LABORATORY SAFETY PEL: Permissible exposure limit

TLV: Threshold limit value

QUALITY CONTROL OEL: Occupational exposure limit

Laboratory Staining: check before issuing to the

pathologist
Special stains: accompanied by a control stain LABELLING
e.g. Giemsa stain for Helicobacter pylori
Standard Operating Procedure (SOP): mandated by - Chemical name, mixture ingredients
accrediting/ regulatory agency (ASCP) - Manufacturer’s name, address/ name of person
who made it
MSDS - Date purchased/ made
- Expiration date
- Detailed procedure for handling hazardous - Hazardous warning and safety procedure
substance
- Personal hygiene practice (handwashing) STORAGE OF HAZARDOUS CHEMICALS
- Records of compliance
- Risk assessment - Conventional cabinet
- Cause and prevention of occupational injury/ illness - Dangerous liquids must be stored below countertop
- Health and safety training for minimum risk of bodily exposure
- PPE - Reagents: stored in plastic containers/ plastic
- Hazardous waste disposal practice coated glass bottle
- Do not store flammable liquids at ref temperature
HEALTH HAZARDS
HANDLING SPILLS
1. Biohazards
o Cause diseases in humans - Gloves
o Infectious agents, contaminated solution, - Disposable plastic aprons (for possible chemical
specimen or object spill)
2. Irritants - Disposable gowns (for biohazard)
o Cause reversible inflammatory effects at site of - Dustpan and brush (for powders)
contact with living tissue - Sponge, towel, mop (for liquid)
3. Corrosive chemicals - Absorbent material (commercial absorbent)
o Cause destruction/ irreversible alterations - Bleach (NaOCl for biohazard)
when exposed to living tissue/ destroy - Baking soda (for acids)
inanimate surfaces (generally metals) - Vinegar (for alkali)
4. Sensitizers - Commercial formalin neutralizing product
o Cause allergic reaction in substantial portion of - Sealable plastic bucket
exposed subjects
o May occur at work (↑exposure levels) **For small amount of spill: wipe, dispose sponge
**Evacuate room if dangerous
5. Carcinogens
**For large spill: call emergency response team
o May induce tumors
FIRST AID
o Chloroform, chromic acid, formaldehyde
6. Toxic materials 1. Skin contact
o Capable of causing death by ingestion, direct o Wash 15-30 mins
contact/ inhalation at certain specific o Emergency shower
concentration
o Soap with water (if not water soluble)
o Remove contaminated clothing
PHYSICAL HAZARDS
2. Splashing on eyes
1. Combustible o Eyewash solution
o Ignite at or above certain temperature (flash o Water temp: 15-350C
point) at which vapors ignite in presence of o Rinsing: 15-30 mins
ignition source
2. Flammables HANDLING POTENTIAL INFECTIOUS SPECIMEN
o Flash points below 1000F/ 37.80C
o Requires specially designed cabinets - Fresh tissue/ body fluids
- Fixed specimen (↓risk, formalin treated)
3. Explosives
- Complete penetration by alcohol destroys
o Chemicals that cause sudden instantaneous
infectious agents except prions
release of pressure, gas and heat when
**Prions cause spongiform encelopathies (CJD,
subjected to sudden shocks, pressure or
scrapie, mad cow disease)
increase in temperature
CJD: Creutzfeld Jakob Disease
4. Oxidizers
Treat with formalin/phenol (48hrs) or formic
o Harmless alone but irritates combustion in
acid (1hr)
other materials
Normal Steam Sterilization: inactivates prions
o Can cause fire (through release of oxygen or
Sodium Hypochlorite & Phenol: effective but cause
other gas) artifacts
 Cutting areas: treat w/ chlorine bleach or with - Excessive exposure may cause disorientation, loss
suitable commercial disinfectant 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 - Irritates skin & eyes

- Should not be mixed with formaldehyde, generates - Corrodes metal
heat that is irritating to respiratory system - Handle under fume hood

Aniline Glutaraldehyde

- Toxic when absorbed by skin - Severe irritation of eyes, skin

- Cause severe irritation of eyes - Toxic by ingestion
- Potential carcinogen
- Excessive exposure may cause drowsiness, Hydrochloric Acid
headache, nausea & cyanosis
- Avoid routine use - Causes severe irritation of skin, eyes, RT
- Corrosive to metals
Chloroform - Conc. acid is dangerous because of its fumes
- Handle under a fume hood, use goggles, apron &
- Toxic when inhaled/ingested gloves
- Carcinogenic
- Affects liver, reproductive organ, CNS, blood & Hydrogen Peroxide
gastrointestinal tract
- Excessive exposure may cause disorientation, loss - Harmless if used in <5%
of consciousness & death
- Avoid use Hydroxide (Na/K)

