Public Site Members. Cancel Share. Written by Sourav Bio. Best Books For Microbial Metabolism. Top 14 books for Industrial Microbiology. Top 15 Books for Food and diary Microbiology. Top 18 books for Environmental Microbiology.
Reply Differences. Difference between innate and acquired immunity. Difference between endocytosis and exocytosis endocytosis vs exocytosis. Close this module. Join the Newsletter. Subscribe to our weekly newsletter below and never miss the latest articles. Email Enter your email address. Acid fast staining procedure involves three different reagents.
Carbol fuschin is used as the primary stain. Acid alcohol is used as the decolourizing agent. Methylene Blue is used as the counter stain.
Staining procedure is performed as follows. Visualization of microorganisms in living state is difficult. Therefore, biological stains and staining procedures are used extensively to study their properties. Differential staining is one type of staining technique used to differentiate bacteria.
Gram stain and acid fast stain are two differential staining techniques. Gram staining differentiates gram negative bacteria and gram positive bacteria based on the thickness of their cell walls. Acid fast staining differentiates acid fast bacteria from non acid fast bacteria based on the mycolic acid content in the cell wall.
This is the difference between acid fast and gram stain. You can download PDF version of this article and use it for offline purposes as per citation note. Aryal, Sagar. Available here. Samanthi Udayangani holds a B. Degree in Plant Science, M.
Your email address will not be published. Figure Gram negative and Gram positive bacteria. Figure 2: Acid Fast Mycobacteria. Leave a Reply Cancel reply Your email address will not be published. Gram Stain vs Acid Fast. Stains, or dyes, contain salts made up of a positive ion and a negative ion.
Depending on the type of dye, the positive or the negative ion may be the chromophore the colored ion ; the other, uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye ; if the negative ion is the chromophore, the stain is considered an acidic dye. Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen.
In most cases, it is preferable to use a positive stain , a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. However, there are scenarios in which it is advantageous to use a negative stain , which is absorbed by the background but not by the cells or organisms in the specimen.
Negative staining produces an outline or silhouette of the organisms against a colorful background Figure 2. Figure 2. Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin , crystal violet , malachite green , methylene blue , and safranin typically serve as positive stains.
On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin , eosin , and rose bengal. Table 2 provides more detail. Some staining techniques involve the application of only one dye to the sample; others require more than one dye.
In simple staining , a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism.
In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. Table 3 provides more detail on these differential staining techniques.
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 1.
Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish between gram-positive and gram-negative bacteria. Step 2: Iodine. Cells remain purple or blue. Step 3: Alcohol. Step 4: Safranin. Gram-negative cells appear pink or red. Figure 3. In this specimen, the gram-positive bacterium Staphylococcus aureus retains crystal violet dye even after the decolorizing agent is added.
Gram-negative Escherichia coli, the most common Gram stain quality-control bacterium, is decolorized, and is only visible after the addition of the pink counterstain safranin. The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative Figure 3. 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 decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative as in Figure 3. This suggests damage to the individual cells or that decolorizer 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 decolorizing 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.
Figure 4. 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 4. 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 carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing 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 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.
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