The Histology of Fishes

Krzysztof Formicki, Frank Kirschbaum

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9780429532887
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Cell Biology
• Index: Biological Sciences

The book is a multi-authored book of 18 chapters comprising the state of the art work of all relevant topics on modern fish histology from 28 authors from ten countries. The topics include Introduction to Histological Techniques, Integument, Fish Skeletal Tissues, Muscular System, Structure and Function of Electric Organs, Digestive System, Glands of the Digestive Tract, Swim Bladder, Kidney, Ovaries and Eggs, Egg Envelopes, Testis Structure, Spermatogenesis, and Spermatozoa in Teleost Fishes, Cardiovascular System and Blood, Immune System of Fish, Gills: Respiration and Ionic-Osmoregulation, Sensory Organs, Morphology and Ecomorphology of the Fish Brain, and Endocrine System. Structural and functional aspects are treated and in a comparative way fish diversity at various taxonomic levels is integrated.

Chapter 1

Introduction to Histological Techniques

Anna Pecio^* and Rafal P. Piprek

The development of histological techniques for the past five centuries was propelled by the invention of the microscope and the improvement of its magnification and resolution. The greatest improvement of methods, allowing observation of plant and animal tissues, dates from the 18th and 19th centuries; whereas in the 20th century mainly the development of electron microscopical and other methods such as fluorescence and freezing techniques, as well as histochemistry and immunohistochemistry proceeded.

Independent of the microscope type used for the observation each sample requires specific histoprocessing techniques including fixation (chemical or physical), dehydration (e.g. in series of alcohols), embedding (e.g. in paraffin wax or plastic media) and staining (e.g. hematoxylin and eosin, trichrome methods, toluidine, and methylene blue etc.).

High resolution and magnification achieved in transmission electron microscopy requires specific methods of fixation (e.g. double fixation with glutaraldehyde and osmium tetroxide), infiltration and embedding in polymerizing plastics like epoxy or acrylic resins, cutting for ultrathin sections with diamond knifes and contrasting with uranyl acetate and lead citrate, whereas samples for scanning electron microscopy require drying after dehydration and vacuum sputtering with carbon or gold before observation.

Early Steps in Histology

Although today’s definition of histology sounds: “the scientific study of fine detail of biological cells and tissues of organisms performed by examining the structure under a light microscope or electron microscope”, we should be aware that the current state is the legacy of five centuries of history and evolution of histology, which goes hand-in-hand with that of the invention of the microscope and the improvement of its resolution.

First compound microscopes were invented probably around 1595 by the Dutch Zacharias Jensen. It was many years before the birth of two known persons: the Englishman Robert Hook (1635–1703) and Antony van Leeuwenhoek (1632–1723), who were making important discoveries with microscopes giving the major milestones in the history of histology in the 17th century. The first cellular observations were documented by Robert Hooke in 1665 in the illustrated book “Micrographia”. This volume described Hooke’s own experiments made with a microscope: flies, feathers and snowflakes, and he correctly identified fossils as remnants of once-living things. Hook was the first who published the record of the word “cell”, while discussing the structure of cork. “Micrographia” as a popular publication has inspired Antony van Leeuwenhoek, who grinded lenses to construct simple microscopes. Leeuwenhoek’s skill enabled him to build microscopes that magnified over 200 times. His curiosity to observe almost anything that could be placed under his lenses gave him the possibility to be the first who had seen living cells and tissues. All his observations were documented in letters, written since 1673 to the newly-formed Royal Society of London. Most of his descriptions of microorganisms are even nowadays recognizable.

Another excellent researcher was the Italian physician, anatomist, botanist, histologist and biologist Marcello Malpighi (1628–1694), who constructed one of the first microscopes for studying tiny biological entities. During his 40 years of research, he developed several methods to study living tissue and structures of plant and animals and initiated the science of microscopic anatomy, giving the foundations of major areas of research in botany, embryology, and human anatomy for future generations of biologists. As a tribute to his great discoveries, many microscopic anatomical structures were named after him: the basal layer, renal corpuscles, as well as insect excretory organs.

