TRIMMING clearance angle between the cutting facet

- Once the tissue has been embedded and and the tissue block
the wax is solidified, the wax is removed
from the block, the identification number is
noted and the excess was is cut off from the Incomplete sections: DISCARDED
block to expose the tissue surface in
Complete Ribbons: picked up at once with a
preparation for actual cutting
camel hair brush or a pair of forceps

 THIN slices: to prevent the block from Successive sections: stick edge-to-edge due to
cracking local pressure with each cutting stroke, forming
 Sides, top & bottom (trimmed) until a RIBBON
perfectly level
 ALL SIDES are parallel (almost to the edge of
the tissue REMOVING PARAFFIN RIBBONS FROM THE KNIFE

 Sections are removed in ribbons of ten

- to allow easy location of serial section
Cutting Rates depends upon:
Serial section
 Types of the tissue
- unbroken sequence of sections throughout
 Size of the block
the tissue block
 Model or type of the microtome
- ALL sections are saved

 These sections are then floated out on a

Actual thickness of the 1st couple of sections in a
water bath set at 45 – 50⁰C, then
ribbon: thicker (because of thermal expansion
approximately 6 – 10⁰C lower than the
when cutting a cold paraffin block
melting point of the wax used in embedding
Cut in a slow & consistent manner, don’t start and  A section is then selected for staining and
stop while the blade is cutting a block: produce a picked onto a clean slide in a vertical
horizontal line across the block and the sections position

SECTIONING
- a process whereby tissues are cut into
uniformly thin slices or “sections” with the
aid of microtome, to facilitate the studies
under the microscope

