standard things easily, no matter which person trains them.

About Joan E. Strassmann

Evolutionary biologist, studies social behavior in insects & microbes, interested in education, travel, birds, tropics, nature, food; biology professor at Washington University in St. Louis
This entry was posted in So you think yours is the best protocol. Bookmark the permalink.

2 Responses to Protocol creep

  1. Sara says:

    Just be aware that you might not have much luck growing the other dictyostelids on that… I know I never had much luck with Polysphondylium until I switched to E. coli and tweaked some other things.

  2. jeff smith says:

    Joan wrote: “That medium does not feed the amoebae; it feeds the bacteria that the amoebae eat. I suppose for us it is like eating those special Spanish hams from pigs that ate only acorns, compared to hams from pigs that ate slop. What they ate matters.”

    The medium and the bacteria’s metabolic response to the medium also create the chemical conditions (pH, etc) under which the amoebae live. They’re not just eating hams—they’re eating hams as they walk around in a vast herd of pigs.

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