SECTIONING Sectioning : cutting tissue uniformly into a thin slice/section with the aid of a machine to facilitate the studies under the microscope. Microtome : a machine/instrument designed for actual cutting of thin section.

KINDS OF MICROTOME

Sliding (celloidin-embedded section)

Base-sledge type - 2 movable pillars holding the knife - chuck/block holder is set on a heavy metal base - block holder is raised towards the knife

Std. sliding type - The block remains stationary - the knife is moved backward and forward during sectioning

Rotary - For paraffin-embedded sections - most common type used for routine & research laboratories

Freezing - For unembedded tissue - a simple lever operated valve release a rapid, intermittent burst of CO2 to freeze the block holder

▪ For rapid Dx

▪ For histological demo of fat

▪ For the study of neurological structure

▪ For the study of sensitive tissue constituent which are

damaged/ destroyed by heat

Rocking - For serial sections of large blocks of paraffin-embedded tissue - produce 60-90sections with ease

▪ Lower arm : resting on pivots and supporting column

▪ Upper arm : carry the block holder on one end by a screw

Ultra thin sectioning microtome

- For electron microscopy

MAJOR PARTS OF A MICROTOME

Block Holder/ chuck Holds the tissue block in place

Knife carrier and knife Actual cutting of tissues

Pawl, ratchet feed wheel, adjs.screw

Line up the tissue in proper position to the knife, Adjust proper thickness of the tissue for successive sections

CARE OF THE ROTARY MICROTOME

▪ after cutting, brush away with soft brush: all accumulated paraffin and tissue

▪ wipe clean all metal parts with xylol

▪ avoid continuous application of xylol to the rest of the machine (can remove

the painted finish)

▪ Dry the machine carefully especially the knife holder

▪ keep the machine well oiled to prevent rust formation

▪ keep the moving parts of microtome oiled

▪ cover the microtome when not in use (prevent accumulation of dust and dirt)

(may interfere the sectioning) MICROTOME KNIVES

PARTS OF MICROTOME KNIFE

Cutting edge Cutting facet, made of good quality steel. Able to cut good section from a paraffin wax block without any serration note on examination.

Wedge

Knife back Spring-loaded semicircular sheet of metal slipped on the knife

Bevel angle Angle between the cutting edge: 27-32

Wedge angle Angle formed by the sides of the wedge knife (5-14)

Clearance angle Angle between cutting facet (5-15)

KINDS OF MICROTOME KNIVES

Plane concave 250mm, 1 side is flat, 1 side is concave

Biconcave 120mm, both sides concave

Plane wedge 100mm, both sides straight

CARE OF MICROTOME KNIFE

Honing ▪ removal of gross nicks on the knife edge (coarse hoaning)

▪ removal of blemishes and irregularities from knife edge

▪ Direction: heel to toe

Stropping ▪ for sharpening

▪ removal of burr or irregularities during honing

▪ final polishing of the knife edge

▪ Direction: toe to heel (reverse of honing)

STROPPING Hone: a natural stone or hard grinding surface for sharpening a knife

TYPES OF HONES

Belgium yellow/ hone ▪ for manual sharpening

▪ usually gives best result

Arkansas ▪ gives more polishing effect

Fine carborundum ▪ much coarser

▪ used for badly nicked knives

Plate glass hone ▪ use diamanthine for final polishing

Machine hone ▪ glass disc or wheel driven by electric motor

COMMON LUBRICANT USED FOR HONING - mineral oil - clove oil - xylene - liquid paraffin - soapy water CARE AND USE OF MICRTOME KNIVES

▪ perfect edge: junction of the smooth plane surface 14°

▪ use fine yellow Belgian water stone to remove large nicks

▪ size 8’’ x 3’’

▪ keep the knife flat when being honed

▪ finish the edge on a leather or linen strop

▪ wipe the knife clean with soft cloth before and after stropping strokes

▪ use gentle pressure when stropping

▪ avoid speed in stropping

▪ leather strop require oiling before use. Use castor oil or vege oil to treat the

strop applied at the back of the strop (not on the surface)

▪ mineral oil destroys the leather strop, done allow to come in contact with the

strop

▪ wax shouldn’t come in contact with the strop

TYPES OF TISSUE SECTIONING

PARAFFIN SECTIONS

▪ Prep: trim the block until all sides are parallel

▪ Adhesive: for entailing significant exposure of section to acids and alkalis, but

cannot be used for protein histochemical investigation

Mayer’s albumin Egg white + glycerin + thymol crystal

Dried albumin Dried albumin + NaCl + thymol crystal

Gelatin Gelatin + water + glycerol +phenol crystal

Gelatin chrome alum

Can be added to water bath.

