Auramine-rhodamine staining is used in microbiology laboratories for the examination of specimens suspected of containing acid-fast bacilli (AFB). The specimens are stained, differentiated and examined under a fluorescence microscope. Auramine stain is also used for the fluorescent examination of AFB.
Auramine-rhodamine staining is used to examine sputum smears from patients suspected of pulmonary tuberculosis (TB). Prepared tissue sections may also be examined for acid-fast organisms. However, the presence of AFB does not confirm Mycobacterium tuberculosis, as other acid-fast organisms may also be present. For adults and adolescents with suspected pulmonary TB, rapid molecular tests are recommended as the initial diagnostic test rather than smear microscopy.
What is Auramine-Rhodamine Staining?
Auramine-rhodamine staining is a fluorochrome acid-fast staining technique used to detect acid-fast bacilli (AFB) by fluorescence microscopy. The staining solution contains two fluorescent dyes, auramine O and rhodamine B. The acid-fast bacilli appear as bright yellow to orange fluorescent rods against a dark background when examined under a fluorescence microscope.
Auramine-rhodamine staining is different from auramine staining, which uses auramine O alone. Both methods are used for the fluorescent detection of acid-fast organisms, including mycobacteria. Fluorescence microscopy allows a larger area of the smear to be examined at lower magnification compared to conventional bright-field acid-fast staining.
The method can detect Mycobacterium tuberculosis and other acid-fast organisms. However, a positive AFB staining result does not identify M. tuberculosis specifically, as other mycobacterial species may also give a positive result.
Purpose of Auramine-Rhodamine Staining
The following are the objectives of auramine-rhodamine staining:
- To detect the presence of acid-fast bacilli (AFB) in clinical specimens.
- To examine sputum smears and other specimens suspected of containing mycobacteria.
- To observe the shape and arrangement of acid-fast bacilli under a fluorescence microscope.
- To detect AFB by examining a larger area of the smear at lower magnification.
- To estimate the number of acid-fast bacilli present in a positive smear.
- To help in the preliminary detection of acid-fast organisms during tuberculosis investigations, without identifying Mycobacterium tuberculosis specifically.
Principle of Auramine-Rhodamine Staining
The principle of auramine-rhodamine staining is based on the acid-fast property of mycobacteria. The mycobacterial cell wall contains a large amount of lipids, particularly mycolic acids, which are responsible for resistance to decolorization by acid-alcohol.
During staining, the fluorescent dyes auramine O and rhodamine B are taken up by the acid-fast bacilli. Auramine O is the main fluorochrome, while rhodamine B is added to improve the contrast of the stained bacilli. The retention of the dyes is associated with the lipid-rich cell envelope. Auramine O can also interact with DNA and RNA present inside the bacterial cells. The exact staining mechanism is not limited to direct binding with mycolic acids.
When the smear is treated with acid-alcohol, the acid-fast bacilli retain the fluorescent stain, while the stain is removed from non-acid-fast cells. Potassium permanganate is used to reduce the background fluorescence of tissue and other materials.
When exposed to suitable ultraviolet or blue-violet light, the retained fluorochrome dyes emit fluorescence. The acid-fast bacilli appear as bright yellow to orange fluorescent rods against a dark background.
Reagents and Materials Required for Auramine-Rhodamine Staining
The following are the materials and reagents required for auramine-rhodamine staining:
Staining Reagents
- Auramine-Rhodamine Staining Solution– It is the primary staining solution containing auramine O and rhodamine B. The solution may also contain phenol and glycerol. Auramine O-only staining solutions do not contain rhodamine B.
- Acid-Alcohol Decolorizer– It is used to remove the fluorescent stain from non-acid-fast cells. A 0.5% acid-alcohol solution is used in some standard protocols.
- Potassium Permanganate Solution– It is used to reduce background fluorescence and provide a dark background for the observation of acid-fast bacilli. A 0.5% potassium permanganate solution is used in some protocols.
- Distilled or Deionized Water– Chlorine-free water is used for washing the slides after staining, decolorization and treatment with potassium permanganate.
The concentrations of the staining reagents depend upon the method used. Commercial staining kits should be used according to the manufacturer’s instructions.
Materials and Equipment
- Clean Glass Slides– Used for the preparation of specimen smears.
- Staining Rack or Coplin Jars– Used to hold the slides during staining and decolorization.
- Dropper or Pipette– Used for adding the staining reagents to the slides.
