Section Cutting
MICROTOMY
- a process whereby tissues are cut into uniformly thin slices
or “sections” with the aid of a machine, to facilitate the
studies under the microscope.
MICROTOME - machine/instrument designed for the accurate
cutting of thin slices sections of tissues.
Types of Microtome:
a. SLIDING (TWO TYPES: STANDANRD SLIDE MICROTOME
AND BASE SLEDGE MICROTOME) - for cutting celloidin
embedded sections – ADAMS – 1789 (PROPONENT AND
DATE)
b. ROTARY - for cutting paraffin embedded sections (MINOT
-1885 – 1886)
- most popular and most common
c. ROCKING - for cutting serial sections of large blocks of
paraffin embedded tissues (CAMBRIGDE) (PALDWELL
TREFALL – 1881) MOST BASIC TYPE OF MICROTOME
d. FREEZING - for cutting unembedded frozen sections
(QUECKETT – 1848)
e. ULTRATHIN - for electron microscopy work. (GERMANS–
1900s)
f. CRYOSTAT - For cutting frozen sections. (WILHELM HIS –
1865-1866)
3 Essential parts
 1. BLOCK HOLDER - where the tissue is held in
position.
 2. KNIFE & KNIFE CARRIER - for actual cutting of
tissue sections. (KNIFE IS STATIONARY)
 3. PAWL, RATCHET FEED WHEEL & MICROMETER
SCREW - to line up the tissue block in proper
position with the knife, adjusting the proper
thickness of the tissue for successive sections.
Whatever the type of microtome is used, the principle
remains essentially the same,
***that is, a spring-balanced PAWL or TEETH is brought into
contact with,
***and turns a RATCHET FEED WHEEL connected to a
MICROMETER screw, which is in turn rotated, moving the
tissue block at a predetermined distance towards the knife
for cutting sections at uniform thickness.
Rocking (Cambridge)
 The MOST BASIC/SIMPLEST among all of the
microtomes.
 It cuts in an ARC OF A CIRCLE motion.
 This consists of a heavy base and two arms; the
lower arm resting on pivots and a supporting
column, and attached to the micrometer screw, at
the base of which is found the ratchet wheel with
feed mechanism.
 A section is cut as the tissue passes to the knife edge
in a slightly curved plane, in 10-12 um thickness.
 The Cambridge rocking microtome, available in two
sizes, has been used to cut SMALL and LARGE blocks
of paraffin tissues.
 It is theoretically not recommended for SERIAL
SECTIONS since tissues are cut in slightly curved
planes.
 It is not currently favored by most laboratories
because of the restrictions in size of tissue block that
can be cut, and the difficulty of reorienting the block.
Rotary (Minot) Microtome
 Most COMMON type used for both routine and
research laboratories, especially for sectioning
paraffin-embedded tissues.
 It can be electrically driven.
 It is different from the rocking microtome in that the
knife and the block holder are brought together by
upward and vertical motions, cutting sections in a
perfectly FLAT PLANE, thereby allowing excellent
SERIAL SECTIONS to be cut.
 It may be used for cutting CELLOIDIN blocks of
tissues although results are better when the SLIDING
MICROTOME is used.
 Usually cuts tissue sections in 3 TO 5 um.
 The knife is placed in a BLADE-UP position and is
therefore relatively dangerous.
 Both manual and electric types can be utilized for
CRYOSTAT & ULTRATHIN CUTS OF SECTIONS
Sliding microtome
 Has 2 types
 BASE SLEDGE - consists of two movable pillars
holding the adjustable knife clamps, allowing the
knife to be set at an angle for cutting celloidin
sections.
 The chuck or block holder is set on a heavy metal
base which can be moved backwards and forwards
under the knife.
 The Base-Sledge microtome is favored in
laboratories where very hard tissue or large blocks
are usually sectioned.
 Can be used for CUTTING SECTIONS EMBEDDED IN
ALL FORMS OF MEDIA
 It was originally designed for cutting sections of very
large blocks. (WHOLE BRAINS).
 Sections are cut in a perfectly FLAT PLANE, thereby
making excellent serial tissue sections.
 STANDARD SLIDING MICROTOME - is different from
the base sledge microtome because with this
instrument, the block remains stationary while the