Starch paste Powdered starch + HCl + thymol crystal

Plasma Outdated blood stored in blood banks

▪ Actual Sectioning -nervous tissue, lymph nodes : slow, gentle motion -the harder the tissue, the harder & cooler the block should be

▪ Floating out bath

45-50C or 6-10C lower than the melting point of paraffin

▪ Drying of sections

-wax oven 56-60C (2hours) -incubator 37C (overnight) -hot plate 45-55C (30-45 min) -blower type electric slide dryer 50-55C (20-30min) -bunsen flame until the wax melts -nervous tissue: incubation overnight 37C

DIFFICULTIES ENCOUTERED IN SECTION CUTTING

FAULTS REASON REMEDY

Brittle/hard tissue

▪ prolonged fixation

▪ prolonged dehydration

▪ prolonged clearing

▪ prolonged paraffin infiltration in over heated paraffin oven

▪ drying out of tissue before fixation

Soften tissue by soaking oil in a smaller dish or bowl (w/water) with detergent, phenol or molliflex

Clearing becomes milky as soon as tissue

is placed in it

▪ water not removed

completely (incomplete dehydration)

Repeat dehydration with absolute alcohol, then clear again

On trimming, tissue smells of clearing

agent

▪ incomplete removal of

clearing agent due to insufficient impregnation

▪Trim the blocks down nearest

to the tissue

▪Melt the remaining wax on embedding oven

▪Repeat paraffin impregnation

Tissue is opaque section cutting is difficult due to

presence of alcohol

▪ insufficient clearing Repeat clearing

Tissue shrinks away from the wax when

▪ insufficient dehydration Repeat whole procedure

trimmed

Tissue is soft when block is trimmed

▪ incomplete fixation Repeat fixation

Airholes found on tissue during trimming

(appear crystalline)

▪ incomplete impregnation

▪ contaminated wax

▪ block not cooled rapid enough

Reembed in freshly filtered wax

Moist & crumbled Paraffin block after

cooling

▪ insufficient paraffin

impregnation

Repeat paraffin impregnation, reembed

Sections fail to form ribbons

▪ surfaces and edges of block are not parallel

▪ horizontal surface of the block is not parallel to the knife

▪ paraffin wax is too hard

▪ knife is tilted too much

▪ sections are too thick

▪ knife is dull

▪Retrim the block

▪Re adjust and reorient the block

▪Coat horizontal edge of block

with wax of lower MP

▪Reduce the tilt of knife

▪Readjust the thickness

Sections roll up on cutting so that they

adhere and get broken and against the knife

edge

▪ knife is dull

▪ knife is tilted too much

▪ knife edge is dirty

Sharpen the knife Reduce the tilt Clean the knife edge

Ribbon is curved, crooked or uneven instead of straight

▪ blunt or dull spot on knife

(irregular knife edge)

▪ edges of the block are not

parallel but round or wedge-shaped

▪ knife is not parallel to the block

▪ paraffin is impure

▪Adjust the knife so that knife

edge will present formly sharp edge to the block or sharpen

▪Retrim the block

▪Readjust the knife and the

block

▪Repeat impregnation using pure wax

Sections are compressed, wrinkled

or jammed

▪knife is blunt or dull

▪ paraffin block is warm and

soft

▪ knife edge is coated with paraffin

▪ sections are too thin

▪ microtome set screw is loose

▪ tilt of knife is too vertical

▪Resharpen the knife

▪Cool the block on ice water

until firm

▪Clean the knife edge

▪Readjust the thickness of

section

▪Thighten the screw

▪Reduce the tilt

Sections are torn and crumble when cut

▪incomplete dehydration, clearing and infiltration of tissue with wax

▪ paraffin is warm and soft

▪ knife is blunt

▪remove paraffin with clearing agent, pass thru decreasing grade of alcohol, then repeat dehydration, clearing and embedding

▪ cool and harden paraffin in

ice water for ¼ to ½ hour.

