Albert's stain: Introduction, Principle, Composition,Procedure and Result

Albert’s Stain: Introduction, Principle, Composition,Procedure and Result Interpretation

Introduction of Albert’s stain

Albert’s stain uses for staining Corynebacterium diphtheriae, an etiological agent of diphtheria and it is a serious infection that can lead to difficulty breathing, heart failure, paralysis, and even death. This stain helps to distinguish C. diphtheriae from most of the short non-pathogenic diphtheroids which lack granules.

Principle of Albert’s Stain

Albert’s meta-chromatic stain demonstrates the presence of metachromatic granules found in C. diphtheriae. The storage granules in this organism are called metachromatic granules because it exhibits the property of metachromasia, wherein the granules appear in a color other than the color used for staining. When stained with polychrome methylene blue, the granules appear violet while the rest of the bacillus appears blue. The granules are made up of polymetaphosphates and are known by various other names such as Babe-Ernst granules, volutin bodies, or polar bodies. The bacterium produces the granules in abundance when grown on nutrient-rich media such as Loeffler’s serum slope. The granules stain purple-black against the light green counter-stained cytoplasm. This helps to distinguish diphtheria bacillus from most of the short non-pathogenic diphtheroids which lack granules.

Composition of Albert’s Stain

There are two reagents that are used in the staining process: Albert’s A solution and Albert’s B solution.

Albert’s A solution

The composition of Albert’s stains contains the following ingredients-

To make 103 ml

Toluidine blue: 0.15 gm
Malachite green: 0.20 gm
acetic acid, glacial : 1 ml
Ethanol or methanol, absolute: 2 ml
Distilled water: 100 ml

Weigh the toluidine blue and malachite green. Transfer these to a brown bottle of just over 100 ml capacity.
Add the acetic acid and about 30 ml of the water. Mix well to dissolve the stains.
Add the remaining water and the ethanol and mix well.
Label the bottle, and store it in the dark at room temperature. The stain is stable for several months.
For use: Filter a small amount of the reagent into a stain dispensing container.

Albert’s B solution (Albert’s iodine solution)

This contains-

To make 300 ml:

Iodine (I) : 2gm
Potassium iodide (KI): 3 gm
Distilled water: 150 ml

Preparation

Weigh the KI, and transfer to a brown bottle premarked to hold 300 ml.
Add 60–80 ml of distilled water, and mix until the potassium iodide is completely dissolved.
Weigh the iodine, and add to the potassium iodide solution. Mix until the iodine is completely dissolved.
Makeup to the 150 ml mark with distilled water and mix well.
Label the bottle, and store it in a dark place at room temperature

Note: Iodine is injurious to health if inhaled or allowed to come in contact with the eyes, therefore handle it with care in a well-ventilated room.

Test Requirements

Specimen
Clean and grease-free glass slide
Inoculating loop or sterile bamboo stick
staining rack
Albert’s A solution
Albert’s B solution
blotting paper
Cedarwood oil
Microscope

The procedure of Albert Staining

Prepare a thin smear on a clean dry and grease-free glass slide.
Air dry and heat fix the smear.
Stain with Albert’s Stain A for 3-5 minutes.
Drain the solution, do not wash.
Flood the smear with Albert’s iodine i.e. Albert’s Stain B for 1 minute.
Rinse with water, blot dry and examine under oil immersion objective.

Result and Interpretation

Diphtheria bacillus ( C. diphtheriae): appears green colored rod-shaped bacteria arranged at an angle to each other, resembling letter V and L also called angular fashion like Chinese lettering along with bluish-black metachromatic granules at the poles.

Diphtheroids: Short and thick evenly stained lacking metachromatic granules

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