knife is moved backward and forward during the
process of sectioning. (MOST DANGEROUS
MICROTOME)
(BLOCK – STATIONARY, KNIFE – MOVING)
 It is inherently more DANGEROUS because of the
movable knife, which makes it difficult to attach
knife guards.
 In both of these machines, the knife can be set
obliquely for CELLOIDIN sections or straight for large
refractory PARAFFIN blocks, cutting both large and
small tissues with ease;
  it is especially recommended for cutting EXTREMELY
HARD and ROUGH tissue blocks.
Freezing Microtome
 The stage for block holder is hollow and perforated
around its perimeter, attached to a reinforced
flexible lead pipe thru which CARBON DIOXIDE
(CO2) passes from a cylinder.
 It is used to cut UNDEHYDRATED thin to semi-thin
sections of FRESH, FROZEN tissues, especially in
instances when RAPID diagnosis is required, when
histological demonstration of FAT is needed, when
certain NEUROLOGICAL structures are to be studied,
and when sensitive tissue constituents to be studied
are damaged or destroyed by heat.
Ultrathin microtome
 An ultrathin microtome equipped with a GLASS or
gem grade DIAMOND knife is used to cut very thin
sections (typically 60 to 100 nanometers) of tissue
embedded in EPOXY resin.
 Sections are stained with an aqueous solution of an
appropriate heavy metal salt and examined with a
TRANSITON ELECTRON MICROSCOPE (TEM)
 The ultrathin microtome is also used with its glass
knife or an industrial grade diamond knife to cut
semi-thin sections prior to thin sectioning.
 These semi-thin sections are generally 0.5 – 1 μm
thick and are mounted on a glass slide and stained to
locate areas of interest under a LIGHT MICROSCOPY
prior to thin sectioning for the TEM.
 Thin sectioning for the TEM is often done with a gem
quality diamond knife.
Care of the Microtome
 ACCUMULATED PARAFFIN – REMOVED WITH A USE
OF CAMEL/SQUIRREL HAIRBRUSH
 AFTER DRYING – WIPE THE KNIFE & MICROTOME
W/ XYLOL (BECAREFUL NOT TO WIPE THE PAINTED
PARTS
 To prevent rusting, movable portions must be OILED.
 To avoid accumulation of dust, COVER THE
MICROTOME.
 The microtome must be away from AIRDRAFTS,
HALLWAYS/CORRIDORS, AND MOVING STAFF.
 Always remove the knife or blade BEFRORE
CLEANING.
 When cleaning the blade avoid dragging anything
along the cutting edge.
 Even CELLULOSE fibers can cause damage to the
blade.
 Have the instrument inspected at least ONCE a year
by a qualified service technician.
SERIAL SECTIONS
– unbroken sequence of sections throughout the tissue
blocks.
- all sections are saved.
Freezing microtomes have been replaced in most
laboratories by the CRYOSTAT which is easier to operate.
╚> faster, and gives thinner sections.
Frozen sections may be cut from fixed or fresh
tissue, but generally fixed enzyme preparation.
Microtome Knives:
1. PLANE CONCAVE (1. LESS CONCAVE [STRAIGHT] –
CELLOIDIN), (2. CONCAVE – PARAFFIN)
– 25mm in length
- one side of the knife is straight while the other is
concave
- APPLICAPLE TO ROCKING, ROTARY, CELLOIDIN,
STANDARD SM, AND BASE SLEDGE MICROTOME)
2. BICONCAVE
– 120mm in length
- with both sides concave
3. PLANE WEDGE - FOR CELLOIDIN SECTIONS (SSM,
BSM)
– 100 mm in length
- have both sides straight
Today, most knives used in section cutting are WEDGE-SHAPE
(CONSIDERED THEORITICAL) The sides of the wedge knives
are inclined at an angle of approximately 150 (ANGLE OF
INCLINATION) and the surfaces of these are highly polished so
that sections will not adhere to them but will move on the
surface, thus minimizing folding, distortion, and sticking and
facilitating good ribbon formation.
* IN PRACTICE – USES DISPOSABLE RAZOR BLADES
 The actual cutting part of the knife forms an angle
w/c is known as the “BEVEL ANGLE”, and is normally
about 27-32 0.
 The angle of these facets is kept constant for each
knife by the provision of a slide-on back for use on
each knife during the processes of honing and
stropping.
 CLEARANCE ANGLE = 5 – 100
Sharpening of Microtome Knives
* The degree of sharpness is proportional to the fineness of
