PROCESSING & DECALCIFICATION
Which group forms an alcohol when it replaces one or more of a hydrocarbon's hydrogen atoms? a) hydroxyl b) ketone c) methyl d) phenol
[A] A hydroxyl group (-OH) placed on a hydrocarbon makes an alcohol. For example, placing a hydroxyl group on the hydrocarbon methane yields methyl alcohol, or methanol. Placing a hydroxyl group on ethane yields ethyl alcohol, or ethanol.
Which of the following dehydrants should be used when speed is important for processing? a) acetone b) ethanol c) isopropanol d) methanol
[A] Acetone dehydrates the fastest of those listed, and is quickly removed by most clearing agents. It also has a greater solvent action on lipids. It does cause brittleness if specimens are left too long due to removing nearly all bound and unbound water from the tissue.
Which of the following epoxy resins penetrates tissue very slowly, requiring a long embedding schedule? a) Araldite b) Epon c) glycol methacrylate d) Spurr
[A] Araldite is an aromatic epoxy resin, which is highly viscous and penetrates very slowly, requiring tissue to be less than a 2 mm cube. Epon and Spurr are aliphatic epoxy resins, generally used for rush biopsies, as they're less viscous and infiltrate more rapidly. Glycol methacrylate is an acrylic resin.
Dehydrating tissue in graded alcohols of increasing concentration, versus going directly to 100%: a) causes less tissue distortion b) is less harmful to the processor c) dehydrates faster d) removes the fixative faster
[A] By incrementally dehydrating tissue with increasing alcohol concentrations, water can be more gradually removed. High concentrations of alcohol will undesirably remove bound water from macromolecules if specimens are dehydrated for too long. This will cause shrinkage, which renders fibrous tissues hard and cellular tissues brittle.
Acid solutions remove which of the following salts? a) calcium b) lithium c) potassium d) sodium
[A] Calcium occurs in bones as either calcium carbonate, or calcium phosphate, both of which are only slightly water-soluble. Acid solutions, however, are capable of dissolving this calcium and combining it with the anion present in the solution, forming a water-soluble salt. For example, when combined with hydrochloric acid, calcium carbonate will react to form water-soluble calcium chloride and carbonic acid. Decalcifying acid solutions are usually diluted to 5%, leaving 95% of the solution's water content to take up the calcium salt.
Which of the following is a chelating agent used in decalcification? a) ethylene diamine tetraacetic acid (EDTA) b) hydrochloric acid c) phenol d) trichloroacetic acid
[A] EDTA is a chelating [chele-: claw, grabbing] agent, meaning it binds metal ions. EDTA slowly binds and removes calcium ions from the bone. Small spicules of bone may take one week to decalcify, while dense cortical bone may take 6 - 8 weeks. Solutions must be changed every 4 - 5 days. EDTA is gentle on tissue, and produces good staining results with minimum artifacts. Glycogen in bone or cartilage is best preserved by EDTA decalcification, as are subsequent immunoenzyme procedures. This method does, however, tend to harden tissue. Hydrochloric and trichloroacetic acids are acid decalcifiers. Phenol is not a decalcifying agent.
Paraffin processing will destroy which of the following, yielding a false-negative stain? a) enzymes b) mucins c) nuclei d) proteins
[A] Many enzymes (e.g. acid and alkaline phosphatases) are inactivated by molten paraffin's high temperature. Instead, frozen sectioning is generally recommended to preserve enzymes. If tissue must be paraffin processed, it's recommended to use a medium with a lower melting point (<58°C) along with vacuum pressure to shorten its time under heat. Most fixatives, dehydrants, and clearants will also deactivate most enzymes.
Which of the following infiltrating media could be used to demonstrate fat or enzymes in pathologic conditions? a) carbowax b) celloidin c) paraffin d) resin
[A] Since alcohol and hydrocarbon solvents can dissolve fat in tissues, infiltrating media that rely on them in previous steps cannot be used to demonstrate fats. Enzymes are also inactivated by most processing reagents and heat. Carbowax is a water-soluble medium used to infiltrate directly after an aqueous fixative where fat and enzymes are preserved. Carbowax, however, won't infiltrate large, fatty tissue specimens. A cool, non-humid lab makes cutting easier, as heat and humidity affect carbowax. Celloidin, paraffin, and resin are inappropriate media for fat or enzyme preservation, as they all use dehydrants and clearing agents during their processing cycle, and paraffin and resin also employ heat.
Methyl alcohol is often used as a dehydrant for: a) blood smears b) cytology smears c) frozen sections d) touch preps
[A] Slides with blood smears can be placed in methanol prior to Romanowsky stains. It can also be used as a blood smear fixative.
A clearing agent must be miscible with: a) dehydrants and infiltrating media b) fixatives and dehydrants c) fixatives and infiltrating media d) water and dehydrants
[A] Solutions used to dehydrate tissues (e.g. alcohol) usually aren't miscible with paraffin-like substances used in infiltration. Clearing agents are miscible with both dehydrants and infiltrating media, and are used as an intermediate step between those two steps. Other terms relating clearing include 'dealcoholization' and 'transitional fluid'.
Heat was used in all stations of an enclosed tissue processor. One possible effect would be that it: a) hardens some tissues b) extends processing time c) damages the processor d) evaporates reagents
[A] Some tissues (esp. spleen, muscle, skin, decalcified tissue, and those containing blood clots) can become too hard if heat is applied at each processing step. Keeping the temperature at or below 37°C at the fixation, dehydration, and clearing stations will speed up the rate of fixation and clearing without harming the tissue. Time in each station will need to be decreased in order to ensure that tissue isn't over-processed. It's often recommended that only vacuum sans heat is used when dehydrating and clearing the above tissues. Heat shortens processing time, and doesn't damage the processor. If using an enclosed processor, there would be no evaporation.
Which of the following methods is used to test for complete decalcification? a) chemical b) electrolysis c) chelating d) ultrasonic
[A] The three usual methods to test for completion of decalcification are through chemical, physical, and radiographic techniques. Of these, physical methods (i.e. bending/squeezing tissue, testing with a needle, or trimming a portion of tissue) aren't recommended, as they can damage the specimen and negatively affect histological examination. Chemical testing relies on the detection of calcium within the decalcifying solution by adding it to a solution of ammonium oxalate and ammonia. If the solution becomes cloudy or has a precipitate, calcium ions are present in the solution, and therefore still in the specimen. If/when the solution is clear, decalcification is complete. Radiography is the most dependable test for completion. Electrolysis, chelating, and ultrasonics are all methods of decalcification.