▪ sharpen the knife

Sections are squashed width of each section less than that of the

block

▪ Bevel of knife is lost due to incorrect sharpening

▪ resharpen, using a knife back or automatic knife sharpener

A Hole is formed in the section

▪ bubble or dirt formed in the embedding medium

▪ hard spot in tissue possibly

due to calcium

▪ reembed in freshly filtered wax if necessary

▪ once embedded in paraffin

wax, decalcification is impractical use a base sledge microtome with a wedge knife

Sections of unequal thickness are

produced

▪ tilt of knife is too great or

bevel is not cleared, hence object is compressed against the knife edge

▪ clamp set screw on the knife

or blockholder is loose

▪ blocks are too large

▪ blocks are too hard

▪ reduce the tilt

▪ tighten screw

▪ cut blocks into smaller fragments

▪ soften blocks in detergent or

phenol

Sections adhere to the knife or other parts of

the machine

▪ static electricity due to low atmospheric humidity

▪ knife edge is dirty

▪ knife edge is dull

▪ knife tilt is too great

Breathe out or blow gently on the block and knife to break up static electricity or boil in water in room to increase the humidity

Ribbon is split or lengthwise vertical

scratches are seen on section

▪ nicks/damage on the knife

edge

▪ dirty embedding medium

▪ knife edge is dirty

▪ tilt of the knife is too great

▪ sharpen the knife

▪ reembed in filetered wax

▪ clean the knife edge with

xylol

Sections are lifted from the knife on

upstrokes

▪ knife tilt is too great

▪ knife is dull

▪ paraffin is too soft r RT is warm

▪ cool paraffin wax in ice water

Resistance is felt on the lower part of

section during cutting

▪ tilt of knife is insufficient, paraffin block is therefore compressed against the base of knife towards the end of the stroke

▪ increase the tilt

Horizontal or parallel lines or forrows across the sections/chatters are seen, forming thin

and thick zones

▪knife edge vibrates due to:

a) hardness of the tissue b) tilt of the knife is too great

▪ treat with phenol during

processing or collodionize

Section cut is sometimes thin, sometimes thick

▪ knife is blunt

▪ knife isn’t clamped properly

▪ tilt of knife is too great

▪ knife or blockholder is loose

▪ knife tilt is too small that block is compressed by bevel

and section isn’t cut

▪ tighten adjusting and locking screws

▪ increase tilt

Knife makes a hard metallic scraping or

ringing sound on backstroke, when

sectioning

▪ tilt of knife is too slant or too big

▪ tissue is too hard

▪ knife blade is too thin

▪ readjust the angulation of the knife

▪ take fresh block

▪ change the knife

Frozen tissue crumbles and comes off the

blockholder when cut

▪ freezing isn’t adequate Refreeze the tissue

Frozen tissue chips into fragments when

cut

▪ tissue is frozen too hard ▪ warm the tissue with fingers

DIFFICULTIES DERIVED FROM THE TISSUES

blood clot, cervix, thyroid tissue

very hard in routine processing

block before cutting, effect with a firm sharp stroke. Use chloroform as clearing agent. Use tetrahydrofuran for dehydrating agent.

fatty tissues (subcutaneous tissue, breast, lipoma)

soft blocks and shredded tissue

Cut thin 2mm, and impregnate with wax in vacuum bath

brain and lymph nodes

very hard and brittle (if using xylol for dealcoholization)

Use chloroform and vacuum impregnation with lymph nodes

soft tissue Tend to expand more when floated out

set and cool the wax block 10-12%shrinkage.. Split the outer rim of wax with a dissecting needle.

