Flagella Regeneration – Data Collection

Sampling at timed intervals will enable us to record the progress of regrowth of flagella. Cultures can be maintained under identical conditions and small representative samples removed for measurement, leaving the rest of the culture to continue. Samples must be fixed and stained to preserve the structures as they were at the exact moment of sampling, and to enable visualization of flagella.

A complete data set of data should include measurements taken at approximately ten minute invervals at the beginning of the experiment, perhaps with wider intervals (e.g., 15 minutes) toward the end if rates of change appear to be slowing. For non-deflagellated samples it should be sufficient to record average flagella length at the beginning and again at the end of the experiment.

Organization

A team of four people sharing two microscopes should have little or no trouble collecting data for this study. Here is how we will set up the experiment for a class of 20 or so.

• Each team will fix and stain a sample of motile Chlamydomonas, prepare a wet mount of the material, and use the wet mount to set up its microscopes
• When most of the teams are ready to fix samples and can readily find/measure flagella, the instructor will carry out the deflagellation; note the type of procedure that was used
• The instructor and teaching assistants will deliver deflagellated culture to each team; team members will distribute the suspension into tubes with pre-measured amounts of colchcine or cycloheximide
• Each team will score the cultures by sampling at preselected intervals and will log data into team members' notebooks
• Each individual is responsible for recording the fixation/staining procedure, methods for finding/observing/measuring flagella, preparation of experimental and control cultures, and the actual data in his/her notebook

We will use Lugol's iodine for fixing the samples. If the colchicine treated samples rupture, then double the ratio of culture to iodine solution for those samples only.

Recommended scoring intervals

To obtain a time course for regeneration (if regeneration takes place), try to sample the untreated deflagellated culture and the experimental culture (deflagellated and treated with 10 µg/ml cycloheximide) at 10 minute intervals. The colchicine treated deflagellated culture is not expected to regenerate flagella, so we don't need to sample as often. One sample at the start, one in the middle of the experiment, and one at the end should be enough.

Non-deflagellated cultures should be examined at the start of the experiment and at the end, at minimum. If you have time, check each of them once or twice during the experiment itself. We are only interested in whether or not there is a significant change in average flagella length over the time course of the study.

Analyzing the data

You'll be evaluated in part on your success as a team in collecting a usable set of data. Others may need your data, so be prepared to share. Details related to data analysis are presented in the talk on quantitative methods.