Chromic Acid (Potassium Dichromate) - Corrosive to eyes & skin

- Toxic to kidneys Isopentane

- Corrosive to skin, mucous membranes
- Carcinogenic - Extremely flammable
- Enviromental toxin - Highly volatile
- Do not subject to drain disposal - Store only in refrigerator/freezer
- Chilled isopentane can cause frostbite
Ethanol - Excessive exposure causes irritation of RT, cough,
irregular breathing
- Skin & eye irritant - Accidental ingestion can cause vomiting,
- Not likely to cause significant toxicity if used under headache, depression & abdominal swelling
standard conditions
Isopropanol
Ether
- Cause mild to moderate irritation of skin & eyes
- Causes mild to moderate irritation of skin & eyes - Toxic by ingestion
- Flammable, extremely volatile
- Do not store in refrigerator/ freezer 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, Helly’s/ Zenker’s 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 - Less toxic substitute for ethylene-based ethers

- Moderate skin & eye irritant Silver Salts

- Toxic by ingestion & inhalation
- May cause blindness/ death if taken excessively - Safe when used in fresh solution
- Explosive when old
Nitric Acid - Iriitates skin & eyes
- Causes severe gastrointestinal discomfort if
- Corrosive to skin, mucous membranes & metals ingested
- Toxic by inhalation - Serious environmental hazard
- Do not subject to drain disposal
Nitrogen (Liquid)
Sodium Azide
- Causes frost bite/ thermal burns
- Excessive inhalation may cause dizziness, loss of - Toxic
consciousness, death from aphyxia - Fatal when swallowed/ absorbed through skin/
mixed with acids
Osmium Tetroxide - Can explode when in contact with metals
- Do not subject to drain disposal
- Corrosive to eyes & mucous membranes
- Vapors are extremely toxic to reproductive, sensory Sodium Bisulfate
& RT
- Vials must be scored, broken & opened under hood - Safe when used in dilute solutions
- Keep away from oxidants
Oxalic Acid
Sodium Hypoclorite
- Safe when used in dilutions prescribed for histology
- Conc. acid is corrosive & causes severe burns of - Liquid chlorine bleach
the eyes, skin & mucous membrane - Strong oxidant
- Repeated skin contact causes dermatitis & slow - Eye irritant
healing ulcers - Corrosive to most metals
- Do not mix with formaldehyde/ DAB
Periodic Acid
Sodium Iodate
- Safe when used in quantities prescribed for
histology - Used to replace mercuric oxide when reconstituting
Harris hematoxylin
Phenol - Little risk when used in laboratory quantities

- Readily absorbed through skin Sodium Thiosulfate

- May cause increased heart rate, convulsions/death
- May burn eyes & skin - Minimal health risk when used in histology under
- Combustible & should be used with extreme normal conditions
caution under a hood
Sulfuric Acid
Picric Acid
- Strong skin, eyes & RT irritant
- Explosive when dry/ combined with metal & - Corrosive to most metals
metallic salts - Dilute solutions are safe
- Should not be disposed by pouring down the drain - Concentrated solutions produces fumes that are
- Toxic when absorbed through skin dangerous, requires use of fume hood, apron,
goggles, gloves
Potassium Ferricyanide & Potassium
Ferrocyanide Toluene

- Safe when handled in conc. prescribed for - Skin & eye irritant
histological use - Toxic by ingestion, inhalation/ skin contact

Potassium Permanganate
- Repeated exposure can cause impaired memory,  Virus
poor coordination, mood swings & permanent  Fungi
nerve damage  Microsporidian parasite
- Should be restricted/ avoided
- Diluent in mounting media for removing coverslips

