VAna 104 - Histology (Laboratory) – March 23, 2020

The Paraffin Technique

-for preparing samples
-reliable and simple
-the processed are reliably accurate and permit inferences to the in-vivo situation
-most common method in preparing specimen (for histology, diagnostic histopathology, etc.
researches using light microscopy)

Cheap procedure and very reliable method of assessing microscopic structures.

Used worldwide
Used for diagnosis

Video Reference: University of Bristol (2018) – Histology Slide Preparation [Video]

paraffin. [ păr′ə-fĭn ] A waxy, white or colorless solid mixture of
hydrocarbons made from petroleum and used to make candles, wax
paper, lubricants, and waterproof coatings.

1. ACQUISITION
– critical step
Obtaining the sample requires knowing gross anatomy
(identify the desired sample) – removal of the sample must be atraumatic (referring
to procedures that causes minimal tissue injury)
A tissue conscience is as important to the histologist as it is to the surgeon
--dull & dirty scalpels or scissors and excessive pressure or traction during
sampling alters tissue components
--most cells and tissues degenerate when separated from their ideal environments.
--MUSTS:
Tissues must be removed rapidly and deftly;
They should reach the fixative in the shortest time possible to inactivate
autolytic enzymes
a.k.a. Tissue Acquisition
tissue conscience – to minimize excessive handling of tissues to lessen artifacts
*artifacts – the results caused by excessive handling of tissues
Traceability – legal implication before acquiring the tissues

2. FIXATION
Chemical fixation’s primary purpose: stopping postmortem autolysis
(breaking down of body)
By denaturing protein, fixatives inactivate the autolytic enzymes responsible for
postmortem change
--FREQUENTLY USED CHEMICAL FIXATIVES:
 Formaldehyde  Picric acid
 Glutaraldehyde  Potassium
 Paraformaldehyde dichromate
 Ethyl alcohol  Mercuric chloride
 Acetic acid  Chromic acid
 Osmic acid
--some chemical agents are coagulative fixatives (induce changes in cells similar
to those occurring when heat is applied to an egg)
 (these fixatives induce marked structural changes)
(macromolecular conformation is altered by coagulative fixatives)
i.e.: ethanol, methanol

--others are described as additive fixatives (achieve fixation by chemically reacting

with cellular biochemical components)
(they do not induce mark morphological changes)
i.e.: aldehydes (fomaldehyde, paraformaldehyde, glutaraldehyde

a 10% solution of neutral-buffered formalin is the most common fixative.

it consists of 10 volumes of commercial formalin (40% formaldehyde in water)
and 90 volumes of phosphate-buffered water
-should be neutralized close to the body pH (normal body pH: 7-7.5) for the result to be
reliable and the tissue is the same as its organism –life-like state
Buffering is done to achieve the life-like state
action of fixatives: 1. prevent postmortem autolysis by inactivating hydrolytic enzymes
2. facilitate sectioning of the tissues by hardening them
3. enhance staining by acting as a mordant (a binding ion)
4. minimize the leaching of many constituents resulting from
subsequent processing
5. stabilize structural components in as near in vivo conditions as
possible
6. protect the histologist through their antiseptic properties

--immediately immerse the sample into the fixative upon removal from the organism
--trim the tissue block only a few millimeters thick (permits through penetration and
fixation)
IDEAL RATIO OF FIXATIVE VOLUME TO TISSUE VOLUME 30:1
a.k.a Tissue Fixation
-duration: 24 hours (formalin)
--the actual time required for fixation varies with the fixative

3. DEHYDRATION AND CLEARING

Water must be removed (to permit sectioning) -water comprises about 75% of most tissues
Staining of tissue is impossible if water is still present on the tissue, that is why
dehydration takes place.

--extensive washing in water is done to remove excess fixative

--the sample is dehydrated in preparation for embedding

DEHYDRATING AGENTS: ethanol, butanol, dioxane, isopropanol

--tissue samples are immersed in increasing alcohol concentrations until totally

dehydration occurs (with absolute ethanol)

--samples are processed through a clearing agent

(because dehydrating and embedding agents are not
miscible with each other)

the clearing agents replaces dehydrating fluid,

embedding agent replaces the clearing substance
 4. EMBEDDING
--the cleared specimen are processed through solutions containing increasing
paraffin concentrations
embedding reagents maintained at 50° to 68° (melting points)
-heat required may induce morphological changes in the tissues
i.e.: commercial mixtures of purified paraffin and plastic polymers
-effective embedding materials

--the infiltrated samples are placed into molds, surrounded with paraffin, and cooled
Use: paraffin wax dispenser, pan with ice, to harden wax
definition of terms:
Dehydration – the process of removing the water from the tissue. The use of
Ethanol is common for this procedure.
Clearing – the process of removing the alcohol from the tissue to allow infiltration of
paraffin wax. The most commonly used chemical is Xylene.
Embedding – this process is done to avoid any change of the architectural feature
of the tissue. This is done using a paraffin wax through infiltration.
This is to make the tissue hard.

5. SECTIONING
--after the paraffin hardens and the molds are removed, the blocks are trimmed to
expose the embedded tissue

fig 1. microtome
--the blocks are mounted on a microtome, thin shavings (the section) are removed
from the cutting surface

Sections forming a ribbon facilitates sample collection

ribbon (trailing edge of one section, adhering to the leading edge of
subsequent section)
-floated in warm water bath, subsequently stretched, and picked up on slides
-portions of the ribbons may be affixed to the slides and placed on a warming table
to minimize compression artifacts

Rotary microtome – cuts thin sections 1 micron/micrometer thick

*most specimen for histological examinations are sectioned between 5 and 7
micrometers and collected on glass slides
Use: floater (expands paraffin)
 6. STAINING AND MOUNTING
Simplified by the sections of paraffin-embedded materials affixed to glass slides

Most tissue stains are soluble in water or alcohol

*the paraffin must be removed before staining
--remove paraffin before staining
--rehydration of specimen
--staining of slides
-- the specimen are dehydrated, cleared, and covered with a mounting medium
*mounting medium – miscible with a clearing agent
--application of coverslip for permanent preparation

Staining is done to differentiate the nucleus from its cytoplasm

Acidophilic – if the tissue takes up the acidic stain (water-soluble)
Basophilic – if the tissue takes up the basic stain (water-soluble)
*to identify which is which, base on the basic-acidic radical colors

fig.2 pH indicator

*in Mounting, drip xylene onto the slide