Fixation

Question 1

Name the chemical components of a cell nucleus and proper fixation methods to preserve these components.

Answer 1

The cell nucleus contains proteins and the two nucleic acids, DNA, and RNA. Some of the proteins are directly attached to the nucleic acids. Fixation generally stabilizes the proteins attached to the nucleic acids, but formalin-fixed nuclei may show irregular staining and clear spaces in the nucleus, which is referred to as nuclear bubbling. Nuclear bubbling results from improper fixation of the nucleic acids. The use of zinc or mercuric chloride in formalin, such as in Zenker solution, improves nuclear details. Commercial zinc formalin is readily available. Bouin solution, which contains picric acid, also enhances nuclear detail.

Question 2

Describe how to identify a poorly fixed tissue sample.

Answer 2

An H&E stained slide of a poorly fixed specimen shows tissue degradation, and the relationship between the various structures in the tissue are poor. Cells lack a clear demarcation between their nucleus and cytoplasm. Often cell membranes are gone. The sample stains poorly. The specimen may show nuclear bubbling (i.e, the nucleus has clumps of chromatin and open, clear spaces within it). In tissue such as intestine or skin, many surface epithelial cells are missing. It is difficult to properly evaluate and diagnose a poorly fixed tissue sample.

Question 3

Discuss why you might see a white deposit in tissue fixed with zinc-formalin.

Answer 3

If you do not wash all the zinc formalin from tissue following fixation, a white precipitate may result. The white deposit is zinc, which precipitates out of solution when the zinc reacts with the carbonates in water. White precipitate may also be caused by a change in the pH. Heat, pressure, and vacuum from automated processing may also cause the precipitate to form. Use buffered formalin to prevent the formation of the white deposit. It will also disappear during H&E staining, because of the acidity of the staining reagents.

Question 4

Discuss the use of picric acid as a fixative.

Answer 4

Picric acid is a coagulating additive fixative. Picric acid penetrates tissue well and leaves the tissues soft, but it does cause extreme shrinkage. It is not a good fixative for nucleic acids, as it leaves DNA soluble. Picric acid will fix proteins well, but tissue left in it will show degradation of other tissue structures, so it must be washed out with 50% alcohol before processing. If picric acid is not washed out, it may also affect staining of the cell. Picric acid is a component of several combination fixatives, such as Bouin solution, Genre solution, Hollande Solution, and Zamboni Solution (Buffered Picric Cid-PAF). Be cautious when using picric acid, because it is toxic and potentially explosive in its dry form at very high temperatures. Aqueous solutions containing picric acid, such as Bouin’s, are not hazardous, but they should be handled according to label directions.

Question 5

Compare the use of osmium tetroxide and aldehydes as the primary fixatives for Electron Microscopy.

Answer 5

Osmium tetroxide is a good fixative for the preservation of cellular ultrastructure. Because it makes lipids insoluble, membrane detail is well preserved. However, tissue cannot be left in osmium longer than four hours because it leaches out protein. Osmium does not penetrate tissue well, so use small pieces of 1mm or less. Osmium cannot be used for histochemical studies. Osmium produces toxic vapors and must only be used under a fume hood. Aldehydes are good general fixatives that can be used for either electron or light microscopy. Tissue preserved in aldehydes can be studied using histochemical techniques because most proteins remain active. Aldehyde fixatives penetrate well, and tissue can remain soaking in most aldehydes indefinitely, although glutaraldehyde-fixed samples should be removed in 2-4 hours. However, detail is not as well preserved with aldehydes as it is with osmium. Membrane lipids are soluble in aldehyde, so membranes are often not visible. Secondary fixation in osmium will demonstrate a small percentage of the lipid structure.

Question 6

Identify the best fixative for the following:
Red Blood cells; fat; collagen and muscle; immunoglobulin in lymphoid tissue; spirochetes; rabies; and uric acid crystals.

