# Albert Staining – Principle, Composition, Steps and Results > Albert staining explained through its principle, Albert A and B composition, laboratory procedure, and microscopic results showing metachromatic granules. Canonical URL: https://biologynotesonline.com/albert-staining/ Author: Sourav Pan Last updated: November 2, 2026 ![Albert Staining – Principle, Composition, Steps and Results](https://biologynotesonline.com/wp-content/uploads/2025/12/principle-of-albert-staining.webp) Albert staining is carried out on a fixed bacterial smear. The smear is first stained with Albert stain A, followed by Albert stain B. After staining, the slide is examined under oil immersion microscopy (National Institute of Communicable Diseases, n.d.). Albert staining is commonly used during the preliminary examination of bacterial specimens suspected to contain [Corynebacterium diphtheriae](https://biologynotesonline.com/corynebacterium-diphtheriae-morphology-pathogenesis-treatment/). The stained smear can be examined for the shape and arrangement of bacterial cells. Microscopic examination provides preliminary information about the bacteria but cannot confirm the presence of toxigenic Corynebacterium diphtheriae. When diphtheria is suspected, additional laboratory tests are required for bacterial identification and confirmation of toxin production (Centers for Disease Control and Prevention, 2026). ## What is Albert Staining? Albert staining is a special bacterial staining method used to demonstrate intracellular metachromatic granules, particularly in Corynebacterium diphtheriae. These granules are rich in polyphosphate and are also known as [volutin granules](https://biologynotesonline.com/storage-granules/) or Babes–Ernst granules (Widra, 1959). The metachromatic granules are present in the bacterial cytoplasm, usually near one or both poles of the cell. General bacterial staining is used to examine the shape and arrangement of bacteria, while Albert staining is used to demonstrate these intracellular granules. Metachromatic granules are also found in other bacteria and are not specific to Corynebacterium diphtheriae (World Health Organization, 2021). ## Objectives of Albert Staining The following are the objectives of Albert staining: - To demonstrate the presence of metachromatic or volutin granules in bacterial cells. - To observe the location and distribution of these granules, particularly at the poles of Corynebacterium diphtheriae. - To examine the shape and arrangement of bacterial cells, including club-shaped bacilli and their characteristic V-shaped or Chinese-letter arrangement. - To distinguish the dark bluish-black metachromatic granules from the green-stained bacterial cytoplasm. - To help in the preliminary identification of corynebacteria during microscopic examination of suspected bacterial cultures. ## Principle of Albert Staining ![Schematic showing volutin granules inside a Corynebacterium cell changing from reddish-purple after Albert stain A to bluish-black after iodine treatment.](https://biologynotesonline.com/wp-content/uploads/2025/12/principle-of-albert-staining-1024x576.webp)Albert stain A stains the bacterial cytoplasm green and the polyphosphate-rich granules with toluidine blue. Subsequent treatment with Albert stain B produces dark purple to bluish-black granules. Albert staining is based on the principle of metachromasia, in which certain cell components take a colour different from the original stain. The volutin granules contain a large amount of polyphosphate with negatively charged phosphate groups. These groups attract the positively charged [toluidine blue](https://biologynotesonline.com/toluidine-blue-staining/) dye. The dye molecules form aggregates at these sites and produce a change in colour (Sridharan & Shankar, 2012). Albert stain A contains two basic dyes, toluidine blue and malachite green. Toluidine blue stains the polyphosphate granules, while malachite green stains the bacterial cytoplasm green. When stained with toluidine blue alone, the metachromatic granules may appear red to reddish-purple. Albert stain B contains iodine, which acts as a mordant and changes the staining appearance of the granules. After iodine treatment, the granules appear dark purple to bluish-black instead of the reddish-purple colour observed with toluidine blue alone (HiMedia Laboratories, 2022). In the completed Albert staining, the metachromatic granules appear dark purple to bluish-black against the green or bluish-green bacterial cells (World Health Organization, 2000). ## Composition of Albert Stain Albert stain consists of two solutions, Albert stain A and Albert stain B. The following composition is based on the Albert Metachromatic Stains Kit (K000495) formulation (Central Drug House, 2023). ### Composition of Albert Stain A Albert stain A contains two basic dyes along with glacial acetic acid, ethyl alcohol and distilled water. The following are the constituents of Albert stain A: - Toluidine blue (0.15 g): It is the primary dye used for staining the metachromatic granules present in bacterial cells. - Malachite green (0.20 g): It is used as a counterstain to stain the bacterial cytoplasm green. - Glacial acetic acid (1.0 mL): It is used to maintain the acidic pH of the staining solution. - Ethyl alcohol, 95% (2.0 mL): It is used as a solvent to dissolve the dyes during the preparation of Albert stain A. - Distilled water (100 mL): It is used as the main solvent for the preparation of the staining solution. ### Composition of Albert Stain B Albert stain B is an iodine-containing solution used after staining with Albert stain A. It is also known as Albert's iodine solution. The following are the constituents of Albert stain B: - Iodine crystals (2.0 g): Iodine is used as a mordant in