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LABORATORY SAFETY

QUALITY CONTROL
Laboratory Staining: check before issuing to the
pathologist
Special stains: accompanied by a control stain
e.g. Giemsa stain for Helicobacter pylori
Standard Operating Procedure (SOP): mandated by
accrediting/ regulatory agency (ASCP)
MSDS
-

Detailed procedure for handling hazardous


substance
Personal hygiene practice (handwashing)
Records of compliance
Risk assessment
Cause and prevention of occupational injury/ illness
Health and safety training
PPE
Hazardous waste disposal practice

HEALTH HAZARDS
1.

2.
3.

4.

5.
6.

Biohazards
o Cause diseases in humans
o Infectious agents, contaminated solution,
specimen or object
Irritants
o Cause reversible inflammatory effects at site of
contact with living tissue
Corrosive chemicals
o Cause destruction/ irreversible alterations
when exposed to living tissue/ destroy
inanimate surfaces (generally metals)
Sensitizers
o Cause allergic reaction in substantial portion of
exposed subjects
o May occur at work (exposure levels)
Carcinogens
o May induce tumors
o Chloroform, chromic acid, formaldehyde
Toxic materials
o Capable of causing death by ingestion, direct
contact/ inhalation at certain specific
concentration

PHYSICAL HAZARDS
1.

2.
3.

4.

Combustible
o Ignite at or above certain temperature (flash
point) at which vapors ignite in presence of
ignition source
Flammables
o Flash points below 1000F/ 37.80C
o Requires specially designed cabinets
Explosives
o Chemicals that cause sudden instantaneous
release of pressure, gas and heat when
subjected to sudden shocks, pressure or
increase in temperature
Oxidizers
o Harmless alone but irritates combustion in
other materials
o Can cause fire (through release of oxygen or
other gas)

PEL: Permissible exposure limit


TLV: Threshold limit value
OEL: Occupational exposure limit

LABELLING
-

Chemical name, mixture ingredients


Manufacturers name, address/ name of person
who made it
Date purchased/ made
Expiration date
Hazardous warning and safety procedure

STORAGE OF HAZARDOUS CHEMICALS


-

Conventional cabinet
Dangerous liquids must be stored below countertop
for minimum risk of bodily exposure
Reagents: stored in plastic containers/ plastic
coated glass bottle
Do not store flammable liquids at ref temperature

HANDLING SPILLS
-

Gloves
Disposable plastic aprons (for possible chemical
spill)
Disposable gowns (for biohazard)
Dustpan and brush (for powders)
Sponge, towel, mop (for liquid)
Absorbent material (commercial absorbent)
Bleach (NaOCl for biohazard)
Baking soda (for acids)
Vinegar (for alkali)
Commercial formalin neutralizing product
Sealable plastic bucket

**For small amount of spill: wipe, dispose sponge


**Evacuate room if dangerous
**For large spill: call emergency response team
FIRST AID
1.

2.

Skin contact
o Wash 15-30 mins
o Emergency shower
o Soap with water (if not water soluble)
o Remove contaminated clothing
Splashing on eyes
o Eyewash solution
o Water temp: 15-350C
o Rinsing: 15-30 mins

HANDLING POTENTIAL INFECTIOUS SPECIMEN


-

Fresh tissue/ body fluids


Fixed specimen (risk, formalin treated)
Complete penetration by alcohol destroys
infectious agents except prions
**Prions cause spongiform encelopathies (CJD,
scrapie, mad cow disease)
CJD: Creutzfeld Jakob Disease
Treat with formalin/phenol (48hrs) or formic
acid (1hr)
Normal Steam Sterilization: inactivates prions
Sodium Hypochlorite & Phenol: effective but cause
artifacts

Cutting areas: treat w/ chlorine bleach or with


suitable commercial disinfectant

Excessive exposure may cause disorientation, loss


of consciousness & death

Ethylene Glycol
HAZARDS AND HANDLING COMMON
HISTOLOGICAL CHEMICALS
-

During process of dilution, conc. acids should be


added to water (never water to acid) to prevent
splashing & should be done under a fume hood

Acetic Acid
-

Direct contact with conc. acid irritates skin, eyes &


repiratory system
1-10% dilute solution: safe to use
Conc. glacial acetic acid should not be mixed with
chromic acid, nitric acid or Na/KOH

Toxic by inhalation/ingestion
Toxic to reproductive, urinary & blood systems &
with additional exposure toxic to skin
Use propylene-based glycol ethers as substitute
Handle under fume hood with butyl gloves

