This document discusses the preparation of histological specimens. It describes the various stages of processing tissues, including fixation, dehydration using ethanol or other agents, clearing with xylene or other substances, and embedding in paraffin wax. Sections are then cut from the embedded tissue using a microtome and stained, often with hematoxylin and eosin, for examination under a microscope. Histology is the study of tissues and how they are arranged into organs at a microscopic level. Proper preparation of specimens is necessary to reflect the actual nature of tissues and enable examination.
We take content rights seriously. If you suspect this is your content, claim it here.
0 ratings0% found this document useful (0 votes)
160 views5 pages
Histology 1
This document discusses the preparation of histological specimens. It describes the various stages of processing tissues, including fixation, dehydration using ethanol or other agents, clearing with xylene or other substances, and embedding in paraffin wax. Sections are then cut from the embedded tissue using a microtome and stained, often with hematoxylin and eosin, for examination under a microscope. Histology is the study of tissues and how they are arranged into organs at a microscopic level. Proper preparation of specimens is necessary to reflect the actual nature of tissues and enable examination.
We take content rights seriously. If you suspect this is your content, claim it here.
You are on page 1/ 5
PREPARATION OF HISTOLOGICAL SPECIMENS rigidity to enable thin sections to be cut, and yet soft
enough not to damage the knife or tissue.
Histology is the study of tissues and how these tissues are arranged into organs Stages of processing: Histo in Greek means tissue or web Dehydration. Tissues consist of cells and extra cellular matrix Clearing. The function of the tissue depends on the Embedding. interaction between cells and extracellular matrix. Dehydration: The small size of cells and matrix content make the study of tissues dependent on microscope To remove fixative and water from the tissue and other advances in biological techniques and replace them with dehydrating fluid. Delicate specimens are dehydrated in a graded TISSUE PREPARATION ethanol series from water through 10%-20%- 50%-95%-100% ethanol to minimize tissue The most widely used method of studying distortion from diffusion currents. tissues is using histological slides. In the paraffin method, dehydration from The tissue in the slide must reflect the actual aqueous fixatives is usually initiated in 60%- nature of the tissue in the body 70% ethanol, progressing through 90%-95% To insure that, tissues to be studied must pass ethanol, absolute ethanol before proceeding to through a series of steps before examination the clearing stage. These steps are in sequential order Fixation is a complex series of chemical events Types of dehydrating agents that differ for the different groups of substance found in tissues. Ethanol Methanol TISSUE FIXATION Acetone Aim of Fixation: Tissues may be held and stored indefinitely in 70% ethanol without harm 1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their Clearing volume and shape during processing. 3- To prepare tissue and leave it in a condition Replacing the dehydrating fluid with a fluid that which allow clear staining of sections. is totally miscible with both the dehydrating 4- To leave tissue as close as their living state as fluid and the embedding medium. possible, and no small molecules should be lost. Choice of a clearing agent depends upon many factors Fixation is a reaction between the fixative and proteins in the specimen which form a gel, so keeping everything Types of Clearing Agents: as their in vivo relation to each Xylene. Types of Fixative: Toluene. Chloroform. Acetic acid Benzene. Formaldehyde 10% Propylene oxide Ethanol Glutaraldehyde Embedding Methanol Is the process by which tissues are surrounded Picric acid by a medium such as agar, gelatin, or wax which Osmic acid (Osmium tetroxide) when solidified will provide sufficient external TISSUE PROCESSING support during sectioning. A substances added alone or in combination to Aim: Is to embed the tissue in a solid medium firm the wax to: enough to support the tissue and give it sufficient o Improve ribboning. o Increase hardness. o Decrease melting point Staining machine o Improve adhesion between specimen and wax Basic dyes stain:
Embedding Moulds: Heterochromatin
Nucleic acids paper boat mould Ribosomes metal boat mould Cartilage Dimmock embedding mould Peel-a-way disposable mould Acidic dyes stain: Base mould used with embedding ring ( F) or Filaments cassette bases (G) Mitochondria CUTTING - using the microtome Collagen Muscle fibers A microtome is a mechanical instrument used to cut biological specimens into very thin Additional Dyes sections for microscopic examination. Many tissue components can not be stained Most microtomes use a steel blade and are with (Hematoxylene and Eosin). used to prepare sections of animal or plant Other dyes are used to specifically stain certain tissues for histology. tissue components Resorcin-Fuchsin for elastic Microtome knives fibers Silver stain for reticular fibers and basement membrane Periodic-Acid Schiff (PAS) Steel knives Reaction for CHO Non-corrosive knives for cryostats Disposable metal blades Glass knives Diamond knives STAINING Hematoxylin and Eosin (H & E)
H & E is a charge-based, general purpose stain.
Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections. Staining Procedure:
Deparaffinize and hydrate to water
If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo) Mayer's hematoxylin for 15 minutes Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counterstain desired Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed Clear in xylene, two changes of 2 minutes each Mount in Permount or Histoclad “HISTOLOGY” - the science of the tissues Purkinje (1787-1869), a Czech biologist, in 1830. histos is Greek for web or tissue Robert Brown (1773-1858), a Scottish botanist logia is Greek for branch of learning introduced the term nucleus in microscopy Tissue was first used to describe the different after the examination of epidermal cell of some textures of body parts being dissected by an orchids and some Asclepiadacea in 1831. anatomist. Cell as the fundamental unit that constitutes all the “Microanatomy” generality of animals and plants the study of the structure (ANATOMY) of small Matthias Schleiden (1804-1881), a German (MICRO) things. botanist, in 1838; He saw, under the small things cells and their arrangement to microscope, thousands of plant specimens, and constitute tissues and the association among inferred that all the vegetables are made of these to form organs. cell. HISTOLOGY Theodor Schwann (1810-1882), a German History and Background zoologist and physiologist, in 1839; He came to the conclusion that all the then known living Who discovered the cell? beings were composed of cells. “CELL THEORY” Robert Hooke (1635-1703) All living cells originated from pre-existing ones. Micrographia, published in 1665 Rudolf Virchow (1821-1902), upon examining a piece of cork with a rudimentary microscope, saw an abundance of a great German pathologist demonstrated that empty small compartments the pathological injuries also had a cellular the “cell” was discovered! structure. cell walls in cork tissue – empty cell the pioneer of the cellular pathology. Latin, cellula;“ CELL” edified the bases of the modern Pathology. In the sequence of the works of Theodor History and Background Schwann, Virchow saw (1855/56) that all the cells of the pathological tissues are derived Transition from "empty cell” to the actual cell from healthy ones and that all living cells 1665 – same year as discovery of cell, Hooke originated from pre-existing ones. and Marcello Malpighi were the first to Law of Virchow (or Fundamental Law of observe the true units that form the tissues of Biology) animals. Omnis cellula e cellula ; that is, each cell comes 1678, after Leeuwenhoek reported of from another cell (1858). discovering "little animals” (animalcules) -- “TISSUE” - - texture bacteria and protozoa to the Royal Society. Hooke confirmed Leeuwenhoek’s findings. “21 Textures” of Marie François Bichat Hooke noted that Leeuwenhoek's simple a French pathologist (1771-1802) microscopes gave clearer images than his Bichat verified that certain textures presented a compound microscope, but found simple thin thickness and were very flat, to the extent microscopes difficult to use. of being compared to pieces of cloth. (French, tissu - means to weave). Discovery of nucleus The first notion of “tissue”. The first description of the nucleus was carried A texture was a “tissue” (a body component) as out by Leeuwenhoek, in 1700, when examining perceived by its macroscopic physical the red blood cells of the salmon. properties. The first description of the nuclear envelope was accomplished by Jan Evangelista The Father of Histology Bichat vs Malpighi
Bichat is considered, by some authors, to be
The first textbook of Histology the founder of Animal Histology. He is virtually, the first histologist. Handbuch der Gewebelehre des BUT…. Menschen (The book for teaching tissues); a landmark Marcello Malpighi (1628-1694), an Italian textbook anatomist considered the true “Father of Author: Rudolph Albert von Kölliker(1817- Histology”. 1905) a Swiss professor of Anatomy published in 1852 WHY??? Leeuwenhoek could not be the inventor of the Malpighi preceded Bichat in a number of years; microscope, nor Malpighi. Bichat never used a microscope; His textures were described based on dissections. Hans Janssen (died about 1595) & Zacharias Although Malpighi used a rudimentary Janssen (1580? -1640) - The first who microscope, he described a series of designed a microscope in the Netherlands late microscopic structures. in the 16th century were working as spectacle He was the first scientist to observe the makers. capillaries. Hans and his son Zacharias Janssen are Marcello Malpighi mentioned in the letters of William Boreel (the Dutch envoy to the Court of France) as also called the "Father of Microscopic having invented a 20X magnification Anatomy" microscope. Malpighi performed his greatest feat when However Jan Swammerdam, Robert Hooke he observed blood circulating through and Malpighi were the first scientists who capillaries and recorded what he saw. used microscopes for their research in Definition of “tissue” histology.
