Printerfriendly preparation and examination of skin smears pdf 225 kb. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Laboratory logregister is legible dates of collection, quality of sputum. The most frequent staining procedure used to detect the presence of afb is ziehlneelsen zn staining. Sincere thanks to members involved from phl for their valuable efforts to revise and update this standard manual and to the joint.
This was developed in 1884 by a danish bacteriologist, hans christian gram. Diagram showing vws in the laboratory with external discharge structure figure 5. The definitive diagnosis of malaria infection and other parasites is still based on finding such organisms in blood films. Procedures of tb microscopy begin with smearing, followed. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. Dna amplification by pcr is a sensitive and specific method for the diagnosis of tuberculosis, and with this simplified dna isolation procedure it can be used in routine clinical practice. 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. Principle, procedure, interpretation, examples and animation last updated. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Lymphadenitis is considered mainly a disease of young children and has a peak age of onset at 20 to 40 years. The stained smear is the easiest and quickest procedure that can be performed for diagnosis of tb and is economical especially in developing countries. Fields stain is a histological method for staining of blood smears. Mycobacteriology laboratory manual world health organization. Standard operating procedure bright field immunohistochemistry manual staining sop i.
Wright stain is intended for use in staining blood films or bone marrow films. Ventilated workstation manual for afb smear microscopy. Laboratory evaluationacidfast bacilli microbiology afb tool. The kinyoun method or kinyoun stain cold method, developed by joseph j.
Efficiency of cold staining method of afb in sputum a comparison with ziehl neelsen method under field conditions article pdf available january 1990 with 436 reads how we measure reads. Cryptosporidium and isospora species have been recognized to cause severe diarrhea in immunocompromised hosts, but it can also cause diarrhea in immunocompetent host. When blood films are treated as herein described, the white. Leishmans stain belongs, as giemsa and wrights stain, to the group of romanovsky stains. Key difference gram stain vs acid fast bacteria are very small microorganisms. Remel tb kinyoun afb stain kit thermo fisher scientific.
Pdf efficiency of cold staining method of afb in sputum. Introduction demonstration of acidfast bacilli afb in a smear made from a clinical specimen provides a preliminary diagnosis of mycobacterial disease, while the isolation of mycobacteria on culture provides a definite diagnosis of tuberculosis or disease due to mycobacteria other. This technique is useful for the identification of oocysts of the coccidian species cryptosporidium, cystoisospora, and cyclospora, which may be difficult to detect with routine stains such as trichrome. Preparation and examination of skin smears official web. This sturdy waterproof poster illustrates the ziehlneelsen afb microscopy technique according to whounion recommendations and highlight quality issues. Cover the smear with 3% vv acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i. Acid fast stain is for the demonstration of mycobacterium tu ber cu lo sis in tissue. The purpose of the handbook is to teach laboratory technicians how to safely collect, process and. Cold kinyoun method modified afb staining procedure principle. To ensure quality control and in compliance with the appropriate quality assurance programs, it is necessary to have detailed standard operating procedures sop. The purpose of afb microscopy is to detect acidfast bacilli afb by. Ziehlneelsen hot, kinyoun cold are still widely used methods in developing countries.
The ziehlneelsen method of staining for tb is known as the hot plate method which means the ziehlneelsen method uses bright field microscopy. Unlike the ziehlneelsen modified acidfast stain, this stain does not require the heating of reagents for staining. The purpose of this study was to detect and compare the presence of acid fast bacilli in lymph node biopsies and their touch preparations using ziehl neelsen and fluorescence staining. June 12, 2018 by sagar aryal gram staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by danish bacteriologist hans christian gram in. Auraminerhodamine fluorochrome staining also known as truant method of staining, is used to visualize acid fast bacilli afb. Use millipore filtered water in the waterbath and staining procedure. The gram staining technique differentiates the mixed culture cells into two terms as gram. They are transparent, and their detection is difficult under living and unstained conditions. Gargi dangremudey abstract biology micro and molecular biology publish your bachelors.
A typical afb stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells. Misdiagnoses of tuberculosis resulting from laboratory crosscontamination of mycobacterium tuberculosis cultures new jersey, 1998. Filter carbol fuchsin stain solution, ziehlneelsen. Although difficult to stain, once the stain goes into the wall, the cell will not destain or decolorize easily. Gram staining technique is the widely used differential staining technique in bacteriology. Quality improvement for an example of how to complete this form. Ventilated workstation manual for afb smear microscopy 5 table of figures figure 1.