Xylene MICROTOME
- Same risk as toluene 1. Rotary Microtome
o Highest performance
Zinc Chloride
o For manual/motorized sectioning of biological
- Corrosive to most metal including stainless steel specimen
- Do not use in tissue processors
Parts
- Skin & eye irritant
- Causes severe gastrointestinal problems when
o Coarse advance hand wheel
ingested
 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
o Sliding clutch or coarse advance
TYPES OF MICROSCOPE  Prevents continued horizontal movement of
specimen arm
1. Bright Field Microscope o Fine advance wheel
o Most common type of light microscope  When turned 3600 it provides vertical
o Can view stained/unstained specimen movement of the specimen arm past the
o Best for stained/naturally pigmented specimen knife area
for tissue section/ living photosynthetic  Compensates for the distance of the
organism retraction plus thickness on the fine
2. Phase Contrast Microscope advance
o For colorless/transparent o Hand wheel locking device
o Difficult to distinguish from their surrounding  Safety lock
 Cell parts of protozoan o Fine advance micrometer dial/ thickness scale
 Bacteria  Sets desired thickness of tissue ribbon
 Sperm trails o LD information display
 Other types of unstainable cells  Read out display
3. Polarizing Microscope  Shows the number of revolution of the
o For studying rocks and minerals under hand wheel
polarized light o Object clamps and adapters
 Crystals in urine  Specimen holder
 Biochemistry o Object orientation adapter
 Biomedical research  Part A: attaches to the specimen arm
4. Fluorescence Microscope  Part B: attaches to the back of object clamp
o Use fluorochrome to stain microorganism o Knife holder
o Thru excitation of fluorescent dye by light o Knife holder base unit
o Light: Xenon arc lamp/ Mercury-vapor lamp o Clearance angle adjustment
 Properties of organic/inorganic substance o Handpad
5. Dark Field Microscope  Power on/off switch
o Objects are illuminated at very low angle from  Emergency stop button
the side  Speed control (0-150 cutting stroke/min)
o Background is dark
 Unstained microorganism suspended in 2. Freezing Microtome
liquid o For cutting thin to semi-thin fresh frozen tissue
 Living microorganism that are invisible in o Freeze using liquid CO2 or ↓ temperature
ordinary light recirculating coolant
6. Dissecting Microscope 3. Rocking Microtome
 Surface of solid specimen o 2-24 microns in steps of 2 microns each
 Carry out close work such as sorting, 4. Sledge Microtome
dissection, microsurgery, watch making, o For cutting large locks of paraffin and resin
small circuit manufacturer/inspection embedded
7. Electron Microscope o For large and hard blocks
o Uses electrons 5. Sliding Microtome
o Can view as small as 0.2 micron (highest o Optimal stability
magnification: 100x) o Consistent high quality sections
 o For hard and large blocks (>80 x 60 mm) 4. Turn over, repeat 1-3
6. Cryostat
o Cold microtome **Edge first, Heel to toe
o Within ref chamber with glass window **Plane concave: only concave side should be rubbed
o -200C on the hone
7. Ultrathin Microtome Precautions
o Can cut up to 0.5 micron
o For electron microscopy  Hone: 8” x 3” to accommodate length of knife
o Knife: fragments of broken plate glass edge
 Hone should be lubricated
MICROTOME KNIVES  Pressure should be gentle and steady
 Hone should be clean
- For trimming and section cutting  After honing, wipe off the oil or soap from the
- Knives bevel angle: 27-320 knife with xylene

1. Plane concave knife B. Stropping

o 25 mm - “burr” formed during honing is removed & cutting
o One side is flat, the other is concave edge of knife is polished
2. Biconcave knife - Knife is stropped before every object is sectioned
o For paraffin embedded sections on rotary - Paddle strop made up of horse leather (attached to
microtome slid back)
o Both side are biconcave - Toe to heel direction
3. Plane wedge knife - 40-120 strokes
o Both sides are straight
o For frozen sections/ cutting hard and tough Precautions
specimen embedded in paraffin
o For base sledge and sliding microtome  Oil/grease to prevent rusting
o Cutting angle/ clearance angle: 150  Use light pressure
 Lesser compression on block  Speed should be avoided
 Leather strops are dry, requires oiling before
HONING AND STROPPING use (vegetable or castor oil)
 Should be used for at least 24-48 hours after
- Sharpening of badly nicked knives to ensure oiling
optimum sectioning of tissue blocks  Do not use mineral oil or wax
A. Honing Disposable blades
- Removal of gross nicks on the knife edge (coarse
honing) - Common
- Cutting the edge of the knife on a stone (honing - Cheaper conventional steel knives
proper) - Can cut up to 2-4 micron thick sections
Types of Hone Glass knives
1. Belgium yellow - For trimming and semi thin sectioning
o For manual sharpening when cutting edge has - For tissue blocks for electron microscopy
been rendered blunt/nicked - Can cut up to 40 x 2.5 cm, plate glass strip
2. Arkansas - Cracked to form 25 x 25 mm square
o More polishing effect than Belgium yellow - Broken down to two triangular shape knives
3. Fine carborundum
o Much more coarser than the two Diamond knives
o Used only for badly nicked knives
- For cutting resin block for electron microscopy
**Hone is wiped clean with xylene then coarsed with - Brittle and expensive
thin film of: (10-20 strokes)