Which schedule should be selected to process routine fixed surgical tissue? a) 2 hrs in Alc. Formalin; 5 hrs in 95%-100% EtOH; 2 hrs in Xylene; 2 hrs in Paraffin ((11 hrs total)) b) 2½ hrs in graded conc. EtOH; 40 min in Toluene; 1 hr in Paraffin ((4 hrs total)) c) 20 hrs in graded conc. EtOH; 9 hrs in Xylene substitute; 6 hrs in Paraffin ((35 hrs total)) d) 2 hrs in Absolute EtOH; 2 hrs in Xylene; 3 hrs in Paraffin ((7 hrs total))
[A] Though the schedule may be too long for some small biopsies, and too short for large specimens, the listed times are adequate for most routine surgical specimens grossed 2-3 mm thick. The first two steps of alcoholic formalin will further fix the tissue, while simultaneously starting dehydration.
Which aqueous infiltrating medium can be used to hold exudate or friable tissue in place? a) agar b) celloidin c) paraffin d) resin
[A] Tissues can be infiltrated from water with liquefied 2% agar at 55 - 60°C for 2 - 4 hours. The mass becomes stiff upon cooling, and sections can be cut on a clinical freezing microtome, or be processed for paraffin embedding. There are many problems with this technique, however. Thin sections and serial sections are difficult to obtain when frozen sectioning on a clinical microtome, and ice crystal artifacts cause interpretation issues. Infiltration takes a long time, and hard tissues are generally poorly infiltrated. Neither celloidin, paraffin, nor resin are aqueous infiltrating media.
Prolonged dehydration in higher grades of alcohol will render the specimen: a) hard b) macerated c) porous d) toxic
[A] Tissues contain two types of water: unbound water molecules, and those bound to proteins. Dehydration should remove only the unbound water, leaving most of the bound molecules as is. Specimens remaining in 95 - 100% alcohol for extended periods of time after aqueous fixation will excessively lose these protein-bound molecules, leading to shrunken, brittle tissue that is difficult to section. Maceration (softening) of a specimen can result from prolonged exposure to very low grades of alcohol. A specimen may be porous and contain open spaces, but not as a result of dehydration. Though dehydrating solutions may be toxic, this characteristic is generally not associated with the specimen itself.
The lab requires faster tissue processing. Which of the following can be done easily while still yielding good results? a) adding vacuum pressure of 400-500 mmHg at each station b) increasing the temperature of each station to 70°C c) using butanol instead of ethanol d) using a harder paraffin for infiltration
[A] Vacuum infiltration is recommended when speed is essential, and to prevent the hardening of tissues by reducing the total time in molten paraffin. Air bubbles will be removed from porous tissue, allowing for more complete infiltration. The amount of vacuum shouldn't exceed 400-500 mmHg in order to prevent tissue damage. Raising the temperature above 60°C will harm tissue specimens. Butanol is one of the slowest dehydrants. Paraffin that contains higher concentrations of plastics will yield harder solid blocks. This provides additional support when the tissue being sectioned is hard, or when thinner sections are required.
To prevent damage to delicate tissues (e.g. embryonic tissue), processing should be started at what concentration of alcohol? a) 30% b) 50% c) 70% d) 90%
[A] When tissue is transferred from an aqueous fixative to alcohol, the tissue is subjected to strong diffusion current. By starting at a lower alcohol concentration, the dehydration process will gradually remove water so as not to disturb cell structures. Cells are generally about 70% water, so starting with a 30% alcoholic solution and incrementally raising its concentration by 10% in subsequent steps, less harm can come to tissue than when raising the concentration by larger degrees. Prolonged treatment in alcohols at concentrations below 70%, however, will macerate most fixed tissues. Fixed tissues can be stored indefinitely in 70% alcohol without the fear of maceration or hardening.
Xylene, toluene, and benzene all belong to what chemical class? a) hydrocarbon b) ketone c) phenol d) sterol
[A] Xylene, toluene, and benzene are all ring (aromatic) molecules composed of hydrogen and carbon atoms. They belong to the chemical class of hydrocarbons. Ketones, phenols, and sterols all have atoms of oxygen in addition to hydrogen and carbon.
Which of the following weak acids is the only one used routinely for bone decalcification? a) acetic acid b) formic acid c) trichloroacetic acid d) picric acid
[B] 5% aqueous formic acid will decalcify most bone in 3 - 5 days, and allows for good nuclear and cytoplasmic staining when performed at room temperature. If decalcification is performed at 37°C, the staining of nuclei, collagen, and bone matrix is impaired. Acetic, trichloroacetic, and picric acids are contained in fixatives such as Carnoy, Susa, and Bouin. Since these are weak acids, they can act as decalcifiers, but are generally deemed unsatisfactory. These fixatives may work well in areas of tissue microcalcification, such as those found in breast biopsies. Used alone, acetic acid can cause considerable swelling. Picric acid is very slow to decalcify, and is only considered usable on small pieces. Trichloroacetic acid is simply too weak to decalcify in a reasonable amount of time.
Selection of a suitable clearing agent must be based on the: a) reagent's color index b) ease of removal by the impregnating medium c) type of fixative used d) amount of vacuum used in the processor
[B] A clearing agent should quickly remove the prior dehydrant, and subsequently be quickly replaced by the embedding medium. Time spent in a clearing agent should be minimal, and there shouldn't be any hardening or distortion of the specimen. Other considerations for suitability should include miscibility with both dehydrants and embedding agents, toxicity, flammability, cost, and disposal. Color index only refers to dyes. Fixatives must be removed from tissue during dehydration before clearing. While use of vacuum pressure will speed up clearing time, it is an optional element.
The basic chemical structure of an aromatic hydrocarbon is: a) aldehyde b) benzene c) ether d) phenol
[B] A hydrocarbon has a basic structure of hydrogens and carbons, with the carbons joined together. Aromatic hydrocarbons have a 6-carbon ring structure known as benzene. Toluene's structure is that of a benzene ring with one methyl (-CH3) group added. Xylene also has a benzene ring structure, but with two methyl groups attached. Compared to xylene's larger structure, benzene's smaller molecular size leads it to clear and evaporate faster, as well as pose a greater danger. The chemical formula for an aldehyde is RCH=O, where R implies a reactive group such as methyl (-CH3). The chemical formula for diethyl ether is (C2H5)2O. The chemical formula for phenol is C6H5OH.
