HUMAN HISTOLOGY

PRELIMS/LABORATORY SECOND SEMESTER

HISTOLOGY & ITS METHODS OF STUDY

HISTOLOGY
STEPS IN TISSUE PROCESSING:
● Is the study of the tissues, their funcstions,and
how these tissues are arranged to constitute
organ.
ROUTINE: FOR HARD/CALCIFIED
(FDCIETS) TISSUES: (FDDCIETS)
TISSUES
1. Fixation 1. Fixation
● Group of cells with specialized to carry on
2. Dehydration 2. Decalcification
interrelated functions and their associated
extracellular matrix.
3. Clearing 3. Dehydration
● Made up of interacting components: CELLS and
EXTRACELLULAR MATRIX
4. Infiltration and 4. Clearing
Embedding
EXTRACELLULAR MATRIX (ECM)
5. Trimming and 5. Infiltration and Embedding
● The extracellular matrix is composed of many kinds
Sectioning
of:
1) Ground substance and
2) Fibers (ECR) 6. Staining 6. Trimming and Sectioning
➢ Elastic Fiber
➢ Collagen Fiber 7. Staining
➢ Reticular Fiber

● Provides support to the cells, transports nutrients, and

eliminates wastes. A. FIXATION
● NOTES: The contents of the ECM may affect the
function of the cells. ● Since cellular decomposition begins immediately
after the death of a human/patient, tissues must
be fixed to the cells to prevent alterations in their
structure through decomposition. (TO PRESERVE)
➔ Purpose: Preserve the structure and
molecular composition.
1. Avoid tissue destruction by digestiv
enzymes (autolysis) or through
bacterial degradation.
2. Terminate cell metabolism.
3. Kill pathogenic microorganisms
such as bacteria, fungi, and viruses.
4. Hardens the tissue by cross-linking
or denaturing proteins.
● Commonly used fixative is FORMALIN.

★ Preferred 10% NEUTRAL-BUFFERED FORMALIN

➢ 75% FORMALIN

PREPARATION OF TISSUE SLIDES B. DECALCIFICATION

● To study tissues, one must prepare thin and

● Only done in specimens such as bone and calcified
translucent histological sections or tissue slices that
tissues.
can be studied with the aid of a microscope.
➔ Purpose: Removal of calcium and lime
salts, done after fixation and before
★ Cross Examination
dehydration and impregnation; calcium might
- Color, size, weight, type of specimen.
interfere with accurate evaluation and
examination.

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 ➔ Significance: Facilitate normal cutting of
tissue in sectioning; prevent obscuring
microanatomical detail of tissue.
C. DEHYDRATION
● Done by successively bathing the specimen in a
mixture of ethanol and water from 70% to 100%.
(increasing concentration of alcohol)
➔ Purpose: Alcohol removes water out from
the tissues.
● Ethanol: Best dehydrant because it is fast-acting,
mixes with water and many organic solvents, and
penetrates tissues easily.
➔ Not poisonous and not very expensive
➔ Clear, colorless, flammable
E. INFILTRATION AND EMBEDDING
● After the clearing procedure, the tissue is placed in a
melted paraffin in an oven set at 52-60 degree
Celsius.
● The heat causes the clearing agent to evaporate so
that the tissue will be filled up with the paraffin.
● The tissue and paraffin will harden after removal from
oven.
● Paraffin wax
➔ Plastic resins- makes use of plastic solution
which hardens tissue by cross-linking
polymers.
● Eliminates the need to use oven and paraffin; little
tissue distortion
F. CUTTING AND SECTIONING

SOLUTION DURATION
● After the specimen is hardened, it is trimmed into
appropriately sized blocks.
1. 70% ethanol 15 min
➔ Cutting- is the removal of excess paraffin.
2. 90% ethanol 15 min ● The block is then mounted in a microtome and cut
with a steel knife.
3. 100% ethanol 15 min ➔ Sectioning- is done with the aid of a
microtome.
4. 100% ethanol 15 min

5. 100% ethanol 30 min

6. 100% ethanol 45 min G. STAINING

● Since paraffin is colorless, staining is a must.

● Before staining, the following should be done:
➔ Removal of paraffin by xylol or toluol.
D. CLEARING ➔ Rehydration of tissue by descending
concentration of alcohol.
● Removal of dehydrating agent by immersing the
● Most dyes act as acidic or basic compounds.
specimen in the solvent that the alcohol and
embedding medium is miscible.
● Tissues with negative charges are readily stained with
● Makes tissue “translucent” or transparent , hence the
basic dyes- BASOPHILIC
term clearing .
➔ Nucleic acids = Nucleus
➔ XYLENE: most commonly used clearing
agent.
● Tissue with positive charges are stained with acidic
dyes- ACIDOPHILIC
➔ Mitochondria, collagen, cytoplasm
SOLUTION DURATION
★ Hematoxylin and Eosin- Most commonly used stain
1. Xylene 1 20 minutes in histology.

2. Xylene 2 20 minutes ● Hematoxylin: basic dye; usually stains nucleus and

RNA- containing portion of cytoplasm.
3. Xylene 3 45 minutes
● Eosin: acidic dye; usually stains cytoplasmic
components and collagen.
● Chemical Stains
➔ Feulgen reaction- DNA
➔ Periodic-acid Schiff- Carbohydrates
➔ Sudan Black- Lipids

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 H. MOUNTING

● Placing cut sections on a slide with adhesives such as

pinene or acrylic resins.
● The last procedure in the series that ends with a
permanent histological preparation on the table, after
the staining.
● Mounting medium: a solution in which the specimen
is embedded, generally under a cover slide.

I. FROZEN SECTIONS

● Fixation is done rapid freezing.

● Compressed carbon dioxide is emitted.
● Sectioning is done through cryostat, a refrigerated
compartment containing a microtome.
● Method is rapid.
● Routinely done in the hospital to study specimens
during surgery
● Lipids and enzymes are best preserved in this
method.