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It should be free flowing and free from air bubbles. Cover glasses used in histopathology Care has to be exercised in selecting cover glasses for mounting, these are available in variable sizes and thickness and are supplied usually in 10 gm packings.
Haematoxylin Haematoxylin as supplied has no staining properties until it has been ripened by oxidation into haematin. This ripening is achieved by two methods: 1.
Exposure of prepared solutions to the air for periods upto weeks, preferably in sunlight or 2. Addition of an oxidizing agent such as sodium iodate, potassium permaganate or mercuric oxide. Sufficient Haematoxylin should be left unoxidized in solution, so that natural oxidation can continue. It prolongs shelf life of the stain. Blueing Alum Haematoxylin stains nuclei and red color, which is converted to blue black color, when the section is washed in weak alkali.
Tap water is usually alkaline enough to produce this color change. Following may be used for rapid blueing of the sections. Carbonate 2 to 3 gm Magnesium sulphate 20 gm Dist. Water ml There are many formulations for preparing haematoxylin stains. Use of many is a matter of personal preference of whether progressive or regressive staining is being used.
In situations where haemotoxylin staining is followed by acidic stains, Iron haemotoxylin is preferred as it resists decolourisation by these counter stains. Various formulations differ mainly in regards to mordant and the shorter oxidiser used. Add a crystal of thymol. Add 0. Promptly remove the flame and plunge into ice cold water. This is the most important part. Leave over night at room temperature and filter. Add 2 or 4 ml of acetic acid before use per ml in the stain.
Note: In place of red oxide of mercury 0. Let it stand for weeks before use. Staining time - hours Note : Quick ripening may be done by adding 0. However the results are not so good. Haematoxyln 1gm Alcohol ml Let it ripen for a week B. Ammonium or potassium alum 50 gms Sodium iodate 0. Allow to stand overnight before use.
Solution is stable for weeks. Staining time minutes to increased after a month. Some basic rules for staining 1. Keep stains and solutions covered when not in use. After the slides are removed from oven these should be cooled before being put in xylene. Filter stains before use. Once the slides have been put in the xylene to remove paraffin they should not be allowed to dry out. Care should be taken that level of any solution used during staining is such as to cover the slides.
Drain the slides well and blot the bottom on filter paper before putting into the next solution. Haematoxylin and Eosin staining Aim The sections, as they are prepared, are colourless and different components cannot be appreciated. Staining them by different coloured dyes, having affinities of specific components of tissues, makes identification and study of their morphology possible.
Principle: Acid base reaction Procedure »» Deparaffinize in hot air oven. In running tap water for 5 Minutes. Poor or inadequate fixation of tissue. Over or under-ripened Haematoxylin. Overused or worked out Haematoxylin. Over or under differentiation of haematoxylin 5. Insufficient blueing following differentiation. Failure to wash blueing agent out of section before counter staining with eosin especially when ammonia is used. Insufficient differentiation of eosin during washing or dehydration.
Insufficient dehydration and clearing of sections. Contamination of stains. Review questions 1. Define the following terms? Dye B. Stain C. Auxochrome D. Chromogen E. Chromophore 2.
Write the different mechanisms of staining? Place the slide under the microscope, and locate the focal plane. Use first an objective lens of 10x, and then 40x magnification. Regressive stain B. Progressive stain 2. Write the different types of hematoxylin? Write the types of eosin? The nuclei will stain blue.
Collagen will stain pink. It supports and connects the other tissues in the body. It differs from epithelium in having few cells and large amount of intercellular substance. Main components of connective tissue are cells, fibers and ground substance. Connective Tissue Cells: 1. Fibroblasts »» They are large cells with branching processes. Histiocytes »» They are as numerous as fibroblasts. Plasma Cells »» They are ovoid or irregularly shaped cells. Mast Cells »» They are large cells with pale nucleus.
Pigment Cells »» They are satellite cells with long processes. Migratory Cells »» Migratory cells from blood and lymph are also seen in connective tissues. Fibers of connective tissue »» Collagen fibers »» Reticular fibers »» Elastic fibers 1. Collagen Fibers »» Collagen fibers are made up of a protein collagen.
Reticular fibers »» They are very thin, 0. Elastic Fibers »» They are made up of a protein elastin. Embryonic connective tissue It is further divided into: A. Mesenchyme B. Mucous »» The mesenchyme is the most primitive connective tissue.
Here the fibroblasts are embedded in a jelly like matrix. It consists of mucoid substance, fine meshwork of type II collagen fibers and few fibroblasts which are highly branched. Loose connective tissue. Dense connective tissue »» It is either irregularly arranged or it may be regular. Adipose tissue »» It is composed of adipocytes contained in loose areolar tissue.
