Histotechniques

HISTOTECHNIQUES:
FINAL TERM DMLT 2018

• Histology:
– Logous – systematic / scientific study
– Histo – tissues
• Branch of science that deals with the systematic study of tissues.
09-10-2020 Dr MANOJ KUMAR PATRO, ASST PROF PATHOLOGY, MKCG MC, BRAHMAPUR 3

• Human body
• Organ systems
• Organs
• Tissues
– Cells [living]
– Extracellular material [non living]

• Cells:

SOME IMPORTANT TERMS:
• HISTOLOGY – microscopic study of normal tissues.
• HISTOPATHOLOGY – microscopic study of disease in a tissue section.
• HISTOTECHNIQUES / MICROTECHNIQUES – the process of making
stained slide containing thin section of the tissue specimen which is
suitable for microscopy.
• HISTOTECHNOLOGY – the study of procedures or stages to reach the
final stained slide of the tissue specimen suitable for microscopy.
• HISTOTECHNOLOGIST – the person responsible for performing these
procedures.
• HISTOCHEMISTRY - study of chemical nature of the tissue components
by histological methods.

Duties and responsibilities of Histotechnologist:
1. Specimen identification & preservation
2. Specimen labeling & logging
3. Preparation of specimen to facilitate their gross & microscopic
examination by the Histopathologist.
4. Record keeping

1. SPECIMEN IDENTIFICATION & PRESERVATION:
• 1st step after receipt of specimen: IDENTIFY IT.
• 2nd step: Check for PROPER FIXATIVE – 15 – 20 times.

2. SPECIMEN LABELING & LOGGING:
• Give ACCESSION NUMBER.
• Large specimen – call the Pathologist to make cuts for fixation.
• Arrange for grossing.
• Keep specimens in order till discarded by the Pathologist.
• Store blocks in order for life long.
• Store slides after 3 days.

Basic requirements for a Histopathology
Lab:
1. Rooms: well lighted & well ventilated
i. 1 for Grossing & Specimen preservation.
ii. 1 for Tissue processing.
iii. 1 for Reporting.
2. Equipments:
i. Grossing station
ii. Tissue processor [Histotechnicon] – Fully / Semi automated.
iii. Embedding station with cold plate.
iv. Microtome.
v. Tissue floatation bath.
vi. Automatic knife sharpener and cover slipper.
3. Glass articles & other consumables.

Histo-techniques:
• Preparation of tissue for microscopic examination
• Series of processes
• Ultimate aim – to make tissue ‘visible’ as it is
• Steps vary:
– Types of tissue & microscopy
– Structure to be seen
– Stains to be used
– Time duration etc.

Types of material received in the Lab:
• Biopsy:
 Incisional.
 Excisional.
• Autopsy:

Steps:
• Tissue procurement and preparation
• Fixation
• Dehydration
• Clearing
• Impregnation
• Embedding
• Section cutting
• Staining and mounting in slide

Tissue preparation & precautions:
• Start fixation a.s.a.p.
• Prevent osmotic damage
 do not dry
 wash with and immerse in NS
• No unnecessary handling
• Remove excess blood, mucus etc.
• Cut with a sharp knife
• Marking of ‘cutting surface’
• Labelling and putting in ‘cassette’
• Instructions for mounting – wall, tube, stained surface etc.

FIXATION:
• Fixation: Fix: to keep at a particular position.
– In this context: maintain structural and functional integrity at
cellular level.
• OBJECTIVES:
1. Preservation of tissue in as life-like condition as possible:
a. Coagulation / Cross linking of proteins – SEMIFLUID –
SEMISOLID STATE.
b. Prevents Autolysis & Putrification.
2. Prevents loss of easily diffusible substances.
3. Render tissue unaffected to the harmful effects of chemicals to
be used in further processing.

FIXATION: effects of:
1. Tissue hardening: EASE OF CUTTING.
2. Mordant action: K-dichromate with Formaline – demonstrates
myelin seath.
3. Optical differentiation: changes the refractive index.

FIXATION: properties of an ideal fixative:
1. Cheap, easily available
2. Nontoxic, non-inflammable, non-irritant
3. Maintains its chemical composition [No degradation]
4. Good tissue penetration
5. Increases tissue consistency
6. Confers optical differentiation
7. Prevents fixation artefacts
8. Prevents structure deformation
9. Preserves cellular constituents
10. Inactivates autolytic enzymes
11. Destroys microorganisms

FIXATION: when to start?
1. As soon as possible after biopsy / autopsy.
FIXATION: what is the ideal amount?
1. 20 times the bulk of tissue.
2. For MUSEUM SPECIMENS: 50 times.
FIXATION: what are the methods?
1. IMMERSION – routinely employed.
2. PERFUSION – Ideal e.g. embalming.

FIXATIVES: classification:
1. PHYSICAL:
a. Heat fixation.
b. Microwave fixation
c. Freeze drying / Cryopreservation
2. CHEMICAL: 5 major groups
a. Aldehydes.
b. Mercurials.
c. Alcohols.
d. Oxidizing agents.
e. Picrates.

FIXATIVES: Physical:
1. PHYSICAL:
a. Heat fixation: Coagulates proteins, Not used for
histopathology.
b. MICROWAVE FIXATION: a type of heat fixation, now
commonly used.
c. Freeze drying / Cryopreservation: Not for routine purpose,
used for enzyme histochemistry.

FIXATIVES: Physical: Microwave fixation:
• Microwave fixation / Microwave stabilization: primary fixation of
tissue in NS.
• Microwave assisted fixation: in the later stage after the tissue is
fixed in a fixative.

FIXATIVES: Chemical: Classification:
1. BASED ON NUMBER OF FIXATIVES USED:
A. Simple fixative: Fixative agent ± Solvent [water / alcohol /
BUFFER] e.g. 10% NBF, Ethanol, etc.
B. Compound fixative: Multiple Fixative agents + Solvent [water /
alcohol / BUFFER] – purpose is the defect in one agent is
compensated by another e.g. Carnoy’s fluid [Ethanol + GAA],
Formal sublimate, etc.

2. BASED ON THE ACTION UPON THE CELL / TISSUE
CONSTITUENTS:
A. Microanatomical fixative: preserve the microscopic anatomy
of the tissue.
B. Cytological fixative: specific action upon a specific part of the
cell structure. They are subdivided into:
1. Nuclear: Fixatives having glacial acetic acid or pH of 4.6
or less.
2. Cytoplasmic: Fixatives having pH above 4.6 or lacking
glacial acetic acid.
C. Histochemical fixative: Cryopreservation is the best.

3. RECENT CLASSIFICATION:
A. Aldehydes: Formaldehyde, Glutaraldehyde
• Functional –CHO group – crosslinks proteins by reacting with basic
amino acids.
B. Oxidizing agents: Chromic acid, OsO4, K2Cr2O7.
• Acts by oxidation reduction reactions.
C. Protein denaturing agents: Ethanol, Methanol, GAA.
• Precipitates structural proteins and lysosomal enzymes.
D. Fixatives of unknown mechanism: HgCl2, Picric acid.

FIXATIVES: Factors affecting fixation:
1. pH:
2. Penetration.
3. Temperature of fixation.
4. Concentration of fixative.
5. Volume of fixative.
6. Additives.

1. pH:
– Fixation is best carried out close to neutral pH - 6-8.
– Common buffers: PO4, HCO3, Cacodylate, and Veronal.
2. Penetration:
– d α t
– d = K √t
Fixative K
3% Pot dichromate 1.33 mm / hour
Ethanol 1 mm / hour
10% Formalin at RT 0.78 mm / hour
1% Picric acid 0.5 mm / hour

3. Temperature of fixation:
– Increasing the temperature will increase the speed of fixation.
– Hot formalin will fix tissues faster - 1st step on an automated tissue
processor.
4. Concentration of fixative:
– should be adjusted down to the lowest level possible.
– Formalin is best at 10%; Glutaraldehyde - 0.25% to 4%.
– Too high a concentration may adversely affect.

5. Volume of fixative:
– Minimum 20:1 but some say ideal is 50:1.
– Change the fixative at intervals to avoid exhaustion.
– Agitation enhances fixation.
6. Additives:
– Electrolytes – CaCl2, (NH4)2SO4, KH2PO4 – improves morphology of
tissue.
– Non-electrolytes – Sucrose, Dextran & Detergent – improves fixation.

