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78 Cards in this Set
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Which of the following groups is available as natural dyes? a. aniline blue, auramine O, brazilin b. carmine, Congo red, ciystal violet c. eosin, indigo, and phloxine d. hematoxylin, orcein, saffron |
THE CORRECT ANSWER IS "D." Originally, dyes were of animal or vegetable origin, but mostly have been replaced in histology by synthetic dyes. Hematoxylin, saffron, carmine and brazilin are dyes known only from natural sources. Hematoxylin is derived from the logwood tree from the West Indies, saffron is from the pistils of a flower, carmine is made from the female cochineal insect, and brazilin is also made from a tree, though little used any more in histology. Orcein (lichen) and indigo (legumes and other plants) can be found as natural or synthetic dyes. Aniline blue, auramine O, Congo red, crystal violet, eosin, and phloxine are examples of synthetic dyes. 1,3,11,16,19 |
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Mordants are generally classified as a/an: a. acid b. gas c. metal d. oxidizer |
THE CORRECT ANSWER IS "C." A mordant is a salt or hydroxide, usually of a divalent (+2) or trivalent (+3) metal. It serves to give the dye a positive charge, thus enabling the dye to attach to a negative component of the tissue. Examples of mordants used with hematoxylin dye are ferric chloride, aluminum ammonium sulfate, and phosphotungstic acid. An acid lowers the pH of a solution. A gas is a state of matter where molecules are widely scattered and free to move about. An oxidizer is a chemical that attaches oxygen to chemicals, or removes hydrogen from chemicals. 1,3,16,19 |
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Which of the following is a method that differentially demonstrates DNA as green and RNA as red? a. Feulgen reaction b. Gomori trichrome c. methyl green-pyronin d. PAS-light green |
THE CORRECT ANSWER IS "C." The methyl green-pyronin Y (MGP) procedure is used to distinguish DNA from RNA. The two dyes are combined in one staining solution. Methyl green will stain the DNA green, while pyronin will stain the RNA red. The MGP stain will also stain cytoplasmic RNA a red color, such as the ribosomal RNA found in plasma cells. Pyronin Y will also stain DNA, but the red color is usually not seen, due to the green color of the methyl green. The Feulgen reaction will stain DNA red, but will not stain RNA. Gomori trichrome is used to differentiate muscle (red) from collagen (green). PAS-light green will stain fungi magenta (and any other PAS positive substances) and the background green. 1, 3, 10, 19 |
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Which of the following chemicals functions as an oxidizer in some hematoxylin solutions? a. acetic acid b. aluminum ammonium sulfate c. ammonium hydroxide d. potassium permanganate |
THE CORRECT ANSWER IS "D." An oxidizer is a chemical that attaches oxygen to chemicals or removes hydrogen atoms from chemicals. Well known oxidizing agents used in the hematoxylin solutions include potassium permanganate, mercuric oxide and sodium iodate. Aluminum ammonium sulfate is a mordant. Acetic acid reduces the pH to increase selectivity of the hematoxylin for nuclei. Ammonium hydroxide can be used to "blue" the hematoxylin after staining the tissue sections in the hematoxylin. 1, 3, 6, |
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Differential staining of nuclei and cytoplasm with Giemsa staining is an example of: a. apochromasia b. hyperchromasia c. metachromasia d. polychromasia |
THE CORRECT ANSWER IS "D." Giemsa solution is a combination of blue and pink dyes. The blue thiazine dyes include methylene blue, which can be further oxidized into other dyes that are other shades of blue. The pink xanthene dyes include eosin and phloxine, either or both of which may be in the Giemsa dye. Different components of the tissue will be stained by the various blue and pink dyes, resulting in a range of shades of blues and pinks. Giemsa is therefore known as a polychromatic dye. Apochromasia is a term used in microscopy which denotes lenses that are free from chromatic aberration. Hyperchromasia refers to increased nuclear staining that is frequently seen in neoplastic cells. Metachromasia involves using a pure dye which will absorb light at different wavelengths, depending upon the dye's concentration and the type of tissue component it is bound to. 1, 3, 10, |
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The pathologist has requested a Feulgen procedure on a lymph node. The staining sequence should be: a. hydrochloric acid, sulfuric acid rinses, Schiff, light green b. hydrochloric acid, Schiff, sulfurous acid, light green c. periodic acid, Schiff, water rinses, light green d. Schiff, hydrochloric acid, light green |
THE CORRECT ANSWER IS "B." DNA contains a sugar known as deoxyribose. The Feulgen reaction depends upon the hydrolysis of the DNA sugar with 1 N (normal) hydrochloric acid, to release aldehyde groups. The 1 N hydrochloric acid is strong enough to hydrolyze the DNA sugar (deoxyribose) so that the newly released aldehyde WILL bind with Schiff reagent. The 1 N hydrochloric acid will also overhydrolyze any background sugars, so that the background sugars will NOT be subsequently stained with the Schiff. After staining with the Schiff, either sulfurous acid rinses or running tap water are used to remove excess Schiff reagent and change the clear Schiff to a magenta color. Light green is an optional counterstain. If periodic acid were used instead of the 1 N hydrochloric acid, the DNA sugar would NOT be hydrolyzed, so NO nuclear staining would be seen. Also, background sugars would have been hydrolyzed to aldehydes, so the background WOULD stain magenta with the Schiff. 1, 3, 10, 19 |
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Which of the following dyes is a known human carcinogen? a. cochineal b. hematoxylin c. orcein d. pararosaniline |
THE CORRECT ANSWER IS "D." Pararosaniline HC1 is a synthetic dye, also known as basic fuchsin, is often used to make the Schiff reagent and other histology red staining solutions. Basic fuchsin, other alternative basic fuchsins, and several other synthetic dyes also made with benzidine or a benzidine derivative, have been declared by OSHA (Occupational Safety and Health Administration) to be known human carcinogens. Since there are other synthetic dyes also made from benzidine or its derivative, all dyes should be handled in a safe manner, such as with gloves at all times. Wearing a mask during the handling of the dry powder has also been recommended. Hematoxylin and cochineal (used to make the mucicarmine stain) are natural dyes. Orcein can be purchased either as a natural dye (made from lichen) or as a synthetic dye. 1, 3 |
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Picric acid, eosin, and tartrazine are examples of which type of dyes? a. basic b. cytoplasmic c. fluorescent d. natural |
THE CORRECT ANSWER IS "B." Cytoplasmic dyes are most often acid/anionic synthetic dyes that have a negative charge. They combine with positively charged basic/cationic components of the cytoplasm. Another name for cytoplasmic dyes is plasma dyes. Basic dyes are cationic/positively charged dyes. They combine with the acidic/anionic/negatively charged components of tissue. Examples of basic/cationic dyes are hematoxylin and methylene blue, which stain nucleic acids. Examples of fluorescent dyes include rhodamine, acridine orange, and fluorescein isothiocyanate (FITC). Examples of natural dyes include hematoxylin, carmine, and indigo. 1, 3, 10, 11, 14, 19 |
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A compound containing a benzene ring with other atomic groupings associated with color is called a/an: a. auxochrome b. chromogen c. chromophore d. dye |
THE CORRECT ANSWER IS "B." A chromogen is a benzene compound containing chromophore radicals, such as C=C, C=O, C=S, C=N, N=N, N=O, and NO2. Chromophores are the atomic groupings associated with the color of the dye. In general, the more chromophores there are in a chromagen, the more pronounced the color. An auxochrome gives the dye a charge, and thus an affinity to attach itself to charges on the tissue. 1, 3, 10, 11, 19 |
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Which of the following is the mordant salt used to make the hematoxylin solutions most commonly used in the routine H&E stain? a. aluminum b. copper c. iron d. tungsten |
THE CORRECT ANSWER IS "A." Aluminum-mordanted hematoxylins, such as Mayer or Gill hematoxylin, are used for routine nuclear staining. Aluminum ammonium sulfate or aluminum potassium sulfate are the salts usually used. Tungsten mordant is used in the PTAH (phosphotungstic acid-hematoxylin) stain, which demonstrates muscle striations and glial cells. Iron, or ferric, mordanted hematoxylins, such as Weigert, are used in special staining procedures. Copper mordanted hematoxylin can be used to demonstrate myelin. 1, 3, 6, 7, 10, 15, 16, 19 |
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A hematoxylin solution that is commonly used progressively and very rarely used regressively is: a. Delafield b. Ehrlich c. Harris d. Mayer |