the ABRASIVE used in sharpening.
* The final sharpening materials used must have a grain size
smaller than the permissible serrations remaining in the edge.
* Sharpening may be carried out either by hand or machine.
HARD SHARPENING – done in 2 ways:
a. By HONES
b. By ABRASIVES and GLASS PLATES (ULTRATHIN)
(EM)
HONE - a natural stone or a hard grinding surface 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.
1. CARBORUNDUM – available in a wide variety of fineness
2. ARKANSAS HONE - a stone of medium fineness
3. YELLOW BELGIAN - the finest
4. BELGIAN BLACK VEIN (blue green)
- also called OIL STONES  because oil is commonly used as
a lubricant
Many oils may be used:
a. light machine oil
b. Liquid paraffin oil (mineral oil)
c. Vegetable oil
d. XYLOL - tends to evaporate too rapidly
e. NATURAL OIL - become messy
Additional hones
 A flat circular glass plate with finely powdered
ALUMINUM OXIDE made into paste with water
(used as an abrasive) may be used for grinding and
removing nicks.
 DIAMANTINE may also be used for final polishing.
The plate glass is usually ¼ - 3/8 inch thick, about 14
inches long and 1-2 inches wider than the length of
the knife blade to be sharpened.
HONING - involves the removal of gross nicks on the knife
(coarse honing) to remove blemishes, and grinding the
cutting edge of the knife on a stone to acquire an even edge.
* Direction: HEEL TO TOE
* Purpose: REMOVE IRREGULARITIES & NICKS ON THE KNIFE
EDGE
STROPPING - the process whereby the “burr” formed during
honing is removed and cutting edge of the knife is polished.
* Direction: HEEL TO TOE
* Purpose: TO SHARPEN & POLISH THE KNIFE
Heel to Toe
 The knife is fitted to its corresponding back, placed
on one end of the hone, and with the cutting knife
edge first, the "heel“ (handle end) is drawn obliquely
or diagonally towards the operator on the stone
until the "toe" (head portion) is reached.
 The knife is then turned over, and the other surface
is again drawn forward, EDGE FIRST, with a HEEL TO
TOE direction.
Toe to Heel
 The procedure is the reverse of honing.
 The knife is first fitted with its appropriate knife
back, then laid obliquely on the strop and with the
cutting edge behind, (EDGE LAST) is pushed
backward and drawn forward in a TOE TO HEEL
direction.
 Around 40 - 120 double strokes are usually required.
Types of Tissue Sections:
1. PARAFFIN SECTIONS
– for paraffin embedded tissue blocks
- may be cut by ROTARY and ROCKING
- - 4-6 µ thick
2. CELLOIDIN SECTIONS
– for celloidin embedded tissues
- usually MADE by means of the SLIDING microtome
- 10-15 µ thick
3. FROZEN SECTIONS may be cut from tissues that have
been fixed and frozen with CO 2 (COLD KNIFE
PROCEDURE) or for fresh or fixed tissues frozen with
the cryostat (COLD MICROTOME)
Other knives
GLASS KNIVES
 are generally used for trimming and semi-thin
sectioning of tissue blocks for electron microscopy.
They are prepared from commercially available 40 x
2.5 cm. plate glass strips that have been washed
with detergent, rinsed in distilled water and alcohol,
and dried with lint-free paper.
DIAMOND KNIVES
 are used to cut any type of resin block for electron
microscopy. When supplied by manufacturers, they
are already mounted in a metal block designed to fit
directly into the knife holder of the ultrathin
microtome.
 These knives are 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.
FLOAT-OUT-BATH/ WATEBATH
 convenient to have such a bath close by 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.
 the thermostat should be set at 45 0C, i.e. about 10 0
below the melting point of the wax used for blocking
out.
 - THIS IS FOR FLOATING OUT OF SECTIONS
- USED TO FLATTEN THE SECTIONS
FORCEPS (fine pointed or curved) and SQUIRREL hair brush
 These tools are needed for handling sections during
cutting, and for removing folds and creases on the
sections during "floating out" in water bath.
PARAFFIN SECTION
Drying Sections on Slides
- after draining, the sections must be fixed to the