Simple fixatives: FORMALDEHYDE
• Commercially available – 40% Formaldehyde gas in water i.e. 40%
gas by weight ≡ 100%.
• 10% solution is prepared from this for use.
ADVANTAGES
Cheap, easy to prepare & stable.
Natural color after fixation.
10% soln no shrinkage / brittleness.
No washing of tissue is required before processing.
Suitable for most of the routine stains.
Easy to prepare compound fixatives of formalin.
Best for Nervous tissue, Good for lipids & proteins.
Antigenicity is not lost, so it is good for IHC.

DISADVANTAGES
Toxic, irritant to skin [dermatitis], irritant to nasal mucosa [sinusitis]. Can cause
asthma.
PARAFORMALDEHYDE: a white precipitate - long periods in cold.
Methanol [an additive] in commercial formalin – denatures proteins – unsuitable
for EM.
Traces of Formic acid, a common impurity present – reduces the nuclear
staining.
Blood + unbuffered formalin – Dark brown refractile pigments – Acid
formaldehyde, hematin.
Medium speed fixative, requires 12 – 24 hours without heat.

Simple fixatives: GLUTARALDEHYDE
• Causes deformation of proteins so is not good for IHC.
• Fixes very quickly so is good for EM.
• Penetrates very poorly, but gives best overall cytoplasmic and
nuclear detail.
• The standard solution is a 2% buffered glutaraldehyde.

Simple fixatives: MERCURIC CHLORIDE
• Unknown mechanism.
• At RT its solubility in water is 7% - used for fixation.
• Zenker’s fluid.
ADVANTAGES
Metallic mercury – mordant – used as 2ndary fixative for
enhanced staining.
Good result in Trichrome stain, Metachromatic staining for
cartilage matrix / mast cell granules & chromaffin reaction.
Give excellent nuclear detail.
Hematopoietic and RE tissues (liver, lung, spleen, lymph nodes,
thymus gland, kidney).

Simple fixatives: MERCURIC CHLORIDE
• Zenker’s fluid.
DISADVANTAGES
Unstable, corrosive, very poisonous.
Produces a lot of shrinkage.
Leaves a black precipitate of mercury in tissues. Washing before processing is
required.

Simple fixatives: PICRIC ACID
• Bouin’s fluid.
ADVANTAGES
Histochemical demonstration of glycogen.
Yellow discoloration prevents misplacing of tiny tissues.
DISADVANTAGES
Yellow discoloration impairs final staining – several washes with Alcohol /
Xylene before staining.
Precipitates all proteins as picrates that are soluble in water – should not come
in contact with water before the picrates are made insoluble with alcohol.

Simple fixatives: POTASSIUM DICHROMATE
• 2 types of fixation:
– pH <4.6 – precipitates proteins and fixes carbohydrates.
– pH >4.6 – cytoplasm is homogeneously preserved and the
mitochondria are fixed.
ADVANTAGES
Myelinated nerve fibres – fixes myelin lipids.
Mordant, Post-chroming – formalin fixed tissue, 8 hours in 3%
potassium dichromate.
DISADVANTAGES
Washing out is required.

Simple fixatives: ETHANOL
• Protein denaturing action.
• Simple fixative – histochemical use.
ADVANTAGES
Preserves glycogen.
Good for enzyme demonstration.
DISADVANTAGES
Lot of hardening & shrinkage.
Highly inflammable.

Simple fixatives: ACETIC ACID
• At 170C it solidifies – Glacial acetic acid.
ADVANTAGES
Nuclear studies & Cytogenetics – precipitates chromatin &
nucleoprotein.
Lytic agent – destroys mitochondria, golgi & RBCs, advantage in
hemorrhagic cytologic smear evaluation.
DISADVANTAGES
Causes cell swelling.
If mixed with Potassium dichromate – lipid fixing ability is lost.

Simple fixatives: TRICHLOROACETIC ACID
• Used in some compound fixatives.
• Causes cell swelling.
• Slow decalcifying agent.

10% FORMAL SALINE:
• Very safe.
• Maintains color – Museum fixative.
• Recommended for CNS material.
• Slow & liable for shrinkage during dehydration.
Formula:
40% Formalin: 100 mL
NaCl: 8.5 Gm
Distilled water: 900 mL

10% NEUTRAL BUFFERED FORMALIN:
• Best overall fixative.
• Storage of surgical specimens.
• Good for BM biopsy.
• Formalin pigment (acid formaldehyde haematin) is not formed.
• Slow fixation – 24 hours.
• Costly & difficult to prepare.
Formula:
NaH2PO4: 3.5 Gm
Na2HPO4: 6.5 Gm

FORMAL CALCIUM: LILLIE : 1965
• Recommended for lipid fixation.
• They have a near neutral pH
• Formalin pigment (acid formaldehyde haematin) is not formed.
Formula:
40% Formalin: 10 mL
Ca-acetate: 2 Gm

BUFFERED FORMAL SUCROSE: HOLT & HICKS: 1961
• This is an excellent fixative for the preservation of fine structure
phospholipids and some enzymes.
• It is recommended for combined cytochemistry and electron
microscopic studies.
• It should be used cold (4°C) on fresh tissue.
Formula:
40% Formalin: 10 mL
Sucrose: 7.5 Gm
M/15 phosphate to 100 mL
buffer (pH 7.4):

10% ALCOHOLIC FORMALDEHYDE:
• Calcium acetate is added for neutrality.
• Fixation / post-fixation of large fatty specimens (particularly breast) –
better identification of lymph nodes [it clears and extracts lipids].
• Dehydration process is initiated so the tissue can be directly transferred
to Absolute alcohol.
Formula:
95% Alcohol: 900 mL
Calcium acetate: 0.05 Gm

FORMOL SUBLIMATE:
• Recommended for routine post-mortem material.
• Cytological details and RBCs are well preserved.
• No hardening / shrinkage.
Formula:
Saturated HgCl2: 900 mL

HEIDENHAIN SUSA:
• Excellent fixative for routine biopsy work
• Allows brilliant staining with good cytological detail
• Rapid and even penetration with minimum shrinkage
• Tissue left in it for > 24 hours - bleached and hardened.
• Removal of mercury pigment .

ZENKER’S SOLUTION:
• Best for nuclei & connective tissue fibers.
• Requires washing out.
• Prolonged immersion causes brittleness.
Formula:
HgCl2: 5 Gm
Pot dichromate: 2.5 Gm
Na2SO4 [optional]: 1 Gm
Add 5 mL GAA just before use.

ZENKER’S FORMAL: HELLY’S FLUID
• Best for pituitary tissue and bone marrow.
• Excellent nuclear fixation and cytoplasmic organelles are well
preserved.
• Requires washing out.
Formula:
HgCl2: 5 Gm
Pot dichromate: 2.5 Gm
Na2SO4 [optional]: 1 Gm
10% Formalin just before use 5 mL

B5 FIXATIVE:
• Fixation of lymph node biopsies both to improve the cytological
details and to enhance immunoreactivity.
B5 stock solution
Mercuric chloride 12 Gm
Sodium acetate 2.5 Gm
Distilled water 200 mL
B5 Working solution
B5 stock solution 20 mL
Formalin (40%) 2 mL

BOUIN’S FLUID:
• Nuclei properly fixed due to GAA.
• Good for Glycogen.
• Good for Testicular biopsy, Embryos, Core needle biopsies of
kidney.
Formula:
40% Formalin: 25 mL
Saturated Aq Picric acid: 75 mL
Glacial acetic acid: 05 mL

GENDER’S FLUID:
• Used for Glycogen.
Formula:
40% Formalin: 15 mL
Picric acid IN 95% alcohol: 80 mL
Glacial acetic acid: 05 mL

CYTOLOGICAL FIXATIVES:
NUCLEAR:
Flemming’s fluid
Carnoy’s fluid
Clarke’s fluid
New Comer’s fluid
CYTOPLASMIC:
Flemming’s fluid without Acetic acid
Helly’s fluid
Formalin with Post-chroming

NUCLEAR FIXATIVES: FLEMMING’S FLUID:
• Best for nuclear structure e.g. chromosomes.
• Only fixative permanently preserve fats.
• Costly but required in small volumes.
• Poorly penetrating.
Formula:
1% Chromic acid: 15 mL
OsO4 [aqueous]: 4 mL
GAA: 1 mL