CELLOIDIN SECTIONS

▪ 10-15u in thickness

▪ don’t require hardening by chilling before cutting

▪ use sliding microtome

▪ use wet method to avoid dehydration and shrinkage FROZEN SECTIONS

2 METHODS OF PREPARATION

Cold knife

▪ knife -40 to -60C

▪ tissue

-5 to -10C

▪ environment 0 to -10C

▪ CO2 (Freezing agent)

▪ different temp

between knife and tissue (latter is colder)

▪ filter paper soaked in gum syrup on microtome stage

▪ apply short burst of CO2

▪ 3-5mm thick

▪ 5 sec interval

▪ dew line: the point at which sections may be cut at 10 u.

Cryostat Near -20 C

▪ (-5 to -15C) brain, lymph nodes, liver, spleen, kidney, testis, uterine, tumor, thyroid

▪ (-15 to -20C) Muscle, conn tissue, pancreas, uterus, cervix, skin without fat, nonfatty breast tissue, ovary, prostate, tongue, gut

▪ (-35C) Fatty tissue, fatty breast, omental

Advantages:

▪ staining (fat demo by oil red O, silver impregnation, CNS methods)

▪ indispensable for rapid diagnosis during operation

▪ enzyme demo

Disadvantage:

▪ sections don’t form ribbons but stick to the knife blade

(remove with camel’s hair

brush)

▪ lack of embedding mass, distorted structural details during cutting

▪ staining of unfixed tissue is rarely unsatisfactory

▪ produce freezing artifact

STAINING Formation of colors of different tissues and cells -LEEUWENHOEK (saffron) -GOPPERT, COHN (carmine) -GERLACH (sel. Nuclear stain) PURPOSES OF STAINING

▪ render different tissue constituents (more visible)

▪ easier optical differentiation for ID of cells and tissue

▪ display various affinities

▪ display physical char and structural relationships

METHODS TO OBTAIN STAINING REACTION:

▪ Capillary osmosis

▪ Solubility

▪ Adsorption

▪ Absorption

TYPES OR METHODS OF STAINING

Direct Use simple aqueous or alcoholic solution of the dye

Indirect ▪Mordant (link/bridge to form color)

▪Accentuator (accelerate/hasten the speed of staining)

Progressive Not washed or decolorized

▪ less favorable than regressive. Difficultly of producing

intense cell structure, diffused and obscured effect

Regressive Gram staining excess stain is decolorized

▪differentiation/decolorization: selective removal of excess

stain from tissue

Counterstaining Apply different color/stain to provide contrast and background. C Y T O P L A S M I C

▪RED ▪Yellow ▪Green

-Eosin Y,B -Picric acid -Light green SF -Phloxine B -Orange G -Lissamine green -Rose Bengal N U C L E A R

▪RED ▪Blue

-Neutral red -Methylene blue -Safranin red -Toluidine blue -carmine -celestine blue -hematoxylin

Metachromatic Use specific dye (metachromasia) Thiazine and triphenylmethane group -methyl violet/crystal violet -cresyl blue -safranin -bismarck brown -basic fuchsin -methylene blue -thionine -toluidine blue -Azure A,B,C

Microanatomical Demostrante general relationship of tissue and cell without emphasizing the inclusion bodies

▪Cytoplasmic staining

-doesn’t differentiate tissue structure in general

▪Negative staining

-demonstrate bacterial morphology in black background (india ink)

Metallic Impregnation

Demonstrate tissue elements not by stains but by colorless solutions of metallic salts. *adsorption The most valuable metal: gold (gold chloride) and silver (silver nitrate)

▪ Explosive

▪ avoid silver glasswares

▪ don’t expose to sunlight

▪avoid metallic instruments

Vital SUPRAVITAL: -neutral red -janus green -tryphan blue -nile blue -thionine -toluidine blue

▪ Selective staining of living cell constituents

▪demonstrate cytoplasmic structures by phagocytosis of

dye particle

▪nucleus is resistant to vital stain

INTRAVITAL STAINING inject the dye into any part of animal body (intravenous/intraperitoneal/subcutaneous) SUPRAVITAL STAINING stain living cells immediately

COMPOSITION OF DYES

NA

TUR

AL

Fro

m p

lan

ts, a

nim

al

(fo

r w

oo

l & c

ott

on

)

Hematoxylin ▪ Low affinity for tissue, must use mordants (alum, iron, chromium,

copper salts)

▪ Extraction from

heartwood of Mexican Tree: Hematoxylin

campechianum

▪ Hematin

Active coloring agent, oxidized

hematoxylin

▪ Ripening

Oxidation process of hematoxylin to

hematin. (exposure of stain to

air and sunlight) OXA: -H2O2 -HgCl2

-KMnO4 -Na perborate

-NaI

Alum Hematoxylin ▪ For progressive

staining

▪ Counterstained with congo red & safranin.

