Cdc recommends fluorochrome staining for detecting afb in primary patient specimens. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. Misdiagnoses of tuberculosis resulting from laboratory. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Tb rapid stain set tb rapid stain primary stain tb rapid. A doctor typically orders the test to determine if a person has tuberculosis tb or another type of mycobacterial infection. Protocol for afb smear staining by cedric cheung 09 oct, 2016 dear all, though it may be old fashioned, we here at our hospital can still only do afb staining for detecting afb in sputum samples. Cold kinyoun method modified afb staining procedure principle.
Jan 03, 2020 periodic acid schiff pas staining is one of the most commonly performed special staining technique in histopathology laboratory which is used to highlight molecules with high percentage of carbohydrate content such as mucin, glycogen, fungi and basement membrane in skin. Standard nurse protocols for registered professional nurses 2018 tuberculosis tb 16. Staining methods ziehlneelsen staining this is an example of insufficient staining with carbolfuchsin during the ziehlneelsen staining procedure andor insufficient flaming of the carbolfuchsin while on the slide. Staining procedure become simple as it does not involve heating of carbolfuschin, which is crucial in the ziehlneelsen zn method. Ziehlneelsen hot, kinyoun cold are still widely used methods in developing countries. A process for pot staining of sputum for detection of acid. Acid fast staining procedure for staining mycobacteria. Summary and explanation the afb stain kit is typically used to identify acidfast bacilli, such as mycobacterium. Acid alcohol is fiammable, therefore use it with care well away from an open fiame. Tb laboratory techniques for diagnosing tuberculosis.
Acid fast staining procedure for staining mycobacteria bruno mmassy. Based on the kinyoun modified staining technique, the acidfast stain is a very useful tool for differentiating acidfast bacteria afb, such as mycobacterium spp. Flood slide with crystal or gentian violet 60 seconds. May cause irritation to skin, eyes and respiratory tract. B staining of blood smear leave it for 12 minutes for fixation of the smear. Cryptosporidium and isospora species have been recognized to cause severe diarrhea in immunocompromised hosts, but it can also cause diarrhea in immunocompetent host. Drain slides, wipe off excess oil, and blot to opacity taking care to remove residual oil. Gram stain and acidfast stain are most popularly known as differential stains. Three types of staining procedures are used for the detection of acidfast organisms. Use fluorescent acidfast staining and microscopic examination goal reporting afb smear result within 24 hrs from receipt of specimen in lab use rapid broth system for primary culture detection of afb goal to reduce culture detection of afb from 34 wks to 12 wks use rapid id, i. Principle, procedure, reporting and limitations by editorial team on january 3, 2020 in bacteriology, microbiology, mycology smear microscopy is the simplest and quickest currently available procedure to detect acid fast bacilli afb in clinical specimens. When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater. Send specimen collected to the georgia public health laboratory gphl in decatur. Mycobacterial cell walls contain a waxy substance composed of mycolic acids.
Protocol for afb smear staining connecting our members. Standard operating procedure bright field immunohistochemistry manual staining sop i. Fields stain is a histological method for staining of blood smears. Neelsen in 1883 used ziehls carbolfuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Qc1 quality control slides for afb, parasitology, bacteriology, and mycology processing, as. Acid fast bacilli staining protocol ihc world, llc. Auramine orhodamine b fluorescent stain is poisonous and may be harmful or fatal if swallowed. This procedure is based on the work of ziehlneelsen 1. Identification of tb strain patterns through molecular typing or. Differential staining is a technique which uses more than one stain to differentiate bacteria.
Your explanation of the principle and procedure for acid fast staining were basically correct. Histology and tissue staining modern techniques in life sciences 09. Overheating of smears when heat fixation is used d. Laboratory evaluationacidfast bacilli microbiology afb. If afb are found on a sputum smear, it often indicates tb disease smear positive however, a positive smear does not confirm a diagnosis of tb because some stained mycobacteria are not m. He developed a stain for the bacterium, although it was not very effective for visualizing this slender bacillus. Module common staining technique microbiology 24 notes indeterminate groups as well. This test is set up in the labcorp computer as a sequential series of tests to comply with customer needs and bill for testing that is performed. Smear, when appropriate, is a separate test along with appropriate specimen processing needed to perform the smear and culture. Acidfast stain principle, procedure, interpretation and.
All reagents in the afb stain kit have been optimally prepared for use on dakos artisan staining system and require no mixing or diluting. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011. Advantage, disadvantage and limitations of microscopy to. Diagnosis of malaria involves identification of malaria parasite or its antigensproducts in the blood of the patient. Pdf files contains the procedure for a special stain, a procedure card, and sample. Using a microscope, laboratory personnel look for afb. If afb are still observed, discard the batches of staining solutions and prepare new ones, making sure to use absolutely clean glassware and distilled water for dissolving the dyes. Auraminerhodamine fluorochrome staining also known as truant method of staining, is used to visualize acid fast bacilli afb. Pdf external quality assessment of afb smear microscopy. Include positive and negative controls with each days reading. Advantage, disadvantage and limitations of microscopy to detect acid fast bacilli afb december 6, 20 acharya tankeshwar bacteriology 0 mycobacteria are distinguished from other microorganisms by thick lipidcontaining cellwalls that retain biochemical stains despite decolourisation by acidcontaining reagents socalled acidfastness. A note to file ntf is a document used to describe an error i. We compared the sensitivity and specificity of a modified ziehlneelsen modifiedzn staining method for acidfast bacilli afb with that of the standard ziehlneelsen standardzn staining method, using culture results with mycobacterium tuberculosis as the gold standard.