Tissue is each of the elementary multicellular Modern: Compound Light MICROSCOPE
components, microscopically and functionally Calibration factors: distinct, that constitute either animals or plants where they associate to form organs and Scanner = 125m systems. LPO = 50 m HPO = 12.5 m “HISTOLOGY” OIO = 5 m August Mayer (1787-1865) coined the term Size of object = no. ocular units X CFused histologie Histological Anatomy from the Greek words (histos, "web" or "warp") and (logos, "word" or "treatise") Advances in chemistry, physiology, to mean "the study of the tissues of the body” immunology, and pathology—and the The term came into common usage in the interactions among these fields—are essential 1840s. for a better knowledge of tissue biology. In 1844, it was recommended for large usage Familiarity with the tools and methods is by Sir Richard Owen (1804-1892), a great essential for a proper understanding of the English palaeontologist. subject. The English word histology probably came from How tissues are prepared for observation the German histologie via the French histologie. The preparation of tissues for microscopic examination For light to pass through, tissues and organs up with a slide coated with a thin film of albumin or must be sectioned to obtain thin, translucent Haupt’s adhesive and allowed to dry. sections. 7. Staining – use of dyes to penetrate into [However, living cells, very thin layers of tissues, translucent tissue section. Basic and acidic dyes or transparent membranes of living animals (Ex: impart contrast to micro-sectioned tissues allowing mesentery, tadpole tail, the wall of cheek) can the observer to study the cells and its ultra- be observed directly in the microscope without structures and the matrix. micro-sectioning.] Most tissues need thin slices using microtome. Behavior of tissues to dyes This method preserves tissues in longer Hematoxylin and Eosin (H&E) – commonly duration under varying physiological or used dyes experimental conditions. Basic dyes – hematoxylin, toluidine blue, Microtome methylene blue stain basophilic structures such as nucleus, free ribosomes, matrix of hyaline Histotechniques for paraffin sections using rotary cartilage. microtome Acid dyes – orange G, eosin, acid fuchsin stain Fixation – prevents autolysis (self-destruction) the acidophilic components of tissues such as or bacterial invasion of tissues leading to tissue mitochondria, secretory granules, cytoplasm distortion and degradation and collagen. Use of fixative chemicals as stabilizers or cross- Trichromes – differentiating stains linkers maintaining the molecular and structural Counterstain – a single stain that is applied to composition of tissues. a section to allow the recognition of nuclei or Formaldehyde and glutaraldehyde are cytoplasm, is used. widely used fixatives which react with the Metallic dyes – silver or gold is ideal for amine groups (NH2) of tissue proteins. nervous tissue. Glutaraldehyde, fixing action is reinforced since it is a dialdehyde, can cross-link proteins. Tissue processing–Involves dehydration (ethanol) and clearing (xylene).
1. Dehydration - It is the first part of the process. It is
usually accomplished by transferring the block of tissue through a series of alcohol-water solutions beginning with 50 percent and running up to water-free or absolute alcohol. 2. Clearing - The alcohol is replaced by xylene or cedar oil, which is readily soluble in alcohol, and in turn, is replaced by melted paraffin. 3. Paraffin Impregnation – melted paraffin infiltrates the tissue gradually. 4. Embedding - paraffin- infiltrated tissue is placed in fresh paraffin and the latter allowed to cool. Paraffin or plastic resins give a rigid consistency to the tissue. Result: Tissue block - solid to facilitate sectioning. 5. Tissue sectioning – mounting the tissue block in the microtome 6. Affixing section on slide – section is floated on a warm water-bath to expand / flatten; and picked