HISTOPATH: TISSUE PROCESSING STEP 6-10

STEP 6: TRIMMING
-Removing excess wax after embedding
-Ideal: four-sided prism/truncated pyramid

Coarse trimming - is done on the microtome at approximately 30 microns at a time until the entire
tissue surface is exposed
Fine trimming - may be done by either setting the thickness adjuster at 15mm or by advancing the
block using the coarse feed mechanism

STEP 7: SECTION CUTTING (MICROTOMY)

-Process by which a previously processed tissue (tissue block), is trimmed and cut into uniformly thin
slices or sections to facilitate microscopic study
-2-4mm: size of average tissue block
Summary of the thickness of the
-2-8micra: size of section cut from the block
section of each microtome:
-5micra: tissue routinely cut
Rocking 10-12micra
-Preparation:
Rotary 4-6micra
✓ Knife should be clean, dry, fully, sharpened
Sliding 7-9micra
✓ Floating out bath must have a temperature (45-50C)
Freezing 10-15
below the melting point of the wax (6-10C lower;
Ultrathin 0.5micra
melting point = 56-65C)

1. ROCKING/CAMBRIDGE (PALDWELL-TREFALL)
-simplest type; both small and large paraffin embedded
sections
-for gelatin and celloidin-embedded tissue
2. ROTARY (MINOT)
-heavier and more stable than Rocking Microtome
-most commonly used for routine paraffin embedded Fig. 12-2. Rotary Microtome
sections; rotation of fly wheel
-for routine and research lab
-uses plane-concave knife
3. SLIDING (ADAMS)
-most dangerous; for celloidin embedded sections
-use biconcave knife
*Standard Sliding: MOST DANGEROUS TYPE OF MICROTOME
*Base Sledge Sliding: has 2 movable pillars that holds
the adjustable knife clamps
4. ULTRATHIN - for electron microscopy
5. FREEZING (QUECKETT)
-frozen Sections (Quenching/Rapid Freezing) & Fat
sections; -20C (storage)
-use plane-wedge knife
6. COLD MICROTOME/CRYOSTAT
-for Fluorescent Ab Staining; Fresh Tissue
Examination; Urgent Biopsies

KINDS OF MICROTOME KNIVES

GLASS KNIVES (40 x 2.5 cm)
-used for trimming and semi-thin sectioning of tissue blocks for electron microscopy
-should be prepared and stored in dust-free boxes with lids, just before use, to avoid
contamination

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 HISTOPATH: TISSUE PROCESSING STEP 6-10

DIAMOND KNIVES
-cut any type of resin block for electron microscopy
-are already mounted in a metal block designed to fit directly into the knife holder of the
ultrathin microtome
-brittle and expensive, but very durable, and the cutting edge must be kept clean to make it cut
longer and to avoid damage during sectioning.

MICROTOME KNIVES LENGTH TYPES OF BLOCK MICROTOME

BICONCAVE 120 mm Paraffin embedded sections CO2 Freezing
- both sides concave Rotary
Rocking
PLANE-WEDGE 100 mm -Celloidin embedded sections Base sledge
-have both sidesstraight & for extremely hard tissues type or Sliding
-RECOMMENDED for FROZEN
SECTIONS

PLANE-CONCAVE 25 mm Paraffin & Celloidin embedded Rotary

-One side of the knife sections Rocking
is flat while the other is concave
-Less concave sides: for cutting
celloidin-embedded tissue blocks
on a sliding microtome.
-More concave sides: to cut
paraffin sections on base-sledge,
rotary or rocking microtome

