When a mixed culture containing both acid-fast and non-acid-fast cells is subjected to this procedure, the acid-fast cells will be stained red and the non-acid-fast cells will be stained blue. The presence of purple-colored cells usually indicates the procedure has been improperly completed; however, some cells are only partially acid-fast and others may contain acid-fast structures. The endospores formed by bacteria in the genus Bacillus will often stain red when subjected to the acid-fast stain.
What is acid-fast staining?
Acid-fast staining is the technique used in discriminating the two types of bacteria. Acid fast bacteria are composed of mycolic acid in their cell wall; hence, they are stained in pink by the primary stain, carbol-fuchsin. Since non acid fast bacteria lack mycolic acid in their cell wall, they are incapable of retaining the primary stain.
What is acid fast in microbiology?
Acid fastness is a physical character of bacteria, which can be described as the resistance to decolorization by acids during staining procedures such as Gram staining. The acid-fast staining is used to differentiate bacteria as acid fast and non acid fast bacteria. The most commonly used acid-fast staining technique is Ziehl-Neelsen stain.
What is the final color of acid and non acid fast bacteria?
Final color. Acid Fast Bacteria: The final color of the acid fast bacteria is pink or red. Non Acid Fast Bacteria: The final color of the non acid fast bacteria is blue.
What is the difference between Gram staining and acid staining?
Two important Differential staining procedures are Gram’s staining method and acid-fast staining method. In the gram staining procedure, gram-positive cells stain purple in color whereas the acid-fast staining procedure differentiates gram-positive bacterial cells into two different types.
What is the color of acid-fast positive organisms at the end of acid-fast staining technique?
Because of the nature of their cell wall, acid-fast bacteria stain red after acid-fast staining. The genus Mycobacterium and the genus Nocardia are among the few bacteria possessing an acid-fast cell wall.
What color are acid-fast cells?
red colorOnly decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.
What color are the cells at the end of the acid-fast staining technique?
reddish-pinkMethylene blue is then applied to counterstain any cells which have been decolorized. At the end of the staining process, acid-fast cells will be reddish-pink, and non-acid fast cells will be blue. (Note: Acid-fast stains are performed on smears that have been heat-fixed.)
What does a blue acid-fast stain mean?
One commonly used counterstain is methylene blue. If the bacteria is acid-fast, it will appear pink. Non-acid fast bacteria will be purple or blue in color, depending on which counterstain you used. A positive result means it has mycolic acid, which helps the cell wall resist acid alcohol and retain the pink color.
What color are non acid-fast cells at the end of the stain?
Non-acid-fast cells are counterstained with a basic dye of a contrasting color (e.g., methylene blue). When a mixed culture containing both acid-fast and non-acid-fast cells is subjected to this procedure, the acid-fast cells will be stained red and the non-acid-fast cells will be stained blue.
Why acid-fast bacteria appears pink?
Acid Fast Bacteria Stain The bacteria are covered with a solution called Carbol-fuchsin. The Carbol-fuchsin dye attaches to the mycolic acids, staining them pink. The excess solution is rinsed out using alcohol and if the bacteria contain mycolic acids, they will appear pink under the microscope.
What color would Staphylococcus aureus be on the slide after acid-fast staining?
The acid-fast Mycobacterium retains carbol fuchsin and stains hot pink. The Staphylococcus epidermidis is decolorized and the counterstain colors them blue.
What is the primary stain in an acid-fast stain?
The primary stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall.
What is the correct order of acid-fast stain?
The correct order of an acid-fast stain is primary staining, iodine fixation, decolorizing, counterstaining.
Which two bacterial genera will usually stain red during the acid-fast staining procedure?
Organisms in the genera Mycobacterium and Nocardia have waxy mycolic acid in their cell walls, as well as a layer of peptidoglycan. (See Fig. 6-1) The mycolic acid layer absorbs the red stain carbolfuchsin.
What does a positive acid-fast bacilli mean?
If your AFB smear was positive, it means you probably have TB or other infection, but an AFB culture is needed confirm the diagnosis. Culture results can take several weeks, so your provider may decide to treat your infection in the meantime.
What bacteria are acid-fast positive?
