![]() It uses a combination of haematoxylin, Orange G, eosin Y, Light Green SF yellowish, and sometimes Bismarck Brown Y. It is frequently used to stain the Pap smear specimens. Papanicolaou staining, or Pap staining, is a frequently used method for examining cell samples from various bodily secretions. Eosin is strongly absorbed by red blood cells, colouring them bright red. Haematoxylin stains cell nuclei blue, while eosin stains cytoplasm, connective tissue and other extracellular substances pink or red. Haematoxylin and eosin staining protocol is used frequently in histology to examine thin sections of tissue. Also, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide. On most Gram-stained preparations, Gram-negative organisms will appear red or pink because they are counterstained due to presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases & hence the CVI complex (Crystal violet -Iodine) can pass through. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria. Gram-positive bacteria stain dark blue or violet. Gram status is important in medicine the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. It is based on the composition of their cell wall. Gram staining is used to determine gram status to classify bacteria broadly. Note: negative staining is a mild technique which may not destroy the microorganisms therefore it is unsuitable for studying pathogens. After drying, the microorganisms may be viewed in bright field microscopy as lighter inclusions well-contrasted against the dark environment surrounding them. This can be achieved simply by smearing the sample on to the slide, followed by an application of nigrosin (indian ink). When excess dye solution is washed away, the mordanted stain remains.Ī simple staining method for bacteria which is usually successful even when the "positive staining" methods detailed below fail, is to employ a negative stain. Many dyes, however, require the use of a mordant: a chemical compound which reacts with the stain to form an insoluble, coloured precipitate. For larger pieces of tissue, thin sections (slices) are made using a microtome these slices can then be mounted and inspected.Īt its simplest, the actual staining process may involve immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation. For samples of loose cells (as with a blood smear or a pap smear) the sample can be directly applied to a slide. In some cases, cells may be grown directly on a slide. Mounting usually involves attaching the samples to a glass microscope slide for observation and analysis. 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. Common fixative include formaldehyde, ethanol, methanol, and/or picric acid. Most chemical fixatives (chemicals causing fixation) generate chemical bonds between proteins and other substances within the sample, increasing their rigidity. Sometimes heat is used to kill, adhere, and alter the specimen so it will accept stains. This treatment will dissolve the cell membranes, and allow larger dye molecules access to the cell's interior.įixation–which may itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Permeabilization involves treatment of cells with (usually) a mild surfactant. The preparatory steps involved depend on the type of analysis planned some or all of the following procedures may be required. A safranin counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well. For example, crystal violet stains only Gram-positive bacteria in Gram staining. A counterstain is stain that makes cells or structures more visible, when not completely visible with the principal stain. Combined with specific protocols for fixation and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools. Certain stains are often to reveal more details and features than a single stain alone. ![]() In vitro staining involves colouring cells or structures that are no longer living. 1.2.3 Haematoxylin and eosin (H&E) staining. ![]()
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