Fixing Chlamydomonas to Preserve and Observe Flagella

The accurate preservation of cellular structures for microscopic examination can be a challenging undertaking. Processing of tissues for electron microscopy has reached the level of an art form. For light microscopy the job is a little easier, but it is not without its pitfalls.

Fixatives and fixation procedures

The word "fix" in this application means "to preserve the structure." The word "fix" is a verb (past tense, "fixed," not "fixated"), "fixation" is the process (a noun), and "fixative," also a noun, is the solution used as the preservative. We have tried two types of fixative and present formulas, procedures, and results. Flagella are extremely fragile, so with either fixative the cells must be examined quickly or else flagella break off and the results are disappointing.

Lugol's iodine

Lugol's iodine consists of 6% potassium iodide and 4% iodine crystals. We prepare the fixative by first dissolving KI in distilled water, then adding metallic iodine and stirring overnight. Before use, we filter the solution through a 0.45 µm syringe filter. One part Lugol's to three parts cell suspension does an adequate job of fixation and staining. Lugol's not only kills and preserves the cells but also stains flagella, which should be observed immediately. Iodine and solutions containing iodine are corrosive and should be handled with care.

One disadvantage of using iodine is that under some circumstances such as in the presence of colchicine, it may cause the cells to rupture.

Fixation with aldehydes

An effective fixative consists of 9% formaldehyde, 3% glutaraldehyde, 0.1% NaCl. It can be made up from stock solutions by mixing 2.4 ml 37.6% formaldehyde, 1.2 ml 25% glutaraldehyde, and 170 µl 1M NaCl, bringing the solution to 10 ml final volume with distilled water. One part fixative to one part cell suspension kills the cells immediately. Aldehyde fixation alone does not stain flagella, so it takes experience and a very good phase contrast microscope in order to see them and make measurements. The fumes from glutaraldehyde and formaldehyde act as irritants and are possible carcinogens, so we must minimize exposure to them.

Observation and measurement of flagella

Complications

Killed cells settle to the bottom of the sample tube. Halos around objects in phase contrast or dark field modes interfere with accurate measurement. An ocular scale may be calibrated to a fraction of a micrometer per division, but it is unrealistic to attempt to report measurements with such implied accuracy, even with the added resolution contributed by oil immersion microscopy. The eye can barely distinguish a single ocular division, in most cases. By now you should have reviewed the document Error analysis and significant figures, perhaps more than once. It should give you a better idea of how to report these data.

Multiple observers contributing to the same data set may apply different criteria to the measurement process, leading to systematic error. For example, one person may line up micrometer divisions such that he/she measures five divisions, while another may measure just four. At 400x, one ocular division may be 2 to 3 micrometers long, thus differences in observing technique can be problematic. When an experiment does require the use of multiple observers, communication among team members is important. One approach is to have each individual examine the same cell (or the same few cells) and record measurements. Comparison of measurements will reveal whether or not a team can expect inconsistency among individuals in reporting of data, and steps can be taken to minimize the problem.

In freshly preserved suspensions (using either fixative) tallies of cells with flagella closely match the estimated percentage of motile cells in the original culture. After several minutes the percentage of cells without flagella increases. Often, in samples kept for more than ten minutes, 50% or more of cells are without flagella. Unattached flagella are readily seen floating in wet mounts of such preparations. Neither fixative appeared to be more effective than the other in preventing the loss of flagella. Any delays in obtaining data will result in measurements that are not representative of the sampled cultures.

In addition to losses that directly result from fixation, there will be variability among measurements from a single sample. Some cells may respond differently to treatments such as deflagellation. Growth rates of flagella may vary from cell to cell. Measurements taken from only a few cells in a sample may not be representative of the whole. Typical cells have two flagella, one of which may be shorter than the other. Flagella may be curved or twisted.

Cells treated with colchicine may swell and rupture in Lugol's iodine. Ruptured suspensions become opaque immediately upon addition of fixative. Examination at 400x reveals the presence of a high concentration of intracellular granules and other debris, especially using dark field. Aside from providing good examples of Brownian motion, such preparations are useless. The few cells that remain intact are swollen and any assessment of presence/absence of flagella is impossible, much less an estimate of average length. This problem has not been evident with aldehyde fixation

Recommendations

In order to be prepared to score samples quickly, inexperienced observers should practice finding cells and measuring flagella, becoming proficient before starting an experiment. The group shoud work as a team to iron out inconsistencies and to double check measurements during the experiment.

Provided that the cells are not treated with colchicine, there is no reason not to use Lugol's iodine as the preferred fixative. The staining of flagella makes the scoring of cultures much more efficient, and Lugol's is less hazardous than aldehyde fixatives. Often, the aldehyde fixative must be used on colchicine-treated cells. With either fixative, cells must be examined immediately.

Gentle agitation of each sample to bring killed cells off the bottom of the sample tube is recommended before taking drops for observation. A sealed wet mount is probably not necessary, since the preparations will have degraded long before an unsealed mount can dry up. However, sealing the coverslip in place reduces water currents, and the vaseline may help hold the coverslip in place when using oil immersion microscopy. To maximize resolution, the sample volume should be just large enough to fill the area beneath a coverslip, and no larger. The greater the space between slide and coverslip, the worse the halo effect and the poorer the contrast, therefore the vaseline layer should be rather thin. To conserve materials, two or three samples should be examined using the same slide.

With either fixative phase contrast microscopy is the method of choice for contrast enhancement. Unstained flagella will not show up at all in bright field mode, but can be readily visualized and measured by an experienced observer in phase contrast or dark field modes. Stained flagella appear thicker than unstained flagella and are readily apparent in phase contrast even to inexperienced observers. They also show up well in dark field, but distortion and glare may interfere with accurate measurement.

It is recommended that one measurement be taken from each cell, from the straighter of the two flagella. A tally should be made of the number of cells found without flagella. An ocular micrometer should be used to measure the straightest of each pair of flagella. Enough cells per sample should be measured in order to obtain a representative sample for calculation of mean length (at least 10 cells per sample, more if there is considerable variation in length).

Some additional observations

Cells that are stained with Lugol's iodine appear brownish with dark flagella in phase contrast, reddish-brown with bright white flagella in dark field. They average 10 µm in diameter, are not perfectly spherical, and display a prominent chloroplast and contractile vacuole if properly illuminated. Oil droplets attached to a glass surface may superficially resemble cells but will have much smaller diameter, are spherical, and show no detail. Unstained cells remain greenish to colorless in all modes. Maximum flagella lengths have ranged from 9 to 12 µm, depending on the culture.

The most common error in making measurements hs been failure to align the phase contrast equipment, so that cells are in sillhouette. The organelles are not apparent under such conditions, and it is impossible to see flagella. All cells, even those without flagella, show a discontinuity in the cell membrane where the base of the flagella should be located. If you can see the "bumps" where the flagella should be, but do not see any evidence of flagella, then chances are you do indeed have a cell without flagella.