NUCLEAR FIXATIVES: CARNOY’S FLUID:
• Good for carbohydrates, glycogen & Nissil substance.
• Dissolves most of the cytoplasmic elements.
• Fixation is usually complete in 1-2 hours.
• Best for lymph nodes & urgent biopsies.
• Excessive shrinkage.
Formula:
Absolute alcohol: 60 mL
Chloroform: 30 mL
GAA: 10 mL

NUCLEAR FIXATIVES: CLARKE’S FLUID:
• Rapid, good nuclear fixation and good preservation of cytoplasmic
elements.
• It in excellent for smear or cover slip preparation of cell cultures or
chromosomal analysis.
Formula:
Absolute alcohol: 75 mL
GAA: 25 mL

NUCLEAR FIXATIVES: NEW COMER’S FLUID:
• Devised for fixation of chromosomes.
• It fixes and preserves mucopolysacharides.
• Fixation in complete in 12-18 hours.
Formula:
Isopropranolol: 60 mL
Propionic acid: 40 mL
Petroleum ether: 10 mL
Acetone: 10 mL
Dioxane: 10 mL

CYTOPLASMIC FIXATIVES: FLEMMING’S FLUID
WITHOUT ACETIC ACID:
• Best for mitochondria.
Formula:
1% Chromic acid: 15 mL
OsO4 [aqueous]: 4 mL

HISTOCHEMICAL FIXATIVES:
• Best - CRYOSTAT.
• Usually such sections are used unfixed but if delay is inevitable
then vapour fixatives are used.
VAPOUR FIXATIVES:
• Formaldehyde vapour:
– Paraformaldehyde 50 – 800C.
– Blocks of tissue - 3-5 hours.
– Section require ½- 1 hour.
• Acetaldehyde vapour: 800C for 1 – 4 hours.
• Glutaraldehyde – 50% aqueous solution: 800C for 2 mins – 4
hours.
• Acrolein /chromyl chloride: at 37°C for 1-2 hours.

HISTOCHEMICAL FIXATIVES:
• Other more commonly used are
1. Formal saline
2. Cold acetone - at 0-4°C is widely used for fixation of tissues
intended to study enzymes esp. phosphatases.
3. Absolute alcohol for 24 hours.

SECONDARY FIXATION:
• Following fixation in formalin it is sometimes useful to submit the
tissue to second fixative e.g. mercuric chloride for 4 hours.
• It provides firmer texture and brilliance to the staining.
POST CHROMATION / POST CHROMATIZATION:
• Mordant action.
• Treated with 3% K-dichromate after normal fixation:
– Tissue blocks before processing – 6 – 8 days.
– Sections – 12 – 24 hours.
• Washing out for 24 hours is required.

Some fixatives of choice:
saline

Fixation : Golden rules
• Sizeof tissue, 3-5 mm1
• Volume of fixative, 20X2
• Time: 12 -24 hrs (formalin)3

Causes of poor fixation:
• Less volume of fixation
• Less time
• Poorly penetrating fixative
• Specimen settling down on container
• Wrong choice of fixative

Artificial Pigments & their removal:
• Natural:
– Exogenous: Carbon particles / Tattoo.
– Endogenous: Hemosiderin – Prussian blue +.
• Artificial:
– Mercuric chloride precipitate.
– Formaldehyde precipitate.

Mercurial Pigments & their removal:

Formalin Pigments & their removal:
• Bring section to 80% alcohol.
• Treat with alcoholic KOH for 10 mins.
• Wash with 2 changes of water.
• Transfer to 80% alcohol for 5 mins.
• Wash in water.
• Stain.
• Deparaffinize with Xylene and wash in absolute alcohol.
• Immerse in saturated alcohol picric acid (85%) for 30 minutes.
• Wash in absolute alcohol to remove the picric acid.
• Bring section to water and continue staining in the normal way.
Barrett’s alcoholic picric acid method:
Verocay’s Method:
1% KOH [aqueous solution]: 1 mL
80% Ethyl alcohol: 100 mL

DECALCIFICATION
6TH DECEMBER 2014

1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
7. TESTS FOR COMPLETION

INTRODUCTION:
1. Bony tissue for tumor / infection.
2. Teeth.
3. Calcified soft tissues – TB lymph nodes, calcified arteries / valves.
• Pose problems in histotechniques:
– Hard to cut by usual microtome knife.
– Small calcified spots – torn & ragged sections.
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

INTRODUCTION:
• Processing of un-decalcified bones:
– Metabolic bone disease investigation.
– Morphometric measurements – Bone density.
1. After fixation, thin sections using a saw & then grinding.
2. Epoxy resin embedding & sectioning using a heavy duty microtome &
diamond knife.
3. Frozen sections – mineralized cancellous bone.
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

DEFINITION:
• Decalcification:
– A process of complete removal of calcium salt from the
tissues like bone and teeth and other calcified tissue following
fixation.
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

PURPOSE:
• To assure that the specimen is soft enough to allow cutting with the
microtome knife.
• Unless the tissues in completely decalcified the sections will be
torn and ragged and may damage the cutting edge of microtome
knife.
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

PRINCIPLE:
• Decalcifying agents: Insoluble calcium salts – Soluble calcium salts.
• Chelating agents: Insoluble calcium salts – Soluble non-ionized
calcium.
• Ion exchange resin: Used with weak acid, removes Ca2+ from solution –
H+ remains available.
• Electrophoretic decalcification: Electric current passed through
formic/HCl acid solution - Ca2+ moves to negative electrode.
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

STEPS:
1. Selection of tissue
2. Fixation
3. Decalcification
4. Check for completion
5. Acid neutralization
6. Washing
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

METHODS:
1. Mineral acids
2. Non-mineral acids
3. Ion exchange resins with non-mineral acids
4. Chelating agents
5. Electrophoresis
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

METHODS: CRITERIA OF GOOD DECALCIFYING FLUID
• Complete removal of calcium salts
• Does not damage cell/fibre structure
• Decalcifies at reasonable speed
• No adverse effects on subsequent staining
• Easy to determine endpoint
• Tissue not removed from bone - no distortion of cells / connective
tissue
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

METHODS: FACTORS AFFECTING DECALCIFICATION
• Temperature of solution
• Bone density
• Strength of solution
• Agitation
• Vaccum
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

MINERALACIDS:
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

MINERALACIDS: Characteristics:
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• Fastest decalcifying agents.
• Cause tissue damage with long exposure.
• May remove iron from bone, resulting in false-negative staining.
• Nitric Acid Disadvantages:
– Overexposure (>48hrs) - hydrolysis of nuclear proteins –poor staining &
Feulgen stain not possible.
– Colours tissues pale brown (corrected by 0.1% urea).
– Dissolves Ca(CO3)2 forming C02.

WEAK ACIDS:
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

WEAK ACIDS: Characteristics
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• Similar mode of action as mineral acids.
• Do not ionize as freely / totally as mineral acids.
• Increased decalcification time, reduced tissue damage.
• Little effect on staining even on over exposure.
• All endpoint methods available .

CHELATING AGENTS:
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS

CHELATING AGENTS: Characteristics
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• Advantages
– No gas produced (no artifact)
– Does not require neutralization (pH 7-7.4)
– Causes little artifacting
– No effect on staining
– Best routine decalcifying agent
• Disadvantages
– Also chelates Mg, Fe - cannot be used to demonstrate Hemosiderin
– More volume – 150 times of tissue changed every 5 – 7 days.
– Extremely slow

DETERMINATION OF COMPLETION:
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• 4 methods:
1. Physical – Probing – not perfect / damage to tissue.
2. X – ray – not suitable for tissues fixed in HgCl2 fixative /
expensive.
3. Chemical.
4. Titration.

1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• Chemical method: Most commonly used.
 Remove the decal fluid – add fresh decal fluid – after 4 hours
take 5 mL of this fluid – add Conc NaOH & 1 mL of (NH)4-
oxalate – see after 5 mins:
 Clear – complete.
 Turbid – incomplete.
 Disadvantage: can not be used with Chelating agents & Ion
exchange resins.

1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• Titration:
 Remove the decal fluid – add fresh decal fluid – after 4 hours
fluid is titrated for calcium:
 No increase in calcium – complete.

SURFACE DECALCIFICATION:
1. INTRODUCTION
2. DEFINITION
3. PURPOSE
4. PRINCIPLE
5. STEPS
6. METHODS
• Small amounts of calcium missed during
grossing, noticed in cutting.
• Rather than reprocessing, surface of wax block can be decalcified.
• Allows for about 10 tissue sections.
• Wax block face-down in decal agent for 2
hours, washed.

