Diagnostic value of polymerase chain reactionacidfast. Carbolfuchsin acid fast stain is used to demonstrate mycobacterium tu ber cu lo sis in tissue sections and on smears. Acid fast stain is for the demonstration of mycobacterium tu ber cu lo sis in tissue sections. Once stained as part of a sample, these organisms can resist the acid andor. Apr 17, 2017 acid fast direct smear microscopy a laboratory training program duration. Acidfast microorganisms will retain this dye and appear red. If you continue browsing the site, you agree to the use of cookies on this website.
What is the difference between gram stain and acid fast. A note on uhlenhuths method for sputum examina tion for tubercle bacilli. If my acid fast species came out blue, instead of fuchsia, then it means that the species i used didnt have a cell wall however staphylococcus auerus for acid fast stained blue meaning it was negative, that it didnt have a cell wall nor mycolic acid. Unlike the ziehlneelsen modified acidfast stain, this stain does not require the heating of reagents for staining. The acid fast stain robert koch was the first person to isolate and identify mycobacterium tuberculosis from a patient with tuberculosis. Prepare bacterial smear on clean and grease free slide, using sterile technique. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. Unlike the ziehlneelsen modified acid fast stain, this stain does not require the heating of.
Acknowledgments uk standards for microbiology investigations uk smis are developed under the auspices of public health england phe working in partnership with the national. This procedure is performed by trained histology laboratory personnel. Make a thin film of the material on a clean glass slide, using a. Ziehlneelsen staining procedure for acidfast bacilli pagina 2 di 4. The ordinary aniline dye solution do not readily penetrate the acid fast bacilli. Most common of the acid fast staining techniques is the ziehlneelsen technique. Certain species of bacteria have a waxy lipid called mycolic acid, in their cell walls which allow them to. In this article we will discuss about the staining procedure used for detecting bacteria. Acid fast stain reagents are for in vitro diagnostic use. Ziehlneelsen n stain acid fast stain linkedin slideshare. Staining techniques for detection of acid fast bacilli herbert. In this lab we will be learning how to stain endospores using bacillus subtilis side. Sep 11, 2009 acid fast stainingsummary microbiology lab 11th septravi a patel slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. 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.
Currently in most laboratories, methods of staining using fluorescence microscopy fm and lightemitting diodes led microscopy for acid fast bacilli afb are. The oil hastens acid fast staining without the use of heat. Both techniques use two stains and one decolourizing. When a single stainingreagent is used and all cells and their structures stain in the same manner, the procedure is called simple staining procedure.
Pdf staining techniques for detection of acid fast bacilli. In the zn acidfast stain is a fairly vigorous staining procedure using carbolfuchsin karbalfyukshun as the main stain and methylene blue as a counter stain other counter stains may be used but we will ignore them for simplicity. Online microbiology notes welcome to, constantly growing and evolving collection of microbiology notes and information. So by use of powerful staining solution that contain phenol and application of heat, the dye.
Acid fast stainingsummary microbiology lab 11th septravi a patel slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. In the 1890s, friedrich neelsen and franz ziehl modified the stain by adding phenol carbolic acid and basic fuschin. Acidfast direct smear microscopy a laboratory training program duration. Place a small strip of blotting or filter paper over the top of the specimen, and place the slide over a boiling hot water bath on a mesh surface. Rinsing slide after application of crystal violet counterstain. Prepare and fix the specimen smear prior to staining. Kinyoun, is a procedure used to stain acidfast species of the bacterial genera mycobacterium and nocardia and the apicomplexan genus cryptosporidium. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes.
Methods assessed include specimen collection, smear preparation, acid fast stain procedure, reading, and reporting. Decolorize in 1% acid alcohol until the slide is a. The evaluator needs to have a good understanding of acid fast bacilli afb smear microscopy and good laboratory practice glp. The ziehlneelsen method is probably the best known and most frequently used procedure for staining tubercle bacilli. 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. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. Kinyoun, is a procedure used to stain acid fast species of the bacterial genera mycobacterium and nocardia and the apicomplexan genus cryptosporidium. The main aim objective of this stain is to differentiate bacteria into acid fast group and nonacid fast groups. Before staining, paraffin blocks were sliced into 8. The acid fast bacterium is mycobacterium stegmatitis. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. Cover the filter paper with the primary stain, carbolfuchsin. Difference between gram stain and acid fast compare the. Acid fast stain principle, reagents, procedure and result.
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. Acid fast stain is for the demonstration of ber cu lo sismycobacterium tu in tissue sections and smears. Partially acid fast organisms show reddish to purple filaments as opposed to non acid fast organisms, which stain only blue. The oil hastens acidfast staining without the use of heat. Acid fastness 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. Applying carbolfuchsin stain to bacterial smear and heating over water bath for 3 5 minutes.
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. They can be stained by aniline dyes using drastic measures such as application of heat and. What are you supposed to be learning about by doing the lab. 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 acidfast.
