Auramine- Rhodamine Staining

Auramine-rhodamine staining is a histological technique that uses fluorescent dyes to visualize acid-fast bacilli, especially Mycobacterium spp, under fluorescence microscopy . The dyes bind with the mycolic acid on the cell wall of the bacteria and make them appear reddish-yellow or orange . Mycolic acid is a long fatty acid that is essential for the survival of Mycobacterium spp and gives them the property of acid-fastness .

Auramine-rhodamine staining was developed as a modification of the Ziehl-Neelsen staining, which is another technique to stain acid-fast bacilli using heat and light microscopy . Auramine-rhodamine staining has several advantages over Ziehl-Neelsen staining, such as being faster, more affordable, more sensitive, and requiring no heat fixation . However, it is not as specific as Ziehl-Neelsen staining and may need confirmation by culture methods or other stains .

Auramine-rhodamine staining can also detect other acid-fast organisms like Cryptosporidium, which is a parasite that causes diarrhea . Therefore, this technique has various applications in microbiology, such as diagnosing tuberculosis, leprosy, and cryptosporidiosis .

In this article, we will compare auramine-rhodamine staining with Ziehl-Neelsen staining, explain the principle and reagents of auramine-rhodamine staining, describe the procedure and result interpretation of auramine-rhodamine staining, and discuss the applications, advantages, and limitations of auramine-rhodamine staining.

Auramine-Rhodamine staining is a modified version of the acid-fast staining technique known as Ziehl-Neelsen staining. Both methods are used to identify and demonstrate the presence of acid-fast bacteria, mainly Mycobacteria, which have a high content of mycolic acid in their cell walls. Mycolic acid makes these bacteria resistant to staining by conventional methods such as Gram stain.

The main difference between Auramine-Rhodamine and Ziehl-Neelsen staining is the type of dyes and the mode of observation used. Auramine-Rhodamine staining uses fluorescent dyes that bind to the mycolic acid and emit bright yellow or orange fluorescence when viewed under a fluorescent microscope with a dark background. Ziehl-Neelsen staining uses basic fuchsin dye that stains all cells pink, and then decolorizes the non-acid-fast cells with acid alcohol. The acid-fast cells retain the red color and are observed under a bright-field microscope with a light background.

Another difference between the two methods is the heat fixation step. Auramine-Rhodamine staining does not require heat fixation of the primary stain, because the fluorescent dyes have a strong affinity for the mycolic acid and bind without heat. Ziehl-Neelsen staining requires heat fixation of the primary stain, because the basic fuchsin dye needs heat to penetrate the cell wall and bind to the mycolic acid.

Auramine-Rhodamine staining has some advantages over Ziehl-Neelsen staining, such as being faster, more sensitive, and less dependent on heat. However, it also has some limitations, such as requiring a fluorescent microscope, being potentially carcinogenic, and fading over time. Therefore, both methods are still widely used in clinical microbiology for the detection and identification of acid-fast bacteria.

Auramine-Rhodamine staining is based on the use of fluorescent dyes that bind to the mycolic acid present in the cell wall of acid-fast bacilli, especially Mycobacterium spp . Mycolic acid is a long fatty acid that makes the cell wall resistant to decolorization by acid-alcohol. Auramine and Rhodamine are nonspecific fluorochrome dyes that have a high affinity for mycolic acid and can penetrate the cell wall when heated . The dyes form a complex that emits bright yellow or orange fluorescence when exposed to ultraviolet light under a fluorescence microscope .

The staining procedure involves four steps: primary staining, decolorization, counterstaining, and examination. In the primary staining step, the smear is flooded with Auramine-Rhodamine stain and heated gently to facilitate the dye uptake by the acid-fast bacilli . In the decolorization step, the smear is rinsed with acid-alcohol to remove the excess dye from the non-acid-fast cells and background . In the counterstaining step, the smear is treated with potassium permanganate, which stains the non-fluorescent tissues and cell debris and reduces the nonspecific fluorescence . In the examination step, the smear is observed under a fluorescence microscope with appropriate filters to detect the acid-fast bacilli as yellow or orange fluorescent rods against a dark background .