TISSUE PROCESSING:
9th January 2015

INTRODUCTION:
• Tissue processing means treatment of the tissue necessary to
impregnate it into a solid medium so that the tissue is rendered
sufficiently firm yet elastic for the tissue sections of desirable
thickness to be cut on microtome.
• Cryostat or freezing microtome on frozen tissues - alternative.
• Other advantages:
– Easy to store and reproducibility of sections at a later date is
easier.

INTRODUCTION:
• Most important step before proceeding on tissue processing as
soon as the tissue is received is: A fool proof labelling system:
– Thin white card with a soft lead pencil, typed or printed labels.
Place tissue and label in tissue processing baskets which are
changed from reagent to reagent.
– Tissue tek system - the tissue identity is written on the cassette
and retained as permanent record during sectioning and storage
of tissue blocks.

PRINCIPLE:
• Aim: To impregnate & embed in a solid medium [paraffin wax]
– to render the tissue firm enough for cutting thin sections.
– At the same time soft enough to be cut by microtome knife.

FACTORS INFLUENCING THE RATE OF PROCESSING:
1. Agitation: can reduce over all processing time by 30%.
2. Heat:
– Increases the rate of penetration & fluid exchange.
– 450C – effective; higher temperature deleterious to IHC staining.
3. Viscosity: low viscosity better penetration.
4. Vacuum:
– increases the rate of infiltration.
– Should not be > 15 inches of Hg – damages the tissue.

STEPS:
1. Fixation
2. Dehydration
3. Clearing
4. Impregnation / infiltration
5. Embedding

DEHYDRATION:
• Purpose: to remove water from the tissue so as to enable the water
immiscible embedding media to enter in to the cell.

DEHYDRATION:
DEHYDRATION:
• It is the process of removing water from tissues.
• Important because paraffin & water are immiscible.
• Complete when <3-4% water remains in the tissue.
• Factors influencing:
1. Permeability of tissues
2. Agitation
3. Temperature
4. Vacuum

DEHYDRATION:
Dehydrants:
• Alcohol: Ethyl alcohol, Methyl / Butyl / Isopropyl.
• Acetone.
• Dioxane.

DEHYDRATION:
Alcohols:
• Increasing graded concentration:
• 70% - 90% - 100% - 100% - 100% - 1 hour each -Routine 4 – 7 mm
thickness.
• Delicate tissues – start at 50%.
• If fixed in Alcoholic fixative / Heidenhain’s Susa – directly to 95%
alcohol.
• WHEN & HOW TO CHANGE ALOHOL:
– After ~ 40 tissues have passed through 1st change of alcohol it is
discarded & all other changes brought one step lower. Absolute
alcohol at the end is always fresh.

DEHYDRATION:
Alcohols: Use of Anhydrous CuSO4
• Hygroscopic.
• Kept at the bottom of the last Absolute alcohol beaker & is covered
with 2- 3 layers of filter paper.
• Indicator: Anhydrous – White; Hydrated – Blue.
• Advantages:
– Accelerates dehydration.
– Prolongs life of alcohol.

DEHYDRATION:
Acetone:
• Clear, colourless, inflammable fluid.
• Used for complete dehydration.
• 4 changes of ½ hour / 2 changes of 1 hour.
• Advantages
– Rapid action
– Easily removed by most clearing agents
– Less expensive
• Disadvantages
– Highly volatile
– Causes shrinkage and brittleness of tissues
– Dissolves lipid more than ethanol

DEHYDRATION:
Dioxane:
• Dehydrates and clears at the same time.
• Tissue is directly transferred to paraffin.
• Advantages
– Less shrinkage – can be left in dioxane for longer time.
• Disadvantages
– Expensive than alcohol.
– Toxic.

CLEARING:
• Purpose:
– to remove dehydrating agent from the tissue so as to enable the immiscible
embedding media to enter in to the cell / tissue.
– Acts as solvent for the mounting media which renders the tissues
transparent and improves the refractive index, making microscopic
examination easier.

CLEARING:
CLEARING:
• After treatment with the clearing agent, as the refractive index of
the agent is equal to that of tissue protein so it becomes
translucent.
• Translucency of the tissue is the end point of clearing.
• Required when impregnating medium is not miscible with the
dehydrating medium.

CLEARING:
Clearing agents:
• Xylene
• Others:
1. Toluene
2. Dioxane
3. Cedarwood oil
4. Cloroform
5. Benzene
6. Carbol-xylene - clears rapidly, it is kept reserved for material
difficult to clear.

Advantage Disadvantage
Xylene Rapid – 3 to 4 mm thickness
in 1 – 2 hours.
Brittleness.
Toluene /
benzene
Similar Less damaging.
Chloroform Less brittleness Slow.
Expensive.
Inflammable.
CCl4 Similar to chloroform, but cheaper.
Cedar wood oil
[Histo]
Least hardening effect –
delicate tissues.
Slow.
Expensive.
CLEARING:
Clearing agents:

CLEARING:
Procedure:
• Xylene / Benzene / Toluene – 1 change 30 to 60 mins.
• Chloroform / CCl4:
– Manual – overnight.
– Automatic tissue processor – 3 changes 1 hour each.

IMPREGNATION:
IMPREGNATION:
• Purpose: to allow paraffin to enter the cell / tissue and
accumulate there so as to give the desired consistency to the
tissue for cutting thin sections yet soft enough not to damage the
microtome knife.

IMPREGNATION:
IMPREGNATION:
• Definition: It is the complete removal of clearing agent by
substitution of paraffin wax or any such similar media in the
tissue.

IMPREGNATION:
IMPREGNATING MEDIA:
• Paraffin wax.
• Paraplast.
• Celloidin.
• Bioloid.

IMPREGNATION:
PARAFFIN WAX:
• Mixture of long chain hydrocarbons of varied melting points –
40 – 700C.
• ↑ melting point wax – makes tissue hard – difficult in ribboning.
• ↓ melting point wax – does not support tissue during cutting.

IMPREGNATION:
PARAFFIN WAX:
• Advantages of using paraffin wax:
– Cheap and easy to use.
– Little difficulty during staining.
• Points to be remembered:
– Free from dust / grit / foreign matter – Filter before use with
ordinary filter paper.
– Water causes crystallization [white] – prevent mixing with
water.

IMPREGNATION:
PARAFFIN WAX: Technique:
• 2 changes – 1 hour each – molten paraffin 2 - 30C above the
melting point.
• Volume of wax – 20 – 50 times of tissue.
• Temperature 50C above the melting point of the paraffin will cause
tissue shrinkage and hardening.
• Overheating / prolonged heating – crystallizes the wax.

IMPREGNATION:
PARAFFIN WAX: Technique:
• Time of impregnation:
1. Size of tissue: Thick tissue – More time & changes.
2. Type of tissue: Bone, Skin, CNS > Liver, Kidney > Muscle,
Fibrous tissue.
3. Clearing agent used:
• Xylene / Benzene / Toluene – easy, 1 change is sufficient.
• Chloroform / CCl4 – 2 – 3 changes.
4. Use of vacuum embedding oven: reduces time to half.

IMPREGNATION:
PARAFFIN WAX: Additives:
• Plasticizers / other resins additives – higher melting point than wax
– create paraffin with the desired hardness.
• Beewax, Rubber, Ceresin, Plastic polymers & Diethylene glycerol
distearate.

EMBEDDING:
EMBEDDING:
• Purpose:
– To obtain necessary orientation of the tissue.
– To provide necessary firmness while section cutting.
• Definition: It is the orientation of tissue in melted paraffin which
when solidified provides a firm medium for keeping intact all parts
of the tissue when sections are cut.

EMBEDDING:
EMBEDDING: Technique:
1. Molten paraffin wax [2-30C above the M.P.] - poured into the mould to
an adequate depth - quickly a thin semi solid layer of wax will be
formed - introduce the tissue with a pre-warmed forceps – press the
tissue in this semisolid wax to orient it at the bottom - Fix the label in
position [usually sides] - As soon as a film of solid wax is formed on the
surface, the whole block with mould are submerged in cold water at
200C.
2. When blocks are set hard they are removed from mould.
3. Trimming with a scalpel.

EMBEDDING:
EMBEDDING: some technical points:
1. Keep tissues in heated wax / in cassettes placed over hot plates
to prevent paraffin wax to cool around the tissue to be blocked.
2. Cutting surface of the tissue - face the bottom of the mould.
3. 2 or more tissues in a block – keep at the same depth.

