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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
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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
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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
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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
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Chemical name, mixture ingredients
Manufacturer’s name, address/ name of person
who made it
Date purchased/ made
Expiration date
Hazardous warning and safety procedure

STORAGE OF HAZARDOUS CHEMICALS
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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
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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
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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
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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

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. irregular breathing Accidental ingestion can cause vomiting. headache. 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.Cutting areas: treat w/ chlorine bleach or with suitable commercial disinfectant - Excessive exposure may cause disorientation. CNS. use goggles. blood & gastrointestinal tract Excessive exposure may cause disorientation. loss of consciousness & death Ethylene Glycol HAZARDS AND HANDLING COMMON HISTOLOGICAL CHEMICALS - During process of dilution. cough. apron & gloves Hydrogen Peroxide - Harmless if used in <5% Hydroxide (Na/K) Chromic Acid (Potassium Dichromate) - - Isopentane Toxic to kidneys Corrosive to skin. eyes. loss of consciousness & death Avoid use Severe irritation of eyes. reproductive organ. 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. 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. acid is dangerous because of its fumes Handle under a fume hood. glacial acetic acid should not be mixed with chromic acid. eyes & repiratory system 1-10% dilute solution: safe to use Conc. skin Toxic by ingestion Hydrochloric Acid - Causes severe irritation of skin. nausea & cyanosis Avoid routine use Chloroform - Toxic when inhaled/ingested Carcinogenic Affects liver. conc. nitric acid or Na/KOH - Toxic by inhalation/ingestion Toxic to reproductive. RT Corrosive to metals Conc. depression & abdominal swelling Isopropanol - Cause mild to moderate irritation of skin & eyes Toxic by ingestion Mercuric Chloride & Mercuric Oxide . 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. headache. acid irritates skin.

prescribed for histological use Potassium Permanganate - Minimal health risk when used in histology under normal conditions Sulfuric Acid - Strong skin. inhalation/ skin contact . loss of consciousness. ammonium hydroxide. death from aphyxia Osmium Tetroxide - Corrosive to eyes & mucous membranes Vapors are extremely toxic to reproductive. 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. 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. 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. sensory & RT Vials must be scored. apron. broken & opened under hood Oxalic Acid - Safe when used in dilutions prescribed for histology Conc. eyes & RT irritant Corrosive to most metals Dilute solutions are safe Concentrated solutions produces fumes that are dangerous. HCl. gloves Toluene - Skin & eye irritant Toxic by ingestion. goggles. do not mix with acetic acid. mucous membranes & metals Toxic by inhalation Nitrogen (Liquid) - Causes frost bite/ thermal burns Excessive inhalation may cause dizziness. formaldehyde. 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.- Cause severe irritation of eyes & skin Corrosive to metal (contains mercury) Solutions will be contaminated if specimen is fixed with B-5. glycerol. requires use of fume hood. acid is corrosive & causes severe burns of the eyes. ethanol. ethylene glycol. hydrogen peroxide.

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. 5. watch making. 4. 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 . poor coordination.- Repeated exposure can cause impaired memory. 7. 2. 5. small circuit manufacturer/inspection Electron Microscope o Uses electrons o Can view as small as 0. 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 o TYPES OF MICROSCOPE 1. 3. 3. 4. 6. microsurgery. dissection.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 o 2.

3. 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. plate glass strip Cracked to form 25 x 25 mm square Broken down to two triangular shape knives 2. Fit knife to corresponding knife back (maintain angle. 3. For trimming and semi thin sectioning For tissue blocks for electron microscopy Can cut up to 40 x 2. 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. hold knife) One end of the hone. Plane concave knife o 25 mm o One side is flat. 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 1.6.5 micron o For electron microscopy o Knife: fragments of broken plate glass 4. 7. Procedure 1. **Edge first. 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. 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. 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. - 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. 3.5 cm. 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 . - 2. A. 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.

b. 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. Epoxy  Epoxy plastic. d. 3.2 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. a. acrylic a. 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.For delicate tissues such as brain. 5. 6. 5. cassette bath. 2. 1-1. 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. 4. mold warmer and work surface Essential Parts 1. Celloidin/ Nitrocellulose o Use to be recommended for embedding hard tissues such as bones. 5. 6. 3. o 3. 2. 4. 3. catalysts + accelerators  Hydrophobic  Reduce antigenicity with VCD (vinylcyclohexane dioxide)  Carcinogenic  Bisphenol A (Arab elite)  Glycerol (Epon)  Cyclohexane dioxide (spurr) b. forceps holder. Leukhart’s 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. polyester. 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. 7. 4. c. 6. lower drying point is used to avoid splitting and cracking of the section o Prone to over heating. 4. 8. 2. 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. recessed area for cassettes and space to reove the lids o Forceps holder is separately heated FRESH TISSUE EXAMINATION .