Auramine-Rhodamine staining is faster, more affordable, and more sensitive than the Ziehl-Neelsen staining, but not as specific . It can also detect other acid-fast organisms like Cryptosporidium. However, it requires a fluorescence microscope and careful handling of the carcinogenic dyes . It also needs confirmation by culture methods or Ziehl-Neelsen staining for negative or doubtful results .

The reagents used in auramine-rhodamine staining are:

• Primary stain: This is a mixture of two fluorescent dyes, auramine O and rhodamine B, dissolved in a solution of glycerol, phenol and distilled water . The primary stain binds to the mycolic acid in the cell wall of acid-fast bacteria and makes them fluoresce under ultraviolet light.
• Decolorizer: This is a solution of acid alcohol, which consists of hydrochloric acid and ethanol . The decolorizer removes the excess primary stain from the non-acid-fast bacteria and the background.
• Counterstain: This is a solution of potassium permanganate, which is a purple oxidizing agent . The counterstain stains the non-acid-fast bacteria and the cell debris, and reduces the nonspecific fluorescence of the background.

Other reagents that may be required for the staining procedure are:

• Distilled water: This is used to rinse the slides after each staining step and to prepare the solutions . Distilled water is preferred over tap water because it does not contain chlorine, which can interfere with fluorescence.
• Glass slides: These are used to prepare thin smears of the specimens to be stained . The slides should be new, clean and unscratched to avoid artifacts and inaccurate results.
• Slide racks: These are used to hold the slides during staining and drying .
• Bunsen burner: This is used to heat fix the smears before staining . Heat fixing kills the bacteria and adheres them to the slide. However, heat fixing is not required for the primary stain, as it can penetrate the cell wall without heat.

The procedure of auramine-rhodamine staining involves the following steps:

• Prepare a thin smear of the specimen on a sterile microscopic glass slide, and gently heat fix the smear avoiding overheating.
• Add enough quantity of the auramine-rhodamine dyes (flooding) on the smear and allow it to stand for 15 minutes and ensure the dyes stain the smear well. Do not apply heat.
• Rinse the stained smear with water until no color appears in the effluent. Ensure the water is chlorine-free water possibly distilled water since chlorine interferes with fluorescence.
• Add the decolorizing agent (acid-alcohol) for 2-3 minutes to destain and wash thoroughly with distilled water and remove excess water by shaking the slide.
• Flood the smears with potassium permanganate (counterstain) for 2 minutes exactly. Note: long periods of counterstaining can quench the fluorescence of the bacilli.
• Rinse thoroughly with distilled water and allow to air dry. Do not blot.
• Examine under a fluorescent microscope at K530 excitation filter and BG12 barrier or G-362 excitation filter and LP 420 barrier or with oil immersion at 400X for verification.

The following table summarizes the reagents used, their functions, and their timings in the procedure:

After staining the specimen with auramine-rhodamine dyes, decolorizing with acid-alcohol, and counterstaining with potassium permanganate, the slide is ready to be examined under a fluorescence microscope. The microscope should have an appropriate filter combination to detect the fluorescence of the dyes. The most commonly used filters are K530 excitation filter and BG12 barrier filter or G-362 excitation filter and LP 420 barrier filter . The magnification should be 400X or higher for optimal visualization.

The result of auramine-rhodamine staining can be interpreted as follows:

• Positive result: The acid-fast bacilli (AFB) appear as bright yellow or orange fluorescent rods against a dark or green background . The fluorescence can vary in intensity depending on the thickness of the smear, the age of the stain, and the exposure to light. A positive result indicates the presence of mycolic acid in the cell wall of the bacteria, which is characteristic of Mycobacterium spp and some other acid-fast organisms .
• Negative result: The non-acid-fast bacilli (NAFB) do not fluoresce and appear as pale yellow or colorless against a dark or green background . A negative result means that no mycolic acid was detected in the cell wall of the bacteria, which excludes most Mycobacterium spp and other acid-fast organisms .

However, a negative result does not necessarily rule out the possibility of infection by Mycobacterium spp, as some strains may not fluoresce well or may be present in low numbers. Therefore, a negative result should be confirmed by Ziehl-Neelsen staining or culture methods .

Similarly, a positive result does not necessarily confirm the diagnosis of tuberculosis or leprosy, as other acid-fast organisms such as Nocardia, Rhodococcus, Corynebacterium, and some parasites may also fluoresce . Therefore, a positive result should be verified by culture methods or molecular techniques .

