IntroductionHistopathology (compound of three Greek words: histos "tissue", pathos "disease-suffering",and -logia) refers to themicroscopicexamination of tissue in order to study

themanifestations of disease. Specifically, in clinical medicine, histopathology refers to theexamination of a biopsyor surgical specimen by a pathologist, after the specimen has been processed andhistological sectionshave been placed onto glass slides. In contrast,cytopathology examines free cells or tissue fragments.Histological techniques provide a visual means for the examination and analysis of cell/tissue physiologyand morphology at the microscopic level. Histology represents a broad technology, invaluable for studying and understanding the microscopic threedimensional organization, structure, and functionof cells and tissues and is especially useful for the diagnosis and understanding of disease at thecellular level. The process first involves isolation and fixation of the cells/tissue of interest. Fixation preserves the structure and morphology of the specimen throughout the harsh conditions of dehydration, clearing, embedding, sectioning and staining. Tissue Processing Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are described below. The persons who do the tissue processing and make the glass microscopic slides are histotechnologists. Specimen Accessioning Tissue specimens received in the surgical pathology laboratory have a request form that lists the patientinformation and history along with a description of the site of origin. The specimens are accessioned bygiving them a number that will identify each specimen for each patient. Gross Examination Tissues removed from the body for diagnosis arrive in the Pathology Department and are examined by apathologist, pathology assistant, or pathology resident. Gross examination consists of describing thespecimen and placing all or parts of it into a small plastic cassette which holds the tissue while it is beingprocessed to a paraffin block. Initially, the cassettes are placed into a fixative.When a malignancy is suspected, then the specimen is often covered with ink in order to mark the marginsof the specimen. Different colored inks can be used to identify different areas if needed. When sections aremade and processed, the ink will mark the actual margin on the slide.1

Gross specimen examinationPREPARATION OF HISTOLOGICAL SPECIMENSPreparation of histologicalspecimens undergo the following steps: 1 . f i x a tion. 2. tissue processing :a. d e h yd r a t io n . b. c l e a r i n g . c. i m p r e g na t io n . 3 . e m b e d d i n g . 4 . t r i m m i n g .5 . c u t t i n g . 6. st a in ing . (1) TISSUE FIXATION

Introduction
Once tissues are removedf rom the body, they undergo a process of self-destruction or autolysis, which is initiated soon after cell death by the action of intracellular enzymes causing the breakdown of protein and eventual liquefaction of thecell.Autolysis is more severe in tissues which are rich in enzymes, such as the liver, brain and kidney, and isless rapid in tissues such as elastic fiber and collagen. The objective of fixation isto preserve cells and tissue constituents in as close a life-lik e state as possible and to allow them to undergo further preparative procedures without change. Fixation arr ests autolysis andbacterial deco mposit io n and stabilizes the cellular and tissue const ituents so that they wit hstand thesubsequent stages of tissue processing.Fixation should also provide for thepreservation of tissue substances and proteins. Fixation is, therefore, the first stepand the foundation in a sequence of events that culminates in the final examination of a tissue section.It is relevant to point out that fixat ionin itself constitutes a major artefact. The living cell is fluid or in a semi-fluidstate, whereas fixat ion produces coagulat io n of t issue proteins and const ituents, a necessary e v e nt t o p r e vent their lo ss or diffusio n during tissue processing; the passage through hypertonic and hypotonic solut ions dur ing tissue processing would otherwise disrupt the cells. For example, if fresh unfixed tissueswere washed for prolonged periods in running water, severe and irreparable damage and cell lysis would result.In contrast, if the t issues were first fixed in formalin, subsequent immersio n in water is gener ally harmless.