In the freeze-substitution technique, the substitution liquid is used in which step? a) clearing b) dehydration c) fixation d) freezing
[B] After tissue has been rapidly frozen at a very low temperature, a dehydrating agent is substituted for the ice in the tissue at a sub-zero temperature. The reagent may be a dehydrant only, or a dehydrant with a fixative. This technique is less expensive than freeze-drying, as less equipment is needed. It produces well-preserved tissues and substances including glycogen, enzymes, and mucoproteins, but does cause considerable shrinkage.
Which schedule should be selected for a fixed liver needle biopsy? a) 2 hrs in Alc. Formalin; 5 hrs in 95%-100% EtOH; 2 hrs in Xylene; 2 hrs in Paraffin ((11 hrs total)) b) 2½ hrs in graded conc. EtOH; 40 min in Toluene; 1 hr in Paraffin ((4 hrs total)) c) 20 hrs in graded conc. EtOH; 9 hrs in Xylene substitute; 6 hrs in Paraffin ((35 hrs total)) d) 2 hrs in Absolute EtOH; 2 hrs in Xylene; 3 hrs in Paraffin ((7 hrs total))
[B] As this is a small tissue specimen, only the minimum amount of time in each reagent is necessary to prevent over-hardening. The liver biopsy must be completely fixed prior to beginning this schedule, as no fixation steps are part of the program. This schedule can also be used for any other small tissue specimens that are already completely fixed, such as small skin biopsies, small endoscopy biopsies, or sections of vas deferens.
Which of the following gases is released during decalcification? a) ammonia b) carbon dioxide c) nitrous oxide d) oxygen
[B] Carbon dioxide is produced during decalcification, forming bubbles on the specimen's surface as long as there is calcium carbonate within it. The presence of these bubbles is often used to determine the decalcification's progress, but shouldn't be used as a reliable method of determining the treatment's end point, as calcium carbonate comprises only about 5% of bone's total mineral composition.
What chemical can be used as an indicator of water in alcohol? a) copper sulfite b) copper sulfate c) sodium sulfite d) sodium sulfate
[B] Copper sulfate's normally grey crystals turn blue when exposed to water, and can be used as an indicator for the presence of water in dehydrating solutions. A layer of copper sulfate can be placed in the final container of absolute alcohol in an open-style tissue processor, but can't be used in an enclosed processor, as the pumping action could pull the chemical through the system.
Which of the following is considered the best routine dehydrant? a) acetone b) ethanol c) isopropanol d) methanol
[B] Ethanol is a reliable dehydrant that's miscible with water in all proportions. It's fast-acting and causes little specimen shrinkage if graded dilutions are used. Pure ethanol is expensive due to high excise taxes. Small amounts of methanol or isopropanol are often added to ethanol as a means to prevent human consumption, thereby abolishing the tax. This mixture is called "reagent alcohol". Acetone, though fast-acting, requires a greater volume-to-tissue ratio, and evaporates quickly. Isopropanol cannot be used with the celloidin technique. Methanol is a poisonous fluid.
Which alcohol is prepared by the fermentation of sugars? a) butanol b) ethanol c) isopropanol d) methanol
[B] Ethanol/ethyl alcohol can be made by the fermentation of the sugar-rich wastes that come from sugarcane, molasses, corn, or grain. These substances contain sugars and starches, which in the presence of water and certain enzymes are converted to ethyl alcohol and CO₂, and is often called 'grain alcohol', and is intended for human consumption. Ethanol may also be made synthetically from ethane, a petroleum gas. Ethanol intended for industrial use, such as in a hospital laboratory, is prepared from ethene. Methanol was originally made by the destructive distillation of wood, and is often called 'wood alcohol'. Most methanol is now made synthetically by either building up from carbon monoxide and hydrogen, or breaking down petroleum hydrocarbons. Normal butanol is made from certain aldehydes, while secondary and tertiary butanols are derived from butene, another petroleum gas. Isopropanol is derived from propylene, net another petroleum gas.
The maximum tissue thickness that still allows adequate xylene clearing in routine processing is: a) 1 - 2 mm b) 3 - 4 mm c) 5 - 6 mm d) 7 - 8 mm
[B] For routine processing, a thickness of 3 - 4 mm is the limit for proper processing. Thicker specimens may clear if given enough time, but their outer portions will end up over-hardened.
A process which can preserve enzymes and prevent the loss of some cellular constituents is called: a) auto-radiography b) freeze-drying c) infiltration d) vaporization
[B] Freeze-drying can be used to demonstrate hydrolytic enzymes destroyed by most fixative solutions. It also prevents the loss of soluble substances (e.g. polysaccharides, mucoproteins, inorganic material, and lipids), the denaturation of proteins, aids in fluorescent antibody and auto-radiography studies, and prepares specimens for scanning electron microscopy. The equipment required for this technique is expensive. After fresh tissue has been quickly frozen at a low temperature (-160°C), water is removed from the tissue through sublimation by increasing its temperature in a vacuum. The temperature must never exceed -30°C, as ice crystals would form and distort the tissue. Auto-radiography is a method of locating radioactive material in tissue sections. Infiltration is the replacement of a clearing agent with molten embedding media. Vaporization is the dispersal of a liquid in the form of a gas.
Excessive exposure to which of the following can cause bone marrow damage in humans? a) amyl acetate b) benzene c) toluene d) xylene
[B] In addition to causing nausea, giddiness, and irritation of the eyes and nose, benzene can also cause damage to bone marrow, and may also be carcinogenic. Its high evaporation rate also makes its use in the general laboratory even more hazardous. There is seldom justification for its use in histology.
In Epon [Epoxy resin], which component is the accelerator? a) DDSA b) DMP 30 c) Epon 812 d) MNA
[B] Most epoxy resin products are a mixture of an epoxy resin, a hardener, a plasticizer, and an accelerator. As these components have different viscosities, they'll penetrate tissue at different rates unless thoroughly mixed prior to use. Epon consists of an epoxy resin (Epon 812), two hardeners (DDSA & MNA), and an accelerator (DMP 30). There is no plasticizer. The hardness of the final block is controlled by changing the amount of the two hardeners.