slides. This may be done in several ways:
a. In a WAX OVEN at 56 – 60 0C for _ hours
b. In an INCUBATOR at 37 0C OVERNIGHT
c. On a HOT PLATE at 45-55 0C for 30-45 MINUTES
preferably inverted, the slide ends resting on tracklike
support 3-4mm above the plate surface.
d. For 20-3- MINUTES at 50-55 0C in a SLIDE DRYER
(BLOW TYPE)
e. By mounting the sections in 0.1 -0.25% aqueous
floating out gelatin solution of or onto albuminized slides,
draining excess fluid, and while still moist, placing them in a
covered coplin jar containing 2-3 mL 40% formalin in the
incubator at 37 0C for 4-18 hours.
f. In urgency, by carefully holding the slide section
upward above a BUNSER BURNER until wax just melts.
FROZEN SECTIONING
Advantages of Frozen and Cryostat Sections
1. For certain staining procedures:
e.g. - demonstration of fat by the OIL RED O method
- SILVER impregnation method
- For certain methods in the CNS
2. Frozen or cryostat sections are indispensable for
rapid diagnosis during operations.
3. All enzymes are destroyed at temperature above
56 0C and although some (phosphatases) may be
demonstrated in paraffin sections, all are best shown in
cryostat or frozen sections of fresh tissues.
CRYOSTAT
- advantage over freezing microtomes: it maintains
the tissue block, knife and section at the same temperature
- optimum working temperature: 18 TO -20 0C
- cuts individual sections  does not RIBBON
- tissues will also adhere easily to the glass slide
without need for any adhesive mixture.
* Fixed tissue may be used.
 the tissue block is selected by the pathologist
 may be immersed in boiling 10 % BUFFERED
NEUTRAL FORMALIN (BNF) for ½ - 2 minutes before freezing
and sectioning for rapid surgical diagnosis.
Staining of Cryostat Sections
for rapid surgical diagnosis, 2 methods are widely used:
a. H&E (HEMOTOXYLIN & EOSIN)
b. POLYCHROME METHYLENE BLUE
Adhesives
 Used in order to promote ADHESION of sections to
slides.
 Spread thinly and evenly on a clean GREASE-FREE
glass slide, which is then gently approximated to the
end of the ribbon and drawn upwards in a near
vertical motion.
 Aside from being grease-free, the slide must also be
DUST FREE
 If staining is to include antigen retrieval
(IMMUNOHISTO CHEMISTRY (IHC), enzyme
pretreatment IN-SITU HYBRIDIZATION (ISH) or
prolonged incubation steps, charged slides or an
adhesive must be used.
 FISH – FLUORESCENCE IN-SITU HYBRIDIZATION
 Adhesives are not necessary for routine staining,
provided that the slides are clean and free from
grease.
 However, they are essential for methods that
require exposure of sections to ACIDS and BASES
(ALKALI) (especially AMMONIACAL SILVER solutions)
during staining.
***If clean grease-free slides are used and sections are
adequately dried, the sections will not float off during staining
and adhesive will not be necessary.
***There are still certain instances when sections may float
from the slide:
 For urgent CRYOSTAT sections to be submitted for
immunocytochemistry
 For CENTRAL NERVOUS SYSTEM (CNS) tissues
 For tissues containing BLOOD CLOTS
 For tissues which have been decalcified
 When sections are to be subjected to high
temperatures
 The most commonly used adhesive is ALBUMIN
 It is prepared by mixing equal parts of glycerin,
distilled water and white of eggs (GLAIR), then
filtered through coarse filter paper and a crystal of
THYMOL is added.
 THYMOL = ANTI-FUNGAL
 One disadvantage of using albumin is that it RETAINS
SOME OF THE STAINS and gives a dirty background.
 Thymol resistant organisms growing in the adhesive
have been known to contaminate gram-stained
sections and cause confusion during microscopic
examination.
 POLY-L-LYSINE, also a favorite adhesive, can be
bought as a 0.1 % solution and further diluted (1 in
10 with distilled water) when ready to use.
 Sections are coated with this dilute poly-L-lysine and
allowed to dry.
 With time, the adhesive ability of this substance
slowly loses its effectiveness.
 Therefore the coated slides should be used within a
few days. (3 DAYS)