Tissue fixation The technique of usingfixatives in the preparation of cytologic, histologic, or pathologic specimens for the purpose of maintaining the existing form and structure of all the constituent elements. Fixatives Agents employed in the preparation of histologic or pathologic specimens for the purpose of maintaining the existing form and structure of all of the const ituent elements. Great numbers of different agents are used; some are also decalcifying and hardening agents. They must quickly kill and coagulate living tissue. A large variet y of fixat ives is now available. Each fixat ive has advantages and disadvantages, so me are restrictive while others are multipurpose. Fixation - types of fixatives The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. There are five major groups of fixatives, classified according to mechanism of action: Aldehydes Mercurials Alcohols Oxidizing agents Picrates Over the years, various classifications of fixatives have been proposed, with major divisions according to f u n c t io n a s c o a g u l a nt s a nd no n- c o a g u l a nt s , o r a c c o r d i ng t o t he i r c h e m i c a l n a t u r e i n t o t h r e e g e n e r a l categories which include alcoholic, aldehydic and heavy metal fixatives. Factors involved in fixation Temperature Size of specimens and penetration of fixative Changes in volume PH and buffers Osmolality Concentration of fixatives Duration of fixation Formulations for various fixatives The details provided relate to commonly used fixatives. Many variations are available and more specialised fixative solutions are not provided. 1) Formaldehyde solutions 10% neutral buffer formalin (4% formaldehyde) Reagents required 40% formaldehyde 100 ml Distilled water 900 ml Sodium dihydrogen orthophosphate 4 gDisodium hydrogen orthophosphate (anhydrous) 6.5 g Method Prepare, using quantities indicated. Fixation time: 24-72 hours. 2) Baker's formol-calcium (modified)Reagents required 40% formaldehyde 100 mlDistilled water 900 ml10% calcium chloride 100 ml7 g of cadmium chloride is sometimes added to the mixture Method

Prepare, using quantities indicated. Fixation time: 16-24 hours. 3) Formo l salineReagents required 1 40% formaldehyde 100 ml2 Sodium chloride 9 g3 Tap water 900 ml Method Prepare, using quantities indicated. 4) Alcoholic formaldehydeReagents required 1 40% formaldehyde 100 ml2 95% alcohol 900 ml3 0.5 g calcium acetate may be added to this mixture to ensure neutrality Method Prepare, using quantities indicated. Fixation time: 16-24 hours. ParaformaldehydeReagents required Solution A26% sodium dihydrogen orthophosphate 41.5 ml52% sodium hydroxide 8.5 mlHeat to 60C-80C in a covered container Paraformaldehyde 2 g Method Add paraformaldehyde to solution A, stirring until the mixture is clear.Filter and cool. Adjust pH to 7.2 7.4.Prepare fresh for use (duration of fixation depends on size of specimen and whether for light or electronmicroscopy). Buffered formaldehyde-glutaraldehyde 200 mOsm 38 REAGENTS REQUIRED 1 Sodium dihydrogen orthophosphate 1.6 g2 Sodium hydroxide 0.27 g3 Distilled water 88 ml4 40% formaldehyde 10 ml5 50% glutaraldehyde 2 ml METHOD Prepare, using quantities indicated. Fixation time: 16-24 hours. Alcoholic fixativesCarnoy's fixativeREAGENTS REQUIRED 1 Absolute ethanol 60 ml2 Chloroform 30 ml3 Glacial acetic acid 10 ml METHOD Prepare, using quantities indicated. Fixation time: 1-5 minutes. MethacarnREAGENTS REQUIRED 1 Absolute methanol 60 ml2 Chloroform 30 ml3 Glacial acetic acid 10 ml METHOD Prepare, using quantities indicated. Fixation time: 5-6 hours. Wolman's solutionREAGENTS REQUIRED 1 Absolute ethanol 95 ml2 Glacial acetic acid 5 ml METHOD Immerse frozen section in solution and microwave at 650 watts for 15 seconds. Acetic alcohol formalinREAGENTS REQUIRED 1 40% formaldehyde 10 ml2 Acetic acid 5 ml3 Ethanol 85 ml METHOD Prepare, using quantities indicated. Fixation time: 24 hours at 4C. Picric acid fixativesRossman's fluid REAGENTS REQUIRED1 100% ethanol saturated with picric acid 90 ml2 Neutralised commercial formalin 10 ml METHOD Prepare, using quantities indicated. Fix for 12-24 hours and wash very well in 95% ethanol. Gendre's fluid REAGENTS REQUIRED 1 90% ethanol saturated with picric acid 80 ml2 40% formaldehyde 15 ml3 Glacial acetic acid 5 ml METHOD Prepare, using quantities indicated. Fixation is normally for 4 hours, followed by washing in 80%, 95%and 100% ethanol. Bouin's fluid REAGENTS REQUIRED

