Zn staining procedure pdf

Fields stain is a histological method for staining of blood smears. Cell staining in microscopy types, techniques, preparations. Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. Once the slides have arrived at the contractors facility, they will be logged in, heat fixed and stained as described. 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. 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 ziehl neelsen stain was first described by two german doctors.

Zn staining and 38 37 sputum, 1 cervical abscess yielded positive results with fm staining. Tb decolorizer tm for staining mycobacteria by the truant, brett 4 x 250 ml 212512. 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. Incomplete removal of mercuric chloride will result in crystals. Zn acid fast stainskit contains s033, s005 and s022 k005l composition arbol fuchsin zn,strongs005. Gently flood smear with crystal violet and let stand for 1 minute. Unlike the ziehlneelsen modified acidfast stain, this stain does not require the heating of reagents for staining. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. 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. Use millipore filtered water in the waterbath and staining procedure. The definitive diagnosis of malaria infection and other parasites is still based on finding such organisms in blood films. However, our investigation of stored zns slides and our prospective.

Reading of slides slides will be read using the available brightfield microscope. Staining, therefore, is of primary importance for the. The bv smears should be packaged so as to protect the glass slides from breaking en route, and maintain ambient temperature during shipping. 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. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Now a days, these methods have been supplanted by more sensitive auraminerhodamine fluorescence staining technique, also called truant method. Tb carbolfuchsin zn for staining mycobacteria by the ziehlneelsen 4 x 250 ml 212511 hot acidfast procedure. It is very important that slides are properly fixed. 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. Standard diagnosis is wet film wf microscopic examination of sputum samples.

Acid fast staining in formalinfixed tissue specimen of. Check out the leishman staining technique used in hematology laboratory for the examination of peripheral blood smear. Jun 12, 2018 acidfast stain principle, procedure, interpretation and examples. 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.

This stain can be used as an alternative to the modified ziehlneelsen stain for staining oocysts of cryptosporidium parvum. Procedure fix the smear of the specimen over the glass slide, either by heating or alcohol fixation. 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. 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 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. 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. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. The skin smear is a valuable, costeffective tool in the routine management of the patient with hansens disease. 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. Do not let the specimens dry at any stage of the staining procedure.

Flood slide with crystal or gentian violet 60 seconds. Microorganism can be identified by microscopic examination. Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until. Acid fast bacilli ziehl neelsen staining principle and procedure. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. 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. Mycobacterial cell walls contain a waxy substance composed of mycolic acids. Demonstration of ziehl neelsen staining, a method of acid fast staining.

This procedure is extensively used in the diagnosis of tuberculosis and leprosy. Alcohols used for wiping the skin may cause hemolysis and artifacts. Ziehlneelsen staining technique can diagnose paragonimiasis. The components of ziehlneelsen stain include primary stain strongconcentrated carbol fuchsin, decolourizer 20% h2so4 and counterstain loefflers methylene blue. It also includes the important aspects of sample estimation for blinded rechecking of slides based. Using forceps, tilt each slide to drain off the staining solution. Nov 06, 2017 ziehl neelsen stain acid fast stainby dr prashant thakur career hub medical coaching bhopal duration. 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. Spread the sputum evenly over the central area of the slide using a continuous rotational movement. Printerfriendly preparation and examination of skin smears pdf 225 kb. Tb decolorizer for staining mycobacteria by the kinyoun cold 4 x 250 ml 212517 and ziehlneelsen hot acidfast procedures. 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. Demonstration of mycobacterium tuberculosis in sputum and.

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. Acidfast stain principle, procedure, interpretation and examples. Both progressive and regressive nuclear staining techniques can be used in papanicolaou stain. For the last fifty years, ziehlneelsen stain zns has been believed to destroy paragonimus eggs. Most common staning technique involve classic ziehlneelsen zn staining method or one of its variants. The zn staining technique is used with ordinary brightfield microscopes. Acid fast bacilli ziehl neelsen staining principle and. Comparison of sensitivity and specificity of zn and.

