Students must locate and observe organisms ranging in diameter from 5 to 10 micrometers to a millimeter or more. Some specimens will have high contrast while others will require special modes of illumination. Students will need to use all available powers of magnification and available modes of light microscopy. It is hoped that they will appreciate the sophistication and utility of the modern research microscope.
Students are presented with two biological models, including Chlamydomonas reinhardi and Naegleria gruberi. The first can be used as a model organism for the study of microtubule assembly and maintenance, and is currently an important model organism in many areas of molecular cell biology. The second is a potentially useful model for cell differentiation.
Students learn how to prepare specimens for microscopy, locate them in a microscope field, fix and stain the cells, and make useful observations on them. Such preparation is essential to the experiments conducted the second week, and to some of the later studies in this and other laboratory courses.
At benches, per pair students:
At the instructor's table:
At the appropriate times prepare:
At a side bench:
Start off having them begin the notebook - title page, contents, page 1 of notes.
Conduct a tutorial on the use of the microscope (this example is for a Nikon Labophot):
Demonstrate how to prepare a wet mount (Vaseline™ mount)
List the day's objectives on board or provide a syllabus (try all modes of illumination and all magnifications):
To keep things moving, a teaching assistant should distribute the first living specimens (Paramecium slides) to the individual students. The instructor must prepare the Chaos/Paramecium slides. The students will be responsible for the remaining preparations themselves, which can be observed in any order. Observations can be made at the students' leisure, however they should be made aware that some studies are timed. For example, the Naegleria do not remain stable for long - in suspension they may either differentiate or encyst. The flagellated stage is transient.
Experienced teaching assistants are a must. As always, the smaller the class size the better. During the tutorial, it is necessary to ensure that students keep up. Each set-up should be checked during focusing to ensure that students focus on the specimen, not on the condenser or something else. They require a great deal of individual attention in using the microscopes.
No formal report is recommended for this first study. However students should keep extensive notes on how to use the microscope and on what modes to use for particular purposes and types of specimens. They should thoroughly record observations made in this laboratory study.
The laboratory study on flagellar regeneration has been adapted in part from Chapter 14 of Bregman, Allyn, Laboratory Investigations in Cell & Molecular Biology (3rd ed.). New York: John Wiley & Sons, 1990. The studies on Naegleria were suggested by Chandler Fulton of Brandeis University.
This study presents a basic experimental design in cell biology, namely use of specific inhibitor substances to interfere with intracellular processes. It also deals with levels of regulation of cellular processes, that is, does flagellar assembly require new protein synthesis? Several experimental principles are emphasized. These include the concept of "blind" sampling, the importance of repeated sampling, the necessity for controls. In fact, most of the samples that are taken in this study are control measurements.
The study also emphasizes the concept of using a simple biological model to study a fundamental cellular process. We ask if flagellar regeneration in Chlamydomonas is dependent on microtubule assembly and/or protein synthesis. These questions can be answered. Next, we ask the same questions about the differentiation of Naegleria gruberi from an anoeba into a flagellate. We cannot answer them because differentiation is such a complex process that we cannot interfere with flagellar assembly alone. We force the cells to encyst, destroy them, or prevent differentiation altogether. Students should take home the message that studies that appear to be unrelated to human biology can be, in fact, fundamental to our understanding of our our own cells' functions.
At benches, per pair students:
For the instructor's use:
At a side bench:
Students should be advised to plan activities before coming to lab. Among other things, each group should have selected someone to do the actual sampling and fixation/staining. Start by going over the experimental design (very briefly) and going through the steps to finding cells and measuring flagella lengths. Have students align/calibrate microscopes and set up data tables while the deflagellation procedure is under way.
To prepare Chlamydomonas first announce that the deflagellation is under way. Group leaders must be available as soon as the deflagellation is complete, and may be asked to take the first sample (an extra one) prior to centrifugation. Deflagellate a large batch of cells as described in the Appendix, part III. Distribute into centrifuge tubes labelled "deflagellated," "deflagellated plus cycloheximide (or emetine)," and "deflagellated plus colchicine." Pool centrifuged cells from two or three tubes to increase the cell numbers. Resuspend in a total of 10 ml each medium.
Nondeflagellated cells from the same culture should be prepared with these same media. We will have, then, a total of six cultures, each in 10 ml medium. These should be kept at a single bench with pipets and bulbs, distilled water in a large vessel for rinsing pipets, and the iodine solution.
Each group leader should take samples from all six cultures at time 0, then from untreated and cycloheximide or emitine treated deflagellated cultures at intervals of 10 minutes. Colchicine-treated and nondeflagellated cultures can be sampled at 30 or 40 min intervals, or just at the end points. Full regeneration typially takes two hours.
If Naegleria transformation experiments are to be performed, the workload
must be adjusted so that students have time to make the additional observations.
They can receive plates or suspensions can be made available to them, having
been prepared by the instructor. Students should be asked to make some kind
of quantitative or semi-quantitative assessment of transformation efficiency,
perhaps by recording number of cysts, flagellates, and amoebae per unit area
per slide. Slides being compared must be prepared identically if quantitative
results are to be meaningful.