Which of the following is a proper disposal method for organic solvents (toluene, xylene, etc.)? a) pour down a sink, flushing with water b) collect in a waste container for future incineration or distillation c) pour into a deep pit in the ground d) collect in old bottles and store in a vacant building
[B] Organic solvents must be burned in an incinerator equipped with an afterburner and scrubber, or in the case of alcohol and certain clearants, may be recovered by distillation. Federal, state, and local laws govern the disposal of hazardous wastes.
When decalcifying specimens, their thickness should be no more than: a) 1 - 2 mm b) 3 - 4 mm c) 5 - 6 mm d) 7 - 8 mm
[B] Specimens shouldn't exceed 3 - 4 mm in thickness for adequate fixation and decalcification. Very dense or hard bone may need to be cut thinner than softer tissue. Specimens should be cut using a fine-toothed hacksaw or a good band saw. Before sawing, all soft tissue should be removed from the bone, particularly tough connective tissue and tendons. The specimen should also not be cut too thin, as very dense bone has limited spaces for embedding medium to penetrate, and thinner pieces are more likely to pop out of the block during sectioning.
Which of the following are used only as dehydrants? a) acetone, chloroform, tetrahydrofuran b) butanol, ethanol, methanol c) carbon tetrachloride, isopropanol, toluene d) cellosolve, dioxane, methyl salicylate
[B] The following are used solely as dehydrants: acetone, butanol, ethanol, methanol, isopropanol, and ethylene glycol monoethyl ether, along with proprietary ethanol substitutes.
What is the usual time needed for the complete, non-vacuum paraffin infiltration of a 3 mm thick tissue sample? a) 1 - 2 hours b) 2 - 3 hours c) 3 - 4 hours d) 4 - 5 hours
[B] The time needed for complete infiltration depends on tissue type and thickness. For most specimens, 2 - 3 hours tends to be sufficient, although brain may require overnight treatment. Some tissues require less time (e.g. muscle, fibrous tissue, bloody organs, thrombi/emboli, scar tissue, and fibromas). Vacuum paraffin infiltration will significantly reduce this time if a volatile clearing agent is used.
Which of the following tissue types would show deleterious effects if placed in a universal solvent? a) bloody b) delicate c) dense d) fatty
[B] Universal solvents aren't suitable for delicate tissues, which become distorted by heavy diffusion currents that result when there are large differences in specific gravity across cell membranes. This can occur at two processing steps: when the tissue is transferred from the aqueous fixative to the universal solvent, and again when going from the universal solvent to infiltration media.
During microtomy, you find difficulty in sectioning a tissue specimen. The tissue is soft and mushy. To correct this in the future, change the processing schedule by: a) decreasing time in formalin b) increasing time in dehydrant c) decreasing time in clearant d) decreasing time in paraffin
[B] When cutting tissue sections thinner than 20µm, the tissue must have a support matrix. This support usually comes from an embedding medium such as paraffin. This embedding medium, however, won't infiltrate tissue containing unbound water. Removing this water is done by dehydrating the tissue, usually with alcohols. Clearing agents, usually organic solvents, are then used to remove the alcohol. Decreasing time in formalin leads to under-fixed tissue, which can lead to the distortion of the tissue's morphology, and can also lead to it being soft and mushy during sectioning. Decreased time in clearant will cause the previous dehydrant to remain in the tissue, preventing paraffin from infiltrating in later steps. If tissue is left too long in paraffin, the tissue may become hard to section.
Which of the following clearing agents is not flammable? a) benzene b) chloroform c) toluene d) xylene
[B] While inflammable, heated chloroform will release an even more toxic gas known as phosgene. The other clearing agents listed are flammable; the gases released upon their combustion are carbon dioxide, and—if oxygen is limited—carbon monoxide.
To help preserve membranes in frozen sections, fixed tissue may be placed in a solution of: a) gum mastic b) salt c) sucrose d) talc
[C] After fixation, tissue can be placed in a 30% sucrose solution containing 1% gum acacia for 2 -24 hours. This solution is believed to help stabilize membranes, especially in the mitochondria. Tissues may be stored indefinitely in this solution if kept at 4°C.
What four chemical families are commonly used as clearing agents? a) acetates, aldehydes, chlorinated hydrocarbons, terpenes b) alcohols, aromatic hydrocarbons, glycol ethers, methacrylates c) aliphatic hydrocarbons, aromatic hydrocarbons, chlorinated hydrocarbons, terpenes d) aldehydes, aliphatic hydrocarbons, aromatic hydrocarbons, methacrylates
[C] Aliphatic hydrocarbons have carbon atoms joined to form a continuous or branching chain, and include such familiar things as paraffin (solid), octane (liquid), and isopentane (gas, or liquid @ room temp), as well as several others that are used as clearants. These are commonly known as "xylene substitutes". They are non-irritating and non-sensitizing to normal human skin, and have low inhalation toxicity. Aromatic hydrocarbons have carbons joined in a ring (cyclic) structure, give off a pleasant odor, and include chemicals such as benzene, xylene, and toluene. They are excellent solvents whose toxicity is a cause for concern. Chlorinated hydrocarbons such as chloroform, trichloroethane, and carbon tetrachloride are also very effective but are highly dangerous to health. Terpenes are represented by limonene—a by-product of the citrus industry—whose toxicity lies between aromatics and aliphatics. Acetates are infrequently used because they are rather poor solvents for wax and are highly odorous. The only alcohol used to any extent for clearing is butanol, but its odor, cost, and slowness all hinder its popularity. Aldehydes are fixatives. Glycol ethers by themselves aren't readily miscible with wax. Methacrylates are plastics used as embedding and mounting media.
The effect of acid decalcification is most noticeable in the staining of: a) collagen b) cytoplasm c) nuclei d) osteocytes
[C] Any acid has some effect on the staining of tissue. The stronger the acid or longer the exposure time, the more the staining is affected. Stronger acid decalcifiers (e.g. nitric, sulfuric, hydrochloric) are generally used in a 5-10% solution. They decalcify quickly, but will impair staining if used for longer than 24-48 hours. This is most noticeable in nuclei, where chromatin will stain poorly with basic dyes such as hematoxylin. Conversely, acid dyes such as eosin may stain more strongly.