Albert staining and is responsible for the iodine treatment of the stained smear. - Potassium iodide (3.0 g): It helps to increase the solubility of iodine in distilled water. - Distilled water (300 mL): It is used as the solvent for preparing the iodine solution. ## Materials and Equipment Required The following are the materials, equipment and staining reagents required for Albert staining: ### Materials and Equipment - Clean, grease-free glass slides for the preparation of bacterial smears. - Sterile inoculating loop for transferring the bacterial culture. - Bacterial specimen or culture to be examined. - Bunsen burner or slide warmer for heat fixation of the bacterial smear. - Staining rack and staining tray for holding the slides during staining. - Wash bottle containing distilled water. - Bibulous paper for drying the stained slides. - Compound light microscope with a 100× oil immersion objective. - Immersion oil for microscopic examination. - Laboratory coat, protective gloves and safety goggles for handling specimens and staining reagents. ### Staining Reagents The following are the staining reagents used in Albert staining (HiMedia Laboratories, 2024): - Albert stain A: It contains toluidine blue and malachite green and is used for staining the bacterial cells and metachromatic granules. - Albert stain B: It is an iodine-containing solution used after Albert stain A. - Distilled water: Used for washing the stained slide after iodine treatment. ## Procedure of Albert Staining The procedure of Albert staining using Albert stain A and B is given below (HiMedia Laboratories, 2024): ![Albert staining procedure showing fixed smear preparation, Albert stain A for 3–5 minutes, drainage without washing, stain B for 1 minute, rinsing, and oil-immersion microscopy.](https://biologynotesonline.com/wp-content/uploads/2025/12/albert-staining-procedure-steps-1024x576.webp)Laboratory sequence for Albert staining using the protocol described in the article. Albert stain A is drained without washing before Albert stain B is applied. ### Preparation and Fixation of Smear - Take a clean, grease-free glass slide. - Transfer a small amount of bacterial culture onto the slide using a sterile inoculating loop and prepare a thin smear. - Allow the smear to air dry completely. - The dried smear is fixed by gentle heating. Avoid overheating the slide. ### Albert Staining Steps - Place the heat-fixed slide on a staining rack. - Cover the smear with Albert stain A and leave it for 3–5 minutes. - Drain off the excess stain. Do not wash the slide with water at this stage. - Cover the smear with Albert stain B and allow it to act for 1 minute. - Wash the slide gently with water to remove the excess iodine solution. - Blot dry the slide using bibulous paper. ### Microscopic Examination - Place a drop of immersion oil on the dried smear. - Examine the slide under the 100× [oil immersion objective](https://biologynotesonline.com/oil-immersion-technique-procedure/) of a compound microscope. - Observe the shape and arrangement of the bacterial cells. - Examine the presence and location of metachromatic granules within the bacterial cells, particularly near the poles (National Centre for Disease Control, 2009). ## Observation and Results of Albert Staining ![Microscopic illustration of green Corynebacterium-like bacilli with dark bluish-black polar metachromatic granules, V-shaped arrangements, and palisades.](https://biologynotesonline.com/wp-content/uploads/2025/12/albert-staining-corynebacterium-diphtheriae-microscopy-1024x768.webp)Typical Albert-stained coryneform morphology showing green bacterial cells with dark polar volutin granules. Some bacilli form V-shaped, L-shaped, or palisade-like arrangements. ### Microscopic Observation The Albert-stained smear is examined under the 100× oil immersion objective. The observations of an appropriately stained Corynebacterium diphtheriae preparation are as follows: - The bacterial cells appear green to bluish-green in colour. - Dark purple to bluish-black metachromatic granules are seen within the bacterial cells. - The granules are usually present at one or both poles of the bacilli. - The bacilli are straight or slightly club-shaped and may vary in size. - The bacterial cells are often arranged in V-shaped or L-shaped pairs. Some cells may also be present in palisade-like arrangements, giving a Chinese-letter appearance (National Centre for Disease Control, 2011). ### Results of Albert Staining Positive Result A positive Albert staining result shows the presence of dark purple to bluish-black metachromatic granules within green-stained bacterial cells. The granules are usually seen near the poles of the bacilli (HiMedia Laboratories, 2024). These microscopic features may be seen in Corynebacterium diphtheriae, but the presence of metachromatic granules alone cannot confirm the bacterial species (World Health Organization, 2021). Negative Result When distinct metachromatic granules are not seen, the result is recorded as metachromatic granules not demonstrated. The absence of visible granules does not exclude Corynebacterium diphtheriae. Granule formation may vary with the culture medium and age of the bacterial culture. ## Interpretation of Albert Staining ![Diagnostic pathway distinguishing preliminary Albert-stained microscopy from bacterial species identification, tox gene PCR, and Elek testing for diphtheria toxin production.](https://biologynotesonline.com/wp-content/uploads/2025/12/albert-staining-diphtheria-diagnostic-interpretation-1024x576.webp)Albert staining provides preliminary microscopic evidence of granule-bearing coryneform bacteria. Additional tests establish bacterial identity and determine whether the isolate produces diphtheria toxin. The presence of metachromatic