Bacteria displaying acid fastness include:Genus Mycobacterium – M. leprae, M. tuberculosis, M. smegmatis, M. Avium complex, M. kansasii.Genus Nocardia – N. brasiliensis, N. cyriacigeorgica, N. farcinica, and N. nova.
What is acid fast bacteria?
Acid-fast bacteria, also known as acid-fast bacilli or simply AFB, is a group of bacteria sharing the characteristic of acid fastness. Acid fastness is a physical property that gives a bacterium the ability to resist decolorization by acids during staining procedures. This means that once the bacterium is stained, it cannot be decolorized using acids routinely used in the process. This important and unique feature of certain bacteria gives us the ability to classify and detect them using relatively easy laboratory procedures such as microscopy. Bacteria displaying acid fastness include:
Is sarcocystis acid fast?
Sarcocystis. Nuclear inclusion bodies in lead poisoning. Even though acid fastness can be attributed to many different bacteria, in clinical practice, correlation with history makes it a fairly unique characteristic of M. tuberculosis. This makes acid-fast staining sensitive and specific, provided clinical correlation is part of the equation.
What is the principle of acid fast stain?
Principle of Acid Fast Stain. Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the cells waxy and resistant to staining with aqueous based stains such as the Gram stain. The primary stain, carbolfuchsin is applied to the cells, and heat and phenol are used to allow the stain to penetrate into the waxy surface ...
How to clean a slide with methylene blue?
Run acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds. Rinse the slide with water. Cover the smear with the secondary or counterstain, methylene blue, for 1 minute. Gently rinse the slide with water. Blot the slide dry with bibulous paper.
What paper to use to dry a slide?
Blot the slide dry with bibulous paper.
How long to leave filter paper on water bath?
Cover the filter paper with the primary stain, carbolfuchsin. Leave the slide on the water bath for 3 to 5 minutes. Continue to apply stain if the filter paper begins to dry.
How to acid fast stain a smear?
Procedure of Acid Fast Stain. Prepare and fix the specimen smear prior to staining. Place a small strip of blotting or filter paper over the top of the specimen, and place the slide over a boiling hot water bath on a mesh surface. Cover the filter paper with the primary stain, carbolfuchsin. Leave the slide on the water bath for 3 to 5 minutes.
Why is filter paper moist?
The filter paper must remain moist and in contact with the specimen during heating to allow for proper penetration of the primary stain. Organisms cultivated on blood agar may experience nutrient deprivation, resulting in a lower lipid content in the outer membrane resulting in poor staining.
What is the difference between acid fast and non acid fast bacteria?
The main difference between acid fast and non acid fast bacteria is that acid fast bacteria resist decolorizing by acid after accepting a stain whereas non acid fast bacteria are readily decolorized by acid ...
How are acid fast and non acid fast bacteria differentiated?
Acid fast bacteria and non acid fast bacteria can be differentiated by acid –fast staining techniques. The cell wall structure of bacteria is involved in the differentiation of both acid fast and non acid fast bacteria.
What is acid fastness in bacteria?
Acid fastness is a physical character of bacteria, which can be described as the resistance to decolorization by acids during staining procedures such as Gram staining . The acid-fast staining is used to differentiate bacteria as acid fast and non acid fast bacteria. The most commonly used acid-fast staining technique is Ziehl-Neelsen stain. Three different reagents are used in acid-fast staining. They are carbol-fuchsin as the primary stain, acid-alcohol as decolorizing agent, and methylene blue as the counterstain. A few genera of bacteria such as Mycobacterium can only be visualized by acid-fast staining. Some protozoa also exhibit acid fastness. The main difference between acid fast and non acid fast bacteria is that acid fast bacteria resist decolorizing by acid after accepting a stain whereas non acid fast bacteria are readily decolorized by acid after staining.
What is the mycolic acid?
The mycolic acid is a long chain of fatty acids, attached to the peptidoglycans. Once the primary stain, carbol-fuchsin is added to the slide containing bacteria, the mycolic acid is attached to carbol-fuchsin. This makes the acid fast bacteria to stain in pink even after decolorization.
What is the most commonly used acid fast staining technique?
The most commonly used acid-fast staining technique is Ziehl-Neelsen stain. Three different reagents are used in acid-fast staining. They are carbol-fuchsin as the primary stain, acid-alcohol as decolorizing agent, and methylene blue as the counterstain. A few genera of bacteria such as Mycobacterium can only be visualized by acid-fast staining. ...
