Introduction to Histology and Histochemistry

Definitions and Historical Context of Histology

Histology is a discipline whose name originates from the Greek words "histos," meaning tissue, and "logia," meaning the study of or knowledge. In a strict sense, it represents the comprehensive knowledge or study of tissues. Formally, histology is defined as the microscopic study of normal tissues, a process achieved by examining thin slices, known as sections, of tissue using either a light microscope or an electron microscope.

The history of histology is inextricably linked to the development of microscopy. In 1665, Robert Hooke became the first individual to observe cells. By examining thin slices of cork under a rudimentary microscope, he noted that the material appeared as a series of small boxes, which he termed "cells." Nearly two centuries later, in 1855, Rudolf Virchow contributed the principle that new cells arise exclusively from the division of pre-existing cells and observed that the chemical reactions necessary for life occur within the cell. In 1883, Mathias Schleiden and Theodor Schwann proposed the foundational concept that all plants and animals are composed of cells, identifying them as the basic building blocks of life. Collectively, these scientific contributions led to the establishment of the cell theory.

Biological Levels of Organization

Life is organized into a hierarchical structure beginning with the cell and progressing toward the whole organism. A cell is defined as the smallest basic structure of higher organisms that is capable of independent existence. When a group of cells with similar functions and origins come together to form functional units, they are classified as a tissue. An organ represents a higher level of complexity, as it is composed of various distinct tissues working together. A system, or organ system, is formed by the combination of several organs. Finally, the sum of these systems constitutes the complete organism.

Tissue Procurement and Initial Preparation

Histological samples are obtained from normal tissues through several clinical or pathological methods including surgery, biopsy, autopsy, or necropsy. A biopsy refers specifically to the examination of tissue removed from a living body. An autopsy is the examination of post-mortem tissue from a human, whereas a necropsy describes the examination of tissue taken from a dead animal.

There are three primary types of histological preparations. The first is a whole mount, which involves the entire organism, such as a fungus or parasite, typically measuring between $0.2\,mm$ and $0.5\,mm$ in thickness. The second type involves sections, where tissue is initially cut into pieces approximately $3-5\,mm$ thick before being processed into much thinner slices. Specifically, sections of $5\,\mu m$ (microns) in thickness are cut using a microtome. Microtomes are specialized instruments equipped with an automatic mechanism designed for cutting extremely thin sections; however, the tissue must be hardened before this is possible. Hardening is achieved through two primary methods: freezing or embedding. The third preparation type is the smear, which is created from blood, bone marrow, or other body fluids.

Tissue Processing: Fixation and Dehydration

The initial stage of tissue processing is fixation, which is the preservation of tissue to avoid autolysis (self-digestion) and putrefaction (bacterial decay). It is mandatory that all tissue specimens are labeled during this stage. Common chemical fixatives include Formaldehyde, Mercuric chloride, Potassium dichromate, Osmium Tetraoxide, $70\%$ alcohol, and picric acid. Fixatives are applied through immersion or perfusion, such as intracardiac perfusion. The procedure involves the dissection of the sample, followed by trimming and orientation, and then immersion fixation within a tissue cassette. Consideration must be given to the sample size and the duration of exposure to the fixative.

Following fixation, the tissue undergoes dehydration, which is the removal of water. This step is a necessary rationale for subsequent paraffin embedding and sectioning. The process begins by washing out the fixative, followed by immersing the tissue in a graded series of alcohol concentrations, specifically $70\%$, $95\%$, and $100\%$. This allows water to be replaced by alcohol via diffusion. This process must be timed precisely, as it should be neither too long nor too short. Dehydration is typically performed overnight using an automated tissue processor featuring successive baths of water and the various alcohol grades.

Clearing, Wax Impregnation, and Embedding

Clearing is the process of removing alcohol from the tissue. This is achieved using a "clearing agent" such as xylene, which also acts as a paraffin solvent. This step is so named because it makes the tissue appear translucent or "clear." Once cleared, the tissue undergoes wax impregnation, where the xylene is replaced with paraffin. The tissue is immersed in two baths of melted paraffin maintained at a melting point of approximately $55^\circ C$. During this stage, it is critical to remove all air bubbles and residual xylene.

Embedding is the step taken to obtain a solid block containing the tissue. While paraffin wax is the most common embedding medium, other materials such as ester wax or cellulose nitrate may be used. The procedure involves placing the tissue cassette into melted paraffin, filling a mold with paraffin, positioning the tissue precisely within that mold, and allowing the assembly to cool until it solidifies.

Sectioning and Mounting

Sectioning involves trimming the solid block to prepare it for the microtome. Various types of microtomes may be used, including the rotary microtome, cryostat, freezing microtome, or vibratome. The rotary microtome is used to produce sections of $5-10\,mm$ (as specified in the protocol), balancing resolution and staining requirements. The procedure requires placing the tissue block in the microtome with the wide edge of the trapezoid shape at the lowest point and parallel to the knife, then advancing the blade toward the block.

Once cut, sections are mounted onto glass slides. The sections are first placed in a $40^\circ C$ water bath, which serves to flatten the paraffin. This enables the section to be picked up onto a glass slide, often with the help of adhesives like gelatin and albumin. Finally, the slides are dried in an oven or air-dried.

Staining, Coverslipping, and Decalcification

Staining is performed to provide contrast and identify cellular structures. Hematoxylin is a basic dye that targets basophilic structures such as DNA and RNA; a differentiation step using sodium bicarbonate is often employed. Eosin is an acid dye that targets acidophilic (or eosinophilic) structures, including mitochondria and collagen. Automated stainers are frequently used for this process.

After staining, coverslipping is performed. This involves covering the stained section with a thin piece of glass to protect the tissue from being scratched and to preserve the section for long-term storage.

Decalcification is a specialized process used for tissues containing calcium salts, such as bone and teeth, following their initial fixation. This process ensures the specimen is soft enough to be cut by a microtome knife. Failure to completely decalcify tissue will result in torn, ragged sections and may damage the cutting edge of the microtome knife. The fixative of choice for bone or bone marrow is Zenker formal or Bouin's fluid. The decalcification process itself takes approximately $2-3$ hours and must be completed before embedding.

Decalcifying agents include strong mineral acids like nitric and hydrochloric acids, which remove large quantities of calcium rapidly but may damage cellular morphology. Organic acids, such as acetic and formic acid, act more slowly, particularly on dense cortical bone. EDTA (Ethylenediaminetetraacetic acid) can remove calcium safely and preserves morphology well, but it works slowly, penetrates tissue poorly, and is expensive when used in large quantities. Electrolysis is another available method. A high-quality decalcifying agent is characterized by the complete removal of calcium, the absence of damage to tissue cells or fibers, no alteration to subsequent staining, and a short required duration for the process.