Methods assessed include specimen collection, smear preparation, acidfast stain procedure, reading, and reporting. What went wrong with your procedure if your acid fast species came out blue instead of fuchsia. Readdownload file report abuse differential staining of bacteria. Nearly all other genera of bacteria are nonacidfast. Jun 12, 2018 the main aim of this staining is to differentiate bacteria into acid fast group and non acid fast groups.
Three types of staining procedures are used for the detection of acidfast organisms. Acid fast stain free microbiology images from science prof online. This file is licensed under the creative commons attributionshare alike 4. Jul 28, 2017 acid fast staining differentiates acid fast bacteria from non acid fast bacteria based on the mycolic acid content in the cell wall. As well as acid staining with our unknown using mycobacterium stegmatitis.
Decolorize in 1% acid alcohol until the slide is a faint pink. Gram stain and acid fast are two differential staining techniques. Paul ehrlich is the first to describe the acid fast properties of the bacterium. Rinse the slide thoroughly with water and then drain any excess from the slides. Examine under high dry magnification and verified under oil immersion. This procedure uses pararosaniline dye in a phenol solution. The method requires controlled heating for its success. 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. Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. Mycobacteriology laboratory manual world health organization. Acid fast staining in formalinfixed tissue specimen of. Make a thin film of the material on a clean glass slide, using a sterile loop or swab for viscous specimens.
This is recommended when the smear has not been prepared from sodium hypochlorite. The pas staining procedure may also be used for the demonstration of fungal organisms in tissue sections. Leave the slide on the water bath for 3 to 5 minutes. This document details the lab protocol for nhanes 20032004 data. International union against tuberculosis and lung disease 5,886 views 31. The kinyoun method or kinyoun stain cold method, developed by joseph j.
In this lab we will be learning how to stain endospores using bacillus subtilis side by side with our unknowns. Whether youre a student, professor, working in the medical field or just curious about microbiology, im sure youll find our articles interesting and informative. 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%. Conclusion gram stain is one of the main bacteriological staining method used to distinguish between grampositive and gramnegative bacteria. However, in developing countries, such as india, where most laboratories rely mainly on spirit lamps as a source of heat, the ziehlneelsen method often cannot be carried. Lab 5 acid fast stain questionsanswers flashcards quizlet. There should be a thin film of oil on the slide at all times during the procedure. The stained smear is the easiest and quickest procedure that can be performed for diagnosis of tb and is economical especially in developing countries. Use millipore filtered water in the waterbath and staining procedure. Acidfast stain definition of acidfast stain by medical. 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. Feb 28, 2018 z n stain is a modification of ehrlichs original method for differential staining of acid fast bacilli by use of aniline gentian violet followed by strong nitric acid. Detection of acid fast bacilli afb in stained and acid washed smears examined microscopically may provide the initial bacteriologic evidence of the. Manual of histopathological and histochemical procedures, 16th ed.
Acidfast stain principle, procedure, interpretation and. Gram positive microorganisms that are resistant to gram stain, acid fast allows us to see this bacteria mycolic acid a waxy substance in cell walls of acid fast bacteria. The evaluator must understand appropriate methods and ensure they are being followed. 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. Endospore staining using bacillus subtilis side by side with our unknowns. Overheating of smears when heat fixation is used d.
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 acidfast stain helps to distinguish grampositive bacteria with waxy mycolic acids in their cell walls. Franz ziehl 1859 to 1926, a bacteriologist and friedrich neelsen 1854 to 1894 a pathologist. This procedure is of two types positive and negative fig. Acid fast staining helps classifying bacteria into two groups. Both techniques categorize bacteria into two groups. Steamers are very hot, and acid alcohol is highly flammable. Acid fast staining differentiates acid fast bacteria from non acid fast bacteria based on the mycolic acid content in the cell wall. Standard operating procedure sop for hematoxylin and. This is the difference between acid fast and gram stain. Furthermore, in gram stain, the grampositive bacteria retain the crystal violet stain, appearing purple while in acidfast stain, the acid. In contrast, in acid fast stain, acid fast bacteria appear in red color while non acid fast bacteria appear in blue color. Paul ehrlich is the first to describe the acidfast properties of the bacterium. The evaluator needs to have a good understanding of acidfast bacilli afb smear microscopy and good laboratory practice glp. Acid fast stain free microbiology images from science prof.
The waxy mycolic acid containing cell wall of mycobacteia are relatively impermeable to ordinary staining techniques. Stain with carbol fuchsin for 20 minutes at room temperature. Flood the slide with the counterstain, methylene blue. Pb specimens for afb were fixed in formalin and embedded in paraffin within 12 hours after ctguided core needle biopsy of pleural lesion. Acidfast stain principle, procedure, interpretation and examples. The nature of the acidfast stain pubmed central pmc. He developed a stain for the bacterium, although it was not very effective for visualizing this slender bacillus. The ziehl neelsen stain was first described by two german doctors. Z n stain is a modification of ehrlichs original method for differential staining of acid fast bacilli by use of aniline gentian violet followed by strong nitric acid. The acidfast stain robert koch was the first person to isolate and identify mycobacterium tuberculosis from a patient with tuberculosis. The main aim of this staining is to differentiate bacteria into acid fast group and nonacid fast groups.