Principle:

Albert stain is done to identify Corynebacterium diphtheriae. This stain differentially stains the metachromatic granules and cytoplasm of this bacterium.

Procedure:

1. Prepare a smear and fix it by gently heat over a flame.
2. Cover the smear with Albert stain (Albert I) staining solution. Allow it for 5 minutes.
3. Pour off the stain after 5 minutes. Do not wash.
4. Cover the smear with Albert’s iodine (Albert II) solution for 2 minutes.
5. Pour off the stain after 2 minutes. Do not wash.
6. Blot to dry the smear.
7. Examine the smear on oil immersion objective lens (x 100) of a microscope.

Note: You might have noticed that we do not wash the stain unlike other staining methods because malachite green is highly soluble in water and quality of stain fades if we wash the stain.  

Observation

Body of Corynebacterium diphtheriae appears green and metachromatic granules appear blue black.

 

Preparation of Albert stain

Toludine blue                      0.15 gm

Malachite green                  0.20 gm

Glacial acetic acid               1 ml

Alcohol (95% ethanol)       2ml

Dissolve the dyes in alcohol and add to the distilled water and acetic acid

Add Distilled water to make the final volume 100ml.

Allow the stain to stand for one day and then filter.

Preparation of Albert’s iodine (Albert II)

Iodine                                    2gm

Potassium iodide (KI)          3 gm

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

 

 

 

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