Histotechnology– LECTURE (1st SEMESTER)
Instructor: Sir CHARLES ANDRE VLILACERAN <3
Transcribed by: FRANCIS ANDREI H. MIRA
LECTURE 4: IMPREGNATION, EMBEDDING, AND MICROTOMY
NOTE: Difference between impregnation and embedding:
Impregnation is infiltration of the paraffin wax to the tissues.
Embedding is when we put the infiltrated tissues into an
embedding mold with the corresponding embedding media.
IMPREGNATION
• AKA: Infiltration
• Clearing agent is completely removed from the tissue and
replaced by a medium that will fill all natural cavities,
spaces, and interstices of the tissues.
• Sets tissue to a sufficient firm consistency to allow cutting
of suitably thin sections without undue distortion and
without alteration of the spatial relationships of the tissue
and cellular elements.
NOTE:
1. Without firm consistency: tissue will collapse during cutting.
2. Ideal Volume: At least 25x the volume of the tissue.
3 METHODS OF IMPREGNATION
• Three methods of impregnation used in histology:
1. PARAFFIN WAX IMPREGNATION
o Simplest, most common, and by far the best for routine
use.
o Melting point is 54-58°C.
2. CELLOIDIN WAX/COLLOIDION IMPREGNATION
3. GELATIN IMPREGNATION
o Most expensive type of impregnation process
NOTE: Automatic Processing makes use of Autotechnicon.
I. CELLOIDIN IMPREGNATION
• Purified form of nitrocellulose soluble in many solvents.
• Suitable for specimens containing large cavities or hollow
spaces which tend to collapse, for hard and dense tissues
(bones and teeth) and for large tissue sections of whole
embryos.
• PROS
o Causes much less shrinkage and distortion than
paraffin wax.
• CONS
o Slow and tedious
o Serial sections are difficult to prepare
o Very thin sections (<10u) are difficult to cut
NOTE: Faster Impregnation for large tissue specimens, use
Celloidin Impregnation.
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II. GELATIN IMPREGNATION
• RARELY USED except when dehydration is to be avoided
and when tissues are to be subjected to histochemical and
enzyme studies.
• It is used for delicate specimens and frozen tissue
sections because it prevents fragmentation of tough and
friable tissues.
• Water-soluble, does not require dehydration and clearing
• Low melting point and does not cause overhardening of
tissues by heating
• Tissues should not be more than 2-3mm thick since
gelatin embedded specimens are harder to freeze than
non-impregnated tissues.
NOTE: 1% PHENOL Solution is used to prevent the growth of
molds in pair with 20% gelatin.
REAGENT TO TISSUE RATIO STEP
Fixation
20:1
Decalcification
Dehydration
10:1
Clearing
25:1 Infiltration
EMBEDDING
• AKA: Blocking/Casting
• Process by which the impregnated tissue is placed into a
precisely arranged position in a mold containing a medium,
which is then allowed to solidify.
• “Orientation” – the process by which a tissue is arranged
in a precise position in the mold during embedding, on the
microtome before cutting, and on the slide before staining.
• After impregnation, the tissue is placed into a mold
containing the embedding medium and this medium is
allowed to solidify – to proceed with the microtomy, it needs
to be solidified along with wax.
• Paraffin embedded tissues are arranged at the bottom of
them old together with their proper labels and immersed in
melted paraffin at a temperature between 5-10°C above its
melting point.
• And then cooled rapidly in a refrigerator at -5°C or
immersed in cold water to solidify, this will allow hardening
of tissues, giving them a firmer consistency and better
support thereby facilitating the cutting of sections.
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OTHER EMBEDDING METHODS

1. CELLOIDIN/NITROCELLULOSE METHOD
• Recommended for embedding hard tissues such as bones
and teeth and for large sections of whole organs like eyes.
• Low Viscosity Nitrocellulose (L.V.N.) is another form of
celloidin, soluble in equal concentration of ether and
alcohol.

2. DOUBLE EMBEDDING METHOD

• The process in which tissues are first infiltrated with
celloidin and subsequently embedded in paraffin mass.
o Combination of Paraffin and Celloidin.
• Used to facilitate cutting of large blocks of dense firm
SEVERAL TYPES OF BLOCKING-OUT MOLDS tissues (brain) and small sections of celloidin blocks.

