Auramine O is a diarylmethane dye used as a fluorescent stain. In its pure form, Auramine O appears as yellow needle crystals, it is 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, 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, it can be used as an antiseptic agent. Auramine O spectra data
Periodic acid–Schiff stain
Periodic acid–Schiff is a staining method used to detect polysaccharides such as glycogen, mucosubstances such as glycoproteins and mucins in tissues. The reaction of periodic acid oxidizes the vicinal diols in these sugars breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide units that are parts of the long polysaccharides, creating a pair of aldehydes at the two free tips of each broken monosaccharide ring; the oxidation condition has to be sufficiently regulated so as to not oxidize the aldehydes further. These aldehydes react with the Schiff reagent to give a purple-magenta color. A suitable basic stain is used as a counterstain. • PAS diastase stain is PAS stain used in combination with diastase, an enzyme that breaks down glycogen. • Alcian blue/periodic acid–Schiff uses alcian blue before the PAS step. PAS staining is used for staining structures containing a high proportion of carbohydrate macromolecules found in e.g. connective tissues, the glycocalyx, basal laminae.
PAS staining can be used to assist in the diagnosis of several medical conditions: Glycogen storage disease. Adenocarcinomas, which secrete neutral mucins. Paget disease of the breast. Alveolar soft part sarcoma. Staining macrophages in Whipple's disease, it can be used to diagnose α1-antitrypsin deficiency. Aggregates of PAS-positive lymphocytes are present in epidermis in Mycosis fungoides and Sezary syndrome, called Pautrier microabscesses. Ewing sarcoma Erythroleukemia, a leukemia of immature red blood cells; these cells stain a bright fuchsia. Pulmonary alveolar proteinosis. Fungal infection, the cell walls of fungi stain magenta. In contrast, Grocott's methenamine silver stain will stain both dead fungal organisms, it is used to identify glycogen in lung biopsy specimens of infants with pulmonary interstitial glycogenosis. It can be used to highlight super cross-linked lipids inclusions in ceroid lipofuscinosis. Presence of glycogen can be confirmed on a section of tissue by using diastase to digest the glycogen from a section comparing a diastase digested PAS section with a normal PAS section.
The diastase negative slide will show a magenta staining where glycogen is present within a section of tissue. The slide, treated with diastase will lack any positive PAS staining in those locations on the slide PAS staining is used for staining cellulose. One example would be looking for implanted medical devices composed of nonoxidized cellulose. If the PAS stain will be performed on tissue, the recommended fixative is 10% neutral-buffered formalin or Bouin solution. For blood smears, the recommended fixative is methanol. Glutaraldehyde is not recommended because free aldehyde groups may be available to react with the Schiff reagent, which may result in false positive staining. PAS Reaction
Acid-fast stain, first introduced by Dr. Paul Ehrlich known as the Ziehl–Neelsen staining, is a bacteriological stain used to identify acid-fast organisms Mycobacteria, it is named for two German doctors who modified it: the bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen. Mycobacterium tuberculosis is the most important of this group because it is responsible for tuberculosis. Other important Mycobacterium species involved in human disease are Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium bovis, Mycobacterium africanum and members of the Mycobacterium avium complex. Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids; these acids resist staining by ordinary methods such as a Gram stain. It can be used to stain a few other bacteria, such as Nocardia; the reagents used for Ziehl–Neelsen staining are – carbol fuchsin, acid alcohol, methylene blue. Acid-fast bacilli are bright red after staining.
A variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular hyphae of certain species of fungi in the genus Russula. It is useful in the identification of some protozoa, namely Cryptosporidium and Isospora; the Ziehl–Neelsen stain can hinder diagnosis in the case of paragonimiasis because the eggs in an ovum and parasite sputum sample can be dissolved by the stain, is used in this clinical setting because signs and symptoms of paragonimiasis resemble those of TB. A typical AFB stain procedure involves dropping the cells in suspension onto a slide air drying the liquid and heat fixing the cells; the slide is flooded with carbol fuchsin, heated to dry and rinsed off in tap water. The slide is flooded with a 1% solution of hydrochloric acid in isopropyl alcohol to remove the carbol fuchsin, thus removing the stain from cells that are unprotected by a waxy lipid layer. Thereafter, the cells are stained in methylene blue and viewed under a microscope under oil immersion.