1 Saturated aqueous picric acid solution 75 ml2 40% formaldehyde 25 ml3 Glacial acetic acid 5 ml METHOD 1 Prepare, using quantities indicated. Fixation may vary from a few hours to 18 hours.2 Washing with 70% ethanol after fixation will remove most of the yellow colour. Sections can also bewashed after removal of paraffin wax. MERCURIC FIXATIVES 1) Buffered formaldehyde sublimateReagents required 1 Mercuric chloride 6 g2 Distilled water 90 ml3 Sodium acetate 1.25 g4 40% formaldehyde 10 ml Method Prepare, using quantities indicated. Fixation time: 16-18 hours.2) Zenker's fluid Reagents required 1 Distilled water 950 ml2 Potassium dichromate 25 g3 Mercuric chloride 50 g4 Glacial acetic acid 50 g Method Prepare, using quantities indicated. Fixation is normally for 4-24 hours followed by an overnight wash.3) Helly's fluid Reagents required 1) Solution ADistilled water 1000 mlPotassium dichromate 25 gSodium sulphate 10 gMercuric chloride 50 g2) Solution B40% formaldehyde 50 ml Method Add solution A to solution B immediately before use. 4) B5 fixat iveReagents required 1) Stock reagent AMercuric chloride 60 gSodium acetate 12.5 gDistilled water l2) Stock reagent B10% buffered neutral formalin Method To prepare a working solution mix 90 ml stock reagent A with 10 ml stock reagent B. Fixation time: 58hours.5) Susa fluidReagents required Distilled water 80 ml40% formaldehyde 20 mlGlacial acetic acid 4 mlTrichloroacetic acid 2 gMercuric chloride 4.5 gSodium chloride 0.5 g Method Prepare, using quantities indicated. Fixation time: 12 hours. (2)TISSUE PROCESSING :Principles of tissue processing the aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue andgive it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue .The most satisfactory embedding material for routine histology is paraffin wax. Most fixatives areaqueous-based and these are not miscible with paraffin wax to enable impregnation with this medium, thetissue must be processed. Each stage must be of sufficient length to ensure completeness.

The stages involved are: Dehydration: to remove fixative and water from the tissue and replace them with dehydrating fluid. Clearing: replacing the dehydrating fluid with a fluid that is totally miscible with both thedehydrating fluid and the embedding medium. Impregnation: replacing the clearing agent with the embedding medium. Embedding Dehydration The first step in processing is dehydration. Water is present in tissues in free and bound (molecular) forms.Tissues are processed to the embedding medium by removing some or all of the free water. During thisprocedure various cellular components are dissolved by dehydrating fluids. For example, certain lipids areextracted by anhydrous alcohols, and water soluble proteins are dissolved in the lower aqueous alcoholsTo minimize t issue distortion from diffusion currents, delicate specimens are dehydrated in a gradedethanol series from water through 10%-20%-50%-95%-100% ethanol.In the paraffin wax met hod, following any necessary post fixat ion treat ment, dehydrat ion fro m aqueousfixat ives is usually init iated in 60%-70% ethanol, progressing through 90%-95% ethano l, then two or three changes of absolute ethanol before proceeding to the clearing stage.Duratio n of dehydrat io n should be kept to the minimum consistent with the t issues being processed.Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require upt o 9 0 m i n u t e s o r lo ng e r i n e a c h c h a ng e . T i s s u e s m a y b e he l d a nd s t o r e d i n d e f i n i t e l y i n 7 0 % e t h a no lwithout harm. Dehydrating agentsAlcohols Ethanol is probably the most commonly used dehydrant in histology. Ethanol is a rapid, efficientand widely applicable dehydrant. Methanol i s a g o o d e t h a no l s u b s t it u t e b u t r a r e l y u s e d fo r r o u t i n e p r o c e s s i n g b e c a u s e o f it s volatility, flammability and cost.

