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Cryptosporidium oocysts in Auramine  Stain

Cryptosporidium parvum oocysts in Auramine- phenol stain as shown above picture.

Introduction of Auramine  Stain

It is a fluorochrome stain and 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), Kinyoun (cold) are still widely used methods to detect acid-fast structures in these organisms in developing countries but 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.

 Principle of Auramine 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 non-fluorescent, 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 

• Auramine -phenol stain (Primary Stain)
• 1 % Acid alcohol  ( Decolorizer) 
•  0.1% Potassium Permanganate
• Test specimen  (e.g. stool )
• Slide ( clean and grease-free)
• Pencil ( diamond if possible)
• Slide racks
• Bunsen burner
• Inoculating loop or sterile bamboo stick

Staining Procedure of Auramine -Phenol Stain

• Smear Preparation

Take 10-20µl of the stool 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 smear with auramine -phenol for 15 minutes. Do not let 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 here  in this case of Cryptosporidium, we used 0.1 % acid alcohol i.e. 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 smear to dry.
6. Rinse thoroughly with distilled water and air dry.
7. Examine the smear by 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).

Result Interpretation

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

Precautions

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

• Acid alcohol is flammable and Corrosive.
• Potassium Permanganate is also corrosive.

Advantages over 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.

Bibliography

• Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
• 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.
• 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.
•  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
•  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
• District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update
• ftp://ftp.cdc.gov/pub/laboratory_info/fluorochrome.ppt
• https://core.ac.uk/download/pdf/82107117.pdf