Formaldehyde & Paraformaldehyde


-

Toxic by inhalation/ ingestion


Severe skin & eye irritant
Carcinogenic
Corrosive to most metals
Workers exposed to formaldehyde must be
periodically monitored for exposure levels
Formadehyde waste can be recycled by
distillation/drain disposal
Can be detoxified by commercial product/ disposed
by licensed waste hauler

Ammonium Hydroxide

Formic Acid

Should be stored away from acids


Should not be mixed with formaldehyde, generates
heat that is irritating to respiratory system

Irritates skin & eyes


Corrodes metal
Handle under fume hood

Aniline

Glutaraldehyde

Toxic when absorbed by skin


Cause severe irritation of eyes
Potential carcinogen
Excessive exposure may cause drowsiness,
headache, nausea & cyanosis
Avoid routine use

Chloroform
-

Toxic when inhaled/ingested


Carcinogenic
Affects liver, reproductive organ, CNS, blood &
gastrointestinal tract
Excessive exposure may cause disorientation, loss
of consciousness & death
Avoid use

Severe irritation of eyes, skin


Toxic by ingestion

Hydrochloric Acid
-

Causes severe irritation of skin, eyes, RT


Corrosive to metals
Conc. acid is dangerous because of its fumes
Handle under a fume hood, use goggles, apron &
gloves

Hydrogen Peroxide
-

Harmless if used in <5%

Hydroxide (Na/K)

Chromic Acid (Potassium Dichromate)

Isopentane

Toxic to kidneys
Corrosive to skin, mucous membranes
Carcinogenic
Enviromental toxin
Do not subject to drain disposal

Ethanol
-

Skin & eye irritant


Not likely to cause significant toxicity if used under
standard conditions

Ether
-

Causes mild to moderate irritation of skin & eyes


Flammable, extremely volatile
Do not store in refrigerator/ freezer

Corrosive to eyes & skin

Extremely flammable
Highly volatile
Store only in refrigerator/freezer
Chilled isopentane can cause frostbite
Excessive exposure causes irritation of RT, cough,
irregular breathing
Accidental ingestion can cause vomiting,
headache, depression & abdominal swelling

Isopropanol
-

Cause mild to moderate irritation of skin & eyes


Toxic by ingestion

Mercuric Chloride & Mercuric Oxide

Cause severe irritation of eyes & skin


Corrosive to metal (contains mercury)
Solutions will be contaminated if specimen is fixed
with B-5, Hellys/ Zenkers fixative
Reagents used to de-zenkerize sections will
release mercury
Must not go through drain disposal
May be replaced with zinc formalin/glyoxal
solutions

Cause irritation of skin & eyes


Accidental ingestion causes severe gastrointestinal
symptoms
Strong oxidant, do not mix with acetic acid,
ammonium hydroxide, ethanol, ethylene glycol,
formaldehyde, glycerol, HCl, hydrogen peroxide,
sulfurix acid

Propylene Glycol

Methanol

Silver Salts

Moderate skin & eye irritant


Toxic by ingestion & inhalation
May cause blindness/ death if taken excessively

Nitric Acid
-

Corrosive to skin, mucous membranes & metals


Toxic by inhalation

Nitrogen (Liquid)
-

Causes frost bite/ thermal burns


Excessive inhalation may cause dizziness, loss of
consciousness, death from aphyxia

Osmium Tetroxide
-

Corrosive to eyes & mucous membranes


Vapors are extremely toxic to reproductive, sensory
& RT
Vials must be scored, broken & opened under hood

Oxalic Acid
-

Safe when used in dilutions prescribed for histology


Conc. acid is corrosive & causes severe burns of
the eyes, skin & mucous membrane
Repeated skin contact causes dermatitis & slow
healing ulcers

Periodic Acid
-

Safe when used in quantities prescribed for


histology

Less toxic substitute for ethylene-based ethers

Safe when used in fresh solution


Explosive when old
Iriitates skin & eyes
Causes severe gastrointestinal discomfort if
ingested
Serious environmental hazard
Do not subject to drain disposal

Sodium Azide
-

Toxic
Fatal when swallowed/ absorbed through skin/
mixed with acids
Can explode when in contact with metals
Do not subject to drain disposal

Sodium Bisulfate
-

Safe when used in dilute solutions


Keep away from oxidants

Sodium Hypoclorite
-

Liquid chlorine bleach


Strong oxidant
Eye irritant
Corrosive to most metals
Do not mix with formaldehyde/ DAB