Of the following, the a cassette or label's ID number for the specimen should be marked with a/an: a) ink pen b) laundry marker c) lead pencil d) wax pencil
[C] Any labeling must be resistant to fixatives, dehydrants, clearing agents, and infiltrating media used during processing. A lead pencil (#2) or xylene-proof pen should be used. Any other labeling marker will be washed away.
Butanol is recommended as a dehydrant for which of the following? a) blood smears b) brain tissue c) plant tissue d) spleen
[C] Butyl alcohol is a slow dehydrant; recommended for tissues that need a long immersion time to dehydrate. Because it doesn't remove bound water, it won't cause shrinkage or hardening of tissue, even with prolonged exposure.
To avoid distortion of soft and delicate structures while still providing enough support during sectioning, which embedding medium is recommended? a) agar b) carbowax c) celloidin d) gelatin
[C] Celloidin requires no heat in any of its processing steps, so there is minimal shrinkage or distortion. Infiltration with celloidin is also performed in small, graded steps; usually 4%, 8%, and 12%. While agar, carbowax, and gelatin are aqueous media that avoid problems with dehydrants and heat, their softness will not provide enough support for thin sectioning.
Which of the following will speed up the rate of decalcification, but not cause harm to tissue? a) increasing the acid concentration b) decreasing the decalcification solution's temperature c) agitating the solution d) grossing a thicker specimen
[C] Decalcification rate varies with the type of acid used, and its pH and ionic strength. The decalcifying solution mustn't be be harmful to the specimen, become depleted, or change in pH by its reaction with the calcium in the specimen. During decalcification, the acid solution around the specimen becomes acid deplete. Through agitation, fresh, undepleted acid solution comes in contact with the tissue, promoting further decalcification. Increasing the percentage of acid in the solution will increase the decalcification rate, but will also harm the tissue. Decreasing its temperature will decrease the rate. A thinner tissue specimen will decalcify faster than a thicker one simply because there's less to react with.
Dehydration refers to the removal of: a) alcohol b) paraffin c) water d) xylene
[C] Dehydration refers to the removal of water.
One indication of the speed of removal of a clearing agent is its: a) color index b) concentration c) molecular size d) toxicity
[C] Diffusion of a fluid through a specimen is a function of its molecular weight/size, generally correlated with its boiling point. Clearing agents with low boiling points (smaller molecular size) will usually be removed from tissue at a faster rate when placed in molten wax than those with higher boiling points (greater molecular size). Concentration and toxicity don't influence a clearing agent's speed of removal. Color index pertains only to dyes.
Which of the following is miscible with water, alcohol, hydrocarbons, and paraffin? a) acetone b) cedarwood oil c) dioxane d) xylene
[C] Dioxane is a universal solvent, and is miscible with all of the solutions listed. Acetone is a dehydrant that isn't soluble in paraffin. Cedarwood oil and xylene are clearing agents, and aren't miscible with water.
Which of the following dehydrating agents must be used in a well-ventilated room due to its odor and cumulative toxic effect? a) acetone b) butanol c) dioxane d) isopropanol
[C] Dioxane is both a dehydrant and a clearing agent, and is also toxic. It's been indicated to cause liver and kidney damage, irritation of the skin, eye, nose and throat, as well as headaches and nausea. It should only be used inside a fume hood. Advantages of its use must be weighed against possible negative effects it can have on people in the lab.
In which of the following dehydrants can tissue remain safely for extended periods of time? a) acetone b) benzene c) ethylene glycol monoethyl ether d) ethanol
[C] Ethylene glycol monoethyl ether is sold under various trade names. It's an efficient dehydrant that needs no graded dilutions and causes very little hardening or distortion. Specimens may remain in it for months without injury. Its use in sequence with toluene as a clearing agent is often recommended for animal tissue. Acetone and ethanol are rapid dehydrants, and will harden tissue if left for too long. Benzene is a clearing agent.
Which of the following is likely the best all-around substitute for ethanol when processing tissues? a) acetone b) butanol c) isopropanol d) methanol
[C] Isopropanol is a good substitute for ethanol. It's neither destructive to tissue, nor dangerous to humans, and is readily available. It does take somewhat longer to dehydrate tissue compared to ethanol, however. Proprietary ethanol substitutes also exist. While acetone can provide rapid dehydration, it also requires a larger dehydrant-to-specimen ratio, and evaporates quickly. Butanol is a very slow dehydrant. Methanol is poisonous.
Which of the following clearants may require prolonged processing time? a) aliphatic hydrocarbons b) chloroform c) limonene d) toluene
[C] Limonenes are extracted from citrus rinds. This oily substance can be used as a xylene substitute, and is considered less toxic than aromatic hydrocarbons (xylene, toluene, benzene), aliphatic hydrocarbons (other xylene substitutes), and other clearants such as chloroform. It has, however, been implicated as a cause of contact dermatitis. Due to its oiliness, it can take up to twice as long to both clear tissue, and be replaced by paraffin impregnation.
Which decalcification method may cause burning of the specimen? a) acid b) chelating c) electrolysis d) ion-exchange
[C] The electrolysis decalcification method involves attaching the bone specimen to an anode, where an electrical current is passed from it to the cathode through an electrolyte decalcifying solution, migrating the calcium ions along with it. This method shortens the time needed for decalcification as heat is also generated by the current. The temperature of the solution must be kept between 30 - 45°C; an increase in temperature beyond this threshold will burn the specimen at its contact point with the anode, and possibly damage or destroy the rest of the tissue, as well. Decalcifying solutions should be changed every 8 hours.
Which of the following are universal solvents? a) benzene and butanol b) cellosolve and methanol c) dioxane and tetrahydrofuran d) ethanol and xylene
[C] The following are used as universal solvents: dioxane, tertiary butanol, and tetrahydrofuran. Universal solvents are dehydrants that are miscible with paraffin, and therefore don't need the additional step of clearing. None can be used with delicate tissue, as the diffusion currents will disrupt the tissue. All are odorous, and dioxane is a known carcinogen.
Which of the following are used only as clearants? a) acetone, methyl salicylate, toluene b) amyl acetate, methyl benzoate, tetrahydrofuran c) benzene, carbon tetrachloride, cedarwood oil d) chloroform, isopropanol, xylene
[C] The following are used solely as clearants: benzene, toluene, xylene, amyl acetate, carbon tetrachloride, essential oils (e.g. cedarwood) methyl benzoate, methyl salicylate, and propylene oxide. Other proprietary xylene substitutes are also included, such as aliphatic hydrocarbons and limonenes. All clearants remove dehydrants from tissue, and are eventually replaced by the infiltrating medium.
