BIOL 441 - Histology

1. BIOL 441 - Histology To get to online lecture notes: Go to USD Biology Web Page (under Arts and Sciences) Click on People link (on left) Click on David Swanson Click on Personal Web Site Click on Courses Click on Histology Syllabus will appear; click on appropriate lecture to get to lecture notes.

2. Definitions Histology (narrow sense) = the study of tissues Cytology = the study of cells In a broader sense, histology encompasses everything learned via microscopic examination = Microscopic Anatomy Basic tools are microscope and prepared microscope slides

3. PREPARATION OF SLIDES Removal of small tissue sample as a block (<1 cm square). If removal is postmortem, this must be done rapidly so as to avoid deterioration Fixation = immersion of small pieces of tissue in fixative solution (e.g., formaldehyde, alcohol, certain acids). Fixative solution used is determined by particular tissue and staining method. Formaldehyde is by far the most common fixative. FUNCTION = prevents deterioration, hardens soft tissues, can increase affinity for certain stains Dehydration = accomplished by passing tissue through successively stronger solutions of alcohol (removes water so hydrophobic embedding medium can be applied). Alcohol then removed with xylol (embedding agent soluble in xylol).

4. PREPARATION OF SLIDES Embedding = tissue passed through melted paraffin (or other embedding agent; e.g., celluloiden, plastic epoxy for E.M.) so that it becomes saturated with embedding agent (paraffin occupies all spaces in tissue originally occupied by water). Paraffin hardens as it cools - forms block. FUNCTION = provides support for tissue so that it can be sliced into very thin sections. Sectioning = excess wax trimmed away, block is mounted in microtome which cuts very thin slices from the tissue block (3-10 �m; micron = 1 X 10-6m = 1/1000 mm). These slices are used in preparation of microscope slides, after staining and mounting.

5. PREPARATION OF SLIDES Staining = in order to stain, paraffin must be removed (since most stains are insoluble in paraffin, but soluble in water). Slice mounted and passed through xylol, toluol or xylene to remove paraffin. Slice passed through 100% alcohol to remove xylol Finally, slice passed through decreasing strengths of alcohol and lastly through water ? ready to stain. Staining Procedure = stain applied to slice for varying amounts of time depending on stain and desired treatment effect. FUNCTION = to enhance contrast and make certain structures more apparent, different stains used for specific structures.

6. PREPARATION OF SLIDES Mounting Excess stain washed away with water (or alcohol for some dyes, depending on dye solvent) Tissue slice dehydrated by passing through increasing strengths of alcohol to absolute alcohol Alcohol removed by a clearing agent (refractive index similar to tissue) so that unstained spaces appear transparent Mounting medium is then added to tissue slice (medium with same refractive index as glass), covered with a coverslip and allowed to dry ? "Ready to View" SEE HANDOUT FOR REVIEW

8. SECTIONING Longitudinal = lengthwise along a structure (Sagittal section = lengthwise splitting structure into two halves) Cross Section = perpendicular to longitudinal plane Oblique Section = any angle between longitudinal and cross sections Tangential Section (Grazing) = only a small portion of the surface removed from a rounded object See Handout for Interpretation

10. STAINING Chemistry of Stains Basic Dyes = carry positive charge, attracted to acidic components of cells Acidic Dyes = carry negative charge, attracted to basic components of cells Neutral Stains = anion (-) and cation (+) provide different colors PURPOSE of staining is to enhance contrast. This is accomplished in two ways: Different colors Coloring to different intensities

11. GENERAL CATEGORIES OF STAINS Acid-Base Combinations = Most sections are stained with both acidic and basic dyes to enhance contrast by providing different colors. The most common combination is Hematoxylin and Eosin (H & E). Hematoxylin = basic dye, stains nuclear structures blue Eosin = acidic dye, stains cytoplasmic and intercellular structures pink Trichrome Methods = provides 3 colors, allows differentiation between cytoplasmic and intercellular components

12. GENERAL CATEGORIES OF STAINS Specific Stains = stain certain structures or molecules specifically Iron hematoxylin = useful in distinguishing finer cytologic details (e.g., subcellular organelles) Mallory-Azan = trichrome method; stains collagen fibers and mucus blue, stains nuclei and cytoplasmic components red Mason = trichrome method; collagen fibers stain green, cytoplasmic components stain purplish-red Periodic-Acid Schiff = selectively stains carbohydrate-containing molecules/substances red (e.g., glycogen, muco- and glycoproteins, glycosaminoglycans) Silver Impregnation = selectively outlines reticular fibers Orcein, Resorcin-Fuchsin = selectively stains elastic fibers Sudan Black B = specifically stains fat

14. ARTIFACTS Consist of imperfections in tissue preparation Shrinkage = separation of portions of tissue that weren't separated in life; gives appearance of empty spaces Folds and Wrinkles = these may occur during cutting or mounting and will appear excessively darkly stained relative to the remainder of the section Nicks = result from defects in microtome knife; appear as pale, straight lines across the section Degeneration = occurs if tissue not removed immediately or not fixed immediately upon removal; results in inferior quality preparation

15. MICROSCOPY LIGHT MICROSCOPE Magnifies 40-1000 times; magnification = Objective lens power X Ocular lens power High power oil immersion lens has refractive index (amount which it bends light) similar to that for oil, this is why oil must be used as the viewing medium Stains provide contrast which light microscope detects (color contrast and intensity contrast)

16. MICROSCOPY ELECTRON MICROSCOPE (2 types) Transmission (TEM) High velocity electron beam shot through a vacuum through the specimen, focused on fluorescent screen or photographic plate Focus of electron beam provided by electric fields "electromagnetic lenses" Contrast provided by differential resistance to electron flow by different structures within the specimen Magnification is dependent upon the wavelength of the electrons in beam; an increase in voltage (at source) causes a decrease in wavelength and an increase in magnification Resolution limit is about 3.5 angstroms (1 X 10-10m), which gives a magnification of about 500,000 times

17. MICROSCOPY Scanning (SEM) Focused electron beam strikes point on surface of object Primary electrons deflected, secondary electrons emitted Both primary and secondary electrons are collected by a detector which displays a 3-D image of the object's surface The focused electron beam scans the surface of the object so that many, many points are involved in the formation of the 3-D image SEM resolution is inversely proportional to the diameter of the electron beam; resolution is on the order of 25-75 angstroms (Magnification about 50,000 X)