Sodium Iodate
-

Used to replace mercuric oxide when reconstituting


Harris hematoxylin
Little risk when used in laboratory quantities

Phenol

Sodium Thiosulfate

Readily absorbed through skin


May cause increased heart rate, convulsions/death
May burn eyes & skin
Combustible & should be used with extreme
caution under a hood

Picric Acid
-

Explosive when dry/ combined with metal &


metallic salts
Should not be disposed by pouring down the drain
Toxic when absorbed through skin

Potassium Ferricyanide & Potassium


Ferrocyanide
-

Safe when handled in conc. prescribed for


histological use

Potassium Permanganate

Minimal health risk when used in histology under


normal conditions

Sulfuric Acid
-

Strong skin, eyes & RT irritant


Corrosive to most metals
Dilute solutions are safe
Concentrated solutions produces fumes that are
dangerous, requires use of fume hood, apron,
goggles, gloves

Toluene
-

Skin & eye irritant


Toxic by ingestion, inhalation/ skin contact

Repeated exposure can cause impaired memory,


poor coordination, mood swings & permanent
nerve damage
Should be restricted/ avoided
Diluent in mounting media for removing coverslips

Virus
Fungi
Microsporidian parasite

Xylene

MICROTOME

1.

Same risk as toluene

Zinc Chloride
-

Corrosive to most metal including stainless steel


Do not use in tissue processors
Skin & eye irritant
Causes severe gastrointestinal problems when
ingested

Parts
o

TYPES OF MICROSCOPE
1.

2.

3.

4.

5.

6.

7.

Bright Field Microscope


o Most common type of light microscope
o Can view stained/unstained specimen
o Best for stained/naturally pigmented specimen
for tissue section/ living photosynthetic
organism
Phase Contrast Microscope
o For colorless/transparent
o Difficult to distinguish from their surrounding

Cell parts of protozoan

Bacteria

Sperm trails

Other types of unstainable cells


Polarizing Microscope
o For studying rocks and minerals under
polarized light

Crystals in urine

Biochemistry

Biomedical research
Fluorescence Microscope
o Use fluorochrome to stain microorganism
o Thru excitation of fluorescent dye by light
o Light: Xenon arc lamp/ Mercury-vapor lamp

Properties of organic/inorganic substance


Dark Field Microscope
o Objects are illuminated at very low angle from
the side
o Background is dark

Unstained microorganism suspended in


liquid

Living microorganism that are invisible in


ordinary light
Dissecting Microscope

Surface of solid specimen

Carry out close work such as sorting,


dissection, microsurgery, watch making,
small circuit manufacturer/inspection
Electron Microscope
o Uses electrons
o Can view as small as 0.2 micron (highest
magnification: 100x)

Rotary Microtome
o Highest performance
o For manual/motorized sectioning of biological
specimen

o
o
o

o
o

o
o
o
o

2.

3.
4.

5.

Coarse advance hand wheel

Turning the wheel in opposite direction


turns the object away from the knife edge

For rapid and simple horizontal movement


of specimen arm

Autotrim feature: control specimen arm


advance
Sliding clutch or coarse advance

Prevents continued horizontal movement of


specimen arm
Fine advance wheel

When turned 3600 it provides vertical


movement of the specimen arm past the
knife area

Compensates for the distance of the


retraction plus thickness on the fine
advance
Hand wheel locking device

Safety lock
Fine advance micrometer dial/ thickness scale

Sets desired thickness of tissue ribbon


LD information display

Read out display

Shows the number of revolution of the


hand wheel
Object clamps and adapters

Specimen holder
Object orientation adapter

Part A: attaches to the specimen arm

Part B: attaches to the back of object clamp


Knife holder
Knife holder base unit
Clearance angle adjustment
Handpad

Power on/off switch

Emergency stop button

Speed control (0-150 cutting stroke/min)

Freezing Microtome
o For cutting thin to semi-thin fresh frozen tissue
o Freeze using liquid CO2 or temperature
recirculating coolant
Rocking Microtome
o 2-24 microns in steps of 2 microns each
Sledge Microtome
o For cutting large locks of paraffin and resin
embedded
o For large and hard blocks
Sliding Microtome
o Optimal stability
o Consistent high quality sections

6.

7.

o For hard and large blocks (>80 x 60 mm)


Cryostat
o Cold microtome
o Within ref chamber with glass window
o -200C
Ultrathin Microtome
o Can cut up to 0.5 micron
o For electron microscopy
o Knife: fragments of broken plate glass

4.

**Edge first, Heel to toe


**Plane concave: only concave side should be rubbed
on the hone
Precautions

MICROTOME KNIVES
-

For trimming and section cutting


Knives bevel angle: 27-320

1.

