HISTOPATHOLGY NOTES: Histopathology The light microscopic study of diseased tissues.

." Histopathology - is the morphological study of cells arranged in tissues. Cytopathology- is the morphological study of dissociated cells. Histopathologic Techniques Study on the basic concepts about the principles and technicalities involved in histopathologic procedures. Purpose For the Medical Technologist to come up with a well-processed tissue to aid in the confirmation and proper evaluation of a disease entity and in the end will determine the proper mode of treatment. SPECIMEN ACCESSIONINGSpecimen received in the lab and given an identity #. GROSS EXAMINATION Done by the pathologist TISSUE PROCESSING EXAMINATION OF THE TISSUES Examination of Tissues methods of tissue examination is based on the ff: 1. Structural & chemical components of the cell to be studied 2. Nature and amount of tissue to be evaluated 3. The need for immediate examination Two types of method: I. Fresh Tissue Examination Teasing, Squash preparing, smear, streaking, Spreading, Pullapart, touch preparation, and frozen section II. Process Tissue Examination 10 processes and the most commonly employed in the lab. Processing of Prepared tissue: 1. FIXATION 2. DEHYDRATION 3. CLEARING 4. INFILTRATION (IMPREGNATION) 5. EMBEDDING 6. TRIMMING 7. SECTION-CUTTING 8. STAINING 9. MOUNTING 10. LABELLING  Fixation: Kills, hrdens and preserve Prevents tissue from: degeneration, putrefaction, decomposition, distortion Substance is Called FIXATIVE Effects of Fixative in general: 1. Harden soft friable tissues

2. Make the cells more resistant to damage & distortion 3. Inhibit bacterial decomposition 4. Increase optical differentiatn 5. Reduce the risk of infection during handling. A good Fixative is: 1. Cheap 2. Stable 3. Safe to handle 4. Kill the cell quickly 5. Inhibit bacterial decomposition 6. Rapid penetration 7. Must be isotonic 8. Produce minimum shrinkage Our 10% Neutral Buffered Formalin is a premixed AFIP formulated product that reduces exposure to toxic fumes. Phosphate buffers are used to stabilize the pH between 6.8 and 7.2. Stringent quality control ensures a formaldehyde concentration of 10% and provides a consistent and reliable product. There are five major groups of fixatives, classified according to mechanism of action: 1. Aldehydes: Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by crosslinkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues. Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for immunoperoxidase staining. However, it fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde 2. Mercurials Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride and include such well-known fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail. Their best application is for fixation of hematopoietic and

reticuloendothelial tissues. Since they contain mercury, they must be disposed of carefully. 3. Alcohols Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well. 4. Oxidizing agents Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive denaturation. Some of them have specialized applications, but are used very infrequently. 5. Picrates Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin. Factors Affecting Fixation: Buffering, Penetration, Volume, Temperature, Concentration, Time interval. Types of Fixatives: Divided into 2: According to composition and action: COMPOSITION 2 types A.) Simple fixative B.) Compound Fixative Action 3 types A.) Microanatomical Fixatve B.) Cytological Fixative C.) Histochemical Fixative General Fixatives & its preparations: I. Neutral buffered formalin - fixation time 12-24 hours. Formalin (40% aqueous solution of formaldehyde) - 100ml Sodium dihydrogen orthophosphate (monohydrate) - 4g

