Acid Fast Stain
Acid-fast stain is a laboratory technique used to identify acid-fast bacteria, such as Mycobacterium species, which have a waxy cell wall that resists conventional staining methods. The stain involves using a lipid-soluble dye, such as carbol fuchsin, which penetrates the cell wall and resists decolorization with acid-alcohol. This allows acid-fast bacteria to retain the dye and appear red or pink under a microscope.
5 Key excerpts on "Acid Fast Stain"
eBook - ePubMicrobiology and Molecular Diagnosis in Pathology
A Comprehensive Review for Board Preparation, Certification and Clinical Practice
- Audrey Wanger, Violeta Chavez, Richard Huang, Amer Wahed, Amitava Dasgupta, Jeffrey K. Actor(Authors)
- 2017(Publication Date)
- Elsevier(Publisher)
...Acridine orange stain can also be used, for these fastidious Gram-negative organisms, which retain counterstain poorly. Acridine orange stain intercalates into the nucleic acid of the organism and stains the organisms orange. Acid Fast Strain Acid Fast Stains are used to differentiate acid fast organisms such mycobacteria. Acid fast bacteria have a high content of mycolic acids in their cell walls. Acid fast bacteria will be red, while nonacid fast bacteria will stain blue/green with the counterstain with the Kinyoun stain. The steps include: Step 1. Apply carbol fuchsin to a fixed slide for 1 minute followed by rinsing. Step 2. The decolorizing agent, 3% hydrogen chloride (HCl), is applied for 2 minutes and remove the primary stain and rinse. Step 3. Apply the counterstain, methylene blue, for 2 minutes then rinse. Step 4. Allow to dry and observe slide with a light microscope. Ziehl Nielson can also be used to stain mycobacteria but uses heat while the Kinyoun method does not. The Kinyoun method can be modified as a weak Acid Fast Stain, which uses 5% sulfuric acid instead of hydrochloric acid. The weak Acid Fast Stain in addition to staining mycobacteria will stain organisms that are not able to maintain the carbol fuchsin after decolorizing with HCl, such as Nocardia spp., Rhodococcus spp., Tsukamurella spp., and Gordonia spp. The weak Acid Fast Stain also helps differentiate among the organisms that appear as Gram-positive branching filamentous rods such as Nocardia spp. and Streptomyces spp. Nocardia will stain positive with a weak Acid Fast Stain and Streptomyces spp. will not. Rhodamine Auramine Strain Rhodamine auramine stain is used for the detection of mycobacteria directly from clinical specimens. The dye binds with the mycolic acids and fluoresces under ultraviolet light. Acid fast organisms (mycobacteria) will appear yellow or orange under ultraviolet light...
eBook - ePub- Luis M. de la Maza, Marie T. Pezzlo, Cassiana E. Bittencourt, Ellena M. Peterson(Authors)
- 2020(Publication Date)
- ASM Press(Publisher)
...Acid‐fast smears are useful not only for assisting with the primary diagnosis but also for monitoring response to antimycobacterial drug therapy. In general, cultures become negative before the smears because the organisms are no longer capable of replicating. Thus, quantitation of the organisms on the smear and the correlation with growth may provide an indication of the effectiveness of the therapy. Figure 41‐24 Acid‐fast stain: auramine fluorochrome. Auramine O can also be used to stain mycobacteria. In this case, fluorochromes dissolved in ethanol and phenol were used to stain the preparation. The specimen was then decolorized with ethanol‐HCl, and potassium permanganate was used as the counterstain. As shown here, mycobacteria appear yellow (golden when rhodamine is used) on a black background when observed under a fluorescence microscope. The advantage of the fluorochrome‐stained smears is that mycobacteria can be seen with a 25× objective, significantly reducing the time required to scan a preparation, whereas a 100× objective is used when examining the Ziehl‐Neelsen stain. However, it is important to emphasize that this type of staining is the direct result of a physicochemical binding of the dye to the lipid‐rich cell of the organisms, and not an antigen‐antibody reaction. Therefore, the staining is not specific. PRIMARY PLATING MEDIA Figure 41‐25 Blood agar. Blood agar is a common medium used for the primary plating of clinical specimens as well as for the propagation of many laboratory bacterial strains. In general, blood agar is nonselective but is differential, in particular for species for which hemolysis is a key characteristic. Shown here is 5% sheep blood agar with a Trypticase soy broth base. There are several variable components of this formulation, including the type of red cells used, the percentage of cells, and the broth base used to prepare the medium...
