CuteChlamyCleaning Up Contaminated Cultures

From bionet.chlamydomonas, composite of several messages 1994-1995

From Olivier Vallon

I have found an easy way of getting rid of fungal infections in cultures of Chlamydomonas reinhardtii. It takes advantage of the fact that C.r. can grow in the presence of L-methionine sulfoximine (MSX, a well known inhibitor of glutamine synthase), provided arginine is added to the medium, while most of our unwanted visitors cannot.

If a fungal infection appears on a plate and it seems difficult to pick a clean Chlamy inoculum, streak on a TAP plate supplemented with MSX (100 microM) and arginine (100 mg/l). If possible, place in bright light for 3-4 days, streak back on your usual medium (or a fresh TARG/MSX plate if you want to be extremely careful). This works fine even with heavily infected cultures. It seems to work also with bacterial infections. Do not maintain your cultures on TARG-MSX, since the drug will have deleterious effects on Chlamy in the long run.

I have no definite explanation why arginine allows resistance to MSX. In its absence, 50 microM MSX are enough to kill C.r. I suspect C.r. has a high ability to use arginine as exclusive ammonium source, bypassing NH3 and the need for glutamine synthase.

Good luck. Let me know of problems you may encounter, or improvements, or explanations.

From Antonio Franco

To get rid of bacterial contaminants, I use antibiograms discs. I stripe a line of Chlamy cells in a TAP solid media and have several of these discs close of such a line. This way, I can use several antibiotics at the same time and in the same Petri dish. Tetracycline, Cefotaxime, Trimethoprim, erythromycin, ceftixozyme are fine. Chlamy cells isolated in this way, remain in pure cultures for a long time.

From Jason Frank

In liquid culture you can clean chlamy by carefully centrifuging them down, washing the bottle walls carefully, repeating a few times, and letting them swim back to the top. I put aluminum foil around the bottle (so they don’t swim down), and nab them with a pipette.

From Elizabeth Harris

We sometimes use ampicillin [50 micrograms/ml], especially in plates to be spread after biolistic transformation experiments where the risk of contamination is pretty high. However I find in general that brute force is a better cleanup technique. Streak cultures at low density on highly nutritive medium (e.g. containing yeast extract), where any contamination will be readily apparent. Using a fine glass needle under a dissecting scope, manipulate individual Chlamy cells away from the mass of contaminated culture. Inspect the plates daily under the microscope, and continue to move the algal cells away from the bacteria (or fungi). When you find a clean, uncontaminated colony, transfer it with a sterile toothpick to a fresh plate.

The Chlamydomonas Sourcebook has some additional suggestions for contamination control on pages 48-50.

Also see the separate article by Cliff Zeyl on Isolating Chlamydomonas from Soil Samples