▪ Salt lakes color: Blue

▪ Blueing: process of

passing the tissue to alkaline solution to

neutralize acid.

▪ Cold water (slows down)

▪ warm water (accelerates)

▪ Very cold water (<10C, produce pink

artifacts)

a) Ehrlich’s hematoxylin

▪ Regressive staining

▪ for muco polysaccharide, cartilage, cement lines of bones b) Harris Hematoxylin

▪ for routine nuclear staining

▪ for exfoliative cytology

▪ for staining sex chromosomes

c) Cole’s hematoxylin

▪ for routine purposes

▪ used in sequence with Celestine blue

d) Mayer’s hematoxylin

▪ used in Celestine blue

▪ for nuclear staining

Iron Hematoxylin ▪ Mordants: ferric ammonium sulfide

(iron alum)

a) Weigert’s hematoxylin

▪ std. for lab

▪ muscle fiber demo

▪ conn.tissue demo

b) Heidenhain’s hematoxylin

▪ cytological stain

▪ for regressive staining of thin sections

▪ for nuclear and cytoplasmic

inclusion (chromatin, chromosome, nucleoli, centrosome, mitochondria) c) Phospho tungstic acid hematoxylin (PTAH)

▪ for structures in paraffin, celloidin, frozen section

Cochineal Dyes ▪ old histologic dye

▪ from extraction of female cochineal bug (coccus cacti)

▪ treated with alum to produce carmine dye

▪ combination with

picric acid/ picrocarmine is used for neutropathological study

▪ combination with

AlCl3/ best’s carmine is used for glycogen demo

Orcein Dye ▪ vege dyes from

lichens

▪ colorless

▪ combination with ammonia and exposure to air produce blue and violet

Syn

the

tic

dye

s/ c

oal

tar

dye

s /A

nili

ne

dye

s (f

rom

C6H

6 h

ydro

carb

on

be

nze

ne)

▪ Chromophores

-produce visible colors

▪ Chromogens

-benzene compounds with chromophore

▪ Auxochrome

-auxiliary radical (props of electrolytic dissociation) -alter the shades of dye -give the props of forming salts

Dyes: a) Acid Dyes

▪ acid: coloring substance

▪ base: sodium

b) Basic Dyes

▪ acid: sulfuric, acetic or HCl

▪ basic: coloring substance c) Neutral dyes

▪ Acid aq + basic aq

▪ for nucleus and cytoplasm

▪ soluble in alcohol

▪ insoluble in water

DIFFERENT STAINS USED IN HISTOPATHOLOGY

Aniline blue Cytoplasmic, counterstain

epithelial section

Basic fuchsin

Plasma stain acid fast organism, mitochondria, smooth muscle Fuelgen’s + schiff’s reagent: detect aldehydes Van gieson’s : conn.tissue, mucin, elastic tissue

Bismarch brown

Contrast stain gram’s technique, acid fast & papanicolau method, diphtheria organism

Carmine Chromatin stain smear prep… Best carmine solution for glycogen Mucicarmine for mucin

Celestine blue

Resistant to strong acid dyes

for routine staining of fixed sections, +alumH = good nuclear staining

Crystal violet

Nuclear/chromatin stain stain amyloid in frozen section, stain platelet in blood

Eosin Eosin B : bluish deeper red color Eosin Y : yellowish

Stain conn tissue and cytoplasm Routine: Hematoxylin – Eosin – methylene blue

Giemsa stain

Stain blood to differentiate leukocyte

Malachite green

Weakly basic, contrast stain, decolorizer, counterstain

Ascaris eggs, erythrocyte,

Methyl green

Chromatin stain, Gives false positive with mucin secretion

Chromatin green (w/acid)