Isopropanol was first suggested as an ethanol subst itute during the prohibit ion era in the Unit edStates. Glycol-ethers Unlike the alcohols, these reagents do not act as secondary fixatives, and apart from solvent effects do notappear to alter tissue reactivity. Ethoxyethanol Dioxane Polyethylene glycols Other dehydrants Acetone Tetrahydrofuran Phenol Clearing Clearing is the transit ion step between dehydration and infiltrat ion wit h the embedding mediu m. Manydehydrants are immiscible with paraffin wax, and a solvent (transition solvent, ante medium, or clearant)miscible wit h both the dehydrant and the embedding medium is used to facilit ate the transit ion betweendehydration and infiltration steps. Shrinkage occurs when tissues are transferred from the dehydrant to thetransition solvent, and from transition solvent to waxChoice of a clearing agent depends upon the following: The type of tissues to be processed, and the type of processing to be undertaken The processor system to be used Intended processing conditions such as temperature, vacuum and pressure Safety factors Cost and convenience. Speedy removal of dehydrating agent. Ease of removal by molten paraffin wax. Minimal tissue damage. Transition solventsHydrocarbons T o lu e n e a n d x y l e n e c l e a r r a p i d l y a n d t i s s u e s a r e r e nd e r e d t r a ns p a r e nt , fa c i l it a t i n g c l e a r i n g e n d p o i nt determination. Petroleum solvents Chlorinated hydrocarbons Chloroform

Carbon tetrachloride Trichloroethane Esters These are colourless flammable solvents miscible with most organic solvents and with paraffin wax. n-Butyl acetate Amyl acetate, methyl benzoate and methyl salicylate9 Terpenes Terpenes are isoprene polymers found in essential oils originally derived from plants, 77 though so me arenow synthesised. Cedarwood oil Limonene Terpineol Infiltration It is the saturation of tissue cavities and cells by a supporting substance which is generally, but not always,the medium in which they are finally embedded. Tissues are infiltrated by immersion in a substance suchas a wax, which is fluid when hot and solid when cold. Alt ernat ively, t issues can be infiltrated with asolution of a substance dissolved in a solvent, for example nitrocellulose in alcohol-ether, which solidifieson evaporation of the solvent to provide a firm mass suitable for sectioning. Parffin wax Properties of paraffin wax are as follows:Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal andmineral oils. It is about two thirds the density and slightly more elastic than dried proteinThe properties of paraffin wax are improved for histological purposes by the inclusio n o f substancesadded alone or in combination to the wax: improve ribboning increase hardness decrease melting point improve adhesion between specimen and wax Embedding Embedding is t he process by which t issues are surrounded by a medium such as agar, gelat ine, or waxwhich when solidified will provide sufficient external support during sectioning.Embedding tissues in paraffin waxTissues are embedded by placing them in amould filled with molten embedding mediumwhich is then allowed to solidify. Embedding requirements and procedures are essentially the same for allwaxes, and only the technique for paraffin wax is provided here in detail. At the completion of processing,tissues are held in clean paraffin wax which is free of solvent and particulate matter.10

General Embedding ProcedureMETHOD 1 Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces arepresent.2 Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mmsurrounding margin of wax.3 Fill the mould with paraffin wax.4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time.5 Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps.This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat.6 Insert the identifying label or place the labelled embedding ring or cassette base onto the mould.7 Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface.8 Remove the block from the mould.9 Cross check block, label and worksheet. ORIENTATION OF TISSUE IN THE BLOCK Correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy.In circumstances where precise orientation is essential tissue should be marked or agar double embedded.Usually tissues are embedded with the surface to be cut facing down in the mould. Some generalconsiderations are as follows: elongate tissues are placed diagonally across the block tubular and walled specimens such as vas deferens, cysts and gastrointestinal tissues are embeddedso as to provide transverse sections showing all tissue layers tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at rightangles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally)

multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.Tissue orientation in the block.(A) elongate tissues;(B) tubular or cystic specimens;(C) hairy skin;(D) multiple tissue fragments.The knife is at the lower block margin.12

