Alternatives to paraffin embedding include various plastics that allow thinner sections. Such plastics include methyl methacrylate, glycol methacrylate, araldite, and epon. Methyl methacrylate is very hard and therefore good for embedding undecalcified bone. Glycol methacrylate has the most widespread use since it is the easiest to work with. Araldite is about the same as methacrylate, but requires a more complex embedding process.
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Concentration Time interval Fixation is best carried out close to neutral pH, in the range of Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a pH of 7. Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant.
Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. One way to get around this problem is sectioning the tissues thinly 2 to 3 mm. Penetration into a thin section will occur more rapidly than for a thick section. The volume of fixative is important. There should be a ratio of fixative to tissue.
Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative.
Agitation of the specimen in the fixative will also enhance fixation. Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor. Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Too high a concentration may adversely affect the tissues and produce artefact similar to excessive heat.
Also very important is time interval from of removal of tissues to fixation. The faster you can get the tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline.
The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur. Fixatives - general usage There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated.
Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly. Most clinicians and nurses can understand what formalin is and does and it smells bad enough that they are careful handling it. However, the mercury deposits must be removed dezenkerized before staining or black deposits will result in the sections.
Glutaraldehyde is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old. The tissue must be as fresh as possible and preferably sectioned within the glutaraldehyde at a thickness no more than 1 mm to enhance fixation.
Alcohols, specifically ethanol, are used primarily for cytologic smears. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness. For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best.
Tissue Processing Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections. The usual way this is done is with paraffin.
Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually routinely. The technique of getting fixed tissue into paraffin is called tissue processing. The main steps in this process are dehydration and clearing. Wet fixed tissues in aqueous solutions cannot be directly infiltrated with paraffin.
First, the water from the tissues must be removed by dehydration. Sometimes the first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major disadvantages. Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has toxic fumes. The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium paraffin.
The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene. Chloroform used to be used, but is a health hazard, and is slow. Methyl salicylate is rarely used because it is expensive, but it smells nice it is oil of wintergreen.
There are newer clearing agents available for use. Many of them are based on limolene, a volatile oil found in citrus peels. Another uses long chain aliphatic hydrocarbons Clearite. Although they represent less of a health hazard, they are less forgiving with poorly fixed, dehydrated, or sectioned tissues.
Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. Paraffins can be purchased that differ in melting point, for various hardnesses, depending upon the way the histotechnologist likes them and upon the climate warm vs.
A product called paraplast contains added plasticizers that make the paraffin blocks easier for some technicians to cut. A vacuum can be applied inside the tissue processor to assist penetration of the embedding agent.
The above processes are almost always automated for the large volumes of routine tissues processed. Automation consists of an instrument that moves the tissues around through the various agents on a preset time scale. The "technicon" tissue processor is one of the commonest and most reliable a mechanical processor with an electric motor that drives gears and cams , though no longer made. Automated tissue processor. Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician who must pick the tissues out of the cassette and pour molten paraffin over them.
This "embedding" process is very important, because the tissues must be aligned, or oriented, properly in the block of paraffin. Tissue embedding. Alternatives to paraffin embedding include various plastics that allow thinner sections.
Such plastics include methyl methacrylate, glycol methacrylate, araldite, and epon. Methyl methacrylate is very hard and therefore good for embedding undecalcified bone. Glycol methacrylate has the most widespread use since it is the easiest to work with. Araldite is about the same as methacrylate, but requires a more complex embedding process. Epon is routinely used for electron microscopy where very thin sections are required. Plastics require special reagents for deydration and clearing that are expensive.
For this reason, and because few tissues are plastic embedded, the processing is usually done by hand. A special microtome is required for sectioning these blocks. Small blocks must be made, so the technique lends itself to small biopsies, such as bone marrow or liver. Sectioning Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it.
There are three important necessities for proper sectioning: 1 a very sharp knife, 2 a very sharp knife, and 3 a very sharp knife. MPEG movie [k] demonstrating sectioning technique with microtome. Knives are either of the standard thick metal variety or thin disposable variety like a disposable razor blade.
The advantage of the disposable blade becomes apparent when sectioning a block in which is hidden a metal wire or suture. Plastic blocks methacrylate, araldite, or epon are sectioned with glass or diamond knives. A glass knife can section down to about 1 micron. Microtomes have a mechanism for advancing the block across the knife. Usually this distance can be set, for most paraffin embedded tissues at 6 to 8 microns.
Sectioning tissues is a real art and takes much skill and practice. Histotechnologists are the artists of the laboratory. It is important to have a properly fixed and embedded block or much artefact can be introduced in the sectioning. Common artefacts include tearing, ripping, "venetian blinds", holes, folding, etc. Once sections are cut, they are floated on a warm water bath that helps remove wrinkles.
Then they are picked up on a glass microscopic slide. Unstained section on glass slide. The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide. If this heat might harm such things as antigens for immunostaining, then this step can be bypassed and glue-coated slides used instead to pick up the sections.
Tray of unstained slides in drying oven. Frozen Sections At times during performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process. The surgeon may want to know if the margins of his resection for a malignant neoplasm are clear before closing, or an unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process.
This is accomplished through use of a frozen section. The piece s of tissue to be studied are snap frozen in a cold liquid or cold environment to Celsius.
In general, microscope and slides are the basic equipment in studying histology. Different Kinds of Microscope There are two main types of microscopes which are optical microscope and electron microscope but it can be classified by lens, cost, application, or in any other aspects. There are various types of microscopes to use depending on your prepared specimen, specifically. Types of Optical Microscope The optical microscopes are first invented in the 17th century. It is the oldest design in terms of model design in microscopy which uses visible light and refract it with optical lenses hence the name light microscope. It is commonly used to observe living cells without staining them. Differential interference contrast microscope — this microscope, similar to the phase contrast, is used to observe minute surface irregularities but at a higher resolution.