Plane concave knife


o 25 mm
o One side is flat, the other is concave
Biconcave knife
o For paraffin embedded sections on rotary
microtome
o Both side are biconcave
Plane wedge knife
o Both sides are straight
o For frozen sections/ cutting hard and tough
specimen embedded in paraffin
o For base sledge and sliding microtome
o Cutting angle/ clearance angle: 150

Lesser compression on block

2.

3.

A.
-

Precautions

Sharpening of badly nicked knives to ensure


optimum sectioning of tissue blocks
Honing
Removal of gross nicks on the knife edge (coarse
honing)
Cutting the edge of the knife on a stone (honing
proper)

Hone: 8 x 3 to accommodate length of knife


edge
Hone should be lubricated
Pressure should be gentle and steady
Hone should be clean
After honing, wipe off the oil or soap from the
knife with xylene

B. Stropping
burr formed during honing is removed & cutting
edge of knife is polished
Knife is stropped before every object is sectioned
Paddle strop made up of horse leather (attached to
slid back)
Toe to heel direction
40-120 strokes

HONING AND STROPPING


-

Turn over, repeat 1-3

Oil/grease to prevent rusting


Use light pressure
Speed should be avoided
Leather strops are dry, requires oiling before
use (vegetable or castor oil)
Should be used for at least 24-48 hours after
oiling
Do not use mineral oil or wax

Disposable blades
-

Common
Cheaper conventional steel knives
Can cut up to 2-4 micron thick sections

Types of Hone

Glass knives

1.

2.
3.

Belgium yellow
o For manual sharpening when cutting edge has
been rendered blunt/nicked
Arkansas
o More polishing effect than Belgium yellow
Fine carborundum
o Much more coarser than the two
o Used only for badly nicked knives

**Hone is wiped clean with xylene then coarsed with


thin film of: (10-20 strokes)

Mineral and clove oil


Xylene
Liquid paraffin/ soapy water (for
lubrication)

Diamond knives
-

2.
3.

Fit knife to corresponding knife back (maintain


angle, hold knife)
One end of the hone, knife heel first
Draw obliquely/ diagonally towards operator until
toe is reached

For cutting resin block for electron microscopy


Brittle and expensive

OTHER EQUIPMENTS USED IN HISTOPATHOLOGY


1.

Procedure
1.

For trimming and semi thin sectioning


For tissue blocks for electron microscopy
Can cut up to 40 x 2.5 cm, plate glass strip
Cracked to form 25 x 25 mm square
Broken down to two triangular shape knives

2.

Paraffin oven
o wax oven
o Maintain temperature of 2-50C above melting
point of wax (routinely 560C)
o Store paraffin in its liquid form
o Allow section to dry
Hot plate
o May be used instead of paraffin oven

For delicate tissues such as brain, lower drying


point is used to avoid splitting and cracking of
the section
o Prone to over heating. Tissue is exposed
o Paraffin oven is preffered for drying
Floatation waterbath
o 5-100C below melting point of the paraffin wax
(45-500C)
o Fish out in less than 30 seconds to prevent
tissue morphology distortion
o Add 20% ethanol/ detergent for easy fishing
out
o Can hold 2L water
Slides
o 76 x 25 mm, 1-1.2 mm thick
o Frosted edge slide
o Label with diamond pencil
Coplin Jar
o Wide mouthed glass jars
o Vertically grooved interior walls
o Used for storage/ staining of slides containing
blood smears or tissue section
Tissue cassettes & embedding molds

c.

3.

4.

5.

6.

a.
b.
c.
d.

Leukharts embedding mold

2 L shaped strips of heavy brass/metal


arranged on a flat metal surface
Compound embedding unit

Made up of series of interlocking plates


resting on flat metal base
Plastic embedding rings and base mold

Consist of special stainless steel base mold


fitted with plastic embedding ring
Disposable embedding mold

Peel away disposable thin plastic


embedding mold (perfect even without
trimming)

Plastic ice trays (for busy routine lab)

Paper boat (for colloid blocks)

Other Embedding Methods:


1.

2.
3.

4.

