Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are used in biology and medicine to highlight structures in biological tissues for viewing. Stains may be used to define and examine bulk tissues, cell populations, in biochemistry it involves adding a class-specific dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging can serve similar purposes, biological staining is used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis. Simple staining is staining with only one stain/dye, there are various kinds of multiple staining, many of which are examples of counterstaining, differential staining, or both, including double staining and triple staining. In vivo staining is the process of dyeing living tissues—in vivo means in life, by causing certain cells or structures to take on contrasting colour, their form or position within a cell or tissue can be readily seen and studied.
In vitro staining involves colouring cells or structures that have removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone, combined with specific protocols for fixation and sample preparation and physicians can use these standard techniques as consistent, repeatable diagnostic tools. A counterstain is stain that makes cells or structures more visible, for example, crystal violet stains only Gram-positive bacteria in Gram staining. A safranin counterstain is applied that stains all cells, allowing identification of Gram-negative bacteria, while ex vivo, many cells continue to live and metabolize until they are fixed. Some staining methods are based on this property and those stains excluded by the living cells but taken up by the already dead cells are called vital stains. Those that enter and stain living cells are called supravital stains, these stains are eventually toxic to the organism, some more so than others.
To achieve desired effects, the stains are used in very dilute solutions ranging from 1,5000 to 1,500000, note that many stains may be used in both living and fixed cells. The preparatory steps involved depend on the type of analysis planned, fixation–which may itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill and alter the specimen so it accepts stains, most chemical fixatives generate chemical bonds between proteins and other substances within the sample, increasing their rigidity. Common fixatives include formaldehyde, methanol, and/or picric acid, pieces of tissue may be embedded in paraffin wax to increase their mechanical strength and stability and to make them easier to cut into thin slices. Permeabilization involves treatment of cells with a mild surfactant and this treatment dissolves cell membranes, and allows larger dye molecules into the cells interior. Mounting usually involves attaching the samples to a microscope slide for observation
A carbohydrate is a biological molecule consisting of carbon and oxygen atoms, usually with a hydrogen–oxygen atom ratio of 2,1, in other words, with the empirical formula Cmn. This formula holds true for monosaccharides, some exceptions exist, for example, deoxyribose, a sugar component of DNA, has the empirical formula C5H10O4. Carbohydrates are technically hydrates of carbon, structurally it is accurate to view them as polyhydroxy aldehydes and ketones. The term is most common in biochemistry, where it is a synonym of saccharide, a group that includes sugars, the saccharides are divided into four chemical groups, disaccharides and polysaccharides. Monosaccharides and disaccharides, the smallest carbohydrates, are referred to as sugars. The word saccharide comes from the Greek word σάκχαρον, meaning sugar, while the scientific nomenclature of carbohydrates is complex, the names of the monosaccharides and disaccharides very often end in the suffix -ose. For example, grape sugar is the glucose, cane sugar is the disaccharide sucrose.
Carbohydrates perform numerous roles in living organisms, polysaccharides serve for the storage of energy and as structural components. The 5-carbon monosaccharide ribose is an important component of coenzymes and the backbone of the genetic molecule known as RNA, the related deoxyribose is a component of DNA. Saccharides and their derivatives include many other important biomolecules that play key roles in the system, preventing pathogenesis, blood clotting. In food science and in informal contexts, the term carbohydrate often means any food that is particularly rich in the complex carbohydrate starch or simple carbohydrates. Often in lists of information, such as the USDA National Nutrient Database, the term carbohydrate is used for everything other than water, fat, ash. This will include chemical compounds such as acetic or lactic acid, carbohydrates are found in wide variety of foods. The important sources are cereals, sugarcane, table sugar, milk and sugar are the important carbohydrates in our diet.
Starch is abundant in potatoes, maize and other cereals, sugar appears in our diet mainly as sucrose which is added to drinks and many prepared foods such as jam and cakes. Glucose and fructose are found naturally in fruits and some vegetables. Glycogen is carbohydrate found in the liver and muscles, cellulose in the cell wall of all plant tissue is a carbohydrate. It is important in our diet as fibre which helps to maintain a healthy digestive system, formerly the name carbohydrate was used in chemistry for any compound with the formula Cm n
Giemsa stain, named after German chemist and bacteriologist Gustav Giemsa, is used in cytogenetics and for the histopathological diagnosis of malaria and other parasites. It is specific for the groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. Giemsa stain is used in Giemsa banding, commonly called G-banding, to stain chromosomes and it can identify chromosomal aberrations such as translocations and rearrangements. It stains the trophozoite Trichomonas vaginalis, which presents with greenish discharge, Giemsa stain is a differential stain, such as when it is combined with Wright stain to form Wright-Giemsa stain. It can be used to study the adherence of bacteria to human cells. It differentially stains human and bacterial cells purple and pink respectively and it can be used for histopathological diagnosis of malaria and some other spirochete and protozoan blood parasites. It is used in Wolbachia cell stain in Drosophila melanogaster, Giemsa stain is a classic blood film stain for peripheral blood smears and bone marrow specimens.
