Rhodamine Stain

Auramine – Rhodamine Stain Purpose, Procedure, Result Interpretation and more

Auraminetain and Rhodamine Stain bind to the cell wall of mycobacteria and emit a bright yellow or orange color under a fluorescent microscope.

Also Known as: Auramine Stain, Rhodamine Stain, Auramine and Rhodamine Stain, Auramine-Rhodamine Stain

Auramine Stain Procedure

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One of the earliest methods devised for the detection of the tubercle bacillus is the microscopic staining technique. Mycolic acid within the mycobacteria cell wall interacts with dyes resulting in the characteristics known as “acid-fastness”. Acid-fast microscopy is the most rapid, initial step in diagnosis and provides information about the number of acid-fast bacilli present. Hagermann described the use of fluorescent dyes for the detection of acid-fast bacilli in clinical specimens. In 1962, Truant, Brett, and Thomas evaluated the usefulness of a fluorescent staining technique and found it to yield a larger number of positive smears than the conventional fuchsin-stained method. In 1966, Bennedson and Larsen also reported a higher yield of positive smears with a reduced time requirement when using the fluorescent technique.

The fluorochrome dye, Auramine O, used in this stain reacts with the mycolic acids in the acid-fast cell wall of the organism and is refractory when rinsed with acid-alcohol (TB Fluorescent Decolorizer). The counterstain, Potassium Permanganate or Thiazine Red, renders tissue and debris non-fluorescent thereby reducing the possibility of artifacts. The cells visualized under ultraviolet light appear bright yellow-green.


The fluorochrome dye, auramine-rhodamine, forms a complex with mycolic acids found in the acid-fast cell wall of organisms that resist discoloration by acid-alcohol. The counterstain, potassium permanganate, renders the tissue and its debris nonfluorescent, reducing the possibility of artifacts. Cells viewed under ultraviolet light appear bright yellow or reddish orange.

Requirements for Testing :

  • Auramine O Fluorescent Stain
  • Fluorescent Decolorizer
  • Potassium Permanganate or Thiazine Red
  • Pipettes or Droppers
  • Glass slides
  • Fluorescent Microscope
  • Immersion Oil
  • Deionized or Distilled Water
  • Bunsen Burner or Slide Warmer

Required Reagents Formulation per 100 mL

Auramine O Stain

Rinse all glassware in distilled water. Place the solution in a 60°C oven overnight, mix on stir plate. Filter amount needed prior to use, discard after use. Label with initials and date, solution is stable for 1 year.

Reagent Quantity
Auramine O0.1 gram
Phenol3.0 gram
Ethanol10 ml
Purified water100 ml


Mix well, label with date and initials. Solution is stable for 1 year.

Reagent 0.5%Quantity
Hydrochloric acid5 ml
Ethanol (70%)995 ml

Auramine Counterstain

Make fresh, discard after use

Reagent 0.5%Quantity
Potassium Permanganate 0.5 Gram
Purified Water100 ml

Auramine and Rhodamine Stain Procedure :

Place the slide containing the fixed smear in a level staining rack. Make sure you have access to distilled or deionized water for the rinsing process before proceeding.

  • Flood slide with Auramine O Stain. Let the smear stain for 15 minutes. Make sure the stain remains on the smear.
  • Rinse slide thoroughly with distilled water and shake off excess liquid.
  • Flood the stain with Auramine Decolorizer. Let the smear decolorize for 2 minutes.
  • Rinse slide thoroughly with distilled water and shake off excess liquid.
  • Flood slide with Auramine Counterstain. Let the smear stain for 2 minutes. (Do not exceed the 2 minute mark as counterstaining may dull the observed fluorescence intensity)
  • Rinse well with distilled water and allow the smear to air dry. Do not erase.
  • Examine microscopically using a fluorescent microscope as soon as possible. Use a 20x or 40x objective for detection and a 100x oil immersion objective to observe the morphology of fluorescent organisms.
  • If desired, the slide can be directly re-stained with one of the other acid-fast stains (Ziehl-Neelsen or Kinyoun Stain) after removing the immersion oil.

Results Interpretations:

  • Positive Reaction: Acid-fast positive organisms fluoresce bright yellowgreen against a dark background with the Potassium Permanganate Counterstain, or dark red with the Thiazine Red Counterstain.
  • Negative Reaction: Nonacid-fast organisms will not fluoresce or may appear a pale yellow or red, quite distinct from the bright acid-fast organisms.

Slide Examin Procedure

The minimum number of fields to examine before reporting a smear as negative for acid-fast organisms.

Final magnification (the magnification of the objective lens multiplied by the magnification of the eyepiece) vs. number of slides

Patterns for Examining the slide

Smear Reporting

  • If fluorescent AFB are seen, report the smear as AFB positive and give an indication of the number of bacilli present in plus signs (+ to +++)
  • If no fluorescent rods are seen, report the smear as NO AFB seen.
Examining and Reporting Acid fastness

Limitations of Procedure :

  • Overheating (burning) during fixation should be avoided.
  • Do not stain smears which have only been air-dried. Smears must also be “fixed”.
  • Smears should not be too thick. After air-drying, examine under a microscope.
  • After staining it is essential that the back surface of the slide is wiped clean.
  • If washing with deionized or distilled water is not done adequately, crystallization of the stain may appear on the slide.
  • A positive staining reaction provides 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.
  • Most strains of rapid growers may not appear fluorescent. It is recommended that all negative fluorescent smears be confirmed with Kinyoun Carbolfuchsin Stain , Ziehl-Neelsen, or Rapid TB Stain. At least 100 fields should be examined before being reported as negative.
  • Excessive exposure to the counterstain may result in a loss brilliance of the fluorescing organism.
  • Turbidity may develop in the stain, but it will not interfere with its effectiveness. Shake the bottle before using.
  • Stained smears should be observed within 24 hours of staining because of the possibility of the fluorescence fading.

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Q: What is auramine stain used for?

Ans: Rhodamine auramine staining is used for the detection of mycobacteria directly from clinical specimens. The dye binds to mycolic acids and fluoresces under ultraviolet light. Acid-fast organisms (mycobacteria) will appear yellow or orange under ultraviolet light.

Q: What is fluorescent staining ?

Ans: The staining method incorporates the use of fluorescein diacetate fatty acid ester (FDA) and ethidium bromide (EB). Nonpolar, nonfluorescent ADF enters living cells where it is enzymatically hydrolyzed by acetyl esterase to polar fluorescent fluorescein, which rapidly accumulates in the cytoplasm.

Q: How does acid-fast staining work ?

Ans: The cells in the sample retain the dye. The slide is then washed with an acid solution and a different stain is applied. Bacteria that cling to the first dye are considered “acid-fast” because they resist acid washing. These types of bacteria are associated with tuberculosis and other infections.

Q: What dye was used in the fluorescent staining method ?

Ans: Fluorescein isothiocyanate (FITC) is an organic fluorescent dye and probably one of the most widely used in immunofluorescence and flow cytometry.

Possible References Used

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