MICROTOME KNIVES INCLINATION/ANGLE

1. CLEARANCE ANGLE: 5–15°
- angle formed between knife and surface of the tissue
-ensures only the cutting edge of the knife touches the
specimen block
-Increasing the angle of clearance angle would give us a
significant difference on the thickness of the tissue being
cut
2. WEDGE ANGLE: 15°
-angle produced between the two plates or both sides of the
knife
3. BEVEL ANGLE/FACET ANGLE/CUTTING ANGLE: 27–32°
-angle formed between the cutting edges A good cutting edge:
-the angles of both sides are reduced so that it can make • Should be made of good quality
a sharp edge that will be used for cutting steel
-SMALLER THE BEVEL ANGLE = SHARPER IS THE KNIFE -Too soft: likely to become dull
4. RAKE ANGLE: edge of the knife at 90° easily
-angle between upper surface of the cutting facet and the -Too hard: likely to produce nicks
surface of the block or jagged edges and irregularities
-the angle produced between the side of the block and the on the knife edge, thereby
side of the knife producing tears or striation on the
tissue sections during cutting
NOTE: Sections are cut between 4-6µ in thickness for routine • Must be able to cut good sections
histologic procedures from a paraffin wax block about 2-3
microns thick, without any
serration noted on examination.

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 HISTOPATH: TISSUE PROCESSING STEP 6-10

HONING (Hard Sharpening)

-Action of sharpening a knife by grinding a cutting edge, either on a stone or with an abrasive
compound.
-Removal of gross nicks and blunts on the cutting edge of knife
-DEGREE OF SHARPNESS = FINENESS OF ABRASIVE USED
DIFFERENT TYPES OF HONING STONES
1. Belgium Yellow 3. Fine carborundum
o Used for manual sharpening o Courser than the first two types
o Gives the best result o Used only for badly nicked knives
o With a very fine abrasive that o (Normally) Followed by either Belgium
produces the best result in terms of yellow or Arkansas
sharpening the edge of the knife
4. Plate Glass
2. Arkansas o Piece of glass about 2 inches wider than
o Has a polishing effect compared to length of knife o Abrasive powder is used to
Belgium yellow remove nicks (Aluminum Oxide made into paste
with water)
o Diamantine is used for final polishing

STROPPING
-process of polishing the cutting edge of the knife on leather or canvas done after honing
-“burr” formed during honing is removed and the cutting edge of the knife is polished
-Purpose: to polish and sharpen the cutting edge

POINT OF DIFFERENCES HONING STROPPING

Use For the removal of gross For the removal of
irregularities/burr

Material Smooth stones, Machine hone Shell horse leather

Lubricant Soapy water, Oil (Mineral oil, Castor Vegetable Oil

oil, Clove oil), Xylene, Liquid Paraffin
Direction Heel-to-Toe Toe-to-Heel

Motion Zigzag Zigzag

STEP 8: STAINING

-process whereby tissue components are made visible
in microscopic sections by direct interaction with
a dye or staining solution
-For contrast; Better optical differentiation;
Improve aesthetic value
NOTE: 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 different color using another stain