Erythrocytes stain pink, platelets show a pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue. It is used to visualize the classic safety pin shape in Yersinia pestis, Giemsa stain is used to visualize chromosomes. Giemsa stains the fungus Histoplasma, Chlamydia bacteria, and can be used to identify mast cells, giemsas solution is a mixture of methylene blue and Azure B. The stain is usually prepared from commercially available Giemsa powder, a thin film of the specimen on a microscope slide is fixed in pure methanol for 30 seconds, by immersing it or by putting a few drops of methanol on the slide. The slide is immersed in a freshly prepared 5% Giemsa stain solution for 20–30 minutes, flushed with tap water, biological stains and staining protocols Histology Leishman stain Microscopy Romanowsky stain Wrights stain
Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red and this is the classic counterstain in both Gram stains, and endospore staining. It can be used for the detection of cartilage, safranin typically has the chemical structure shown at right. There is trimethyl safranin, which has a methyl group in the ortho- position of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin do not distinguish between the two, commercial safranin preparations often contain a blend of both types. Safranin is used as indicator in analytical chemistry. Safranines are the compounds of symmetrical 2, 8-dimethyl-3, 7-diamino-phenazine. They are crystalline solids showing a green metallic lustre, they are readily soluble in water. They are strong bases and form stable monacid salts and their alcoholic solution shows a yellow-red fluorescence.
Phenosafranine is not very stable in the state, its chloride forms green plates. It can be readily diazotized, and the salt when boiled with alcohol yields aposafranine or benzene induline. F. Kehrmann showed that aposafranine could be diazotized in the presence of concentrated sulfuric acid. Aposafranone, C18H12N2O, is formed by heating aposafranine with concentrated hydrochloric acid and these three compounds are perhaps to be represented as ortho- or as para-quinones. The safranine of commerce is an ortho-tolusafranine, the first aniline dye-stuff to be prepared on a manufacturing scale was mauveine, which was obtained by Sir William Henry Perkin by heating crude aniline with potassium bichromate and sulfuric acid. Mauveine was converted to parasafranine by Perkin in 1878 by oxidative/reductive loss of the 7-N-para-tolyl group, another well known safranin is phenosafranine widely used as histological dye and redox probe. This article incorporates text from a now in the public domain, Hugh
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is commonly performed by examining cells and tissues under a microscope or electron microscope, the specimen having been sectioned, stained. Histological studies may be conducted using tissue culture, where human or animal cells are isolated and maintained in an artificial environment for various research projects. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of histological stains, histology is an essential tool of biology and medicine. Trained physicians, frequently licensed pathologists, are the personnel who perform histopathological examination and their field of study is called histotechnology. In the 17th century, Italian Marcello Malpighi invented one of the first microscopes for studying tiny biological entities, Malpighi analysed several parts of the organs of bats and other animals under the microscope. Malpighi, while studying the structure of the lung, noticed its membranous alveoli and his discovery established how the oxygen we breathe enters the blood stream and serves the body.
In the 19th century, histology was a discipline in its own right. The French anatomist Bichat introduced the concept of tissue in anatomy in 1801, the 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had dueling interpretations of the structure of the brain based in differing interpretations of the same images. Cajal won the prize for his theory and Golgi for the staining technique he invented to make it possible. There are four types of animal tissues, muscle tissue, nervous tissue, connective tissue. All tissue types are subtypes of these four basic tissue types and their structure is very different from animal tissues. The most common fixative for light microscopy is 10% neutral buffered formalin, for electron microscopy, the most commonly used fixative is glutaraldehyde, usually as a 2. 5% solution in phosphate buffered saline. These fixatives preserve tissues or cells mainly by irreversibly cross-linking proteins and this can be detrimental to certain histological techniques.