Studies have shown. An AFB culture should be performed along with an AFB stain. Carbol fuchsin stains every cell; when they are de-stained with acid-alcohol, only non-acid-fast bacteria get de-stained since they do not have a thick, waxy lipid layer like acid-fast bacteria. When counter stain is applied, non-acid-fast bacteria pick it up and become blue or green when viewed under the microscope. Acid-fast bacteria retain carbol fuchsin. 1% sulfuric acid alcohol for actinomycetes, nocardia. 0.5–1% sulfuric acid alcohol for oocysts of isospora, cyclospora. 0.25–0.5% sulfuric acid alcohol for bacterial endospores. Brucella differential stain – glacial acetic acid used, no heat applied, secondary stain is Loeffler's methylene blue. Kinyoun modification is available. A protocol in which a detergent is substituted for the toxic phenol in the fuchsin staining solution. Lowenstein–Jensen medium Gram stain "Microbiology with Diseases by Body System", Robert W. Bauman, 2009, Pearson Education, Inc. Morello, Josephine A. Paul A. Granato, Marion E. Wilson, Verna Morton.
Laboratory Manual and Workbook in Microbiology: Applications to Patient Car. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print. Ziehl–Neelsen protocol. Media related to Ziehl-Neelsen stain at Wikimedia Commons
Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. Antiseptics are distinguished from antibiotics by the latter's ability to safely destroy bacteria within the body, from disinfectants, which destroy microorganisms found on non-living objects; some antiseptics are true germicides, capable of destroying microbes, while others are bacteriostatic and only prevent or inhibit their growth. Antibacterials include antiseptics. Microbicides which destroy virus particles are called antivirals. Antifungals known as an antimycotics, are pharmaceutical fungicides used to treat and prevent mycosis; the widespread introduction of antiseptic surgical methods was initiated by the publishing of the paper Antiseptic Principle of the Practice of Surgery in 1867 by Joseph Lister, inspired by Louis Pasteur's germ theory of putrefaction. In this paper, Lister advocated the use of carbolic acid as a method of ensuring that any germs present were killed.
Some of this work was anticipated by: Ancient Greek physicians Galen and Hippocrates and Sumerian clay tablets dating from 2150 BC that advocate the use of similar techniques. Medieval surgeons Hugh of Lucca, Theoderic of Servia, his pupil Henri de Mondeville were opponents of Galen's opinion that pus was important to healing, which had led ancient and medieval surgeons to let pus remain in wounds, they advocated draining and cleaning the wound edges with wine, dressing the wound after suturing, if necessary and leaving the dressing on for ten days, soaking it in warm wine all the while, before changing it. Their theories were bitterly opposed by Galenist Guy de Chauliac and others trained in the classical tradition. Oliver Wendell Holmes, Sr. who published The Contagiousness of Puerperal Fever in 1843 Florence Nightingale, who contributed to the report of the Royal Commission on the Health of the Army, based on her earlier work Ignaz Semmelweis, who published his work The Cause and Prophylaxis of Childbed Fever in 1861, summarizing experiments and observations since 1847 Alcohols, including ethanol and 2-propanol/isopropanol are sometimes referred to as surgical spirit.
They are used to disinfect the skin. Chlorhexidine gluconate is used as a skin antiseptic. Hydrogen peroxide is used as a 6 % solution to deodorize wounds and ulcers. More 3% solutions of hydrogen peroxide have been used in household first aid for scrapes, etc. However, the strong oxidization causes scar formation and increases healing time during fetal development. Iodine is used in an alcohol solution or as Lugol's iodine solution as a pre- and postoperative antiseptic; some studies do not recommend disinfecting minor wounds with iodine because of concern that it may induce scar tissue formation and increase healing time. However, concentrations of 1% iodine or less have not been shown to increase healing time and are not otherwise distinguishable from treatment with saline. Novel iodine antiseptics containing povidone-iodine are far better tolerated, do not negatively affect wound healing, leave a deposit of active iodine, thereby creating the so-called "remnant", or persistent, effect; the great advantage of iodine antiseptics is their wide scope of antimicrobial activity, killing all principal pathogens and, given enough time spores, which are considered to be the most difficult form of microorganisms to be inactivated by disinfectants and antiseptics.
Octenidine dihydrochloride increasingly used in continental Europe as a chlorhexidine substitute. Polyhexanide is an antimicrobial compound suitable for clinical use in critically colonized or infected acute and chronic wounds; the physicochemical action on the bacterial envelope prevents or impedes the development of resistant bacterial strains. Balsam of Peru is a mild antiseptic. Dakin's solution is a sodium hypochlorite solution also containing boric acid to lower pH, it is used on live tissues for cleaning wounds of bacteria and viruses. Because of practicality of preparation and lower cost, it is used in Veterinary Medicine treatments, it does not stain the animal's fur or affect it's aesthetic or commercial value. Super-oxidized solutions contain hypochlorous acid and are stabilised at a neutral pH. SOS are acting, broad spectrum antiseptics that are clinically effective at non-cytotoxic concentrations that in contrast to many cytotoxic antiseptics, support wound healing There is now growing consensus that modern SOS are more effective for healing wounds faster After continued exposure to antibiotics, bacteria may evolve to the point where they are no longer harmed by these compounds.