Answer 6

Red Blood cells- Methanol
fat- Formalin
collagen and muscle- Bouin
immunoglobulin in lymphoid tissue- B-5
spirochetes- NBF
rabies- Acetone
uric acid crystals- Absolute alcohol

Question 7

Explain how formalin works as a fixative

Answer 7

Formalin is an additive fixative. It reacts primarily with the amino groups on the amino acids of proteins to form bonds, which cross-link the protein chains together. It is a non-coagulating fixative, forming a gel, that makes the proteins insoluble and keeps them in place and able to withstand further processing. Formalin penetrates the tissue quickly, although there is evidence that complete fixation is not rapid. The formation of cross-linkage, which stabilizes the protein, is a slow process that may take up to seven days for complete fixation to occur.

Question 8

Define paraformaldehyde

Answer 8

Paraformaldehyde is a polymerized form of formaldehyde. It is often used in electron microscopy when a very pure formaldehyde solution is required. Several fixatives are prepared by heating paraformaldehyde, which depolymerizes or dissociates into pure formaldehyde. A common preparation for an EM fixative is 4% paraformaldehyde in cacodylate buffer. Depolymerize the paraformaldehyde by warming it to 60 deg C. Add sodium hydroxide and mechanically stir the solution until it clears. Add the cacodylate buffer to adjust the pH from 7.2 to 7.4. If the final solution is cloudy, it is probably because depolymerization was incomplete.

Question 9

Discuss the use of glutaraldehyde as a fixative.

Answer 9

Glutaraldehyde acts like a formaldehyde in that it acts as a cross-linking agent on protein. It does not penetrate tissue as well as formalin, so it is best used on smaller samples. Glutaraldehyde is commonly used in electron microscopy because it preserves ultrastructural details. Let tissue remain in glutaraldehyde for less than 2 hours to avoid overhardening, then transfer the tissue to a buffer solution. Do not use glutaraldehyde with PAS (periodic-acid Schiff) stain, as remaining free aldehyde groups will cause false positive reactions.

Question 10

Discuss considerations for preparation of tissue for a muscle biopsy.

Answer 10

The majority of enzyme stains done in Histology are on muscle. It is very important that you freeze the muscle according to established procedures, to prevent the formation of ice crystals in the tissue, which could confuse the interpretation of the staining. Orient the tissue to get a cross-section, so that the muscle fibers and the pattern of staining can be observed. The best frozen sections are obtained by freezing tissue in isopentane and liquid nitrogen. Let the isopentane reach -150 deg C before submerging the tissue, to prevent ice crystals from forming. If isopentane is not available, dust the tissue with talc and then submerge it into the liquid nitrogen. The liquid nitrogen prevents the formation of bubbles. After you remove the tissue from the isopentane, allow it to warm to -20 deg C to evaporate any excess isopentane before you cut it. If the tissue is too cold, it will be very hard to section.

Question 11

Explain why a cytology specimen might be fixed in Carnoy solution.

Answer 11

Carnoy solution is a rapid-acting fixative made from a combination of absolute alcohol, chloroform, and acetic acid. It preserves glycogen and maintains the nuclear features of the cells. Carnoy solution lyses red blood cells, which is desirable in cytology samples, where the red blood cells do not need to be examined because they are not of interest. Carnoy solution causes excessive shrinkage and hardens the tissue, so do not use it routinely. Make a modified Carnoy solution by substituting methyl alcohol for ethyl alcohol. This modification decreases shrinkage and hardening of tissues.

Question 12

Discuss the formation of formalin pigment as a fixation artifact.

Answer 12

Formalin pigment is an acid hematin pigment that can be produced during formalin fixation of tissue. The brown, crystalline pigment forms in tissue that is rich in blood when the pH of the formaldehyde becomes acidic, usually below a pH of 6.0. It is an undesirable artifact because the fine particles can be confused with more diagnostically important features, such as pathologically relevant pigments or microorganisms. Prevent the formation of formalin pigment by using neutral solutions of formalin. If formalin pigment forms, remove it by using alcoholic picric acid or alkaline alcohol.

Question 13

Identify components of the fixative B-5. Describe when B-5 should be used as a fixative.

Answer 13

B-5 has become the fixative of choice for preservation of nuclear detail in bone marrow and lymph tissue. It is also useful for special stains, such as immunofluorescence and IHC, because it does not destroy tissue antigens. B-5 contains mercuric chloride and sodium acetate in formaldehyde. Prepare it immediately before use. Wash the tissue before staining it to remove the mercury pigment. Modifications of B-5 that use non-toxic zinc salts, instead of neurotoxic mercury, have been developed and are available commercially. Studies show comparable results to the original formula. Zinc-based B-5 is advantageous because technicians do not have to deal with handling and disposing of the toxic mercury.