Methylyne blue

Basic nuclear stain, plasma cells, cytological evaluation

Fresh sputum, malignant cells, bacterial stain, milk grading, diphtheria, nervous tissue,

Methylene violet

Metachromatic dye Leukocytes’s nuclei: reddish purple with methylene blue

Orcein Dermatological study Excellent for elastic fibers

Picric acid Contrast stain Cytoplasmic stain Counterstain Tissue fixative Decalcifying agent

Acid fuchsin, conn.tissue (van gieson) crystal violet

Prussian blue

Colored salt of ferric ferrocyanide Microanatomical color contrast

Manufacture of paints Circulatory system

Toluidine blue

Nuclear stain Substitute Recommended for:

Fixed tissue Thionine in frozen section -nissl granules -chromophilic bodies

COMMONLY USED SPECIFIC TISSUE STAIN

Routine histologic study H & E

Conn. Tissue/ collagen Methylene blue

reticulum Gold method

Elastic tissue Orcein

Muscle tissue Van gieson’s

fibrin H & E

Amyloid Methyl violet

Glycogen Best carmine

Mucin Meyer’s mucicarmine

Acid mucopolysaccharide Toluidine blue

Nissl bodies H & E

Neurons, axons, neurofibrils Bielschowsky’s technique

Myeline sheath Sudan black

Astrocytes Mallory’s PTAH

Argentaffin granules Masson Fontana silver method

Phospholipids Baker’s technique

Neutral fat Sudan black

Fatty acids Lillie’s sulfuric acid nile blue

Hb Amido black

Hemosiderin Prussian blue

Hematoidin Gmelin’s

Bile pigment Gmelin’s

Hemofuchsin pigment Mallory’s fuchsin stain

Melanin Masson Fontana

Bacteria Gram staining

Actinomyces Brown breen’s

Acid fast Ziehl neelsen

Spirochetes Giemsa stain

Fungi Ziehl neelsen

Nocardia Gram staining

Amoeba Heidenhain’s iron hematoxylin

Inclusion bodies/ negri Schleifsten’s method

Calcium Calcium Prussian blue

Urates De galantha stain

ROUTINE HEMATOXYLIN STAINING PROCEDURE

DEPARAFFINIZATION

Xylol I 5 min

Removes paraffin Xylol II

Acetone-alcohol 3 min Removes xylol

Acetone

HYDRATION

Alcohol 95%

3 min Hydrates Alcohol 80%

Alcohol 70%

Alcohol 50%

Wash with distilled water

STAININIG Hematoxylin 5 min Stains nuclei

Rinse with water

DECOLORIZATION Acid alcohol 1 dip Decolorize

BLUEING

Ammonia water

Dip to blue Blues nuclei Alcohol 80%

Alcohol 95%

COUNTERSTAINING Eosin 8 min Stains cytoplasm

DEHYDRATION

Alcohol 95%

3 min Dehydrates and removes excess

stain

Alcohol 95%

Acetone

Acetone-xylol

CLEARING Xylol I

5 min Clears out

alcohol Xylol II

RESULTS

Basophil cytoplasm, plasma cells, osteoblast Purplish

Calcium and calcified bone Purplish blue

Cartilage Pink/light blue to dark blue

Cement line of bones Blue (ehrlich)

Collagen, osteod Light pink

Cytoplasm Pink

Decalcified bone matrix Deep pink

Karyosome Dark blue

Muscle fibers, thyroid colloid, thick elastic fibers Deep pink

Nuclei Blue to blue black

RBC, Eosinophil granules, keratin Bright orange red

DIFFICULTIES IN STAINING

Stains on the skin ▪ poor technique

▪ health hazards

Flake or float off tissue section

▪ dirty or greasy slide

▪ inadequate fixation on the slide

▪ torn section

▪ sections contain air bubbles

▪ inadequate infiltration

▪ albumin adhesive is too old

▪ unthorough spreading

Section doesn’t take up the stain

▪ insufficient/improperly ripened hematoxylin

▪ impurities in the dye/water solvent

▪ inadequate paraffin washing off

▪ loss of staining ability (ppt)