Disodium hydrogen orthophosphate (anhydrous) - 6.5g Distilled water - 900ml This fixative is suitable for most histological purposes. It is to be preferred to formol-saline (a single 10% solution of formalin in 9% aqueous NaCl) as formalin pigment is avoided. Specimens may be stored in this fluid. The solution is isotonic. II. Acetic alcohol - fixation time 1 minute. (For smears, cytospin preparations or frozen sections). 95% methanol - 100ml Glacial acetic acid - 3ml. The sections should be washed in water before staining III.Bouin's fluid - fixation time 6 hours. Saturated aqueous solution of picric acid - 75ml Formalin - 25ml Glacial acetic acid - 5ml Fixed tissue should be transferred to 70% alcohol IV. Carnoy's fluid - fixation time 1-3 hours. Ethanol - 60ml Chloroform - 30ml Glacial acetic acid - 10ml Fixed tissue should be processed immediately or transferred to 80% alcohol V. Formol sublimate - fixation time 4-6 hours. Formalin - 100ml Mercuric chloride (saturated aqueous) - 900ml Fixed tissue should be transferred to 80% alcohol VI. Helly's fluid - fixation time 12-24 hours. Stock solution:Potassium dichromate - 25g Mercuric chloride - 50g Sodium sulphate - 10g Distilled water - 1000ml For use:Stock solution - 100ml Formalin - 5ml The fixative solution should be made up just before use. Fixed tissue must be washed for 24 hours in running tap water prior to processing. VII. Michel's fixative for immunoflourescence fixation time 24-48 hrs. Buffer : 0.81g potassium citrate

I. II.

0.0625g N-ethylmaleimide - HANDLE WITH CARE! 0.123g magnesium sulphate 100mls distilled water Before use add 55g ammonium sulphate and allow to dissolve. Adjust pH to 7.0-7.2 with 1M KOH. Place tissue biopsies in fixative for 24-48 hours. Wash tissues in buffer, three times over 10 minutes, and freeze at -70oC. Paraformaldehyde - fixation time depends on technique to follow. Sodium dihydrogen orthophosphate 2.26% 41.5ml Sodium hydroxide 2.52% - 8.5ml Heat to 60-80oC in a covered container. Add 2g `Analar' paraformaldehyde and stir until dissolved. Filter. VIII. Zenker's fluid - fixation time 4-24 hours. Distilled water - 950ml Potassium dichromate - 25g Mercuric chloride - 50g Glacial acetic acid - 50g Fixed tissue should be washed overnight in running tap water before processing.  Decalcification Removal of Calcium or Lime Salts ( most espcly bones & teeth) following fixation I. A good decalcifying agent must Be capable of removing Calcium Salts from tissues completely w/o Producing considerable destruction Of cells and tissue components The rate of decalcification will Depend upon the structures, Temperature, and volume of the Solution to be used Ideal time is 24-48 hours Methods of Calcium removal: Acid Decalcifying Agents Nitric Acid- most common: HCL Formic acid TCA Sulphurous acid Chromic acid Citric Acid

Flex is a patented formulation of methyl and isopropyl alcohol. Use of Flex results in complete dehydration and brilliant cytoplasmic staining. Moreover, it minimizes overdehydration in smaller biopsies. This product is excellent for lipid extraction allowing for increased paraffin infiltration and may be used as a solvent for special stains  Clearing: - Removal of Alcohol or - De-alcoholization Characteristics: 1. Should be miscible with alcohol 2. Should be miscible w/ paraffin & or mounting med. 3. It should not produce excessive tissue shrinkage 4. It should make tissue transparent Commonly Used Clearing Agents: 1. Xylene- (most common) 15-30 mins. 2. Toluene-best but 3X more expensive 3. Benzene- carcinogenic 4. Chloroform-slow & health hazardous 5. Cedarwood oil 6. Aniline oil 7. Clove oil 8. Carbon tetrachloride 9. Methyl salicylate- rarely used,xpensiv,nice smell 10. Limoline-volatile oil in citrus fruit (newer ones) 11. Aliphatic hydrocarbons (Clearite)-sensitive 3 ways by w/c Paraffin wax impregnation & embedding of Tissues maybe performed: 1. by Manual Processing 2. by Automatic Processing 3. by vacuum embedding  Impregnation (Infiltration) - Is the process of removing Clearing agent and replaced by a medium that will completely fill all the tissue cavities. Thereby giving firm consistency & allowing easier handling & Suitable thin section. 3 Types of medium: 1. Paraffin wax impregnation 2. Celloidin impregnation 3. Gelatin Impregnation Richard-Allan Scientific Xylene can be used in all tissue processors and automatic stainers. Strict quality assurance, including gas chromatography, is performed on each lot to guarantee a consistent product. Xylene offers superior lipid extraction, optimal paraffin infiltration, and excellent deparaffinization and clearing during staining. Richard-Allan Scientific