FixationNeutral formalin (acid fixatives and potassium dichromate should be avoided).SectionsThin (3-5 ) paraffin sections. SAFETY NOTE: Turn on the exhaust system before staining.Wear protective clothing, gloves and safety glasses during the staining procedure. Procedure1. Take sections to water 2. Rinse well in distilled water.3. Transfer sections to a mixture of equal parts of 2% Potassium Ferrocyanide and 2% Hydrochloric Acidfor 20-30 min.4. Wash in tap water and then rinse in distilled water.5. Counterstain in filtered 1% neutral red for 1 minute.6. Rinse in tap water.7. Rapidly dehydrate in absolute alcohol, clear and mount.ResultsHaemosiderin and ferric salts: deep blueTissues and nuclei: redCytoplasm pink Erythrocytes yellowNotes1 More pronounced staining is obtained by heating the ferrocyanide to 37oC.2 If the stain fades it may be revived by treating with 10 vol water.3 Tap water must be avoided at all times. The distilled water must be iron-free, and the hydrochloric acidmust be of analytical grade, or it will contain iron.Reagents 1. 2% Aqueous Solution of Hydrochloric Acid 2. 2% Aqueous Solution of Potassium Ferrocyanide 3. Perls Working Solution : Mix equal parts of 2% hydrochloric acid and 2% potassium ferrocyanidesolution just before use. 4. 1% Neutral Red Neutral red (CI 50040) 1.0 gDistilled water 99.0 mLGlacial acetic acid 1.0 mL Coverslipping The stained section on the slide must be covered with a thin piece plastic or glass to protect thetissue from being scratched, to provide better optical quality for viewing under the microscope,and to preserve the tissue section for years to come. The stained slide must go through the reverseprocess that it went through from paraffin section to water. The stained slide is taken through aseries of alcohol solutions to remove the water, then through clearing agents to a point at which apermanent resinous substance beneath the glass coverslip, or a plastic film, can be placed over thesection34

Decalcification Some tissues contain calcium deposits which are extremely firm and which will not section properly withparaffin embedding owing to the difference in densities between calcium and parffin. Bone specimens arethe most likely type here, but other tissues may contain calcified areas as well. This calcium must beremoved prior to embedding to allow sectioning. A variety of agents or techniques have been used todecalcify tissue and none of them work perfectly. Mineral acids, organic acids, EDTA, and electrolysishave all been used.Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because theywill remove large quantities of calcium at a rapid rate. Unfortunately, these strong acids also damagecellular morphology, so are not recommended for delicate tissues such as bone marrow.Organic acids such as acetic and formic acid are better suited to bone marrow, since they are not as harsh.However, they act more slowly on dense cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fixand decalcify tissues at the same time.EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue poorly and worksslowly and is expensive in large amounts.Electrolysis has been tried in experimental situations where calcium had to be removed with theleast tissue damage. It is slow and not suited for routine daily use. Artefacts in Histologic Sections A number of artefacts that appear in stained slides may result from improper fixation, from the type of fixative, from poor dehydration and paraffin infiltration, improper reagents, and poor microtomesectioning.The presence of a fine black precipitate on the slides, often with no relationship to the tissue (i.e., theprecipitate appears adjacent to tissues or within interstices or vessels) suggests formalin-heme pigment hasformed. This can be confirmed by polarized light microscopy, because this pigment will polarize a brightwhite (and the slide will look like many stars in the sky). Formalin-heme pigment is most often seen invery cellular or bloody tissues, or in autopsy tissues, because this pigment forms when the formalin buffer is exhausted and the tissue becomes acidic, promoting the formation of a complex of heme (from redblood cells) and formalin. Tissues such as spleen and lymph node are particularly prone to this artefact.Making thin sections and using enough neutral-buffered formalin (10 to 1 ratio of fixative to tissue) willhelp. If the fixative solution in which the tissues are sitting is grossly murky brown to red, then place thetissues in new fixative.The presence of large irregular clumps of black precipitate on slides of tissues fixed in a mercurial fixativesuch as B-5 suggests that the tissues were not "dezenkerized" prior to staining. These black precipitateswill also appear white with polarized light microscopy.Tissues that are insufficiently dehydrated prior to clearing and infiltration with paraffin wax will be hardto section on the microtome, with tearing artefacts and holes in the sections. Tissue processor cyclesshould allow sufficient time for dehydration, and final ethanol dehydrant solution should be at 100%concentration. In humid climates, this is difficult to achieve. Covering or sealing the solutions from35 ambient air will help. Air conditioning (with refrigerants, not with evaporative coolers) will also reducehumidity in the laboratory. Toluene as a clearing agent is more forgiving of poorly dehydrated tissues, butit is more expensive and presents more of a health hazard than other non-xylene clearing agentsThough alcohols such as ethanol make excellent fixatives for cytologic smears, they tend to make tissuesections brittle, resulting in microtome sectioning artefacts with chattering and a "venetian blind"appearance.Bubbles under the coverslip may form when the mounting media is too thin, and as it dries air issucked in under the coverslip. Contamination of clearing agents or coverslipping media may alsoproduce a bubbled appearance under the microscope. Problems in Tissue Processing "Floaters" are small pieces of tissue that appear on a slide that do not belong there--they have floated induring processing. Floaters may arise from sloppy procedure on the cutting bench-- dirty towels,instruments, or gloves can have tissue that is carried over to the next case. Therefore, it is essential thatyou do only one specimen at a time and clean thoroughly before opening the container of the next case.The best way to guard against unrecognized floaters is to always separate like specimens in the numberingsequence. For example, if you have three cases with prostate chips, separate them in accessioning