 3 – APES is a better section adhesive and coated
slides can be stored for a long time.
APES – AMMINOPROPYL TRIETHOXYSILANE
 Slides are dipped in 2% APES in acetone drained then
dipped in acetone, drained again and finally dipped
in distilled water.
 It is invaluable in cytology particularly for cytosine
preparation of PROTEINACEOUS or bloody material.
Adhesives
Commonly used adhesives:
1. MAYER’S EGG ALBUMIN
2. DRY ALBUMIN
3. GELATIN
4. STARCH PASTE
5. PLASMA
Mounting Medium
- a.k.a. MOUNTANT
- a syrupy fluid applied between the section and the
coverslip, setting the section firmly, preventing the
movement of the coverslip.
- MOUNTANT – CERTAIN TYPE OF FLUID
* If an unmounted stained section is examined under the
microscope, very little detail can be made out because of the
great difference in the REFRACTIVE INDICES (RI) of the glass
slide, the tissue component, and air.
 for best results with stained tissue sections, they must be
impregnated by a transparent medium having a REFRACTIVE
INDEX (1.518) close to that of glass.
- necessary to protect the stained section from PHYSICAL
INJURY and from BLEACHING or deterioration of the stain as
a result of OXIDATION.
Mounting
Properties of the Best Mounting Medium:
1. will have a R.I. close to that of glass (1.518)
2. will be freely miscible with XYLENE and TOLUENE
3. will be non-reactive and will not change in pH or
COLOR
4. will set without DISTORTION or SHRINKAGE of
tissue sections
5. will set hard without GRANULARITY or CRACKS
6. will not LEACH out any stain or cause any loss of
staining over long periods
Two main groups of Mounting media:
1. AQUEOUS – designed to mount water- miscible
preparation directly from water in cases where the
stain is removed or decolorized with alcohol or
xylene
a. WATER - low refractive index (1.333)
b. GLYCERINE (R.I 1.46) (GLYCERIN JELLY [KAISER 1880]-
R.I. 1.47) - also used as preservative, has high index of
refraction
c. GUM SYRUP (FARRANT’S) R.I. 1.43 – does not solidify
upon storage
d. APATHY’S R.I. 1.52 – used for Methylene Blue-stained
nerve preparations and as general purpose Aqeous Mountant
e. BRUN’S FLUID – recommended for mounting frozen
sections from water
2. RESINOUS
a. CANADA BALSAM (1.524) – natural resin extracted
from the Canadian Tree, Abus Balsamea
- recommended for whole mounts and for thick
sections because it does not shrink much
- miscible with xylene and is quite expensive
b. DPX (1.532) – for small tissue
DPX – DIBUTYL PHTHALATE XYLENE
c. XAM (1.52) – synthetic resin mixture in xylene in
pale yellow or colorless solution
d. CLARITE (1.544)- usually diluted to 60% wit xylene
I. Permanent Mounting Media
- these are resins, either natural or synthetic (soluble in
alcohol)
E.g.
CANADA BALSAM
CLARITE
ENTELLAN
DPX
EUKIT
* Only experience will teach one how much mounting
medium to place on the cover glass.
 If too much is used, it will ooze at the sides of the cover
glass and should be carefully wiped away with the fingernail
covered by a fine cloth dipped in xylol.
 If too little (or too diluted) mounting medium is used, as
the medium sets, it will draw away from the edges of the
cover glass and the section must be remounted, removing the
cover glass by soaking in xylol.
II. Semi-Permanent & Temporary Mounts
* For some preparations, it is imperative not to use
the permanent xylol-resin mounting media, because xylol
dissolves out the essential staining, an example is fat stains
(neutral fats stained in frozen sections by OIL RED O, SUDANS
), these may be mounted in one of the following:
1. water
2. glycerin (glycerol)
3. mineral oil (Liquid Paraffin)
4. Glycerin Jelly (Glycerol Gelatin)
5. Von Apathy’s Gum Syrup Medium
6. Farrant’s Medium
7. Levulose (Fructose) Syrup
RINGING
- process of sealing the margins of the coverslip to
prevent escape of fluid or semi-fluid mounts and evaporation
of mountant, to immobilize the coverslip, and to prevent
sticking of slides upon storage.
 What is the temperature of the hot oven utilized for
KRONIG CEMENT mounting sections that are not properly dehydrated?
- made up of 2 parts PARAFFIN WAX mixed with 4-9 ________________
parts powdered COLOPHONIUM RESIN, heated and filtered.