Question 14

Identify cellular features that can be used to assess fixation in electron microscopy.

Answer 14

Electron microscopy demonstrates ultrastructural details that cannot be seen in light microscopy, even at the highest magnifications. It is very important to fix tissue properly to preserve the most detail. Fixation should begin immediately after the tissue is excised, to limit the amount of autolysis and putrefaction. Properly fixed tissue shows a complete plasma membrane. The space between the two layers that make up the nuclear membrane should be apparent. Cytoplasmic organelles should remain intact. Mitochondria are a good monitor of proper fixation, as they should show no swelling or disruption. The endoplasmic reticulum should appear as distinct channels in the cytoplasm. Nuclear detail will vary, depending on the fixative used.

Question 15

Describe a method for transporting kidney for immunofluorescence studies.

Answer 15

To transport kidney tissue short distances, wrap the tissue in saline-dampened gauze, place it in a tight container, and keep the container on ice. Do not expose the kidney tissue directly to ice. For longer times in transport, use Michel Transport Medium. Michel’s will keep the tissue at physiological pH to preserve the antigens for immunofluorescence studies.

Question 16

Discuss microwave radiation as a method of fixation. Include its advantages and disadvantages.

Answer 16

Microwave radiation is less destructive to tissue structure and greatly increases the speed of fixation. No chemicals are involved to react with key cell components. However, it can cause excess tissue shrinkage, and cause the tissue to look spongy. Red blood cells may be destroyed. Excess heat (over 55 degrees) can cause pyknotic nuclei, which overstain. It is often difficult to control the temperature of commercial microwave ovens. Special laboratory models are available. Combination methods have been developed that use microwaves to stabilize the tissue prior to chemical fixation. Microwaves increase the permeability of the cell to further fixatives, and reduce the amount of time required for fixative penetration. Increasingly, microwaves are used to speed up fixation in automated processors. Use caution, because carcinogenic formalin vapors are produced in the microwave oven. Wear goggles (because formalin can fix contact lenses to your eyes), and a mask (because formalin can cause nosebleeds).

Question 17

Zinc salts are used increasingly in formalin fixatives instead of mercury salts. Tell why zinc is preferable and what advantage a fixative containing zinc may have over other fixatives.

Answer 17

Zinc salts are far less toxic than mercury salts. Zinc is normally present in the body, so the zinc formalin solution is safe for you to use as a fixative in a well-ventilated area with proper PPE. Zinc is only toxic if you take in more than 100 mg per day or are pregnant, have stomach ulcers, hemochromatosis, or glaucoma. Mercury is a powerful neurotoxin. Zinc salts are not corrosive and may be disposed of in laboratory plumbing systems. Zinc salts do not require any additional washing prior to staining, as do the mercury salts, and fit into the routine lab better, because they save an additional step. Evidence is that zinc sulfate formalin solutions preserve the reactivity of antigens, making zinc more suitable for IHC (antibody/antigen staining process), in situ hybridization (nucleotide chain staining process), and PCR. The zinc formalin solutions also show excellent nuclear preservation and better paraffin infiltration than with formalin alone. Zinc sulfate is preferable to zinc chloride in formalin, as it is less corrosive to automated processors.

Question 18

Discuss the use of non-aqueous fixatives.

Answer 18

The most common non-aqueous fixatives used for fixation are acetone, ethyl alcohol, or methyl alcohol. These are organic solutions that are coagulating, non-additive fixatives. they stabilize proteins by disrupting the bonds that maintain the tertiary structure of the protein. Primary and secondary structure remains intact. Therefore, these organic compounds are often used to demonstrate the presence of enzymes, or for frozen sections that demonstrate cell surface proteins by IHC techniques, because the proteins maintain their antigenicity. Methanol is used to fix red blood cells and cytology samples. Acetone is used to preserve enzymes and is also the fixative to choose for demonstrating rabies in brain tissue. Organic fixatives shrink tissue and overburden it, so they are not recommended for routine use. Organic solvents are very flammable and must be stored in fireproof cabinets.

