Acidfast stain principle, procedure, interpretation and. Primary stain is carbol fuchsin ziehlneelson zn requires heat during staining requires higher magnification. Slides should be drained on a paper towel to remove excess. The cold staining method has the distinct advantage of being a simple procedure which can be carried out easily without the need of trained personnel and it does not require spirit for heating purposes. Standard diagnosis is wet film wf microscopic examination of sputum samples. Before staining, wet fixation immediately with cytology spray fixative 96% ethanol for minimum 30 min is required. The definitive diagnosis of malaria infection and other parasites is still based on finding such organisms in blood films. Pour carbol fuschin over smear and heat gently until fumes appear. Please note that zn procedure it was early introduced by paul elich and later it was modified by two german scientists ziehl and nelsen thats why the procedure clinch as zn staining but not developed by ziehl and modified by nelsen as mentioned by writer of this page. An intro to routine and special staining in histopathology. Pdf stamps modified ziehlneelsen staining for brucella. 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 1884. Gram staining is still the cornerstone of bacterial identification and taxonomic division. For the last fifty years, ziehlneelsen stain zns has been believed to destroy paragonimus eggs.
Author summary lung fluke paragonimus infection causes similar symptoms to pulmonary tb and is an important differential diagnosis in endemic areas. The ziehlneelsen stain, also known as the acidfast stain, widely used differential staining procedure. Application the zn staining technique is used with ordinary brightfield microscopes. Zn acid fast stainskit contains s033, s005 and s022 k005. Now a days, these methods have been supplanted by more sensitive auraminerhodamine fluorescence staining technique, also called truant method. 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. Stain a positive control and a negative control slide using the new batch of staining reagent and observe for reagent quality.
Staining of slides will be done with the same staining solutions used under routine conditions. The kinyoun staining procedure is often referred to as cold carbolfuchsin because no heat is applied during the staining process unlike the ziehlneelsen procedure. Zn staining is a simple and inexpensive method for presumptive identi cation of stamps modi ed ziehlneelsen staining for brucella. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Do not let the specimens dry at any stage of the staining procedure. This procedure is extensively used in the diagnosis of tuberculosis and leprosy. Prepare a thin smear using approximately 10 l of 10% formalin fixed stool suspension unconcentrated on a glass slide.
This staining method was developed at cdc using various components of the trichrome staining method to differentiate microsporidia spores from background fecal elements. Cell staining in microscopy types, techniques, preparations and procedures. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Demonstration of mycobacterium tuberculosis in sputum and. Smear microscopy is the simplest and quickest currently available procedure to detect acid fast bacilli afb in clinical specimens. Flood slide with crystal or gentian violet 60 seconds. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis tb along with some others of this. Conventional smear microscopy with the ziehlneelsen zn stain is a rapid and practical method for detecting acidfast bacilli afb, especially in lowincome countries, due to its rapidity, low cost, and high positive predictive value for tuberculosis. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. Heat gently to steaming and allow to steam for 5 min. Acid fast bacilli ziehl neelsen staining technique principle, procedure and reporting. Both progressive and regressive nuclear staining techniques can be used in papanicolaou stain.
The primary stain for the kinyoun procedure is the aniline dye, basic fuchsin, that stains all the cells present red. Make a thin smear of the material for study and heat fix by passing the slide 34 times through the flame of a bunsen burner or use a slide warmer. Nov 06, 2017 ziehl neelsen stain acid fast stainby dr prashant thakur career hub medical coaching bhopal duration. The recommended size of the smear is about 20 mm by 10 mm. As microscopy is the mainstay of tuberculosis diagnosis in our country, in this study we wanted to compare the sensitivity of zn staining method and auraminerhodamine ar staining method in sputum and saliva smears obtained from pulmonary tuberculosis patients and to introduce a routine screening test using saliva as media and fluorescent microscopy as a tool in the diagnosis of. Acid fast staining in formalinfixed tissue specimen of.
