Auramine-Phenol Fluorochrome Staining: Introduction, Principle, Test Requirements, Test Procedure, Result- Interpretation, Limitations, and Keynotes

Introduction of Auramine-Phenol stain

Auramine-Phenol is a fluorochrome stain used to visualize acid-fast structures of various microorganisms, especially Mycobacterium tuberculosis and in modified form for Mycobacterium leprae, Nocardia species, Cryptosporidium parvum, Cyclospora cayetanensis , Isospora belli, and fungal spores. Ziehl-Neelsen (hot), and Kinyoun (cold) are still widely used methods to detect acid-fast structures in these organisms in developing countries but the sensitivity is high of fluorochrome stain. The acid fastness of Mycobacterium tuberculosis is due to having a thick cell wall composed of waxes and lipids that has a high content of mycolic acid.

Auramine-phenol stained smear of sputum fluorescence microcopy
Fig. Auramine-phenol stained smear of sputum fluorescence microscopy

 Principle of Auramine-Phenol Stain

Auramine is the fluorochrome dye that forms a complex with mycolic acids found in the acid-fast cell wall of organisms that resist decolorization by acid-alcohol.  Potassium permanganate, counterstain renders tissue and its debris nonfluorescent, therefore reducing the possibility of artifacts. The cellular structures visualized under U-V appear bright yellow or brilliant greenish-yellow against a dark background.

Test Requirements for Auramine-Phenol Stain

  • Auramine-phenol stain (Primary Stain)
  • 1 % Acid alcohol  ( Decolorizer) 
  •  0.1% Potassium Permanganate
  • Test specimen
  • Slide ( clean and grease-free)
  • Pencil ( diamond if possible)
  • Slide racks
  • Bunsen burner
  • Inoculating loop or sterile bamboo stick
  • Two positive slides, one for a heavy load and another for a weak load of AFB to maintain quality control

Staining Procedure of Auramine-Phenol Stain

  • Smear preparation from sputum specimen

Take a purulent part of the sputum to a slide and make a thin smear using an inoculating loop or bamboo stick. Cover an area of approximately 2 cm square and spread the smear using circular movements. Allow it to air dry. Finally,  perform heat-fixing passing the dried slide, smear facing upward, 2 to 3 times through the blue cone of a burner flame.

  • Staining
  1. Put the fixed smear on a staining rack and flood the smear with auramine-phenol for 15 minutes. Do not let the smear dry.
  2. Wash off the stain with clean water.
  3. Decolorize the smear by covering it with acid-alcohol for 3-5 minutes. ( But in this case, weak acid-fast  organisms like Nocradia, Cryptosporidium, etc  use 0.1 % acid alcohol i.e. a modified form of auramine-phenol stain.)
  4. Wash off the acid alcohol with clean water.
  5. Now cover the smear with potassium permanganate for 15 seconds. Do not allow the smear to dry.
  6. Rinse thoroughly with distilled water and air dry.
  7. Examine the smear with a fluorescence microscope and use the 10X objective to focus the smear. Finally, observe the smear using the 40 X objective for acid-fast structures or acid-fast bacilli (AFB).
Staining Procedure of Auramine-Phenol Stain
Fig. Staining Procedure of Auramine-Phenol Stain

Result and Interpretation of Auramine-Phenol Stain

Test Positive: Acid-fast organisms fluoresce bright yellow or reddish-orange against a dark background.
Negative Test: Non-acid-fast organisms will not fluoresce

Reporting Sputum Smear

According to IUATLD (International Union against Tuberculosis and Lung Diseases) and WHO guidelines-

Report using 40 x objective and 10 x eyepiece

Scanty  AFB  1-19 AFB in 40 fields

+                         20-199 AFB in 40 fields

++                       5-50 AFB per field (examine 20 fields)

+++                   >50 AFB per field ( examine 8 fields)

To say no AFB seen: Examine 40 fields that should be free from AFB.

Acid fast bacilli (AFB) of Mycobacterium tuberculosis in Auramine-phenol fluorochrome Staining
Fig. Acid-fast bacilli (AFB) of Mycobacterium tuberculosis in Auramine-phenol fluorochrome Staining (Mag.400X)


Take precautions during handling the following reagents due to the following reasons-

  • Acid alcohol is flammable and corrosive.
  • Potassium permanganate is also corrosive.

Advantages of Z-N Stain

  1. Nearly 10% more sensitive than Z-N stain
  2. It does not require the use of oil immersion fields and thus no need for cedarwood oil.
  3. Heat is not required for auramine-phenol staining.
Acid fast bacilli (AFB) of Mycobacterium tuberculosis in Auramine phenol staining
Fig. Acid-fast bacilli (AFB) of Mycobacterium tuberculosis in Auramine phenol staining (Mag.1600X)


  1. False-positive result: The possible reasons for false-positive results are as follows- re-use of containers or positive slides; contaminated stain prepared with water containing environmental mycobacteria;  use of scratched slides; AFB floated off one slide and became attached to another during the staining procedure because there was no space between adjacent slides; inadequate decolorization;  lack of experience, confusion with artifacts (especially if stains are not or poorly filtered);  microscope (lamp) in poor condition or poorly adjusted: interpreting glitter as AFB;  poor quality of staining solutions.
  2. False-negative results: Among the possible reasons for false-negative results are as follows- poor quality of specimen; not taking a proper portion of specimen for smear preparation; excessive decolorization; poorly prepared staining solution; too little time staining with auramine; over-staining with permanganate; overheating during fixing; reading less than one length; slide exposed to daylight for too long; too long an interval between staining and reading, particularly if slides were poorly stained or not kept in the dark.
  3. Restaining smears for Ziehl-Neelsen staining: When required auramine-phenol stained smears can be restained after first treating the smear with 5 % oxalic acid for 2 minutes, followed by washing in water.
  4. Rhodamine is being carcinogenic, and Auramine-phenol stain is common nowadays.
  5. The method of examination of smears for AFB is given below-
Method of examination of AFB smear

Limitations of Auramine-Phenol Stain

  1. This type of stain needs a fluorescent microscope which is costly, and cumbersome to handle ( needs trained staff).
  2. A positive result only provides presumptive evidence of the presence of mycobacteria and a negative result does not indicate that the specimen will be culturally negative. Thus, cultural methods must be employed.
  3. Acid–alcohol, and potassium permanganate are also strong irritants to the skin, eyes, and respiratory system, and therefore precaution is required while handling and staining using such reagents.
  4. Most strains of rapid growers may not appear fluorescent.
  5.  All negative fluorescent smears should be confirmed with the Ziehl-Neelsen stain.
  6. Excessive exposure to the counter (potassium permanganate) stain may result in a loss of the brilliance of the fluorescing organism.
  7. Stained smears slides should be observed within 24 hours of staining because of the possibility of fluorescence fading.
Nocardia in fluorochrome (Auramine o) stained smear of sputum microscopy
Fig. Nocardia in fluorochrome (Auramine o) stained smear of sputum microscopy

Further Readings

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Betty A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  4.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  5.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  8. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update

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