Stain rehydrated sections in hematoxylin solution for 2040 minutes. Artisan acidfast bacillus afb stain kit english code. The stain must not be allowed to evaporate and dry on the slide. Ziehlneelsen staining is a type of acid fast stain, first introduced by paul ehrlich.
The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from nonspore formers. Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. Mycobacteriology laboratory manual world health organization. Mordants substance that causes certain staining reactions to take place by forming a link between the tissue and the stain. This removes excess dye, allowing nuclear details to emerge. This is a unique staining procedure to identify some characteristic groups of bacteria termed acid fast bacillus, which are extremely pathogenic to man. At the end of staining remove the paper with tweezers and wash the slide thoroughly. Primary stain carbol fuchsin basic fuchsin and dilute phenol mixture. Microbiology acid fast staining technique and its details. Acid fast stains or other procedures discussed later are necessary for demonstrating the bacilli, which are not visible in hematoxylin and eosinstained preparations. It is named for two german doctors who modified the stain.
Acid fast stain principle, reagents, procedure and result. 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. However, staining within a smear and between specimens can vary from unstained, to partial red staining, to complete staining. Acid fast staining in formalinfixed tissue specimen of. Grow cultured cells overnight on sterile glass cover slips at 37 c. Simple staining implies the use of only a single stain, which is usually sufficient to. Procedure of spore stain schaefferfulton method prepare smears in the usual manner using aseptic technique. 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%. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Once stained as part of a sample, these organisms can resist the acid andor ethanolbased decolorization procedures common in many staining protocols, hence the name acid fast.
The presence of acid fast bacilli in multiple specimens was investigated comparatively with ziehl. Once the slides have arrived at the contractors facility, they will be logged in, heat fixed and stained as described. There are two procedures commonly used for acid fast staining. This is due to the highly pathogenic nature of certain members of the genus mycobacteriumm.
Another variation on this staining method is used in mycology to differentially stain acidfast incrustations in the. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Mycobacterium and many nocardia species are called acid fast because during an acid fast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acidalcohol. Non acid fast organisms and background material will stain blue or green depending on the counterstain used. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. He developed a stain for the bacterium, although it was not very effective for visualizing this slender bacillus. Acid fast vs non acid fast bacteria the difference between acid fast and non acid fast bacteria is in their cell wall, basically. The waxy mycolic acid containing cell wall of mycobacteia are relatively impermeable to ordinary staining techniques. Acid fastness is a physical character of bacteria, which can be described as the resistance to decolorization by acids during staining procedures such as gram staining. Difference between acid fast and non acid fast bacteria. Fetal cell stain kit modified kleihauerbetke acid elution.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Spore stain principle, procedure and result interpretation. Acid fast staining differentiates acid fast bacteria from non acid fast bacteria based on the mycolic acid content in the cell wall. Most common of the acid fast staining techniques is the ziehlneelsen technique.
Acid fast stain principle, procedure, interpretation and examples. So this method is also called ziehlneelsen staining techniques. Why must heat or a surfaceactive agent be used with application of the primary stain during acid fast staining. Flood slide with crystal or gentian violet 60 seconds. What is the difference between gram stain and acid fast.
It is a type of differential staining method, which is used to distinguish between the acid fast and non acid fast bacteria. Acid fast staining was developed by robert koch in 1882 and later modified by other scientists. The procedure for kinyoun staining is similar to the. This procedure is extensively used in the diagnosis of tuberculosis and leprosy. Nearly all other genera of bacteria are nonacid fast. Acidfast staining and petroff hausser chamber counting of. 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.
Lessons from a proficiency testing event for acidfast microscopy. Learn acid fast staining microbiology with free interactive flashcards. Usa home product directory microbiology microscopy reagents and kits staining solutions staining solutions for acid fast staining. After you have stained your bacterial smears, you will examine them with the oil immersion lens, noting the morphological and staining characteristics of each species. The acid fast stain is a differential stain used to identify acid fast organisms such as members of the genus mycobacterium acid fast organisms are characterized by waxlike, nearly impermeable cell walls. Due to high lipid content, most dyes cannot enter easily through the cell wall, so mycobacterium species cannot be stained by gram staining. Thus ziehlneelsen staining techniques was developed. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. Do acid fast or non acid fast stain with carbolfuchsin. The ziehl neelsen stain was first described by two german doctors.