Preparation and examination of skin smears official web. The grading of sputum smears was done as per rntcp guidelines for zn staining 14 and a quantitation scale for acidfast bacillus smears according to stain was used 15. Ziehlneelsen is a hot method of acid fast staining. The procedure of leishman staining may vary as per the purpose of staining that means whether the staining is done for the examination of blood cells morphology, toxic granules in leucocytes, type of anemia etc. Using forceps, tilt each slide to drain off the staining solution. Zn staining and 38 37 sputum, 1 cervical abscess yielded positive results with fm staining. The components of ziehlneelsen stain include primary stain strongconcentrated carbol fuchsin, decolourizer 20% h2so4 and counterstain loefflers methylene blue. Tb decolorizer for staining mycobacteria by the kinyoun cold 4 x 250 ml 212517 and ziehlneelsen hot acidfast procedures. Staining methods histology and cytology 152 notes sources of errors irregular distribution of the blood smear on a glass slide may result in an erroneous cell counts. The ziehlneelsen method of staining for tb is known as the hot plate method which means the ziehlneelsen method uses bright field microscopy. Gently flood the smear with grams iodine and let stand for 1 minute. Microscopy refers to the practice that involves the use of a microscope for the purposes of observing small scale structures that cannot be viewed using the naked eye and often cell staining is necessary as s tructures are difficult to discern due to insufficient contrast.
Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Ziehl neelsen stain is one of those differential stain used in identifying acid fast bacteria such as mycobacterium tuberculosis and leprae. However, our investigation of stored zns slides and our prospective. Printerfriendly preparation and examination of skin smears pdf 225 kb. Fields stain is a histological method for staining of blood smears. Acid fast bacilli ziehl neelsen staining principle and procedure. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Ziehlneelsen staining technique can diagnose paragonimiasis. Phenol auramine stain3 this stain can be used as an alternative to the modified ziehlneelsen stain for staining oocysts of cryptosporidium parvum. These microorganisms are very small, transparent, and invisible to our naked eyes so it is very difficult to observe these microorganisms. This stain can be used as an alternative to the modified ziehlneelsen stain for staining oocysts of cryptosporidium parvum. Once the slides have arrived at the contractors facility, they will be logged in, heat fixed and stained as described.
Tb carbolfuchsin zn for staining mycobacteria by the ziehlneelsen 4 x 250 ml 212511 hot acidfast procedure. Place slide with heat fixed smear on staining tray. Principle, procedure, interpretation, examples and animation last updated. Principle, procedure, reporting and modifications by editorial team on january 11, 2020 in bacteriology, microbiology the ziehlneelsen stain zn stain, also called the hot method of afb staining, is a type of differential bacteriological stain used to identify acidfast organisms, mainly mycobacteria. The zn staining technique is used with ordinary brightfield microscopes. The procedure for kinyoun staining is similar to the ziehl neelsen stain, but does not involve heating the slides being stained. As microscopy is the mainstay of tuberculosis diagnosis in our country, in this study we wanted to compare the sensitivity of zn staining method and auraminerhodamine ar staining method in sputum and saliva smears obtained from pulmonary tuberculosis patients and to introduce a routine screening test using saliva as media and fluorescent microscopy as a tool in the diagnosis of cases. Pdf efficiency of cold staining method of afb in sputum a. Efficiency of cold staining method of afb in sputum a comparison with ziehl neelsen method under field conditions. 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. Franz ziehl 1859 to 1926, a bacteriologist and friedrich neelsen 1854 to 1894 a pathologist.
The ziehl neelsen stain was first described by two german doctors. Zn acid fast stainskit contains s033, s005 and s022 k005l composition arbol fuchsin zn,strongs005. Reading of slides slides will be read using the available brightfield microscope. Introduction as bacteria consist of clear protoplasmic matter, differing but slightly in refractive index from the medium in which they are growing, it is difficult with the ordinary microscope, except when special methods of illumination are used, to set them in the unstained condition. Feb 28, 2018 the procedure for kinyoun staining is similar to the ziehl neelsen stain, but does not involve heating the slides being stained. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. Incomplete removal of mercuric chloride will result in crystals. Do not allow the staining solution to dry on the slides add fresh solution as required. Procedure of progressive papanicolaou staining method.
