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
PREPARATION OF SLIDES
1. Removal of small tissue sample as a block (less than 1 cm square). If
removal is postmortem, this must be done rapidly so as to avoid
deterioration
2. 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
3. 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 is soluble in xylol).
4. 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.
5. 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.
6. Staining = in order to stain, paraffin must be removed (since
most stains are insoluble in paraffin, but soluble in water).
A) Slice mounted and passed through xylol, toluol or xylene to
remove paraffin.
B) Slice passed through 100% alcohol to remove xylol
C) 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.
7. Mounting
a) excess stain washed away with water (or alcohol for some
dyes, depending on dye solvent)
b) tissue slice dehydrated by passing through increasing
strengths of alcohol to absolute alcohol
c) alcohol removed by a clearing agent (refractive index
similar to tissue) so that unstained spaces appear transparent
d) 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
SECTIONING - How to interpret microscopic sections (SEE HANDOUT Fig1 and Fig2)
1) Longitudinal = lengthwise along a structure
(Sagittal section = lengthwise splitting structure into two
halves)
2) Cross Section = perpendicular to longitudinal plane
3) Oblique Section = any angle between longitudinal and cross
sections
4) Tangential Section (Grazing) = only a small portion of the
surface removed from a rounded object
STAINING
Chemistry of Stains
1) Basic Dyes = carry positive charge, attracted to acidic
components of cells
2) Acidic Dyes = carry negative charge, attracted to basic
components of cells
3) Neutral Stains = anion (-) and cation (+) provide different
colors
PURPOSE of staining is to enhance contrast. This is accomplished
in two ways:
1) Different colors
2) Coloring to different intensities
GENERAL CATEGORIES OF STAINS:
1) 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
2) Trichrome Methods = provides 3 colors, allows differentiation
between cytoplasmic and intercellular components
3) Specific Stains = stain certain structures or molecules
specifically
a) Iron hematoxylin = useful in distinguishing finer cytologic
details (e.g., subcellular organelles)
b) Mallory-Azan = trichrome method; stains collagen fibers and
mucus blue, stains nuclei and cytoplasmic components red
c) Mason = trichrome method; collagen fibers stain green,
cytoplasmic components stain purplish-red
d) Periodic-Acid Schiff = selectively stains
carbohydrate-containing molecules/substances red (e.g., glycogen,
muco- and glycoproteins, glycosaminoglycans)
e) Silver Impregnation = selectively outlines reticular fibers
f) Orcein, Resorcin-Fuchsin = selectively stains elastic fibers
g) Sudan Black B = specifically stains fat
ARTIFACTS = imperfections in tissue preparation
1) Shrinkage = separation of portions of tissue that weren't
separated in life; gives appearance of empty spaces
2) Folds and Wrinkles = these may occur during cutting or mounting
and will appear excessively darkly stained relative to the
remainder of the section
3) Nicks = result from defects in microtome knife; appear as pale,
straight lines across the section
4) Degeneration = occurs if tissue not removed immediately or not
fixed immediately upon removal; results in inferior quality
preparation
MICROSCOPY
I. LIGHT MICROSCOPE
A) magnifies 40-1000 times; magnification = Objective lens power
X Ocular lens power
B) 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
C) stains provide contrast which light microscope detects (color
contrast and intensity contrast)
II. ELECTRON MICROSCOPY - 2 types:
1) Transmission (TEM)
a) high velocity electron beam shot through a vacuum through
the specimen, focused on fluorescent screen or photographic plate
b) focus of electron beam provided by electric fields
"electromagnetic lenses"
c) contrast provided by differential resistance to electron
flow by different structures within the specimen
d) 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
e) resolution limit is about 3.5 angstroms (1 X 10-10m), which
gives a magnification of about 500,000 times
2) Scanning (SEM)
a) focused electron beam strikes point on surface of object
b) primary electrons deflected, secondary electrons emitted
c) both primary and secondary electrons are collected by a
detector which displays a 3-D image of the object's surface
d) 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
e) SEM resolution is inversely proportional to the diameter
of the electron beam; resolution is on the order of 25-75
angstroms