Sections don’t appear clear under microscope

▪ xylol should be replaced

▪ water in absolute alcohol

▪ moisture in the coverslip

▪ too much egg albumin on the slide

▪ decolorizer wasn’t completely removed

RESTAINING OLD SECTIONS (OLD/BLEACHED/FADED)

Xylol (heat until mounting media bubbles) 24 hr

Remove coverslip by dissecting needle

Xylol to remove remaing mounting media 30 min

Wash with water

Potassium permanganate 5-10 min

Wash with water

5% oxalic acid until decolorized 5 min

Wash with water 5 min

Restain

Mount

MOUNTING OF SECTIONS Purpose:

▪ protect spn from physical injury

▪ protect section from bleaching or deterioration

▪ preserve slide for permanent keeping MOUNTING MEDIUM syrupy fluid applied between section and coverslip, preventing movement of the coverslip CHARACTERISTICS OF GOOD MOUNTING MEDIUM

▪ Ref.Index : near to the glass 1.518

▪ shouldn’t dry quickly

▪ shouldn’t dissolve out or fade tissue section

▪ should not cause shrinkage and distortion

▪ should set hard

▪ shouldn’t have granularity/ cracking

▪ should be miscible with xylene or toluene

▪ should be nonreactive, no pH or color change

CLASSIFICATION

Temporary/ Aqueous ▪ for water miscible prep

▪ made of gelatin to solidify medium

▪ made of glycerol to prevent cracking

▪ made of sugar to increase ref. index

▪ made of a prsv. Solution

Permanent/ Resinous ▪ for dehydrated prep and cleared prep

▪ natural/synthetic

PERMANENT MOUNTING MEDIA

Canada balsam ▪ from Canadian tree (abus balsamea)

▪ dissolved in xylene

▪ recommended for whole mounts and thick section

▪ darken slightly with age

DPX/ Kirkpatrick and lendrum

▪Ref. index = 1.52

▪ recommended for small tissue section

Clarite X ▪ most widely used in north America

▪Synthetic resin

▪ ref index = 1.544

XAM ▪ synthetic resin mixture in xylene

▪ pale yellow/colorless

▪ dries quickly without retraction, prsv. Stains well

SEMIPERMANENT OR TEMPORARY MOUNTING MEDIA

Water ▪ low ref index

▪ good visibility

▪ least permanent

▪ not for OIO

Glycerin ▪ last for months

▪ ref index 1.46

Glycerin jelly ▪ good for fat stains (ref index 1.4-1.47)

Farrant medium ▪ ref index 1.44

Von apathy’s gum syrup ▪ for methylene blue stained nerve prep

▪ gen.purpose aqueous mountant

▪ ref index 1.52 Brun’s fluid ▪ recommended for mounting frozen sections

from water

Levulose/ fructose ▪ high ref index 1.5

▪ doesn’t leach metachromatic stain

Mineral oil/ liquid paraffin ▪ for romanowsky stained smear

S E A L I N G

RINGING process of sealing the margins of coverslip to prevent escape of fluid. Ringing media:

-paraffin wax -kronig’s/Dunoyer’s mixture

-nail polish -Durofix (cellulose adhesive) REASONS FOR RESTAINING SECTION -faded section -superimposed staining BROKEN SLIDES -unfragmented slide, unimportant section may be reassembled -vital part section, unavailable replacement transfer to another slide

SPECIAL PROCESSING TECHNIQUE

when chemical constituents of tissues shouldn’t have been altered/removed/

displaced Frozen Section most ideal tissue prsv. To avoid complete or partial loss of enzyme General Principle

▪ Quenching/freezing = produce instant cessation of cellular activities, prevent

chemical alteriation

▪ rapid freezing = prevent ice crystal artifacts formation

▪ freezing agents: Liquid nitrogen

▪ isopentane, pentane, propane = cooled to very low temp, to retain fluidity

METHODS TO AVOID CHEMICA FIXATION OF BLOCKS

FREEZE DRYING -quenching/rapid freezing -dessication (removal of water) DISADVANTAGE -time consuming -expensive -diff.to section -brittle -not for routine

