The most important step in the preparation of high quality microscopic slides of tissue is:
B. ensuring adequate fixation
The others are all admirable and desired features; however, without proper fixation, other procedures are difficult and often impossible, and the results of poor or improper fixation cannot be corrected in the subsequent steps.2,6"
The breakdown of tissue due to enzyme activity is called:
Autolysis is the breakdown of tissue due to enzyme activity from within the cell. When the blood supply is interrupted, some enzymes cease synthesizing amino acids into protein, while other enzymes act to split protein into amino acids. This results in a general dissolution of proteins, rendering them incapable of coagulation by heat or chemical agents. Polymerization is the process of forming a polymer, or larger molecule, by the combination of simpler molecules. Putrefaction is the breakdown of tissue by bacterial action. Osmosis is the passage of a fluid through a semi-permeable membrane in the direction of the more concentrated solution in an effort to equalize the concentration of the solutions on both sides of the membrane.2,6"
The breakdown of tissue by bacterial action is called:
Putrefaction is the breakdown of tissue by bacterial action. This is common in the intestinal tract where bacterial content is normally high. Autolysis is the breakdown of tissue due to enzyme activity. Denaturation refers to rendering proteins insoluble. Oxidation refers to the loss of electrons and does not pertain to the breakdown of tissue by bacterial action.6"
A physical agent used in fixation is:
The only physical agents used in fixation are heat and desiccation. Heat coagulates protein rapidly, producing extreme distortion. It may be used, as with microwave fixation, but extreme care is recommended. The stabilization of protein is a result of fixation. Coagulation, referring to proteins, is a result of fixation. Emulsification is the suspension of small globules of one liquid in a second liquid.2,6"
The volume of fixing fluid employed should be:
A. 15 - 20 times that of the tissue
For good fixation the volume of fixative should be 15-20 times that of the tissue.2,4,6"
The rate of fixation varies with the fixative and is also dependent upon the:
A. temperature of fixative
Chilled fixatives penetrate more slowly than those at room temperature, thus retarding both the rate of fixation and the stopping of autolytic changes. Higher temperatures (above room temperature) will accelerate fixation, but will also increase autolytic changes in the tissues. It takes approximately 6 to 48 hours to fix tissues adequately at room temperature.2,4"
Fixation of a piece of lung occurs when the fixative:
C. penetrates and kills very slowly
Good fixation requires that various cell constituents be preserved in as life-like manner as possible by rendering the proteins of the cell insoluble. With the denaturation of protein in the cells, the tissues become more resistant to the effects of subsequent processing. Good fixation also stops autolysis, brings out differences in refractive indices of various tissue elements, and penetrates and tills the tissue rapidly.2,3,6"
The primary function of a general purpose fixative is:
B. stabilization of proteins
The stabilization of protein is the primary aim of fixation. As a result of the protein-stabilizing action of fixatives, enzymes, which are proteins, are inactivated. This is a very important fixative characteristic, as enzymatic action causes tissue autolysis. Some fixatives will help preserve or retain lipid and carbohydrates initially, but much of the time, these substances will be lost in the subsequent processing.1,2,3,6"
"A fresh, unfixed liver biopsy can be held safely for a short time by placing it:"
C. in saline-moistened gauze and then in a refrigerator
Moist gauze keeps the tissue from drying out, while refrigeration retards the effects of autolysis and putrefaction. Tissues that have been frozen may display artifacts due to the separation of tissue layers by ice crystals. Tissue biopsies should not be placed in physiological saline, because even though the solution is isotonic, some undesirable changes will be produced in the tissue. Room temperature should not be used as a ""holding"" temperature, and tissue should never be placed on dry filter paper as this rapidly removes moisture from the tissue.2,6"