The time needed for paraffin infiltration is most dependent upon the: a) fixative used b) style of processor c) type of tissue d) number of cassettes
[C] The time needed for molten wax infiltration is dependent upon a specimen's thickness, tissue composition, clearing agent type, and assistance of a vacuum. When lacking a vacuum, paraffin infiltration should take place for only 2 - 4 hours. Use of a vacuum is recommended to shorten this time. Specimens left in molten paraffin for too long will harden and shrink, even at the correct temperature, and higher temperatures will produce the same effect. If infiltrated for too short a time, a specimen's center won't have the appropriate support matrix needed for proper sectioning. Though tissue must be fixed before processing, the fixative type doesn't influence the time needed for paraffin infiltration. Processor type may affect infiltration rate to some degree, but only on the basis of the above variables (i.e. duration, temperature, vacuum).
A tissue specimen inadvertently dried out prior to the paraffin step during processing, and is now very difficult to section. The tissue could be re-softened by placing it in a solution containing: a) decalcifier b) formalin c) glycerol d) phenol
[C] Tissue can dry out during processing if the cassettes are at some point left out of a solution, removing bound and unbound water. Further processing steps will make make the tissue even harder, as they'll continue to remove water. While not completely reversible, the tissue can be softened and slightly rehydrated by placing it in a solution of 70% ethanol and 30% glycerol for several hours or overnight before re-processing at the first alcohol step.
Which of the following infiltrating media requires tissue to be dehydrated? a) agar b) carbowax c) celloidin d) gelatin
[C] Tissues must be dehydrated when using celloidin, as nitrocellulose isn't miscible with water. The largest problem with celloidin is probably water contamination; even water vapor from the atmosphere must be kept from the solutions. Absolute alcohol, anhydrous ether, and air-tight containers should be used during processing. Tissue meant for celloidin embedding cannot be dehydrated with isopropanol, as the two chemicals aren't miscible. Agar, carbowax, and gelatin are all water-soluble infiltrating media, so tissue will not be dehydrated in these circumstances.
Which schedule should be selected to process a 1 cm thick section of fixed brain? a) 2 hrs in Alc. Formalin; 5 hrs in 95%-100% EtOH; 2 hrs in Xylene; 2 hrs in Paraffin ((11 hrs total)) b) 2½ hrs in graded conc. EtOH; 40 min in Toluene; 1 hr in Paraffin ((4 hrs total)) c) 20 hrs in graded conc. EtOH; 9 hrs in Xylene substitute; 6 hrs in Paraffin ((35 hrs total)) d) 2 hrs in Absolute EtOH; 2 hrs in Xylene; 3 hrs in Paraffin ((7 hrs total))
[C] Using this prolonged schedule would adequately process a large specimen. Additionally, brain is primarily composed of fatty material, requiring a longer time to thoroughly process. The tissue must be completely fixed before processing begins, as no fixation steps are included in this program.
The property that all fluids have of resisting change in shape is known as: a) refraction b) stress c) viscosity d) voltage
[C] Viscosity is a physical property caused by the friction of molecules in a solution as they slide past one another. The larger the molecules present, the larger the intermolecular forces occurs between them, making the solution more viscous and slower-moving. Fluids with small molecules will have weaker intermolecular forces, lessening their viscosity. Viscosity is measured in 'poises', or the more frequently used 'centipoises' (1/100 poise). The viscosity of all solutions, gases, and solids is compared to the viscosity of water, which is 1.002 centipoise @ 20°C. A thicker, more viscous substance flows slower than water and therefore has a higher viscosity value, while thinner, less viscous substances move faster than water, therefore having a lower viscosity value.
If the clearing agent is cloudy, it's probably contaminated with: a) absolute alcohol b) bacteria c) water d) yeast
[C] Water isn't miscible with clearing agents such as toluene and xylene. A clearing agent contaminated with water will appear cloudy, and should be changed. Absolute alcohol is miscible with clearing agents, and won't cause a cloudy appearance. However, if the clearing agent is mixed with graded alcohols (which contain water), the clearing agent may have a cloudy appearance. Bacteria and yeast do not proliferate in clearing agents.
After finishing decalcification, the specimen should be placed directly in: a) acetone b) alcohol c) fixative d) water
[D] Acids that aren't removed from tissue post-decalcification will continue to affect the specimen in the following processing and embedding steps. Before continuing with processing, decalcified tissue should be washed in running water for at least 30 minutes. The acid may also be neutralized by placing the specimen in a solution of lithium or sodium carbonate overnight, then washing with water. Failure to wash out the decalcifying or neutralizing solutions will also cause problems with staining.
To ensure adequate tissue processing while safely decreasing the time spent, one can: a) reuse reagents all week b) begin dehydration using stronger solutions c) skip clearing stages d) use agitation throughout all processing stages
[D] Agitation increases the flow of fresh reagents around and through tissue. To further increase processing speed, dehydration steps can begin earlier through the use of: 70 - 80% alcoholic formalin, vacuum and heat, faster-acting reagents, and thinner specimen samples.
Why should the alcohols in an automated tissue processor be changed on a regular basis? a) the alcohols become saturated with bile from gallbladder specimens, contaminating other tissues b) increased formalin concentrations in these alcohols can create a potentially explosive situation c) gram negative organisms can grow in overlong, unchanged alcohol d) the alcohols can absorb moisture and become dilute
[D] Alcohols absorb moisture from both tissues and the atmosphere, diminishing their dehydrating properties.
To prevent formation of a pigment during processing, fixatives containing which of the following must be thoroughly removed from a specimen prior to dehydration? a) alcohol b) mercuric chloride c) picric acid d) potassium dichromate
[D] All dichromate-based fixatives must be removed from a fixed specimen by washing with water before processing, as the dichromate can react with the dehydrating alcohols to form an insoluble green-brown pigment. Alcoholic fixatives can usually be started in a higher concentration. The precipitate from a mercuric chloride fixative is removed later on from already-cut sections with an iodine solution. The yellow of a picric acid fixative is also later removed from cut sections by washing the slide with either 50% alcohol, or a saturated solution of lithium carbonate in 70% alcohol.