EMBEDDING:
EMBEDDING: some technical points:
4. If small biopsy fragments - the largest piece should be first
blocked.
5. The tissue should have at least 2 mm wax around its edges.
6. Smear mineral or machine oil on the inner surface of the mould
for facilitating easy removal of block.
7. Whitish areas around tissue in block denotes crystallization
which may be due to moisture / incomplete removal of clearing
agent.

EMBEDDING:
EMBEDDING: tissues requiring special orientation:
• Tubular structures – Arteries / Veins / Tubes – cut in cross-
section.
• Epithelial biopsies – Skin / GB / etc. – cut in a plane right angles
to the epithelial surface, epithelium is cut last.
• Muscle – both transverse & longitudinal planes.

TISSUEPROCESSOR:
TISSUE PROCESSOR:
• Manual:
• Automatic:
1. Tissue transfer type:
Reagents are constant, tissues
move.
2. Enclosed type: Tissues are at
one place, reagents change.

TISSUEPROCESSOR: TISSUE PROCESSOR:
Auomatic:
• Parts:
– Tissue containers [Baskets]
– Beakers
– 2 wax baths – thermostatically controlled at 560C + 40C.
– Stirring mechanism – one rotation per minute.
– Timer.

Auomatic – some Important points:
1. Fill beakers and wax baths up to appropriate mark.
2. Check for their correct position in the machine.
3. Keep it clean – wipe any spillages & remove accumulated wax on
covers, lids and surrounding areas.
4. Wax bath thermostats - usually 2 - 30C above the M.P. of wax.
5. Timing should be set with utmost care when loading the machine.
6. Paraffin wax baths should be checked to ensure that the wax is molten.

Processing Schedules:
• Manual
• Automatic

Processing Schedules:
10% Formal-saline 3 Hours
10% Formal-saline 3 Hours
70% Alcohol 1 Hour
70% Alcohol 1 Hour
95% Alcohol 1 Hour
100% Alcohol 1 Hour
100% Alcohol 1 Hour
100% Alcohol 1 Hour
Xylene ½ Hour
Xylene ½ Hour
Paraffin 1 Hour
Paraffin 1 Hour09-10-2020 Dr MANOJ KUMAR PATRO, ASST PROF PATHOLOGY, MKCG MC, BRAHMAPUR 129

SECTIONCUTTING:
SECTION CUTTING / MICROTOMY:
• The technique of making thin sections of 3 – 4 µm from the tissue
blocks prepared by tissue processing using a microtome.

SECTIONCUTTING:
MICROTOME:
• Greek: Mikros – small, Temenin – to cut.
• A microtome is a tool used to cut extremely thin slices of
material [known as sections] suitable for microscopy.

SECTIONCUTTING:
MICROTOME: Types
1. Hand microtomes – Botanical sections.
2. Sliding microtome.
3. Rocking microtome.
4. Rotary microtome.
5. Automatic microtome.
6. Ultracut microtome.
7. Freezing microtome - Cryostat.
8. Base sledge microtome.
9. Vibrating knife microtome.

SECTIONCUTTING:
MICROTOME: Types
• Sliding microtome.
• Block is stationary, knife moves horizontally against it.

SECTIONCUTTING:
MICROTOME: Types
• Base sledge microtome.
• Block is stationary, knife moves horizontally against it.

SECTIONCUTTING:
MICROTOME: Types
• Rocking microtome.
• The knife is fixed, block of tissue moves through an arc and strikes
against the knife.

SECTIONCUTTING:
MICROTOME: Types
• Rotary microtome.
• The knife is fixed, block of tissue moves up and down and strikes
against the knife.

SECTIONCUTTING:
MICROTOME: Types
• Automatic microtome.
• The knife is fixed, block of tissue moves up and down and strikes
against the knife.

SECTIONCUTTING:
MICROTOME: Types
• Vibrating knife microtome.
• Sections are obtained without fixation / impregnation / freezing, useful
for histochemistry.
• Sections are thick and not suitable for routine staining.

SECTIONCUTTING:
MICROTOME: Types
• Ultracut microtome.
• Used for cutting 500 to 600 A° thin sections for EM.

SECTIONCUTTING:
MICROTOME: Parts
• Block holder
• Knife clamps screws
• Knife clamps
• Block adjustment
• Thickness gauge
• Angle of tilt adjustment
• Operating handle
• Internal and external lock

SECTIONCUTTING:
MICROTOME: KNIVES
• Greatest single factor for making good sections.
• Developed to fit specific types of microtomes and cope with different degrees
of hardness of tissues and embedding media.
• Material:
– Metal – Steel.
– Non-metal:
• Glass.
• Diamond.
• Paraffin-wax embedded tissues – Steel knives.
• Resin-embedded tissue - Glass knives.

SECTIONCUTTING:
MICROTOME: KNIFE PARTS
• Larger knives with detachable handle ranging from (8-24 cm).8cm
for freezing microtomes and 24 cm for base sledge microtomes.
• HEEL-Angle formed by the cutting edge and end of the knife
nearest the handle.
• TOE-Angle formed by the cutting edge and end of the knife
farthest from the handle.

SECTIONCUTTING:
MICROTOME: KNIFE ANGLES
• “B” – Bevel / Facet angle [27 – 320C]: Smaller – Sharper knife but easy
distortion of cutting edge.
• “C” – Clearance angle [3 – 80C]: 50C is commonly used.
• “R” – Rake angle.
B
C
R

SECTIONCUTTING:
MICROTOME: KNIFE SHARPENING
• Manual.
• Automatic.
• Two steps:
– Honing.
– Stropping.

SECTIONCUTTING:
MICROTOME: KNIFE SHARPENING – Honing:
• Honing - grinding the cutting edge of the knife on a hard
abrasive surface to sharpen the knife.
• Microtome knives are sharpened against a special stone k/a
“Hone”.

SECTIONCUTTING:
• Types of Hone:
– Belgian black vein / Belgian yellow: Best hone.
– Arkansas.
– Aloxide.
– Carborundum.
– Plate glass: used by applying an abrasive [Aluminium oxide].

SECTIONCUTTING:
• Abrasives:
– Aluminum oxide (alumina)
– Iron oxide (Jeweler's rouge)
– Silicon carbide
– Diamond

SECTIONCUTTING:
• Lubricants for Hone:
– Soap water.
– Liquid paraffin.
– Castor oil.
– Clove oil.

SECTIONCUTTING:
• Procedure:
• The hone is placed on a bench on a non-skid surface.
• Pour & smear light lubricant oil and then apply abrasive powder.
• Place the complete knife i.e. with handle and honing guide on the hone
with the cutting edge facing away from the operator, and the heel in the
centre of the nearest end of hone.

SECTIONCUTTING:
• Positioning of the fingers - by holding the handle of the knife between
the thumb and forefinger with the cutting edge facing away from the
operator, the tips of finger and thumb of other hand rest on the other end
of knife ensuring even pressure.
• Knife is pushed forward diagonally from heel to toe, turned over on its
back and moved across the hone until the heel is in the centre with the
cutting edge leading and then brought back diagonally. It is then turned
over on its back and moved across the hone to its original position
completing figure of eight movement.

SECTIONCUTTING:

SECTIONCUTTING:
• The process is continued until all jagged edges have been
removed.
• The knife is ready for stropping.

SECTIONCUTTING:
MICROTOME: KNIFE SHARPENING – Stropping:
• Process of polishing an already fairly sharp edge.
• Should be kept free from grit and dust.
• Types: best strops made from hide from the rump of the horse.
– 2 types: flexible (hanging) and rigid.

SECTIONCUTTING:
• Flexible type:
• Back of the strop is made of canvas and is intended to support the leather during
stropping.
• Applying a small quantity of vegetable oil into the back of the leather to keep it
soft.
• Rigid type:
• Single leather strop stretched over a wooden frame to give a standard tension or
a block of wood about 12x2x2 inches in size having a handle at one end with
four grades of leather or even a soft stone cemented on each side.
• The sides of these strops are numbered and the knife is stropped on No1, then
No2 and so on finishing on the finest leather.

SECTIONCUTTING:
• Knife is laid on the near end of the strop with the cutting edge
towards the operator (opposite direction to that used in honing).
• Knife held with forefinger and thumb to facilitate easy rotation at
end of each stroke.
• Action is exact opposite to that used in honing, using full length of
the strop and stropping evenly the whole of the blade.