THE CORRECT ANSWER IS "D." Mayer hematoxylin can be used progressively, requiring no differentiator. The tissue section is kept in the hematoxylin solution until the desired amount of staining is achieved. Delafield, Ehrlich and Harris hematoxylins are usually used regressively with a differentiator. The tissue section is overstained, and then the excess background hematoxylin is removed by dipping the slide briefly in a weak acid solution. Mayer hematoxylin may also be used regressively. |
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In the Feulgen reaction for nucleic acids, slides are placed in a 1 N (normal) solution of hydrochloric acid to promote: a. alkalinity b. autolysis c. hydrolysis d. oxidation |
THE CORRECT ANSWER IS "C." In this step, the purine and pyrimidine bases in the DNA of the nucleus are split off from the sugar-phosphate groups of the DNA. The exposed deoxyribose sugar is converted to an aldehyde. The uncovered aldehyde groups form a stable colored compound with the Schiff reagent. Hydrochloric acid is acidic, not alkaline, in pH. Autolysis occurs when the tissue is not placed immediately into a fixative upon removal from the body. Oxidation is the addition of oxygen or the removal of hydrogen |
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An amphoteric substance is one that can act as a/an: a. acid or base b. acid or oxidizer c. base or salt d. oxidizer or salt |
THE CORRECT ANSWER IS "A." An amphoteric dye is one that can act as an acid or as a base, depending upon the pH of the staining solution. An acid is a solution that has a pH less than 7, while a base is a solution that has a pH greater than 7. An oxidizer is a chemical that adds oxygen and/or removes hydrogen. A salt is a compound made of a positively charged metal and the negatively charged portion of an acid |
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A stain using a silver salt that requires a developer is known as which of the following reaction types? a. argentaffm b. arginine c. argyria d. argyrophil |
THE CORRECT ANSWER IS "D." Stains using silver nitrate deposit the silver salt onto the component being stained. If the component can naturally reduce the silver ion (Ag+2) to black metallic silver (Ag°), it is known as an argentaffm, or argentaffinic, reaction. If the addition of a developer solution is required to reduce the silver ion to black metallic silver, it is known as an argyrophil, or argyrophilic, reaction. |
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A methyl green-pyronin procedure is done on a formalin-fixed section of tissue. There is no evidence of RNA. More desirable results would have been realized if the tissue had been fixed in: a. Bouin solution b. Carnoy fluid c. Zinc-formalin d. Zamboni PAF |
THE CORRECT ANSWER IS "B." For best results, RNA should be preserved with Carnoy fluid if the methyl green-pyronin procedure is to be done. If tissue has been fixed in formalin, a positive result may be realized after soaking the sections for one hour in absolute methyl alcohol. |
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Weigert iron hematoxylin becomes too weak to use after one to three: a. days b. weeks c. months d. years |
THE CORRECT ANSWER IS "A." Weigert hematoxylin contains ferric chloride, which acts as a mordant and an oxidizer. When combined with hematoxylin dye, it makes a black dye that is ready for use immediately. However, the Weigert hematoxylin solution will continue to oxidize due to the ferric chloride, as well as air and light, and will become overoxidized rapidly. Within a few hours to days, the Weigert hematoxylin will no longer stain the nuclei black. 1, 3, 10, 19 |
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Malignant tumors of connective tissue are known as: a. carcinoma b. fibroma c. lipoma d. sarcoma |
THE CORRECT ANSWER IS "D." Sarcomas are malignant tumors of connective tissues. A fibrosarcoma is a malignant tumor of fibrous elements (connective tissue), as opposed to a fibroma, which is a benign tumor of connective tissue. In the same light, a liposarcoma is a malignant tumor of adipocytes (fat cells), while a lipoma is a benign tumor of the fat cells. Carcinoma is a malignant cancer of epithelial cells. An adenocarcinoma is a malignant epithelial tumor with glandular elements, while an adenoma is a benign epithelial tumor composed of glandular tissue. 17, 20 |
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In humans, nonkeratizing stratified squamous epithelium would be found lining: a. esophagus b. skin c. trachea d. urinary bladder |
THE CORRECT ANSWER IS "A." Squamous epithelium is a flat, scaly epithelium. Stratified squamous epithelium is multiple layers of squamous epithelium. Stratified epithelium is found covering areas of tissues/organs where epithelial cells are easily removed due to materials rubbing across the area. In humans, the esophagus is covered by non-keratinized squamous epithelium. Skin is covered with keratinized squamous epithelium. The keratin is a strong fibrous protein that gives structural and protective properties to epidermis, hair, nails, and horny tissues. The esophagus in some animals, such as rat, has keratinized stratified squamous epithelium, which is why the rat can eat rougher texture food than humans. Trachea is lined with tall, pseudostratified, ciliated epithelium and goblet cells. Urinary bladder is lined with transitional epithelium. The bladder appears to have a single layer of cuboidal epithelium when the bladder is extended with urine. However, when the bladder is void of urine, the cuboidal epithelial cells appear to pile on top of each other, to look like a stratified cuboidal epithelium. Other types of epithelium include: simple squamous, as found in the lining of lungs and blood capillaries; columnar, such as found in the small and large intestine; cuboidal, such as found in the tubules of the kidney and the collecting ducts of the salivary gland and pancreas. |
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Ciliated epithelium can be found in the: a. gastrointestinal tract b. male reproductive tract c. renal tubules d. respiratory system |
THE CORRECT ANSWER IS "D." The surfaces of some epithelial cells may be specialized, and contain cilia, microvilli or stereocilia. Cilia are specialized epithelial surface structures involved in movement. They are usually found in areas where mucous or fluid must be rhythmically moved over the epithelial surface in a consistent direction. In the airways, mucus from the goblet cells traps debris from the air. The cilia move the mucus upwards towards the throat, where it is swallowed, thus keeping the lungs clean. In the oviduct, cilia help to move the ovum from the ovary to the uterus. The epithelium in small intestine and proximal renal tubules contains microvilli. These specialized projections are involved in absorption. They are often referred to as the brush border. Stereocilia are extremely long microvilli, found in some areas of the male reproductive tract, such as the epididymis. Their exact function is not known. 2, 18, 20 |
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Which of the following muscle types is characterized by striations, nuclei in the center of the muscle, and intercalated discs? a. cardiac b. involuntary c. skeletal d. visceral |
THE CORRECT ANSWER IS "A." Muscle cells specialize in movement. Cardiac muscle cells are the muscles in the heart. Cardiac muscle cells are long, cylindrical, contain one or two centrally located nuclei, and are striated in appearance. The ends of cardiac muscles split into several branches, which join up with other branches, creating a junction known as an intercalated discs. Skeletal muscle is also known as striated muscle or the voluntary muscle. Skeletal muscle cells are long, unbranched, cylindrical, have a striated appearance, and have numerous nuclei located on the periphery of the muscle fibers. Skeletal muscles are involved in the movement of the skeleton and are under voluntary control. Visceral muscle is known as involuntary muscle or smooth muscle. Smooth muscle cells are short, spindle-shaped with pointed ends, without any visible striations, and have a single nucleus in the center. Smooth muscles are involved in continuous, low force contractions that are not under voluntary control, such as that found in the GI tract. Cardiac muscles are the heart muscles |
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The staining mechanism in which metallic substances are selectively deposited over structures and made visible by reduction of the metal, is called: a. adjective b. impregnation c. progressive d. substantive |
THE CORRECT ANSWER IS "B." Impregnation involves attaching a particulate metal salt, such as silver nitrate, onto or around a tissue structure. This metallic salt is then made black by one of several methods. Impregnation differs from traditional histology staining, in that "staining" involves the penetration of a dye into a tissue component. An adjective stain involves a substance, such as a mordant, that attaches a dye to the tissue, eg. hematoxylin and aluminum salts. In progressive staining, tissue remains in the dye solution only until the desired color is achieved, eg. alcian blue. A substantive stain is one that acts directly on the tissue without the intervention of another substance, such as eosin staining. |