withtotally different specimens such as uterus or stomach. That way, if numbers are transposed or labelswritten wrong or tissue carried over, then you will have an obvious mismatch. Carrying over one prostateto another, or transposing the numbers of identical tissues may never be recognized.If reusable cassettes are employed, you must be aware that tissue may potentially be carried over andappear as "floaters" even several days later, when the cassette is re-used. The problem arises when, duringembedding, not all the tissue is removed from the cassette. Then, in the cleaning process, not all of thewax is removed. Then, the next person using the cassette does not pay attention to the fact that there istissue already in the cassette and puts his specimen in it. The floater that appears on the slide will look well-preserved--it should, because it was processed to paraffin.Always be sure that you properly identify the tissue! This means that you make sure that thepatient label on the specimen container matches that of the request slip. An accession number isgiven to the specimen. This number must appear with the tissue at all times. You must never submit a cassette of tissue without a label. You must never submit a cassette of tissue with thewrong label. Mislabelling or unlabelling of tissues is courting disaster. Safety in the Lab The lab should be well-ventilated. There are regulations governing formalin and hydrocarbonds such asxylene and toluene. There are limits set by the Occupational Safety and Health Administration (OSHA)that should not be exceeded. These limits have recently been revised to reduced levels.Every chemical compound used in the laboratory should have a materials safety data sheet on file thatspecifies the nature, toxicity, and safety precautions to be taken when handling the compound.The laboratory must have a method for disposal of hazardous wastes. Health care facilities processingtissues often contract this to a waste management company. Tissues that are collected should be stored in36 formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to alarge sink (similar to a large garbage disposal unit).Every instrument used in the laboratory should meet electrical safety specifications (be U.L. approved)and have written instructions regarding its use.Flammable materials may only be stored in approved rooms and only in storage cabinets that are designedfor this purpose.Fire safety procedures are to be posted. Safety equipment including fire extinguishers, fire blankets, andfire alarms should be within easy access. A shower and eyewash should be readily available.Laboratory accidents must be documented and investigated with incident reports and industrial accidentreports.Specific hazards that you should know about include: Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, itbecomes explosive. Many reagent kits have sodium azide as a preservative. You are supposed to flush solutions containingsodium azide down the drain with lots of water, or there is a tendency for the azide to form metal azides inthe plumbing. These are also explosive. Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not beused. Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers.Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed.37 formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to alarge sink (similar to a large garbage disposal unit).Every instrument used in the laboratory should meet electrical safety specifications (be U.L. approved)and have written instructions regarding its use.Flammable materials may only be stored in approved rooms and only in storage cabinets that are designedfor this purpose.Fire safety procedures are to be posted. Safety equipment including fire extinguishers, fire blankets, andfire alarms should be within easy access. A shower and eyewash should be readily available.Laboratory accidents must be documented and investigated with incident reports and industrial accidentreports.Specific hazards that you should know about include:

 Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, itbecomes explosive. Many reagent kits have sodium azide as a preservative. You are supposed to flush solutions containingsodium azide down the drain with lots of water, or there is a tendency for the azide to form metal azides inthe plumbing. These are also explosive. Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not beused. Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers.Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed. http://www.scribd.com/doc/44698015/Histology-Procedure#archive