Differential staining procedures require more than one dye for staining. Two important differential staining procedures are Gram’s staining method and the acid-fast staining method. In the gram staining procedure, gram-positive cells stain purple in color, whereas the acid-fast staining procedure differentiates gram-positive bacterial cells into two different types. But there is a certain type of gram-positive bacteria that contains a waxy layer in their cell wall. This waxy layer is due to high lipid content made from a complex hydrocarbon known as mycolic acid. Because of this waxy layer, bacterial cells don’t take up stain during staining. A separate staining method is used to stain such bacteria with the waxy layer in the cell wall.
Principal:
In Acid-fast bacteria, the cell wall is waxy with high lipid content. This lipid is made of a complex hydrocarbon known as mycolic acid. The wax layer in the cell wall doesn’t allow the stain to penetrate. Thus staining acid-fast bacteria is difficult. But once stained with carbol fuchsin then it is difficult to remove the stain even with an acid alcohol solution. Such bacteria are known as acid-fast.
To understand the acid fast bacteria watch this simple and easy video given below
- Those bacteria who retain the primary dye carbol fuchsin and this dye is not removed with acid in alcohol and don’t take up the counterstain methylene blue are known as Acid-fast bacteria
- Non-acid-fast bacteria lose primary dye carbol fuchsin with acid alcohol treatment and take up the counterstain methylene blue and appear in blue colour
- The acid-fast staining method is a very important tool in diagnostic microbiology.
- Pathogenic Bacteria which cause diseases such as tuberculosis, and leprosy are acid-fast bacteria. Bacteria belonging to the genus Mycobacteria are acid-fast.
- There are two different methods of acid-fast staining. The first acid-fast staining method is the Ziehl-Neelsen technique. The second acid-fast staining method is the Kinyoun technique
Reagents for acid-fast staining:
1. Primary stain— Carbol fuchsin
2. Decolorizing agent —Acid–alcohol (3% HCl + 95% ethanol)
3. Counterstain— Methylene blue
Acid-fast staining procedure:
A. Preparation of smear on a glass slide:
Take a loopful of bacterial culture samples on a clean and grease-free slide. Make a smear on the slide, Air dry, and heat fix the smear. (Gently pass the slide through the flame 2-3 times)
B. Staining the fixed smear using dyes:
1. Add carbol fuchsin on smear and steam the slide on of water bath for 1 min.
2. Allow the slide to cool down and gently wash the slide using tap water.
3. Add the decolourizer acid alcohol gram’s Iodine solution for 1 min
4. Gently Wash the slide using tap water
5. Add counterstain Methylene blue stand for 2 min.
6. Gently Wash the slide using tap water and air dry or blot dry the slide.
3. Observation of stained smear under the microscope:
C. Observe the slide under the microscope with a 100 X objective oil immersion lens.
Role of reagents used for Acid-fast staining:
1. Carbol fuchsin:
It is the primary dye in the acid-fast staining method. Carbol fuchsin is red dye. This dye is more soluble in lipids that are why it binds to waxy cell wall containing more amount of lipid. Acid-fast bacteria don’t stain easily with Carbol fuchsin. However a gentle heat or steam helps in penetration and retention of dye. After staining with Carbol fuchsin, acid-fast bacteria and non-acid-fast bacteria both appear in red colour.
2. Acid–alcohol solution
The solution of acid alcohol acts as a decolorizing agent in the acid-fast staining method. Acid alcohol solution removes the carbon fuchsin from the cell wall of non-acid fast bacteria. Therefore non-acid fast bacterial cells become colorless. An acid-fast bacteria, the carbol fuchsin binds strongly to mycolic acid in the cell wall. Therefore, the acid alcohol solution does not remove the carbon fuchsin from the cell wall of non-acid-fast bacteria. After the decolorization step, acid-fast bacteria retain the primary dye and appear red color. Only non-acid fast bacteria become colorless after the decolorization step.
3. Methylene blue :
Methylene blue acts as a counterstain in the acid-fast staining method. This dye stains only the colorless non-acid-fast bacterial cells. After staining with methylene blue non-acid-fast bacteria appear in blue color. Since acid-fast bacteria have the Methylene blue does not stain. Acid-fast bacteria
To get clear understanding on acid fast staining procedure watch this simple and easy video given below
The following table describes each steps in acid fast staining method.
| Acid fast staining step | Acid fast bacteria | Non acid fast bacteria |
| Staining with carbol fuchsin | Take up the dye and appear red | Take up the dye and appear red |
| Decolorization with acid alcohol | No effect of acid alcohol Bacteria retain red color | acid alcohol washes out the red color. Bacterial cells become colorless |
| Staining with counterstain methylene blue | Bacterial cells already has red dye So doesn’t take counterstain | Bacterial cells take up counterstain and appear blue. |
| Final result under microscope | appear in red color | appear in blue color |
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Thank you for sharing this important information