Albert Staining Principle, Reagents, Procedure, Results, Interpretation

Albert stain is a specialized bacterial staining technique designed to identify organisms containing metachromatic granules, such as Corynebacterium diphtheriae. These granules serve as intracellular storage bodies for complexed inorganic polyphosphate (poly-P) and enzymes within certain bacterial cells. When subjected to methylene blue staining, these granules assume a reddish-purple hue rather than the typical blue coloration.

Corynebacteria, including the pathogenic species C. diphtheriae, possess these metachromatic granules. Cultivation of the bacteria on selective media like Loeffler agar, Mueller-Miller tellurite agar, or Tinsdale tellurite agar, followed by isolation and preparation of liquid cultures, precedes the application of Albert stain. It is essential to note that Albert stain exclusively targets volutin granules, limiting its applicability to bacteria lacking these structures. The technique primarily functions as a confirmatory test for the presence of C. diphtheriae.

Principle of Albert Staining

Albert staining is a specialized microbiological technique used to identify bacteria that contain metachromatic granules, also known as volutin granules. The principle behind Albert staining is based on the differential affinity of these granules for specific stains.

  • Metachromatic Granules:

Certain bacteria, such as Corynebacterium diphtheriae, possess metachromatic granules within their cytoplasm. These granules are composed of complexed inorganic polyphosphate (poly-P) and enzymes. They serve as storage sites for these essential cellular components.

  • Specific Staining Reaction:

Metachromatic granules have a unique property – they display a differential affinity for stains. When subjected to certain stains, such as methylene blue, these granules react in a distinctive manner.

  • Color Change of Granules:

In the presence of methylene blue, the metachromatic granules within the bacterial cells take on a reddish-purple coloration. This is in contrast to the blue coloration typically observed with other cellular components.

  • Confirmation of Corynebacterium diphtheriae:

Albert staining specifically identifies Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria. This staining technique acts as a confirmatory test for the presence of C. diphtheriae in a sample.

  • Exclusion of Bacteria without Metachromatic Granules:

Since Albert staining is designed to target metachromatic granules, bacteria lacking these structures will not be stained or identified using this technique. It is selective for organisms possessing these specialized inclusion bodies.

  • Cultivation and Preparation:

Prior to Albert staining, Corynebacterium species are typically cultured on selective media, isolated, and prepared in liquid culture. This liquid culture is then used for the staining procedure.

Procedure of Albert Staining

The Albert staining procedure is a specialized technique used to identify bacteria that contain metachromatic granules, such as Corynebacterium diphtheriae. Here is a step-by-step procedure for Albert staining:

Materials Needed:

  • Prepared liquid culture of the bacterial sample (e.g., Corynebacterium species)
  • Methylene blue stain
  • Microscope slides
  • Bunsen burner or heat source
  • Microscope


  • Preparation of Smear:

Take a clean microscope slide and prepare a bacterial smear from the liquid culture of the target organism. Allow the smear to air dry.

  • Heat Fixation:

Pass the slide, smear-side up, through the flame of a Bunsen burner or use a heat source to heat-fix the bacteria. Heat fixation helps to attach the bacterial cells firmly to the slide, preventing them from being washed away during subsequent staining steps.

  • Application of Methylene Blue Stain:

Flood the heat-fixed smear with methylene blue stain. Ensure that the entire smear is covered with the stain.

  • Incubation:

Allow the methylene blue stain to act on the smear for a specific period (usually about 2-5 minutes). The exact incubation time may vary and should be determined through prior optimization.

  • Rinsing:

Gently rinse the stained smear with distilled water to remove excess stain.

  • Blotting:

Blot the slide with absorbent paper to remove excess water. Be careful not to disturb the bacterial smear.

  • Examination under Microscope:

Place the slide on the microscope stage and examine it under low-power (10x) and high-power (40x) objectives.

  • Observation:

Focus on the bacterial cells and look for reddish-purple metachromatic granules within the cytoplasm. These granules are indicative of the presence of Corynebacterium species, particularly Corynebacterium diphtheriae.

  • Record and Interpretation:

Record your observations and make note of the presence or absence of metachromatic granules. Positive identification of metachromatic granules confirms the presence of Corynebacterium diphtheriae.

  • Cleanup:

Clean and rinse the microscope slide for future use. Dispose of any waste materials properly.

Interpretation of Albert Staining

The interpretation of Albert staining involves examining the stained bacterial smear under a microscope and looking for the presence or absence of metachromatic granules within the bacterial cells. Here’s how to interpret the results:

  • Presence of Reddish-Purple Granules:

If metachromatic granules are observed within the cytoplasm of bacterial cells, they will appear as reddish-purple structures. This is a positive result for the presence of metachromatic granules.

  • Absence of Reddish-Purple Granules:

If no reddish-purple granules are observed, it indicates the absence of metachromatic granules in the bacterial cells.

  • Confirmation of Corynebacterium diphtheriae:

A positive result with reddish-purple granules confirms the presence of metachromatic granules, which is characteristic of Corynebacterium species. In particular, this staining technique is used to identify Corynebacterium diphtheriae.

