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Albert Stain Test - Use, Principle, Procedure, and Result

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Albert stain test is used to identify and stain metachromatic granules in Corynebacterium diphtheriae. Read below to learn more about it.

Medically reviewed by

Dr. Shubadeep Debabrata Sinha

Published At May 22, 2023
Reviewed AtJanuary 10, 2024


A corynebacterium is a group of bacteria that are gram-positive, non-acid-fast, non-sporing, and non-motile. The most significant member of this group is Corynebacterium diphtheriae, the pathogen responsible for causing diphtheria in children. The bacterium was first identified by Klebs, but Loffler was the first to grow it in the laboratory and named it Klebs-Loffler's bacillus (KLB). These bacteria form metachromatic granules, which are also produced by many other types of bacteria. These granules are composed of polyphosphate and are considered to be the bacteria's food reserve, involved in all metabolic processes. On a sputum slide, Albert's staining is used to distinguish gram-positive bacteria that form meta-positive chromatic or volutin granules. These bacteria are arranged in the shape of Chinese letters, or CLUB.

What Is the Use of Performing Albert Stain?

The Albert stain is commonly used in histology and pathology to visualize collagen fibers in tissue samples. It has various uses in different fields of research and medicine, including:

  • Diagnosis of Connective Tissue Disorders: The Albert stain is used to identify and diagnose disorders that affect collagen fibers, such as Ehlers-Danlos syndrome, Marfan syndrome, and scleroderma.

  • Evaluation of Tissue Repair: The Albert stain is used to study tissue repair processes in different organs, such as the skin, liver, and lungs.

  • Research on Fibrosis: The Albert stain is used to study the formation of excessive collagen fibers in organs and tissues, which is a characteristic of fibrosis.

  • Study of Wound Healing: The Albert stain is used to evaluate the healing process of wounds, especially those that involve collagen-rich tissues, such as skin and tendons.

  • Development of Biomaterials: The Albert stain is used to evaluate the biocompatibility and tissue response to various biomaterials, such as artificial skin, bone grafts, and implants.

What Is the Principle of the Albert Stain Test?

Corynebacterium diphtheriae and Yersinia pestis bacterial cells contain volutin granules in their cytoplasm, which are highly acidic while the cytoplasm is neutral. The Albert stain uses two basic dyes, toluidine blue O and malachite green, which have a strong affinity for neutral tissue components such as cytoplasm. To adjust the pH of the Albert stain to 2.8, glacial acetic acid is added, which is acidic for the cytoplasm (as it is neutral) but basic for volutin granules (as their pH is highly acidic). Therefore, when the Albert stain is applied to the cell, the volutin granules are stained red by toluidine blue O, while the cytoplasm appears green due to malachite green. The metachromatic property of volutin granules causes them to appear red when stained with toluidine blue O dye.

What Is the Composition of the Albert Stain Test?

The Albert stain is made up of two solutions, namely Albert’s A solution and Albert’s B solution. Albert’s A solution is composed of the following:

  • Toluidine blue (0.15 grams).

  • Malachite green (0.20 grams).

  • Glacial acetic acid (1 milliliter).

  • Alcohol (2 milliliters of 95 percent ethanol).

Dissolve the dyes in alcohol and add them to the distilled water and acetic acid. Allow the stain to stand for one day and then filter. Add distilled water to make the final volume 100 milliliters. On the other hand, Albert’s B solution is composed of:

  • Iodine (2 grams).

  • Potassium iodide (3 grams).

Dissolve it in water by first dissolving the KI and then adding the iodine. The staining process requires a smear on a glass slide, a staining rack, Albert’s A and B solutions, blotting paper, immersion oil, and a microscope.

How Is the Albert Stain Test Performed?

The procedure for Albert staining is as follows:

  • Materials Required:

  • Tissue sample.

  • Formalin or other fixatives.

  • Alcohol (70 percent and 95 percent)

  • Xylene or other clearing agents.

  • Glass slides.

  • Albert’s stain A and B solutions.

  • Distilled water.

  • Microscope.


  • Fixation: The tissue sample is first fixed in formalin or another fixative to preserve its structure.

  • Dehydration: The sample is then dehydrated in a series of alcohol solutions of increasing concentration, starting with 70 percent and ending with 95 percent ethanol.

  • Clearing: The sample is cleared in xylene or another clearing agent, which removes the alcohol and prepares the tissue for mounting on a glass slide.

  • Staining: The tissue sample is placed on a glass slide and then stained with Albert's A solution for ten minutes. The slide is then rinsed with distilled water.

  • Differentiation: The slide is then immersed in Albert's B solution for five to ten minutes. This solution differentiates the tissue and allows for the visualization of the nuclei and other cellular components. The slide is then rinsed with distilled water.

  • Dehydration and Clearing: The slide is dehydrated again in a series of alcohol solutions of increasing concentration, starting with 95 percent and ending with 100 percent ethanol. Finally, the slide is cleared in xylene or another clearing agent.

  • Mounting and Observation: The slide is mounted with a coverslip and observed under a microscope.

The resulting image will show the collagen fibers in red, while the nuclei and other cellular components will appear blue. The Albert stain is a useful tool for identifying and studying various connective tissue disorders and tissue repair processes.

What Is the Interpretation of the Albert Stain Test Results?

The interpretation of the Albert stain test results involves looking for the presence of Corynebacterium diphtheriae in the sample being examined. When this bacterium is present, it appears as green-colored, rod-shaped bacteria arranged at angles to each other, similar to the letters L or V in the English alphabet. Additionally, the bacteria will have blue-black metachromatic granules at the poles. This distinguishes C. diphtheriae from short, non-pathogenic diphtheroid, which lack these granules.

Is It Necessary to Take Any Precautions During Albert Stain Test?

Before conducting Albert's staining test, it is recommended to take some basic precautions, such as refraining from using an antiseptic mouthwash before the sample is collected and informing the doctor about any medication a person is currently taking, including antibiotics. Failure to take these precautions may compromise the accuracy of the test results.


The gram-positive bacilli group Corynebacterium includes Corynebacterium diphtheriae, which is the cause of diphtheria in children. Toluidine blue and malachite green are used in the Albert stain, a histology and pathology procedure, to make collagen fibers visible and spot conditions that damage them. It can also assess how well wounds and tissues heal, as well as use red staining to find Corynebacterium diphtheriae and volutin granules.

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Dr. Shubadeep Debabrata Sinha
Dr. Shubadeep Debabrata Sinha

Infectious Diseases


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