Further fixatives are often used for electron microscopy such as osmium tetroxide or uranyl acetate Formalin fixation leads to degradation of mRNA, miRNA and DNA in tissues, extraction and analysis of these nucleic acids from formalin-fixed, paraffin-embedded tissues is possible using appropriate protocols. Frozen section procedure is a way to fix and mount histology sections using a refrigeration device called a cryostat. It is often used after surgical removal of tumors to allow determination of margin
Auramine O is a diarylmethane dye used as a fluorescent stain. In its pure form, Auramine O appears as yellow needle crystals and it is very soluble in water and soluble in ethanol. Auramine O can be used to stain acid-fast bacteria in a way similar to Ziehl-Neelsen stain and it can be used as a fluorescent version of Schiff reagent. Auramine O can be used together with Rhodamine B as the Truant auramine-rhodamine stain for Mycobacterium tuberculosis and it can be used as an antiseptic agent
Methyl violet is a family of organic compounds that are mainly used as dyes. Depending on the number of attached methyl groups, the color of the dye can be altered and its main use is as a purple dye for textiles and to give deep violet colors in paint and ink. Methyl violet 10B is known as violet and has medical uses. The term methyl violet encompasses three compounds that differ in the number of groups attached to the amine functional group. They are all soluble in water, diethylene glycol, methyl violet 2B is a green powder which is soluble in water in ethanol and water, but not in xylene. It appears yellow in solution of low pH and changes to violet with pH increasing toward 3.2, methyl violet 10B has six methyl groups. It is known in medicine as Gentian violet and is the ingredient in a Gram stain. It is used as a pH indicator, with a range between 0 and 1.6, the protonated form is yellow, turning blue-violet above pH levels of 1.6. Gentian violet destroys cells and can be used as a disinfectant, compounds related to methyl violet are potential carcinogens.
Methyl violet 10B inhibits the growth of many Gram positive bacteria, when used in conjunction with nalidixic acid, it can be used to isolate the streptococci bacteria for the diagnosis of an infection. Methyl violet is a mutagen and mitotic poison, therefore concerns exist regarding the impact of the release of methyl violet into the environment. Methyl violet has been used in vast quantities for textile and paper dyeing, numerous methods have been developed to treat methyl violet pollution. The three most prominent are chemical bleaching and photodegradation, chemical bleaching is achieved by oxidation or reduction. Oxidation can destroy the dye completely, e. g. through the use of sodium hypochlorite or hydrogen peroxide, reduction of methyl violet occurs in microorganisms but can be attained chemically using sodium dithionite. Biodegradation has been investigated because of its relevance to sewage plants with specialized microorganisms. Two microorganisms that have studied in depth are the white rot fungus.
Light alone does not rapidly degrade methyl violet, but the process is accelerated upon the addition of large band-gap semiconductors, TiO2 or ZnO
Bacteria constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods, Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, acidic hot springs, radioactive waste, Bacteria live in symbiotic and parasitic relationships with plants and animals. Most bacteria have not been characterised, and only half of the bacterial phyla have species that can be grown in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology, There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water. There are approximately 5×1030 bacteria on Earth, forming a biomass which exceeds that of all plants, Bacteria are vital in many stages of the nutrient cycle by recycling nutrients such as the fixation of nitrogen from the atmosphere. The nutrient cycle includes the decomposition of bodies and bacteria are responsible for the putrefaction stage in this process.
In March 2013, data reported by researchers in October 2012, was published and it was suggested that bacteria thrive in the Mariana Trench, which with a depth of up to 11 kilometres is the deepest known part of the oceans. Other researchers reported related studies that microbes thrive inside rocks up to 580 metres below the sea floor under 2.6 kilometres of ocean off the coast of the northwestern United States. According to one of the researchers, You can find microbes everywhere—theyre extremely adaptable to conditions, the vast majority of the bacteria in the body are rendered harmless by the protective effects of the immune system, though many are beneficial particularly in the gut flora. However several species of bacteria are pathogenic and cause diseases, including cholera, anthrax, leprosy. The most common fatal diseases are respiratory infections, with tuberculosis alone killing about 2 million people per year. In developed countries, antibiotics are used to treat infections and are used in farming, making antibiotic resistance a growing problem.