In the mid-18th century, Ernst Abbe working in Germany in the Zeiss Factory improved microscopes with spherical and chromatic aberration. The researchers, who studied until this time with the help of rudimentary microscopes, received new opportunities to study cellular details. At that time, new techniques using a variety of chemicals to preserve tissues in a life-like state and to prepare them for later staining had developed.

Further great milestones in the development of histology as an academic discipline occurred in the 19th century, when Marie François Xavier Bichat (1771–1802), a French anatomist and pathologist, known as the father of histology, introduced in 1801 the concept of “tissue” in anatomy. He distinguished 21 types of elementary tissues of which the organs of the human body are composed (Nafziger 2002). The term “histology” appeared nearly twenty years after that, in 1819, when a German anatomist and physiologist, Karl Mayer (1787–1865), published a book: “Ueber Histologie und eine neue Eintheilung der Gewebe des menschlichen Körpers” (in English: On histology and a new classification of tissues of the human body).

In 1906, the Nobel Prize in Physiology or Medicine was awarded to histologists: to the Italian pathologist Camillo Golgi (1843–1926) and the Spanish neuroscientist Santiago Ramon y Cajal (1852–1934), who gave the first descriptions of brain structure. Cajal won the prize for his original pioneering investigations of microscopical structures and Golgi for a revolutionary method of staining individual nerve and cell structures, which is termed “black reaction”. This method uses a weak solution of silver nitrate and is particularly valuable in tracing the processes and the most delicate ramifications of cells.

Histological Techniques

The examination of the details of cells and tissues using microscopes requires a proper preparation of the tissues dissected from the organisms. This special preparation of tissue is called “histological techniques” or histoprocessing. A process of tissue preparation allowing the examination of normal tissue structure or cell details on the slides under the microscope takes place in the following steps: fixation, dehydration, clearing, infiltration, embedding, microtomical cutting, staining, mounting.

Fixation is the process of preservation of tissues in its normal condition; it means to preserve the cell volume and fix all chemical components: proteins, carbohydrates, fats, etc. The material may be fixed using physical or chemical methods. The simplest physical method is heating specimens immersed in boiling saline or by cooking in a microwave oven, which causes the coagulation of proteins and melting of lipids (Bernhard 1974). The opposite method, by freezing, requires specimens to be immersed in isopentane cooled to its freezing points (−170ºC), in liquid nitrogen (−196ºC) or solid carbon dioxide (−75ºC). Fixation by freezing is a rapid method especially useful in histochemistry and immunochemistry because of preserving proteins (Pearse 1980, Bald 1983).

Chemical methods are very popular and used in laboratories for most histological and histochemical purposes. They were used also by early histologists, who used potassium dichromate, alcohol and the mercuric chloride to harden tissues. All nowadays used chemicals have additional properties such as: preventing shrinkage or swelling, rapid rate of penetration, which allows the fixing of a specimen of 3–5 mm within 24 hours and preserves tissue from degradation. The appropriate rate of penetration allows the maintaining of the structure of the cell and of sub-cellular components such as cell organelles (e.g., nucleus, endoplasmic reticulum, mitochondria, etc.).

The chemical fixatives are administered in two ways: (1) through perfusion (fixatives are infused in the animals’ body through diffusion in a very short time) or (2) through immersion of the prepared tissue in fixative solution. Fixatives may be simple, e.g.: aldehydes (formaldehyde, glutaraldehyde), acetone, alcohols (ethanol or methanol), acids (glacid acetic acid, picric acid, trichloroacetic acid) and many others such as mercuric chloride, chromium trioxide, osmium tetroxide, etc., and compound fluids (e.g.: Bouin’s fluid, Carnoy’s fluid, Zenker’s fluid).

The main action of aldehyde fixatives is to cross-link amino groups in proteins through the formation of methylene bridges (−CH₂−), in the case of formaldehyde, or by C5H10 cross-links in the case of glutaraldehyde. Buffered aqueous 2–5% solution of formaldehyde at pH 7.2–7.4 is used in routine practice for most histological and histochemical examination in light microscopy. This solution is an appropriate fixative as well for immersion or perfusion fixation (Gerrits and Horobin 1996). Formaldehyde reacts with several parts of protein molecules and preserves most lipids and carbohydrates because it does not react with them (Kiernan 1990). However, formaldehyde fixation leads to degradation of mRNA, miRNA and DNA in tissues. For nucleic acids, the recommended fixative is acetic acid, but it does not fix proteins though.