Bacteria can develop a resistance to antiseptics, but the effect is less pronounced. The mechanisms by which bacteria evolve may vary in response to different antiseptics. Low concentrations of an antiseptic may encourage growth of a bacterial strain, resistant to the antiseptic, where a higher concentration of the antiseptic would kill the bacteria. In addition, use of an excessively high concentration of an antiseptic may cause tissue damage or slow the process of wound healing. Antiseptics are mo
In biology and biochemistry, a lipid is a biomolecule, soluble in nonpolar solvents. Non-polar solvents are hydrocarbons used to dissolve other occurring hydrocarbon lipid molecules that do not dissolve in water, including fatty acids, sterols, fat-soluble vitamins, diglycerides and phospholipids; the functions of lipids include storing energy and acting as structural components of cell membranes. Lipids have applications in the food industries as well as in nanotechnology. Scientists sometimes broadly define lipids as amphiphilic small molecules. Biological lipids originate or in part from two distinct types of biochemical subunits or "building-blocks": ketoacyl and isoprene groups. Using this approach, lipids may be divided into eight categories: fatty acids, glycerophospholipids, sphingolipids and polyketides. Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids encompass molecules such as fatty acids and their derivatives, as well as other sterol-containing metabolites such as cholesterol.
Although humans and other mammals use various biosynthetic pathways both to break down and to synthesize lipids, some essential lipids can't be made this way and must be obtained from the diet. In 1815, Henry Braconnot classified lipids in two categories and huiles. In 1823, Michel Eugène Chevreul developed a more detailed classification, including oils, tallow, resins and volatile oils. In 1827, William Prout recognized fat, along with protein and carbohydrate, as an important nutrient for humans and animals. For a century, chemists regarded "fats" as only simple lipids made of fatty acids and glycerol, but new forms were described later. Theodore Gobley discovered phospholipids in mammalian brain and hen egg, called by him as "lecithins". Thudichum discovered in human brain some phospholipids and sphingolipids; the terms lipoid, lipin and lipid have been used with varied meanings from author to author. In 1912, Rosenbloom and Gies proposed the substitution of "lipoid" by "lipin". In 1920, Bloor introduced a new classification for "lipoids": simple lipoids, compound lipoids, the derived lipoids.
The word "lipid", which stems etymologically from the Greek lipos, was introduced in 1923 by Gabriel Bertrand. Bertrands included in the concept not only the traditional fats, but the "lipoids", with a complex constitution. In 1947, T. P. Hilditch divided lipids into "simple lipids", with greases and waxes, "complex lipids", with phospholipids and glycolipids. Fatty acids, or fatty acid residues when they are part of a lipid, are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty acid synthesis, they are made of a hydrocarbon chain. The fatty acid structure is one of the most fundamental categories of biological lipids, is used as a building-block of more structurally complex lipids; the carbon chain between four and 24 carbons long, may be saturated or unsaturated, may be attached to functional groups containing oxygen, halogens and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which affects the molecule's configuration.
Cis-double bonds cause the fatty acid chain to bend, an effect, compounded with more double bonds in the chain. Three double bonds in 18-carbon linolenic acid, the most abundant fatty-acyl chains of plant thylakoid membranes, render these membranes fluid despite environmental low-temperatures, makes linolenic acid give dominating sharp peaks in high resolution 13-C NMR spectra of chloroplasts; this in turn plays an important role in the function of cell membranes. Most occurring fatty acids are of the cis configuration, although the trans form does exist in some natural and hydrogenated fats and oils. Examples of biologically important fatty acids include the eicosanoids, derived from arachidonic acid and eicosapentaenoic acid, that include prostaglandins and thromboxanes. Docosahexaenoic acid is important in biological systems with respect to sight. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Fatty esters include important biochemical intermediates such as wax esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines.
The fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide. Glycerolipids are composed of mono-, di-, tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides; the word "triacylgl
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 with the aid of different microscopes. Stains may be used to define and examine bulk tissues, cell populations, or organelles within individual cells. 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, 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. Staining is not limited to biological materials, it can be used to study the morphology of other materials for example the lamellar structures of semi-crystalline polymers or the domain structures of block copolymers.