Improper preservation of tissue may be due to:
A. a delay in fixation
Rapid penetration of the fixative is very important for the proper preservation of tissue more than a few cell layers in thickness. Any delay in fixation will permit putrefaction and autolysis. 70% alcohol is recommended for the long-term storage of tissue. Eight hours is adequate time for processing fixed tissue of the recommended thickness.1,2,6"
"For the best preservation of staining properties during long term storage, tissues should be stored in:"
C. 70% ethanol
For storage of fixed tissues, 70% ethanol is best because the staining qualities of the tissue will be unaffected. Although buffered formalin is usually used for storage of tissues fixed in formalin, this solution and any of the other formalin solutions will cause the tissue to gradually lose the basophilic staining properties.4,6"
Chemical fixation of tissue begins at the:
B. periphery and proceeds inward
Fixation begins at the periphery of the tissue and proceeds inward. Slow penetrating fixatives allow postmortem autolysis to occur in the center of the specimen.1"
"For good fixation, it is recommended that the tissue be no larger than:"
B. 2 cm square and 3 - 4 mm thick
Tissue should be no more than 4 mm thick; 3 mm is better for total penetration of the reagents. Sections larger than a 2 cm square are difficult to handle and process, and require a larger slide.6"
A good fixative is one that:
B. makes tissues more permeable to subsequent reagents
A good fixative will make tissues more permeable to fluids than they are in their natural state, thus enhancing the penetration of the subsequent reagents. A good fixative will preserve tissue as near its living state as possible, will inhibit putrefaction, and will harden tissues quickly.6"
A POOR fixative can be defined as one that:
C. penetrates tissue slowly
A poor fixative penetrates tissues slowly, retarding the rate of fixation and permitting autolytic changes to occur in the center of the tissue. The other distractors are characteristics of a good fixative.6"
A universal fixative which is used for routine purposes and which permits a broad spectrum of staining methods is:
D. 10% neutral buffered formalin
A wider variety of special stains can be done on formalin fixed tissue than on tissue fixed with any other fixative. Zenker solution was used extensively in the past but many stains, especially neuropathology stains, are not satisfactory following Zenker fixation. The hazards associated with the use of Zenker solution are quickly making this fixative obsolete. Zamboni solution is recommended for specimens for electron microscopy, but it is not recommended for routine use. Carnoy solution also is not recommended for routine use.1,2,6"
10% formalin contains what percentage of formaldehyde?
The stock solution is a 37-40% solution of formaldehyde gas dissolved in water. When given as formalin, the solution is considered as 100%, or when given as formaldehyde the true concentration of 37-40% is used. A 10% formalin solution is prepared by diluting one part of the stock solution with nine parts water; this is equivalent to a 4% solution of formaldehyde.2,6"
"To make a 10% formalin solution, how many mL of water should be added to 300 mL of 37-40% formaldehyde solution?"
37-40% formaldehyde is considered a stock solution of 100% formalin. To prepare 10% formalin, one part of stock (100%) formalin is diluted with 9 parts of water. Therefore if one has 300 mL (1 part) of stock (100% formalin or 37-40% formaldehyde) then 2700 mL (9 parts, or 9 x 300) of water would be needed. This makes a total of 3000 mL.2,3,6"
The formic acid present in commercial 37-40% formaldehyde solutions may:
D. create formalin pigment
Formic acid is present in commercial 37-40% formaldehyde solutions as either an impurity or as a product of oxidation of formaldehyde. Its presence is undesirable and detrimental to tissue sections. Acidic formalin solutions promote the formation of formalin pigment, may leach hemosiderin from tissue sections, and may reduce the quality of routine staining. Formic acid has no shrinking effect on tissue.2,6,7"
Which of the following fixatives is used for specimens to be mailed?