Once regarded as constituting the class Schizomycetes, bacteria are now classified as prokaryotes. Unlike cells of animals and other eukaryotes, bacterial cells do not contain a nucleus and these evolutionary domains are called Bacteria and Archaea. The ancestors of modern bacteria were unicellular microorganisms that were the first forms of life to appear on Earth, for about 3 billion years, most organisms were microscopic, and bacteria and archaea were the dominant forms of life. In 2008, fossils of macroorganisms were discovered and named as the Francevillian biota, gene sequences can be used to reconstruct the bacterial phylogeny, and these studies indicate that bacteria diverged first from the archaeal/eukaryotic lineage. Bacteria were involved in the second great evolutionary divergence, that of the archaea, eukaryotes resulted from the entering of ancient bacteria into endosymbiotic associations with the ancestors of eukaryotic cells, which were themselves possibly related to the Archaea
Connective tissue is one of the four types of biological tissue that supports, connects or separates different types of tissues and organs in the body. The other three types are epithelial and nervous tissue, Connective tissue is found in between other tissues everywhere in the body, including the nervous system. In the central system, the three outer membranes that envelop the brain and spinal cord are composed of connective tissue. All connective tissue consists of three components, ground substance and cells. Blood and lymph lack the fiber component, all are immersed in the body water. The cells of connective tissue include fibroblasts, macrophages, mast cells, Connective tissue can be broadly subdivided into connective tissue proper, and special connective tissue. Connective tissue proper consists of connective tissue and dense connective tissue Special connective tissue consists of reticular connective tissue, adipose tissue, bone. Other kinds of connective tissues include fibrous and lymphoid connective tissues, new vascularised connective tissue that forms in the process of wound healing is termed granulation tissue.
Fibroblasts are the responsible for the production of some CT. Type I collagen, is present in forms of connective tissue. Characteristics of CT, Cells are spread through an extracellular fluid, ground substance - A clear and viscous fluid containing glycosaminoglycans and proteoglycans to fix the body water and the collagen fibers in the intercellular spaces. Ground substance slows the spread of pathogens, not all types of CT are fibrous. Examples of non-fibrous CT include adipose tissue and blood, adipose tissue gives mechanical cushioning to the body, among other functions. Although there is no dense collagen network in adipose tissue, groups of cells are kept together by collagen fibers. The matrix of blood is plasma, both the ground substance and proteins create the matrix for CT. Types of fibers, Connective tissue has a variety of functions that depend on the types of cells. They allow organs to resist stretching and tearing forces, elastic fibers, made from elastin and fibrillin, provide resistance to stretch forces.
They are found in the walls of blood vessels and in certain ligaments
Fuchsine or rosaniline hydrochloride is a magenta dye with chemical formula C20H19N3·HCl. There are other similar chemical formulations of products sold as fuchsine and it becomes magenta when dissolved in water, as a solid, it forms dark green crystals. As well as dying textiles, fuchsine is used to stain bacteria, in the literature of biological stains the name of this dye is frequently misspelled, with omission of the terminal -e, which indicates an amine. American and English dictionaries give the spelling, which is used in the literature of industrial dyeing. It is well established that production of fuchsine results in development of bladder cancers by production workers, production of magenta is listed as a circumstance known to result in cancer. Fuchsine was first prepared by August Wilhelm von Hofmann from aniline, françois-Emmanuel Verguin discovered the substance independently of Hofmann the same year and patented it. An 1861 article in Répertoire de Pharmacie said that the name was chosen for both reasons, Acid fuchsine is a mixture of homologues of basic fuchsine, modified by addition of sulfonic groups.
While this yields twelve possible isomers, all of them are satisfactory despite slight differences in their properties, basic fuchsine is a mixture of rosaniline, new fuchsine and Magenta II. Formulations usable for making of Schiff reagent must have content of pararosanilin. The actual composition of basic fuchsine tends to vary by vendor and batch. In solution with phenol as an accentuator it is called carbol fuchsin and is used for the Ziehl–Neelsen and other similar acid-fast staining of the mycobacteria which cause tuberculosis, leprosy etc. Basic fuchsine is used in biology to stain the nucleus. The crystals pictured at the right are of basic fuchsine, known as basic violet 14, basic red 9 and their structure differs from the structure shown above by the absence of the methyl group on the upper ring, otherwise they are quite similar. They are soft, with a hardness of less than 1 and they possess a strong metallic lustre and a green yellow color. They leave dark greenish streaks on paper and when these are moistened with a solvent, the strong magenta colour appears.
The two magenta stains on the paper were made by placing one drop of ethanol-water azeotrope, the crystals were replaced and the photograph taken. Fuchsine is a salt and has three amine groups, two primary amines and a secondary amine. If one of these is protonated to form ABCNH+, the charge is delocalized across the whole symmetrical molecule due to pi cloud electron movement