Why staining is often required for microscopic works

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. However, they must be even drier than is necessary for a TEM.

Critical point drying with inert liquid carbon dioxide under pressure is used to displace the water from the specimen. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium target, with energetic particles. Additionally, this species has not been successfully cultured in the laboratory on an artificial medium; therefore, diagnosis depends upon successful identification using microscopic techniques and serology analysis of body fluids, often looking for antibodies to a pathogen.

Since fixation and staining would kill the cells, darkfield microscopy is typically used for observing live specimens and viewing their movements. However, other approaches can also be used. For example, the cells can be thickened with silver particles in tissue sections and observed using a light microscope. It is also possible to use fluorescence or electron microscopy to view Treponema Figure Figure Though the stain kills the cells, it increases the contrast to make them more visible.

In clinical settings, indirect immunofluorescence is often used to identify Treponema. Multiple secondary antibodies can attach to each primary antibody, amplifying the amount of stain attached to each Treponema cell, making them easier to spot Figure Indirect immunofluorescence can be used to identify T. Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes.

Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells immunofluorescence ; others may attach to DNA molecules in a process called fluorescence in situ hybridization FISH , causing cells to be stained based on whether they have a specific DNA sequence. Sample preparation for two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes.

Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au Toluene vapor is a common fixative.

After some additional testing, the technician determines that these bacteria are the medically important species known as Staphylococcus aureus , a common culprit in wound infections. Because some strains of S. After testing several antibiotics, the lab is able to identify one that is effective against this particular strain of S.

This reduces the risk that any especially resistant bacteria could survive, causing a second infection or spreading to another person. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample. Cells may also be enumerated by staining cells to determine biomass in an environment of interest. Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample:.

There are several types of staining media, each can be used for a different purpose. Commonly used stains and how they work are listed below. All these stains may be used on fixed, or non-living, cells and those that can be used on living cells are noted. In order to reveal structural detail using brightfield microscopy, some form of staining is required. With this method, cell nuclei are stained blue, and cytoplasm and many extra-cellular components in shades of pink.

However, sometimes additional information is required to provide a full differential diagnosis, and this requires furthermore specialized staining techniques. These methods can all be applied to paraffin sections, and in most cases, the slides produced are completely stable and can be kept for many years. After staining, the sections are covered with a glass coverslip and are then sent to a pathologist who will view them under a microscope to make an appropriate diagnosis and prepare a report.

The content, including webinars, training presentations and related materials is intended to provide general information regarding particular subjects of interest to health care professionals and is not intended to be, and should not be construed as, medical, regulatory or legal advice.

Any links contained in the content which provides access to third party resources or content is provided for convenience only. For the use of any product, the applicable product documentation, including information guides, inserts and operation manuals should be consulted. Leica Biosystems and the editors hereby disclaim any liability arising directly or indirectly from the use of the content, including from any drugs, devices, techniques or procedures described in the content.

All rights reserved. Geoffrey Rolls is a Histology Consultant with decades of experience in the field. We are looking for more great writers to feature here. Send us a submission and we'll be in touch! Get more Knowledge Pathway content like this delivered directly to your inbox.

Unsubscribe at any time. Back Leica SM R. Back View All. Featured Products. Learn more. Reagent Alcohol, Isopropyl Alcohol Learn more. It's New. Service Back Aperio Training. All Shop. You won't be able to use the quotation basket until you enable cookies in your Web browser. Your Web browser is no longer supported.

Please upgrade to a modern browser. To use the quotation basket you have to enable JavaScript in you Web browser. Apply for self-reported educational credits.

Preparation options Because of the microscopy requirements, options for preparing specimens are limited to: Whole-mounts, where an entire organism or structure is small enough or thin enough to be placed directly onto a microscope slide e. Sections, where specimens are supported in some way so that very thin slices can be cut from them, mounted on slides, and stained. Section preparation. Figure 1: A diagnostic section being prepared with a cryostat microtome.

The section, which has been cut from snap-frozen tissue, is being picked up onto a warm slide where it will be immediately fixed and stained. Figure 2: A rotary microtome being used to cut paraffin sections.