ACID FAST STAIN: ZIEHL-NEELSEN

Technical Data #ZIEHLa / 98.07.22

The Ziehl-Neelsen Stain is used to stain acid-fast organisms as well as Mycobacterium sp. in specimens and in culture. Ziehl-Neelsen Stain uses an hot procedure to wax content of Mycobacterium. Acid fast microorganisms are resistant to acid decolorizer, with a remaining reddish color stained and blue background.

FORMULA
in grams per liter purified filtered water

Carbol Fuchsine, Stain
Basic Fuchsine	3,0
Phenol	50,0
Ethanol, Denatured	100,0 ml
Water, deionized	950,0 ml
Acid-Alcohol, Decolorizer
Ethanol, denatured	970,0 ml
Hydrochloric Acid, concentrated	30,0 ml
Methylene Blue, Counterstain
Methylene Blue	3,0
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 an overnight growth.
2. Fix smears by passing through a Bunsen burner for 10 seconds.
3. Flood slides with Carbol Fuchsine and heat slowly to steaming. Maintain steaming for 5 minutes.
4. Rinse thouroughly with tap water and decolorize with Acid-Alcohol until pink color flow disappears.
5. Rinse with tap water and add counterstain with Methylene Blue for 30 seconds.
6. Rinse with tap water. Blot dry and examine microscopically.

QUALITY CONTROL

Organisms	ATCC	Color	Morphology
Escherichia coli	25922	blue	bacilli
Mycobacterium tuberculosis	25177	reddish	large 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

Basic Fuchsine	1 Kg	QB-46-0714
Methylene Blue	1 Kg	QB-46-3004
Solutions	500 ml	1 Liter	4 Liters	6x250 ml
Carbol fuchsine	8511	8510	8512	8519
Decolorizer	8545	8585	8522	8520
Methylene Blue	8544	8531	8532	8521
Complete Set	3 x 100 ml	8542
	3 x 250 ml	8502

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