ACID FAST STAIN: AURAMINE RHODAMINE

Technical Data #AURAMINEa / 98.07.21

The Auramine-Rhodamine is used to stain acid-fast organisms as well as Mycobacterium sp. in specimens and in culture. This fluorescent method, actually the best procedure, stains selectively mycobacteria by binding dye to the mycolic acid of the cell wall.

FORMULA
in grams per liter

Auramine Rhodamine, Stain
Auramine O	11,1
Rhodamine B	5,6
Phenol	74,0
Glycerol	556,0 ml
Water, deionized	370,0 ml
Acid-Alcohol, Decolorizer
Ethanol, denatured	700,0 ml
Hydrochloric Acid, concentrated	5,0 ml
Water, deionized	300,0 ml
Potassium Permanganate, Counterstain
Potassium Permanganate	5,0
Water, deionized	1000,0 ml

PRECAUTIONS
This medium is for IN VITRO diagnostic use only.

STORAGE
Store in tightly-sealed containers at 15-30ºC in the dark.

SIGN OF DETERIORATION
Stains should not be used if they show signs of deterioration or if the expiration date has passed.

PROCEDURE

1. Prepare the smears as described in previous standardized methods directly from specimens or from culture.
2. Fix smears by passing through a Bunsen burner for 10 seconds.
3. Flood slides with Auramine-Rhodamine and stain for 15 minutes.
4. Rinse thouroughly with tap water and decolorize with Acid-Alcohol for 2 minutes.
5. Rinse with tap water and counterstain with 0,5% Potassium permanganate for 3 minutes.
6. Rinse with tap water. Blot dry and examine microscopically under a fluorescent microscope at low and high magnification.

QUALITY CONTROL

Organisms	ATCC	Color	Morphology
Escherichia coli	25922	green	bacilli
Mycobacterium tuberculosis	25177	fluorescence	bacilli

LIMITATIONS OF METHOD
Slides of acid-fast should be stained individually to prevent acid fast bacilli carry-over from one slide to another. This is a part of identification. Other methods may be required.

REFERENCES

1. Finegold, S.M., E.J. Baron 1986 Bailey and Scott's Diagnostic Microbiology 7th ed. C.V. Mosby St. Louis
2. Lennette, E.H., A. Balows, W.J. Hausler Jr., H.J. Shadomy 1985 Manual of Clinical Microbiology 4th ed. American Society for Microbiology, Washington, D.C.
3. Vestal, A.L., 1969 Procedures for the isolation and identification of mycobacteria. USDHEW, Public Health Service No. 1995, U.S. Government Printing. Washington, D.C.

CATALOGUE NUMBERS

Solutions
Auramine Rhodamine, Stain	500 ml	8537
Acid-Alcohol, Decolorizer	500 ml	8541
Potassium Permanganate, Counterstain	500 ml	8538
Complete Set
Auramine-Rhodamine	3x250 ml	8540
Auramine	3x250 ml	8543

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