The most rapid of the commonly available agents used to freeze fresh tissue is: a) aerosol sprays b) dry ice c) gaseous carbon dioxide d) liquid nitrogen/isopentane
[D] At -160°C, isopentane cooled with liquid nitrogen is usually used for enzyme histochemistry. It is a very rapid freezing agent—faster than liquid nitrogen alone. Liquid nitrogen on its own forms gas bubbles at the tissue's surface, which insulates the specimen. One disadvantage of freezing at -160°C is that the rapid expansion of ice within soft tissue can cause it to crack. The other methods listed will freeze tissue, but at a much slower rate, potentially creating freezing artifacts.
Which of the following clearing agents is preferred when processing brain sections? a) amyl acetate b) cedarwood oil c) dioxane d) toluene
[D] Brain tissue can be left in toluene overnight to allow the removal of lipids. Because there already is little bound water in brain tissue, it's less apt to harden than other tissues, although over-processing is still possible.
Which schedule should be selected to process tissue fixed in Carnoy solution? a) 2 hrs in Alc. Formalin; 5 hrs in 95%-100% EtOH; 2 hrs in Xylene; 2 hrs in Paraffin ((11 hrs total)) b) 2½ hrs in graded conc. EtOH; 40 min in Toluene; 1 hr in Paraffin ((4 hrs total)) c) 20 hrs in graded conc. EtOH; 9 hrs in Xylene substitute; 6 hrs in Paraffin ((35 hrs total)) d) 2 hrs in Absolute EtOH; 2 hrs in Xylene; 3 hrs in Paraffin ((7 hrs total))
[D] Carnoy solution contains absolute alcohol, acetic acid, and chloroform; no water is present. After complete prior fixation, processing can begin either at the 100% alcohol steps to help remove the acetic acid and chloroform, or at the clearing agent steps.
How long can tissues remain safely stored in cedarwood oil? a) minutes b) hours c) days d) months
[D] Cedarwood oil is the least harmful of known clearing agents. Since it penetrates slowly, tissue must remain in the oil for a long time to clear completely. Specimens may be stored in cedarwood oil for months with little evaporation. To ensure good sectioning, the oil must be removed from the tissue with extra changes of subsequent clearing agents (e.g. xylene) and molten waxes.
A bone marrow biopsy is decalcified, but the following H&E section reveals the loss of nucleic acids. To best prevent this in the future, first fix the tissue in: a) formalin b) glutaraldehyde c) osmium tetroxide d) Zenker solution
[D] Dichromate, mercuric chloride, zinc, and other heavy metal fixatives will help counteract the hydrolysis of nucleic acids caused by decalcifying agents, thus helping to preserve nuclear staining. Examples include Orth, Mueller, Zenker, Helly, B-5, and zinc-formalin. Formalin and glutaraldehyde are fair nuclear fixatives, but aren't able to withstand the hydrolysis caused by acid. Osmium tetroxide fixes lipids, not nuclei.
If ingested, which of the following solutions can be oxidized to formaldehyde in the body? a) acetone b) butanol c) ethanol d) methanol
[D] If methanol is absorbed through the skin, inhaled, or ingested, it is carried through the bloodstream to the liver, where it is oxidized to created formaldehyde and formic acid. As these highly toxic compounds accumulate, they're carried by the blood system to all parts of the body. Methanol poisoning is known to cause vision disturbances and blindness. Other symptoms include headache, drowsiness, nausea, vomiting, and disturbances of the digestive tract. Reagent alcohol, which has methanol added, can produce the same problems.
A fresh, fatty breast biopsy was received late in the day and placed in the processor, beginning immediately. During embedding, the tissue appears very yellow, mushy, and greasy. It's decided that the tissue needs to be re-processed. Which of the following solutions should it be placed in first? a) formalin b) 70% alcohol c) 100% alcohol d) xylene
[D] If tissue isn't thoroughly fixed or hasn't been in any of the processing solutions long enough, it won't process correctly, and will remain soft—particularly in the center. In this case, fatty tissue will still feel greasy. To correct this, the tissue needs to be sent back to either the beginning absolute alcohol step in the processing schedule, or all the way back to fixation. To do this, all paraffin in the tissue must be removed through several changes of a clearing agent such as xylene (30 - 60 mins each). The tissue then goes through absolute alcohol to remove the xylene, again with several changes, for a total reverse-treatment spanning several hours. If the problem is under-dehydration, these last alcohol changes will serve as a remedy, allowing the tissue to then go forward again through several prolonged clearant changes, followed by several prolonged paraffin changes, and re-embedding.
Which of the following decalcifying methods rejuvenates itself? a) acid b) calcium oxalate c) chelating d) ion-exchange
[D] In an ion-exchange method, calcified tissue can be treated in a mixture of formic acid and a commercially available ion-exchange resin where calcium is rapidly removed from the specimen without depleting said acid. In a mixture of formic acid and 10-20% resin, a 2-3mm thick bone specimen can be treated in 2-3 hours. As calcium is removed, calcium formate is produced, and then converted back formic acid as the resin absorbs the calcium ions, releasing hydrogen in its place. Until the resin is exhausted, the formic acid will remain at full strength. The resin can be reused through periodic regeneration with a strong acid. It's important to use the correct type of resin, as most replace calcium with sodium, preventing the formic acid from keeping its original concentration. Advantages of using this decalcification method include well-preserved cellular detail, the elimination of solution changes, and the resin's reusability. Determining a specimen's end point of decalcification must be performed via x-ray; chemical tests cannot be applied due to the resin's ammonium form of sulphonated polystyrene. In the acid and chelating decalcifying methods (which take anywhere between 12 hours to 8 weeks), the solution must be replaced several times to prevent reagent depletion. The calcium oxalate method is a chemical test for the end point of decalcification.
The process of saturating tissue with the same medium that will later be used for embedding is called: a) clearing b) dehydration c) fixation d) infiltration
[D] Infiltration/impregnation is the complete replacement of the clearing agent with a support medium. For complete tissue penetration, the infiltrating material must be liquid at a temperature low enough not to harm the tissue, while hard enough at room temperature for adequate sectioning. The infiltrating material should only cause minimum tissue distortion, and not dissolve tissue components.
The best overall method to demonstrate enzymes in tissue sections is: a) agar embedding b) celloidin embedding c) paraffin embedding d) unfixed frozen sectioning
[D] Many enzymes are inactivated by fixatives, processing solutions, and heat. Thus, many enzyme methods require the use of fresh frozen tissue sections. Frozen sections may be obtained in a manner that isn't time consuming or expensive.