Question 19

Discuss the following factors in electron microscopy (EM) tissue fixation: Temperature; pH; osmolality; soaking time; and size of the specimen.

Answer 19

Fixation for electron microscopy can take place at room temperature. Fixatives for EM specimens should have an approximate pH of 7.2 to 7.4. Use buffered solutions, such as phosphate, cacodylate, and veronal acetate. Take precautions when handling cacodylate buffer, as it contains arsenic. Because EM studies ultrastructure, the osmolality of the fixative is critical to ensure that cellular structures do not swell or shrink. Osmolality, a measure of salt concentration, should be approximately 300 mOsm. Buffer salts contribute to the osmolality. Add dextrose or sucrose to the fixative to adjust its osmolality, if necessary. The soaking time must be long enough to ensure penetration of the fixative into the sample. Osmium tetroxide leaches tissue proteins. Do not leave tissue in osmium tetroxide for more than four hours. The size of the tissue sample is critical for EM, because if it is too large, fixative penetration will not be complete. For osmium fixation, cut tissue cubes of about 1 millimeter.

Question 20

Discuss factors that influence the choice of fixative used to preserve tissue.

Answer 20

When selecting a fixative, consider what stains will be used on the tissue later, and what components need to be demonstrated in the tissue. If only a routine H&E is needed, routine fixation in formalin may suffice. However, if the tissue will be stained to demonstrate proteins using IHC methods, then the antigens must be preserved and remain active so they will react with the antibody reagent. Many antibodies will not work in formalin-fixed, paraffin-embedded sections. Similarly, for an enzyme stain such acid or alkaline phosphatase, the enzyme must remain reactive. It may be that fixatives are inappropriate, and you should prepare a frozen section, instead. Consider the correct fixative for bacteria or viral studies. If you use an improper fixative, it may be possible for you to perform a post-fixation correction. However, it is not always possible. Choose the correct fixative when the specimen arrives in the lab.

Question 21

Discuss the difference between formaldehyde and formalin and describe the components of commercial formalin.

Answer 21

Formaldehyde is an organic compound known as a simple aldehyde. Although it occurs naturally as a colorless gas, it is most commonly dissolved in a 37-40% aqueous solution. The terms formaldehyde and formalin are often used interchangeably. Commercial grade preparations of the formaldehyde are considered 100% formalin. For fixation, the 100% formalin is diluted to 10% formalin, which means that the concentration is really 3.7% to 4.0% formaldehyde. To prepare 10% formalin, dilute 1 part of formalin with 9 parts of water. Commercial grades of formalin often contain methanol as a stabilizer, which prevents the precipitation of paraformaldehyde. 10% NBF is most commonly used, which maintains pH at 6.8. Other combinations of formalin are also used, and may contain sodium chloride (saline), calcium chloride, ammonium bromide, or ethyl alcohol.

Question 22

Discuss OSHA requirements for labs that use formaldehyde or formalin.

Answer 22

OSHA requires the plant operations manager at your facility to ensure that the air is exchanged at least six times every hour, and ideally twelve, where formaldehyde is used in your laboratory. This includes Autopsy, Histology, and Parasitology. Your employer must fit you for an N100 respirator mask that at least covers your nose and mouth, and ideally, a shield that also covers your eyes, if you will be exposed to more than 0.5 ppm of formaldehyde in the air during an eight-hour shift. Your employer must train you in proper use of the respirator. You must shave your beard if it interferes with a close face-fit. OSHA requires laboratories to monitor workers’ exposure to formaldehyde. Occupational Health may ask you to wear a monitor badge and require you to get yearly blood tests.

Question 23

Discuss the use of mercuric chloride as a fixative. Include safety issues.

Answer 23

Pure mercury is not as commonly used today for fixation as it was in the past, because it is highly toxic to the central nervous system (CNS). Although mercury does not distort cells, it penetrates poorly and causes shrinking in subsequent processing steps. A mercury pigment is formed during fixation, which can be removed by treating slides with iodine, followed by sodium thiosulfate. Mercury is still found in compound fixatives. Mercuric chloride acts on tissue to coagulate proteins. Mercuric chloride is a highly corrosive chemical and quickly reacts with metals. Mercuric salts are additive fixatives, and leave tissue very receptive to staining. Use extreme caution when using pure mercury or mercury compounds. Dispose of mercury as a hazardous waste material. Use non-toxic zinc salts as substitutes for mercury whenever possible.

