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Week 1: Basic Lab Protocol for Flagella identification in Chlamydomonas reinhardtii.

1. To start, remove 20 uL of the culture and add to a microscope slide to make a wet mount (place a cover slip over the droplet of Chlamydomonas).

a. First, view under 4X, direct field (A) . You should see small green structures that are MOVING.

b. Now, view under 40X, direct field (A). In your notebook, estimate (roughly) how many of them are actually moving.

2. Now, while on 40X, move the setting to Ph1. How does the image change? Can you see any more structures? (yes, they are still moving here!). Record this in your notebook.

3. Now, while on 40X, move the setting now to Ph2. How does the image change? Can you see any more structures? (yes, they are still moving here!). Record this in your notebook.

4. Now, while on 40X, move the setting now to Ph3. How does the image change? Can you see any more structures? (yes, they are still moving here!). Record this in your notebook.

· At this point, record which setting was the best for you to clearly see structures such as flagella.

Part II: Staining Chlamydomonas with Lugol’s Iodine

Lugol’s iodine typically stains starches, and can reveal structures such as flagella, cilia without destroying them.

1. Remove 20 uL of Chlamydomonas and pipet gently into a small glass test tube. Add 2 uL of Lugol’s iodine to the sample in the tube. The Lugol’s should kill the Chlamydomonas.

2. Add the Lugol’s treated sample (22 uL) to a slide and make a wet mount as you did before.

a. First, view under 4X, direct field (A).

b. Now, view under 40X, direct field (A). Under 40X, note what structures you are able to see— can you see both flagella? Any Internal structures?

3. Now, while on 40X, move the setting to Ph1. How does the image change? Can you see any more structures? Record your observations.

4. Now, while on 40X, move the setting now to Ph2. How does the image change? Can you see any more structures? Record your observations.

5. Now, while on 40X, move the setting now to Ph3. How does the image change? Can you see any more structures? Record your observations

At this point, record which setting—direct or phase contrast-- was best for you to see stained flagella. This is important because we will be measuring the length of the flagella, so you need to be able to see it.

Part III: Estimating size (length) of flagella

In the field of view, each of you should be able to see an ocular micrometer through one of your eyepieces. We will use this to estimate the length of the flagella that you are seeing.

Converting ocular micrometer measurements to “real” measurements.

Under 40X, you can use the following relationship: 10 “ocular” units= 0.1 mm (or 100 um)

1. Using the same slide as you did in part II, identify stained flagella under 40X using the Ph2 setting.

2. Estimate the length of the flagella for 10 individual organisms using the micrometer, and record each observation, and calculate the average flagellar length.

Part IV: Data Analysis

For next week, we will design an experiment to test the effects of certain chemicals on the process of flagellar regeneration. Some of this work has been done before, as summarized from this graph shown below (this data chart represents a classroom project evaluating the effects of certain drug treatments). Answer the questions based on the graph below.

· Describe how the three drugs used—colchicine, actinomycin D, and cycloheximide—normally work. Your answer should indicate whether they affect microtubules directly or not. (some do, some do not). You will need to look this up—you must provide the website or citation for the source that you use. Failure to cite will result in zero points for this question.

· Based on this graph, which drug has the strongest effect on flagellar growth? Explain your answer.

· If the “M” line represents the non-drug control, about how long does it take to fully regenerate the flagella?