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To achieve three shades of eosin in an H&E stained piece of tissue, the pH of the eosin solution should be: a. 3 b. 5 c. 7 d. 9 |
THE CORRECT ANSWER IS "B." In a good H&E stain, there should be at least three shades of eosin: red blood cells and eosinophils should be the darkest, muscle a medium shade, and connective tissue should be the lightest shade of eosin. To achieve this, the pH of the eosin solution must be lowered to around a pH of 4 to 5, usually using dilute acetic acid. An eosin solution NOT in this range will reveal one or two shades of eosin, plus the intensity of the color may also be affected. |
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Nuclei stained with a new Harris hematoxylin solution are predominately red and imprecisely outlined. Of the following, the most probable cause is that the: a. slides were not differentiated long enough after staining in the hematoxylin b. slides were placed in an alkaline solution too long after being stained in the hematoxylin c. hematoxylin had not ripened long enough d. hematoxylin contained no acetic acid |
THE CORRECT ANSWER IS "C." Microscopically, in a good H&E stain, the nuclei should be blue, have white unstained areas in the nuclei, and have distinct borders around the nuclei. The cytoplasm of most cells should not be stained blue. The dye hematoxylin needs to be oxidized from a red to a blue color, creating a new compound called hernatein. This oxidation can take place either with chemicals, such as sodium iodate, or by natural means, such as air and light. Insufficiently oxidized, or "ripened", hematoxylin will appear red, and will stain the nuclei red. If the hematoxylin is old, and therefore over-ripened, the hematein will have become oxidized (ripened) to a colorless compound, and the nuclei may also appear red. If a drop of properly oxidized hematoxylin is placed on a filter paper, a maroon center will appear, with purple around the edges. If a hematoxylin stained section was not placed in the differentiator long enough, the cytoplasm of the cells would appear blue after "bluing", as would the white space in the nucleoplasm. Extending the time in the alkaline solution, i.e., bluing agent, will not change the color of the hematoxylin stained nuclei. However, the tissue may fall off the slides, due to the alkaline nature of the bluing agent. If the hematoxylin solution did not contain acetic acid, the pH would be too high. This would cause other components in the tissue, such as goblet cells and white space in the nucleoplasm, to pick up the blue color of the hematoxylin. |
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In metachromatic staining, tissue components are stained a different color from that of the dye solution. Which of the following methods is an example of metachromatic staining? a. Congo red b. Giemsa solution c. Schiff reagent d. toluidine blue |
THE CORRECT ANSWER IS "D." Metachromatic dyes are pure dyes. Due to the nature of the dye and the components in the tissue, the background of the tissue will stain one color (orthochromatic), while the specific structure being stained is a different color (metachromatic). When the dye molecule is in a large concentration in a structure, such as with toluidine blue in cartilage, the dye molecules are close to each other. Since they are so close to each other, their molecules can interact and bind with each other. This will cause a change in the spacing between the dye molecules. This new spacing will cause a change in the absorption of light, creating a different color. Of the above dyes listed, only toluidine blue is a pure dye. Depending upon the formulation of the stain procedure, toluidine blue can be used to metachromatically stain amyloid, cartilage, or mast cell granules. Though amyloid will stain metachromatically with toluidine blue, Congo red is NOT a metachromatic dye. Schiff reagent is a leuco dye, while Giemsa is a polychromatic dye. |
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Hematoxylins that are useful for demonstrating muscle striations and myelin, as well as nuclei, are those that have as their mordant: a. aluminum b. lead c. molybdenum d. tungsten |
THE CORRECT ANSWER IS "D." Tungsten hematoxylins, of various formulations, can be used to demonstrate muscle striations, myelin, and nuclei, in addition to glial cells. Aluminum hematoxylins are used in the tradition H&E stain to demonstrate nuclei (chromatin). They usually do not demonstrate the other structures. Lead hematoxylin can be used to demonstrate the granules in the endocrine tract. Molybdenum hematoxylins have been used to demonstrate collagen, coarse reticulin, and argentaffin cell granules. Two other hematoxylin mordants are iron or chrome salts. Iron hematoxylins have been used in the past, depending upon their formulation, to demonstrate nuclear chromatin, amoebae, myelin, elastin or muscle striations. Chrome hematoxylin has been used to demonstrate myelin. 1, 3, 10 |
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To achieve clarity and good detail with a staining procedure, it is important that the counterstain be: a. acidic b. dilute c. heated d. unfiltered |
THE CORRECT ANSWER IS "B." A dilute counterstain should be used, to avoid masking the primary staining reaction. Another alternative is to use an undiluted counterstain, but greatly reduce the time. However, using a dilute counterstain allows for greater control of the staining procedure. Counterstains may be acidic or basic, depending upon the dye and what tissue component is being stained. Some counterstains do require heating, to facilitate dissolving into solution. However, for some dyes, heating would cause degradation of the dye. Many dye solutions need to be filtered before using, to remove precipitated dyes. |
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If NO chemical oxidizer has been added to Delafield hematoxylin, the solution can be aged with: a. alcohol and acetic acid b. heat and storage in the dark c. light and air d. ferric chloride and citric acid |
THE CORRECT ANSWER IS "C." The "dye" hematoxylin must be oxidized to hematein, which is a weakly anionic (negatively charged) dye. Reagents such as sodium iodate, mercuric oxide or potassium permanganate may be used to chemically oxidize the hematoxylin dye. Chemically oxidized (also known as aged or ripened) hematoxylin can be used almost immediately after being made. Hematoxylin may also be naturally ripened, using heat, light or air. Natural ripening may take several months before enough hematoxylin dye has been oxidized to hematein, so that the hematoxylin solution can be used. Whether the hematoxylin has been chemically or naturally oxidized, the hematoxylin will continue to be oxidized by air, heat and light. Overoxidized hematoxylin converts to oxyhematein, and will stain very poorly, or not at all. To slow down the continuing oxidation, the ripened hematoxylin is stored in dark or translucent bottles at room temperature. Acetic acid and citric acid can be used to adjust the pH. Alcohol is a solvent, along with water, that can be used to dissolve the hematoxylin dye. Ferric chloride is a mordant. Sometimes, the mordant can also serve as an oxidizer, such as in the iron hematoxylins |
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After H&E staining, the cytoplasm is dark pink and contrasts poorly with the nuclei. The most likely cause is that the: a. dye eosin B was used instead of eosin Y b. rack of slides was overdeparaffinized in the first xylenes c. slides did not spend enough time in alcohol after the eosin d. tissues were fixed in neutral buffered formalin |
THE CORRECT ANSWER IS "C." The primary purpose of the graded alcohols following eosin is that of differentiating out, or removing, excess eosin. Correctly stained and differentiated sections will demonstrate three shades of eosin: red blood cells and eosinophils the darkest, muscle next darkest, and collagen the lightest. Other causes of overstaining include: sections stained too long in eosin; sections underdifferentiated through the graded alcohols; thick sections; and the pH of the eosin being below 5.0. Eosin B and Eosin Y are both pink cytoplasmic dyes, with variations in grades of pink. Eosin Y is a more yellow-pink, while Eosin B is a more red-pink. Regardless of which is used, there should still be three shades of pink in the tissues. Leaving the slides too long in the deparaffinization step will not harm the tissue; too short of a time will leave paraffin on the section, so staining will be impaired. If the tissue is well fixed, regardless of the type of fixation, multiple shades of eosin should result. Depending upon the type of fixative, the overall color of eosin may be lighter or darker, but there will still be variations of shades. Failure to fix completely may result in poorly preserved components, which would interfere with staining. |