Question 24

Name three aldehyde fixative solutions that can be used for EM and differentiate between them.

Answer 24

Zamboni fixative is a phosphate-buffered picric acid solution (PAF) that can be used to fix tissue for both light and electron microscopy. Glutaraldehyde preserves ultrastructure better than any other aldehyde. Although it does not penetrate tissue well, it tends to harden tissue, so specimens cannot be left in it for more than two hours before transferring them to a buffer solution. Prepare it at a concentration of 2-4% in a phosphate or cacodylate buffer. Millonig formalin can be also used in light microscopy and EM. This is formaldehyde in a phosphate buffer. The buffer allows for a wide variation in pH changes without changing the osmolality of the solution. It is a stable solution that can be prepared in large volumes.

Question 25

Discuss reasons why tissue for enzyme testing should be fixed when possible. Identify a good storage method for post-fixation sections.

Answer 25

When you receive biopsy tissue, consider its source and collection time to estimate the amount of enzyme autolysis. Ideally, biopsy tissue should be fixed prior freezing, if possible, to minimize the diffusion of enzymes. However, some enzymes are soluble in fixatives, and will either be lost during the fixation step or the amount of enzyme greatly reduced. Blood cell preparations should be fixed as soon as they are dried to prevent loss of enzymes. Muscle biopsies are frozen unfixed, although they may be post fixed. Slides for esterase techniques can be fixed in absolute methanol/formaldehyde. Following fixation, the best storage medium is 30% sucrose that contains 1% gum acacia. Slides can be stored in this medium at 4 deg C for several weeks.

Question 26

Define four physical factors that influence fixation.

Answer 26

Four factors affecting fixation are: (1) Temperature; (2) tissue size; (3) the ratio of tissue volume to fixative volume; and (4) the length of time for fixation. Fixation occurs fastest at high temperatures, but if the temperature is too high, proteins break down or diffuse from their original location into the cell. Historically, manual fixation was done either at cold temperatures or room temperatures to best preserve cell structure. However, many modern, automated tissue processes use higher temperatures to speed up fixation, without significant changes in cell morphology. The size of the tissue, coupled with how long that tissue is in fixative, will determine how complete fixation will be. The goal is for the fixative to penetrate completely. The fixative volume should be at least 15-20 times greater than the volume of the tissue itself. Poor fixation will result from insufficient volume of fluid, and may also result in poor staining.

Question 27

Define autolysis and putrefaction, and tell why they are important to fixation.

Answer 27

Autolysis is the breakdown of tissue that occurs when enzymes normally within the tissue start to attack it and break it down. Enzymatic activity does not stop when the blood supply to the tissue is stopped, after excision or death. Only when a fixative is added will the process be halted; therefore, it is important to begin fixation as soon as possible after excision or death to prevent tissue deterioration. Autolyzed tissue does not stain properly, appears pale, and lacks structural detail. Putrefaction refers to the decay of tissue that result from exposure to bacteria or mold. All tissue contains some bacteria. Begin fixation quickly so that the bacteria do not break down tissues. Putrefaction makes it difficult to handle the resulting mushy tissue, and greatly limits the pathologist’s ability to identify tissue and cellular structures.

Question 28

Fixatives are defined as coagulating or non-coagulating. Discuss the difference and give examples of both.

Answer 28

Coagulation is the transformation of a liquid or a solid into a semisolid or solid mass. In fixation, coagulation is the process that renders proteins insoluble and localized in the tissue. A coagulating fixative forms a semisolid network of molecules that allows other preservative solutions to penetrate the tissue deeply. Coagulating fixatives are preferred when paraffin infiltration and embedding is required. Examples of coagulating fixatives are alcohol, zinc salts, and picric acid. Non-coagulating fixatives form a solid gel, which does not allow for easy penetration of solutions, and are not used for paraffin embedding. Examples of non-coagulating fixatives are glutaraldehyde, osmium tetroxide, potassium dichromate, and acetic acid. Fixative solutions are formulated and selected depending on the required processing, staining components desired, and the nature of the tissue. This accounts for the wide variety of fixatives used in the histology laboratory.

