Acid-Fast Staining
Acid-fast staining is used to stain bacteria that are resistant to classical staining methods. Acid-fast bacteria retain their carbol fuchsin staining even after decolorization, unlike non-acid-fast bacteria. In this procedure, a few drops of carbolic-fuchsin dye are added to a thermally fixed sample prepared on a slide, and the sample is heated without boiling. After cooling, the stained sample is washed with water to remove residues and then decolorized with an acid-alcohol solution. It is then stained with methylene blue; acid-resistant bacteria will not turn blue and retain the carbolic-fuchsin staining.
Capsule Staining
This type of staining typically employs crystal violet, which initially stains the protective bacterial capsule, followed by copper sulfate for decolorization. Without heating, the sample fixed on the slide is stained with crystal violet and washed with water after 1-2 minutes. The decolorization is due to copper sulfate, which removes the dye from the capsule but does not affect the stained cell. As a consequence, the capsule becomes visible as a transparent shell around the stained cell body.
Simple Staining
This is one of the most classic staining methods, used to study cell morphology and behavior. The process involves fixing the specimen onto a slide, followed by staining it with a basic dye such as crystal violet, safranin, or methylene blue. The slide is then washed with water to remove the excess stain. Finally, the resulting stained cells are observed under a microscope to analyze the desired trait.
Basic and Acid Staining
Differential staining is used to identify cells according to their cell wall composition. A thermofixed sample is filled with crystal violet, methylene blue, or safranin, the primary pigments, and then washed with water. An acid-alcohol solution is added to the stained stain, which is designed to discolor. After rinsing, the specimen is stained with eosin or nigrosin contrast solutions for microscopic examination. This type of staining forms the basis for Gram staining, which distinguishes between Gram-positive and Gram-negative cells.
Differences in Staining Results
Based on staining patterns, scientists classify cells according to the procedure used and the cell’s reaction. For example, Gram-positive cells retain their purple color after staining, whereas Gram-negative cells do not. The reason for the difference in coloration is the composition of the cell wall of each type of bacteria: Gram-positive cells have a thick layer of peptidoglycan, whereas Gram-negative cells have a relatively thin peptidoglycan surrounded by an outer shell, which prevents the violet pigment from attaching.
In addition, in acid-fast staining, acid-fast bacteria, unlike non-acid-fast bacteria, remain stained with carbol fuchsin, which gives them a pinkish color (Hartline, 2023a). The reason for this is the presence of a broad, waxy lipid layer in acid-tolerant bacteria, which binds the carbol fuchsin molecules. The difference between Capsule+ and Capsule- is determined by the ability of bacteria to form capsules, outer protective shells of polysaccharides or glycoproteins, which makes the use of classical staining methods difficult. Capsule+ bacteria are identified by contrast staining, whereby the cell itself is stained, but the capsule is not, which looks like a transparent halo around the cell, and which is not observed for Capsule- (Tankeshwar, 2022).
Ultimately, the main distinction is that Endospore+ organisms can create protective outer envelopes, a trait that simultaneously hinders the use of classical staining techniques (Hartline, 2023b). In cell staining, heat allows the pigment to penetrate the endospore structure. Thus, the actual difference between Endospore- and Endospore+ bacteria is whether they are stained after application of the procedure (Endospore+) or not (Endospore-).
References
Hartline, R. (2023a). Acid-fast stain. LibreTexts Chemistry.
Hartline, R. (2023b). Endospore stain. LibreTexts Chemistry.
Tankeshwar, A. (2022). Capsule stain: Principle, procedure, results. Microbe Online.