Ziehl-Neelson Acid Fast Staining

Introduction

Although no definitive evidence has been presented, the high lipid content (especially the mycolic acid component ) of mycobacteria is thought to be related to the mechanism of acid fastness. It has long been known that even light mechanical injury to the cell wall will cause them to lose the acid fast characteristic, suggesting that permeation through cell membranes might be an important part of the mechanism. Carbol fuchsin is used to stain the slide. Acid alcohol is used to decolorize the slide. It has been suggested that the dye replacement power of the counter stain could be used to decolorize and counter stain at the same time. It has been shown that acid alcohol increases the differentiation obtained, and gives superior results. After decolorization, the slide may be counter stained with methylene blue or brilliant green. The acid fast organisms will appear red while non acid fast organisms will stain blue or green.

Reagents

PRODUCT #
Description
PACKAGING
484K
8 oz staining
484A-8oz
Z-N Carbol Fuchsin
8 oz.

484A-1gl

Z-N Carbol Fuchsin
1 gallon
311A-8oz
Acid Alcohol Decolorizer
8 oz.
311A-1gl
Acid Alcohol Decolorizer
1 gallon

 

675A-8oz  
Methylene Blue 1%
8 oz.
675A-1gl
Methylene Blue 1%
1 gallon
460A-8oz
Brilliant Green 1%
8 oz.
460A-1gl
Brilliant Green 1%
1 gallon

Specimen Collection

Organisms being stained by an acid fast method are usually taken from a solid or liquid medium on (in) which they have been cultured from their original source (e.g. wounds, throat, swabs, sputum, etc.). An aqueous suspension is made, in the case of the solid medium, by taking a small amount of the material and suspending it in a drop of distilled water on a microscope slide. Care should be taken not to make the smear too thick. In the case of a liquid medium, a drop is used directly from the culture container. However, due to the solids from the medium, this method is not always satisfactory. The suspension made by either method is air dried, then "fixed" by passing rapidly through a Bunsen burner flame two or three times. Allow the smear to cool before staining.

Procedure

  1. Place the "fixed" smear on a staining rack and flood slide with Ziehl-Neelson stain. Heat underside of slide for 3 minutes. Do not allow stain to boil.
  2. Wash off the stain with distilled water.
  3. Decolorize with acid alcohol until no more color runs from the smear.
  4. Rinse thoroughly with distilled water.
  5. Flood slide with methylene blue or brilliant green for 1-2 minutes.
  6. Rinse thoroughly with distilled water and air dry.
  7. Examine under high dry magnification and verify under oil immersion.

Note: Staining times may vary to suit the individual.

Sources of Error

  1. Overheating (burning) during fixation can be avoided by just touching the back of the slide to the back of the hand each time the slide has been passed though the flame.
  2. Do not stain smears which have only been air dried. Smears must also be "fixed".
  3. Smears should not be too thick. After air drying, examine under a microscope. If there are no areas of bacteria separation, more water should be added to dilute the smear.
  4. After staining it is essential that the back surface of the slide is wiped clean.
  5. If washing with distilled water is not done adequately, crystallization of the stain may appear on the slide.