Question 29

Discuss two ways time is important to fixation.

Answer 29

The first way time affects fixation is with regard to how long it takes to get the excised tissue into the fixative. The longer it takes to get the tissue completely immersed after biopsy or post mortem, the more changes will be seen. Optimally, the tissue should be fixed immediately following surgery to prevent autolysis. Submersion time is the second critical fixation step. When tissue is not fixed long enough, the relationship between tissue structure is not preserved, or important details are lost. Subsequent processing steps require adequate fixation. For example, colon tissue in which the fixative has not penetrated adequately may not preserve the epithelial layer. Autolysis will be visible and the tissue will appear distorted. there does not appear to be a negative effect on leaving tissue in fixatives for storage over a long period of time.

Question 30

Discuss the fixation of lipids.

Answer 30

Lipids (fats and oils) can be fixed in tissue with formalin, but it is difficult to retain lipids in tissue because of the effect of organic solvents used in processing, which dissolve lipids. Osmium tetroxide and chromic acid will make the lipids insoluble and fix them better than other fixatives, but they will still diffuse into the processing solvents. One way to show lipids is to fix the tissue in formalin and then make frozen sections of it, followed by special stains for lipids.

Question 31

Define osmolality and explain why it is important in a discussion of fixatives.

Answer 31

Osmolality is a measure of the number of ions dissolved in a solution, based on the weight of the solution. A solution with the same osmolality as body fluids is said to be isosmotic or isotonic. A solution that is hypertonic has a greater concentration of particles outside the tissue than inside it; hypotonic solutions have a lower concentration of particles outside the tissue than inside it. The cell membrane is semipermeable and allows water to cross it. When a cell is placed in a hypertonic solution, water will move across the membrane to the more concentrated fluid in an attempt to equalize the concentration. The hypertonic effect on the cell is to remove water and the cell shrinks. Conversely, a cell in a hypotonic solution will take in water and the cell will swell. If too much water enters, the cell can burst, called lysis. Non-reactive salts are added to a fixative solution to adjust the osmolality and prevent shrinking or swelling of the tissue.

Question 32

Fixatives can be classified as additive or non-additive. Discuss the meaning of these terms.

Answer 32

In an additive fixative, the chemicals combine with the protein molecules to change them. In most cases, this means the protein is made insoluble by the addition of the fixative, and then becomes immobilized, remaining at its original location in the cell. Additive fixatives change the tertiary structure of a protein. Most common additive fixatives contain salts, such as mercuric chloride, zinc sulfate, or zinc chloride. Non-additive fixatives do not combine with the protein. They are usually non-aqueous, organic compounds, such as alcohols or acetone. They act on the proteins to coagulate them and precipitate them, but do not change their chemical structures. Non-additive fixatives are beneficial for preserving tissues that are soluble in aqueous solutions. Organic fixatives are not used routinely, as they tend to stiffen tissue, but are reserved for special needs. For example, acetone is the organic fixative of choice for preserving brain tissue for rabies investigations. Methyl alcohol is used as an organic fixative for peripheral blood smears.

Question 33

Discuss how chemical and physical fixatives are different. Give examples of both.

Answer 33

Chemical fixatives are solutions added to the tissue to prevent degradation by denaturing proteins, making them insoluble, thus preventing autolysis (breakdown of the tissue by enzymatic action). Most fixations in the histology lab laboratory are chemical fixation. The most common chemical fixative for routine use is 10% neutral buffered formalin (NBF). Physical fixation occurs when the tissue is treated with heat or desiccation. Heat and desiccation will denature proteins and fix them in place, although neither method is good at tissue preservation as chemical fixation heat can destroy tissue structure, and affects the way proteins absorbs stain. Microwaving tissue is a method of physical fixation, and is becoming more commonly used, especially in automated processors. Microwave radiation is less destructive to connective tissue and greatly increases the speed of fixation, although it may have other negative effects on morphology. Desiccation is not commonly used for fixation, except for peripheral blood smears.