What is acid fast staining?
Acid-fast staining is the technique used in discriminating the two types of bacteria. Acid fast bacteria are composed of mycolic acid in their cell wall; hence, they are stained in pink by the primary stain, carbol-fuchsin. Since non acid fast bacteria lack mycolic acid in their cell wall, they are incapable of retaining the primary stain.
What is acid fast?
Acid fastness is a physical property of bacteria, which rely on the structure of the bacterial cell wall. Typically, the cell wall of bacteria is made up of proteins, carbohydrates, and lipids. Acid fast bacteria comprise a thin layer of peptidoglycans. The mycolic acid is a long chain of fatty acids, attached to the peptidoglycans.
What is the decolorizing reagent used in Gram stain?
Acetone-alcohol is the decolorizing reagent used in the Gram stain. If this material is used to decolorize cells stained with carbol fuchsin, the red color will not come out of any cells, and non-acid-fast cells will appear to be acid-fast, i.e., will stain red. It is important to use the correct decolorizing reagents when preparing different types of differential stains.
What is mycolic acid?
Mycolic acid is the wax-like lipid present in the walls of acid-fast cells. This material makes cells resistant to staining, but also makes them resistant to being decolorized once they have been successfully stained.
What is the solution used to remove primary stain?
2. The stained cells are subjected to decolorizing with an acid-alcohol solution ; 95% ethyl alcohol containing 2.5% concentrated nitric or hydrochloric acid (HNO3 or HCl). This step will remove the primary stain from cells that are not acid-fast, rendering them colorless.
What color is used to color cells?
1. Application of the primary stain, carbon fuchsin, will color all the cells present a deep red or fuchsia color. 2. Application of a decolorizing solution, will remove color from cells that are not acid-fast. 3. Application of a counterstain reagent, methylene blue, will color the cells that are not acid-fast, blue.
What color do acid fast cells stain?
Acid-fast cells stain red, while other cells appear blue (when methylene blue is used as the counterstain).
Is acid fast stain a basic stain?
Both the Gram stain and the acid-fast stain are basic stains and both are differential stains that cause differences in cell wall composition to be visible as color differences.
Is acid fast a direct stain?
The acid-fast stain is a direct stain and a differential stain, what does this mean?
Why is it so hard to stain bacteria?
The acid-fastness property of these bacteria is correlated with high lipid contents, which makes them difficult to stain. Hence for staining of these bacteria heating with strong dye is required. Once the acid-fast bacteria are stained, it is difficult to decolourize them even with acid and alcohol.
What is the dormant structure of bacteria?
This dormant structure is called an endospore since it develops within the cell. Endospore morphology and location vary with species and often are valuable in identification. Endospores are not easily stained well by most dyes.
What is the procedure used to detect bacteria?
In this article we will discuss about the staining procedure used for detecting bacteria. 1. Simple Staining Procedure : When a single staining-reagent is used and all cells and their structures stain in the same manner, the procedure is called simple staining procedure. This procedure is of two types – positive and negative (Fig. 17.5).
What is a smear on a slide?
A thin film of young culture (smear) of endosporous bacteria is fixed on a clean slide. ii. The smear is heat-fixed on to the slide by gentle warming. iii. Smear is covered with the solution of malachite green which is a very strong stain that can penetrate the spore-coat of an endospore.
What is the name of the technique used to differentiate a gram positive from a gram negative?
This procedure is called Gram- staining technique. This technique has experienced numerous modifications from time to time and proves to be valuable for staining smears of pure cultures of bacteria.
What is acid fast stain?
The acid-fast stain is a differential stain developed first by Paul Ehrlich in 1882 and later on modified by Ziehl- Neelsen, and is in use even today by microbiologists. Majority of the bacteria are stained with simple stain and Gram-stain but certain bacteria do not do so because they have waxy components of the cell wall, hence their cell wall has limited permeability.
Why are bacteria motile?
Many bacteria are motile due to the presence of flagella that originate in the cytoplasm and project out from the cell wall. Bacterial flagella are fine, threadlike organelles that are so slender (about 10 to 30 nm in diameter) that they can only be observed directly using electron microscope.