Why stain specimens

There are several different methods to do this, sometimes heat is used to fix a sample, chemicals may be applied to make the specimen accept a dye or stain, other times a stain may be applied directly. The stains and dyes used on microscope slides are numerous and the chosen stain will produce a different outcome. Stains are selected based on the type of sample being observed and what organisms or cells need to be observed.

Most commonly, a positive stain is used as the stain is readily absorbed by the cell or organism making it stand out against the background. In some cases, negative stains are more advantageous — negative stains have the opposite effect, staining the background and leaving the cell or organism as a negative silhouette.

Commonly used dyes such as crystal violet , basic fuchsin, malachite green , and methylene blue serve as positive stains. Acidic dyes, such as rose bengal and eosin serve as negative stains as these are repelled by negatively charged cell walls.

On the opposide side of the cover slip place a paper towel or cloth to draw the liquid out from the cover slip. As the liquid is drawn out, the stain will be pulled in under the cover slip. Microscope Slide Staining Information Microscope cell staining is a technique used to enable better visualization of cells and cell parts under the microscope.

By using different stains, a nucleus or a cell wall are easier to view. Most stains can be used on non-living fixed cells, while only some types of stain can be used on living cells. Bismarck Brown - colors a type of protein called acid mucins yellow. Carmine - colors animal starch glycogen , red. Coomassie Blue - stains proteins a bright blue, and is often used in gel electrophoresis Crystal Violet - stains cell walls purple when combined with mordant.

This stain is used in Gram Staining. A secondary counterstain, methylene blue, is then applied, which renders non—acid-fast cells blue.

The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria AFB.

If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells Figure 5.

Figure 5. Ziehl-Neelsen staining has rendered these Mycobacterium tuberculosis cells red and the surrounding growth indicator medium blue. Mycobacterium tuberculosis , the bacterium that causes tuberculosis , can be detected in specimens based on the presence of acid-fast bacilli. If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification.

Variations of this approach can be used as a first step in determining whether M. An alternative approach for determining the presence of M. In this technique, fluorochrome-labeled antibodies bind to M. Antibody-specific fluorescent dyes can be used to view the mycobacteria with a fluorescence microscope.

Certain bacteria and yeasts have a protective outer structure called a capsule. Capsules do not absorb most basic dyes; therefore, a negative staining technique staining around the cells is typically used for capsule staining. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell.

The specimen does not need to be heat-fixed prior to negative staining. One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or nigrosin to a specimen.

Other capsular stains can also be used to negatively stain encapsulated cells Figure 6. Alternatively, positive and negative staining techniques can be combined to visualize capsules: The positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving halo around each cell.

Figure 6. The halos surrounding the cells are the polysaccharide capsules. Encapsulated cells appear to have a light-blue halo. Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell.

The Schaeffer-Fulton method the most commonly used endospore-staining technique uses heat to push the primary stain malachite green into the endospore. Washing with water decolorizes the cell, but the endospore retains the green stain.

The cell is then counterstained pink with safranin. The resulting image reveals the shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible Figure 7. Figure 7. A stained preparation of Bacillus subtilis showing endospores as green and the vegetative cells as pink. Endospore-staining techniques are important for identifying Bacillus and Clostridium , two genera of endospore-producing bacteria that contain clinically significant species.

Among others, B. Figure 8. A flagella stain of Bacillus cereus, a common cause of foodborne illness, reveals that the cells have numerous flagella, used for locomotion. Flagella singular: flagellum are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. Because they are so thin, flagella typically cannot be seen under a light microscope without a specialized flagella staining technique. Flagella staining thickens the flagella by first applying mordant generally tannic acid, but sometimes potassium alum , which coats the flagella; then the specimen is stained with pararosaniline most commonly or basic fuchsin Figure 8.

Though flagella staining is uncommon in clinical settings, the technique is commonly used by microbiologists, since the location and number of flagella can be useful in classifying and identifying bacteria in a sample.

When using this technique, it is important to handle the specimen with great care; flagella are delicate structures that can easily be damaged or pulled off, compromising attempts to accurately locate and count the number of flagella. Samples to be analyzed using a TEM must have very thin sections. But cells are too soft to cut thinly, even with diamond knives. The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the cell, where it solidifies.