Wipe the back of the slide clean, and place it in a draining rack for the smear to airdry do not blot dry. Wright stain is a modified romanowsky stain intended for differentially staining the cellular elements of blood. The study describes a cold staining technique that uses the same staining solutions as the conventional ziehlneelsen method. Analysis of specimens for the detection of acid fast bacilli by microscopy comprises 2 steps. Acidfast stain stain kit for the detection of acid fast organisms in cultures and clinical samples kit includes carbol fuchsin kinyoun, decolorizer, and methylene blue counter stain 8.
An intro to routine and special staining in histopathology. The skin smear is a valuable, costeffective tool in the routine management of the patient with hansens disease. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. Diagram showing appropriatelyinstalled vws in the laboratory figure 4.
Each procedure lists the materials, equipment, and forms required for the test, along with the applicable quality control procedures. Afb staining kinyoun kit, methylene blue azer scientific. If observed results vary from expected results, please contact sigmaaldrich technical service for assistance. Acid fast staining procedure for staining mycobacteriaa presentation byisaac. Periodic acidschiff pas staining system procedure no. Extraction slide will have no visible pas staining of glycogen when compared to the undigested glycogen positive control slide. An appendix k form is required when a qc test fails to give proper results. Overheating of smears when heat fixation is used d. Sop for microbial staining procedures standard operating procedure to stain the bacterial cells, bacterial spores and fungus.
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. Cover the smear with methylene blue or malachite green stain for 12 minutes. In thin films the red blood cells are fixed and parasitized cells can be identified by. Laboratory diagnosis of tuberculosis by sputum microscopy. A diagnosis of tuberculosis tb is rarely disputed if mycobacterium tuberculosis is isolated from a clinical specimen. Sensitivity and specificity, microscopy for afb, zn staining, fluorescent stain, concentration of sputum 1. Flood slide with crystal or gentian violet 60 seconds. The most common form of extrapulmonary tuberculosis is lymphadenitis. In some cases, a situation may require both an appendix k and a ntf. Examine the smear microscopically, using the 100x oil immersion objective. In this staining technique acid fast bacilli take bright red colour and background is blue. Acid alcohol is fiammable, therefore use it with care well away from an open fiame. 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. Full text get a printable copy pdf file of the complete article 994k, or click on a page image below to browse page by page.
For the detection of malaria parasite leishman staining seems more sensitive than e. Currently in most laboratories, methods of staining using fluorescence microscopy fm and lightemitting diodes led microscopy for acid fast bacilli afb are. It is a variation of a method developed by robert koch in 1882. Thus, various staining methods are developed to facilitate bacterial detection. All newcomer supply stain procedures are designed to be used with coplin jars filled to 40 ml following the staining procedure provided below. 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. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. Acid fast staining procedure for staining mycobacteria. Misdiagnoses of tuberculosis resulting from laboratory. Module common staining technique microbiology 24 notes indeterminate groups as well. When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater. This technique was introduced by ziehlneelsen in year 18821883.
Zn method is still the most extensively used procedure for afb staining. Manual of histopathological and histochemical procedures, 16th ed. Detection of acid fast bacilli in tuberculous lymph node. It is considered as an easy to do technique which gives a fairly acceptable contrast. Quality control slides for microbiology polysciences, inc. Acid fast staining in formalinfixed tissue specimen of. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Introduction direct examination of sputum for acidfast bacilli afb by ziehlneelsen zn stained smears using conventional microscopy is a standard procedure in the. It is also used to document any quality improvements made by the laboratory. Cold kinyoun method modified afb staining procedure. By colouring otherwise transparent tissue sections, these stains allow highly trained pathologists and researchers to view, under a microscope, tissue morphology structure or to look for the presence or prevalence of particular cell types, structures or even microorganisms such as bacteria. Detection of mycobacterium tuberculosis in sputum samples.
Afb microscopy examination of smears is a rapid, convenient and inexpensive test all types of specimens can be evaluated sputum, tissue, body fluids, etc. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Kinyoun, is a procedure used to stain acidfast species of the bacterial genera mycobacterium and nocardia and the apicomplexan genus cryptosporidium. For direct smears, the correlation of results of the cold staining procedure with those of the ziehlneelsen method was 97% and for concentrated smears was 99%. Acidfast bacteria, of which there are very fewthe major genus mycobacterium, have a high concentration of mycolic acid, a lipid, in their walls. Comparison of sensitivity and specificity of zn and. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011.
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