II. Chelating Agents (EDTA) III. Ion exchange Resin IV. Electrophoresis ( Electrical Ionization )

Xylene is a para-depleted product which reduces processing artifacts. We also ensure the lowest level of benzene, a known carcinogen. Clearing consist of removal of dehydrant With a substance that will be miscible w/ The embedding medium. -xylene most common -toluene is good but 3x expensive -chloroform used to be but health hazard -Methyl salicylate is rarely used but smell so nice( it is oil of wintergreen) -others based on limoline(oil found in citrus peels. -aliphatic hydrocarbons (Clearite) are less hazardous but poor result Our signature series paraffin is a unique blend of polymers and highly-refined paraffin with narrow carbon chain distribution that minimize compression and offer rapid infiltration. Both Type L and Type H can be used for infiltration and embedding and have a melting point of 55-57C. Type H allows for ultra-thin sections, while Type L may reduce infiltration times during processing. Both Type L and Type H paraffins are triple-filtered to eliminate impurities and are packaged in easy-to-open, resealable 2-pound bags. I.Paraffin Wax Impregnation- simplest, most common and best embedding medium used for routine work Substitute for Paraffin Wax: 1. Paraplast 2. Ester wax 3. Water Soluble Waxes II. Celloidin ImpregnationSuitable for Specimens with large hollow cavities w/c Tend to collapse, for hard & dense tissues Such as bones and teeth, & whole embryo III. Gelatin Impregnation- is rarely used Except when dehydration is to avoided & When tissues are to be subjected to histochemical and enzyme studies. Three ways of Paraffin Wax and Embedding Of tissues maybe performed: 1. By Manual Method 2. By Automatic Processing 3. By Vacuum Embedding Our disposable base molds are designed for use with all standard-size cassettes and embedding rings. Molded from rigid plastic, the block design provides greater base support and a smaller cutting surface. Cleaning associated

with metal molds is not required. Transparency facilitates tissue orientation. Available in 5 sizes. The TurbOflow metal base molds: Our embedding rings are manufactured from precision molded plastic to fit most base molds. Available in 5 colors for easy identification and packaged in convenient dispenser boxes of 250 rings  The 6th Step Is Trimming of Section  The 7th Step is Section Cutting with the use of Microtome Knives. 3 Basic Shapes: 1.Plane-concave Knife 2.Biconcave Knife 3.Plane-wedge knife The microtome: function and design Steel microtome knives: profile A, strongly planoconcave or biconcave; profile B, planoconcave; profile C, wedge-shaped; profile D, plane-shaped Edge-Rite low profile microtome blades are manufactured from surgical grade steel and will fit any low profile blade holder. Our microtome blade is 20% thicker than traditional blades. This feature increases knife edge stability, which allows for excellent ribboning and also facilitates usage in the cryostat. The blades are specially coated to increase longevity and optimize section quality. Additionally, the low profile blade is 80 mm in length, 5% longer than other blades thus allowing for more sections per blade. The Edge-Rite high profile microtome blade is also coated. All dispensers are translucent and include a used blade receptacle for safe disposal. A portable and precise automatic Microtome knife sharpening equipment for fine sharpening of all Microtome knives upto 140 mm in length. Sharpening duration can be adjusted with timer from 0-60 minutes.  7 Step Sectioning Once the tissues have been embedded they must be cut into sections that can be place on a slide. There are 3 important necessities for proper sectioning: * A very sharp knife, * A very sharp Knife, * A very sharp Knife DIFFERENT TYPES OF MICROTOMES I. Rocking Microtome- For cutting serial sections of large blocks of Paraffin embedded Tissues.
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II.Rotary Microtome- For cutting paraffin embedded section. III.Sliding Microtome- For cutting celloidin embedded sections IV.Freezing Microtome- For cutting unembedded frozen sections. V. Ultrathin Microtome- For cutting sections for Electron Microscopy Actual Cutting of Sections: Sections are allowed to float In the water bath w/ temp.45-50 Degrees centigrade. Fishing out the Paraffin Section Fishing is carried out with the use Of slides that was smeared with adhesives: Mayers egg albumin Dried albumin Gelatin Starch paste Plasma

 -

Dirt on the knife edge Sections crumble or tear Tissue is incompletely dehydrated Tissue has not cleared properly Paraffin wax denatured from excessive heating Blunt knife Wrong melting point wax used for the conditions - wax too soft Wax has crystallised from slow cooling Wax is contaminated with clearing agent or water

1. 2. 3. 4. 5.