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Which of the following is an acid commonly used to differentiate excess Gill hematoxylin from tissue? a. ammonia b. hydrochloric c. periodic d. picric |
THE CORRECT ANSWER IS "B." Differentiation is a process used to selectively remove excess dye from tissue components. A solution of hydrochloric acid in 70% alcohol maybe used, or a solution of hydrochloric acid in water. The aqueous hydrochloric acid will differentiate out the hematoxylin faster than the alcoholic hydrochloric acid. Therefore, care must be taken when using the aqueous acid, as it may overdifferentiate the tissue. In comparison, the aqueous acid is usually more dilute than the alcoholic acid, or the time in the aqueous acid is decreased. Acetic acid could be used. However, it is much weaker than hydrochloric acid. Therefore, the time must be prolonged in the dilute acetic acid solution, or the percentage of acetic acid must be increased as compared to the percentage of hydrochloric acid. Ammonia is a base, not an acid. Periodic acid is used to create aldehyde groups during the PAS staining procedure. Picric acid is used to make fixatives or staining solutions |
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Fixation with which of the following will cause IMPAIRED nuclear staining in sections of average thickness? a. alcoholic formalin over the weekend b. buffered formalin for one month c. Bouin solution overnight d. Zenker solution for two week |
THE CORRECT ANSWER IS "D." Prolonged fixation in Zenker solution, or in most mercuric fixatives, can impair nuclear staining. The recommended fixation time is 6-8 hours for sections of average thickness, or 2-3 hours for biopsies. Tissues fixed hi alcoholic formalin or neutral buffered formalin can remain in the fixatives indefinitely, provided the fixative to tissue ratio is adequate, and the fixative solutions do not evaporate. Overnight fixation in a picric acid solution is recommended for average size tissues, while biopsies should be fixed no more than 4 hours. Prolonged fixation in a picric acid fixative could also cause impaired nuclear staining. |
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Which of the following will stain nuclei blue, but no other tissue component? a. alcian blue b. aniline blue c. celestine blue d. Nile blue A |
THE CORRECT ANSWER IS "C." Celestine blue, when mordanted with an iron salt, will stain nuclei. It is often recommended as an alternate for hematoxylin in a routine H&E stain when hematoxylin is unavailable, or when a double nuclear stain is required. Alcian blue is used to demonstrate acid mucopolysaccharides. Aniline blue is a cytoplasmic stain, usually used in trichrome stains. Nile blue A is a dye used to demonstrate lipids in frozen sections of tissues. 1, 3, 19 |
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Following hematoxylin and eosin staining, slides are dehydrated through ascending strengths of alcohol and cleared in xylene. After several racks of slides, the first xylene in the clearing series is turning milky-white. The most logical action is to: a. air dry the slides, then start them in absolute alcohol b. change only the xylenes at the end of the series c. change only the absolute alcohols and xylenes after the eosin d. change all dehydrating and clearing solutions in the staining series |
THE CORRECT ANSWER IS "C." After staining in eosin, the slides are placed in a diluted alcohol, usually somewhere between 70 to 95% alcohol. The slides are then placed in successively stronger percentages of alcohol, until they are in 100% alcohol. This action removes all the water from the slides. The slides are then placed into xylene, or a xylene substitute. If the slides are not drained well between solutions, there may be a lot of water carried over into successive solutions. A milky-white xylene is an indication that water has been introduced into it. To prevent this, drain the racks well between each solution. Also, running many racks of slides through the same solutions will also eventually cause excessive water carry over. A good indication that there may be water contaminating the absolute alcohols is the presence of eosin in them. When the absolute alcohols begin to turn pink due to the presence of eosin, properly dispose of the alcohol, and use fresh. |
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Microscopic evaluation of an H&E section reveals dark blue-black precipitate irregularly dispersed over the tissue. This could most likely be corrected in the future if the hematoxylin solution was: a. adjusted to pH 4.0 b. filtered before use c. mordanted with ferric chloride d. ripened longer |
THE CORRECT ANSWER IS "B." On standing, traditional aluminum hematoxylin solutions may develop a sheen of oxidized dye on top of the solution. The aluminum-hematoxylin component in the solution, upon further exposure to air, is converted to aluminum-hematein. If the concentration of the aluminum-hematein surpasses its solubility limits in water, the excess aluminum-hematein will appear as scum. This sheen must be removed by filtering before using, to prevent a blue-black precipitate on the sections. To prevent the formation of the scum, manufacturers have either: diluted the hematoxylin solution, which increases the amount of water so that the aluminum-hematein solubility limit will not be reached; decreased the amount of hematoxylin, which reduces the amount of aluminum-hematein so that the aluminum-hematein solubility limit would not be reached; or add a chemical such as ethylene glycol to the hematoxylin solution, which will hold the aluminum-hematein compound in solution. Aging, or ripening, converts the hematoxylin dye to hematein. If the hematoxylin solution had not been aged long enough, the staining of the tissue would have been light but no precipitate would have formed. Routine hematoxylins are mordanted with aluminum salts, not ferric chloride. The pH of hematoxylin should be in the range of around 2.4. 1, 3, 5, |
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A method sometimes used to increase the rate of staining is to: a. decrease the concentration of the dye solution b. differentiate the stain using an alkaline solution c. increase the temperature of the dye solution d. use an acid to raise the pH of the dye solution |
THE CORRECT ANSWER IS "C." In order for a dye molecule to bind to a tissue component, the dye must be in close proximity to the tissue on the slide. Once the dye molecules that are next to the tissue bind to the tissue, no other staining could occur unless dye molecules that are far away from the tissue are moved closer to the tissue. All molecules oscillate/jiggle, thus slightly moving on their own through a solution. This is known as Brownian movement. The warmer the solution, the more the molecules oscillate, and the more likely they are to move into proximity with the tissue. Conversely, cooling a solution will slow down the Brownian movement, thus slowing the movement of the dye molecules, and thereby slowing down the staining rate. In addition, heating the tissue may also cause the tissue components to swell. This may create "windows" through which dye molecules can more easily penetrate into the tissue. Another method that would increase the rate of staining would be to stir the solution or jiggle the slides. This would physically move the molecules around, again increasing the chances of the molecules coming into contact with the tissue. A third method would be to increase the concentration of the dye molecules in the staining solution. By increasing the number of dye molecules, the odds of a dye molecule coming into proximity with the tissue are also increased, which speeds up staining. Decreasing the concentration of a dye solution will slow down the rate of staining. A differentiator removes stain from a tissue section. The pH that is required for a staining solution is dependent upon the type of component being stained, the isoelectric point of the component, and the types of charges in the dye molecule. The rate of staining maybe increased or decreased by changing the pH of the staining solution. However, adding an acid will lower the pH, not raise it |
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Microscopic examination of an H&E stained section reveals a pink stained artifact surrounding the tissue and in the tissue spaces. The most probable cause is excess: a. acetic acid in the eosin b. gelatin in the flotation bath c. phloxine dye in the eosin solution d. time in the alcohol after the counterstain |
THE CORRECT ANSWER IS "B." Egg albumin and gelatin are proteinaceous materials and will take up acid/anionic dyes such as eosin and phloxine. This problem is compounded by heating the sections, which causes coagulation of these adhesives and increases their affinity for these acid/anionic dyes. Excessive acetic acid in eosin would drop the pH of the eosin, causing a loss of three tones of eosin. Phloxine can be added to an eosin solution, resulting in red blood cells that are stained slightly darker pink than if eosin had been used alone. Increased time in the dehydrating alcohols would decrease the eosin staining |