Glutaraldehyde is the most widely used fixative for electron microscopy, usually as a 2.5% solution in phosphate buffered to pH 7.2–7.4. As single fixative, it is useful only when small pieces (0.5–1.0 mm) are processed. In larger specimens, glutaraldehyde causes a tightly cross-linked proteinaceous matrix that is difficult to penetrate (Gerrits and Horrobin 1996). This process, while preserving the structural integrity of the cells and tissue, can damage the biological functionality of proteins, particularly enzymes, and can also denature them.

Aldehyde fixation does not add electron density to tissues, so post fixation in osmium tetroxide is usually practiced. Additionally, osmium tetroxide is used as secondary fixative, increasing electron density and is also effective as stain.

Methanol, ethanol, and acetone may be used alone for fixing films and smears of cell or unfixed cryostat sections, but they are not suitable for blocks of tissue because they cause considerable shrinkage and hardening.

Trichloroacetic and picric acids are widely useful by biochemists for the demonstration of proteins because they precipitate them and cause coagulation forming salts with the basic group of proteins. Acetic acid does not fix the proteins, but it coagulates nucleic acids and is included in fixative mixtures to preserve chromosomes.

All above mentioned liquids are used as individual fixatives, but histologist and biochemists more often employ a mixture of different agents in order to offset undesirable effects of individual substances and to obtain more than one of the chemical fixation.

For general histology and for the preservation of nucleic acids and macromolecular carbohydrates, the best result is given by non-aqueous, Carnoy’s fluid. The others, Bouin’s and Zenker’s fluids, are aqueous fixatives excellent for preserving morphological features, but are not compatible with histochemical techniques.

The choice of fixative depends first of all on the structural or chemical components of the tissue that are to be demonstrated. However, some fixatives (e.g., Zenker’s fluid, osmium tetroxide) are unsuitable for glycol methacrylate embedding procedures (GMA), since they contain substances which interfere with the polymerization reaction (Gerrits and Horobin 1996).

Dehydration is the processing of removing all water from tissues. This process is followed by clearing and infiltration, which allows for the penetration by a medium, in which the tissue is finally embedded (paraffin wax, celloidin, plastic media). Paraffin and celloidin infiltration developed at the end of the 19th century (Klebs 1869, Duval 1897). Celloidin is also a common embedding medium, which allows the assessment of excellent morphologic details, but is difficult to remove and puts significant restrictions on success with immunostaining.

Nowadays, the most frequently used medium for light microscopy is paraffin wax. Dehydration in alcohol is followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax. The most commonly used paraffin wax is usually a mixture of straight chain or n-alkanes with a carbon chain. Paraffin wax can be purchased with melting points at different temperatures, the most common for histological use being about 56ºC–58ºC, but often the temperature is increased to 60ºC to decrease viscosity and improve infiltration. Today’s companies offer a wide assortment of paraffin waxes: e.g., Paraplast, Paramat, in which added plastic polymers increased the paraffin elasticity and improved tissue penetration or Carbowax Polyethylene Glycol, which is an excellent embedding medium for histochemistry.

Embedding in paraffin allows immunostaining to be performed, but preservation of cellular details is relatively poor. The sections embedded in paraffin media can be cut typically at 5 μm, but it does not provide a sufficiently hard matrix for cutting very thin sections. To enhance cutting of thin sections of the tissues, plastic embedding media (methacrylates, polyester resins, epoxy resins) were introduced in the mid-20th century (Litwin 1985). These media facilitate obtaining semithin sections (0.5–2.0 μm) for light microscopical examination, but also allow ultrathin sectioning of biological specimens for electron microscopy. The epoxy resins are definitely the most popular embedding media for electron microscopy at present, whereas the glycol methacrylate has remained in use for light microscopy because of its large number of advantages, when compared to embedding media such as paraffin or celloidin (Litwin 1985, Gerrits and Horobin 1996, Titford 2009). Glycol methacrylate is polar, which means it is permeable to aqueous solutions of stains and reagents and provides the possibility to perform histochemical reactions without removing it (Leduc and Bernhard 1962). Shrinkage of tissues after is very limited and Glycol methacrylate is therefore suitable for quantitative investigations as well for embedding of heterogeneous tissues, including uncalcified bone (Chappard et al. 1983, Hanstede and Gerrits 1983). Glycol methacrylate embedding can be carried out in the cold or at ambient temperature, and thus allows to demonstrate enzymatic activity on sections in contrast to paraffin embedding performed at 60ºC (Higuchi et al. 1979).