▪ 2mm tissue + freezing agent + liquid nitrogen

▪ solidification 2-3sec = prevent ice crystal artifacts

▪ high vacuum drying apparatus

▪ water sublimation

▪ dessication 24-48hr

▪ infiltration, impregnation in vacuum embedding oven

▪ sectioning

▪ staining

ADVANTAGES: -min. shrinkage -fresh tissue -min. chemical change -less displacement -for enzyme studies

FREEZE SUBSTITUTION -rapid freezing -subsequent infiltration, embedding

▪ fixation with rossman’s fluid or osmium tetroxide in 1%

acetone 4-6 days (-60 to -70C)

▪ infiltration, embedding

▪ more economical

▪ recommended for routine

FRESH FROZEN TISSUE SECTIONING

▪ maintain fresh and solid state

DECALCIFICATION Process of removing calcium salts or lime salts and its deposits from hard tissue for softening of tissue to facilitate embedding and sectioning. FIXATION DECALCIFICATION IMPREGNATION **Inadequate decalcification = poor cutting of hard tissue, damage to knife edge Tissues for decalcification: -bones -teeth -atheromatous aorta -calcified tuberculous foi BEFORE CALCIFICATION

▪ cut tissue into small pieces 5 mm = permit complete penetration ,

▪ adequate fixation

▪ place tissue in buffered neutral formalin for 2-4 days or helly’s fluid 15-24hr

GOOD DECALCIFYING AGENT:

▪ can remove calcium salts completely

▪ doesn’t produce destruction of cells

DECALCIFYING AGENTS

Acid ▪ most widely used

▪ stable

▪ easily available

▪ relatively

inexpensive

The rate is affected by:

▪ tissue structure

▪ temp

▪ volume and conc.

Of solution

▪ external factor

(accelerate)

Nitric acid HCl Formic acid Trichloroacetic acid Sulfurous acid Chromic acid Citric acid

Chelating agents

Combines Ca2+ to form soluble non ionized complex, facilitate removal of calcium.

Advantage: -forms min. histological artifacts Disadvantage: -slow reacting

▪ EDTA/

Versene

Ion exchange resin

Ammonium form of plystyrene resin

▪ remove calcium

ions from formic acid , increase solubility

Advantages: Cellular detail is well preserved

Electrical ioinization/ electrophoresis

▪ Ca2+ are

attracted to negative electrode, then removed from decalcifying solution.

Advantage:

▪Faster due to heat and electrolyte reaction

▪ recommended for

small bone fragments Disadvantage: Not well preserved

NITRIC ACID: most common, very rapid, min. distortion, inhibits nuclear staining, destroy tissue

Aquous nitric acid solution 10%

12-24 hr ▪ rapid

▪ for urgent biopsy

▪ prolonged may

distort tissue Formol nitric acid 1-3 days

Perenyi’s fluid 2-7 days ▪ no maceration ▪ slow

Phloroglucin nitric acid

12-24 hr ▪ most rapid ▪ poor nuclear

staining

HCl : slow, great distortion, good nuclear staining

FORMIC ACID: Moderate, beter nuclear staining, for postmortem studies. ▪

fixative + decalcifying agent in one ▪ slow

Formic acid sodium citrate

3-14 days

▪ excellent nuclear staining

▪ slow

▪ not routine

TRICHLOROACETIC ACID : 4-8 days, good nuclear staining, weak, not for dense tissue

SULFUROUS ACID: very weak, only for min. bones

CHROMIC ACID/FLEMMING’S FLUID: fixative + decalcifying agent, inhibit nuclear

staining

CITRIC ACID-CITRATE BUFFER 4.5pH: use chloroform as prsv., excellent nuclear and cytoplasmic staining

VON EBNER’S FLUID: good cytologic staining, for teeth and small bones

DEGREE OF DECALCIFICATION:

▪ shortened/prolonged = unsatisfactory

▪ prolonged = maceration, destruction

▪ underdecalcification = interfere normal cutting

WAYS TO DETERMINE THE EXTENET OF DECALCIFICATION

Physical/mechanical test ▪Touching/bending tissue with fingers

▪determine consistency

X-RAY or radiological method ▪can detect smallest focus of Ca

▪ very expensive

Chemical method ▪ simple, reliable, convenient

CLOUDY: -Incomplete -presence of Ca NOT CLOUDY: -complete -no more Ca