On handling Chlamydomonas

As part of my doctoral research, I'm working with the unicellular algae Chlamydomonas reinhardtii as one model genetic species in a predator-prey system (the predator being the model ecological species, Daphnia pulex).  Recently,  Kyle Hernandez, a post-doc in the department who is interested in the genetics of function-valued traits, has started working with me.  One of our goals is to generate a large QTL mapping population starting with wild-type strains that differ in some respect, in this case, average cell size through the day.  Algae cell size important for two major reasons: first because small-bodied Daphnia selectively consume the algae based on cell size, but large Daphnia do not; and second because cell size--hence surface-area to volume ratios--is central to various physiological processes, such as nutrient uptake rates.

Generating a mapping population requires crossing the parental strains, separating out individual zygospores, then individually separating the four daughter cells produced when the zygospores mature, to start each line in the mapping population.  While there is an excellent Journal of Visualized Experiments article on making Chlamydomonas crosses, Kyle and I have run across many questions during our trials of generating crosses:

1. Is plating the cells out on COMBO media with 10% normal nitrate (i.e., very low concentration) sufficient to trigger sexual reproduction.
• Yes, it is
2. After the one-week period of incubation in the dark, will we actually be able to tell the zygospores from vegetative cells?
• Yes, at least with the lines we are crossing, the zygospores are larger and a glowing golden color distinct from the smaller, green vegetative cells...super obvious
3. Are you kidding me?  How the world do people handle individual Chlamydomonas cells???
• No, seriously, is this actually possible?
• Yes.

So that last point has been our big learning experience over the past couple of days.  The process requires practice moving very slowly and carefully, but it also requires making the glass needles correctly.  Specifically, the first needles we pulled were far too short and far too inflexible to be of any real use: the slightest twitch and the needle tip will plunge right through the agar (taking with it any cells with which you might be trying to work).  The key is to pull glass needles that look something like this:

Yes, it is difficult to see the needle, because it's ~20-30 microns across out towards the hooked end (the hook is nice for pulling cells along).  The more important point is noting the length of the thin portion: in this example it's over 2cm long.  The long, very thin portion allows a lot of flex to the needle, to the point that the surface tension of fluid on the surface of the agar is enough to hold the tip down and deliberate pulling up is required to overcome the spring to lift the hook.  Once I got the appropriate needle length pulled, it became much easier to cut a little bunch of zygospores from the incubation plate; place them on the maturation plate; then slide the cluster around repeatedly until individual cells popped off and could be slid into place.  

Kyle and I could not find this information anywhere on the Internets, and so hope that this information is helpful to anyone starting to work with Chlamy (or other single-cell species that require such techniques and tools)!