Unlike the ziehlneelsen modified acidfast stain, this stain does not require the heating of reagents for staining. Institute clsi, the foundation for innovative new diagnostics find mgit procedure manual, and laboratory procedures from the national institutes of health tb research unit, as well as biosafety recommendations from the world health organization who and the us centers for disease control and prevention cdc. Semiquantitative results for zn will follow the scale used by the local ntp. Procedure fix the smear of the specimen over the glass slide, either by heating or alcohol fixation. Another narrowspectrum fungus stain is ziehlneelson zn. It also includes the important aspects of sample estimation for blinded rechecking of slides based. Staining, therefore, is of primary importance for the. The kinyoun staining method uses carbol fuchsin as a primary stain, followed by decolorization with an acidalcohol solution and. Zn staining with the increase in the concentration of primary stain carbol fuchsin from 0.
Gram stain reagents are checked weekly and also when a new lot of stain is to be put into use, as per the clia quality control regulatory notice published july 18, 1997. Acid fast staining in formalinfixed tissue specimen of patients with extrapulmonary tuberculosis. Mycobacterial cell walls contain a waxy substance composed of mycolic acids. Tb decolorizer tm for staining mycobacteria by the truant, brett 4 x 250 ml 212512.
This staining technique divides bacteria into two groups namely acidfast and non acidfast. Alcohols used for wiping the skin may cause hemolysis and artifacts. Most common staning technique involve classic ziehlneelsen zn staining method or one of its variants. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Ziehlneelsen staining procedure method stepwise when you see this icon click on it to view what the slide would appear like if that step of the staining procedure was not performed correctly. The kinyoun staining method uses carbol fuchsin as a primary stain, followed by decolorization with an acidalcohol solution and methylene blue as a counter stain. Demonstration of ziehl neelsen staining, a method of acid fast staining. Microorganism can be identified by microscopic examination.
Sep 08, 2018 acid fast bacilli ziehl neelsen staining technique principle, procedure and reporting 2 likes 2 comments share acidfastness refers to a physical property of certain bacterial or group of bacteria and eukaryotic cells, as well as some subcellular structures found in a organism, which makes it resistant to decolorization by the acid alcohol. Check out the leishman staining technique used in hematology laboratory for the examination of peripheral blood smear. 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 present instruction is applicable within the whole advanced mycobacterial diagnosis dma area and whenever positive cultures or possible sources of contamination must be checked out for the presence of mycobacteria. Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011 novaultra special stain kits principle.
The document covers the methods for the staining procedures commonly used in clinical microbiology laboratories for the identification of pathogens and dyes stains used for the differentiation of blood cells, for example, methylene blue stain used for white blood cell wbc differentiation. Zn staining of nss showed presence of both extracellular and intracellular afb. Acid fast bacilli ziehl neelsen staining principle and. Use millipore filtered water in the waterbath and staining procedure. Jun 12, 2018 acidfast stain principle, procedure, interpretation and examples. Acidfast stain principle, procedure, interpretation and examples. 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. The skin smear is a valuable, costeffective tool in the routine management of the patient with hansens disease. Overheating of smears when heat fixation is used d.
Spread the sputum evenly over the central area of the slide using a continuous rotational movement. It is very important that slides are properly fixed. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. The cold staining method has the distinct advantage of being a simple procedure which can be carried out easily without the need of trained personnel and it. Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until. Cell staining in microscopy types, techniques, preparations. Scope this instruction describes the ziehlneelsen zn staining technique for the detection of acidfast bacilli afb by microscopy. Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of the slide by. Zn acid fast stainskit is used for staining of acid fast bacteria.
In thin films the red blood cells are fixed and parasitized cells can be identified by their morphology. Gently flood smear with crystal violet and let stand for 1 minute. Comparison of sensitivity and specificity of zn and. The sensitivities of the zn and fm staining were 61% and 83% respectively p zn and fm contained between 1 to 9 afb per fields.