ACID FAST STAIN: ZIEHL-NEELSEN

Technical Data #ZIEHLa / 2013.07.22

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

 

 

 


 

 

2325, Dandurand, Suite 300 Phone Fax Internet
Montréal (Quebec) (514) 277-2558 (514) 277-4714 http://www.quelab.com
Canada H2G 1Z9 1 (800) 579-0998 1 (800) 579-8177 info@quelab.com