Albert’s Staining - Principle, Reagents, Procedure, Result and uses

 

Overview:

Albert’s staining technique is a unique technique to detect a special structure in bacteria. It is mostly application tool for the detection of metachromatic granules or volutin granules in Corynebacterium diphtheriae. Yersinia pestis and Mycobacterium species both have Metachromatic granules. The name Corynebacterium diphtheriae is derived from the Greek term “Coryne” which describes the club shape of bacteria common in ancient culture. C. diphtheriae is Gram-positive, Nonmotile, and non -spore-forming bacilli, which cause the disease diphtheria, is a nasopharyngeal infection (affecting the nose and throat) and also affects the skin. Bacterium-stored granules are known as metachromatic granules because they possess the ability to metachromasia; the granules look violet when stained with polychrome methylene blue, whereas the bacilli remain appears blue. The granules are made up of poly-metaphosphates and are also called various other names such as volutin bodies, Babe- Ernst granules or polar bodies Nessers’s stain and Pugh’s stain are the further staining method that is employed to identify the presence of granules in the bacterial cytoplasmic membrane. As the bacterium grows on Loeffler’s serum slant, the granules are produced.

Purpose:

To detect and primary identification of Corynebacterium diphtheriae based on the presence of metachromatic granulated bodies from a clinical specimen.

 Principle:

Alberts stain is performed with two stains that are Alberts stain A and Alberts stain B. Stain A contains toluidine blue and malachite green which are basic dyes with a high affinity for acidic tissue components like cytoplasm. The addition of Albert satin A to the smear volutin granules stained toluidine blue stain which is an acidic part of the cell and malachite green stains the cytoplasm Blue-green. Then adding Alberts stain B, the presence of Alberts’s iodine effect the blue volutin granules to change blue-black.

Sample container:

Sterile cotton swab

Sample collection procedure:

A sample for Albert’s staining test is taken on a sterile cotton swab by gently rubbing it over the throat and tonsils.

Required equipment and reagents:

Equipment:

        Bunsen burner

        Biological safety cabinet

        Light Microscope - 1000 magnification (oil immersion – lens)

Reagents:

Albert’s Stain A:  Composition of Glacial acetic acid, ethyl alcohol, Toluidine blue, Malachite green, and Distilled water.

Albert’s Stain B: Composition of Iodine crystals, Potassium iodide, and Distilled water.

Procedure:

1.    Prepare a thin smear on a clean dry glass slide.

2.    Allow it to dry and fix it with gentle heat

3.    Stain with Alberts stain A for 3-5 Minutes

4.    Drain the solution, do not wash, as it gets washed off and fades the stains, affecting the results.

5.    Apply with Alberts stain B for 1 Minute

6.    Rinse with water, air dry, and observe under oil immersion.

Interpretation:

The metachromatic granules of Diphtheria bacilli appear as black colored round shaped dots and the cytoplasm appears ‘L or V‘shaped green color.

Result:

If the presence of Corynebacterium diphtheriae appears green-colored rod-shaped bacteria arranged as ‘L, V ‘or Chinese letters along with black metachromatic granules at the poles.

 

Corynebacterium diphtheriae 

Uses:

This is used to distinguish Corynebacterium diphtheriae from nonpathogenic diphtheroid, which lacks granules.

References:

1.    Practical Medical Microbiology by Mackie & McCartney 14^th Edition, Page No-796 – 798.

2.    Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth edition by Washington et al., 2006.