Which of the following is soluble in various fat solvents? a) agar b) carbowax c) gelatin d) paraffin
[D] Paraffin is a mixture of solid hydrocarbons, and is soluble in aromatic hydrocarbons such as xylene, toluene, and benzene. It's also soluble in aliphatic hydrocarbons (i.e. xylene substitutes) and chlorinated hydrocarbons such as chloroform. All of these solutions also dissolve fat, and are therefore known as fat solvents. Paraffin is insoluble in water and alcohol. Agar, carbowax, and gelatin are all water soluble embedding media, and aren't soluble in fat solvents.
Which of the following infiltrating media is routinely used in electron microscopy? a) agar b) celloidin c) ester wax d) resin
[D] Routine histology embedding media are inadequate when it comes to ultra-thin sections and high-resolution microscopy. A resin embedding media provides enough support for the tissue to allow sectioning at 30 - 80 nm, and identification of certain nuclear/cytoplasmic elements via high-res microscopy.
A specimen was fixed in Carnoy solution. Dehydration should begin at which percentage of alcohol? a) 70% b) 80% c) 90% d) 100%
[D] Specimens fixed in alcoholic fixatives such as Carnoy or Clark solutions should be transferred to absolute alcohol, having already been partially dehydrated by the alcoholic fixative.
Which of the following is miscible with acetone, chloroform, and ether? a) agar b) Bouin's solution c) paraffin d) tetrahydrofuran
[D] Tetrahydrofuran (THF) is a universal solvent, and is therefore miscible with water and all dehydrants, clearing agents, and molten waxes. Agar is an aqueous embedding media, and won't dissolve in the listed solutions. Bouin solution, aqueous fixative, would be miscible with dehydrants such as acetone and ether, but not with clearants such as chloroform. Paraffin, while miscible with chloroform, isn't miscible with the dehydrants acetone or ether.
In an ion-exchange resin decalcifying system, how often should the acid solution be changed? a) twice per day b) once per day c) 1 - 2 times per week d) no changing is necessary
[D] The acid solution in an ion-exchange resin setup doesn't need to be changed, as the system allows it to regenerate on its own.
When placed in a solution whose refractive index is similar to that of tissue proteins, the tissue becomes: a) fragile b) hard c) small d) translucent
[D] The refractive index (RI) is the ratio of the velocity of light in a vacuum to its velocity in any substance of the same temperature. If two substances have the same RI, light rays will pass from one to the other in a straight line, rendering them indiscernible from each other. When RIs differ, the light's path will be altered, rendering an object visible. The RI of most clearing agents is similar to that of tissue proteins; the closer these two RIs are in similarity, the more translucent the tissue will become. Of the clearing agents available, chloroform's RI differs the most from that of tissue proteins, rendering tissue more opaque compared to other agents, thus preventing a change in opacity from determining its endpoint of clearing.
Which freezing method is preferred to demonstrate muscle enzymes? a) cryostat freezer plate b) dry ice c) liquid nitrogen d) pre-chilled isopentane
[D] The transverse section of muscle tissue should be mounted onto a chuck or piece of cork, then immersed in pre-chilled (-160°C) isopentane until frozen. Specimens freeze more quickly with this method due to the heat-conducting nature of isopentane. Faster freezing yields less diffusion and disruption caused by ice crystal artifacts. A tissue specimen small enough to freeze in under 30 seconds should be used to minimize the danger of it cracking. When freezing a specimen, liquid water just above the freezing point expands while frozen water below -20°C contracts. In larger pieces of tissue, the outer tissue contracts while the inner tissue is still expanding. Rapid freezing of smaller tissue aims to lessen this effect and avoid cracking the tissue. The other methods listed have a slower freezing rate, posing a greater risk of ice crystal artifacts.
Which schedule should be selected to process a gouty tophus? a) 2 hrs in Alc. Formalin; 5 hrs in 95%-100% EtOH; 2 hrs in Xylene; 2 hrs in Paraffin ((11 hrs total)) b) 2½ hrs in graded conc. EtOH; 40 min in Toluene; 1 hr in Paraffin ((4 hrs total)) c) 20 hrs in graded conc. EtOH; 9 hrs in Xylene substitute; 6 hrs in Paraffin ((35 hrs total)) d) 2 hrs in Absolute EtOH; 2 hrs in Xylene; 3 hrs in Paraffin ((7 hrs total))
[D] Tissue containing gout must be fixed in 100% alcohol, as its uric crystals dissolve in water. Schedule D is the only one available with no presence of water. After the tissue has been completely fixed in alcohol prior to processing, the processing cycle could either begin at the initial alcoholic steps, or be moved directly to the clearing stages.
When using epoxy resins during processing, which of the following is usually the transitional fluid between alcohol and the epoxy resin? a) acetone b) chloroform c) dioxane d) propylene oxide
[D] Tissue to be infiltrated with epoxy resin has usually already been dehydrated with ethanol, which isn't miscible with most resins. Bound water molecules also still reside within the tissue. Resins will correctly polymerize only with the complete absence of water. To remedy this problem, a transitional fluid such as propylene oxide is used after the dehydration step to remove any remaining excess water, helping the epoxy resin to properly penetrate the tissue. Since it may cause contact dermatitis, propylene oxide should be worked with while wearing protective gloves.
Penetration of a solution into tissue is most dependent on which of the solution's characteristics? a) evaporation b) sublimation c) toxicity d) viscosity
[D] Viscosity is a measure of how thick a solution is. The larger the solution's molecular size, the higher its viscosity (thicker), and the slower the rate of penetration. Smaller molecular size lends to lower viscosity (thinner) and faster penetration rate. Heat reduces a solution's viscosity, and is why it's often used in all processing stations. Evaporation and toxicity are also somewhat related to molecular size. In general, smaller molecular size lends to faster evaporation and a higher likelihood of toxicity. These two factors, however, don't directly determine a solution's penetration rate into tissue. Sublimation is the ability of a substance to change from a solid state to a gaseous state, skipping the liquid phase.
How much 95% alcohol is needed to prepare 1000 mL of 70% alcohol? a) 136 mL b) 316 mL c) 665 mL d) 737 mL
[D] V₁C₁ = V₂C₂ 95x = (1000)(70) 95x = 70000 x = 736.84 ≈ 737 mL
