Volutin granule staining

Volutin granule staining

Aim

To stain volutin granules by Albert’s staining method.

Principle

Certain bacteria have stored reserve food material in the form of granules which are utilized by the organism during starvation.  These granules are mainly of four types, Volutin granules, Sulphur granules, Nitrogenous reserve food material and Non-nitrogenous reserve food material.  Volutin granules are made up of polymetaphosphates and are known by various other names such as volutin bodies, Babe-Ernst granules, polar bodies, Metachromatic granules, etc.  Volutin granules are an intracytoplasmic storage form of complexed inorganic polyphosphate.  These granules are called as Volutin granules because these granules were first observed in Spirillum volutans.  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. Volutin granules are mainly present in bacteria such as Corynebacterium diphtheria, algae, fungi and protozoa.  These granules are stained by using Albert’s staining method.  When we apply Albert’s stain to the bacterial smear the stain Toluidine blue stains the most acidic part of cell that is Volutin Granules and Malachite green stains the cytoplasm.

Materials required

Lactobacillus (curd sample), Glass slide, staining rack, Albert’s A solution, Albert’s B solution, blotting paper, immersion oil, microscope

Albert’s A stain solution

1.      Toludine blue                                        0.15 gm

2.      Malachite green                                    0.20 gm

3.      Glacial acetic acid                                1 ml

4.      Alcohol (95% ethanol)                          2ml

5.      Distilled water                                      100 ml

Albert’s B solution consist of

1.          Iodine                                                  2gm

2.          Potassium iodide (KI)                         3 gm

3.          Distilled water                                    100 ml

Procedure

1.      A thin smear of curd was prepared on clean grease free slide.

2.      The smear was air dried and heat fixed.

3.      The smear was flooded with Albert’s stain for about 7 mins.

4.      The excess stain was drained off, but not washed with water.

5.      The smear was flooded with Albert’s iodine for 2 minutes.

6.      The slide was washed with water, air dried and observed under oil immersion lens.

Observation

The volutin granules appeared bluish black in colour while cytoplasm appeared light green in colour.

Result

Volutin granules were observed in bacteria present in curd.

 

 

 

 

 

 

 

 

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