- Wash Bottle– Contains distilled or deionized water for washing the slides.
- Slide Warmer– Used for heat fixation of prepared smears according to the laboratory protocol.
- Fluorescence Microscope– A microscope equipped with a suitable ultraviolet or blue-violet light source and appropriate excitation and barrier filters. Fluorescence-compatible immersion oil is required when an oil-immersion objective is used.
- Positive Control Slide– A prepared slide containing known acid-fast bacilli. It is used to check the staining reaction and fluorescence.
- Negative Control Slide– A prepared slide containing known non-acid-fast bacteria. It is used to check for unwanted staining.
- Personal Protective Equipment– Laboratory coat, protective gloves and safety goggles.
Procedure of Auramine-Rhodamine Staining
The procedure of auramine-rhodamine staining using the combined auramine O and rhodamine B solution is given below. Auramine O-only staining uses a different dye formulation. The staining and decolorization times may differ for commercial staining kits, which should be used according to the manufacturer’s instructions.
Preparation of Specimen and Smear
- Take a clean, grease-free glass slide and label it with the specimen identification number.
- Mix the processed sputum sediment inside a biological safety cabinet. Place approximately 100 µL of the specimen on the slide and spread it over an area of about 1 × 2 cm to prepare a thin smear.
- Allow the smear to air dry completely inside the biological safety cabinet.
- The dried smear is heat-fixed on a slide warmer at 65–75°C for at least 2 hours. Do not expose the slides to ultraviolet light. Heat fixation does not always kill the mycobacteria.
- Include known positive and negative control smears with each batch of slides.
Staining Procedure
- Place the heat-fixed slides on a staining rack with the smear side facing upward. Keep a distance of at least 1 cm between the slides.
- Cover the entire smear with auramine-rhodamine staining solution for 20 minutes. Heating of the stain is not required.
- Wash the slides gently with chlorine-free distilled water and drain off the excess water.
- Add 0.5% acid-alcohol solution to cover the smears and allow them to decolorize for 2 minutes.
- Wash the slides with distilled water to remove the acid-alcohol.
- Cover the smears with 0.5% potassium permanganate solution for 1–2 minutes. Treatment for more than 2 minutes may reduce the fluorescence of acid-fast bacilli.
- Wash the slides thoroughly with distilled water to remove the potassium permanganate.
- Allow the slides to air dry on the staining rack. Do not blot or wipe the smears. Keep the stained slides away from direct light.
Microscopic Examination
- Place the dried slide on the stage of a fluorescence microscope equipped with a suitable light source and fluorescence filters.
- Examine the smear first under a 20× objective with a 10× eyepiece, giving a total magnification of 200×.
- Scan the entire smear systematically for fluorescent bacilli. A 40× objective may be used for closer examination of suspected acid-fast organisms.
- The acid-fast bacilli appear as bright yellow to reddish fluorescent organisms against an almost black background.
- Examine the positive and negative control slides and check their staining results before reporting the specimen.
- Record the number of acid-fast bacilli observed according to the laboratory grading system.
- The slides should preferably be examined immediately after staining. If examination is delayed, keep them in a covered slide box away from light and examine within 24 hours.
Results and Interpretation of Auramine-Rhodamine Staining
Positive Results
The observations of a positive auramine-rhodamine staining result are as follows:
- The acid-fast bacilli are seen as bright yellow to orange fluorescent rods against a relatively dark background.
- The bacilli may also give yellow-green fluorescence. The colour depends upon the staining solution and the microscope filters used.
- The bacilli are usually slender, straight or slightly curved. Some may show a beaded appearance.
- They may be present singly, in pairs or in groups.
- Fluorescent debris and stain deposits may also be seen in the smear. These should not be mistaken for acid-fast bacilli.
Negative Results
The following are the observations of a negative auramine-rhodamine staining result:
- No characteristic fluorescent rods of acid-fast bacilli are seen in the examined smear.
- The background is relatively dark. Some particles may show nonspecific fluorescence.
- If no acid-fast bacilli are found after examining the required number of microscopic fields, the smear is reported as AFB-negative.
A negative smear does not exclude tuberculosis or other mycobacterial infections.
Interpretation and Reporting of AFB Smears
The stained smears are examined for the presence and number of acid-fast bacilli. The results are reported as follows:
- A smear is reported as AFB-positive when characteristic fluorescent acid-fast bacilli are present.
- The number of acid-fast bacilli is recorded according to the laboratory grading system. Positive smears may be graded as 1+, 2+, 3+ or 4+ depending upon the number of bacilli observed.