 Section is usually cut between 4 – 6 micra in

thickness: Routine Histologic Procedure
 The knife is usually tilted at 0 – 15⁰
angulation on a microtome, to allow a
STAINING Histologic stain: purified form of a coloring agent or
crude dye that is generally applied in an aqueous
- A process of applying dye on the sections to
solution
see and study the architectural patterns of
the tissue and the physical characteristics of
the cells
Acidic in character (nucleus) = greater affinity for
- A process whereby tissue components are
basic dyes
made visible in microscopic sections by
Basic constituents (cytoplasm) = take more of the
direct interaction with a dye or staining
acid stains
solution
- Colored compound: used to produce a
contrast between different tissues and Mordant – a chemical compound that reacts with
cellular components based in their varying the stain to form an insoluble, colored precipitate
affinities for most dyes and stains on the tissue and make the staining reaction
 Morphological changes are more possible
easily identified  When excess dye solution is washed away,
 Physical characteristics and the mordant stain remains.
structural relationships of tissues
and their cells can be evaluated
 Presence or absence of disease can NOTE!
be established The colors of stains are not the real color of a
particular tissue, and that a structure that appears
as one color using one stain, may be a quite
COMMON FAULTS OCCURING DURING TISSUE different color using another stain.
PROCESSING
FAULTS REASONS REMEDY TISSUE STAINING
Prolonged Soak the
fixation, tissue in bowl 1. Histological staining
Brittle or - staining whereby the tissue constituents
dehydration, containing
hard tissue and general relationship between cell and
clearing, water with
embedding phenol tissue are demonstrated in sections by
Repeat direct interaction with a dye or staining
Clearing dehydration solution
Incomplete
agent turns with absolute
dehydration  producing coloration of the active
milky alcohol and
tissue component
clear again
Contaminated Examples:
On trimming wax Re-embed in
wax appears Block is not freshly filtered o Micro-anatomic stains
crystalline cooled rapidly wax o Bacterial stains
enough o Specific tissue stains (e.g., muscles,
Frozen tissue
Warm the connective tissue and neurologic stains)
chips into Tissue is frozen
tissue with
fragments too hard/much
fingers 2. Histochemical staining (Histochemistry)
when cut
 - process by which various constituents of - current recommendation:
tissues are studied thru chemical reactions o maximum of 4% Neutral Buffered
that will permit microscopic localization of a Formaldehyde Solution (NBS)
specific tissue substance o fixation time: up to maximum of
 Chemical ions: calcium, molecules 48hrs (some antibodies)
(bile pigments), and biopolymers
(cellulose), DNA and specific
enzymes METHODS OF STAINING
 In enzyme histochemistry: active
1. Direct (Simple) staining
staining reagent – substrate (which
- process of giving color to the sections by
the enzymes act, and the final
using aqueous or alcoholic dye solutions
coloration produced)
 only 1 dye is used (washed away after 30 –
Examples: 60 seconds, prior to drying and
examination)
o Perls’ Prussian Blue reaction with Iron
 basic dye: positive charge
(hemoglobin)
o cell wall and cytoplasm (bacterial
o Periodic Acid Schiff (PAS) reaction staining
cells): negative charge
for Carbohydrates
o positively charge dye is attracted to
the negatively charged cells,
enhancing the ability of the stain
3. Immunohistochemical staining
stick to and color the cells
- a combination of immunologic and
histochemical techniques using a wide Example:
range of polyclonal or monoclonal,
 Methylene blue
fluorescent labeled or enzyme-labeled
- blue stain will color all cells blue, making
antibodies to detect and demonstrate tissue
them standout against the bright
antigens (e.g., proteins) and phenotypic
background of the light microscope
markers under the microscope
- Widely used in the diagnosis of:
 abnormal cells (such as those found in
2. Indirect staining
cancerous tumors)
- a process whereby the action of the dye is
 localization of biomarkers and
intensified by adding another agent known
differentially expressed in proteins in
as MORDANT which serves as a link or
different parts of biological tissue
bridge between the tissue and the dye, to
 detection of specific molecular markers
make the staining reaction possible
that are characteristic of particular
- Dye may stain only weakly (if at all)
cellular events such as proliferation or
cell death (apoptosis)
MORDANT
- based on antigen-antibody bindings, which
- serves as a link or bridge between the tissue
can be affected by inappropriate fixative
and the dye, to make the staining reaction
selection and duration
possible
 - combines with the dye to form a colored REGRESSIVE staining
“lake”, then this lake will combine with the - The tissue is first overstained to obliterate
tissue forming the “tissue-mordant-dye- the cellular details, and the excess stain is
complex” that is rendered insoluble in removed or decolorized from unwanted
ordinary aqueous and alcoholic solvents parts of the tissue, until the desired
- allows subsequent counterstaining and intensity of color is obtained
dehydration to be carried out easily Example:
 Microanatomical Studies of Tissues
It may be: - Routine Hematoxylin and Eosin (H&E)
 applied to the tissue before the stain staining (most common method): using RS
 included as part of the staining techniques which consists of overstaining the nuclei,
 added to the dye solution itself followed by removal of superfluous and
excessive color of the tissue constituent by
Example: acid differentiation
o Potassium alum with Hematoxylin in
Ehrlich’s hematoxylin Differentiation (decolorization)
o Iron in Weigert’s hematoxylin - selective removal of excess stain from the
tissue during regressive staining in order
that a specific substance may be stained
ACCENTUATOR distinctly from the surrounding tissues
- not essential to the chemical reaction or
union of the tissue and the dye DIFFERENTIAL staining
- does not precipitate in the staining reaction, - uses more than one chemical stain to better
but merely accelerates or speeds up the differentiate between various
staining reaction by increasing the staining microorganisms or structures/cellular
power and selectivity of the dye components of a single organism
- done by washing the section in simple
solution (e.g., water or alcohol), or by the
PROGRESSIVE staining
use of acids and oxidizing agents
- process whereby tissue elements are
- Gram stain (commonly recognizable):
stained in a definite sequence, and the
Crystal violet & Fuchsin or Safranin
staining solution is applied for specific
(counterstain) to differentiate between
periods of time or until desired intensity of
Gram-positive bacteria (large Peptidoglycan
color is attained
layer on outer surface of cell) and Gram-
- Once the dye is taken up by the tissue, it is
negative bacteria
NOT washed or decolorized
- Differentiation or distinction of tissue detail:
COUNTER staining
relies solely on the selective affinity of the
- the application of a different color or stain
dye for different cellular elements
to provide contrast and background to the
staining of the structural components to be
demonstrated
ORTHOCHROMATIC staining by the living cells but taken up by the already
- color shades of the dye itself will stain the dead cells
tissues similarly
Example: Color of the dye (RED) = tissue after SUPRAVITAL staining
staining (RED) - when living cells are stained after being
removed from the body
METACHROMATIC staining - used in microscopy to examine living cells
- dyes that stains tissue with different color that been removed from an organism
from the dye - very dilute solution: 1:5,000 to 1:50,000
- Entails the use of specific dyes which Example:
differentiate particular substances by o New Methylene Blue
staining them with a color that is different o Brilliant Cresyl Blue (reticulocyte staining)
from that of the stain itself (metachromasia)
- Tissue components combine with these Common Dyes:
dyes to form a different color from the  Neutral red – best vital dye
surrounding tissue  Janus green – mitochondria
- Particularly employed for staining cartilage,  Trypan blue – 1 g of dye is dissolved 100ml
connective tissues, epithelial mucins, mast of sterile distilled water (to be used
cell granules, and amyloid immediately); dangerous to allow the
suspension to stand for than 1 hr., because
Examples: it is likely to become toxic to the cell
o Methyl violet  Nile blue
o Azures or Toluidine blue (more effective)  Thionine
o Cresyl blue  Toluidine blue
o Safranin
o Bismarck Brown INTRAVITAL staining
- staining of cells while still part of the body
- done by injecting the dye into any part of
VITAL staining the animal body (either intravenous,
- when inclusions of live cells or tissues are intraperitoneal or subcutaneous), producing
stained specific coloration of certain cells,
- selective staining of living cell constituents, particularly those of the reticuloendothelial
demonstrating cytoplasmic structures by system
phagocytosis of the dye particle
(cytoplasmic phagocytosis), or by staining Common Dyes:
pre-existing cellular components (true vital  Lithium
staining), as in the staining of mitochondria  Carmine
by Janus green  India ink

Reticuloendothelial system with trypan blue or

Propidium iodide for Eukaryotic cells: excluded