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 seen and studied; the usual purpose is to reveal cytological details. In vitro staining involves colouring cells or structures that have been removed from their biological context. Certain stains are 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, when not visible with the principal stain. 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. Those stains excluded by the living cells but taken up by the dead cells are called vital stains; those that enter and stain living cells are called supravital stains. However, these stains are toxic to the organism, some more so than others. Due to their toxic interaction inside a living cell, when supravital stains enter a living cell, they might produce a characteristic pattern of staining different from the staining of an fixed cell. To achieve desired effects, the stains are used in 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; this treatment dissolves cell membranes, allows larger dye molecules into the cell's interior. Mounting involves attaching the samples to a glass microscope slide for observation and analysis. In some cases, cells may be grown directly on a slide. For samples of loose cells the sample can be directly applied to a slide. For larger pieces of tissue, thin sections are made using a microtome. Most of the dyes used in microscopy are available as BSC-certified stains; this means that samples of the manufacturer's batch have been tested by an independent body, the Biological Stain Commission, found to meet or exceed certain standards of purity, dye content and performance in staining techniques. These standards are published in the Commission's journal Histochemistry.
Many dyes are inconsistent in composition from one supplier to another. The use of BSC-certified stains eliminates a source of unexpected results; some vendors sell stains "certified" by themselves rather than by the Biological Stain Commission. Such products may not be suitable for diagnostic and other applications. A simple staining method for bacteria, successful when the "positive staining" methods detailed below fail, is to use a negative stain; this can be achieved by smearing the sample onto the slide and applying nigrosin or India ink. After drying, the microorganisms may be viewed in bright field micros
A carbohydrate is a biomolecule consisting of carbon and oxygen atoms with a hydrogen–oxygen atom ratio of 2:1 and thus with the empirical formula Cmn. This formula holds true for monosaccharides; some exceptions exist. The carbohydrates are technically hydrates of carbon; the term is most common in biochemistry, where it is a synonym of saccharide, a group that includes sugars and cellulose. The saccharides are divided into four chemical groups: monosaccharides, 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 often end in the suffix -ose, as in the monosaccharides fructose and glucose and the disaccharides sucrose and lactose. Carbohydrates perform numerous roles in living organisms. Polysaccharides serve 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 immune system, preventing pathogenesis, blood clotting, development, they are found in a wide variety of processed foods. Starch is a polysaccharide, it is abundant in cereals and processed food based on cereal flour, such as bread, pizza or pasta. Sugars appear in human diet as table sugar, lactose and fructose, both of which occur in honey, many fruits, some vegetables. Table sugar, milk, or honey are added to drinks and many prepared foods such as jam and cakes. Cellulose, a polysaccharide found in the cell walls of all plants, is one of the main components of insoluble dietary fiber. Although it is not digestible, insoluble dietary fiber helps to maintain a healthy digestive system by easing defecation. Other polysaccharides contained in dietary fiber include resistant starch and inulin, which feed some bacteria in the microbiota of the large intestine, are metabolized by these bacteria to yield short-chain fatty acids.
In scientific literature, the term "carbohydrate" has many synonyms, like "sugar", "saccharide", "ose", "glucide", "hydrate of carbon" or "polyhydroxy compounds with aldehyde or ketone". Some of these terms, specially "carbohydrate" and "sugar", are used with other meanings. In food science and in many informal contexts, the term "carbohydrate" means any food, rich in the complex carbohydrate starch or simple carbohydrates, such as sugar. In lists of nutritional information, such as the USDA National Nutrient Database, the term "carbohydrate" is used for everything other than water, fat and ethanol; this includes chemical compounds such as acetic or lactic acid, which are not considered carbohydrates. It includes dietary fiber, a carbohydrate but which does not contribute much in the way of food energy though it is included in the calculation of total food energy just as though it were a sugar. In the strict sense, "sugar" is applied for sweet, soluble carbohydrates, many of which are used in food.
The name "carbohydrate" was used in chemistry for any compound with the formula Cm n. Following this definition, some chemists considered formaldehyde to be the simplest carbohydrate, while others claimed that title for glycolaldehyde. Today, the term is understood in the biochemistry sense, which excludes compounds with only one or two carbons and includes many biological carbohydrates which deviate from this formula. For example, while the above representative formulas would seem to capture the known carbohydrates and abundant carbohydrates deviate from this. For example, carbohydrates display chemical groups such as: N-acetyl, carboxylic acid and deoxy modifications. Natural saccharides are built of simple carbohydrates called monosaccharides with general formula n where n is three or more. A typical monosaccharide has the structure H–x–y–H, that is, an aldehyde or ketone with many hydroxyl groups added one on each carbon atom, not part of the aldehyde or ketone functional group. Examples of monosaccharides are glucose and glyceraldehydes.
However, some biological substances called "monosaccharides" do not conform to this formula and there are many chemicals that do conform to this formula but are not considered to be monosaccharides. The open-chain form of a monosaccharide coexists with a closed ring form where the aldehyde/ketone carbonyl group carbon and hydroxyl group react forming a hemiacetal with a new C–O–C bridge. Monosaccharides can be linked togeth