granules in coryneform bacilli suggests the presence of granule-bearing bacteria. The shape and arrangement of the bacterial cells may help in the preliminary recognition of Corynebacterium species. Similar granules are also found in other corynebacteria and some Gram-positive bacteria. Albert staining alone cannot identify Corynebacterium diphtheriae (World Health Organization, 2021; Mitchell & Markantonis, 2025). The interpretation of Albert staining depends on the quality of the bacterial smear and staining conditions. A thick smear may make the granules difficult to observe. The presence of granules also depends upon the age of the bacterial culture and the growth medium used. Poor staining or insufficient granule formation may result in the absence of visible granules. Bacterial culture and additional identification tests are required to confirm the species. Albert staining cannot determine whether the bacteria produce diphtheria toxin. PCR can detect the presence of the diphtheria toxin gene, but toxin production must be confirmed separately by a test such as the Elek test (Centers for Disease Control and Prevention, 2026). ## Applications of Albert Staining The following are the applications of Albert staining: - It is used to demonstrate the presence of metachromatic or volutin granules in bacterial cells. - To observe the location and distribution of metachromatic granules, particularly at the poles of the bacterial cells. - It is used for the preliminary identification of suspected Corynebacterium species from bacterial cultures. - To examine the shape and arrangement of coryneform bacteria under the microscope. - It helps to distinguish granule-bearing Corynebacterium diphtheriae from some nonpathogenic [diphtheroids](https://biologynotesonline.com/diphtheroids/) that lack visible metachromatic granules. - It is used in microbiology practical classes to demonstrate metachromatic staining and bacterial storage granules. ## Limitations of Albert Staining The following are the limitations of Albert staining: - Albert staining is not specific for Corynebacterium diphtheriae. Metachromatic granules are also found in other corynebacteria (World Health Organization, 2021). - The staining method cannot confirm the identity of Corynebacterium diphtheriae. Bacterial culture and additional identification tests are required. - It cannot distinguish between toxigenic and non-toxigenic strains of Corynebacterium diphtheriae. Diphtheria toxin production must be confirmed by a separate test, such as the Elek test (Centers for Disease Control and Prevention, 2026). - Metachromatic granules may not be visible in every bacterial cell. The absence of visible granules does not exclude Corynebacterium diphtheriae. - The formation of metachromatic granules depends upon the age of the bacterial culture and the growth medium used. - A thick or uneven bacterial smear may make the metachromatic granules difficult to observe under the microscope. - Improper fixation or excessive heating may affect the morphology of the bacterial cells. - The staining result may be affected by the quality of staining reagents, staining time and improper washing of the slide. ## Precautions of Albert Staining The following are the precautions of Albert staining: - Use clean, grease-free glass slides for the preparation of bacterial smears. - Prepare a thin and uniform bacterial smear. A thick smear may make the metachromatic granules difficult to observe. - Use a young bacterial culture grown on a suitable medium for the demonstration of metachromatic granules. - Allow the smear to air dry completely before heat fixation. Avoid excessive heating, as it may distort the shape of bacterial cells. - Use properly stored staining solutions within their expiry date. Keep Albert stain A and B in tightly closed containers away from bright light. - Cover the smear completely with Albert stain A and follow the recommended staining time. - Drain off the excess Albert stain A without washing the slide. Washing at this stage may remove the stain and affect the staining result (HiMedia Laboratories, 2024). - Apply Albert stain B for the recommended time. Do not allow the staining solution to dry on the slide. - Wash the slide gently with water only after Albert stain B treatment. Avoid excessive washing. - Blot dry the slide using clean bibulous paper. Do not rub the stained smear. - Wear laboratory gloves, a lab coat and eye protection while handling bacterial specimens and staining reagents. - Suspected Corynebacterium diphtheriae cultures should be handled using appropriate containment and biosafety practices. Procedures that may produce aerosols should be performed in a [biological safety cabinet](https://biologynotesonline.com/biosafety-cabinet/) (Public Health Agency of Canada, 2011). - Decontaminate contaminated slides and other laboratory materials before disposal. ## References - Mitchell, B. I., & Markantonis, J. E. (2025). An underestimated pathogen: Corynebacterium species. Journal of Clinical Microbiology, 63(10), e01552-24. [https://doi.org/10.1128/jcm.01552-24](https://doi.org/10.1128/jcm.01552-24) - Sridharan, G., & Shankar, A. A. (2012). Toluidine blue: A review of its chemistry and clinical utility. Journal of Oral and Maxillofacial Pathology, 16(2), 251–255. [https://doi.org/10.4103/0973-029X.99081](https://doi.org/10.4103/0973-029X.99081) - Widra, A. (1959). Metachromatic granules of microorganisms. Journal of Bacteriology, 78(5), 664–670. [https://doi.org/10.1128/JB.78.5.664-670.1959](https://doi.org/10.1128/JB.78.5.664-670.1959) - World Health Organization. (2021). WHO laboratory manual for the diagnosis of diphtheria and other related infections. [https://iris.who.int/handle/10665/352275](https://iris.who.int/handle/10665/352275)