- B. M. Mitruka(Author)
- 2017(Publication Date)
- CRC Press(Publisher)
...The bacterial cells are fixed to the slide by heat and stained with a basic dye (e.g., crystal violet) which is taken up by all bacteria in similar amounts. The specimen is treated with an I 2 -KI mixture (Gram’s iodine) to fix (mordant) the stain. It is then washed with a 1:1 mixture of acetone and ethanol and finally is counterstained with a paler dye of a different color (e.g., safranin). The slide is washed gently with water, blotted dry, and examined microscopically under the oil immersion lens. Gram-positive organisms retain the initial violet stain, while Gram-negative organisms are decolorized by the organic solvent and hence show the counter-stain. Young growing cultures showing a Gram-positive reaction may tend to lose this characteristic with age. Acid-fast stains – Acid-fast organisms retain stain when treated with dilute acid followed by alcohol wash. The Ziehl-Neelsen carbolfuchsin stain is usually used. Fluorescent dyes such as auramine O and rhodamine B are also used for acid-fast organisms, particularly Mycobacterium tuberculosis and Runyon’s Groups I, II, and III of the atypical mycobacteria which retain the stain and fluoresce when the stain is excited by light of suitable wavelength. Granule stains – Granule stains such as methylene blue are used for throat and nose smears. These stains are used to accentuate granules in bodies of organisms such as Coryne-bacterium diphtheriae. Capsule stains — These are used by the Anthony and Muir staining methods. With the Anthony method, the capsule is unstained against deep purple-stained cells and background. The Muir method uses Ziehl-Neelsen carbolfuchsin followed by mordant, tannic acid, mercuric chloride, and potassium alum. Ethanol is used to decolorize the stained specimen to a faint pink color. The specimen is counterstained with 0.3% methylene blue. Capsules stain blue and cells stain red. Flagella are visualized with the Leifson or Gray method...
- Kim S Suvarna, Christopher Layton, John D. Bancroft(Authors)
- 2018(Publication Date)
- Elsevier(Publisher)
...The avoidance of defatting agents or solvents, such as alcohol and xylene in methods for Mycobacterium leprae, is an attempt to conserve this fragile fatty capsule. Mycobacteria are PAS positive due to the carbohydrate content of their cell walls. However, this positivity is evident only when large concentrations of the microorganisms are present. When these organisms die, they lose their fatty capsule and consequently their carbol fuchsin positivity. The carbohydrate can still be demonstrated by Grocott’s methenamine silver reaction, which may prove useful when acid-fast procedures fail, particularly if the patient is already receiving therapy for tuberculosis. A possible source of acid-fast contamination may be found growing in viscous material sometimes lining water taps and any rubber tubing connected to them. These organisms are acid- and alcohol-fast but are usually easily identified as contaminants by their appearance as clumps, or floaters, above the microscopic focal plane of the section. Ziehl-Neelsen (ZN) stain for Mycobacterium bacilli (Kinyoun, 1915) Sections Formalin or fixative other than Carnoy’s, paraffin wax embedded. Solutions Carbol fuchsin commercially available, or Basic fuchsin 0.5 g Absolute alcohol 5 ml 5% aqueous phenol 100 ml Mix well and filter before use. Acid alcohol Hydrochloric acid 10 ml 70% alcohol 1000 ml Methylene blue solution (stock) commercially available, or Methylene blue 1.4 g 95% alcohol 100 ml Methylene blue solution (working) Methylene blue (stock) 10 ml Tap water 90 ml Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain in carbol fuchsin solution for 30 minutes. 3. Wash well in tap water. 4. Differentiate in acid alcohol until solutions are pale pink. (This usually only takes 2–5 dips.) 5. Wash in tap water for 8 minutes, then dip in distilled water. 6. Counterstain in working methylene blue solution until sections are pale blue. 7. Rinse in tap water, then dip in distilled water. 8...
eBook - ePub- Lynne Shore Garcia(Author)
- 2016(Publication Date)
- ASM Press(Publisher)
...Oocysts in clinical specimens may be difficult to detect without special staining. Modified acid-fast stains are recommended to demonstrate these organisms. Application of heat to the carbol fuchsin assists in the staining, and the use of a milder decolorizer allows the organisms to retain their pink-red color (37). With continued reports of diarrheal outbreaks due to Cyclospora, it is also important to remember that these organisms are acid fast and can be identified by using this staining approach (42, 43). Although the microsporidial spores are also acid fast, their size (1 to 2 µm) makes identification very difficult without special stains or the use of monoclonal antibody reagents. Concentrated sediment of fresh, formalin, other non-mercury single-vial fixative-preserved stool, or those preserved in Universal Fixative may be used. Other types of clinical specimens such as duodenal fluid, bile, and pulmonary specimens (induced sputum, bronchial washings, or biopsy specimens) may also be stained. Carbol Fuchsin 1. To make basic fuchsin (solution A), dissolve 0.3 g of basic fuchsin in 10 ml of 95% ethanol. 2. To make phenol (solution B), dissolve 5 g of phenol crystals in 100 ml of distilled water. (Gentle heat may be needed.) 3. Mix solution A with solution B. 4. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label. 1–3% Sulfuric Acid 1. Add 1 to 3 ml of concentrated sulfuric acid to 99 or 97 ml of distilled water. 2. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label. Methylene Blue 1. Dissolve 0.3 g of methylene blue chloride in 100 ml of distilled water. 2. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label. Quality Control for the Modified Ziehl-Neelsen Acid-Fast Staining Method QC guidelines are the same as those for the Kinyoun’s acid-fast stain and are given on p...