 Mineral and clove oil

 Xylene OTHER EQUIPMENTS USED IN HISTOPATHOLOGY
 Liquid paraffin/ soapy water (for
lubrication) 1. Paraffin oven
o “wax oven”
Procedure o Maintain temperature of 2-50C above melting
point of wax (routinely 560C)
1. Fit knife to corresponding knife back (maintain o Store paraffin in its liquid form
angle, hold knife) o Allow section to dry
2. One end of the hone, knife heel first 2. Hot plate
3. Draw obliquely/ diagonally towards operator until o May be used instead of paraffin oven
toe is reached
 o For delicate tissues such as brain, lower drying c. Acrylic plastic
point is used to avoid splitting and cracking of  Esters of acrylic or methacrylic acid
the section  Used for LM
o Prone to over heating. Tissue is exposed  GMA (Polyglycol methacrylate)
o Paraffin oven is preffered for drying  MMA (Methyl methacrylate)
3. 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 TISSUE EMBEDDING CENTER
o Add 20% ethanol/ detergent for easy fishing
out - Leica EG1160
o Can hold 2L water - Compact bench top TEC which enables the user to
4. Slides produce paraffin embedded tissues that can later
o 76 x 25 mm, 1-1.2 mm thick be successfully sectioned with ease
o Frosted edge slide - Features digital program interface for individual
o Label with diamond pencil temperature setting for the paraffin reservoir,
5. Coplin Jar cassette bath, mold warmer and work surface
o Wide mouthed glass jars
o Vertically grooved interior walls Essential Parts
o Used for storage/ staining of slides containing
blood smears or tissue section 1. Refrigerating system
6. Tissue cassettes & embedding molds 2. Paraffin melting chamber
3. Microscreen (filters particle/sediments)
a. Leukhart’s embedding mold 4. Hot and cold orientation platforms
 2 L shaped strips of heavy brass/metal 5. Waste drawer
arranged on a flat metal surface 6. Hot well (for preheating forceps)
b. Compound embedding unit
Components and Features
 Made up of series of interlocking plates
resting on flat metal base
1. Paraffin reservoir
c. Plastic embedding rings and base mold
o Holds 3L paraffin
 Consist of special stainless steel base mold
o 45-700C (paraffin liquid temp)
fitted with plastic embedding ring
2. Paraffin dispenser with illumination
d. Disposable embedding mold
o Dispenser is separately heated and always has
 Peel away disposable thin plastic
the same temp as paraffin reservoir
embedding mold (perfect even without o Dispenser handle is used for manually
trimming)
operating the paraffin flow with a dispenser
 Plastic ice trays (for busy routine lab) handle and extension clamp
 Paper boat (for colloid blocks) 3. Mold warmer
o 33-700C
Other Embedding Methods: 4. Cassette bath
o 45-700C
1. Celloidin/ Nitrocellulose o Can hold more than 100 cassettes
o Use to be recommended for embedding hard
5. Cold plate
tissues such as bones, teeth and for large o -50C
sections
o Optimal consistency of the blocks minimizes
2. Double embedding method
the risk of brittleness as a result of rapid
o Process in which tissues are first infiltrated with
cooling and high level of productivity
celloidin and then embedded in paraffin mass
6. Refrigeration spot
3. Resin embedding
o Integrated in the cold plate ensuring consistent
o For electron microscopy
low temp
o For undecalcified bone and for high resolution
o Mold containing the sample filled with liquid
light microscopy of tissue sections thinner than
paraffin (1/3) are placed in refrigeration spot to
usual 2-6um such as renal biopsy
allow partial solidifying
4. Plastics: Epoxy, polyester, acrylic
7. Paraffin collecting tray
a. Epoxy
o Located under the heated work area to collect
 Epoxy plastic, catalysts + accelerators
excess paraffin derived from surface
 Hydrophobic
8. Work area
 Reduce antigenicity with VCD o 45-700C
(vinylcyclohexane dioxide) o Embedding area, forceps holder, recessed area
 Carcinogenic
for cassettes and space to reove the lids
 Bisphenol A (Arab elite) o Forceps holder is separately heated
 Glycerol (Epon)
 Cyclohexane dioxide (spurr)
b. Polyester plastic
 For EM FRESH TISSUE EXAMINATION
  Not done in muscle
Examination may be done on fresh or preserved biopsy
tissues depending upon necessity.  Isopentane - cooled by liquid
nitrogen – for muscle biopsies
Methods of Fresh tissue examination  Carbon dioxide gas –
1. Teasing or Dissociation freezing microtome, cylinder