Analysis of specimens for the detection of acid fast bacilli by microscopy. Staining procedures scope of document this test procedure contains the methods for the staining procedures commonly used in clinical microbiology laboratories for the identification of pathogens. Scope this instruction describes the ziehlneelsen zn staining technique for the detection of acidfast bacilli afb by microscopy. Acid fast stain mycobacterium and many nocardia species are called acid fast because during an acid fast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent. True to its name, the simple stain is a very simple staining procedure involving single solution of stain. 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. The study describes a cold staining technique that uses the same staining solutions as the conventional ziehlneelsen method. Principle of ziehlneelsen method of acid fast staining. Oocysts in clinical specimens may be difficult to identify with out special staining. Counterstaining with methylene blue, which renders non acid fast cells blue. Main difference acid fast vs non acid fast bacteria.
Such staining procedure is called negative staining. The nature of the acidfast stain pubmed central pmc. This is the difference between acid fast and gram stain. Full text full text is available as a scanned copy of the original print version. A sputum stain for mycobacteria is a laboratory test performed on a sample of your sputum, or phlegm.
Acid fast bacillus afb stain kit english code ar162 intended use for in vitro diagnostic use. Cryptosporidium and isospora species have been identified as causes of severe diarrhea in immunocompromised hosts, but they can also cause diarrhea in the immunocompetent host. Although this staining method will stain t he microsporidia, the range of stain intensity and the small size of the spores will cause some difficulty in identifying these organisms. Mycobacterium species contains large amount of mycolic acid in its cell wall. How is this stain procedure similar to the spore stain procedure. Neelsen zn and fluorescence microscopy fm staining in. Wash in tap water for 15 minutes, until sections turn blue bluing. This staining technique divides bacteria into two groups namely acid fast and non acid fast. The main difference between gram stain and acid fast stain is that gram stain helps to distinguish bacteria with different types of cell walls whereas acid fast stain helps to distinguish grampositive bacteria with waxy mycolic acids in their cell walls. Ziehlneelsen staining is a bacteriological stain used to identify acid fast organisms, mainly mycobacteria. Heat is used in acid fast staining to drive the carbolfuchsin lipoidal cell wall and into the cytoplasm. While acid fast and gram staining are both differential stains, the acid fast stain is much more specific.
Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Some bacteria contain a waxy lipid, mycolic acid, in there cell wall. Allow the preparation to stain for 5 min, heat gently. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results.
Get a printable copy pdf file of the complete article 809k, or click on a page image below to browse page by page. The steps of the gram stain procedure are listed below and illustrated in table 1. Bacteria are generally identified and observed with differential staining procedures. For staining such organism ziehlneelsen staining method is used. This staining technique was discovered by scientist paul ehrlich in 1883. Methods applying fluorochromes have been used for acidfast staining since the introduction of an auramine obased method by hagemann and richards et al. Place a small piece of bibulous paper over the smear. Stains and dyes are frequently used in histology the study of tissue under the microscope and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level.
Acid fast stain a differential stain which distinguishes bacteria based on cell wall properties. Comparison between the ziehlneelsen staining method for acid fast bacilli, applied with and without heating, was carried out in a controlled investigation using smears prepared from 306 sputum. Ziehlneelsen staining is a type of acidfast stain, first introduced by paul ehrlich. Fall 2018 jackie reynolds, richland college, biol 2421. Application the zn staining technique is used with ordinary brightfield microscopes. 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. After decolorization, the acid fast bacteria appear red in color whereas the non acid fast bacteria appear as colorless bodies. Here, a method using modified kinyoun acid fast staining was adapted for use with a petroffhausser sperm and bacteria cell counting chamber by using a liquid suspension staining technique. Acid fast bacteria picks up the primary stain pink or red while non acid fast bacteria picks up the blue counterstain. Stains may be used to define biological tissues highlighting.
Most practitioners choose either the ziehlneelsen or the kinyoun stain, neither of which will stain nocardia unlike the fite. It makes use of a primary stain carbol fuchsin and methylene blue as counterstain. Under heating the slide will fail to stain acid fast cells. The kinyoun stain is a method of staining acidfast microorganisms, specifically. How to survive alone in the wilderness for 1 week eastern woodlands duration. 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. Fields stain is a histological method for staining of blood smears. The definitive diagnosis of malaria infection and other parasites is still based on finding such organisms in blood films. Without it, dye is not capable of binding to and staining the tissue. Use millipore filtered water in the waterbath and staining procedure. The acid fast genera have lipoidal mycolic acid in their cell walls.
Flood smears with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. 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 acid fast organisms, mainly mycobacteria. Any basic dye such as methylene blue, safranin, or. Franz ziehl 1859 to 1926, a bacteriologist and friedrich neelsen 1854 to 1894 a pathologist. Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. Ziehlneelsen acidfast staining procedure medical laboratories.