Auramine-rhodamine staining is a useful technique for detecting and identifying acid-fast bacteria, especially Mycobacterium spp. that cause tuberculosis and leprosy. Some of the applications of this technique are:

• Diagnosis of tuberculosis and leprosy: Auramine-rhodamine staining can be used to screen sputum, urine, tissue, or other specimens for the presence of Mycobacterium tuberculosis or Mycobacterium leprae. The stained smears can be examined under a fluorescent microscope and the acid-fast bacilli can be seen as bright yellow or orange against a dark background. This method is faster and more sensitive than the conventional Ziehl-Neelsen staining technique. However, it cannot differentiate between different species of Mycobacterium or between viable and non-viable bacilli. Therefore, it should be followed by culture or molecular methods for confirmation and further characterization.
• Drug susceptibility testing: Auramine-rhodamine staining can also be used to assess the drug susceptibility of Mycobacterium spp. by observing the changes in fluorescence intensity after exposure to different antibiotics. For example, rifampicin-resistant strains of Mycobacterium tuberculosis show reduced fluorescence compared to rifampicin-susceptible strains. This method can provide rapid results and help guide the appropriate treatment of tuberculosis patients.
• Environmental monitoring: Auramine-rhodamine staining can also be applied to monitor the environmental contamination by Mycobacterium spp. For example, it can be used to detect the presence of Mycobacterium avium complex (MAC) in water samples or biofilms. MAC is a group of opportunistic pathogens that can cause respiratory and disseminated infections in immunocompromised individuals. Auramine-rhodamine staining can help identify the sources and routes of transmission of these bacteria and prevent outbreaks.
• Research purposes: Auramine-rhodamine staining can also be used for research purposes to study the morphology, physiology, and pathogenesis of Mycobacterium spp. For example, it can be used to visualize the cell wall structure and composition of acid-fast bacilli, which is important for understanding their resistance to antibiotics and host defenses. It can also be used to track the intracellular fate and survival of Mycobacterium spp. in host cells or animal models by using fluorescent microscopy or flow cytometry.

Auramine-rhodamine staining has several advantages over the conventional Ziehl-Neelsen staining for detecting acid-fast bacilli. Some of these advantages are:

• It is a rapid staining technique that can be completed within 30 minutes, compared to the Ziehl-Neelsen technique that takes about an hour .
• It is more sensitive than the Ziehl-Neelsen stain, as it can detect as few as 10^3^ bacilli per milliliter of specimen, while the Ziehl-Neelsen stain requires at least 10^4^ bacilli per milliliter .
• It does not require heat for fixation of dyes, which reduces the risk of damaging the specimen or the microscope slide .
• It allows for screening of large numbers of specimens in a short time, as multiple slides can be examined simultaneously under a fluorescent microscope .
• It can also stain parasites with sporozoa, such as Cryptosporidium and Isospora, which are important causes of diarrhea in immunocompromised patients .

Although auramine-rhodamine staining is a rapid and sensitive technique for detecting acid-fast bacilli, it also has some limitations that should be considered :

• A positive staining reaction provides only presumptive evidence of the presence of mycobacteria. A negative staining reaction does not indicate that the specimen will be culturally negative. Therefore, cultural methods must be employed to confirm the identification and speciation of the organisms.
• Most strains of rapid growers (non-tuberculous mycobacteria) may not appear fluorescent or may show weak fluorescence. Therefore, auramine-rhodamine staining may not be suitable for detecting these organisms.
• The fluorescent dyes (auramine and rhodamine) are possibly carcinogenic and should be handled with care. The decolorizer (acid-alcohol) and the counterstain (potassium permanganate) can also irritate the skin, eyes, and respiratory tract. Proper safety precautions should be followed when using these reagents.
• Excessive exposure to the counterstain may result in a loss of the brilliance of the fluorescing organisms. Therefore, the counterstaining time should be strictly controlled and the slides should be examined within 24 hours of staining before the fluorescence fades away.
• The use of a fluorescent microscope requires special equipment, maintenance, and training. The quality of the microscope and the light source can affect the detection and interpretation of the results. The microscope should be checked regularly for alignment, focus, and cleanliness.

We are Compiling this Section. Thanks for your understanding.