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The type of staining in which tissue binds the dye and is completely penetrated by the dye is called: a. absorption b. adsorption c. impregnation d. vital |
THE CORRECT ANSWER IS "B." In adsorption staining, dye molecules bind to components in the tissue based on the charge of the dye and the charge of the tissue component. Most histology stains are adsorption stains. In absorption staining, a dye molecule is placed in a solvent into which it does NOT dissolve very well. The dye molecule will then readily move into a solvent in which it is more soluble. Lipid stains such as oil red O and Sudan black B are examples of absorption staining because the dye moves from the alcohols or propylene glycol solvents into the lipid in which it is more soluble. Impregnation staining involves heavy metals, such as silver nitrate, that bind to the surface of the component, rather than penetrating the tissue component. Vital staining refers to staining of living cells, where the uptake of the dye is due to osmosis. |
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When histotechs speak of "acid dyes," they are generally referring to the: a. hydrogen-ion concentration of the dye solution b. commercial products that are actually acids c. salts of a colored dye d. acetic acid added to staining solutions |
THE CORRECT ANSWER IS "C." Acid dyes are the salts of colored acids, using the term "acid" in the non-modern-chemist's vocabulary, hi modern chemistry, if a solution has more hydrogen ions (+) than hydroxyl ions (-), yielding a net result of more positive charges, the solution is said to be acidic. Conversely, if the solution has more hydroxyl ions (-) than hydrogen ions (+), with a net result of more negative charges, the solution is said to be basic. When an acid (+) and a base (-) are combined, a salt is made. Much of the terminology used in histology staining comes from textile dyes. The terms acid dyes and basic dyes do NOT pertain to the pH of the dye or its solution, nor to the amount of hydrogen (H+) or hydroxyl (OH") ions in the solution, hi textile and histology dye terminology, an acid dye is an ionic (charged) dye whose colored component is anionic, which is a negative charge. When a base such as ammonia is added to the acid dye, a salt will form. Nearly all acid dyes contain sulfonic or carboxylic acids on the dye molecules, which are negatively charged. Basic dyes are ionic dyes which contain cationic, or positively charged, components. When an acid such as hydrochloric acid is added to a basic dye, a salt is formed. Nearly all basic dyes contain an amino group, which is positively charged. |
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A common acidic auxochrome is: a. -NH2 b. -COOH c. -N=N- d. NO2 |
THE CORRECT ANSWER IS "B." An auxochrome is a chemical group added to a dye to impart a charge to the dye molecule. Acidic dyes, also known as anionic dyes, have a negative charge due to the added auxochromes. The carboxyl ion (-COO-) has a negative charge. Other acidic/anionic auxochromes are the sulphonic ion (-SO3-) and the phenolic ion (benzene ring -O-) The amino group (-NH3+ ) is an example of a positively charged, basic/cationic auxochrome. The azo group (-N=N-) is an example of a basic/cationic chromophore, while the nitro group (NO2) is an example of an acidic/anionic chromophore. A chromophore is any chemical group which is NOT an auxochrome, that when added to a dye, imparts color to the dy |
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An orthochromatic dye will stain tissue: a. different from the dye color b. several different colors c. nuclei only d. the color of the stain used |
THE CORRECT ANSWER IS "D." An orthochromatic dye colors the tissue in a predictable way. For example, blue dyes staining tissue blue. The dye binds to the tissue. As white light passes through the tissue-dye complex, some of the wavelengths of the light will be absorbed by the tissue-dye complex. If all colors are absorbed except blue, then the blue is passed along to the eye, so the color observed is blue. If the dye-tissue complex absorbs different wavelengths than the color of the dye, a different color will be observed. This is known as metachromasia. |
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Select the type of staining that results in extreme sharpness of differentiation. a. absorption b. physical c. progressive d. regressive |
THE CORRECT ANSWER IS "D." Regressive staining is done by overstaining the tissue, then differentiating out the stain, until the sharpest degree of differentiation is achieved, or only the desired structure is left stained (the background is no longer stained). Verhoeff and Luxol fast blue are examples of a regressive stain. Absorption is a type of physical staining, in which the dye dissolves easier in the tissue component, rather than in the solvent used to make the staining solution. Sudan black B is an example of an absorption and physical type of staining mechanism. In progressive staining, the tissue is left in the staining solution, until the desired intensity of staining is achieved. The Prussian blue stain for iron is an example of a progressive stain. |
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During microscopic review of old H&E slides, it is noted that the eosin has faded. This was probably due to which of the following? a. extended time in the acid rinse after hematoxylin staining b. inadequate rinsing with water after the bluing step c. insufficient dehydration through alcohols after the eosin d. clearing in a xylene substitute instead of xylene |
THE CORRECT ANSWER IS "B." Dilute ammonia, lithium carbonate and Scott's tap water are often used as "bluers" after staining tissue with hematoxylin. After bluing, the slides are rinsed in running tap water, to remove these alkaline solutions. Failure to remove these alkaline solutions could result in either the failure of the eosin to bind to the tissue, due to the pH of the tissue now being too alkaline, or the fading of dye intensity over time, following coverslipping, due to "bleaching." (Think of the effect of bleach being accidentally spilled on colored clothes.) The mounting media used to coverslip could also cause fading of staining over time. To prevent this, most modem mounting media contain anti-oxidants. Extended time in the acid rinse would cause the fading of hematoxylin staining, not eosin staining. Insufficient dehydration after eosin staining would result in water droplets remaining on the tissue. The tissue sections would probably appear cloudy. Clearing with either xylene or a xylene substitute should not cause the fading of staining. However, care must be taken to use a compatible mounting media, since droplets or cloudiness could result if they are not compatible |
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For a particular stain to work, the procedure states that the tissue MUST be fixed in Bouin solution. The tissue was, originally placed in 10% neutral buffered formalin and routinely processed. After microtomy, and, prior to staining, the sections can be deparaffinized, rehydrated, and: a. placed in the appropriate fixative b. rinsed in running water for ten minutes c. soaked in a solution of lithium carbonate d. washed in a solution of sodium bisulfite |
THE CORRECT ANSWER IS "A." If tissue is not initially fixed in the fixative most appropriate for a staining procedure, cut sections can be post-mordanted in the appropriate fixative prior to staining. An example of this would be the use of Bouin solution to post-mordant sections before doing a trichrome stain. Slides are usually rinsed in water before most staining procedures, as most dye solutions are aqueous. However, the time interval usually does NOT need to be as much as 10 minutes. Sections that have been differentiated with acid-alcohol, or have been decalcified with an acid, might be soaked in a lithium carbonate solution to neutralize them prior to staining. A solution of sodium bisulfite or periodic acid is sometimes recommended to revitalize the staining of tissue that has been stored in formalin for a long time |
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Microscopically, sections of a particular patient's tissue stained with H&E show a lack of nuclear staining, while the H&E control slides show good nuclear staining. A possibility is that the patient's tissue was: a. fixed in a zinc-based fixative b. placed in the fixative immediately c. stored in unbuffered formalin d. underdecalcified after fixation |
THE CORRECT ANSWER IS "C." Unbuffered formalin has an acidic pH. On long term storage, the formaldehyde can combine with oxygen from the air, creating formic: acid, which makes the solution even more acidic. This acidity can destroy the protein in the tissue, including the DNA in the nuclei. Once the proteins are destroyed, staining will be poor to none. Overdecalcification could also destroy the proteins in the tissue, yielding poor to no staining. Underdecalcification, however, will result in poor sectioning due to calcium remaining in the tissue. However, nuclear staining will still be good. Decalcification should always occur after fixation, so that the proteins are somewhat stabilized to resist the denaturing effects of the acids on the proteins. Decalcifying tissue prior to fixation could also destroy the unstabilized proteins, which would yield poor to no staining. Placing tissue in the fixative immediately prevents loss of proteins due to autolysis. Delaying the placement of the tissue into a fixative could result in autolysis and loss of proteins, which would again yield poor to no staining. Zinc fixatives are recommended for preservation of nuclear detail. 1, 3, 7, 9, 10, l6, 19, 21 |