Connective tissue stains: Trichrome stains »» Is selective demonstration of muscle, collagen fibers, fibrin, and erythrocytes. Factors affecting trichrome staining 1. Tissue permeability and dye molecular size 2. Heat 3. In the Van Gieson stain, collagen and most reticulin stain selectively with acid aniline dyes acid fuchsin. Picric acid acts as counter stain for muscle and cytoplasm and form complex with the dyes. This complex has special affinity for collagen.
Reagents 1. Solution A A. Haematoxylin 1. Solution B A. Hydrochloric acid 1. Weight's iron heamatoxylin solution :mix equal quantities of solution A and solution B. Colour of this reagent should appear violet black. Saturated aqueous picric acid — 10 ml B. Deparaffinize with xylene 2. Hydration take sections to water 3. Wash in distilled water 5. Van Gieson stain min 6. Rinse well in distilled water 7.
Dehydrate in absolute alcohol 2 changes 8. Clear in xylene 2 changes 9. Collagen - Red 2. Muscle and Cornified epithelium - Yellow 3. Nuclei - Blue to Black Clinical significance Several pathological changes are associated with cellular changes in connective tissues. Histological diagnosis of collagen diseases is based on the study of the section of various connective tissues.
Aim: Elastic fiber techniques are used for the demonstration of pathologic changes in elastic fibers. This include atrophy of the elastic tissue, thinning or loss that may result from arteriosclerotic changes, and reduplication, breaks, or splitting that may result from other vascular diseases. The techniques also may be used to demonstrate normal elastic tissue, as in the identification of veins and arteries, and to determine whether or not the blood vessels have been invaded by tumor.
Principle The tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine. Both ferric chloride and iodine serve as mordants, but they also have an oxidizing function that assists in converting hematoxylin to hemtein. The mechanism of dye binding is probably by formation of hydrogen bonds, but the exact chemical groups reacting with the hematoxylin have not been identified.
The dye will be attracted to the larger amount of mordant in the differentiating solution and will be removed from the tissue. The elastic tissue has the strongest affinity for the iron hematoxylin complex and will retain the dye longer than the other tissue elements, this allows other elements to be decolorized and the elastic fibers to remain stained.
Deparaffinize and hydrate to distilled water. Verhoeff's hematoxylin for 30 minutes save solution until stain is completed.
Wash in tap water. Rinse in water. Hypo for 1 minute to remove iodine. Wash in water. Counterstain in Van Gieson's for 5 minutes. Dehydrate, clear in xylene, and coverslip. Results Elastic fibers and nuclei………. Moreover toluidine blue staining is an interesting method for the early diagnostic of cancer because DNA labeling in cancer cells is more intense than in healthy cells.
Principle: Toluidine blue is a basic or acidophilic staining based on the principle of metachromasia due to molecular properties, the stain intensity varies in different tissue components depending on their pH, temperature and concentration variations. The components stained in purple or red are called metachromatic while the other components are orthochromatic because they keep the blue staining. Deparaffinize and hydrate to distilled water 2.
Toluidine blue solution for 2 minutes. Rinse in distilled water. Cover slip section with distilled water. Blot around edge of cover glass and seal with fingernail polish. Examine the sections as soon as possible after preparation. Results Metachromatic tissue- pink Review questions 1. Define metachromatic? Mention uses of toluidine blue? Classification of carbohydrates Carbohydrates are broken down into two broad categories: simple carbohydrates or those molecules composed purely of carbohydrates, and glycoconjugates, those molecules composed of carbohydrates and other molecules such as protein or lipid.
The simple carbohydrates are further categorized as monosaccharides, oligosaccharides, or polysaccharides. Monosaccharide The most basic or simple form of a carbohydrate is the monosaccharide.
Typical monosaccharides are of the empirical formula CH2O n, where n is a value between 3 and 9. The basic monosaccharide is the six-carbon. Glucose is not charged or ionized and for this reason is referred to as a neutral sugar. Other neutral sugars include mannose, galactose, and fructose. Monosaccharides contain asymmetric carbons referred to as chiral centers.
The letters D or L at the beginning of a name refer to the conformation of one of the chiral carbons within the molecule. The high number of hydroxyl OH groups present on the monosaccharide renders most of them extremely water soluble. Monosaccharides within a tissue specimen are lost during fixation and tissue processing due to the small size and the water solubility of these molecules.
As a result, the monosaccharides are not easily demonstrated by most histochemical techniques. Polysaccharides A polysaccharide is a large macromolecule composed of multiple monosaccharides joined by covalent bonds referred to as glycosidic linkages. Glycogen is the only polysaccharide found in animals that frequently is evaluated by histochemical techniques. Glycogen serves as a major form of stored energy reserves in humans.
There are a number of disease processes or pathological conditions in which histochemical assessment of glycogen content or accumulation may be of value diagnostically.