- When only a few acid-fast bacilli are seen, the actual number may be recorded or the finding may require confirmation, depending upon the reporting system.
- The grading also depends upon the magnification used and the number of microscopic fields examined.
- A positive AFB smear does not identify Mycobacterium tuberculosis specifically. Other mycobacterial species may also give a positive staining result.
- Molecular testing or culture is required for the identification of M. tuberculosis.
Applications of Auramine-Rhodamine Staining
The following are the applications of auramine-rhodamine staining:
- It is used to detect acid-fast bacilli (AFB) in sputum smears from patients suspected of pulmonary tuberculosis.
- To examine other respiratory specimens, such as bronchial washings and bronchoalveolar lavage fluid, for the presence of acid-fast bacilli.
- It is used for the detection of acid-fast organisms in tissue sections and biopsy specimens from suspected mycobacterial infections.
- The staining method is also used to detect nontuberculous mycobacteria in clinical specimens. However, it cannot identify the mycobacterial species.
- To examine smears prepared from mycobacterial cultures for the presence of acid-fast bacilli.
- It is used for screening a large number of smears, as the specimens can be examined at lower magnification under a fluorescence microscope.
- To estimate the number of acid-fast bacilli present in positive smears using the laboratory grading system. It may also be used to examine follow-up sputum smears during tuberculosis treatment.
Advantages of Auramine-Rhodamine Staining
The following are the advantages of auramine-rhodamine staining:
- It is generally more sensitive than the conventional Ziehl-Neelsen staining method for the detection of acid-fast bacilli.
- The bright fluorescent bacilli can be seen clearly against a dark background.
- A larger area of the smear can be examined at lower magnification compared to Ziehl-Neelsen staining.
- Less time is required for the examination of smears. This is useful when a large number of specimens are examined in the laboratory.
- It helps in the detection of acid-fast bacilli even when only a small number of organisms are present in the smear.
- The examination of stained smears at lower magnification does not require the use of an oil-immersion objective.
- Heating of the primary stain is not required during the staining procedure.
Limitations of Auramine-Rhodamine Staining
The following are the limitations of auramine-rhodamine staining:
- Auramine-rhodamine staining cannot identify Mycobacterium tuberculosis specifically. Other mycobacterial species may also give positive staining results.
- A smear containing a small number of acid-fast bacilli may give a negative result. A negative smear does not exclude mycobacterial infection.
- Living and dead acid-fast bacilli cannot be differentiated by this staining method.
- The drug resistance of mycobacteria cannot be determined by auramine-rhodamine staining.
- Fluorescent debris, stain deposits and other materials may be mistaken for acid-fast bacilli, giving false-positive results.
- The sensitivity of the method depends upon the quality of the specimen, smear preparation and staining procedure.
- Excessive treatment with potassium permanganate may reduce or completely remove the fluorescence of acid-fast bacilli.
- The fluorescence of stained bacilli may decrease with time, particularly when the slides are exposed to light.
- A fluorescence microscope with suitable filters is required. These microscopes are more expensive than conventional light microscopes and require proper maintenance.
- Trained laboratory personnel are required to distinguish acid-fast bacilli from fluorescent artifacts.
Precautions of Auramine-Rhodamine Staining
The following are the precautions of auramine-rhodamine staining:
- Prepare the specimen smears within a biological safety cabinet. Appropriate PPE must be worn and care taken to avoid the generation of any aerosols.
- Use clean, grease free glass slides and prepare thin smears. Allow the smears to air dry completely prior to heat fixing.
- Heat fix the smears in accordance with the lab procedure. Heat fixation doesn’t always kill the mycobacteria so the slides need to be handled with care.
- Do not heat the auramine-rhodamine stain solution during staining.
- Keep adequate space between the slides on the staining rack. They should not touch one another at all as this would lead to cross contamination.
- Use the correct auramine-rhodamine staining solution (one containing both auramine O and rhodamine B). Auramine O only solutions should not be used in place of the correct solution unless you’re following the appropriate protocol.
- Cover the smear completely with the staining solution and allow it to stain for the required time.
- Use chlorine free distilled or deionised water for washing the slides. Wash gently to avoid removing the smear and splashing onto the other slides.
- Use the acid alcohol decolouriser at the recommended concentration and for the stated time.
- Do not treat the smears with potassium permanganate for more than 2 minutes as per the above procedure. Over treatment may result in a reduction of the fluorescence of the acid fast bacilli.
