Albert Stain: Principle, Procedure and Results

Albert stain is a type of differential stain used for staining high-molecular-weight polymers of polyphosphate known as metachromatic granules or volutin granules found in Corynebacterium diphtheriae. Metachromatic granules are also found in Yersinia pestis, and Mycobacterium species.

It is named metachromatic because of its property of changing color i.e when stained with blue stain they appear red in color. When grown in Loffler’s slopes, C. diphtheriae produces a large number of granules.

Fig: Albert Staining -Corynebacterium diphtheriae
Fig: Albert Staining –Corynebacterium diphtheriae

Principle of Albert Staining:

Albert stain is basically made up of two stains that are toluidine blue’ O’ and malachite green both of which are basic dyes with high affinity for acidic tissue components like cytoplasm. The pH of Albert stain is adjusted to 2.8 by using acetic acid which becomes basic for volutin granules as the pH of volutin granule is highly acidic.

Therefore on applying Albert’s stain to the smear, toluidine blue’ O’ stains volutin granules i. e the most acidic part of cell and malachite green stains the cytoplasm blue-green. On adding Albert’s iodine due to effect of iodine, the metachromatic property is not observed and granules appear blue in colour.

Composition of Albert Stain

Albert stain is composed of two reagents:

Albert’s A solution consist of

  1. Toludine blue                      0.15 gm
  2. Malachite green                  0.20 gm
  3. Glacial acetic acid               1 ml
  4. Alcohol (95% ethanol)       2ml
  • Dissolve the dyes in alcohol and add 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 100ml

Albert’s B solution consist of

  1. Iodine                                    2gm
  2. Potassium iodide (KI)          3 gm

Dissolve KI in water and then add iodine. Dissolve iodine in potassium iodide solution 

Requirements: Smear on a glass slide, staining rack, Albert’s A solution, Albert’s B solution, blotting paper, immersion oil, microscope

Procedure of Albert Staining

  1. Prepare a smear on clean grease free slide.
  2. Air dry and heat fix the smear.
  3. Treat the smear with Albert’s stain and allow it to react for about 7 mins.
  4. Drain of the excess stain do not water wash the slide with water.
  5. Flood the smear with Albert’s iodine for 2 minutes.
  6. Wash the slide with water, air dry and observe under oil immersion lens.


If Corynebacterium diphtheriae is present in the sample it appears green colored rod-shaped bacteria arranged at an angle to each other, resembling English letter ‘L’, ‘V’, or Chinese letter pattern along with bluish-black metachromatic granules at the poles.


Albert stain helps to distinguish Corynebacterium diphtheriae from most of the short nonpathogenic diphtheroid which lack granules.


  1. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
  2. Review of Medical Microbiology and Immunology, Warren E. Levinson, 15th edition
  3. Microbiology: An Introduction. Gerald J. Tortora, Berdell R. Funke, and Christine L.Case. Pearson Education.
  4. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

3 thoughts on “Albert Stain: Principle, Procedure and Results

  1. what is the source of both stains organic or inorganic

    how get spores are seprated of corynebacterium diphtheriae

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