Celloidin/ Nitrocellulose
o Use to be recommended for embedding hard
tissues such as bones, teeth and for large
sections
Double embedding method
o Process in which tissues are first infiltrated with
celloidin and then embedded in paraffin mass
Resin embedding
o For electron microscopy
o For undecalcified bone and for high resolution
light microscopy of tissue sections thinner than
usual 2-6um such as renal biopsy
Plastics: Epoxy, polyester, acrylic
a. Epoxy

Epoxy plastic, catalysts + accelerators

Hydrophobic

Reduce antigenicity with VCD


(vinylcyclohexane dioxide)

Carcinogenic

Bisphenol A (Arab elite)

Glycerol (Epon)

Cyclohexane dioxide (spurr)


b. Polyester plastic

For EM

Acrylic plastic

Esters of acrylic or methacrylic acid

Used for LM

GMA (Polyglycol methacrylate)

MMA (Methyl methacrylate)

TISSUE EMBEDDING CENTER


-

Leica EG1160
Compact bench top TEC which enables the user to
produce paraffin embedded tissues that can later
be successfully sectioned with ease
Features digital program interface for individual
temperature setting for the paraffin reservoir,
cassette bath, mold warmer and work surface

Essential Parts
1.
2.
3.
4.
5.
6.

Refrigerating system
Paraffin melting chamber
Microscreen (filters particle/sediments)
Hot and cold orientation platforms
Waste drawer
Hot well (for preheating forceps)

Components and Features


1.
2.

3.
4.
5.

6.

7.
8.

Paraffin reservoir
o Holds 3L paraffin
o 45-700C (paraffin liquid temp)
Paraffin dispenser with illumination
o Dispenser is separately heated and always has
the same temp as paraffin reservoir
o Dispenser handle is used for manually
operating the paraffin flow with a dispenser
handle and extension clamp
Mold warmer
o 33-700C
Cassette bath
o 45-700C
o Can hold more than 100 cassettes
Cold plate
o -50C
o Optimal consistency of the blocks minimizes
the risk of brittleness as a result of rapid
cooling and high level of productivity
Refrigeration spot
o Integrated in the cold plate ensuring consistent
low temp
o Mold containing the sample filled with liquid
paraffin (1/3) are placed in refrigeration spot to
allow partial solidifying
Paraffin collecting tray
o Located under the heated work area to collect
excess paraffin derived from surface
Work area
o 45-700C
o Embedding area, forceps holder, recessed area
for cassettes and space to reove the lids
o Forceps holder is separately heated

FRESH TISSUE EXAMINATION

Examination may be done on fresh or preserved


tissues depending upon necessity.
Methods of Fresh tissue examination
1. Teasing or Dissociation
Is a process whereby a selected tissue
specimen is immersed in a watch glass
containing isotonic salt solution, carefully
dissected or separated
2. Squash Preparation (Crushing)
Process whereby small pieces of tissue not
more than 1mm in diameter are places in a
microscopic slide and forcibly compressed with
another slide.
3. Smear preparation
Is the process of examining sections or
sediments whereby cellular materials are
spread lightly over slide by means of a wire
loop or applicator or by making a apposition
smear with another slide.
o Streaking w/ an applicator stick or a
platinum loop, the material is rapidly
and gently applied in a direct or zigzag
line throughout the slide
o Spreading a selected portion of the
material is transferred to a clean slide
and gently spread into a moderately
thick film by teasing the mucous apart
with an applicator stick
o Pull Apart done by placing a drop of
secretion or sediment upon slide and
facing it to another clean slide
o Touch Preparation (impression
smear) whereby the surface of a
freshly cut piece of tissue is brought
into contact and perused on to the
surface of a clean slide
4. Frozen Section
This method is normally utilized when a rapid
diagnosis of the tissue in question is required
and is especially recommended when lipids
and nervous tissue elements are to be
demonstrated.
o Temperature: -10 to -20 C
o Applications in histotechnology:

Rapid pathologic diagnosis


during surgery

Diagnostic and research


enzyme histochemistry

Demonstration of soluble
substances such as lipids and
carbohydrates

Immunofluorescent and
immunohistochemical staining

Some specialized sliver stains ,


particularly in neuropathology
o More commonly used methods for
freezing:

Liquid nitrogen most rapid


freezing agent

Formation of vapor
phase (uneven pulling
of tissue

Not done in muscle


biopsy
Isopentane - cooled by liquid
nitrogen for muscle biopsies
Carbon dioxide gas
freezing microtome, cylinder
Aerosol sprays widely used

PROCESSING OF TISSUES
Fixation
Dehydration
Clearing
Infiltration (Impregnation)
Embedding
Trimming
Section-Cutting
Staining
Mounting
Labeling
FIXATION AND FIXATIVES
Fixation the most and critical step in
histotechnology involves fixing or preserving fresh
tissue for examination.
Aim:
o To preserve the morphologic and
chemical integrity of the cell in as life
like a manner as possible.
o To harden and protect the tissue from
the trauma of further handling.
Fixatives have the property of forming cross
links between proteins.
Soluble proteins are fixed to structural proteins
and thus rendered insoluble.
Fixation Prevents:
Degeneration
Decomposition
Putrefaction
Distortion of tissues
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Two Basic Mechanisms in Fixation:


