Staining protocol listed below is the protocol for the use of cntst100 stain kit to stain fixed cells growing on different cell culture surfaces, e. Evaluation of a dualstaining method for acidfast bacilli ncbi nih. Cite the purpose of each following reagents in the differential staining procedure. Key difference gram stain vs acid fast bacteria are very small microorganisms. Ziehlneelsen method for acidfast staining carbol fuchsin stain. A fluorescent stain is used in this method and the method is more sensitive than ziehlneelsens method.
Acid fast staining procedure for staining mycobacteria. The evaluator needs to have a good understanding of acidfast bacilli afb smear microscopy and good laboratory practice glp. The evaluator must understand appropriate methods and ensure they are being followed. Background the purpose of this experiment is to illustrate the differentiation of bacteria in acidfast and nonacid fast staining groups. Acidfast staining and petroff hausser chamber counting of.
Nearly all other genera of bacteria are nonacidfast. Paul ehrlich is the first to describe the acid fast properties of the bacterium. Guidelines for intensified tuberculosis casefinding and isoniazid preventive therapy. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. Pdf a fast silver staining protocol enabling simple and. Acid fast stain principle, reagents, procedure and result. Acid fast stainingsummary microbiology lab 11th septravi a patel slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Most of the bacteria can be stained, either by simple basic staining or by gram staining, as their cells take up stains easily. Carbolfuchsin acid fast stain is used to demonstrate mycobacterium tu ber cu lo sis in tissue sections and on smears. Adding too much stain will cool the slide and drip on the bench. Wash in tap water for 15 minutes, until sections turn blue bluing.
Choose from 500 different sets of acid fast stain flashcards on quizlet. Principle cryptosporidium and isospora species have been recognized as causes of severe diarrhea in immunocompromised hosts, but they can also cause diarrhea in the immunocompeten t host. Stain rehydrated sections in hematoxylin solution for 2040 minutes. Ziehlneelsen staining is a type of acidfast stain, first introduced by paul ehrlich. Without it, dye is not capable of binding to and staining the tissue. Fluorescent antibody staining procedure for detection of viral antigens uncontrolled copy 3. 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. Under heating the slide will fail to stain acidfast cells. If you continue browsing the site, you agree to the use of cookies on this website. This is due to the highly pathogenic nature of certain members of the genus mycobacteriumm. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. The main aim of this staining is to differentiate bacteria into acid fast group and non acid fast groups.
Add sufficient staining solution to cover the cells. This staining technique divides bacteria into two groups namely acidfast and non acidfast. As stated above acid fast staining is used to identify bacteria that contain an outer membrane high in mycolic acid. The pas staining procedure may also be used for the demonstration of fungal organisms in tissue sections. These organisms have waxy envelopes that make them difficult to stain and decolourise. The acid fast stain the acid fast stain is one of the most medically important stains, second only to gram staining. Decolorize the smear with 1% sulfuric acid until no more colour appears in the washing approx. In addition, the stain is used to determine the presence of af bacteria from. Gram stain is a very important differential staining technique used in the initial characterization and classification of bacteria in microbiology. Learn acid fast stain with free interactive flashcards. The acidfast stain is a differential stain used to identify acidfast organisms such as members of the genus mycobacterium acidfast organisms are characterized by waxlike, nearly impermeable cell walls. Answer to if you failed to perform the decolorization step during the acidfast staining 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. This method is valuable for the rapid initial step. Primary stain, counterstain, decolorizing agent, mordant. Acidfast staining techniques are the most rapid way to verify the presence of mycobacteria, however, cells are smeared onto a glass slide and thus, are difficult to accurately quantify weyer, 1998. Overheating the slide or letting it dry will distort the cells.
Subscribe to our email newsletter to receive updates. At the end of staining remove the paper with tweezers and wash the slide thoroughly. In 1915, kinyoun published a method that has become known as the cold staining method because the heating step was removed in favor of using a higher concentration of the carbol fuchsin primary stain. The acidfast genera have lipoidal mycolic acid in their cell walls. Get a printable copy pdf file of the complete article 240k, or click on a page image. 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. A dualstaining procedure for acidfast bacilli was found to have poor. The primary stain, carbolfuchsin is applied to the cells, and heat and phenol are used to allow the stain to penetrate into the waxy surface of acid fast microorganisms. Use millipore filtered water in the waterbath and staining procedure. Acid fast staining procedure for staining mycobacteriaa presentation byisaac.
What is the specific diagnostic value of the acidfast. A note to file ntf is a document used to describe an error i. Organisms exhibiting the property of acidfastness, once stained, are not easily decolourized by alcohol. This procedure is extensively used in the diagnosis of tuberculosis and leprosy. Zn acid fast stainskit contains s033, s005 and s022 k005. The characteristics displayed between mycobacteria and other microorganisms are very different because of the presence of a thick, waxy wall that makes. New or revised documents should be controlled within the laboratory in. Methods assessed include specimen collection, smear preparation, acidfast stain procedure, reading, and reporting. This procedure uses pararosaniline dye in a phenol solution. Non acid fast organisms and background material will stain blue or green depending on the counterstain used. Mycobacteriology laboratory manual world health organization. This file is licensed under the creative commons attributionshare alike 4.