SECTIONCUTTING: MICROTOME: KNIFE SHARPENING –
AUTOMATIC:
• Two basic designs available.
– Knife is held vertically with revolving sharpening wheels grinding
the cutting edge.
– Knife is held horizontally against the surface of a slowly rotating flat
plate.
• Plates – glass , copper or bronze charged with an abrasive.
• Glass plates need to be roughened before use to allow the abrasive
particles to be held more easily in place.
• Copper and bronze plates used in conjunction with diamond paste.

SECTIONCUTTING: MICROTOME: KNIFE SHARPENING –
AUTOMATIC:

SECTIONCUTTING:
MICROTOME: KNIFE SHARPENING
• Assessment of sharpening: examine the edge of the knife under
microscope / reflected light.
• Care of knife:
– Keep the knife covered in the box when not in use.
– Oil the knife to prevent corrosion.
– Always clean knife with xylene cloth before and after use.
– It should always be stropped before use.
– Knife should be sharpened as and when required.

SECTIONCUTTING:
PARAFFIN SECTION CUTTING:
• Equipments required:
– Microtome
– Water bath preferably thermostatically controlled.
– Hot plate / drying oven thermostatically controlled.
– Fine pointed forceps.
– Small hair brush.
– Seeker
– Scalpel
– Clear cloth or paper towel.
– Slide rack
– Clean glass slides
– Section adhesive
– Fluff less blotting paper
– Ice cubes
– Diamond marker pencil .

SECTIONCUTTING:
• Water bath / Tissue floatation bath:
• The temperature of the water should be about 5-6°C below the melting
point of the paraffin wax.
• Alcohol or small quantities of detergent may be added for reducing
surface tension and allowing the section to flatten out with greater ease.

SECTIONCUTTING:
• Hot plate / Drying oven:
• Small drying ovens are now available, incorporating a fan, especially designed
for drying tissue section on slides.
• With a temperatures setting at the melting point of the wax no obvious damage is
done to the sections and drying is complete in 30 minutes.
• On Hot plate, temperature is maintained at 45-50 degree 30 minute is sufficient.

SECTIONCUTTING:
• Brush, seeker, forceps: needed to remove folds and creases in
sections after floating out.
• Slides - Majority of sections fit comfortably on a 76 x 25 x 1.2
mm slide.
• Diamond pencil – needed to write the identification details like
name or specific number.

SECTIONCUTTING:
• Section adhesives:
• An adhesive is a substance which can be smeared on to the slides so that the
sections stick well to the slides.
• Required to prevent lifting of the tissue from the slide staining.
• Routinely practised, but not a must for H & E staining of all tissues except brain,
spinal cord, blood clot and decalcified tissues.
• Must for histochemical methods requiring alkaline solutions e.g. ammonia tend
to remove sections from slide.

SECTIONCUTTING:
• Types of adhesives:
– Albumin
– Gelatin
– Starch
– Cellulose
– Sodium silicate
– Resin
– Poly L Lysine
– Charged / Plus slides

SECTIONCUTTING:
• Mayer’s Glycerol Albumin:
– This is the most popular adhesive for routine use:
• Fresh egg white 50 mL
• Glycerol 50 mL
• Sodium salicylate 1 mL
– Mix and agitate - filter through coarse filter paper. A crystal of thymol is
added to inhibit the growth of moulds.
– Smear fluid over the slide.
– While manipulating the albumenized slide under water in the floatation bath
to pick up the section, avoid dipping the entire slide as the albumin may
wash off.

SECTIONCUTTING:
• BLOCK TRIMMING:
– Wax is removed with a sharp knife until 1/8th inch remains on
all sides of the tissue.
– Only small flakes of wax should be trimmed at a time.
– Attempts to trim large pieces can lead to splitting and exposure
of tissue.

SECTIONCUTTING:
• TECHNIQUE OF CUTTING:
• Insert the knife in the knife-holder & screw tightly.
• Fix the block in the block holder & ensure it’s secure.
• Feed mechanism is adjusted until the wax block is almost touching
the knife. Ensure that the whole surface of the block will move
parallel to the knife so that straight ribbon of sections is obtained.
• All screws should be tight to avoid faulty sectioning

SECTIONCUTTING:
• For block trimming section thickness of 15µ with a rough knife is
taken
• Sharp knife is used for sectioning
• Reset the thickness gauge to required thickness. 4-5 μ
recommended for routine work
• Apply ice to the block surface to make the wax hard which would
have become soft by frictional heat.
• There should be a smooth continuous plastic flow of the sections in
the form of a ribbon.

SECTIONCUTTING:
• When the ribbon comes off it is held gently with a fine moistened brush
or forceps and then transferred to water bath.
• Section is then float on water bath (temp 5-6°C below melting point of
wax) to remove creases.
• Clean albumenised slide is half submerged in water and section is picked
up using a dissecting needle.
• The slide is then set in an upright position to drain.
• Slides are kept in incubator (37°C overnight for plain slides and 60°C for
2 hours for albuminised ones).

STAINING:
• Purpose:
– The sections obtained after microtomy are colorless –
morphology can not be studied.
– To color different cellular components differently using
different colored dyes – suitable for morphological study.

STAINING:
Some important terminologies:
• Basophilic: Baso – Basic + Phillic – Loving: Substances stained with
basic dyes.
• Acidophilic: Acido – Acidic + Phillic – Loving: Substances stained with
acidic dyes.
• Vital staining: Staining of structures in living cells - Janus green is
taken up by living cells and stains the mitochondria.
• Supravital staining: Staining of structures in living cells outside the
body – New methylene blue is taken up by living cells and stains the
RNA in Reticulocytes.

STAINING:
• Metachromatic staining:
– Meta – Beyond; Chroma – Color.
– Staining of tissue components in a different color to that of the
color of the dye – Metachromasia.
– Mechanism: Presence of polyanions in the tissue – polymerization
of the basic dye.
– Metachromatic dyes: Basic dyes of the aniline group: Toluidine blue,
Methylene blue, Methyl violet, Crystal violet, Thionin.
– Tissue components often demonstrated by metachromatic stains:
Amyloid, Mast cell granules, Mucin, Cartilage.

STAINING:
• Direct staining: Application of simple dye to stain the tissue in varying shades
of colours.
• Indirect staining: Use of mordant to facilitate a particular staining method or
the use of accentuator to improve either the selectivity or the intensity of stain.
• Mordants: Substance that causes certain staining reactions to take place by
forming a link between the tissue and the stain. The link is referred as lake.
Without it, the dye is not capable of binding to and staining the tissue. E.g.
Ammonium and Potassium alum for haematoxylin.
• Accentuators: Substances that causes an increase in the selectivity or in the
staining power of dye - more intense staining. E.g. Phenol in Carbol fuchsin,
KOH in Methylene blue.

STAINING:
• Progressive staining: Stain applied to the tissue in strict sequence
and for specific times. The stain is not washed out or
decolorized because there is no over staining of tissue
constituents. Staining is controlled by frequent observation
under microscope.
• Regressive staining: Tissue is first over stained and then the
excess stain is removed from all but the structures to be
demonstrated. This process is called differentiation and should
always be controlled under microscope.

STAINING:
• Histochemical staining: Stain which is used to indicate the
chemical composition of the tissue or cellular elements.
• Counterstain: a stain with colour contrasting to the principal stain,
making the stained structure more easily visible.

STAINING:
DYES USED IN STAINING: Classification
• According to Source:
– Natural.
– Synthetic.
• According to Affinity to tissues:
– Acidophilic.
– Basophilic.
• According to Chemical composition:
– Thiazines.
– Azo-dyes.
– Rosailins.

STAINING:
DYES USED IN STAINING: NATURAL DYES
• Very few.
• 2 MC used are:
• HEMATOXYLIN:
– extracted from the bark of a tree, “Hematoxylon campechianum” –
bloodwood tree. Barks are boiled in water – Orange red solution –
turns yellow – then black on cooling.
– Can only stain after oxidation: Sunlight – 3 / 4 months, Chemical
(NaI) – instantaneous.
– Weak dye – needs a mordant for staining.
• CARMINE: ground bodies of cochineal beetles.

