Auramine rhodamine stain procedure

Auramine-Rhodamine Fluorochrome Staining: Principle, Procedure, Results and Limitations

Smear microscopy is the simplest and quickest currently available procedure to detect Acid Fast Bacilli AFB in clinical specimens. Now a days, these methods have been supplanted by more sensitive Auramine-Rhodamine Fluorescence staining technique, also called Truant method for acid-fast staining.

Auramine and Rhodamine are nonspecific fluorochrome dyes that have an affinity for acid fast organisms. In case of Mycobacteria, the dyes can bind specifically to the mycolic acid contained in the cell wall allowing the penetration of the stain. This complex resists decolorization by acid-alcohol decolorizer solution. The counterstain, potassium permanganate helps to prevent nonspecific fluorescence, thus reducing the possibility of artifacts. When observed under the microscope with UV illumination, acid fast cells are yellow or bright orange against dark background.

Examine microscopically using a fluorescent microscope as soon as possible. Use a 20x or 40x objective for screening, and a x oil immersion objective to observe the morphology of fluorescing organisms.

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If desired, the slide can be directly restained using one of the other acid-fast stains Ziehl-Neelsen or Kinyoun Stain. The minimum number of fields to examine before reporting a smear as negative for acid-fast organisms at specific magnification are as follows :. Are these stains have expiration date or how many months can you store this inorder to have a reliable results.

Name required. Email will not be published required. I have got more information from this slide. You are One of my best website. The informations you provide are really excellent. Kaye April 8, at pm. Leave a Reply Click here to cancel reply. Comment Name required Email will not be published required Website.Smear microscopy is the quickest and easiest procedure that can be performed.

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Mycobacteria, which do not stain well by Gram stain, are stained with carbol fuchsin combined with phenol. The stain binds to the mycolic acid in the mycobacterial cell wall. After staining, an acid decolorizing solution is applied.

This removes the red dye from the background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain hold fast to the dye and are therefore referred to as acid fast bacilli AFB. Following decolorization, sputum smear is counterstained with malachite green, or methylene blue which stains the background material, providing a contrast colour against which the red AFB can be seen.

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Among the Mycobacterium species, M. Note: 0. Mycobacterium leprae, Nocardia asteroides are much less acid and alcohol fast than Mycobacterium tuberculosis bacilli.

Heat Fixation of smear Upper: using electric heater, lower: using burner Heat fix dried smear. Cover the smear will carbol fuchsin stain Heat the smear until vapour just begins to rise i. Do not overheat boil or dry. Add additional stain if necessary.

Auramine-Rhodamine Staining for AFB : Principle, Procedure, Reporting and Limitations

Allow the heated stain to remain on the slide for 5 minutes. Wash off the stain with clean water. Note: Check to see that no more red color runs off the surface when the slide is tipped. Examine the smear microscopically, using the x oil immersion objective 10X eye piece for a total of X magnifi cation and scan the smear systematically. Procedural note:. It is revolutionizing TB control with aids in prompt diagnosis and treatment selection of appropriate TB regimen.

It contains inspissated eggs, malachite green, and glycerol or pyruvate. LJ medium […]. The Mantoux tuberculin skin test is the standard method of determining whether a person is infected with Mycobacterium tuberculosis or have exposure to tubercle bacilli. It was developed by Koch in but the intradermal […].

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Please, kindly check the reporting of your AFB positive. Dear Naseeb Khan Thank you so much for your query. The sputum smear grading differs among countries some are using their national criteria, not globally accepted WHO criteria. You seems of suffering from smear positive pulmonary tuberculosis take preventive measure so that you do not transmit the disease to others.

I strongly encourage you to visit your physician and follow his recommendation. Tuberculosis can be cured with proper medication. So applying this logic, the bacteria still seen are possibly dead. But as treatment progresses, one normally expects numbers to decrease.

Hope that answers your query and clears your mind of doubts, plus reinforces the close link between smears and cultures, in follow-up. Sir can we use heated carbol fushion over the fixed smear??? Instead of heating the smear with it?? I mean. Dear Pranay Ji This is not the standard procedure and it does not give better result. If you are determined to test, whats about performing the staining in laboratory using both standard method as well as the method you have suggested methods?