Alcoholic fixative) Main factors involved in fixation: 1. o Streaking – w/ an applicator stick or a platinum loop. 7. 5. 10. Hydrogen ion concentration o pH: 6 to 8 2. 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. 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.formalin. Thickness of section o 1 to 2 mm2 – electron microscope o 2cm2 – light microscope 4. 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. 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. 8. Examination may be done on fresh or preserved tissues depending upon necessity. Non Additive Fixation . carefully dissected or separated 2. 6. 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.cooled by liquid nitrogen – for muscle biopsies Carbon dioxide gas – freezing microtome. 2. 4. Additive Fixation . 3.Chemical constituent of fixative becomes part of the tissues by forming cross-links (ex. Methods of Fresh tissue examination 1. 3.Not incorporated but alters the composition of tissues by removing the bound water attached to H bonds (ex. Aim: o To preserve the morphologic and chemical integrity of the cell in as life like a manner as possible. Temperature o room temperature o electron microscopy and histochemistry: 0-40oC o Rapid Fixation: 60oC o Tissue with tuberculosis: 100oC 3. Soluble proteins are fixed to structural proteins and thus rendered insoluble. Two Basic Mechanisms in Fixation: 1. and osmium tetroxide) 2. 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 . 9. Teasing or Dissociation Is a process whereby a selected tissue specimen is immersed in a watch glass containing isotonic salt solution. 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 . o To harden and protect the tissue from the trauma of further handling. Fixatives have the property of forming cross links between proteins. Fixation Prevents: Degeneration Decomposition Putrefaction Distortion of tissues 1. Osmolality o Hypertonic solutions: cell shrinkage o Isotonic solutions: cell swelling . mercury.

Compound fixatives. 2.o o 400-40 mOsm (recommended) Sucrose is commonly added to osmium tetroxide fixatives in electron microscope 5.5gm 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.25) ideal for immune-electron microscopy 6. Speed – specimen should be fixed immediately 2. Preparation: Sodium Dihydrogen phosphate (anhydrous) 3. 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. - - 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. and is soluble in water to the extent of 37-40% weight in volume o Commonly used as a 4% solution. Penetration – formalin diffuses into the tissues at the rate of 1mm per hour 3. giving 10% formalin for tissue fixation o Buffered to pH 7 with phosphate buffer. - - - DECALCIFICATION Is it a process whereby calcium or lime salts are removed from tissues (most especially bone and teeth: tuberculous organs and arteriosclerotic vessels). agitation or microwave Types of fixative ACCORDING TO COMPOSITION 1. Aldehydes b. Duration of fixation – fibrous organs take longer fixation *fixation time can be cut down by using heat. 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. Chromate fixatives iii. Heat Formalin – A gas produced by the oxidation of methyl alcohol. 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. vacuum. Metallic Fixatives i. Cytological fixatives – are those that preserve specific parts and particular microscopic elements of the cell itself. easily available and relatively inexpensive. Simple Fixatives – are made up of only one component substance a. 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. Lead fixatives iv. post mortem and research specimens. 1. Aqueous Nitric Add solution 10%  Decalcification time: 12 – 24 hours B. Type of fixative ACCORDING TO ACTION 1.5gm Disodium hydrogen phosphate (anhydrous) 6. Formol-Nitric Acid  Decalcification time: 1 – 3 days .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. Mercuric chloride ii. Volume – 10-25 times the volume 4. Concentration o Formaldehyde: 10% o Glutaraldehyde :3% o Glutaraldehyde: (0. o 4% for large tissues less than 4mm thick in 6-8 hrs up to 24hrs o Especially used for central nervous tissues 2. A.