The acidfast stain robert koch was the first person to isolate and identify mycobacterium tuberculosis from a patient with tuberculosis. The usual bacteria that you see range in size from 0. Fluorescent antibody staining procedure for detection of. Types of staining techniques used in microbiology and. Acidfast stain principle, procedure, interpretation and.
Most bacterial organisms can be stained by either simple or gram staining procedures. They retain the dye when treated with acid, which is. Mycobacterium and many nocardia species are called acidfast because during an acidfast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acidalcohol. Acidfast stain mycobacterium and many nocardia species are called acidfast because during an acidfast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acidalcohol 95% ethanol with 3% hcl.
This method is used for those microorganisms which are not staining by simple or gram staining method, particularly the member of genus mycobacterium, are resistant and can only be visualized by acid fast staining. Differentiate sections in 70% ethanolcontaining 1% hclfor 5 seconds. Immunocytochemistry and immunofluorescence protocol procedure for staining of cell cultures using immunofluorescence. Paul ehrlich is the first to describe the acidfast properties of the bacterium. A fast silver staining protocol enabling simple and efficient detection of ssr markers using a nondenaturing polyacrylamide gel article pdf available in journal of visualized experiments 2018. They stain with carbol fuschin, which is a red dye. This removes excess dye, allowing nuclear details to emerge. Oocysts in clinical specimens may be difficult to detect without special staining. Periodic acid schiff pas reagents are for in vitro diagnostic use. This method is used for those microorganisms which are not staining by simple or gram staining method, particularly the member of genus mycobacterium, are resistant and can only be visualized by acidfast staining. Acid fast stain reagents are for in vitro diagnostic use. While microscopic enumeration of bacteria must be verified by more dependable colony forming unit cfu counts, a.
The purpose of the handbook is to teach laboratory technicians how to safely collect, process and. If you failed to perform the decolorization step during. Do not let the specimens dry at any stage of the staining procedure. Immunocytochemistry and immunofluorescence protocol. All species in the genus mycobacteria are acid fast positive. One of the earliest methods devised for the detection of the tubercle bacillus is the microscopic staining technique. Some counterstains which are acidic may lighten or remove the nuclear stains. These acidfast staining techniques and other different procedures of afb staining. Mordants substance that causes certain staining reactions to take place by forming a link between the tissue and the stain. The main aim of this staining is to differentiate bacteria into acid fast group and nonacid fast groups. Modified kinyouns acidfast stain cold preanalytical considerations i. Acidfast bacilli afb can be identified with ziehlneelsen zn staining method in resourcelimited settings 912. Gram staining helps to identify bacterial pathogens in specimens and cultures by their gram reaction grampositive and gramnegative and morphology coccirod.
Acid fast staining procedure involves three different reagents. Nonacid fast organisms are decolourized by acid fast decolourizer and take up the counter stain. Ziehl neelsen staining principle, procedure and interpretations. This is a unique staining procedure to identify some characteristic groups of bacteria termed acid fast bacillus, which are extremely pathogenic to man. Fulltext staining techniques for detection of acid fast bacilli. Determine grampositive retain the purple primary color or gramnegative undergo decolorization.
Experiment on performing acidfast staining of a bacteria. Acid fast stain is for the demonstration of ber cu lo sismycobacterium tu in tissue sections and smears. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Analysis of specimens for the detection of acid fast bacilli by microscopy comprises 2 steps. Franz ziehl then altered ehrlichs staining technique by using carbolic acid as the mordant.
Alcohols used for wiping the skin may cause hemolysis and artifacts. Because of this feature, this stain is extremely helpful in identification in diseases caused by acid fast bacteria, particularly tuberculosis and leprosy. Acid fastness is an uncommon characteristic shared by the genera mycobacterium and nocardia weakly acid fast. Experiment on performing acidfast staining of a bacteria to find out whether it is acidfast or nonacidfast. Browse our database of protocols for cell culture, nucleic acid, protein analysis, rna and dna applications, cloning, cell analysis, and drug discovery research. This staining technique is used to demonstrate the presence of acid fast bacilli. Microbiology lab midterm questions and study guide. Primary stain carbol fuchsin basic fuchsin and dilute phenol mixture.
However, some bacteria possess a thick waxy cell wall made of lipid. Acid fast mycobacteria contain mycolic acid in their outer membrane, making the cells waxy and resistant to staining with aqueous based stains such as the gram stain. This procedure is used to stain mycobacterium tuberculosis and mycobacterium leprae. He developed a stain for the bacterium, although it was not very effective for visualizing this slender bacillus.
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