1. Additive Fixation
- Chemical constituent of fixative becomes part of the
tissues by forming cross-links (ex.formalin, mercury,
and osmium tetroxide)
2. Non Additive Fixation
- Not incorporated but alters the composition of tissues
by removing the bound water attached to H bonds
(ex. Alcoholic fixative)
Main factors involved in fixation:
1. Hydrogen ion concentration
o pH: 6 to 8
2. Temperature
o room temperature
o electron microscopy and
histochemistry: 0-40oC
o Rapid Fixation: 60oC
o Tissue with tuberculosis: 100oC
3. Thickness of section
o 1 to 2 mm2 electron microscope
o 2cm2 light microscope
4. Osmolality
o Hypertonic solutions: cell shrinkage
o Isotonic solutions: cell swelling

o
o

400-40 mOsm (recommended)


Sucrose is commonly added to osmium
tetroxide fixatives in electron
microscope

5. Concentration
o Formaldehyde: 10%
o Glutaraldehyde :3%
o Glutaraldehyde: (0.25) ideal for
immune-electron microscopy
6. Duration of fixation
o Primary fixation in buffered formalin is
usually carried out for 2-6 hours but
can remain in fixative over the
weekend
o Electron microscopy: 3 hours then
placed in a holding buffer
Practical consideration of Fixation:
1. Speed specimen should be fixed
immediately
2. Penetration formalin diffuses into the
tissues at the rate of 1mm per hour
3. Volume 10-25 times the volume
4. Duration of fixation fibrous organs take
longer fixation
*fixation time can be cut down by using heat, vacuum,
agitation or microwave
Types of fixative ACCORDING TO COMPOSITION
1. Simple Fixatives are made up of only one
component substance
a. Aldehydes
b. Metallic Fixatives
i. Mercuric chloride
ii. Chromate fixatives
iii. Lead fixatives
iv. Heat
Formalin A gas produced by the oxidation of
methyl alcohol, and is soluble in water to the
extent of 37-40% weight in volume
o Commonly used as a 4% solution,
giving 10% formalin for tissue fixation
o Buffered to pH 7 with phosphate buffer.
-

10% Formol Saline saturated formaldehyde


(40% by weight volume diluted to 10% sodium
chloride)
o For central nervous tissues and general
post mortem tissues for histochemical
staining

10% Neutral Buffered Formalin or


Phosphate buffered formalin (pH7)
o Prevents precipitation of acid formalin
pigments
o Recommended for preservation and
storage of surgical, post mortem and
research specimens.
Preparation:
Sodium Dihydrogen phosphate (anhydrous)
3.5gm
Disodium hydrogen phosphate (anhydrous)
6.5gm
Formaldehyde 40%
100ml

Distilled h2o
-

900ml

Formal corrosive (formal sublimate)


o Formol mercuric chloride solution for
routine post mortem
Gendres Fixative
o Alcoholic formalin

Glutaraldehyde 2 formaldehyde residues


linked by 3 carbon chain
o 2-5% for small tissue fragments and
needle biopsies fixed in 2-4hrs at room
tempt.
o 4% for large tissues less than 4mm
thick in 6-8 hrs up to 24hrs
o Especially used for central nervous
tissues

2.

Compound fixatives- are those that are made


up of two or more fixatives which have been
added together to obtain the optimal combined
effect of their individual actions upon the cells
and tissue constituents.

Type of fixative ACCORDING TO ACTION


1. Microanatomical fixatives are those that
permit the general microscopic study of tissue
structures without altering the structural
pattern and normal intercellular relationship of
the tissues in question.
2. Cytological fixatives are those that
preserve specific parts and particular
microscopic elements of the cell itself.
-

DECALCIFICATION
Is it a process whereby calcium or lime salts
are removed from tissues (most especially
bone and teeth: tuberculous organs and
arteriosclerotic vessels).
It should be done after fixation
Calcium may be removed by the following:
o Acid
o Chelating agents
o Ion exchange resins
o Electrical ionization (electrophoresis)
ACID DECALCIFYING AGENTS
They are the most widely used agents for
routine decalcification of large amounts of
bony tissues because they are stable, easily
available and relatively inexpensive.
1. NITRIC ACID

This is the most common and


the fastest decalcifying agent
used so far

Recommended concentration:
5-10%

Disadvantage: inhibiting
nuclear stains and destroying
tissues especially in
concentrated solutions.
A. Aqueous Nitric Add solution
10%

Decalcification time: 12 24
hours
B. Formol-Nitric Acid

Decalcification time: 1 3 days

C. Perenyis fluid

Decalcification time: 2 7 days


D. Phloroglucin-Nitric Acid

Decalcification time: 12 24
hours
2. HYDROCHLORIC ACID

Is inferior compared to nitric


acid in its role as a decalcifying
agent because of its slower
action and greater distortion of
tissue.