- Just allow the stained slides to air dry. Don’t blot or wipe the smears.
- Protect the stained slides from direct light and look at them as soon as possible. If you have to delay the examination then keep the slides in a covered box and examine them within 24 hours.
- Use positive and negative control slides with each staining batch and the results of the staining should be checked prior to looking at the patient smears.
- Use a fluorescence microscope with appropriate excitation and barrier filters to view the acid fast bacilli.
- Handle the staining reagents with care and strictly adhere to the manufacturer’s instructions for their preparation, storage and use.
Auramine-Rhodamine Staining vs Ziehl-Neelsen Staining
Auramine-rhodamine staining and Ziehl-Neelsen staining are two acid-fast staining methods used for the detection of acid-fast bacilli (AFB). Auramine-rhodamine staining uses fluorescent dyes, while Ziehl-Neelsen staining uses carbol fuchsin as the primary stain.
The differences between auramine-rhodamine staining and Ziehl-Neelsen staining are given below:
| Features | Auramine-Rhodamine Staining | Ziehl-Neelsen Staining |
|---|---|---|
| Type of staining | It is a fluorochrome acid-fast staining method. | It is a conventional acid-fast staining method. |
| Primary stain | Auramine O and rhodamine B are used together as the primary staining solution. | Carbol fuchsin is used as the primary stain. |
| Microscopy | The stained smears are examined under a fluorescence microscope. | The stained smears are examined under a bright-field light microscope. |
| Decolorization | Acid-alcohol is used for decolorization, commonly at a lower acid concentration than in the Ziehl-Neelsen method. | Acid-alcohol is used for decolorization. The concentration depends upon the protocol used. |
| Counterstaining | Potassium permanganate is commonly used to reduce background fluorescence. | Methylene blue is commonly used as the counterstain. |
| Heating | Heating of the primary stain is not required during staining. | Heating of carbol fuchsin is required in the conventional Ziehl-Neelsen method. |
| Appearance of AFB | The acid-fast bacilli appear as bright yellow, yellow-green or orange fluorescent rods, depending upon the dye formulation and microscope optics. | The acid-fast bacilli appear as red or pink rods against a blue background. |
| Background | The background is relatively dark due to the reduction of background fluorescence. | The background appears blue due to methylene blue staining. |
| Magnification | The smears are commonly examined at 200× or 400× magnification. | The smears are commonly examined at 1000× magnification using a 100× oil-immersion objective. |
| Screening time | A larger area of the smear can be examined in less time due to the use of lower magnification. | More time is generally required to examine the same area of the smear at higher magnification. |
| Sensitivity | It is generally more sensitive for the detection of acid-fast bacilli, particularly in smears containing fewer organisms. | It is generally less sensitive than fluorochrome staining for detecting small numbers of acid-fast bacilli. |
| Equipment | A fluorescence microscope with a suitable light source and filters is required. | A conventional light microscope with an oil-immersion objective is required. |
| Interpretation | A positive result indicates the presence of acid-fast organisms but does not identify Mycobacterium tuberculosis specifically. | A positive result indicates the presence of acid-fast organisms but does not identify Mycobacterium tuberculosis specifically. |
The staining reagents, decolorization times and counterstaining procedures may differ between validated laboratory protocols. The heating requirement in the table refers to the primary staining solution and not to the heat fixation of the specimen smear.
References
- European Centre for Disease Prevention and Control. (2026). Handbook on tuberculosis laboratory diagnostic methods in the European Union: Updated 2026. https://doi.org/10.2900/8334202
- Forbes, B. A., Hall, G. S., Miller, M. B., Novak, S. M., Rowlinson, M.-C., Salfinger, M., Somoskövi, A., Warshauer, D. M., & Wilson, M. L. (2018). Practical guidance for clinical microbiology laboratories: Mycobacteria. Clinical Microbiology Reviews, 31(2), e00038-17. https://doi.org/10.1128/CMR.00038-17
- Global Laboratory Initiative. (2014). Mycobacteriology laboratory manual (1st ed.). https://stoptb.org/wg/gli/assets/documents/gli_mycobacteriology_lab_manual_web.pdf
- Procop, G. W. (2016). Laboratory diagnosis and susceptibility testing for Mycobacterium tuberculosis. Microbiology Spectrum, 4(6), TNMI7-0022-2016. https://doi.org/10.1128/microbiolspec.TNMI7-0022-2016