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Oil red O staining is based on the principle of: a. adsorption b. impregnation c. regression d. solubility |
THE CORRECT ANSWER IS "D." The oil red O dye molecule is more soluble in fat than in the dye solvent in which the staining solution is prepared. This solvent is usually either isopropyl alcohol or propylene glycol. Consequently, the dye will preferentially move from the stain solvent to the fat or lipid in the tissue. Sudan black B also stains in this manner. This phenomenon is also known as absorption. In adsorption phenomenon, the dye binds to the components, substance or structures in the tissue based on the charge of the dye and the charge of that component, substance or structure, such as positive binding to negative. Most histology staining dyes bind to tissues due to charges. Impregnation involves the staining of tissue with a metal, such as with silver nitrate. In regressive staining, tissue is overstained with a dye, and the dye is then differentiated out of the background. |
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Generally, the most desirable coverglass thickness to use for photomicrography is: a. 0.15 millimeters b. 0.18 millimeters c. 0.21 millimeters d. 0.24 millimeters. |
THE CORRECT ANSWER IS "B." 0.18 millimeters |
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Although hematoxylin solutions generally require a mordant, they may be used without a mordant for the demonstration of: a. amyloid b. copper c. glycogen d. lipids |
THE CORRECT ANSWER IS "B." Freshly prepared hematoxylin solutions without mordants have been used by Mallory to demonstrate deposits of lead, copper and iron in tissue sections. This is due to the ability of unmordanted hematoxylins to form blue-black dye lakes with these metals. 1, 15, 19 |
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The main function of auxochromes in artificial dyes is to: a. change the shade of the color b. give the dye affinity for the tissue c. increase solubility in alcohols d. stabilize the dye compound |
THE CORRECT ANSWER IS "B." Although auxochromes may alter the shade of the compound, their main function is the salt-forming property of electrolytic dissociation that allows the dye to attach itself to the tissue |
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A pathologic condition characterized by liver cirrhosis caused by abnormally high deposits of iron in the liver is called: a. hematocytosis b. hematotoxicosis c. hemochromatosis d. hemosiderosis |
THE CORRECT ANSWER IS "C." Hemochromatosis is a disorder of iron metabolism characterized by excess deposits of iron in the tissues, skin pigmentation, and cirrhosis of the liver. It is also called iron storage disease. |
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A rhabdomyosarcoma is suspected in a biopsy submitted to the laboratory. To verify this diagnosis, the most helpful histology stain would be which of the following procedures? a. Oil red O (ORO) b. periodic acid - Schiff (PAS) c. phosphotungstic acid - hematoxylin (PTAH) d. Verhoeff-van Gieson (VVG) |
THE CORRECT ANSWER IS "C." A rhabdomyosarcoma is a malignant tumor arising from striated (voluntary, skeletal) muscle, as opposed to a leiomyosarcoma, which is a malignant tumor of smooth (involuntary) muscle. PTAH is an excellent stain for demonstration of the cross-striations in striated muscle. Though nowhere near as good as a PTAH, a well stained H&E or trichrome can also demonstrate muscle striations. However, the use of immunohistochemistry procedures for the demonstration of actin and myosin are most commonly used today to distinguish between these two tumors. ORO demonstrates lipids. PAS demonstrates glycogen, mucin, basement membrane and fungus. VVG demonstrates elastin fibers |
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A cholecystectomy for cholelithiasis would involve the removal of a/an: a. colon segment b. gallbladder c. liver lobe d. spleen |
THE CORRECT ANSWER IS "B." "Chol" or "chole" is a prefix that refers to bile. "Ectomy" is a suffix meaning removal or excision. "Litho" is a prefix denoting stone. Therefore, a cholecystectomy for cholelithiasis would be the removal of the gallbladder for gall stones. Colectomy is the removal of the colon. Hepatectomy is the removal of the liver. Splenectomy is the removal of the spleen |
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A hematoxylin solution mordanted with which of the following salts would be most appropriate to use in a lengthy staining procedure that uses very acidic stains? a. aluminum b. iron c. lead d. tungsten |
THE CORRECT ANSWER IS "B." Iron hematoxylin, such as Weigert, use ferric chloride as its mordant. The resulting black color is more resistant to decolorization when succeeding stains are lengthy or very acidic. |
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The specific function of a mordant in combination with a dye is to: a. act as a differentiator b. decolorize during dehydration c. function as a neutralizer d. provide a charge |
THE CORRECT ANSWER IS "D." A mordant is a salt of a divalent or trivalent metal, such as aluminum (Al+3), ferric iron (Fe+3), or tungsten (W+5 or W+6). Some dyes, such as hematoxylin, have very little net charge of their own. The addition of the positively charged salts to the hematoxylin imparts a positive charge onto the dye. This positive charge can now bind with negatively charged areas on the tissue, such as the phosphoric acid on the DNA in the nucleus |
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The reddish tint sometimes seen in old aluminum hematoxylin solutions is generally caused by the: a. formation of sulfuric acid from the aluminum salt b. concentration of acetic acid used to establish the pH c. creation of additional auxochromes on the hematoxylin d. oxidation of the hematoxylin molecule to oxyhematein |
THE CORRECT ANSWER IS "D." When the hematoxylin dye molecule is oxidized to hematein and then bound to an aluminum salt, the resultant aluminum-hematein complex has a purple color. If this complex is further oxidized, it changes to a redder, non-staining material which is given the name oxyhematein, although it is probably not a single molecular entity. Sulfuric acid is not created from aluminum salts. Acetic acid is used to adjust the pH of the solution. Auxochromes add charges to a dyes, allowing binding to tissue. In general, the addition of auxochromes may change the color shade, though usually does not change the color |
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If no chemical oxidizer is used in the PTAH solution, the solution must be oxidized naturally for: a. 1 week in total darkness, tightly covered b. 1 month on the counter, lightly covered c. 3 months in the sunlight, lightly covered d. 1 year in the refrigerator, tightly covered |
THE CORRECT ANSWER IS "C." Hematoxylin dye molecules contain two hydroxyl ions (-OH). The hydrogen must be removed from one, so that a double-bonded oxygen will result (=O). This action of removing a hydrogen is known as oxidation. This new double-bonded oxygen changes the hematoxylin to hematein. To remove the hydrogen from the hematoxylin dye molecule, oxygen must be present in the air around the solution. The oxygen from the air combines with the hydrogen from the hematoxylin, creating water (H2O) in the solution and a double-bonded oxygen on the dye. To provide the oxygen for the solution, it is recommended that the solution be lightly covered. To accelerate the reaction with the oxygen, it is recommended that the solution be placed in light. Once the PTAH solution has oxidized to the correct color, the solution should be transferred to a dark bottle, and stored in the dark away from heat to slow down the overoxidation |
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An entire rack of slides are stained with H&E. It is noticed that one of the slides has increased staining of eosin in muscle and red blood cells. This slide was most likely fixed in: a. Camoy fluid b. Hollande c. Zamboni PAF d. Zenker fluid |
THE CORRECT ANSWER IS "D." After Zenker fixation, muscle and red blood cells stain more intensely with eosin. Mercuric chloride is the mordant used in mercuric fixatives. It is positively charged, and will bind to negatively charge amino acids in the cytoplasm, such as the carboxyl groups (-COO"). This leaves more positively charged amino acids available for subsequent staining. Eosin is negatively charged in solution, and will bind to positively charge amino acids. Since there are more positively charged amino acids available after fixation in mercuric fixatives, eosin staining will be more intense. Formaldehyde, in comparison, is negatively charged. It will bind to positively charged amino acids in the cytoplasm, such as the amino groups (-NH3+). This leaves more negatively charged amino acids available for staining. However, since eosin is also negatively charged, the number of eosin dye molecules that will bind to formaldehyde-fixed cytoplasmic amino acids is fewer than the mercuric chloride fixed tissue. If the staining is too intense in mercuric fixed tissues, it is recommended that a dilute solution of eosin be used, that the time in routine eosin be reduced, or that the time in the differentiating alcohols after eosin be increased. |