Question 34

Identify the following cell components: Mitochondria; Endoplasmic Reticula; and ribosomes. State if they can be seen under the light microscope in an H&E stained section.

Answer 34

The amount of cytoplasm in a cell, and the structures seen within the cytoplasm, differ depending on the activity and function of the cell. Some organelles can be seen on the light microscopy; others only by electron microscopy. mitochondria are small organelles within the cytoplasm that are responsible for energy production. They are generally not seen in an H&E. Endoplasmic reticula (ER, singular is reticulum) are channels within the cytoplasm that carry materials through the cell, either to be used within the cell, or to be transported out of it. There are two types of endoplasmic reticula: Rough (granular) and smooth (agranular). In an H&E, the exact structure of the ER may not be seen. However, basophilic (purple) staining within the cytoplasm is due to the ER. Very basophilic cytoplasm may be seen in highly active cells, such as the acinar cells of the pancreas. Ribosomes are the site of protein synthesis. Ribosomes contribute to basophilic staining, but are not seen as distinct structures in H&E. They can be found attached to ER, or free in the cytoplasm.

Question 35

Discuss the difference between primary, secondary, and tertiary protein structure.

Answer 35

Proteins are made up of base units called amino acids. The primary structure of a protein is determined by its sequence of amino acids, which are linked by covalent bonds. The secondary structure of a protein is determined by hydrogen bonding between amino acids. The tertiary structure of a protein is defined by other types of chemical bonds between amino acids, such as ionic bonds, hydrogen bonds, and linkages between sulfur atoms. The primary, secondary, and tertiary structures together, determine the size and shape of the protein molecule. Fixatives stabilize proteins to stop them from degrading before histological examination and in storage. Additive and non-additive fixatives affect the tertiary structure of proteins. Additive fixatives combine with the protein. Non-additive fixatives precipitate the proteins.

Question 36

Discuss the purpose of fixation.

Answer 36

Fixation stabilizes tissue, and minimize the fact of post mortem degradation so that the tissue can be viewed with as little distortion as possible. Fixation prevents the breakdown of tissue caused by the enzymatic activity that remains following death. It will also prevent decay of the tissue, which can be caused by bacteria and mold. Fixation stabilizes proteins, retains cell morphology, and keeps the cells in normal relation to their connective tissue. By selecting the proper fixative, the histologist maximizes the retention of key cellular components, such as lipids or carbohydrates. Fixation hardens the tissue and makes further handling easier. It also makes the tissue more receptive to stains, which allows cellular structures to be visualized. Fixation enhances differences in the refractive index of the tissue, which will make it easier to view under the microscope.

Question 37

Discuss the role enzymes play in tissue degradation. Name three organs that are extremely rich in enzymes.

Answer 37

Enzymes are proteins that function in cells and tissues as catalyst in cellular chemical reactions. They accelerate and facilitate metabolic processes. Following tissue death, enzymes remain active for a period of time. If they are not rendered inactive by fixation, they will continue to affect cellular processes and cause the breakdown of cell structures. This process of degradation from enzymatic activity is called autolysis. Fixation stops autolysis and allows the cells and tissue to maintain their structure. All cells contain enzymes, but the liver, pancreas and brain are particularly enzyme-rich. Tissues, rich in enzymes break down more quickly than other tissues, so start fixating the liver, pancreas, and brain slices first, as soon as possible, following the loss of blood supply (perfusion), to prevent loss of structure in these tissues.

Question 38

Identify the following cell components: Nucleus; nuclear membrane; nuclear pores; and nucleolus. State if they can be seen under the light microscope in an H&E stained section.

Answer 38

The cell is divided into two major parts: the nucleus and the cytoplasm. The nucleus is contained within nuclear membrane. An H&E stain will show a dark blue or purple nucleus, and a pink cytoplasm. The nuclear membrane can sometimes be seen under the light microscope at high magnification, but what we know of its structure has been learned using electron microscopy. The nuclear membrane is actually made up of two membranes that are separated by a narrow space. The membrane has small openings called nuclear pores, which transport molecules in and out of the nucleus. The nucleolus, which means small nucleus, is made up of mostly protein and some RNA. Active cells have many nucleoli, which can be seen in an H&E.