Histology and tissue staining modern techniques in life sciences 09. Cover the smear with 3% vv acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i. Pdf external quality assessment of afb smear microscopy. Advantage, disadvantage and limitations of microscopy to. Drain slides, wipe off excess oil, and blot to opacity taking care to remove residual oil. Acidfast stain principle, procedure, interpretation and. Acid fast bacilli staining protocol ihc world, llc. A comparison of heat versus methanol fixation for gram.
Standard operating procedure bright field immunohistochemistry manual staining sop i. Its also known as an acidfast bacillus afb stain or a tuberculosis tb smear. Smear, when appropriate, is a separate test along with appropriate specimen processing needed to perform the smear and culture. B staining of blood smear leave it for 12 minutes for fixation of the smear. The primary stain for the kinyoun procedure is the aniline dye, basic fuchsin, that stains all the cells present red. Stains for acid fast bacilli can be performed on specimens collected as detailed below. Cryptosporidium and isospora species have been recognized to cause severe diarrhea in immunocompromised hosts, but it can also cause diarrhea in immunocompetent host. 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.
Standard nurse protocols for registered professional nurses 2018 tuberculosis tb 16. Gram staining is a differential staining technique, which separates. Mycobacterial cell walls contain a waxy substance composed of mycolic acids. Acid fast staining in formalinfixed tissue specimen of.
Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011. Based on the kinyoun modified staining technique, the acidfast stain is a very useful tool for differentiating acidfast bacteria afb, such as mycobacterium spp. Acid fast staining in tuberculosis freelance clinical microbiologist. 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. The evaluator needs to have a good understanding of acidfast bacilli afb smear microscopy and good laboratory. Misdiagnoses of tuberculosis resulting from laboratory cross. A process for pot staining of sputum for detection of acid. Contains material which may cause cancer based on animal data. Auraminerhodamine fluorochrome staining also known as truant method of staining, is used to visualize acid fast bacilli afb. Sep 11, 2009 acid fast staining procedure for staining mycobacteria bruno mmassy.
A sputum stain for mycobacteria is a laboratory test performed on a sample of your sputum, or phlegm. Some bacilli show bipolar staining, with the ends staining deeply and the middle portion very little. The acidfast stain robert koch was the first person to isolate and identify mycobacterium tuberculosis from a patient with tuberculosis. Acid fast staining procedure for staining mycobacteria. Now a days, these methods have been supplanted by more sensitive auraminerhodamine fluorescence staining. General algorithm mgit 960 cultures 43 flow chart 2. A doctor typically orders the test to determine if a person has tuberculosis tb or another type of mycobacterial infection.
Misdiagnoses of tuberculosis resulting from laboratory. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. Three types of staining procedures are used for the detection of acidfast organisms. Clinical practice guideline standard operating procedure title. A diagnosis of tuberculosis tb is rarely disputed if mycobacterium tuberculosis is isolated from a clinical specimen. The slides from the same specimen demonstrated a high correlation between manual staining methods and aerospray staining methods with. Misdiagnoses of tuberculosis resulting from laboratory crosscontamination of mycobacterium tuberculosis cultures new jersey, 1998.
Flood slide with crystal or gentian violet 60 seconds. Fields stain is a histological method for staining of blood smears. Acidfast stain kit afb staining for mycobacteria and. Module common staining technique microbiology 24 notes indeterminate groups as well. Make a thin film of the material on a clean glass slide, using a sterile loop or swab for viscous specimens. Staining procedure become simple as it does not involve heating of carbolfuschin, which is crucial in the ziehlneelsen zn method. He developed a stain for the bacterium, although it was not very effective for visualizing this slender bacillus. The mechanism for acidfastness is not clearly understood, however it is suspected that it is determined by. When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.
Overheating of smears when heat fixation is used d. 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. Acid alcohol is fiammable, therefore use it with care well away from an open fiame. 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. Include positive and negative controls with each days reading. Protocol for afb smear staining connecting our members. Add leismans stain drop by drop till it covers whole of the smear. Acid fast staining, a presentation for biotechnic and biotechnology class. 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. Acid fast staining procedure for staining mycobacteria bruno mmassy. Clinical practice guideline standard operating procedure.
Ziehlneelsen hot, kinyoun cold are still widely used methods in developing countries. After collection, specimens are sent to a laboratory for examination. 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. The stained smear is the easiest and quickest procedure that can be performed for diagnosis of tb and is economical especially in developing countries. Procedures of tb microscopy begin with smearing, followed by staining, and lastly, reading. 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. 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. Send specimen collected to the georgia public health laboratory gphl in decatur. Auramine orhodamine b fluorescent stain set, auramine o.
Qc1 quality control slides for afb, parasitology, bacteriology, and mycology processing, as. Tb rapid stain set tb rapid stain primary stain tb rapid. Jun 12, 2018 it is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. 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. Filter carbol fuchsin stain solution, ziehlneelsen. Your explanation of the principle and procedure for acid fast staining were basically correct.
Although this seems simple, the efficacy of the diagnosis is subject to many factors. 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. Identification of tb strain patterns through molecular typing or. The kinyoun staining procedure is often referred to as cold carbolfuchsin because no heat is applied during the staining process unlike the ziehlneelsen procedure. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. All reagents in the afb stain kit have been optimally prepared for use on dakos artisan staining system and require no mixing or diluting. Using a microscope, laboratory personnel look for afb. 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. The definitive diagnosis of malaria infection and other parasites is still based on finding such organisms in blood films. Diagnosis of malaria involves identification of malaria parasite or its antigensproducts in the blood of the patient. May cause irritation to skin, eyes and respiratory tract. To ensure quality control and in compliance with the appropriate quality assurance programs, it is necessary to have detailed standard operating procedures sop. 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. 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.
Pdf files contains the procedure for a special stain, a procedure card, and sample. In thin films the red blood cells are fixed and parasitized cells can be identified by their morphology. A note to file ntf is a document used to describe an error i. Introduction to special stains introduction to special stains special stains special stain is a term that is mostly used in a laboratory setting. Neelsen in 1883 used ziehls carbolfuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. The purpose of the handbook is to teach laboratory technicians how to safely collect. There are a several types of bacteria that may be detected in this manner. 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. This procedure is based on the work of ziehlneelsen 1. 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. Laboratory evaluationacidfast bacilli microbiology afb. Use millipore filtered water in the waterbath and staining procedure. I am not a lab person, but as i understand it, the gold standard protocol involves digestion, decontamination, and centrifugation using a 50 ml.
Tb laboratory techniques for diagnosing tuberculosis. Auramine orhodamine b fluorescent stain is poisonous and may be harmful or fatal if swallowed. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. Cdc recommends fluorochrome staining for detecting afb in primary patient specimens. Mycobacteriology laboratory manual world health organization. Most common staning technique involve classic ziehlneelsen zn staining method or one of its variants. 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. Cold kinyoun method modified afb staining procedure. Summary and explanation the afb stain kit is typically used to identify acidfast bacilli, such as mycobacterium. Jul 10, 2011 acid fast staining procedure for staining mycobacteriaa presentation byisaac. 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. Cold kinyoun method modified afb staining procedure principle. Remel tb kinyoun afb stain kit thermo fisher scientific. Differential staining is a technique which uses more than one stain to differentiate bacteria.
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