Methods of Staining:
HISTOLOGICAL STAINING
• Direct/Simple staining
-tissue constituents and general relationship
-process of giving color to the sections by
between cell and tissue are demonstrated in
sections by direct interaction with a dye or using aqueous or alcoholic dye solutions
-only one dye is used
staining solution, producing coloration of the
• Indirect staining
active tissue component
-process whereby the action of the dye is
-Micro-anatomic stains, bacterial stains and
intensified by adding another agent or a
specific
MORDANT (serves as a link or bridge between
tissue stains
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 HISTOPATH: TISSUE PROCESSING STEP 6-10
the tissue and the dye) to make the staining
reaction possible
• Progressive staining
-tissue elements are stained in a definite
sequence, and the staining solution is
applied for specific periods of time or
until the desired intensity of coloring of
the different tissue elements is attained
• Regressive staining
-tissue is first overstained to obliterate
the cellular details, and the excess stain
is removed or decolorized from unwanted
parts of the tissue, until the desired
intensity of color is obtained
• Differentiation (decolorization)
- selective removal of excess stain from
the tissue during regressive staining in
order that a specific substance may be
stained distinctly from the surrounding
tissues
• Counterstaining
- application of a different color or stain
to provide contrast and background to the
staining of the structural components to
be demonstrated
• Metachromatic staining
- technique entails the use of specific dyes
which differentiate particular substances
by staining them with a color that is
different from that of the stain itself
(metachromasia)
• Metallic Impregnation
-specific tissue elements are demonstrated,
not by stains, but by colorless solutions of
metallic salts which are reduced by the
tissue, producing an opaque, usually black
deposit on the surface of the tissue or
bacteria
• Vital staining
-selective staining of living cell
constituents, demonstrating cytoplasmic
structures by phagocytosis of the dye
particle (cytoplasmic phagocytosis), or
-by staining of pre-existing cellular
components (true vital staining), as in the
staining of mitochondria by Janus green
➢ Intravital staining - done by
injecting the dye into any part of the
animal body (either intravenous,
intraperitoneal or subcutaneous),
producing specific coloration of
certain cells, particularly those of
the reticulo-endothelial system.
Common dyes used: lithium, carmine
and India ink
➢ Supravital staining - examine living
cells that have been removed from an
organism
-enter and stain living cells
Common dyes used are:
1 Neutral red -probably the best vital
dye
2. Janus green-especially recommended
for mitochondria.
3. Trypan blue -one gram of dye is
dissolved in 100 ml. of sterile
distilled water to be used
immediately; it is dangerous to
allow the suspension to stand for
more than one hour, because it is
likely to become toxic to the
cell.
4. Nile blue
5. Thionine
6. Toluidine blue
HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
-process whereby various constituents of tissues
are studied thru chemical reactions that will
permit microscopic localization of a specific
tissue substance
-Tissue components that can be identified:
✓ Chemical ions such as calcium
✓ Molecules such as bile pigments
✓ Biopolymers such as cellulose, DNA and
specific enzymes
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 HISTOPATH: TISSUE PROCESSING STEP 6-10
IMMUNOHISTOCHEMICAL (IHC) STAINING
-combination of immunologic and histochemical
techniques using a wide range of polyclonal or
monoclonal, fluorescent labeled or enzyme-labeled
antibodies
-detect and demonstrate tissue antigens (e.g.,
proteins) and phenotypic markers under the
microscope
STAINS AND STAINING SOLUTIONS
-Divided into 2 categories:
1. NATURAL DYES - derived from plants and
animals, previously used for dyeing wool and
cotton
Ex. Hematoxylin, Cochineal dyes, Orcein,
Saffron
a.) Hematoxylin
- From Hematoxylin campechianum
- Most valuable staining reagent used by the
cytologist due to its powerful nuclear and
chromatin staining capacity
- It is not a true basic dye
- Active coloring agent: hematin
- Oxidation of hematoxylin through the
process of “Ripening”
- Bridge between stain and tissue (Mordant)
and is a requirement for the coloring
reaction
-Copper hematoxylin solutions are utilized
for the study of spermatogenesis
- Examples: Alum, Iron, Chromium, Copper
b.) Cochineal dyes
- Extracted from cochineal bug (Coccus cacti)
- Carmine dye: Cochineal dye with alum;
chromatin and nuclear stain for fresh and
smear preparation
- Picrocarmine: Cochineal dye with picric
acid; for neuropathological stain
- Best’s carmine: Cochineal dye with
aluminum chloride; for demonstration of
glycogen
c.) Orcein
- Colorless, vegetable dye (lichens) used for
staining elastic fibers
- When treated with ammonia and exposed to
air, it produces a blue or violet color
- Weak acid, soluble in alkali
Note: Litmus is also obtained from lichens,
treated with lime and soda, and exposed to
ammonia and air. It is, however, not used as
a cytological stain because of its poor
staining property. It is instead, used mainly
as an indicator
2. SYNTHETIC DYES - Also known as “coal tar
dyes”
- Derived from hydrocarbon benzene
- Collectively known as “Aniline dyes”
- Chromophore: are capable of producing
visible color but is not permanent and can be
easily removed
-Auxochrome: dyeing property; added to a
chromogen, altering its shade, enabling it to
form salts with another compound and enables
it to retain its color in the tissue
a.) Acid Dyes
- coloring substance is found in the acid
component and the inactive base is usually the
sodium salt of a sulfonate of rosaniline
- Basic cell structures have an affinity for
acid dye ions and are called acidophilic
b.) Basic Dyes
- coloring substance is found in the basic
component that combines with the acid radical
(usually taken from sulfuric, acetic or
hydrochloric acid)
- Acidic structures (chromatin, mucus) have an
affinity for basic dyes and are called
basophilic
c.) Neutral Dyes
- formed by combining aqueous solutions of acid
and basic dyes
- stains the cytoplasm and nucleus
simultaneously and differentially
- Insoluble to barely soluble in water, soluble
in alcohol
-Examples: Romanowsky dyes used in hematology,
Giemsa's stain, Irishman's stain for leukocyte
differentiation
LIST OF COMMON HISTOLOGICAL STAINS:
• IODINE
-Oldest stain
-Stains amyloid for microscopic study of starch
granules
• HEMATOXYLIN
-Natural dye derived from the heartwood of a
Mexican tree, Hematoxylin Campechianum
-RIPENING: Hematoxylin ---[o]---→ Hematin
-Karyosome: dark blue, nucleus: blue; cytoplasm:
pink
• EOSIN
-Most valuable stain
-used as a counterstain
-Eosin B: blue-deep red; Eosin Y: yellow-green
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 HISTOPATH: TISSUE PROCESSING STEP 6-10