Next, thin sections are cut using a specialized device called an ultramicrotome Figure 9. Finally, samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms. Figure 9. Fish and Wildlife Service Northeast Region.

When samples are prepared for viewing using an SEM, they must also be dehydrated using an ethanol series. Several staining techniques have been established to improve the staining methods. The histopathology lab today is laden with a great work load and different types of histological assignments Musumeci, In the history of histology, a great shift and development in histologic stains were shaped by improved technologic development of microscopes and the establishment of the histologic stains factory aniline dye in in Germany which manufactured variety of new-histological stains Godwin, It is during this time that the paraffin infiltration staining technique was devised Titford, While these changes have taken place, there are old stain procedures that are still in use today and many others have been replaced with new immunal or staining techniques.

Additionally, the complexity of stains has been enhanced for the purpose of efficient and consistent staining processes that show fine and differentiated tissues Ntziachristos, Histological staining is a commonly used medical process in pathological diagnosis and forensic studies.

The process of histological staining takes five key stages, and they include fixation, processing, embedding, sectioning and staining. Early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to hard cellular tissues. These fixatives and staining agents were ingenious and after a period colored staining agents were developed which are still applicable in the laboratory staining techniques today.

Staining techniques used were; carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin among others. There have been great changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques collectively referred to us histochemistry and have facilitated greatly in the study of organs and tissues.

Hematoxylin is a basic dye that is commonly used in this process and stains the nuclei giving it a bluish color while eosin another stain dye used in histology stains the cell's nucleus giving it a pinkish stain Victor, While these changes have taken place, there are old stain procedures that are still in use today and many others have been replaced with new immunalstaining or staining techniques Sine, Similarly, there have been great changes in workload requiring more advanced technics of staining.

The case studies indicate that, in the modern histology a combination of different stain techniques are used to enhance the effectiveness of the staining process.

In the modern histologic as a way of improving histological stains, several stains have been modified and combined with other stains to improve their effectiveness. National Center for Biotechnology Information , U. Glob J Health Sci.

Published online Jun Author information Article notes Copyright and License information Disclaimer. E-mail: moc. Received May 7; Accepted May This article has been cited by other articles in PMC. Abstract The history of histology indicates that there have been significant changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques, collectively referred to as histochemistry.

Keywords: histological staining, histology, histopathology, histochemistry. Introduction Histology is the microscopic study of animal and plant cell and tissues through staining and sectioning and examining them under a microscope electron or light microscope.

Specific Aspects of Histopathology 2. Methodology The research used an extensive exploration and review of historical, recent and current medical research studies and case studies in order to collect quantitative and qualitative data in regard to histological stains used in the past and recent cases Silverman, Literature Review 4. Hematin and Hematoxylin These are naturally occurring substances that have been in use in the history of histopathology Titford, Silver Nitrate Silver Nitrate has had a long usage in historical staining techniques and is still used in modern pathology.

Romanowsky Stains—Giemsa Stains They were developed in the by Dimitri Romanowsky and popular for its multicolor in identifying blood parasites. Gram Stain The Gram staining method was named after a Danish inventor Hans Christian Gram, who invented it as an approach to differentiating bacteria species in Musumeci, Trichrome Stains Historical assessment on the use of various stains in histology indicates that most pathologists were attracted by stains that gave multicolored results on the tissue specimens.

Case Study 2 The aim was to investigate the difference in capacity among different stains: Hematoxylin and Eosin, toluidine blue Stain, neuron-specific enolase NSE immunostaining and the S protein. Results and Discussion The literature review on staining techniques indicates that there has been great improvement in the histopathology and histotechnology.

Summary Histological staining is a commonly used medical process in pathological diagnosis and forensic studies. References Anderson J. An introduction to Routine and special staining.

Bancroft J. D, Layton C. The Hematoxylin and eosin. In: Suvarna S. K, Layton C, Bancroft J. D, editors. Theory Practice of histological techniques. Philadelphia: Churchill Livingstone of El Sevier; Microbiology: Principles and exploration.

John Wiley Sons; L, Prescott J. March Veterinary Pathology. X, Brito K. O, Gomes M. A, Caliari M. Histopathological and immunohistochemical study of the hepatic lesions experimentally induced by Entamoeba. Histochemical uses of haematoxylin-a review. From www.