 Sections show areas of varying thickness (chatters) - Loose knife or block - Knife clearance angle too great - Excessively hard tissue or wax - Areas of calcification present within tissue    Sections will not ribbon Knife clearance angle too fine or too great Dirty knife edge Wax surface too cold Sections roll up on cutting Blunt knife Knife clearance angle too great Micrometer set too great Wrong melting point wax for the conditions Sections disintegrate on water bath Water temperature too high Wax contaminated with clearing The 8 step is them Highlight & Mostly ask in the BOARD! Be ready  8th Step Staining Is rendering the different Tissue constituent more visible Thru variation in colors thereby Promoting optical differentiatn & identificatn of the cells & Tissues. 2 Types of Dyes: 1. Natural Dyes- obtain fm plants & animals e.g Cochineal, log-wood, and vegetable extract. 2. Synthetic Dyes-sometimes known as Coal Tar Dyes derived fm Hydrocarbon benzene and collective known as Aniline Dyes. I. Mordant- Bidge the stain & the tissue e.g alum II. Chromophone- coloring property III. Auxochrome- the dyeing property IV. Acid dye- coloring subs. Found in the acid component e.g Picric acid V. Basic Dye- coloring subs. Is found in the basic component e.g methylene blue
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PROBLEMS and their CAUSES:  Crooked or uneven ribbons *Edges of knife and block are not parallel *The four sides of the block are not trimmed parallel *The knife edge is not regularly sharpened *Impurities in the wax causing dense areas *Varying densities in the tissue  Wrinkled or compressed sections - Blunt knife - Warm block or knife - Clearance angle too great - Micrometer set too thin for wax hardness - Loose knife - Bevel of knife too deep making cutting angle too shallow  Thick and thin sections - Loose knife or loose block - Block too large for the microtome in use - Knife clearance angle too fine - Tissue or wax excessively hard - Areas of calcification present in tissue  Sections adhere to block on upstroke of microtome - Knife clearance angle too fine - Blunt knife - Dirty knife edge or dirty underside of block  Ribbons split vertically or scratch lines appear - Small nicks in the knife edge - Knife clearance angle too great - Hard material embedded in the wax (grit, dirt, crystals, talc) - Hard material in the tissue (calcium, crystals, sutures)

VII. Neutral Dye- are formed by combining aqueous solns acid and base e.g Giemsas stain VIII. Accentuator- hastens the staining process e.g Phenol, KOH Different Methods of Staining: 1.Progressive Staining- low concentration to high concentration e.g. Wright Stain 2.Regressive Staining- high concentration to low concentration e.g. AFB Staining 3.Metachromatic Staining- is the used of specific dye w/c diffrntiate particular substance by staining them w/ a color that is diffrnt from that of the stain itself (Metachromasia). These are basic dyes belonging to the thizine & Triphenylmethane groups. E.g. Methyl Violet, Cresyl Blue,Safranin, Bismarck brown, Basic fuchsin,toluidine blue Thionine, Methylene blue, Azure A,B,C 4.Counterstaining-application of a different color or stain to provide contrast or background to the staining of the structural components to be demonstrated. Common Counter stain & there Colors: COMMON COUNTERSTAINS USED IN THE LABORATORY: a. CYTOPLASMIC STAINS: Red : Eosin Y, Eosin B, Phloxine B, Rose Bengal. Yellow: Picric Acid, Orange G Green: Light Green SF, Lissamine Green b. NUCLEAR STAINS Red: Neutral Red, Safranin O, Carmine Blue: Methylene Blue, Toluidine Blue, Celestine Blue, Hematoxyline More Methods On Staining: 5.Direct Staining- sections are stained w/ simple aqueous solutions of the dye. e.g Methylene blue, Eosin 6. Indirect Staining- staining is made is possible with the used of a Mordant to form tissuemordant-dye-complex.e.gPotassium Alum 7. Metallic impregnation- specific tissue element is not stained but colorless solutions of metallic salts w/c are reduced by the tissue producing an opaque, usually black deposits on the surface of the tissue or bacteria. E.g. Ammonical Silver, Silver nitrate, gold chloride 8.Vital Staining- selective staining of living cell constituent, demonstrating structures by phagocytosis of the dye particle e.g RES with Trypan Blue