HEMATOXYLENE
• Hematoxylon campechianum
• Boiling
• Precipitated out by urea
• Hematin (Oxidation)
– Natural. eg., Ehlrich’s, Delafield’s
– Chemical. eg., Mayer’s (NaI), Harris’ (Mercury oxide/ NaI)
• Hematoxylene
– Alum
– Iron (Weigert’s)
– Tungsten (PTAH)
– Lead
– Molybdenum (Phosphomolybdic acid)

STAINING:
DYES USED IN STAINING: SYNTHETIC DYES
• Most are Aniline base & are derived from coal tar.
• Aniline dyes offer wide range of color and action.
• Chemically – Basic / Acidic / Amphoteric (Neutral).
– Basic – Cationic – Nuclei, Basophilic granules, Bacteria.
– Acidic – Anionic – Cytoplasm, Eosinophilic granules.

STAINING:
THEORIES OF STAINING:
• PHYSICAL THEORIES:
– Simple solubility – Fat stains are more soluble in fat than
alcohol.
– Adsorption.
• CHEMICAL THEORIES: acidic dyes stain basic elements
(Cytoplasm) & basic dyes stain acidophilic material (Nucleus). Not
always true – Hematoxylin.

STAINING:
MOUNTANTS:
• Two types:
1. Aqueous - Used for material which is unstained, stained for fat, or
metachroamtically stained.
2. Resinous – For routine staining.

STAINING:
MOUNTANTS:
• AQUEOUS:
1. Apathy’s medium – Fluorescent microscopy.
2. Farrant’s medium – Fat stains.
3. Glycerin jelly – Fat stains.
4. Highman’s medium – Metachromatic dyes especially Methyl
violet.

STAINING:
MOUNTANTS:
• RESINOUS: Natural / Synthetic resins dissolved in Benzene /
Toluene / Xylene.
1. Canada balsam – natural.
2. D.P.X. – synthetic.

STAINING:
MOUNTANTS:
• Criterias of a mountant:
1. Refractive index should be as close as possible to that of glass i.e.
1.5.
2. It should not cause the stain to diffuse or fade.
3. It should not crack or appear granular on setting.
4. It should be dry to a non sticky consistency and harden relatively
quickly.
5. It should not shrink back from edge of cover-glass.
6. It should be free flowing and free from air bubbles.

STAINING:
CYTOPLASMIC STAINS:
• EOSIN:
• Eosin 1% stock - Dissolve 1 Gm of eosin Y [water soluble] in 20
mL of distilled water and 80ml of 95% alcohol.
• Eosin working
– Stock Eosin - 1 Part
– Alcohol 80% - 3 Parts
– Add 0.5 mL of Acetic acid just before use per 100 Ml.

STAINING:
CYTOPLASMIC STAINS:
• EOSIN PHLOXINE:
• Eosin Phloxine working
– Stock Eosin 1% in D. W. - 100 mL
– Stock Phloxine1% in D. W. - 10 mL
– Alcohol 95% - 780 mL
– Acetic acid – 4 mL
– Working solution to changed weekly.

STAINING:
NUCLEAR STAINS:
• HEMATOXYLIN EHRLICH’S:
– 2% Haematoxylin in alcohol 100 mL
– 3% ammonium or potassium alum in distilled water 100 mL
– Mix the above two and add the following in order
– Glycerol 100 mL (Prevent overoxidation & rapid evaporation)
– Acetic acid 10 mL (Accentuator)
– Keep the bottle loosely plugged
– Let it ripen for 1-3 months

HEMATOXYLIN HARRIS

STAINING:
NUCLEAR STAINS:
• HEMATOXYLIN HARRIS:
– 10% ammonium or potassium alum in D.W. 100 mL
– 10% alcoholic hematoxylin 10 mL
– Bring the alum to boiling point and add the haematoxylin solution
carefully till the solution is deep red. Add 0.5 gm of red oxide of
mercury - solution becomes deep purple - Promptly remove the
flame and plunge into ice cold water. This is the most important part.
Leave over night at room temperature and filter. Add 2 or 4 mL of
acetic acid per 100 mL of the stain before use.
– Note: In place of red oxide of mercury, 0.177 gm of potassium
permanganate can be used but should added after cooling the solution
and never while boiling.

STAINING:
NUCLEAR STAINS:
• HEMATOXYLIN PHOSPHOTUNGSTIC ACID
MALLORY’S:
– Haematoxylin 1.0 Gm
– Phosphotungstic acid 20 Gm
– Distilled water 1000 mL
– Dissolve haematoxylin and phosphotungstic acid separately in
distilled water with the aid of heat, when cool, combine the
solution and make up to 1 litre with distilled water. Let it stand
for 5-6 weeks before use. Staining time - 12-24 hours
– Note : Quick ripening may be done by adding 0.177 gm of
potassium permanganate. However the results are not so good.

STAINING:
NUCLEAR STAINS:
• WEIGERT’S IRON HEMATOXYLIN:
A. Haematoxyln 1 Gm
– Alcohol 100 mL
– Let it ripen for a week
B. 30% solution of ferric chloride 4 mL
– Hydrochroloric acid (conc.) 1 mL
– Immediately before use mix equal parts of A & B add B to A
and not vice versa.
– Staining time 20-30 minutes

STAINING:
NUCLEAR STAINS:
• MAYER’S HEMATOXYLIN:
– Hematoxylin 1 Gm
– Heat distilled water to 55 to 60°C and add hematoxylin rotate till
dissolved.
– Ammonium or potassium alum 50 Gms
– Sodium iodate 0.20 Gm (to be weighed exactly)
– Add the above in order given.
– Citric acid 1 Gm
– Chloral hydrate 50.0 Gm
– Above must added in order given. Allow to stand overnight before
use. Solution is stable for 6-8 weeks.
– Staining time 6-8 minutes.

STAINING:
STAINING SOME BASIC RULES:
• Keep stains and solutions covered when not in use.
• After the slides are removed from oven these should be cooled
before being put in xylene.
• Filter stains before use.
• Once the slides have been put in the xylene to remove paraffin they
should not be allowed to dry out. Particular care must be taken not
to let the sections dry at the time of mounting as the xylene easily
evaporates and if the section dried before mounting preparation
would become useless.

STAINING:
STAINING SOME BASIC RULES:
• Care should be taken that level of any solution used during staining
is such as to cover the slides.
• Drain the slides well and blot the bottom on filter paper before
putting into the next solution. This is particularly necessary in
transferring from 95% to absolute alcohol and absolute alcohol in
xylene.
• Xylene used to remove paraffin should not get mixed up with the
clearing xylene. It also should be frequently changed as it tends to
get saturated.
• If for blueing an alkali e.g. ammonia has been used, it should be well
washed out. Failure to do that will lead to disagreeably hazy blue
colour of nuclei.

H&E STAINING
1. XYLENE- 10M DEPARAFFINAZION
2. XYLENE- 5M
3. 90% ALCOHOL- 1M
4. 70% ALCOHOL- 1M HYDRATION
5. 50% ALCOHOL- 1M (BROUGHT TO WATER)
6. WATER- 3M
7. HEMATOXYLENE- 5-10M PRIMARY (NUCLEAR) STAIN
8. WATER- 3M
9. 1% ACID ALCOHOL- 1 DIP DIFFERENTIATION
10. RUNNING TAP WATER- 20-30M BLUEING
11. 1% AMMONIA SOLUTION- 2 DIPS
12. 70% ALCOHOL- 1M
13. EOSIN- 2DIPS COUNTER (CYTOPLASMIC) STAIN
14. ABSOLUTE ALCOHOL- 3M
15. ABSOLUTE ALCOHOL- 3M DEHYDRATION
16. ABSOLUTE ALCOHOL- 3M
17. XYLENE- 10M CLEARING
18. XYLENE- 10M

Mount in DPX
Results
• Nucleus - blue
• Cytoplasm and background - pink

STAINING:
H & E STAINING:
• Causes of poor quality of staining
1. Poor or inadequate fixation of tissue.
2. Over or under-ripened Haematoxylin.
3. Overused or worked out Haematoxylin.
4. Over or under differentiation of haematoxylin
5. Insufficient blueing following differentiation.
6. Failure to wash blueing agent out of section before counter staining with eosin
(especially when ammonia is used).
7. Insufficient differentiation of eosin during washing or dehydration.
8. Insufficient dehydration and clearing of sections.
9. Contamination of stains.

STAINING:
SPECIAL STAINS:
• Van Gieson stain for connective tissue.
• Gomori’s stain for reticulin.
• PAS (Periodic Acid Schiff) for glycogen / Mucin / etc.
• Mayer’s mucicarmine for mucin.
• Fite’s stain for AFB.
• Von Kossa’s stain for calcium.