• METHYLENE BLUE -Contrast stain for Gram’s stain, In Acid Fast,

-Common basic nuclear stain Papanicolau method & Diphtheria organism
-valuable for plasma cells
• VON KOSSA SILVER NITRATE
• METHYLENE VIOLET -Calcium: BLACK
-Metachromatic dye
-for leukocytes • PRUSSIAN BLUE
-Colored Salt of Ferric ferrocyanide, normally
• TOLUIDINE BLUE used for the manufacture of paints
-Nuclear stain substitute for thionine for fresh
frozen tissue • ORCEIN
-Nissl/Tigroid granules and chromophilic bodies -Excellent stain for elastic fiber
-Orcein + Ammonia ---exposed to air→ Blue or
• CRYSTAL VIOLET Violet color
-For amyloid, fungi, platelets in blood
• PICRIC ACID
• ANILINE BLUE -For the demonstration of connective tissue
-Counterstain for epithelial cells (fixative and stain)

• BASIC FUSCHIN • CARMINE

-Is a plasma stain -Used as a chromatin stain for fresh materials in
-deep staining for acid fast organisms smear preparations

• VAN GIESON • ALCIAN BLUE

-Mixture of picric acid and acid fuschin -Stains mucopolysaccharide; more specific for
-for the demonstration of connective tissue connective tissue and epithelial mucins
-SIMPLEST method of differential staining of
collagen • NEUTRAL RED
-Cell granules & vacuole phagocytic cells
• GIEMSA
-Used for staining blood to differentiate • CONGO RED
leukocytes -Best known as an indicator
-Utilized as a stain for axis cylinder in embryos
• CELESTINE BLUE -4% aqueous solution: elastic tissues, amyloid,
-Resistant to strong acids myelin
-Recommended for routine staining of fixed
sections • JANUS GREEN B
-ALTERNATIVE to Iron Hematoxylin nuclear stain -For demonstrating mitochondria during intravital
staining
• MALACHITE GREEN
-Stains Ascaris eggs, RBCs, Bacterial spore • VICTORIA BLUE
-both a decolorizer and a counterstain -Used for demonstration of neuroglia in frozen
sections
• METHYL GREEN
-Stains chromatin green • NIGHT BLUE
-It gives false positive reactions with certain -Is used as a substitute for Carbol Fuschin
secretions such as mucin
• METHYL GREEN PYRONIN
• FEULGEN’S STAIN -DNA: green to blue green
-Most reliable and specific histochemical -RNA: rose red
staining technique for DNA
• ACRIDINE RED 3B
• BISMARCK BROWN -Demonstrates deposits of calcium salts and
possible sites of phosphatase activities

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• ACRIDINE ORANGE
-Permits discrimination between dead and living • GMELIN’S TEST - Diagnostic for Bile pigments
cells ---DNA: green fluorescence
-RNA: red fluorescence • PERL’S PRUSSIAN BLUE - Hemosiderin (iron-
containing pigment of hemoglobin)
• RHODAMINE B
-Stains blood and glandular tissues • BENZIDINE METHOD - For Hemoglobin