It will produce good nuclear


staining and if used in 1%
solution with 70% alcohol may
be recommended for surface
decalcification of the tissue
blocks.
3. FORMIC ACID

Is a moderate acting
decalcifying agent which
produces better nuclear
staining

It is recommended for routine


decalcification of postmortem
research tissues

Formula: Formic acid (SG: 1.20)


10ml / Formal saline 10% 90ml

Decalcification time: 2 7 days


A. Formic Acid-Sodium Citrate
Solution

Formula:

Aqueous sodium citrate


20% - 50 ml

Formic Acid 45% - 50


ml

Decalcification time: 3 14
days
4.

TRICHLOROACETIC ACID

Formula:

Trichloroacetic acid 5
g

Formol saline 10 % - 95
ml

Decalcification time: 4 8 days

CHELATING AGENTS
Substances which combine with calcium ions
and other salts
The most common chelating agents: EDTA
Tissue is placed in EDTA from 1 3 weeks for
small specimens, but it may take 6 8 weeks
or longer to totally decalcify dense cortical
bone.
pH : 7 7.4
Formula:
o EDTA disodium salt 5.5 gm
o Distilled water 90 ml
o Formaldehyde 10 ml
DEHYDRATION
Process or removing intercellular and
extracellular water from the tissue following
fixation and prior to wax impregnation.

Dehydrating agents are alcohols of various


types that are generally used in increasing
strengths to remove aqueous tissue fluids with
little disruption to the tissue
Commonly used dehydrating agents:
o Alcohol
o Acetone
o Dioxane 4- cellosolve
o Triethyl phosphate
o Tetrahydrofuran

A. Alcohol
o Ethyl alcohol is the alcohol
recommended for routine dehydration
of tissues.
o It is a clear, colorless and flammable
fluid
o Fast acting
o Methyl Alcohol is a toxic dehydrating
agent, primarily employed for blood
and tissue films and for smear
preparations
o Butyl alcohol is utilized in plant and
animal micro-techniques, Slow
dehydrating agent
o Temperature: 37C will hasten
dehydration
B. Acetone
o It is a cheap, rapid acting dehydrating
agent utilized for most urgent biopsies
which it dehydrates in to 2 hours.
o Limited only to small pieces of tissues
due to its extreme volatility and
inflammability
C. Dioxane
o Is an excellent dehydrating and
clearing agent readily miscible in
water, melted paraffin, alcohol and
xylol.
o Ribbon poorly
D. Cellosolve
o It dehydrates rapidly
o Ethylene glycol ethers are combustible
ar 110 120F
o Toxic in inhalation, skin contact, and
ingestion
E. Triethyl phosphate
o It is soluble in alcohol, water, ether,
benzene, chloroform, acetone and
xylene
F. Tetrahydrofuran
o It both dehydrates and clears tissue
since it is miscible in both water and
paraffin.
o May cause conjunctival irritation
*when 4% phenol is added to each 95% ethanol baths
part of dehydration process, it acts as a softener for
hard tissues such as tendon, nail, dense fibrous tissue.
-

CLEARING
Is the process whereby alcohol or a
dehydrating agent is removes from the tissue

A.

B.

C.

D.

E.

and replaced with a substance that will


dissolve the wax.
It is should be miscible also with paraffin in
order to facilitate the penetration of this
embedding medium.
Xylene
o Is colorless clearing agent that is
commonly used in histology lab.
o Clearing time is usually to 1 hour.
Toluene
o Used as substitute to xylene or
benzene
o Clearing time: 1-2 hours
Benzene
o Is preferred by some as clearing agent
in embedding process of tissues
because it penetrates and clears
tissues rapidly.
o It is rapid acting (15 -30 minutes)
Chloroform
o Causes less brittleness
o Thicker tissue blocks, even those up to
1cm can be processed.
o Tissues placed in chloroform do not
become translucent
Cedarwood oil
o Used to clear both paraffin and
celloidin sections during embedding
process.
o It is especially recommended for
central nervous system and cytological
studies.
o Clearing time: 2 3 days
IMPREGNATION AND EMBEDDING
Impregnation (Infiltration): is the process
whereby the clearing agent is completely
removed from the tissue and replaced by a
medium that will completely fill the tissue
cavities
Embedding (Casting or Blocking): is the
process by whichh the impregnated tissue is
places into a precisely arranged position in a
mold containing a medium which is then
allowed to solidify.