Unlike the ziehlneelsen modified acid fast stain, this stain does not require the heating of. Immunofluorescence staining procedure sigmaaldrich. Difference between gram stain and acid fast compare the. The objective is to differentiate bacteria into acid fast group and non acid fast. The acid fast stain is a differential stain used to identify acid fast organisms such as mycobacterium from non acid fast organisms. Acid fast cell walls are so durable that the stain carbol fuschin must be driven into the cells with heat. Perform a bacterial smear of bacillus or the organism you want to stain, as discussed in figure 352 on page 150 of your lab manual.
Acid fast organisms will appear red and non acid fast. Mycobacterium and many nocardia species are called acid fast because during an acid fast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acid alcohol. To differentiate between acid fast and non acid fast bacteria. Koch used the method to observe the tubercle bacilluswhat we now call mycobacterium tuberculosis, in sputum samples. Acid fast stain mycobacterium and many nocardia species are called acid fast because during an acid fast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acidalcohol 95% ethanol with 3% hcl. Some counterstains which are acidic may lighten or remove the nuclear stains. Choose from 500 different sets of acid fast staining microbiology flashcards on quizlet. Jun 12, 2018 acidfast stain principle, procedure, interpretation and examples. Links to pubmed are also available for selected references. What is chemically unique about the mycobacterium genus that causes it to be acid fast.
Acidfastness is a physical property of certain bacterial and eukaryotic cells, as well as some subcellular structures, specifically their resistance to decolorization by acids during laboratory staining procedures. Acid fast bacteria, of which there are very fewthe major genus mycobacterium, have a high concentration of mycolic acid, a lipid, in their walls. Ziehl neelsen stain is one of those differential stain used in identifying acid fast bacteria such as mycobacterium tuberculosis and leprae. Introduction quality assurance many of the stains that are described in this national standard method nsm are commercially available. This lipid makes the cells more durable and is commonly associated with pathogens. The acid fast staining is used to differentiate bacteria as acid fast and non acid fast bacteria. Acid fast staining is one of such methods to distinguish a. Acidfast stain principle, procedure, interpretation and.
Overheating of smears when heat fixation is used d. The acid fast stain the acid fast stain is one of the most medically important stains, second only to gram staining. The bv smears should be packaged so as to protect the glass slides from breaking en route, and maintain ambient temperature during shipping. Acid fast staining is a differential staining technique which differentiate acid fast and non acid fast bacteria. Xylene blot excess xylene before going into ethanol. Alternatively, a surfaceactive agent can be used to reduce the surface tension between cell wall of the bacteria and the stain. Acid fast bacillus afb stain kit is intended for laboratory use to identify, by light microscopy, acid fast bacilli, such as mycobacterium, in tissue samples. Using the modified ziehlneelsen stain, the oocysts are acid fast. Alum acts as mordant and hematoxylin containing alum stains the nucleus light blue. Differentiate sections in 70% ethanolcontaining 1% hclfor 5 seconds. Methylene blue is applied which stains the non acid fast bacteria and they appear as blue colored bodies under the microscope. The usual bacteria that you see range in size from 0. This is the key difference between gram stain and acid fast stain. Thus, acid fast staining sometimes refers to ziehl and neelson staining.
They can be stained by aniline dyes using drastic measures such as application of heat and phenol. Because of a high concentration of mycolic acid in their. Permeabilize cells by incubating for 15 minutes on ice with 2 ml of pbs with 0. Cover the slide with filtered carbol fuchsin and heat until steam rises. A comparative study on the different staining methods. Acid fast staining technique helps us to differentiate the organism as acid fast and non acid fast organisms. Accurate and rapid cell counts of mycobacterial species in culture are difficult to obtain. Its also known as an acid fast bacillus afb stain or a tuberculosis tb smear. A secondary counterstain, methylene blue, is then applied, which renders nonacid fast cells blue. The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria. This uses a combination of two dyes, hematoxylin and eosin used for demonstration of nucleus and cytoplasmic inclusions in clinical specimens. Acid fast stain principle, reagents, procedure and result interpretation.
Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. Detection of acid fast 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. The main read more acidfast stain principle, procedure, interpretation and examples. Staining solutions for acid fast staining sigmaaldrich. Acid fast staining technique is a differential staining technique in bacteriology. In thin films the red blood cells are fixed and parasitized cells can be identified by their morphology. The acid fast stain robert koch was the first person to isolate and identify mycobacterium tuberculosis from a patient with tuberculosis.
Endospore staining principle, reagents, procedure and result. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. Mycobacterium is an acid fast bacteria which retains the colour of. Acid fast bacilli ziehl neelsen staining principle and. Paul ehrlich is the first to describe the acid fast properties of the bacterium. Place slides containing paraffin sections in a slide holder glass or metal deparaffinize and rehydrate sections. Carbolfuchsin methods which include the ziehlneelsen and kinyoun methods light bright field microscope fluorochrome procedure using auramineo or auraminerhodamine dyes fluorescent microscope.
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