After that you will have result to compare. Ihank you for your assistance ,my question is what are the factors that can give false positive results during the stain hot technique.Iman F. Gaafar, MD, Auramine-Phenol vs. The comparison was based on the number of detected cases, sensitivity, specificity, time required for the procedure, ease of use, interpretation, and cost. A total of fecal specimens were examined by conventional methods: Direct wet saline smear, iodine smear, and formol ether sedimentation technique.

Duplicate smears of the fecal concentrates were stained by both procedures. The control group 27 immunocompetent relatives showed a high incidence of Giardia lamblia infection.

Kinyoun smears were difficult to interpret, while auramine smears were extremely easy to read, thus requiring less time. Artifacts were readily recognizable. Although the overall ranking of both staining techniques was high, the auramine-phenol stain was a more desirable test despite its higher cost.

The increased number of documented human coccidia infections that are often indistinguishable from other forms of community-acquired diarrhea, together with the possibility of treating some of them, suggests a need for proper diagnostic techniques to recover and identify the organism. Human intestinal coccidia, including Cryptosporidium parvum C. Most of these parasites cause major problems in the presence of acquired immunodeficiency syndrome AIDS and other causes of immunodeficiency.

These organisms also cause disease in immunocompetent individuals. These coccidia are intracellular cyst-forming apicomplexan protozoa that predominantly infect intestinal epithelial cells. Most are transmitted by the fecal-oral route or via contaminated water or food, although Sarcocystis spp is transmitted by improperly cooked meat. Infection is associated with intestinal inflammation, pathological lesions such as villus blunting, and abnormal function such as malabsorption.

Mild to moderate self-limiting diarrhea is common in healthy individuals during the infective stage of these organisms; however, asymptomatic infection can occur. Patients with immune dysfunction can have severe intestinal injury, prolonged diarrhea, extreme weight loss, and generalized wasting. Both C. Their oocysts are immediately infective. Ingesting cryptosporidial oocysts can lead to intestinal cryptosporidiosis.

However, the lifecycle of Sarcocystis spp requires an alternating infection between intermediate hosts—cattle or pigs—and definitive hosts, which can be carnivores or omnivores, including humans. Ingesting Sarcocystis spp oocysts causes cysts to form in muscles. Intestinal Sarcocystis spp. Both infections are acquired by ingesting oocysts, which require moisture and moderate temperature to sporulate and become infective.

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Fluid replacement and supportive care are essential in the treatment of coccidial diarrhea.It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen.

So this method is also called Ziehl-Neelsen staining techniques. Thus Ziehl-Neelsen staining techniques was developed. The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups. This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacteriumare resistant and can only be visualized by acid-fast staining.

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red. The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless.

Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color. Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis. The shape of the acid-fast stain is a rod? Please how is the biochemical composition of acid fat bacteria is used in the identification and diagnosis of a named AFB.

This is because it retains the primary color cabolfuchsin due to its large lipoidol membrane which does not allow decolorization by either the acid or alcohol hence acid-alcohol fast. If we skipped the heat during carbol fuchsin staining step, what is the end result? Will acid-fast bacteria be clear? And will non-acid fast bacteria be colored blue with methylene blue? No colour will be realized because there will be no stain penetration through the thick cell wall.

The heating process allows the primary stain, cabolfuchsin to penetrate further especially when the lipoidol membrane is large which otherwise could not permit penetration.

Omitting the step could lead to false results since the cabolfuchsin might be de-colorized leading to false negative results. Both acid fast and non-acid fast cells could take the counter stain and interfere with the end results. Nice explanation of the principle. You just boost my understanding of this topic. But what are the acid-alcohol fast bacilli? These have athuck cell wall. Have more mycolic acids and wax. Studying Medical Lab Technician.

You did well in your explaination, very helpful. B Mycobacterium, since its staining meth is for differenciation, and so, is it a gram pos or neg? OK Thanks you sir.

But you mean no need of malachites green when methylene blue is available??? James Adekeye thanks for your explanation. What happens if we overheat the slide? Heating simple helps in the enhancement on the penetration of the carbol fuchsin primary dye into the cytoplasm. This then makes the organism colour red.Don't have a profile?