  • Identification of Other Corynebacteria:

Besides C. diphtheriae, other Corynebacterium species may also exhibit metachromatic granules and appear reddish-purple with Albert staining. However, additional tests may be needed for precise species identification.

  • Potential Sources of Error:

False negatives may occur if the staining process is not carried out correctly or if the bacterial smear is over-decolorized. Additionally, improper fixation or inadequate staining time can lead to inaccurate results.

  • Confirmation with Additional Tests:

While Albert staining is a valuable confirmatory test for the presence of C. diphtheriae, it is often used in conjunction with other laboratory tests and clinical information for a comprehensive diagnosis.

Remember that the interpretation of Albert staining results should be conducted by trained laboratory personnel in adherence to established protocols and quality assurance measures. It is crucial to consider the staining technique’s limitations and confirmatory nature when using it in clinical or research settings.

Applications of Albert Staining

  • Identification of Corynebacterium diphtheriae:

The primary application of Albert staining is to confirm the presence of metachromatic granules in Corynebacterium diphtheriae, the causative agent of diphtheria. This staining technique aids in the specific identification of this pathogenic bacterium.

  • Confirmation of Metachromatic Granules:

Albert staining provides a visual confirmation of the presence of metachromatic granules within bacterial cells. This characteristic feature is useful for distinguishing certain bacterial species.

  • Supplementary Diagnostic Test:

Albert staining is often used as a supplementary diagnostic test alongside other laboratory methods to confirm the presence of C. diphtheriae, especially in cases of suspected diphtheria infections.

  • Microbiological Research:

In microbiological research, Albert staining is employed to study the morphology and unique intracellular structures of bacteria, including metachromatic granules. This aids in characterizing bacterial species.

  • Training and Education:

Albert staining is a valuable tool for teaching microbiology and laboratory techniques in educational institutions. It provides hands-on experience in bacterial identification methods.

  • Epidemiological Surveillance:

In situations where diphtheria outbreaks or clusters of cases are suspected, Albert staining can be used as a rapid confirmatory test to identify C. diphtheriae in clinical specimens.

  • Quality Control in Diagnostic Laboratories:

Diagnostic laboratories may use Albert staining as part of their quality control procedures to ensure accurate identification of Corynebacterium species in clinical samples.

  • Public Health Interventions:

In public health settings, particularly in regions with a history of diphtheria, Albert staining may be employed to quickly identify and confirm cases, allowing for timely implementation of control measures.

  • Comparative Studies:

Albert staining may be used in comparative studies to assess the presence of metachromatic granules in different strains or isolates of Corynebacterium species.

  • Research on Metachromatic Granules:

Studies focused on the biology and function of metachromatic granules may utilize Albert staining to visualize and analyze these structures in bacterial cells.

  • Reference Laboratories:

Reference laboratories that specialize in the identification of specific bacterial pathogens, including C. diphtheriae, may employ Albert staining as part of their comprehensive diagnostic procedures.

Limitations of Albert Staining

  • Specificity for Corynebacterium diphtheriae:

Albert staining is primarily designed to identify Corynebacterium diphtheriae, and it may not be as effective for identifying other species of Corynebacterium or bacteria with similar granules.

  • Limited Application to Other Bacterial Species:

The staining technique is specific to bacteria containing metachromatic granules, making it unsuitable for the identification of bacteria lacking these structures.

  • Confirmation, not Quantification:

Albert staining confirms the presence of metachromatic granules but does not provide quantitative information about their abundance within the bacterial cells.

  • Subjective Interpretation:

The interpretation of Albert staining results relies on visual assessment, which can be subjective. This may lead to variability in results depending on the observer’s experience.

  • Possibility of False Negatives:

If the staining process is not carried out correctly or if the bacterial smear is over-decolorized, false-negative results may occur, leading to an inaccurate assessment.

  • Dependence on Proper Fixation:

Adequate heat fixation of the bacterial smear is crucial for the success of the staining process. Improper fixation can lead to suboptimal results.

  • Limited Information on Other Bacterial Characteristics:

While Albert staining provides information about the presence of metachromatic granules, it does not provide additional information about other bacterial characteristics or the presence of other specific antigens.

  • Not Suitable for High-Throughput Screening:

Albert staining is a time-consuming process that requires manual handling and interpretation. It may not be suitable for high-throughput screening applications.

  • Requires Specialized Expertise:

Performing Albert staining accurately requires a certain level of expertise and experience in microbiological techniques, particularly in the preparation of bacterial smears and the staining process.

  • May Require Confirmation with Other Tests:

While Albert staining is a useful confirmatory test, it is often used in conjunction with other laboratory tests and clinical information for a comprehensive diagnosis.

Despite these limitations, Albert staining remains a valuable tool in the identification of Corynebacterium diphtheriae and in research involving bacteria with metachromatic granules. It is important to consider these limitations when interpreting results and to use the technique in conjunction with other appropriate tests for accurate bacterial identification.

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