D. 10% neutral buffered formalin
Tissue may remain in only one of the listed fixatives indefinitely, a definite requirement for mailed tissues; that fixative is 10% neutral buffered formalin. All other listed fixatives require that the; tissue be removed within approximately 24 hours and washed (water - Zenker and Helly; 50% alcohol - Bouin) and then either processed or stored in 70% alcohol.2,6,7"
"When neutral solutions of formalin are prepared by storing the solution over calcium or magnesium carbonate, the fluid drawn off for fixation promptly becomes:"
The addition of an excess of calcium carbonate or magnesium carbonate neutralizes formalin as long as it is in the storage container, but the formalin becomes acid when drawn off and used for fixation. This is due to oxidation of formalin to formic acid by atmospheric oxygen. Proteins are amphoteric; they can develop either positive or negative charges depending on the pH of solution. Polychromatic refers to a stain that exhibits many colors on tissue.2,6"
Formalin pigment is generally formed in tissues fixed in formalin when the pH:
B. falls below 6
Formalin pigment is generally formed in tissues, especially those rich in blood, when the pH drops below 6.0. This may occur with any formalin containing fixative with a pH below 6.0. Large quantities of pigment are formed at pH levels below 5.0.2,4"
An unknown pigment in a tissue section that can be bleached with a saturated alcoholic solution of picric acid is most likely:
Formalin pigment is formed after tissue is fixed or stored in non-buffered (acidic) or in acetate-buffered formalin. Formalin pigment is an artifactual, dark brown, crystalline, birefringent pigment. The pigment usually is not formed in tissues that have been fixed in formalin solution with a pH greater than 6.0.2,4,6"
10% neutral buffered formalin is prepared with which of the following constituents?
A. "37-40% formaldehyde, sodium phosphate monobasic and dibasic"
The constituents of 10% neutral buffered formalin are 1 part of 37-40% formaldehyde, 9 parts water, sodium phosphate monobasic, and sodium phosphate dibasic. The phosphate salts serve to buffer the solution.2,5,6"
Helly solution is prepared by combining mercuric chloride with which of the following constituents?
D. potassium dichromate and 37-40% formaldehyde
Helly solution (Zenker-formol) does NOT contain acetic acid, potassium permanganate, or sodium bisulfite. Sodium sulfate is an optional ingredient in Zenker and Helly solutions.4 The 37-40% formaldehyde must be added just before use to prevent turbidity and a dark brown precipitate, due to the reducing action on potassium dichromate.2,5,6"
Zenker solution is prepared by combining mercuric chloride with which of the following constituents?
C. potassium dichromate and glacial acetic acid
Zenker solution does NOT contain formaldehyde, potassium permanganate, or sodium bisulfite. Sodium sulfate is an optional ingredient in Zenker and Helly solutions.4 Zenker solution is stable, even after the addition of glacial acetic acid.2,5,6"
Carnoy solution is prepared with which of the following constituents?
C. "absolute alcohol, chloroform, and glacial acetic acid"
Carnoy solution is a non-aqueous fixative, containing only absolute alcohol, chloroform, and glacial acetic acid. Carnoy solution is recommended as a fixative if nucleic acid are to be studied in paraffin sections. Never use this fixative if acid-fast bacilli are to be demonstrated, as Carnoy fixation renders the organisms non-acid-fast.1,2,3,5,6"
Bouin solution is prepared by combining picric acid with which of the following constituents?
D. 37-40% formaldehyde and glacial acetic acid
Absolute alcohol, 95% alcohol, and IN HC1 are NOT constituents of Bouin solution. Bouin solution is an aqueous fixative containing picric acid, 37-40% formalin, and glacial acetic acid. Gendre solution is comparable to Bouin solution except that it is an alcoholic solution containing picric acid, 37-40% formaldehyde, and glacial acetic acid.2,3,5,6,7"
Flemming solution is prepared with which of the following constituents?
A. "1% chromic acid, 2% osmium tetroxide, and glacial acetic acid"
Flemming solution does NOT contain formaldehyde or absolute alcohol. Chromic acid will be reduced by formaldehyde and is incompatible (a hazard if mixed) with alcohol. Flemming solution does contain 1% chromic acid, 2% osmium tetroxide, and glacial acetic acid.4,6"