Mycobacteriology laboratory manual world health organization. Its also known as an acidfast bacillus afb stain or a tuberculosis tb smear. Acid fast staining in formalinfixed tissue specimen of. The purpose of the handbook is to teach laboratory technicians how to safely collect. Clinical practice guideline standard operating procedure title. In this study, we examined whether the method of fixation heat versus methanol that is used to adhere bacteria to the slide prior to staining might influence the staining results.
So this method is also called ziehlneelsen staining techniques. Gram staining is a differential staining technique, which separates. Although this seems simple, the efficacy of the diagnosis is subject to many factors. Jul 10, 2011 acid fast staining procedure for staining mycobacteriaa presentation byisaac. History of gram stain the gram stain procedure was developed in 1883 by hans christian gram, a danish physician, who was working as a pathologist on a method he hoped would differentiate all bacteria from mammalian cells. Cold kinyoun method modified afb staining procedure. A diagnosis of tuberculosis tb is rarely disputed if mycobacterium tuberculosis is isolated from a clinical specimen. I am not a lab person, but as i understand it, the gold standard protocol involves digestion, decontamination, and centrifugation using a 50 ml. They get their name because they can be seen and counted under the microscope when smeared on a slide and treated with a special acidfast staining procedure.
Most common staning technique involve classic ziehlneelsen zn staining method or one of its variants. Cover the smear with 3% vv acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i. The primary stain for the kinyoun procedure is the aniline dye, basic fuchsin, that stains all the cells present red. Smear microscopy is the simplest and quickest currently available procedure to detect acid fast bacilli afb in clinical specimens. The stained smear is the easiest and quickest procedure that can be performed for diagnosis of tb and is economical especially in developing countries. Problems encountered as result of over heating such as disfigurations of acid fast bacilli afb in smear and drying of stain if unattended for a longtime by the technicians can be prevented. The kinyoun staining procedure is often referred to as cold carbolfuchsin because no heat is applied during the staining process unlike the ziehlneelsen procedure. Jun 12, 2018 it is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. Clinical practice guideline standard operating procedure. Thermo scientific remel tb kinyoun afb stain kit is for use in qualitative procedures to differentiate acidfast from nonacidfast bacteria and for the detection of cryptosporidium spp. The acidfast stain robert koch was the first person to isolate and identify mycobacterium tuberculosis from a patient with tuberculosis. A comparison of heat versus methanol fixation for gram. Artisan acidfast bacillus afb stain kit english code ar162.
Introduction to special stains introduction to special stains special stains special stain is a term that is mostly used in a laboratory setting. Some bacilli show bipolar staining, with the ends staining deeply and the middle portion very little. The evaluator needs to have a good understanding of acidfast bacilli afb smear microscopy and good laboratory. To ensure quality control and in compliance with the appropriate quality assurance programs, it is necessary to have detailed standard operating procedures sop.
Use millipore filtered water in the waterbath and staining procedure. A sputum stain for mycobacteria is a laboratory test performed on a sample of your sputum, or phlegm. Now a days, these methods have been supplanted by more sensitive auraminerhodamine fluorescence staining. Paul ehrlich is the first to describe the acidfast properties of the bacterium. Stains for acid fast bacilli can be performed on specimens collected as detailed below. In your procedure however, the preparation of the smear needs not be sterile but aseptically done to prevent unwanted materialsorganisms on your slide and assuring that only the specimen you intend to examine is on your slide. Staining techniques in microbiology guddeti prashanth kumar. Acid fast staining in tuberculosis freelance clinical microbiologist. The word gram is always spelled with a capital, referring to hans christian gram, the inventor of gram staining gram staining principles gram staining is used to determine gram status to classify bacteria broadly. General algorithm mgit 960 cultures 43 flow chart 2.
Auramine orhodamine b fluorescent stain set, auramine o. After collection, specimens are sent to a laboratory for examination. Make a thin film of the material on a clean glass slide, using a sterile loop or swab for viscous specimens. Remel tb kinyoun afb stain kit thermo fisher scientific. Procedures of tb microscopy begin with smearing, followed by staining, and lastly, reading. The mechanism for acidfastness is not clearly understood, however it is suspected that it is determined by. Sep 11, 2009 acid fast staining procedure for staining mycobacteria bruno mmassy. There are a several types of bacteria that may be detected in this manner. Misdiagnoses of tuberculosis resulting from laboratory crosscontamination of mycobacterium tuberculosis cultures new jersey, 1998. Filter carbol fuchsin stain solution, ziehlneelsen. Detection of acidfast bacilli afb in stained and acidwashed smears examined microscopically may provide the initial bacteriologic evidence of the presence of mycobacteria in a clinical specimen. Add leismans stain drop by drop till it covers whole of the smear. Contains material which may cause cancer based on animal data. Acidfast stain kit afb staining for mycobacteria and.
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