- Is a process whereby a selected tissue  Aerosol sprays – widely used
specimen is immersed in a watch glass
containing isotonic salt solution, carefully PROCESSING OF TISSUES
dissected or separated 1. Fixation
2. Dehydration
2. Squash Preparation (Crushing) 3. Clearing
- Process whereby small pieces of tissue not 4. Infiltration (Impregnation)
more than 1mm in diameter are places in a 5. Embedding
microscopic slide and forcibly compressed with 6. Trimming
another slide. 7. Section-Cutting
8. Staining
3. Smear preparation 9. Mounting
- Is the process of examining sections or 10. Labeling
sediments whereby cellular materials are FIXATION AND FIXATIVES
spread lightly over slide by means of a wire Fixation – the most and critical step in
loop or applicator or by making a apposition histotechnology involves fixing or preserving fresh
smear with another slide. tissue for examination.
o Streaking – w/ an applicator stick or a - Aim:
platinum loop, the material is rapidly o To preserve the morphologic and
and gently applied in a direct or zigzag chemical integrity of the cell in as life
line throughout the slide like a manner as possible.
o Spreading – a selected portion of the o To harden and protect the tissue from
material is transferred to a clean slide the trauma of further handling.
and gently spread into a moderately - Fixatives have the property of forming cross
thick film by teasing the mucous apart links between proteins.
with an applicator stick - Soluble proteins are fixed to structural proteins
o Pull Apart – done by placing a drop of and thus rendered insoluble.
secretion or sediment upon slide and Fixation Prevents:
facing it to another clean slide - Degeneration
o Touch Preparation (impression - Decomposition
smear) – whereby the surface of a - Putrefaction
freshly cut piece of tissue is brought - Distortion of tissues
into contact and perused on to the
surface of a clean slide Two Basic Mechanisms in Fixation:
1. Additive Fixation
4. Frozen Section - Chemical constituent of fixative becomes part of the
- This method is normally utilized when a rapid tissues by forming cross-links (ex.formalin, mercury,
diagnosis of the tissue in question is required and osmium tetroxide)
and is especially recommended when lipids
and nervous tissue elements are to be 2. Non Additive Fixation
demonstrated. - Not incorporated but alters the composition of tissues
o Temperature: -10 to -20 C by removing the bound water attached to H bonds
o Applications in histotechnology: (ex. Alcoholic fixative)
 Rapid pathologic diagnosis
during surgery Main factors involved in fixation:
 Diagnostic and research 1. Hydrogen ion concentration
enzyme histochemistry o pH: 6 to 8
 Demonstration of soluble 2. Temperature
substances such as lipids and o room temperature
carbohydrates o electron microscopy and
 Immunofluorescent and histochemistry: 0-40oC
immunohistochemical staining o Rapid Fixation: 60oC
 Some specialized sliver stains , o Tissue with tuberculosis: 100oC
particularly in neuropathology
o More commonly used methods for 3. Thickness of section
freezing: o 1 to 2 mm2 – electron microscope
 Liquid nitrogen – most rapid o 2cm2 – light microscope
freezing agent
 Formation of vapor 4. Osmolality
phase (uneven pulling o Hypertonic solutions: cell shrinkage
of tissue o Isotonic solutions: cell swelling
 o 400-40 mOsm (recommended)
o 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
- Gendre’s 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. Perenyi’s 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 o It dehydrates rapidly
 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 ½ 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 Ethylene glycol ethers are combustible
ar 110 – 120F
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
 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.
A. Xylene
o Is colorless clearing agent that is
commonly used in histology lab.
o Clearing time is usually ½ to 1 hour.
B. Toluene
o Used as substitute to xylene or
benzene
o Clearing time: 1-2 hours
C. 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)
D. 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
E. 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
- 56C – 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. Leuckhart’s 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