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In the Feulgen procedure, the splitting of purine bases from the sugar and phosphate groups of the DNA is called: a. autolysis b. biolysis c. hydrolysis d. synthesis |
THE CORRECT ANSWER IS "C hydrolysis |
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When acetic acid is added to solutions of eosin, it acts as an: a. accelerator b. accentuator c. acceptor d. activator |
THE CORRECT ANSWER IS "B." The term "accentuator" means "to heighten the effect of." The addition of acetic acid, or any other dilute acid, reduces the pH of the eosin solution. To obtain the best staining with eosin, the pH of the solution should be approximately 4.6 to 5. If the pH is above 7, the tissue proteins will remain negatively charged and will not bind with the negatively charged eosin dye molecule. If the pH is below 4, the eosin dye molecule is converted to a free acid which can nonspecifically bind to the tissue. At pH 4.6 to 5, the tissue proteins become more positively charged, and thus can bind best with the negatively charged eosin. At this pH, a minimum of three shades of eosin should be seen in the tissue, with RBC's and eosinophils being the darkest, muscle the next darkest, and collagen the lightest shade. An accelerator is a solution that speeds up the rate of reaction. An acceptor is a substance that unites with another substance. An activator is a substance the promotes enzyme activity in histochemical reactions. |
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Of the following, the reagent that will extract both DNA and RNA from tissue sections is. a. deoxyribonuclease b. perchloric acid c. ribonuclease d. trichloroacetic acid |
THE CORRECT ANSWER IS "D." A 4% solution of trichloroacetic acid extracts either RNA only, or RNA and DNA, depending upon the procedure used. Hydrochloric acid can also be used. Deoxyribonuclease extracts only DNA. Perchloric acid, usually used in a 10% solution, extracts only RNA. Ribonuclease extracts only RNA |
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Which of the following are acidophilic tissue components, as distinguished by light microscopy following H&E staining? a. basement membrane, fibrin and nuclei b. collagen, erythrocytes, and muscle c. elastin, eosinophils and plasma cell cytoplasm d. Nissl granules, Paneth cells granules and reticulin |
THE CORRECT ANSWER IS "B." The term "acidophilic" is used in histology to denote a tissue substance or component that is easily stained with acid (anionic) dyes, such as eosin. Of the components listed above, the following are termed acidophilic: all connective tissues (collagen, elastic, reticulin, elastin, fibrin, basement membrane), as well as erythrocytes (RBC's), eosinophils, and Paneth cell granules. The term "basophilic" refers to a tissue substance or component that is easily stained with basic (cationic) dyes, such as-hematoxylin. Of the components listed above, the following are termed basophilic: nuclei (due to the DNA), plasma cell cytoplasm (due to the RNA), and Nissl granules (due to RNA). |
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Of the following preservative methods, which is considered the LEAST acceptable when nuclei are to be stained with acridine orange? a. alcohol b. Carnoy c. formalin d. freezing |
THE CORRECT ANSWER IS "C." When staining with acridine orange, fresh frozen sections, or tissues fixed in Carnoy solution or alcoholic fixatives are preferred to formalin or any other fixative. |
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Light microscopy examination of a brain section stained with cresyl violet acetate shows basophilic material in the cytoplasm of neurons. This material is most likely: a. glycogen b. lipofuscin c. melanin d. RNA |
THE CORRECT ANSWER IS "D." Neurons contain Nissl bodies, which are composed primarily of rough endoplasmic reticulum with high ribosomal content, thus high RNA (ribonucleic acid) content. Nissl granules can be stained with thionine or cresyl violet acetate, also known as cresyl echt violet. |
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The selectivity for nuclear staining with Gill hematoxylin can be increased by adding an excess of which of the following to the hematoxylin solution? a. aluminum salts b. ethanol c. hematoxylin d. sodium iodate |
THE CORRECT ANSWER IS "A." Aluminum salts bind to hematoxylin to make a positively charged dye lake. In the cell, DNA and RNA have a strong affinity for this positive dye lake. This is due not only to the charges on the DNA/RNA and the dye lake, but also to a 3-dimensional complex ionic/non-ionic binding between them |
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The Romanowsky color range in staining results from a combination of which of the following dyes? a. alcian blue and orange G b. aniline blue and basic fuchsin c. luxol fast blue and phloxine d. methylene blue and eosin |
THE CORRECT ANSWER IS "D." Methylene blue dye, over time, can oxidize to azure A, azure B, methylene violet, and various thionine. These give a range of colors in the blue to purple range. A dye containing a variety of dyes and their derivatives is know as a polychromatic dye. The pink color originates from the addition of a xanmene dye such as eosin Y, eosin B, or phloxine B. |
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A dye solution is made colorless by reduction, and subsequently used to react with a specific tissue component. The next step is oxidation of the reaction product, which restores the chromophoric groups and thus the color. This dye solution is known as: a. leuco b. mordant c. peroxidase d. phosphatase |
THE CORRECT ANSWER IS "A," Leuco compounds are staining solutions that are made colorless by reduction of the chromophoric groups. After the dye reacts with the tissue component(s), the color is restored by the restoration of the chromophores by oxidation. Schiff reagent, used to demonstrate glycogen and mucin, is an example of a leuco dye solution. |
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The majority of the oxazines, azines and thiazines dyes are examples of: a. natural dye counterstains b. natural nuclear stains c. synthetic dye counterstains d. synthetic nuclear stains |
THE CORRECT ANSWER IS "D. Synthetic nuclear stains |
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In a routine staining series, slides are placed in xylene for clearing prior to coverslipping. Macroscopically, the slides have an opaque rather than a clear appearance. This is most likely caused by insufficient: a. dehydrating b. deparaffinization c. differentiation d. staining |
THE CORRECT ANSWER IS "A." Insufficient dehydration would leave water on the slides. This water would be carried over into the xylene, which would inhibit clearing. |
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The most common basic auxochrome encountered in dye chemistry is the: a. amino group (- NH2) b. azo group ( -N=N- ) c. carboxyl group (- COOH) d. hydroxyl group (-OH) |
THE CORRECT ANSWER IS "A." An auxochrome is a compound added to a dye to give that dye a charge. This charge, either positive or negative, helps to give the dye an affinity for the charges found on the amino acids in the tissue proteins. Auxochromes may be positively charged, such as the amino group (-NH3+), which is considered to be cationic or basic. Auxochromes may change the shade of the dye, but are not the primary cause of the color of the dye. |
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A bone marrow aspirate is stained with Giemsa. A microscopic examination reveals blue-gray erythrocytes and blue leukocytes. The most likely explanation for this result is that the: a. cells are stained correctly b. fixative used was B-5 c. sample is from an anemic patient d. staining solution is too alkaline |
THE CORRECT ANSWER IS "D." The Giemsa procedure is generally used for the differentiation of various types of leukocytes (white blood cells) in the bone marrow, rather than to distinguish leukocytes from erythrocytes (red blood cells). The pH of the Giemsa staining solution must be between 6.4 and 6.9 in order for proper staining to occur. Red blood cells should be a dark pink, while leukocytes should have a variety of colors, depending upon the type of white blood cell. If the pH is too high (alkaline), the cells will stain as described above. If the pH is too low (acidic), the red blood cells will stain pale pink and the white blood cells will be understained or not stained at all. Use of a mercuric fixative, such as B-5, will reveal better chromatin patterns. An aspirate from an anemic patient would reveal red blood cells that are decreased in size or number. However, the staining of all blood components would remain the same. |