There are several well-characterized glycogen storage diseases which are the result of inherited defects of one or more of the enzymes involved in the synthesis or breakdown of glycogen. In most of these disorders, the liver shows massive accumulation of glycogen. In some diseases, glycogen accumulation is also observed in skeletal and cardiac muscle. Connective tissue glycoconjugates Proteoglycans also are commonly referred to as connective tissue mucins or mucopolysaccharides.
These molecules are large glycoconjugate complexes that are found in high concentrations within the extracellular matrix of connective tissues. The carbohydrate components of proteoglycans are known as glycosaminoglycans. Glycosaminoglycans are large polysaccharide polymers that are covalently bound to the protein core of proteoglycans. The typical glycosaminoglycan is a long unbranched polysaccharide composed of repeating disaccharide units each made up of two different monosaccharides.
This property accounts for the gel-like consistency of the extracellular matrix and connective tissues such as cartilage. The proteoglycans of cartilage in particular contain many glycosaminoglycan chains and thus are capable of binding a large volume of water.
Proteoglycans act in stabilizing and supporting fibrous elements of connective tissue. Tissues that contain high concentrations of proteoglycans include cartilage, tendons, ligaments, blood vessels, heart valves, and skin. Hyaluronic acid is found in high concentrations in synovial fluid and in the ground substance and connective tissue matrices where other proteoglycans are found. Several pathological conditions involve accumulation of glycosaminoglycans or proteoglycans.
The mucopolysaccharidoses are a group of genetic disorders that result from a deficiency of one or more of the enzymes that are involved in the degradation of heparin sulfate and dermatan sulfate.
This results in the abnormal accumulation of glycosaminoglycans in connective tissues as well as cell types such as neurons, histiocytes, and macrophages. Typically, the carbohydrate component is attached via an O- glycosidic linkage to serine or threonine. Mucins are categorized into functionally distinct families muc1, muc2, muc3, etc.
In contrast to the glycosaminoglycans, which are strongly acidic polyanions, the polysaccharide chains of the mucins vary from neutral or weakly acidic to strongly acidic sulfomucins. Function of Mucin The function of the mucins depends in part upon the tissue location of the mucin- producing cell as well as the mucin type. Detection of mucin in a tumor may be a valuable clue in the identification of a malignancy. Malignancies derived from simple epithelial tissues carcinomas frequently contain detectable mucin.
In contrast, melanomas, lymphomas, and sarcomas rarely exhibit significant levels of mucins. In addition, determining the type of mucin i. The detection of acid or sulfomucins within the gastric mucosa may aid in the detection and characterization of intestinal metaplasia, a lesion associated with gastric carcinoma. Dissolve 1. Add 20 ml of 0. Keep in the dark for hours. When the solution becomes straw coloured, add mg of activated charcoal, shake vigorously, filter and store. Harris haematoxylin stain Procedure 1.
Oxidize in periodic acid solution for 5 minutes 3. Rinse in distilled water 4. Wash in running water for 10 minutes for pink colour to develop. Harris haematoxylin for 6 minutes or light green counter stain for a few seconds.
Wash in running water. Dip in ammonia water to blue the sections Wash in running water for 10 minutes Mount in DPX. Nuclei — blue 2. Glycogen, mucin, hyaluronic acid, reticulin, colloid droplets, amyloid infiltration, thrombi.
Other chapters focus on amyloids, pigments, minerals, phosphatases, and esterases. The principles of enzyme histochemistry are also considered, with emphasis on preservation and factors affecting enzyme activity.
The final chapter is devoted to ultra-histochemistry, the application of histochemistry to electron microscopy. This monograph will be of interest to histochemists, histopathologists, and technologists involved in histochemical work.
Bancroft is a one-stop reference for all those involved with histological preparations and applications, from student to highly advanced laboratory professional"--Publisher's description.
Also, they aimed to produced a book as a practical companion of Theory and Practice of Histological Techniques Bancroft and Stevens, and to produce a laboratory manual containing a full repertoire of standard and non-standard, well-known and not-so-well-known histological techniques. Score: 5.
It is an resource suited to all those involved with histological preparations and applications, from the student to the highly experienced laboratory professional. New to this edition: Brand new co-editor.
Self assessment questions and answers. Will help reinforce all of the basics in order to pass course exams, professional certification exams. It encompasses three advances that have taken place since , providing a balance between the new and the traditional techniques.
Score: 1. Ships with Tracking Number! Advanced Book Search Browse by Subject. This reflects the percentage of orders the seller has received and filled. In this event, there may be a slight delay in shipping and possible variation in description. Add to want list. Social responsibility Did you know that sinceBiblio has used its profits to build 12 public libraries in rural villages of South America?
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