Catalog No. Due to product restrictions, please Sign In to purchase or view availability for this product. Stains smears prepared from specimens and cultures suspected of containing mycobacteria for early presumptive diagnosis of mycobacterial infection and characterization of bacterial isolates.

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Fluorescent staining of AFB

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Serological Pipettes. View All Cell Culture.Ziehl-Neelsen hotKinyoun cold are still widely used methods in developing countries. The acid fastness of Mycobacteria is due to their thick cell wall composed of waxes and lipids that have a high content of mycolic acid. Fluorescent dyes like Auramine-Rhodamine binds to the mycolic acid present in them and impart bright yellow or orange fluorescence against a greenish background when viewed using a fluorescent microscope.

It is also used to stain all Acid fast organisms including the sporozoan parasites. The fluorochrome dye, Auramine-Rhodamine, forms a complex with mycolic acids found in the acid-fast cell wall of organisms which resist decolorization by acid-alcohol. The counterstain, potassium permanganate, renders tissue and its debris nonfluorescent, thus reducing the possibility of artifacts.

The cells visualized under ultraviolet light appear bright yellow or reddish orange. Smears that are too thick may flake during staining and may be difficult to decolorize.

auramine rhodamine stain procedure

Acid-fast organisms that might be present may be obscured. Smears that are too thin may not contain enough sample. Either condition—too thick or too thin—can lead to erroneous results, particularly false negatives. Here image 1 the smear in the center is of the proper thickness.

auramine rhodamine stain procedure

Allow to air dry and heat fix the specimen. Use of an electric slide warmer is usually the preferred method for heat-fixing smears. An alternate method of heat-fixing is to pass the dried slide, smear facing upward, 2 to 3 times through the blue cone of a burner flame. The minimum number of fields to examine before reporting a smear as negative for acid-fast organisms.

Final magnification the objective lens magnification multiplied by the eyepiece magnification Vs. Number of slides. I am a pathologist. I am using IF microscope for anti nuclear antibody testing.

I am a student of MLT. How does it differ from selective media. Dear D. Wathukarge thank you for your comment. You can find differences between enrichment media and selective media in this blog post about bacteriologic media.

If Fluorochrome stain is negative is not necessary to do Kinyoun stain for confirmation, because Fluorochrome stain is more sensitive. Kindly mention how to prepare aura mine and rhodamine. Hello Tankeshwar, this is Pham Khaniam South Sudanese medical laboratory technicianworking in national public health laboratory as TB lab technician in bacteriology department. This is Abid Khan from Pakistan. This site uses Akismet to reduce spam. Learn how your comment data is processed.

Culture Media Tests microbes Difference Between. News Ticker. Patterns for Examining the slide. About Acharya Tankeshwar Articles. Hello, thank you for visiting my blog. I am Tankeshwar Acharya.

Auramine–rhodamine stain

Blogging is my passion.What were the most popular topics in ? Click on our Statistics page to find out. Page views in 4, Cite this page: Pernick N. Accessed January 30th, Ziehl-Neelsen classic : common method; bacteria stain bright red due to retention of carbol-fuchsin dye; background is methylene blue counterstain; procedure involves heat 12 Ziehl-Neelsen modified bleach : may be more sensitive than classic stain Acta Cytol ;J Cytol ; Ziehl-Neelsen modified for stool specimens : does not require heating Centers for Disease Control Kinyoun: common method; uses more concentrated fuchsin dye and lipid solvent, but no heat; bacteria stain bright red against green background 12 Fite: to detect M.

Microscopic histologic images. Images hosted on other servers: Cryptosporidium: Oocysts: modified acid-fast stain.

Stool specimen Ziehl-Neelsen. Oocysts: auramine-rhodamine stain. Acid-fast stain. Modified acid-fast stain. Ziehl-Neelsen stains. Auramine stain of lung. Skin biopsies. Site-unknown, breast and colon Ziehl-Neelsen. Liver Fite stain. Fite-Faraco modified acid fast stain of lung. Tuberculous lymphadenopathy Ziehl-Neelsen. Pleural fluid.

auramine rhodamine stain procedure

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