9. Intravital Staining- staining of living cells is done by injecting the dye into part of the animal body. E.g RES with Lithium,Carmine and India Ink. ( Supravital Staining- stain living cells immediately after removal from the living body. E.g Neutral red (best vital dye), Janus green for mitochondria,Trypan Blue, Nile blue, Thionine, Toluidine Hematoxylin 7211 is a uniquely formulated product yielding results which exceed that of Gill or Harris Hematoxylins. A distinct characteristic of 7211 is that there is no affinity for acid mucopolysaccharide (mucin) staining. Slides stained with Hematoxylin 7211 are crisp with clear, well-defined nuclear chromatin delineation Richard-Allan Scientific Modified Harris Hematoxylin is a regressive nuclear stain. This formulation contains no mercury and is more stable than conventional Harris Hematoxylin. This stain can be used in both histology and cytology. Commonly requested Histologic Stains: H&E (hematoxylin and eosin): H&E is the most commonly requested histologic stain. Hematoxylin and eosin staining is used for general morphology and reference and is recommended on all samples. We use Harris Hematoxylin to stain anionic or negatively charged elements (DNA and RNA) purple-blue and alcoholic Eosin Y as a secondary stain to stain non-nuclear components like cytoplasm, muscle, and connective tissue. b. Trichrome: Trichrome stains use a combination of three different dyes. Tissue stained with a trichrome stain shows muscle tissue, fibrin, and collagen dyed with three different colors (the colors are dependent on the variation of this technique that is used). Trichrome stains are used to differentiate between fibrous and normal tissue. This is a good stain for fibrotic changes due to inflammation. c. PAS (periodic acid Schiff): The PAS stain is used for staining polysaccharides. This stain is often used to stain glomeruli in kidney tissue and to demonstrate glycogen in the liver. Fungi with carbohydrate capsules stain PAS positive. It is a very versatile stain and can be used for a variety of other tissue components such as mucin, hyaluronic acid, reticulin, fibrin of thrombi, colloid droplets, hyalin of arteriosclerosis, granular cells in renal arterioles where preserved, most basement membranes, colloid of pituitary stalks and a.

thyroid. All positive PAS components stain rose to purplish red (magenta) d. Silver Stains: Silver stains can be used to stain a variety of structures. Methods which rely on silver solutions are used primarily to identify molecules with strong reducing groups (e.g. melanin), to stain carbohydrates (e.g. glycoproteins), or localize cytoplasmic or extracytoplasmic structures that absorb silver ions e.g. secretory granules of neuroendocrine cells). Silver techniques are commonly employed to demonstrate melanin, reticulin fibers, fungus, spirochetes, nerve cells and processes, or basement membranes. e. Verhoff's Elastic Stain: This stain selectively stains the elastin fibers in tissue. The stains are useful for examining lung tissues or arteries to detect the loss of elasticity (e.g. emphysema or arterial thickening). Other Special Stains worth noting: Congo Red (stains amyloid), Alcian Blue (stains acid mucopolysaccherides), Mucicarmine (stains mucin), Cresyl Violet (stains nerve cells and glia), Luxol Fast Blue (myelin), Prussian Blue (iron), Giemsa or Wright's (stains hematopoietic cells).  MOUNTING The final stage in the preparation of tissues for microscopy is mounting. Occasionally tissue samples are examined dry, but in almost every other situation a liquid medium or 'mountant' is applied. Mountants with a refractive index (RI) as close as possible to that of the tissue (usually taken as the RI of fixed protein, between 1.53 and 1.54) will effectively render a section transparent. To be effective, a mountant should possess certain characteristics. These include the following1-2: it should be colourless and transparent it should be able to completely permeate and fill tissue interstices it should have no adverse effect on tissue components it should be resistant to contamination (particularly microorganism growth) it should protect the section from physical damage and chemical activity (oxidation and changes in pH) it should be completely miscible with dehydrant or clearing agent