Now, the commercially available plastic media such as Technovit 7100 and HistoResin are widely used and less toxic (Gerrits and Smid 1983).

Staining is a technique used in microscopy to enhance contrast and highlight colors of various components of the tissue for viewing in the microscopic image. Antony van Leeuwenhoek was the first, who in 1673 used dye extracted from the saffron crocus bulb to study his specimens. In the 18th century, dyes used in histological staining were the naturally occurring dyes of plants (madder, saffron, indigo), animals (shellfish purple, cochineal), and minerals. Researchers found numerous ways to use dyes to stain tissues because of their extremely universal character. For example, carmine is one of the first dyes used by early botanists (e.g., John Hill who published his work in 1770) and it is still used for staining chromosomes and nuclei (Clark and Kasten 1983, Bracegirdle 1986). The stained compounds—cochineal (= carminic acid) is obtained from the dried Mexican insect Coccus cacti. The dried and grinded bodies of females treated with alum and calcium salts result in carmine—a bright red color (Dapson 2007). In nowadays’ histology, carmine with potassium salt is used to stain glycogen in popular Best’s carmine method (published in 1906) or used for counterstaining of fat in frozen sections by modified Grenatcher’s and Orth methods (Clark 1981). Another dye, still useful in laboratories today because of its versatility, is haematoxylin. It is known since 1863, when it was used by Wilhelm van Waldeyer, a German anatomist. Haemtoxylin is obtained from the logwood tree, Haematoxylon campechianum, and is a weak dye applied in different solutions, based on its oxidized form, haematein. Haematein with an oxidizer as a mordant can be used to identify a variety of cellular acidic structures, staining them blue-purple (Titford 2005).

The development of histological staining was accelerated by the invention of the first synthetic dye in 1856 by William Perkin. These aniline dyes became more and more popular in the industry and many of them proved to be useful in histological staining techniques (Titford 2009).

A dye molecule contains two elements: a chromogen and an auxochrome. The chromogen is a colored part and consists of a specific arrangement of atoms—a chromophore, which is responsible for the absorption of visible light. The auxochrome, responsible for attaching the chromogen to the substrate, is a part of the ionizable substituent, or a substituent that reacts to form a covalent bond or coordinate with metal (mordant) ion. When auxochromes are amines, they are classified as basic (cationic) dyes, whereas when they are derived from carboxylic, sulphonic acids or from phenolic hydroxyl groups, they are acid (anionic) dyes.

Staining of sections, after removing the paraffin wax, is performed in large excess of staining solution at room temperature. When a solution of a dye is allowed to act slowly until the desired effect is obtained the staining is progressive, whereas in regressive staining the tissue is overstained, and is followed by the process of differentiation, which is a controlled removal of the dye in ...

Table of contents

• Cover
• Title Page
• Copyright Page
• Preface
• Table of Contents
• 1. Introduction to Histological Techniques
• 2. Integument
• 3. Fish Skeletal Tissues
• 4. Muscular System
• 5. Structure and Function of Electric Organs
• 6. Digestive System
• 7. Glands of the Digestive Tract
• 8. Swim Bladder
• 9. Kidney
• 10. Ovaries and Eggs
• 11. Egg Envelopes
• 12. Testis Structure, Spermatogenesis, and Spermatozoa in Teleost Fishes
• 13. Cardiovascular System and Blood
• 14. Immune System of Fish
• 15. Gills: Respiration and Ionic-Osmoregulation
• 16. Sensory Organs
• 17. Morphology and Ecomorphology of the Fish Brain: The Rhombencephalon of Actinopterygians
• 18. Endocrine System
• Index
• Family Names Index
• English Names Index
• Species Index
• Color Plate Section