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Sections of brain begin to float off the slides as they are being deparaffinized and run down through alcohols. To adhere the sections to the slides before staining, coat the slides and sections with: a. carbowax b. celloidin c. polyethylene glycol d. paraffin |
THE CORRECT ANSWER IS "B." Often, to better adhere sections to slides, the slides are coated with either a proteinaceous or a chemically charged material before the sections are attached to the slide. Or, the protein or charged chemical can be added to the flotation bath. However, if the sections have already been dried onto the slides, and are now in the process of being deparaffinized and hydrated through graded alcohols to water when they start to fall off the slides, then these methods will not help at this point. One method to prevent the section from falling off the slides would be to return the slide to absolute alcohol, and then to place the slides for 1 to 2 minutes in a solution of 0.5% celloidin made with ether-acetone mixture. The slides are then placed in 70-80% alcohol for a few minutes, to harden the celloidin. The slides are then rinsed in water and are ready to be stained. There are several disadvantages with this method. The celloidin coating is a carbohydrate (cellulose), and will stain with carbohydrate stains, such as periodic acid-Schiff (PAS), mucicarmine and alcian blue. Celloidin also must be dissolved in an ether-alcohol mixture, which is hazardous since ether is very explosive and flammable. Carbowax, polyethylene glycol and paraffin are types of embedding media that must be removed from tissue sections prior to staining. |
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After staining slides in hematoxylin and eosin, the sections appear very pink with, pale reddish- brown nuclei. The most logical step to take is to: a. change all the solutions and restain the sections b. replace the hematoxylin with fresh solution and restain c. restain longer in the same hematoxylin solution d. switch to a different bluing agent |
THE CORRECT ANSWER IS "B." A solution of hematoxylin contains a mixture of unoxidized hematoxylin, oxidized hematoxylin (hematein), hematein bound to an aluminum salt (Al-hematein), over-oxidized hematein (oxy-hematein), and overoxidized hematein bound to an aluminum salt (Al-oxy-hematein). The Al-hematein produces the blue color on nuclei (after the bluing step). If the Al-hematein becomes over-oxidized to Al-oxy-hematein, nuclei will stain a reddish-brown. Changing all the solutions is a waste of solutions and tech time, since only the hematoxylin solution is bad. Restaining longer in the same hematoxylin solution will not cause the dye to change back to a blue color. Using a different bluing agent will not reduce the reddish-brown color. If the nuclei remain a reddish-blue color, then possibly the bluing agent is inadequate and should be changed, or at the very least, made again. 1, 3, 5, 6, 7, 9, 10, 13, 16, 21 |
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A lymph node has been processed, sectioned at 2 micrometers, and stained with H&E to verify a diagnosis of malignant lymphoma. The sections show strong nuclear staining, but very little cytoplasmic staining around the lymphoid cells. Which of the following would be the best course of action? a. recut and restain the sections for a shorter period of time in hematoxylin b. destain the sections and restain them for a shorter time in hematoxylin c. destain the sections and restain them for a longer time in eosin d. give the slides to the pathologist, as the sections are stained correctly |
THE CORRECT ANSWER IS "D." Lymph nodes, as well as other lymphoid tissues such as spleen, contain a type of cell called a lymphocyte. Unreactive lymphocytes have very small, dense nuclei, which tend to stain very darkly with hematoxylin. Unreactive lymphocytes also contain very little cytoplasm, often appearing as just a rim around the nucleus, if at all. Sections of tissue and tumors with unreactive lymphocytes would therefore appear to contain very dark nuclear staining with very little cytoplasmic staining. Thinner sections, such as 2-3 micrometers, are often used to obtain single layers of the lymphocytes, thus making the nuclei look less dark. |
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A special stain was requested on the only extra slide from the second level of a biopsy. However, this slide was accidentally stained in H&E. To decolorize this slide, into what two solutions should the slide be placed? a. acetic acid and ammonia water b. Lugol iodine and sodium thiosulfate c. picric acid and distilled water d. running water and acetone |
THE CORRECT ANSWER IS "A. |
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Which of the following groups of dyes are generally used for counterstaining? a. carmine, eosin, hematoxylin b. light green, methylene blue, nuclear fast red c. metanil yellow, neutral red, orcein d. methyl green, phloxine, Prussian blue |
THE CORRECT ANSWER IS "B." Counterstains are pale dyes that contrast the color of the primary dye. The counterstains are used to highlight the background, so that the area of positivity can be seen in relationship to the rest of the tissue. Light green is commonly used as a counterstain with the Grocott silver stain for fungus. Methylene blue is commonly used as a counterstain for Kinyoun or other AFB procedures. Nuclear fast red is often used as the counterstain in the iron reaction. |
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Staining with an aluminum-hematein dye molecule is dependent upon all of the following bonding types EXCEPT: a. covalent b. hydrogen c. ionic d. van der Waal's |
THE CORRECT ANSWER IS "A." Covalent bonds are rare in histology staining. |
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Which of the following nuclear dyes is easily removed by placing the stained slide in alcohol? a. azure A b. celestine blue c. hematoxylin d. nuclear fast red |
THE CORRECT ANSWER IS "A." The azure dyes, such as those used in the Giemsa stains, are very soluble in alcohol. The affinity of the dye for the nuclei (strength of the bonds) is much lower than the affinity of the azure dyes for the alcohol. This means that the dye will want to remain in the alcohol, and can be easily removed from the nuclei. After staining in a Giemsa solution, the slide is quickly differentiated through alcohols. This removes excess azure dyes from the tissue. Prolonged differentiation through the alcohols removes even more dye from the nuclei, and could result in overdifferentiation and thus poor nuclear staining. |
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DNA has an affinity for cationic dyes. Which of these is a cationic dye? a. acridine orange b. orange G c. ponceau S d. tartrazine |
THE CORRECT ANSWER IS "A." Cationic dyes have positive charges on them, and bind to negatively charged (anionic) components in the tissue. Cationic dyes are often used to stain nuclei. Acridine orange usually has a zinc chloride salt bound to it, giving it a positive charge. Other cationic, nuclear dyes include hematoxylin, toluidine blue, the azures, and methylene blue |
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Which of the following microscopes is used to examine unstained living tissue? a. fluorescence b. light c. phase contrast d. transmission electron |
THE CORRECT ANSWER IS "C." Phase contrast microscopes are often used to study living, unstained cells or tissues, which are nearly invisible when viewed with a standard light microscope. In a phase contrast microscope, the light passes through the specimen. Some of this light is scattered. All the light passes through the objective, then onto a special phase plate. This phase plate has two areas on it: one that is normal glass, and one that has a chemical compound placed on part of the glass. The scattered light rays pass through the normal glass. Some of the light rays pass through the chemically treated areas, which slows down the light rays. The normal light rays combine with the slowed down light rays. The two light rays are now in different phases. These differences in phase help to highlight the details of the stained specimen components, such as the cell walls and the nuclei. |
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In the H&E set-up, the container with the dilute ammonia water has a coating of white gritty material. To remove this material, fill the container with fresh cold water and add a small amount of which of the following? a. ammonium hydroxide b. buffered formalin c. hydrochloric acid d. sodium hypochlorite |
THE CORRECT ANSWER IS "C." Aluminum hematoxylins, such as Mayer or Gill, contain an aluminum sulfate salt (Al2(SO4)3). Slides must be thoroughly rinsed after staining with the hematoxylin, to remove the excess aluminum sulfate. If the slides are not rinsed enough, the excess aluminum sulfate will combine with the ammonium hydroxide (NH4OH) in the dilute ammonia water, forming aluminum hydroxide (Al(OH)3). |