• BENZIDINE • CAJAL’S GOLD SUBLIMATE - Astrocyte

-Used for staining haemoglobin
• BIELSCHOWSKY’S TECHNIQUE - Neurons, Axons,
• Sudan Black Neurofibrils
-Phospholipids
• WEIGERT-PAL TECHNIQUE - Normal Myelin Sheaths
• SUDAN IV/SCHARLACH R
-Triglycerides and Neutral lipids (deep red) • LINDQUIST’S MODIFIED RHODAMINE - Copper

• SUDAN III • GRAM-TWORT - Bacteria

-Fats (orange)
• BROWN AND BRENN (B&B) - Bacteria, Nocardia,
• SILVER NITRATE Actinomyces
-Used in Spirochetes reticulum and fiber stains
• CRESYL VIOLET ACETATE - Helicobacter
• AZOCARMINE
-Connective tissues • DIETERLE - Legionella pneumophilia

• PERIODIC ACID SCHIFF (PAS) • WARTHIN-STARRY/LEVADITI’S METHODS -Spirochetes

-Histochemical stain used for the demonstration
of carbohydrates (GLYCOGEN) • GROCOTT METHAMINE SILVER (GMS) - Fungi

• ALDEHYDE FUSCHIN STAIN (GOMORI) • ORCEIN METHOD - Hepatitis B Surface antigen

-Used for differential staining of pancreatic
islets of Langerhans • GORDON AND SWEET’S METHOD - Reticular Fibers

• MALLORY’S PHOSPHOTUNGSTIC ACID HEMATOXYLIN CHIEF SOLVENTS USED FOR STAINS

(PTAH) 1. WATER - should always be distilled unless
-Staining for muscles and bones; Astrocytes otherwise stated.
2. ALCOHOL - Ethyl alcohol may be used in
• LISSAMINE FAST RED TARTRAZINE METHOD various concentrations.
-Muscle demonstration -Methyl alcohol, if to be used, is usually
absolute, and is indicated especially in the
• OSMIC ACID/OSMIUM TETROXIDE STAIN preparation of blood stains, for which
-Used for fats reason, it should be acetone free.
3. ANILINE WATER -10 ml. of aniline is added to
• LEVADITI’S METHOD every 1/2 to 1 liter of hot distilled water,
-Spirochetes shaken, cooled, and filtered.
4. PHENOL - is used in aqueous solution of 0.5
• MASSON FONTANA TECHNIQUE - 5%
-Melanin (Silver Modification): BLACK

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STEP 9: MOUNTING & ADHESION

-Process that involves the use of a medium and a coverslip to facilitate the ease of handling and storage
of the slide and to prevent damage to the section
-Characteristics of a good mounting medium:
1. Refractive index near to that of the glass 9. Does not leach out any stain or affect
(1.518) staining (or form reaction products including
2. It should be immiscible with xylene and those from enzyme histochemical,
toluene hybridization, or immunohistochemical
3. Should be freely miscible with xylene and procedures)
toluene (clearing and dehydrating agent) 10. Does not change in color or pH (should be
4. Does not dry quickly colorless or transparent)
5. Does not produce artifacts on the slides 11. Sets hard and produces permanent mounting of
6. Does not dissolve out or fade tissue sections sections
7. Does not cause shrinkage and distortion of 12. Should set without crystallizing, cracking
tissues or shrinking.
8. Should also completely permeate and fill
tissue interstices.