4 general types of tissue impregnation and


embedding medium:
1. Paraffin wax - rotary
2. Celloidin sledge microtome
3. Gelatin
4. Plastic
-

PARAFFIN WAX IMPREGNATION


Paraffin: is the simplest, most common and
best embedding medium used for routine
tissue processing.
Disadvantages:
o Prolonged impregnation can cause
excessive tissue shrinkage
o Paraffin must be free from dust, water
droplets
56C is the normally used for routine work
Three ways by which paraffin wax
impregnation and embedding of tissues may
be performed:
o By manual Processing

o By automatic processing
o By vacuum embedding
MANUAL PROCESSING
o At least four changes of wax are
required at 15 minutes intervals in
order to insure complete removal of the
clearing agent from the tissue.
o Immersed the specimen for another 3
hours in the paraffin wax to insure
complete embedding or casting of
tissue.
o FIXATION

10% buffered formalin 24hrs


o DEHYDRATION

70% Alcohol- 6 hours

95% alcohol 12 hours

100% alcohol- 2 hours

100% alcohol- 1 hour

100% alcohol- 1 hour


o CLEARING

Xylene/ Toluene- 1 hour

Xylene/Toluene- 1 hour
o IMPREGNATION

Paraffin Wax- 15 minutes

Paraffin Wax- 15 minutes

Paraffin Wax- 15 minutes

Paraffin Wax- 15 minutes


o EMBEDDING

Paraffin Wax- 3 hours


AUTOMATIC PROCESSING
o Only 2 or 3 changes of paraffin wax are
required to remove the clearing agent
and properly impregnate the specimen
VACUUM EMBEDDING
o Involves the wax impregnation under
negative atmospheric pressure inside
an embedding oven to hasten removal
of air bubbles and clearing agent from
the tissue block thereby promoting a
more rapid wax penetration of tissue.
o Prevent: brittleness, shrinkage and
hardening of tissues.
EMBEDDING
Orientation : is the process by which a tissue is
arranged in precise positions in the mold
during embedding, on the microtome before
cutting and on the slide before staining.

Several types of Blocking out Molds:


1. Leuckharts Embedding Mold- consists of L
shaped strips of heavy brass or metal arranged
on flat metal plate and which can be moved to
adjust the size of the mold to the size of
specimen.
2. Compound Embedding unit is made up of
a series of interlocking plates resting on a flat
metal base
3. Plastic Embedding Rings and Base Mold
consists of a special stainless steel base mold
fitted with a plastic embedding ring.
4. Disposable Embedding Mold
a. Peel Away- disposable thin plastic
embedding molds available in 3
different sizes. Giving perfect block
even without trimming
b. Plastic Ice Trays

c.

Paper boats are normally utilized for


embedding celloidin blocks but are
equally for paraffin wax blocks.

Other embedding methods:


1. Celloidin or Nitrocellulose Method
o Used to be recommended for
embedding hard tissues such as bones,
teeth and for large sections of whole
organs.
2. Double Embedding Method
o Is the process in which tissues are first
infiltrated with celloidin and
subsequently embedded in paraffin
mass.
3. Plastic or Resin Embedding
o Used in hard tissues such as
undecalcified bone and for high
resolution light microscopy of tissue
sections thinner than usual 4- 6 um.
o Plastic are classified as: epoxy,
polyester and acrylic
o EPOXY: are made up of a carefully
balanced mixture of epoxy plastic,
catalysts and accelerators.

3 types:

Bisphenol A (araldite)

Glycerol (Epon)

Cyclohexene dioxide
(spurr)

Disadvantages:

Hydrophobic

Reduce antigenicity

Compromise the result


of
immunohistochemistry
staining

Vinylcyclohexane dioxide
carcinogenic
o POLYESTER: were originally introduced
for electron microscopy.
o ACRYLIC PLASTICS: are made up of
esters of acrylic of methacrylic acid.

Used extensively for light


microscopy
Polyglycol methacrylate (GMA) *hydrophilic* and
Methyl Methacrylate widely used because of its
hardness as the ideal embedding medium for
undecalcified bone