Adhesives STAINING PROCEDURES

Question:
The main constituent of a Mayer’s egg albumin is glycerin INTRODUCTION
and? * If unstained sections of tissue are examined under the
a. Thymol microscope with transmitted light, very little detail other than
b. Egg yolk the nuclear and the cell boundaries can be identified.
c. Dried albumin
d. Albumin STAINING PROCEDURES
e. Glair
DYEING/STAINING
Question: - enables one to study and to see any physical
What solution is added to dried albumin to form a dried characteristic/s and relationship of the tissues and
albumin adhesive? components of the cell
a. Saline Solution - made possible through CAPILLARY OSMOSIS,
b. Potassium dichromate SOLUBILITY, ABSORPTION & aDSORPTION of stains or dyes
c. Potassium chloride by tissue.
d. Sodium chloride - display differing affinities for most dyes or stains
e. Sugar solution - ABSORPTION – DYE IS TAKEN IN BY THE TISSUE
- ADSORPTION – DIE USUALLY ATTACHES ON THE
The amount of gelatin utilized for gelatin adhesive SURFACE OF THE TISSUE
preparation.
a. 1 gram Nucleus + BASE ---------- BLUE
b. 2 grams (Acid) (HEMATOXYLIN )
c. 3 grams Cytoplasm + ACID -------- RED
d. 4 grams (Basic) (EOSIN)
e. 5 grams
FIXATION - ACID = ACID
What is added to the starch paste in order to prevent mold - BASE = BASE
formation? STAINING = “OPPOSITE ATTRACTS”
a. Phenol Crystals
b. Thymol crystals Staining Principle and Example/results
c. Celloidin procedure Characteristics
d. Paraffin Specific * It is the basis of *Hemosiderin with
e. None of these Staining HISTOCHEMISTRY PERL’S PRUSSIAN
*It is accomplished by BLUE (FERRIC ION)
The amount of outdated plasma dispensed into sterile tubes controlled, specific Fe3+
for plasma usage in section adhesion? chemical reactions - (TURN BULL) Fe2+
1. 0.05 ml designed to give a FINAL
2. 0.5 ml COLOR (staining) at the *Polysaccharides
3. 1 ml site/location of the with the PAS
4. 2 ml structure of the technique
5. 3 ml substances in the cells or PAS – PERIODIC ACID
tissues SCHIFF
Mounting
The following contribute to the Refractive index EXCEPT: Simple or - the staining of tissue by Methylene BLUE
a. Glass slide Direct means of SIMPLE DIPTHERIA
b. Cover slip Staining SOLUTIONS OF A DYE ORGANISM
c. Syrupy fluid (AQUEOUS OR “BABES ERNST
d. All of these ALCOHOLIC) GRANULES”
e. None of these
Indirect -Here the action of the For mordants:
What are the two mounting media commonly used today? Staining dye is intensified by 1. ALUM with
__________________ some other agents: Hematoxylin
 a. MORDANT in Ehrlich’s agents.
b. ACCENTUATOR 2. IRON in 2. Basic is to
Weigert’s acid and
Hematoxylin vice-versa.
3. Alcohol is a
For Accentuator: differentiat
1. POTASSIUM or for both
HYDROXID basic and
(KOH) in acid dyes
Loeffler’s Metachro -a.k.a. METACHROMASIA  Metachrom
Methylene matic -entails the use of the atic dye-
Blue staining specific dyes that stains basic dyes
tissues with a color that is belonging
MORDANT different from that of the to
 a substance which, when taken up by the tissue, stain color itself. - Thizine
helps make the dye in return serving as a LINK or - particularly employed for - Triphenylm
BRIDGE to make the staining reaction possible. staining of : (CECAM) ethane
 combines with a dye forming a colored “LAKE” w/c • CARTILAGE groups
combines with tissue to form an insoluble “TISSUE- • EPITHELIAL Ex: BRILLIANT
MORDANT DYE COMPLEX” RESIN CRESYL BLUE (BCB)
 an integral part of the staining reaction itself, • COLLAGEN for reticulocytes.
without which, no staining could possibly occur. • AMYLOID ***All
• MAST CELL metachromatic dyes
ACCENTUATOR GRANULES are cations and lost
 chemical substances that do not participate but if dehydrated in
merely HEIGHTEN or INCREASE the color intensity, ALCOHOL after
CRISPNESS and SELECTIVITY of the stain. staining, hence
 - differ from mordants in that they do not bind or WATER is necessary
link the tissue to the dye. Counter -the application of a EOSIN - for the
staining different color or stain to cytoplasm
Staining Principle and Example/results provide contrast and
procedure Characteristics background to the
Progressiv - staining is continued (IN Any stain is possible staining of the structural
e Staining A DEFINITE SEQUENCE) as long as no components to be
until the desired intensity DIFFERENTIATION is demonstrated.
of coloring of the different done. Vital – the selective staining of TRYPAN BLUE vital
tissue elements is Staining living cell constituents, stain of RES
attained. demonstrating (RETICULO
- No washing/ cytoplasmic structures by ENDOTHELIAL SYS)
decolorization in between phagocytosis of the dye JANUS GREEN - true
is required solely relies on particle (CYTOPLASMIC vital staining of
the SPECIFICITY of dyes PHAGOCYTOSIS). mitochondria
for different cellular - the nucleus of the living
elements. cell is resistant to vital Other info:
Regressiv - the tissues are AFS and Gram stains, and therefore is – demonstration of
e Staining OVERSTAINED and the Staining not demonstrated. nuclear structures
excess dye is then Other Info: during vital staining
REMOVED until the 1. This is suggests
desired intensity is usually permeability of the
obtained. done by membrane by the
- 2 principles: washing the dye, signifying the
a. OVERSTAINING section in death of the cell
(FLOODING) simple
b. DIFFERENTIATION, solutions METALLIC IMPREGNATION
DECOLORIZATION, OR (water or
WASHING-OUT alcohol),  makes use of heavy metals which are precipitated
-selective removal of acids or with selectivity of certain cellular and tissue
excess stain from tissue. oxidizing components.
  has its greatest application in tissue from the
CENTRAL NERVOUS SYSTEM (CNS)
 but is also used for the demonstration of RETICULIN
 differs from staining in that it consists of an OPAQUE
BLACK PARTICULATE PRECIPITATE
 displays many of the characteristics of true staining.

