practical exercises

Albert's staining technique


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. 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 is 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 bacillus 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.

When stained with Albert's stain, the bacillus stains green whereas the granules stain bluish black. There are two reagents that are used in the staining process: Albert's A solution and Albert's B solution. Albert's A solution consists of Toluidine blue, malachite green, glacial acetic acid, and ethyl alcohol. Albert's B solution contains Iodine and Potassium iodide in water.

Requirements: Smear on glass slide, staining rack, Albert's A solution , Albert's B solution, blotting paper, immersion oil, and microscope.

Place the slide on the staining rack with smear facing upwards. Cover the smear with Albert's A solution and allow it to act for 7 minutes. Pour off the stain and wash the slide in Albert's B solution (NOT tap water). Place the slide back on the rack and cover the smear with Albert's B solution and let it act for two minutes. Pour off the stain and wash the slide in tap water. Dry the slide using blotting paper, place a drop of immersion oil on the smear and observe under oil immersion objective.

Green coloured, rod shaped bacteria that are arranged at angles to each other resembling English letter 'L', 'V' or Chinese letter pattern along with bluish black metachromatic granules at the poles seen.

The given smear contains bacteria morphologically resembling Corynebacterium diphtheriae.

gram stain

Note: You will have search many fields to spot Acid Fast Bacilli.

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