Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.
The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.
The steps of the Gram stain procedure are listed below and illustrated in Table 2. The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative Figure Table 2. However, there are several important considerations in interpreting the results of a Gram stain. First, older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive.
Thus, it is best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on de-colourizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative as in Figure 2. This suggests damage to the individual cells or that de-colourizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture. Besides their differing interactions with dyes and decolourizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance.
For example, Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. Gram-negative bacteria tend to be more resistant to certain antibiotics than gram-positive bacteria. We will discuss this and other applications of Gram staining in more detail in later chapters. However, more information is needed to make a conclusive diagnosis. The technician decides to make a Gram stain of the specimen.
This technique is commonly used as an early step in identifying pathogenic bacteria. After completing the Gram stain procedure, the technician views the slide under the brightfield microscope and sees purple, grape-like clusters of spherical cells Figure 2. Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box. Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool.
An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique.
Both use carbol fuchsin as the primary stain. The waxy, acid-fast cells retain the carbol fuchsin even after a decolourizing agent an acid-alcohol solution is applied. A secondary counterstain, methylene blue, is then applied, which renders non—acid-fast cells blue. The fundamental difference between the two carbo lfuchsin-based methods is whether heat is used during the primary staining process.
The Ziehl-Neelsen method uses heat to infuse the carbol fuchsin 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 colour can be seen clearly against the blue background of the surrounding tissue cells Figure 2.
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 2. Alternatively, positive and negative staining techniques can be combined to visualize capsules: The positive stain colours the body of the cell, and the negative stain colours the background but not the capsule, leaving halo around each cell.
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 decolourizes 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 2. Endospore-staining techniques are important for identifying Bacillus and Clostridium , two genera of endospore-producing bacteria that contain clinically significant species.
Among others, B. 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 2. 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 2. Finally, samples are fixed to fine copper wire or carbon-fibre grids and stained—not with coloured dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.
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 2. 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 2.
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.
Fluorescence in situ hybridization FISH can get around this problem. Following the seminal work of Carl Woese, taxonomically-unique oligonucleotide sequences complementary to the small subunit rRNA or its gene, can be synthesized and conjugated to fluorescent stains to make a probe for identification and quantification of that microorganism in environmental or clinical samples Figure 2.
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 vapour is a common fixative.
He found differences in the colouring of bacteria that is now known to be Streptococcus pneumoniae and Klebsiella pneumoniae. The differences Gram observed are a result of the composition of the bacterial cell wall.
Some bacteria have a cell wall composed of peptidoglycan, a polymer of sugar and amino acids. Others, that do not contain peptidoglycan, are not stained and are referred to as gram-negative, and appear red. Its popularity peaked between and More recently, Gram staining has been used to help identify new antibiotics, which are key in the battle against antimicrobial resistance.
Teixobactin — one of two new antibiotics released to the pharmaceutical market in — was identified by employing a new twist on a tried and tested method of screening soil for bacteria that have evolved to kill their competitors. A team at Northwestern University in Boston, Massachusetts screened 50, types of soil-dwelling bacteria for antibiotics that killed bugs like the hospital acquired infection MRSA and the bacteria that cause multi-drug resistant TB.
If used correctly, the researchers behind the discovery of teixobactin could be a viable treatment option for bacterial diseases — and safe from the threat of resistance — for at least 30 years.
Nonetheless, the Gram stain remains one of the most commonly performed tests in the clinical microbiology laboratory, and a foundational technique in treating bacterial infections and saving lives.
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