it should set cracking or shrinking

without

crystallising,

Types of mountants: I. Hydrophobic mountants A. CANADA BALSAM -This is an oleoresin obtained from the bark of the fir Abies balsamea (of the family Pinaceae), native to North America. B. DPX (DISTRENE, PLASTICISER, XYLENE)- One of the most commonly used mountants, DPX is a colourless, neutral medium in which most standard stains are well preserved. It is prepared by dissolving the common plastic, polystyrene, in a suitable hydrocarbon solvent (usually xylene). C. EUPARAL5- Euparal is a mixture of eucalyptol, sandarac (a resin from the tree, Tetraclinin articulata grown in north west Africa), paraldehyde and camsal (camphor and phenyl salicylate). Its relatively low RI (which is usually given as 1.483 but ranges from 1.478 at 20C to 1.535 when solid) makes it useful for mounting unstained sections. D. RESIN-EMBEDDED TISSUE -Sections of tissue embedded in plastic compounds (such as epoxy resins) can be successfully mounted in liquid resin of the same type E. PHOTOSENSITIVE RESINS- Light polymerising resins have the advantage of very short setting times, requiring in the order of 1030 seconds exposure to UV light to harden completely. II. hydrophilic (aqueous) mountants A. WATER -Although of low RI (1.333), water serves as a convenient temporary mountant for some whole specimens13 for examining certain microorganisms live (saline mount) and particularly when checking sections during staining procedures. B. GLYCEROL-Glycerol is also a useful temporary mountant but with a higher RI (1.460) and longer drying time than water. C. GLYCERINE (GLYCEROL) JELLY- This commonly utilised aqueous mountant is a mixture of glycerol and gelatine and has a RI of 1.47 D. APATHY'S MEDIUM- Sucrose added to gum arabic preparations increases the RI and prevents overdrying. The inclusion of potassium acetate will prevent leaching of metachromatic dyes.21 E. POLYVINYL ALCOHOL-Polyvinyl alcohol, often used as a mountant in immunofluorescence microscopy, has been recommended as an alternative for glycerine

jelly.22 Adding paraphenylenediamine to the preparation is effective in retarding photo fading.23-24  Labelling and storage of slides Slide mounted sections are identified during preparation by inscribing the slide with the tissue accession number or suitable code using a diamond marker or pencil (frosted slides). Care should be exercised when using characters such as A, I, O, T, V and the like as these appear the same when viewed from either side of the slide; incorrectly identifying the side upon which the section is mounted may lead to poor staining or section damage. Safety in the Lab 1. The lab should be well-ventilated. There are regulations governing formalin and hydrocarbonds such as xylene and toluene. 2. Every chemical compound used in the laboratory should have a materials safety data sheet on file that specifies the nature, toxicity, and safety precautions to be taken when handling the compound. 3. The laboratory must have a method for disposal of hazardous wastes. Health care facilities processing tissues often contract this to a waste management company. Tissues that are collected should be stored in formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to a large sink (similar to a large garbage disposal unit). 4. Every instrument used in the laboratory should meet electrical safety specifications (be U.L. approved) and have written instructions regarding its use 5. Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose. 6. Fire safety procedures are to be posted. Safety equipment including fire extinguishers, fire blankets, and fire alarms should be within easy access. A shower and eyewash should be readily available. 7. Laboratory accidents must be documented and investigated with incident reports and industrial accident reports. 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, it becomes explosive. *Many reagent kits have sodium azide as a preservative. You are supposed to flush solutions containing sodium azide down the

drain with lots of water, or there is a tendency for the azide to form metal azides in the plumbing. These are also explosive. *Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not be used. *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.