MUSEUM TECHNIQUES
All teaching hospitals and colleges of Pathology have Museums
which serve many functions:
• permanent exhibition of common specimen for undergraduate and
postgraduate teaching purposes
• illustrating specimens of rarity
• Permanent source of histologic material
• gross and microscopic photography

BASIC MUSEUM TECHNIQUES
1. Reception
2. Preparation
3. Fixation
4. Restoration
5. Preservation
6. Presentation

Reception of the Specimen
• Any specimen received in the museum should be recorded in a
Reception book and given a number followed by year (e.g.
32/2013).
• This number will stay with specimen even after it is catalogued in
its respective place.
• This number is written on tie-on type label in indelible ink and is
firmly attached or stitched to the specimen.
• The reception book should contain all necessary information about
the specimen (clinical, gross and microscopic findings).

Preparation of the specimen
• An ideal specimen is received fresh in unfixed state.
• Usually will already be formalin fixed.
• If planning to use a specimen for museum, part of it can be kept
without disturbing for museum, e.g. in kidney it can be bisected
and one half kept aside for museum.

Fixation of the specimen
• The objective of fixation is to preserve cells and tissue constituents in as
close a life-like state as possible and to allow them to undergo further
preparative procedures without change.
• Fixation arrests autolysis and bacterial decomposition and stabilizes the
cellular and tissue constituents.
• The fixatives used in museums all over the world are based on formalin
fixative technique, and are derived from Kaiserling technique and his
modifications.
• Kaiserling recommended that the initial fixation be a neutral formalin
(KI) solution and then transferred to a final preserving glycerin solution
(KIII) for long term display.
• Colour preservation is also maintained with these solutions.

Kaiserling’s Technique
Fixation of specimen:
• The specimen needs to be kept in a large enough container which
can accommodate specimen along with 3-4 times volume of
fixative.
• Specimen is stored in the Kaiserling I Solution for 1 month
depending on the size of the specimen.
• The specimen should not rest on bottom or an artificial flat surface
will be produced on hardening due to fixation.

Kaiserling I Solution:
• Formalin 1L
• Potassium acetate 45 g.
• Potassium nitrate 25 g.
• Distilled water Make up to 10 litres
Restoration of specimen
• It is required to restore the specimens, as they lose their natural color on
fixation.
• The recommended method is the Kaiserling II method.
• It involves removing the specimen, washing it in running water and
transferring to 95% alcohol for 10 minutes to 1hour depending on the
size of specimen.
• The specimen is then kept and observed for color change for around 1-
1.5 hrs.
• After this step, specimen is ready for preservation.

Kaiserling II Solution:
• Alcohol 95%
*Store specimen in this solution for 10 minutes to 1 hour depending on size
of specimen.
Rejuvenator Solution:
• Pyridine 100 ml
• Sodium hydrosulphite 100 gm
• Distilled water 4 litres
*Formalin decreases the natural colour of the specimen. However,
rejuvenator solution restores the colour.
Preservation of specimen
• The recommended solution for this step is Kaiserling III.
• This is the final solution in which the specimen will remain for display. It
is based on glycerine solution.

Kaiserling III Solution:
• Potassium acetate 1416 g.
• Glycerine 4 litres
• Distilled water Make up to 10 litres
• Thymol crystals added to prevent moulds.
*Leave solution to stand for 2 – 3 days before using to ensure proper
mixing of chemicals.
• Add 1% pyridine as stabilizer.
• This solution acts as permanent fixative.
• This solution easily turns yellowish and needs to be replaced to
restore colour of the specimen.
• The specimen will initially float to surface but later sink to bottom.

Presentation of the Specimen
• Initially all museum specimens were mounted in cylindrical jars
and sealed with sheep bladder walls.
• Later they were replaced by rectangular glass jars.
• They were better than cylindrical ones as the flat surfaces afforded
a clear view of specimens without any distortion.
• They are covered by rectangular glass plates.
• These jars can be purchased readymade or assembled in museum
itself, as per need.
• Nowadays, Perspex jars are also available, which are lighter than
glass jars.
• However, they cannot be used to store specimens fixed in alcohol
or methyl salicylate as they react with plastics.

Mounting the Specimens
• To support the specimen within its jar, it is attached to the specimen
plate or rectangular bent glass rods.
• It can be done by tying the specimen with nylon threads.
• Double knots should be made by threads, on the specimen surface.

FROZEN SECTION
• Sections are prepared quickly for histological examination by
freezing the tissue.
• The section should be thin, and without water crystals.
• It is an important procedure for quick diagnosis.

ADVANTAGES
􀁺 Quick diagnosis
􀁺 Study the margins of cancer
􀁺 Enzyme histochemistry
􀁺 Immunohistochemistry
􀁺 Detection of lipid
􀁺 Silver impregnation (neuropathology)
DISADVANTAGES
􀁺 Morphology is distorted
􀁺 Cellular details are not well seen
􀁺 Staining is not very good
􀁺 Some specials stains cannot be performed

Handling of specimen
• Tissue must reach histopathology laboratory immediately.
• To avoid tissue being dried it should be kept in saline.
• The size of the tissue should be small thin (ideally about 3mm to
4mm).
• The tissue can directly be taken to cryostat or can be fixed with
10% formalin or formol –alcohol.

Embedding media
• Sucrose (20%) or a drop of water may be applied on the chuck.
• Optimum Cooling temperature (OCT) compounds or 20% sucrose
gives good result.
• Completion of freezing is observed by the change of color of tissue
which turns glossy white.
• Freezing should be done fast (this will prevent ice crystal
formation, morphology is better preserved and artifacts are less).
• Lower the temperature, harder the block.

FREEZING SUBSTANCES
Different freezing substances are used depending upon the availability
and feasibility.
• Carbon Dioxide gas (most common)
• Liquid Nitrogen (expertise required)
• Aerosol sprays (easiest)
• Isopentane (best)

CRYOSTAT
• Essentially a microtome, placed in a freezer.
• Cooling by electricity/ liquid nitrogen.
• Electronic temperature control.
• Electronic retraction/ advance of block.
• Digital temperature and section thickness visualization.
• Automatic defrost.

PROCEDURE
• Specimens that are soft at room temperature are mounted on a cutting
medium (often made of egg white) on a metal “chuck”, and frozen to
cutting temperature (for example at -20 degrees C).
• Once frozen, the specimen on the chuck is mounted on the microtome.
• The crank is rotated and the specimen advances toward the cutting blade.
• Once the specimen is cut to a satisfactory quality, it is mounted on a
warm (room temperature) clear glass slide, where it will instantaneously
melt and adhere.
• The glass slide and specimen are air dried, and stained.
• The entire process from mounting to reading the slide takes from 10 to 20
minutes.

Coated slides
• Poly-L-lysine
• Gelatin-formaldehyde
Different temperatures
• Brain, liver -(7 – 10)
• Heart, lung -(15 – 20)
• Bone marrow -(20 – 25)
• Fat -(25 – 30)
QUENCHING (Freeze drying)
• Rapid freezing (-160 C)
• Removal of water by sublimation in vaccum (-40 C)
• Used in research labs.

AUTOPSY
• Investigative dissection of a dead body primarily to find out cause
of death.
• Derived from the word “autopsia”, meaning is to see for oneself.
• Performed primarily to determine the cause of death, to identify or
characterize the extent of disease states that the person may have
had, or to determine whether a particular medical or surgical
treatment has been effective.
• Two types:
– Medico legal autopsy
– Pathological autopsy

A forensic autopsy technician is responsible for providing technical assistance when diagnosing
disease or trauma within the realm of medicolegal autopsies.
Essential duties of forensic autopsy technicians may include:
• Positioning and photographing bodies and organs
• Opening and closing bodies
• Drawing and spinning blood samples
• Taking cultures
• Collecting latent and cast fingerprints
• Removing organs, tissues and fluids
• Performing diagnostic imaging activities, including body and dental x-rays
• Photographing and documenting wounds and areas of injury
• Preparing and preserving forensic evidence
• Collecting and preserving clothing, physical and biological evidence for forensic analysis
• Assisting forensic scientists in specialty forensic examinations, such as dental or anthropology
examinations
• Preparing preliminary summaries of the deceased’s clinical history
• Maintaining detailed records of all collected evidence and examinations
• Overseeing the release of bodies to funeral homes

Histotechniques

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