Albert's stain - Allied Guru

Table of Contents

Principle

Albert’s stain works based on the unique ability of Corynebacterium species to accumulate metachromatic granules in their cytoplasm.
These granules are a result of the accumulation of polyphosphate.
The staining method relies on the use of a special stain that binds to these granules, turning them blue-black, while the rest of the cell takes on a greenish hue.
The principle of Albert’s staining involves the use of a combination of dyes that selectively stain the bacterial cells and their granules.
The metachromatic granules of Corynebacterium species will appear as dark blue-black inclusions under the microscope, making them easily distinguishable from the rest of the cell.

To perform Albert’s staining, you will need the following materials:

Microscope: A compound light microscope, preferably with an oil immersion lens (100x) for high magnification.
Glass slides: For preparing bacterial smears.
Inoculating loop: To transfer the bacterial sample onto the slide.
Heat source: To fix the smear onto the slide.
Staining rack: To hold the slides during staining.
Distilled water: For rinsing the slides.
Staining containers: To hold the staining reagents.

Albert’s Stain Solution: This is a mixture of two primary components:
- Alberts’ Solution A: Contains a combination of methyl violet and iodine, which stains the bacteria and binds to the granules.
- Alberts’ Solution B: Contains potassium iodide, which aids in the iodine staining and helps to intensify the color of the granules.
Acid-alcohol solution: This is used as a decolorizing agent to remove excess stain from non-granular areas.

Bacterial Culture: Albert’s stain is most commonly used to detect Corynebacterium diphtheriae in clinical specimens. The sample can be collected from various sources such as:
- Throat swabs: For suspected diphtheria cases.
- Tissue biopsies: For diagnosing infections caused by Corynebacterium species.
- Sputum and other respiratory samples: For patients with respiratory symptoms suggestive of diphtheria.
Smear Preparation: A small sample of the bacteria is spread onto a glass slide to create a thin smear. This is then air-dried and heat-fixed to ensure that the bacteria stay attached to the slide during the staining process.

Prepare the smear:
- Place a small amount of the bacterial sample on a clean glass slide.
- Using an inoculating loop, spread the sample into a thin, even layer.
- Allow the smear to air dry completely.
- Heat-fix the smear by gently passing the slide through a flame 2-3 times to ensure that the bacteria stick to the slide.
Stain with Albert’s Solution A:
- Apply a few drops of Albert’s Solution A (the methyl violet and iodine mixture) onto the smear. Ensure the entire smear is covered with the stain.
- Allow the stain to sit for 5-10 minutes at room temperature.
Decolorize with acid-alcohol:
- Gently rinse the slide with an acid-alcohol solution to remove excess stain from the background. Be careful not to wash away the granules.
- Rinse with distilled water to stop the decolorization process.
Apply Albert’s Solution B:
- Apply Albert’s Solution B (containing potassium iodide) onto the smear and leave it for an additional 5 minutes.
- This step intensifies the staining of the granules and helps in further enhancing their visibility.
Final rinse and drying:
- Rinse the slide with distilled water to remove any excess stain.
- Allow the slide to air dry completely.
Microscopic examination:
- Examine the stained slide under a compound light microscope using an oil immersion lens (100x).
- The metachromatic granules will appear as dark blue-black granules within the bacterial cells, while the rest of the bacterial cell will appear greenish.

Positive Results:
- The bacteria will show characteristic metachromatic granules that stain dark blue-black.
- The rest of the bacterial cell will appear greenish.
- Corynebacterium diphtheriae and other Corynebacterium species will have these darkly stained inclusions, making them easily identifiable.
Negative Results:
- If the bacteria do not contain metachromatic granules, they will not show the characteristic blue-black staining.
- The cells will appear as greenish, with no dark inclusions.

Diagnosis of Diphtheria: Albert’s staining is primarily used for identifying Corynebacterium diphtheriae, the bacterium responsible for diphtheria. The presence of metachromatic granules is a key diagnostic feature of this pathogen.
Identification of Corynebacterium Species: Albert’s staining helps to identify various Corynebacterium species, especially those that cause opportunistic infections in immunocompromised individuals.
Bacterial Research: This method is used in microbiological research to study Corynebacterium species, their morphology, and granule formation.
Educational Purpose: Albert’s stain is also used in educational settings to demonstrate the presence of metachromatic granules in bacteria, which serves as an excellent example of a bacterial feature under the microscope.
Confirmation of Infection in Clinical Specimens: It is an effective method for confirming the presence of diphtheria-causing bacteria in throat swabs and other clinical samples.