acetone and xylene F. but it may take 6 – 8 weeks or longer to totally decalcify dense cortical bone. - - 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. rapid acting dehydrating agent utilized for most urgent biopsies which it dehydrates in ½ to 2 hours. Cellosolve o It dehydrates rapidly o Ethylene glycol ethers are combustible ar 110 – 120F o Toxic in inhalation. - CLEARING Is the process whereby alcohol or a dehydrating agent is removes from the tissue . skin contact. chloroform. 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. and ingestion E.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.cellosolve o Triethyl phosphate o Tetrahydrofuran A. melted paraffin. Tetrahydrofuran o It both dehydrates and clears tissue since it is miscible in both water and paraffin. Alcohol o Ethyl alcohol is the alcohol recommended for routine dehydration of tissues. nail. Acetone o It is a cheap. Dioxane o Is an excellent dehydrating and clearing agent readily miscible in water. Triethyl phosphate o It is soluble in alcohol. o It is a clear. o Ribbon poorly D.  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. colorless and flammable fluid o Fast acting o Methyl Alcohol is a toxic dehydrating agent. Phloroglucin-Nitric Acid  Decalcification time: 12 – 24 hours 2.C. 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. 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.4 Formula: o EDTA disodium salt – 5. ether. o Limited only to small pieces of tissues due to its extreme volatility and inflammability C. benzene. dense fibrous tissue.50 ml  Formic Acid 45% . Formic Acid-Sodium Citrate Solution  Formula:  Aqueous sodium citrate 20% .20) – 10ml / Formal saline 10% 90ml  Decalcification time: 2 – 7 days A. water. 3. alcohol and xylol. Slow dehydrating agent o Temperature: 37C will hasten dehydration B. pH : 7 – 7.50 ml  Decalcification time: 3 – 14 days 4. Perenyi’s fluid  Decalcification time: 2 – 7 days D. primarily employed for blood and tissue films and for smear preparations o Butyl alcohol is utilized in plant and animal micro-techniques. - - - 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.

1 hour  Xylene/Toluene. 2. o Immersed the specimen for another 3 hours in the paraffin wax to insure complete embedding or casting of tissue.rotary 2. Disadvantages: o Prolonged impregnation can cause excessive tissue shrinkage o Paraffin must be free from dust.disposable thin plastic embedding molds available in 3 different sizes. shrinkage and hardening of tissues. 4 general types of tissue impregnation and embedding medium: 1. E. D.A. Plastic Ice Trays . Paraffin wax . o It is rapid acting (15 -30 minutes) Chloroform o Causes less brittleness o Thicker tissue blocks. 4. o Clearing time is usually ½ to 1 hour. o It is especially recommended for central nervous system and cytological studies. Plastic Embedding Rings and Base Mold – consists of a special stainless steel base mold fitted with a plastic embedding ring. Several types of Blocking out Molds: 1. on the microtome before cutting and on the slide before staining.15 minutes  Paraffin Wax.1 hour  100% alcohol. Xylene o Is colorless clearing agent that is commonly used in histology lab. Compound Embedding unit – is made up of a series of interlocking plates resting on a flat metal base 3.3 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. Plastic - - PARAFFIN WAX IMPREGNATION Paraffin: is the simplest. Peel Away. B. o Tissues placed in chloroform do not become translucent Cedarwood oil o Used to clear both paraffin and celloidin sections during embedding process.15 minutes  Paraffin Wax.15 minutes  Paraffin Wax. Leuckhart’s Embedding Mold.2 hours  100% alcohol.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. It is should be miscible also with paraffin in order to facilitate the penetration of this embedding medium. EMBEDDING Orientation : is the process by which a tissue is arranged in precise positions in the mold during embedding.1 hour o IMPREGNATION  Paraffin Wax. 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. Gelatin 4.15 minutes o EMBEDDING  Paraffin Wax. C. Giving perfect block even without trimming b. o FIXATION  10% buffered formalin – 24hrs o DEHYDRATION  70% Alcohol. Celloidin – sledge microtome 3. most common and best embedding medium used for routine tissue processing. o Prevent: brittleness. 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. even those up to 1cm can be processed. Disposable Embedding Mold a. 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. - - and replaced with a substance that will dissolve the wax.6 hours  95% alcohol – 12 hours  100% alcohol.1 hour o CLEARING  Xylene/ Toluene.

o Plastic are classified as: epoxy. 2. 3.  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  . teeth and for large sections of whole organs. polyester and acrylic o EPOXY: are made up of a carefully balanced mixture of epoxy plastic. Celloidin or Nitrocellulose Method o Used to be recommended for embedding hard tissues such as bones. 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. Paper boats – are normally utilized for embedding celloidin blocks but are equally for paraffin wax blocks.c. 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.6 um. catalysts and accelerators. o ACRYLIC PLASTICS: are made up of esters of acrylic of methacrylic acid. Double Embedding Method o Is the process in which tissues are first infiltrated with celloidin and subsequently embedded in paraffin mass. Other embedding methods: 1.