METALLIC IMPREGNATION
E.g.
SILVER NITRATE – most commonly used agent for
impregnation
- can behave as a stain and can
outline the tissue elements in a
non- particulate union
OSMIUM TETROXIDE/OSMIC ACID – used for
demonstration of lipids
(YELLOW)

A dye should consist of 2 substances:

1. Chromophores
- Grk. “COLOR BEARERS”
- substances capable of producing visible colors.
2. Auxochromes
- Grk. “COLOR INCREASERS” -> SALT FORMING
MOLECULE
- substances which impart to the compound the
property of electrolytic dissociation, thereby altering the
shade of the dye, giving it the property of forming salts with
another compound and ultimately retaining its color.

• The introduction of a chromophoric group into an

uncolored molecule will cause it to be colored; it will
then be a CHROMOGEN, which is colored and not a
dye.
• For a chromogen to be a dye, it must be composed
of an ACID and a BASE, and therefore have SALT
forming properties, ultimately retaining its color.
• The coloring property is attributed to the
CHROMOPHORES and the dyeing property to the
salt forming AUXOCHROMES

Classification of Dyes:

1. NATURAL DYES – e.g. Cochineal dyes, logwood dyes

& vegetable extract
a. HEMATOXYLIN – the most important
b. ORCEIN – a vegetable dye extracted from lichens
which are normally colorless
c. COCHINEAL DYES – a dye derived from an extract
from the female Cochineal Bug (Coccus cacti)
d. SAFRON – a plant with orange stigmas yielding a
dye

*COCHINEAL + ALUM = CARMNE

*CARMINE+ ALUMINUM CHLORIDE= BEST CARMINE
*CARMINE + PICRIC ACID = PICROCARMINE