د. أديب عبد العالي الازبجي Histology Lecture :- 1
Tissue processingHistology is the study of the tissues of the body and how these tissues are arranged to constitute organs. This subject involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ.
The most common procedure used in histologic research is the preparation of tissue slices or “sections” that can be examined visually with transmitted light. Because most tissues and organs are too thick for light to pass through, thin translucent sections are cut from them and placed on glass slides for microscopic examination of the internal structures.
The basic steps used in tissue preparation for light microscopy are :-
1-Fixation
Preserves tissues in situ as close to the lifelike state as possible
Ideally, fixation will be carried out as soon as possible after removal of the tissues, and the fixative will kill the tissue quickly, thus preventing autolysis.
Chemical substances like formalin, mercuric chloride, acetic acid, picric acid and glutaraldehyde are used as fixatives to preserve tissues.
Small pieces of fresh tissues are placed in common fixatives like 10% neutral formal saline for 24 hours.
The purpose of fixation is
– to preserve the morphology and chemical composition of the tissue,
– to prevent autolysis and putrefaction,
– to harden the tissue for easy manipulation,
– to solidify colloidal material, and
– to influence staining.
Factors affecting fixation
Buffering
• Fixation is optimal at a neutral pH, in the range of 6 to 8 .
Penetration .
Fixative solutions penetrate at different rates, depending on the diffusibility of each individual fixative
Volume
Should be at least a 10:1 ratio of fixative to tissue .
Temperature
Increasing the temperature, as with all chemical reactions, increases the speed of fixation
Concentration
Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4%
Time interval
Formalin should have 6 to 8 hours to act before the remainder of the processing is begun .
Decalcification
Tissue calcium deposits are extremely firm and do not section properly with paraffin embedding .
After fixation, some hard tissues like bone and tooth, which contain large amount of calcium salts, require an additional step called decalcification before they are subjected for dehydration.
Decalcification makes the hard tissues soft, enabling them to be cut with microtome. For decalcification, several decalcifying agents are used, namely 10% nitric acid, 5% trichloro-acetic acid and ethylene diamine tetra acetic acid (EDTA).
2-Dehydration
Fixed tissue is too fragile to be sectioned and must be embedded first in a non-aqueous supporting medium (e.g., paraffin)
Water from the tissues is removed in a gradual manner by immersing the tissues in ascending grades of alcohol, 50%,70%, 90% and absolute alcohol, in order to embed it in paraffin wax which is not miscible in water.
Tissue remains in each of these grades for 30–60 minutes.
3-Clearing
After dehydration the tissue is treated with a paraffin solvent (clearing agent) like xylene or toluene for 2-3 hours. These agents penetrate and replace the alcohol from the tissue and make it translucent (clear).
Ethanol is not miscible with paraffin, so nonpolar solvents (e.g., xylene, toluene) are used as clearing agents; this also makes the tissue more translucent .
4-Embedding
In order to obtain thin sections with microtome, tissue is infiltrated with embedding medium which gives a rigid consistency to the tissue.
The various embedding media are paraffin wax, celloidin, gelatin, plastic resins (for electron microscope), etc.
Paraffin is the routinely used embedding medium for light microscopy.
Embedding involves two steps, namely, impregnation and casting or block making.
5-Section Cutting (Trimming)
The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome.
5–7 μm-thick sections are cut with a rotary microtome.
The slides with sections are either air dried or dried in an incubator overnight at 37 °C and stored for staining at room temperature.
6-Staining
Allows for differentiation of the nuclear and cytoplasmic components of cells as well as the intercellular structure of the tissue .
7-Cover-slipping
The stained section on the slide is covered with a thin piece of plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years .
STAINING PROCEDURE :-
Staining is done routinely by using a basic and an acidic dye that stain tissue components selectively.
Tissue components that stain more readily with basic dyes are termed basophilic and are blue in color and those with an affinity for acid dyes are termed acidophilic and are pink/orange in color.The basic dyes are haematoxylin, toluidine blue and methylene blue. The acidic dyes are eosin, orange G and acid fuchsin.
The combination of haematoxylin and eosin (H&E) is most commonly used in histological staining procedure.
However, special stains like periodic acid Schiff reagent (PAS), osmic acid, Mallory and Masson’s, trichrome stains are being used to selectively identify certain tissue components.
Haematoxylin usually stains the acid component (nucleus) of the cell, blue or black, whereas eosin stains the basic components present in the cytoplasm, pink.
• Hematoxylin does not directly stain tissues but needs a “mordant” or link to the tissues; this is provided by a metal cation such as iron, aluminum, or tungsten
Conversely, eosin stains the more basic proteins within cells (cytoplasm) and in extracellular spaces (collagen) pink to red (acidophilic) .