Acid fast staining - Ziehl Neelsen Method, Introduction, Reagent, Principle, Procedure, Result

Introduction

Acid-fast staining, also known as Ziehl Neelsen Method, is a differential staining procedure used to differentiate acid-fast bacteria from non-acid-fast bacteria. Many genera of microorganisms are easily stained by staining techniques such as negative staining, simple staining, or gram staining.

However, some bacteria, especially the members of the genus Mycobacterium are highly resistant and can only be observed after staining by acid-fast methods. In a clinical laboratory, species such as Mycobacterium tuberculosis and Mycobacterium leprae are visualized by acid-fast staining methods.

Reagents

Reagents used for acid-fast staining include:

• Primary stain: Carbol Fuchsin

• Decolorizing agent: Acid-Alcohol (HCl + 95% ethyl alcohol)

• Counterstain: Methylene Blue

Principle

The main difference between Mycobacterium and other groups of bacteria such as gram-positive Staphylococcus and gram-negative E. coli is the presence of a thick waxy lipodial wall in Mycobacterium. This component makes penetration by stains very difficult. However, once stained, it is equally difficult to remove such a stain - even through vigorous use of the decolorizing agent (95% ethyl alcohol).

Primary stain

The primary stain used for the acid-fast staining method is carbol fuchsin. It is a red phenolic stain that is soluble in lipoidal materials. Since Mycobacterium cell walls contain a significant amount of lipoidal materials, the stain can penetrate the bacteria and is retained inside the bacterial cell.

When heat is applied, the penetration of carbol fuchsin through the lipodial wall and into the cytoplasm of the Mycobacterium. Thus, coloration is further amplified.

At this stage, all cells appear red.

Decolorizing agent

Acid-Alcohol (HCl + 95% ethyl alcohol) is used as the decolorizing agent for the acid-fast staining method. When acid-alcohol is introduced to the bacterial smear, acid-fast bacteria will resist its decolorizing nature as carbol fuchsin is more soluble in lipodial wall than acid-alcohol. On the other hand, non-acid-fast microorganisms are decolorized under its effects.

The acid-fast bacteria will remain red while the non-acid-fast microorganisms will be colorless during this step.

Counterstain

The counterstain methylene blue used for the acid-fast staining method is able to stain previously decolorized bacterial cells and takes its blue color. Methylene blue is unable to enter the acid-fast bacteria so will retain the color of carbol fuchsin and appears red in color.

Procedure

The procedure for acid-fast staining method is as follows:

1. Take a clean, grease-free slide

2. Prepare a bacterial smear of the test organism

3. Allow the smear to air dry

4. Heat fix the dried smear by quickly passing the smear through the bunsen burner 2-3 times

5. Flood the smear with the primary stain (carbol fuchsin), place it in a water bath, and let it sit for 5 minutes

6. Let the glass slide cool and wash the smear with slow-running water

7. Pour decolorizing agent (HCl + 95% ethyl alcohol) on the smear drop by drop

8. Wash the smear with slow-running water

9. Flood the smear with methylene blue (counterstain) for 30 seconds to 1 minute

10. Wash the smear with slow-running water

11. Gently tap the slide with blotting paper to remove the remaining water/dye

12. Examine under a microscope (oil immersion at 100x)

Heat fixation

Heat fixation is done in most staining procedures (except capsule staining, and negative staining) because, during the washing steps, the microbial smear could wash away. During heat fixation, the bacterial proteins are coagulated and fixed to the glass slide surface.

Heat fixation for the acid-fast staining method is done by rapidly passing the air-dried smear 2-3 times over a bunsen burner's flame.

* Instead of heat fixation, several methods of heat fixation have been recognized. One such commonly used procedure is the alcohol-based fixative. Ethanol or methanol can also be used as they also function by precipitating proteins.

Result

Acid-fast microorganisms for the acid-fast staining method will be seen as red, rod-shaped bacteria. Non-acid-fast bacteria will appear blue in color.