3. PLASTIC OR RESIN METHOD

• Has superior results for light microscopic studies,
particularly in hard tissues (undecalcified bone) and for high
resolution light microscopy of tissue sections (renal and
bone marrow biopsies).
o A. Epoxy Plastics (recommended for Electron
Microscopy
o B. Polyester Plastics
o C. Acrylic Plastics

MICROTOMY
(TRIMMING/SECTION CUTTING)
• MICROTOMY
o Process whereby processed tissues are trimmed and
cut into uniformly thin slices or sections to facilitate the
studies under the microscope.
• TRIMMING
o The excess wax is cut off from the block to expose the
tissue surface in preparation for actual cutting.
• SECTIONING/SECTION CUTTING
o Tissues are cut into uniformly thin slices with the aid of
machine to facilitate the studies under the microscope.
• MICROTOME
o Machine/instrument designed for the accurate cutting
of thin slices/sections of tissues

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 CLINICAL CHEMISTRY – LECTURE

STROPPING
• The process whereby the “burr” (debris) formed during
honing is removed and the cutting edge of the knife is
polished.
o DIRECTION: HEEL-TO-TOE (Edge Last)
o PURPOSE: To polish and sharpen the knife.

FLOATING-OUT BATH
• Used to flatten sections.
• Convenient to have such a bath
close to the microtome.
• A circular, thermostatically
controlled bath 10-12 inches in
diameter and 3-4 inches in depth is
desirable.
• The inside surface should be black, thus enabling easier
visualization of sections (visualization of folds and creases
in sections).
• The thermostat should be set at 45°C (45-50°C) about 10°C
(6-10°C) below the melting point of the wax used for
blocking out.
CRYOSTAT/COLD MICROTOME
• Used for:
o Fluorescent Antibody Staining Techniques
o Histochemical Studies
NOTE: Most common solution used for Frozen Sections is your • For rapid preparation of urgent tissue biopsies
Liquid Nitrogen. • The microtome inside the cryostat:
o Rotary Microtome
HONING • STAINING OF CRYOSTAT SECTIONS FOR RAPID
• Involves the removal of gross nicks and irregularities on the SURGICAL DIAGNOSIS, 2 METHODS ARE WIDELY
knife edge (coarse honing) to remove blemishes, and USED:
grinding the cutting edge of the knife on a stone (honing o H&E Staining Technique
proper) to acquire an even edge. o Polychrome Methylene Blue (Loeffler’s Polychrome
o DIRECTION: HEEL-TO-TOE (Edge First) Methylene Blue)
o PURPOSE: To remove the gross nicks and
irregularities on the knife.
• Hone – a natural sharpening stone or a hard grinding
surface (carborundum) for sharpening a knife or other
cutting tools.
• Good quality hones are expensive, but only the best quality
should be used.
• The finer the grain in a hone, the harder the hone.

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 CLINICAL CHEMISTRY – LECTURE

FROZEN SECTIONS
• This method is normally utilized when a rapid diagnosis of
the tissue in question is required, and is essentially
recommended when lipids and nervous tissue elements are
to be demonstrated.
• Requires that the tissue be maintained in the solid frozen
state during cutting of section.
• ADVANTAGES
o 1. For Certain Staining Procedures
▪ a. Demonstration of fats by Oil Red O Method
▪ b. Silver Impregnation Method
(CNS/Neuropatholgy)
▪ c. Immunofluorescence and Immunocytochemical
Staining
o 2. Frozen or Cryostat Sections are essential for rapid
diagnosis during operations.
o 3. All enzymes are destroyed at temperatures
above 56°C and although some (phosphatases) may
be demonstrated in paraffin sections, all are best
shown in cryostat or frozen sections of fresh tissues.
o 2 Methods of Preparing Frozen Sections
▪ a. Cold Knife Procedure
▪ b. Cryostat Procedure (Cold Microtome)
o Commonly used methods of freezing
▪ a. Liquid Nitrogen
▪ b. Isopentane cooled by Liquid Nitrogen
▪ c. Carbon Dioxide Gas
▪ d. Aerosol Sprays

SPECIAL PROCESSING TECHNIQUES

1. FREEZE DRYING
• Preserving tissues by rapid freezing (quenching) and
removing water (desiccation) by a physical process from
the still-frozen tissue block without the use of any chemical
fixative.
• TISSUE SIZE: 2mm Thick
• COMPLETE PROCESSING TIME: 24 – 48 hours
• ADVANTAGES:
o Produces minimum shrinkage
o Allows tissues to be processed in a fresh state
o Less displacement

2. FREEZE SUBSTITUTION
• Similar to freeze-drying
• DIFFERENCE:
o Tissue is fixed in Rossman’s Fluid or in 1% Acetone
and Dehydrated in Absolute Alcohol.
• ROSSMAN’S FLUID COMPONENTS:
o Saturated Picric Acid
o Formaldehyde

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