ADHESIVES
-Adheres the tissue onto the glass slide -It has also been reported to improve protein
1. MAYER’S EGG ALBUMIN coating of ELISA plates
-Most common, easy and convenient, relatively 7. APES (3-aminopropyltriethoxysilane)
inexpensive -Used for cytological studies
-Proteinaceous or bloody material
-Compositions: 50 cc of Egg white, 50 cc of -Slides dipped with 2% APES in acetone
Glycerin -Corrosive
-THYMOL: prevents the growth of mold
-Is used to adhere fecal specimens to glass MOUNTING SECTIONS
slides in the Modified Iron Hematoxylin -Helps to prevent the distortion of image during
2. DRIED ALBUMIN microscopic examination
-Compositions: Dried albumin, NaCl, 1. Koplin Jar: 5-9 slides
Distilled water, Crystals of Thymol 2. Slotted Staining Dishes: 5-19 slides
3. GELATIN 3. Metal/Glass Stain Racks/Carriers: 10-30 slides
-Compositions: Gelatin, Phenol crystals
(prevents the growth of mold), Glycerol, Summary of Refractive Index
Distilled water MOUNTANT RI
-Traditionally, it is used in warm and in a Aqueous Mountant
liquid state Glycerin Jelly 1.47
Farrant’s medium 1.43-1.44
4. STARCH PASTE Apathy’s medium 1.52
-Compositions: Powered starch, Distilled Brun’s fluid -
water (10cc cold, 20cc boiling), N/1 HCl, Resinous Mountant
Thymol crystals Canada Balsam 1.524
-Recommended for use with paper artifacts DPX 1.532
5. PLASMA Clarite 1.544
-Readily available from outdated blood stored XAM 1.52
in blood banks, dispensed into sterile tubes,
0.5 mL Aqueous Mountant
-Hydrophilic and Nonadhesive
6. POLY-L-LYSINE -Generally suitable for all enzymatic
-0.1% detergent solution label/chromogen combinations and fluorescent
-1:10 with distilled water labels
-Final dilution: 0.01%
-Utilized for Immunohistochemistry
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 HISTOPATH: TISSUE PROCESSING STEP 6-10

• GLYCERIN JELLY • CANADA BALSAM

-best for long term preservation
-standard mounting medium used when
dehydration and clearing with xylene cannot
be made
-maybe used as a preservative
-4.0°C ref; 60OC: dissolved/melted before
use (requires ringing)
• FARRANT’S MEDIUM
-Does not solidify upon storage and does
not need to be heated before use
-Requires ringing
-It is utilized for fat & fat derivative
sources
• APATHY’S MEDIUM
- has higher R.I
-Used for methylene blue stained nerve
preparations and as a general aqueous
mountant
-Does not require ringing, sets quite hard
and
-It is generally used for fluorescent
staining
• BRUN’S FLUID
-Recommended for mounting frozen sections
from water
Resinous Mountant
-for preparations that have been dehydrated
and cleared in xylene or toluene, and are
recommended for majority of staining methods
-Hydrophobic adhesive, Organic, and non-aqueous
-A natural resin extracted from the Canadian
Tree (Abus Balsamea)
-Recommended for whole mounts and for thick
sections (does not shrink)
-It sets hard without any granulation &
quite expensive
• Dibutylphthalate Polystyrene Xylene (DPX)
-Recommended for small tissue sections but
not for whole mounts
-Available in a neutral colorless solution
which dries rapidly
• CLARITE
-Usually diluted to 60% with xylene
• XAM
-Synthetic resin mixture of xylene
-Available in colorless or pale-yellow
solution
-Preserves stains well
RINGING
-Process of sealing the margin of coverslip
o To prevent the escapage of fluid or semi-
fluid mounts
o Prevents the evaporation of mountant
o To immobilize the coverslip
o Prevent the sticking of the slides upon
storage
-Kronig Cement: 2 parts of paraffin wax mixed with
4-9 parts of powdered Cophonium Resin
-“Cellulose Adhesive”: Durofix

STEP 10: LABELLING

➢ Process of indicating the year, specimen number on one end of the prepared slide for proper
identification
➢ When it comes to labelling, the slide should be properly labelled with an identifying case number
o The case number on the side of the mounted coverslip o As a general rule, it should be a paper
label bearing
▪ The patient’s name
▪ The section number
▪ preferable the staining method used o avoid causing any damage to the section when labelling
▪ This tends to happen when you wipe the wrong side of the slide
➢ We have the name of the hospital, accession number, the specimen type, what stain was used, and
what date the slide was processed.
➢ In some institutions the name of the patient is placed.
➢ It usually takes 2 days to process a slide.

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