Chapter 1. Methods of Study

This chapter should help the student to:

• Know the mathematical relationships among the units of measure used for histologic specimens.
• Name the instruments and techniques used to prepare and study histologic specimens; know the attendant advantages and limitations of each.
• Know and compare the basic steps in preparing specimens for light and electron microscopy.
• Select appropriate methods to reveal specific microscopic features and substances in cells and tissues.
• Describe the basic principles of histochemistry.
• Know the substances of biologic interest that can be localized by histochemical techniques.
• Name the classes of histochemical techniques and describe the advantages and limitations of each.
• Name and describe methods appropriate for the isolation and study of specific cells and tissues from intact organisms.
• Name and describe methods appropriate for the isolation and identification of specific proteins and nucleic acid sequences from cells and tissues.

1. List, in order, the basic steps in preparing histologic sections for microscopy (Fig 1–1) and briefly describe the purpose of each step (Table 1–1).

2. Name the type of microscopy that best visualizes unstained living cells and tissues and serves as a basic tool for tissue culture (IV.D.3).

3. Compare light and electron microscopy in terms of:

1. Methods of fixation, embedding, sectioning, and staining (Tables 1–1, 1–2, and 1–3)

2. Thickness of sections (Table 1–1)

3. The support on which sections are mounted (Table 1–1)

4. The type, source, wavelength, and path of illuminating beams (IV.A and B; V.A and B)

5. Magnification (V.A)

4. Compare transmission and scanning electron microscopy in terms of:

1. Methods of preparing tissue for observation (Table 1–1; Fig. 1–1; V.D.2)

2. Electron path (V.A, B, and D.2)

3. Image obtained (V.D.1 and 2)

5. Explain cryofracture, and its advantages over conventional tissue preparation for electron microscopy (Table 1–4)

6. Describe autoradiography, and the type of information it provides (Table 1–4)

7. List the types of cell and tissue components that can be identified by histochemistry (VII.A-F).

8. The Periodic acid–Schiff (PAS) reaction allows the localization of which substances (VII.E)?

9. Describe a typical enzyme histochemical reaction (VIII) in terms of:

1. How the location of a particular enzyme is marked

2. Special considerations in preparing tissue for sectioning

10. Name three types of markers used to make antibodies visible with a microscope (IX.B).

11. List the advantages of studying isolated cells, tissues, or organ rudiments in culture rather than in the intact organism (XI).

12. What is the purpose of cell fractionation and how is it accomplished (XII)?

13. Name three types of column chromatography and describe the protein characteristics targeted by each (XIII; Table 1–5).