Ziehl neelsen stain is one of those differential stain used in identifying acid fast bacteria such as mycobacterium tuberculosis and leprae. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. 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. The kinyoun staining method uses carbol fuchsin as a primary stain, followed by decolorization with an acidalcohol solution and. 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. Feb 28, 2018 the procedure for kinyoun staining is similar to the ziehl neelsen stain, but does not involve heating the slides being stained. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of the slide by. In thin films the red blood cells are fixed and parasitized cells can be identified by their morphology. Another narrowspectrum fungus stain is ziehlneelson zn. 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. Prepare a thin smear using approximately 10 l of 10% formalin fixed stool suspension unconcentrated on a glass slide. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Stain a positive control and a negative control slide using the new batch of staining reagent and observe for reagent quality.

Ziehlneelsen is a hot method of acid fast staining. Zn staining of nss showed presence of both extracellular and intracellular afb. These microorganisms are very small, transparent, and invisible to our naked eyes so it is very difficult to observe these microorganisms. The recommended size of the smear is about 20 mm by 10 mm. The procedure for kinyoun staining is similar to the ziehl neelsen stain, but does not involve heating the slides being stained. Scope this instruction describes the ziehlneelsen zn staining technique for the detection of acidfast bacilli afb by microscopy. A typical afb stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells.

Preparation and examination of skin smears official web. 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. Overheating of smears when heat fixation is used d. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011 novaultra special stain kits principle. The kinyoun staining procedure is often referred to as cold carbolfuchsin because no heat is applied during the staining process unlike the ziehlneelsen procedure. 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. Franz ziehl 1859 to 1926, a bacteriologist and friedrich neelsen 1854 to 1894 a pathologist. 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. Zn staining is a simple and inexpensive method for presumptive identi cation of stamps modi ed ziehlneelsen staining for brucella. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

Before staining, wet fixation immediately with cytology spray fixative 96% ethanol for minimum 30 min is required. Acid fast staining in formalinfixed tissue specimen of patients with extrapulmonary tuberculosis. Mar 28, 2010 demonstration of ziehl neelsen staining, a method of acid fast 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. Pour carbol fuschin over smear and heat gently until fumes appear. Efficiency of cold staining method of afb in sputum a comparison with ziehl neelsen method under field conditions. Phenol auramine stain3 this stain can be used as an alternative to the modified ziehlneelsen stain for staining oocysts of cryptosporidium parvum. Smear microscopy is the simplest and quickest currently available procedure to detect acid fast bacilli afb in clinical specimens. Application the zn staining technique is used with ordinary brightfield microscopes.

The primary stain for the kinyoun procedure is the aniline dye, basic fuchsin, that stains all the cells present red. Do not allow the staining solution to dry on the slides add fresh solution as required. Zn acid fast stainskit is used for staining of acid fast bacteria. Zn acid fast stainskit contains s033, s005 and s022 k005. This staining technique divides bacteria into two groups namely acidfast and non acidfast. Procedure of progressive papanicolaou staining method. Gently flood the smear with grams iodine and let stand for 1 minute.

Cell staining in microscopy types, techniques, preparations and procedures. This staining method was developed at cdc using various components of the trichrome staining method to differentiate microsporidia spores from background fecal elements. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. The ziehlneelsen stain, also known as the acidfast stain, widely used differential staining procedure. Leishman stain is a mixture of methylene blue, and eosin dye, prepared in alcohol medium and diluted with buffer or distilled water during staining procedure. Pdf efficiency of cold staining method of afb in sputum a. Primary stain is carbol fuchsin ziehlneelson zn requires heat during staining requires higher magnification. Acidfast stain principle, procedure, interpretation and. Staining of slides will be done with the same staining solutions used under routine conditions. Slides should be drained on a paper towel to remove excess. 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. The ziehlneelsen method of staining for tb is known as the hot plate method which means the ziehlneelsen method uses bright field microscopy. Principle, procedure, interpretation, examples and animation last updated.

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. Gram staining is still the cornerstone of bacterial identification and taxonomic division. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. An intro to routine and special staining in histopathology. Heat gently to steaming and allow to steam for 5 min. Zn staining with the increase in the concentration of primary stain carbol fuchsin from 0. Semiquantitative results for zn will follow the scale used by the local ntp. Pdf stamps modified ziehlneelsen staining for brucella. Author summary lung fluke paragonimus infection causes similar symptoms to pulmonary tb and is an important differential diagnosis in endemic areas.