METACHROMATIC GRANULES STAINING

METACHROMATIC GRANULES STAINING

AIM

            To stain the Metachromatic granule of Corynebacterium diphtheriae by Albert’s staining procedure.

PRINCIPLE

            Metachromatic granule staining or Albert's staining technique is a type of Special staining technique since it is used to demonstrate a special structure in bacteria. It is chiefly used to demonstrate Metachromatic granules found in Corynebacterium diphtheriae (Gram positive bacilli). This bacterium is responsible for the disease Diphtheria. The name Corynebacterium is derived from the Greek word "Coryne", which refers to the Club shape of the bacteria seen in old cultures. The storage granules in this bacterium are called Metachromatic granules because it exhibits the property of Metachromasia, wherein the granules appear in a colour other than the colour used for staining. When stained with polychrome Methylene blue, the granules appear violet while the rest of the bacteria appears blue. The granules are made up of Polymetaphosphates and are known by various other names such as Volutin bodies, Babe-Ernst granules or Polar bodies. The bacterium produces the granules in abundance when grown on nutrient rich medium such as Loeffler's serum slope. Two types reagents that are used in the Metachromatic granule staining process. They are Albert's solution - A and Albert's solution - B. The Albert's solution - A consists of Toluidine blue, Malachite green, Glacial acetic acid and Ethyl alcohol. It acts as the staining solution. The Albert's solution - B contains Iodine and Potassium iodide in Water.  It acts as the Mordant, i.e an ion element that binds and holds a chemical dye, to make it stuck on the bacteria. When stained with Albert's stain, the Corynebacterium diphtheriae stains Green whereas the Granules stain Bluish black. Metachromatic granule staining helps to distinguish Corynebacterium diphtheriae from most of the short non-pathogenic diphtheroides which lack granules.

MATERIALS AND REAGENTS REQUIRED

i.       24 hours culture of Corynebacterium diptheriae.

ii.     Bunsen burner

iii.   Inoculating loop

iv.   Glass slide

v.     Hot plate

vi.   Microscope.

vii. Albert Stain – A (Toludine Blue – 0.15 g, Malachite Green – 0.2 g, Glacial acetic acid – 1 ml, 95 % Alcohol – 2 ml and Distilled water – 100 ml).

viii.  Albert Stain - B (Potassium Iodide – 3 g, Iodine – 2 g & Distilled water – 300 ml).

PROCEDURE

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

b)     Allow it to dry and fix with gentle heat.

c)     Stain with Albert Stain - A for 5 to 7 minutes.

d)     Drain the solution, do not wash.

e)     Apply Albert Stain - B for 1 to 2 minute.

f)      Rinse with water, blot dry and examine under oil immersion objective.

OBSERVATION AND RESULTS

            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.

Figure – 1: Green coloured Corynebacterium diptheriae rods and Bluish black Metachromatic granules (Source: paramedicsworld.com)