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Course syllabus

Introductory lab manual (Bios 211)

Bios 314 Course Syllabus

***THIS COURSE IS NOT OFFERED THE CURRENT ACADEMIC YEAR***

All meetings and due dates for assignments will be established by consensus among students, sponsoring investigator(s), and instructor. The schedule below describes the planned sequence of events. Below the schedule you will find the basis for performance evaluation and course grade.

Weeks 1-2

Organizational meeting

We will meet briefly as a group, preferably the first week of classes. We will discuss course content, try to establish a tentative meeting schedule, and go over performance and grading criteria.

Meeting with sponsoring investigator(s)

The class and instructor will meet with each investigator prior to beginning project work. It will be up to each investigator whether to conduct an informal meeting or present an informal introduction to the project. If more than one investigator is sponsoring the course we will try to schedule a single meeting so that all of us are familiar with the rationale for each sponsored project.

First talk – introduction to TEM

Topics will include: basic principles of electron microscopy and design of an electron microscope; suitable specimens for electron microscopy.

Weeks 2-4

Research proposal

Each project will begin with a research proposal that describes the rationale behind the study, including significance, specific objectives, a research plan, methods, and a lay summary. We will determine by consensus whether to prepare individual proposals or a single group proposal.

Second talk — specimen preparation

The talk will cover the rationale behind "routine" fixation, dehydration, and embedding for transmission electron mircoscopy, and some practical issues. If any research proposal requires a special type of sample preparation, the rationale behind the methodology will be covered.

First experiment(s)

Obviously the timing of the experimental work is completely project-dependent. Members of the team must agree on when to do the experiment and coordinate with the investigator. Some experiments can be done in an afternoon, while others may require days or weeks of preparation. With rare exceptions an experiment will terminate with sample fixation, dehydration, and embedding, a process that requires one full day or two half days on consecutive dates.

The experimental objectives may require special treatment during sample processing, requiring an extra talk or informal meeting to discuss the rationale.

Weeks 4-?

Second talk – sectioning and staining

Topics will include: introduction to the ultramicrotome; rough trimming of specimens; precision trimming; rationale behind ultrathin sectioning; glass vs. diamond knives; cutting ribbons; interference colors; grids and support media; storing grids.

Training session – sectioning

We will try to organize so as to demonstrate use of the microtome to the entire group. Practice samples will be provided. Each participant will rough- and precision trim a block and obtain sections using a glass knife. If necessary, experienced personnel will prepare sections using a diamond knife.

Staining sections

Staining methodology will be demonstrated in one or more informal training sessions. Students will then be responsible for staining their own materials.

Observations

We will schedule time on the biological TEM, observe specimens, and record data if possible.

Round 2

There are no guarantees that an experiment will work the first time, or any time for that matter. If your specific objectives cannot be met with the samples from the first experiment, you will have to go back to the proposal, modify the methodology or possibly even the research plan, and repeat the study. It might be apparent while conducting the first study that a second try will be required. In that case you might proceed with round 2 while finishing up round 1.

Final presentation

We will decide how you will report the study by consensus. Whatever the format, you may choose to submit the work as a group or individually. You may decide to write a paper, prepare a poster (with accompanying talk), or present the results as you would a meeting paper using PowerPoint or similar medium.

Performance criteria and grading

Let's base the course grade 50% on performance and 50% on proposal and final presentation. The performance grade will be based upon (1) a self evaluation, (2) peer evaluation(s), (3) comments from the sponsoring investigator, and (4) observations by the instructor. General criteria for a good performance in any laboratory course are described in the introductory lab manual (Bios 211) under Expectations and Assessment. In addition, a solid performance in this project means giving timely attention to your goals. Past students have jumped on the project from day one, completed their objectives including collecting usable data, and have presented their work in poster sessions or had some of their work published. Publishability of your work is highly dependent on the scope and success of the overall project, so we won't hold up publication as a requirement for excellence. Some students in the past have put off completing the "hard" parts such as sectioning/staining, or even completing the experiment and sample preparation in order to start the project. Course grades have ranged from D to A+; we're serious about performing at this level.

The Bios 318 (microbiology lab) web site includes links to evaluation forms in pdf format. You might take a look at the criteria we used for self and peer performance evaluations as well as criteria used for evaluating posters and presentions. For writing criteria look again at the Bios 211 manual under Expectations and Assessment. Your instructor will probably be saddled with responsibility for evaluating written work, although every effort will be made to coerce a sponsoring investigator into providing comments and recommended scores.

Research proposal

The proposal should be prepared in sections as follows.

• Title and significance of the study
• Specific objectives
• Research plan
• Methods
• Summary

To describe the significance of a study means to defend it, that is, describe how it might contribute to the appropriate field. Your goals might be to shed light on some aspect of the basic biology of cells, for example, or might ultimately be related to understanding a specific disease, developmental problem, etc. Significance includes overall objectives of this area of research. A specific objective is something that will be directly accomplished by a planned experiment. For example, you might study the distribution of myosin light chain isoforms in cultured skeletal muscle tissue toward the overall goal of understanding the role of such proteins in muscular dystrophy. A specific objective of an experiment might be: to determine if the proportion of LC2 is altered in cultured myotubes that are unable to contract due to poisioning with tetrodotoxin when compared with contracting myotubes.

The research plan should describe the experiment in some detail, including the model, how the experiment will be conducted, numbers of replicate samples if applicable, how data will be collected and analyzed, etc. Methods are the details of specific procedures. In the above example the research plan would describe the culturing of skeletal muscle cells including species, type of muscle, method used to assay for light chain isoforms. It would describe how the experimental design will accomplish the objective. Methods would include sources of materials, complete details on how cells were obtained for culture, media components, timing of sampling, method for extracting protein from cultured cells, method of conducting light chain assays.

The summary should be a one paragraph abstract that very briefly describes the overall goals and specific objectives of the study and how the study will accomplish its objectives. It should be relatively nontechnical.